ISEV2024 Abstract Book

The International Society for Extracellular Vesicles is the leading professional society for researchers and scientists involved in the study of microvesicles and exosomes. With nearly 1,000 members, ISEV continues to be the leader in advancing the study of extracellular vesicles. Founded in 2012 in Sweden, ISEV has since moved its Headquarters to the United States. Through its programs and services, ISEV provides essential training and research opportunities for those involved in exosome and microvesicle research.

Advancing extracellular vesicle research globally.

Our vision is to be the leading advocate and guide of extracellular vesicle research and to advance the understanding of extracellular vesicle biology.

The International Society for Extracellular Vesicles is the is the premier international conference of extracellular vesicle research, covering the latest in exosomes, microvesicles and more. With an anticipated 1,000+ attendees, ISEV2024 will feature presentations from the top researchers in the field, as well as providing opportunities for talks from students and early career researchers.

IOC Chairs: Cherie Blenkiron (New Zealand), David Greening (Australia)

IOC Members: Randy Carney (USA), Leslie Cheng (Australia), Eisuke Dohi (Japan), Qing-Ling Fu (China), Charles Lai (Taiwan), Metka Lenassi (Slovenia), Andreas Moeller (China), Jisook Moon (South Korea), Natalie Turner (Australia)

Jan Lötvall (Sweden)

0T04.O02 Cellular interaction and uptake of human endogenous retrovirus (HERV) envelope-displaying EVs

Dr. Zach Troyer, Sarah Marquez, PhD Olesia Gololobova, PhD Kenneth Witwer

0T04.O03 Functionalized engineered extracellular vesicles for targeted delivery to intervertebral disc cells

Ms Mia Kordowski, Dr Ana Salazar-Puerta, Ms María Rincon-Benavides, Mr Justin Richards, Dr Nina Tang, Dr Safdar Khan, Dr Elizabeth Yu, Dr Judith Hoyland, Dr Devina Purmessur, Dr Natalia Higuita-Castro

0T04.O04 Phospholipid scrambling: a novel regulator of extracellular vesicle cargo packaging and function

Ms Akbar Marzan, Ms Monika Petrovska, Professor Suresh Mathivanan, Sarah Stewart

0T04.O05 Quantitative features of extracellular vesicle-mediated crosstalk in multi-cellular 3D tumor models

Dr. Maria Harmati, Akos Diosdi, Ferenc Kovács, Ede Migh, Gabriella Dobra, Timea Boroczky, Matyas Bukva, Edina Gyukity-Sebestyen, Peter Horvath, Krisztina Buzas

FA01 Extracellular vesicles in human body fluids compete with virus particles for binding of phosphatidylserine receptors to prevent infection and transmission

Dr. Ruediger Gross, Hanna Reßin, Pascal von Maltitz, Dan Albers, Laura Schneider, Hanna Bley, Markus Hoffmann, Mirco Cortese, Dhanu Gupta, Miriam Deniz, Jae-Yeon Choi, Jenny Jansen, Christian Preußer, Kai Seehafer, Stefan Pöhlmann, Dennis R Voelker, Christiane Goffiniet, Elke Pogge-von Strandmann, Uwe Bunz, Ralf Bartenschlager, Samir El Andaloussi, Konstantin MJ Sparrer, Eva Herker, Stephan Becker, Frank Kirchhoff, Jan Münch, Janis A Müller

FA02 Machine learning models detect blood ‘fingerprints’ for accurate glioblastoma tumour surveillance

Dr Susannah Hallal, Dr Ágota Tűzesi, Dr Abhishek Vijayan, Dr Laveniya Satgunaseelan, Associate Professor Hao-Wen Sim, Associate Professor Brindha Shivalingam, Associate Professor Michael Buckland, Associate Professor Fatemeh Vafaee, Dr Kimberley Alexander

FA03 Barcoding of small extracellular vesicles with CRISPR-gRNA enables high-throughput, subpopulation-specific analysis of their release regulators

Prof. Dr. Ryosuke Kojima, Mr. Koki Kunitake, Professor Tadahaya Mizuno, Professor Yasuteru Urano

FA04 In vivo visualization of endothelial cell-derived extracellular vesicle formation in steady state and malignant conditions

Dr Georgia Atkin-Smith, Jascinta Santavanond, Amanda Light, Joel Rimes, Andre Samson, Jeremy Er, Joy Liu, Darryl Johnson, Melanie Le Page, Pradeep Rajasekhar, Raymond Yip, Niall Geoghegan, Kelly Rogers, Catherine Chang, Vanessa Bryant, Mai Margetts, Cristina Keightley, Trevor Kilpatrick, Michele Binder, Sharon Tran, Erinna Lee, Doug Fairlie, Dilara Ozkocak, Andrew Wei, Edwin Hawkins, Ivan Poon

LB01.O01 Fetal Exposure to Extracellular Vesicles. Is it safe?

Dr Ishmael Inocencio², Mr Naveen Kumar², A/Prof Rebecca Lim², Dr Tamara Yawno²

¹Hudson Institute Of Medical Research, Clayton, Australia, ²The Ritchie Centre, Clayton, Australia

LB01.O02 Engineered EVs as mRNA cancer vaccine delivery platform conferring immune modulation in HCC

Lecturer Cong He^1,2, Guangxin Shao², Dr. Yumin Li⁴, Dr. Xiao Yun⁵, Dr. Bo Sun⁴, Prof. Zhongdang Xiao⁴, Prof. Beicheng Sun³

¹Jiangsu Key Laboratory for Biofunctional Molecules, College of Life Science and Chemistry, Jiangsu Second Normal University, Nanjing, China, ²Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China, ³Department of Hepatobiliary Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, China, ⁴State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China, ⁵Department of General Surgery, The Affiliated Changzhou No. 2 People's Hospital of Nanjing Medical University, Changzhou, China

LB01.O03 Extracellular vesicles derived from human umbilical cord mesenchymal stem cells attenuate septic acute kidney injury by delivering miR-125a-5p and miR-125b-5p to inhibit inflammation and glycolysis

Dr. Feng Chen^1,2, Dr Tao-Tao Tang², Dr. Zhi-qing Chen², Prof. Zhong Wang¹, Dr Bi-Cheng Liu²

¹Tsinghua University, Beijing, China, ²Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, China

LB01.O04 Extracellular vesicles derived from clonally expanded, immortalized mesenchymal stromal cells lower Alzheimer's pathology in mice

Dr Lien Van Hoecke¹, Yanis Mouloud², Tobias Tertel², Prof Bernd Giebel², Prof Roosmarijn E Vandenbroucke¹

¹VIB-UGent, Gent (Zwijnaarde), Belgium, ²University Hospital Essen, University Duisburg-Essen, Essen, Germany

LB02.O01 Lipid droplets are packaged within extracellular vesicles during virus infection

Dr Ebony Monson¹, Miss Irumi Amarasinghe¹, Mr William Phillips², Dr Amy Baxter², Ms Camille Braganca¹, Ms Abbey Milligan², Dr Donna Whelan², Dr Eduard Willms², Professor Andrew Hill², Professor Karla Helbig¹

¹Department of Microbiology, Anatomy, Physiology & Pharmacology, La Trobe University, Melbourne, Australia, ²La Trobe Institute for Molecular Sciences, La Trobe University, Melbourne, Australia, ³Institute for Health and Sport, Victoria University, Melbourne, Australia

LB02.O02 Breast cancer sEVs binding to CCL2 and other cytokines directs cancer metastasis organotropism

Dr Luize Lima¹, Dr Sunyoung Ham^1,2,3, Professor Andreas Möller^1,2,3

¹Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, Australia, ²Department of Otorhinolaryngology, Head and Neck Surgery, Chinese University of Hong Kong, Shatin, Hong Kong, ³School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia

LB02.O03 Tubule derived exosomal Integrin reside in ECM microenvironment activates latent TGF-β1 in renal fibrosis

Phd Anran Shen^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}, Phd Xin Zhong^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}, Phd Ning Li^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}, Phd Yuqi Fu^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}, Professor Linli Lv^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}

¹Medical School Of Southeast University, Nanjing, China

LB02.O04 Enhanced Packaging of U6 Small Nuclear RNA and Splicing-Related Proteins into Extracellular Vesicles During HIV Infection

Dr Yiyao Huang^1,2, Ahmed Abdelgawad³, Dr Olesia Gololobova¹, Zhaohao Liao¹, Xinyu Cong⁴, Associate Professor Mona Batish³, Prof. Lei Zheng², Dr Kenneth Witwer^1,5,6

¹Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, United States, ²Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China, ³Department of Medical and Molecular Sciences, University of Delaware, Newark, United States, ⁴Division of Biostatistics and Bioinformatics, Department of Environmental and Public Health Sciences, University of Cincinnati, Cincinnati, United States, ⁵Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, United States, ⁶Richman Family Precision Medicine Center of Excellence in Alzheimer's Disease, Johns Hopkins University School of Medicine, Baltimore, United States

LB03.O01 Protein mutation identification and monitoring using exosomes-SERS-AI

Master's degree Kim Seungmin, Doctor of Philosophy (Ph.D.) ByeongHyeon Choi, Hyunku Shin, Master's degree Kihun Kwon, Doctor of Philosophy (Ph.D.) Sung Yong Lee, Doctor of Philosophy (Ph.D.) Hyun Koo Kim, Doctor of Philosophy (Ph.D.) Yeonho Choi

¹Department of Biomedical Engineering, Korea University, Seoul, South Korea, ²Korea Artificial Organ Center, Korea University, Guro, Republic of Korea, ³Department of Thoracic and Cardiovascular Surgery, Korea University, Guro, Republic of Korea, ⁴Exopert Corporation, Seoul, Republic of Korea, ⁵Department of Internal Medicine, Korea University, Guro, Republic of Korea, ⁶School of Biomedical Engineering, Korea University, Seoul, Republic of Korea

LB03.O02 Proteomic investigations of mechanisms underlying high-dose sodium ascorbate in sepsis in circulating plasma extracellular vesicles

Dr Samantha Emery-Corbin^{1. Division of Advanced Technology and Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia 2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia}, Dr Jumana Yousef^{1. Division of Advanced Technology and Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia 2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia}, Professor Yugeesh R Lankadeva^{3. The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia 4. Department of Critical Care, University of Melbourne, Parkville, Victoria, Australia}, Professor Rinaldo Bellomo^{4. Department of Critical Care, University of Melbourne, Parkville, Victoria, Australia 5. Department of Intensive Care, Austin Hospital, Melbourne, Victoria, Australia 6. Australian and New Zealand Intensive Care Research Centre (ANZIC-RC), Monash University, Melbourne, Victoria, Australia 7. Department of Intensive Care, Royal Melbourne Hospital, Parkville, Victoria, Australia 8. Data Analytics Research and Evaluation Centre, Austin Hospital, Melbourne, Victoria, Australia}, Dr Fumitaka Yanase^{5. Department of Intensive Care, Austin Hospital, Melbourne, Victoria, Australia 6. Australian and New Zealand Intensive Care Research Centre (ANZIC-RC), Monash University, Melbourne, Victoria, Australia}, Associate Professor Mark P Plummer^{9. Department of Intensive Care, Royal Adelaide Hospital, Adelaide, South Australia, Australia}, Professor Clive N May^{3. The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia 4. Department of Critical Care, University of Melbourne, Parkville, Victoria, Australia}, Dr Laura F Dagley^{1. Division of Advanced Technology and Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia 2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia}

¹Division of Advanced Technology and Biology Division, Walter And Eliza Hall Institute Of Medical Research, Melbourne, Australia, ²Department of Medical Biology, University of Melbourne, Melbourne, Australia, ³The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Australia, ⁴Department of Critical Care, University of Melbourne, Melbourne, Australia, ⁵Department of Intensive Care, Austin Hospital, Melbourne, Australia, ⁶Australian and New Zealand Intensive Care Research Centre (ANZIC-RC), Monash University, Melbourne, Australia, ⁷Department of Intensive Care, Royal Melbourne Hospital, Melbourne, Australia, ⁸Data Analytics Research and Evaluation Centre, Austin Hospital, Melbourne, Australia, ⁹Department of Intensive Care, Royal Adelaide Hospital, Adelaide, Australia

LB03.O03 Framework for isolating EVs from neurons and measuring their cargo

Senior Staff Scientist Dmitry Ter-Ovanesyan¹, Sara Whiteman, Tal Gilboa, Siddharth Iyer, Bogdan Budnik, Aviv Regev, George Church, David Walt

¹Wyss Institute, Harvard University, Boston, United States

LB03.O04 Identification of a circulating HNSCC proteomic biomarker signature from peripheral and local tumor plasma before/after tumor removal surgery

Dapi Menglin Chiang, Dr. Christina Ludwig, Dr. Chen Meng, PD Dr. Marlene Reithmair, Laura Benecke, Yannik da Silva, PD Dr. Laurent Müller, Prof. Dr. Michael W. Pfaffl

LB04.O01 Detection of EVs in hepatotoxicity using CD9-mEmerald reporter mice

Section Chief Ryuichi Ono¹, Mie Naruse², Makiko Kuwagata¹, Yusuke Yoshioka³, Yoko Hirabayashi¹, Takahiro Ochiya³, Masahito Ikawa⁴, Satoshi Kitajima¹

¹National Institute Of Health Sciences, Japan, Kawasaki, Japan, ²National Cancer Center Research Institute, Tokyo, Japan, ³Tokyo Medical University, Tokyo, Japan, ⁴Osaka University, Osaka, Japan

LB04.O02 Drafting the proteome and lipidome atlas of circulating small EVs in humans: Universal protein and lipid features

Dr Alin Rai¹, Prof David Greening

¹Baker Heart and Diabetes Institute, MELBOURNE, Australia

LB04.O03 Enhanced endosomal escape by amphipathic-charged engineered extracellular vesicles

Dr Hema Saranya Ilamathi^1,2, Dr Doste Mamand^1,2, Anna Maria Zimbo³, Dr Samir El Andaloussi^1,2, Dr Oscar Wiklander^1,2

¹Division of Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden, ²Center for Cell Therapy and Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital, Stockholm, Sweden, ³Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Italy

LB04.O04 Depletion-zone isotachophoresis of extracellular vesicles: a separation method suitable for biomarker discovery applications that can increase purity over current techniques

Dr. Andrea Capuano¹, Meia Numan², Prof. Thomas Hankemeier¹

¹University of Leiden, Leiden, The Netherlands, ²EXIT071 B.V., Leiden, The Netherlands

OF09.O02 Enhancing breast cancer diagnosis and subtyping: multiplexed profiling of dual surface protein-expressing single extracellular vesicles via nano-flow cytometry

Phd Yunyun Hu, Haonan Di, PhD Ye Tian, Yiyin Weng, Jujiang Guo, Professor Xiaomei Yan

OF09.O03 Analyzing copy number variation status in extracellular vesicles as novel clinical biomarkers of high-grade serous ovarian carcinoma

MD. Ryosuke Uekusa, Dr. Akira Yokoi, Dr. Kosuke Yoshida, Dr. Jyuntaro Matsuzaki, Dr. Yusuke Yamamoto, Dr. Hiroaki Kajiyama

OF09.O04 Extracellular vesicles as biomarkers of endometriosis – a comparison between peritoneal fluid and peripheral blood

Ms Chloe James, Ms Johanna Farley, Ms Natasha Borash, Dr Hannah Nazri, Ms Anna Tresso, Dr Shima Bayat, Dr Anup Shah, Dr Joel R Steele, Prof Ralf B Schittenhelm, Dr Shanti Gurung, Prof Caroline E Gargett, Prof Christian M Becker, Prof Beverley Vollenhoven, Dr Thomas Tapmeier

OF09.OWP01 Paracrine miRNA communication via extracellular vesicles: regulating glucose metabolism and fetal growth in gestational diabetes mellitus between maternal adipose tissue and placenta in vivo

Dr Nanthini Jayabalan, Dr Andrew Lai, Dr Dominic Guanzon, Dr Soumyalekshmi Nair, Mrs Katherin Scholz-Romero, Valeska Ormazabal, Professor Aasa Handberg, Dr Flavio Carrion, Professor Harold McIntyre, A/ Professor Martha Lappas, Professor Carlos Salomon

OF09.OWP02 Profiling of single-vesicle surface proteins via droplet digital immuno-PCR for multi-subpopulation extracellular vesicles counting towards cancer diagnostics

Dr. Huixian Lin, Dr. Chunchen Liu, Prof. Bo Li, Prof. Lei Zheng

OF09.OWP03 Proteomics of salivary exosomes: a potential breakthrough for screening of oral cancer among tobacco consumers

Ms Afsareen Bano, Dr. RASHMI BHARDWAJ

OF10.O02 Are the extracellular vesicles released by Parabacteroides goldsteinii anti-inflammatory?

Dr Simon Swift, Yevetta Xiang, Dr Priscila Dauros-Singorenko, Professor Hsin-Chih Lai

OF10.O03 Physicochemical and immunomodulatory properties of Bifidobacterium- derived extracellular vesicles with anti-allergic potential

Ms Dominika Kozakiewicz, Dr Agnieszka Razim, Dr Sabina Górska

OF10.O04 Bacterial growth conditions and mechanisms of biogenesis alter the composition and functions of bacterial extracellular vesicles

Dr Ella Johnston, Dr Lauren Zavan, Associate Professor David Greening, Professor Andrew Hill, Associate Professor Maria Kaparakis-Liaskos

OF10.O05 Klebsiella pneumoniae-derived extracellular vesicles facilitate bacterial translocation from the gastrointestinal tract to the liver by inducing the M2-like macrophage phenotype

Hitoshi Tsugawa, Student Shogo Tsubaki, Dr Takuma Araki, Dr. Yusuke Yoshioka, Dr. Juntaro Matsuzaki, Dr Hitoshi Tsugawa

OF11.O02 Endothelial cell-derived extracellular vesicles modulate bone marrow in cardiovascular disease

Mr Lewis Timms, Dr Daniel Radford Smith, Prof Daniel C. Anthony, Associate Prof Naveed Akbar, Prof Robin P. Choudhury

OF11.O03 Renal tubule-derived EVs carrying complement 3 aggravate vascular calcification of CKD by downregulating autophagy in vascular smooth muscle cells

Yuxia Zhang, Associate Researcher Taotao Tang, Professor Rining Tang, Professor Bicheng Liu

OF11.O04 Small extracellular vesicles (sEV) mediate tubular ferroptosis propagation in the transition from acute to chronic kidney disease

Ms Xiangju Wang, A/Prof Chang Seong Kim, Mr Benjamin Adams, Dr Monica Ng, A/Prof Helen Healy, Dr Andrew Kassianos

OF11.O05 Role of cardiomyocyte-derived extracellular vesicles in post-ischemic cardiac remodeling

Phd Lélia Borowski, Cécile Devue, Paul Alayrac, Jean Sébastien Silvestre, Chantal M. Boulanger, Xavier Loyer, Stéphane Camus

OF12.O02 Ex vivo imaging of exosomes in Drosophila secondary cells reveals a novel membrane microdomain involved in exosome biogenesis

Mr Adam Wells, Dr Pauline Marie, Dr Claudia C. Mendes, Dr Shih-Jung Fan, Dr Mark Wainwright, Dr. Preman Singh, Dr. Bhavna Verma, Professor Clive Wilson, Dr Deborah Goberdhan

OF12.O03 Mutation in ESCRT-II component VPS25 alters small extracellular vesicle processing in lethal neonatal encephalopathy

Ioannis Isaioglou, Lama AlAbdi, Yossef Lopez de los Santos, Muhammad Tehseen, Mansour Aldehaiman, Gloria Lopez-Madrigal, Norah Altuwaijri, Maya Ayach, Ashraf Al-Amoudi, Rachid Sougrat, Vlad-Stefan Raducanu, Amani Al-Amodi, Hessa Alsaif, Firdous Abdulwahab, Amal Jaafar, Tarfa Alshidi, Adriana Montaño, Kara Klemp, Ellen Totten, Wesam Kurdi, Samir Hamdan, Stephen Braddock, Fowzan Alkuraya, Jasmeen Merzaban

OF12.O04 Expressing the human proteome in Saccharomyces cerevisiae as a model for advancing extracellular vesicle biology

Mr. Joseph Trani, Dr. Aashiq H. Kachroo, Dr. Christopher L. Brett

OF12.O05 The unexpected formation of the footprint of death during apoptosis

Ms Stephanie Rutter, Ms Amy Hodge, Ms Dilara Ozkocak, Dr Julian Ratcliffe, Dr Taeyoung Kang, Dr Niall Geoghegan, Dr Pradeep Rajasekhar, Dr Georgia Atkin-Smith, Dr Ivan Poon

OF13.O02 Shear Stress-Induced Extracellular Calcium Influx: A Pivotal Trigger Amplifying Mesenchymal Stem Cell-derived Extracellular Vesicle Production

Ph.D candidate Youngju Seo¹, Ibio hyejin Kang, Ibio, Mechanical Engineering Jaesung Park

¹Postech, South Korea

OF13.O03 Acellular therapy with umbilical cord mesenchymal stem-derived small extracellular vesicles or mitochondria, as a new treatment for osteoarthritis

Miss Cynthia Garcia Guerrero, Patricia Luz-Crawford, Ana Maria Vega-Letter, Carolina Pradenas, Alexander Ortoff, Jose Barraza, Fernando Figueroa, Maroun Khoury, Aliosha Figueroa, Yeimi Herrera

OF13.O04 Improved neurological recovery in a rodent ischemic stroke model using human GMP compatible embryonic vascular progenitor cell exosomes

Scientist Jieun Lee

OF13.O05 Examining the efficacy of Immortalised Human Amniotic Epithelial Derived Extracellular Vesicles in a rodent model of perinatal brain injury.

Mr Naveen Kumar¹, Dr Ishmael Inocencio, Dr Tamara Yawno, Dr Dandan Zhu, Associate Professor Rebecca Lim

¹Hudson Institute Of Medical Research, Clayton, Australia

OF13.O06 Small extracellular vesicles from metabolically reprogrammed mesenchymal stem/stromal cell as a potential immunosuppressive mechanism

Miss Eliana Lara Barba, Miss Yesenia Flores Elías, Mr Felipe Bustamente Barrientos, Miss María Jesús Araya, Miss Yeimi Herrera Luna, Miss Noymar Luque Campos, Ms Ana María Vega Letter, Ms Patricia Luz Crawford

OF14.O02 Biomarkers from neuronal-enriched EVs predict resilience to Alzheimer's disease in the presence of APOE ε4 allele: findings from a large longitudinal study

Dimitrios Kapogiannis, Maja Mustapic, Carlos Nogueras-Ortiz, Apostolos Manolopoulos, Francheska Delgado-Peraza, Pamela Yao, Krishna Pucha, Mark A Espeland, Luigi Ferrucci, Stephen R. Rapp, Susan M. Resnick

OF14.O03 Identification of PECAM1+ and ITGB1+ plasma extracellular vesicle as biomarkers of unruptured intracranial aneurysm based on single extracellular vesicle proximity barcoding assay

Dr. Hao Tian, Dr. Yanling Cai, Mrs. Fang Wang, Professor Chuanzhi Duan, Dr. Haitao Sun

OF14.O04 A liquid biopsy approach: Neural networks-based identification of brain tumor exosomes via their SERS signatures

Hülya Torun^{Stanford and Koç University}, PhD Ugur Parlatan, Chris Nguyen, BS Tim Valencony, MS Furkan Kaysin, PhD Ozgur Albayrak, MD Ibrahim Kulac, MD, PhD Candidate Oguz Baran, MD, PhD Candidate Goktug Akyoldas, MD Ihsan Solaroglu, PhD Utkan Demirci, PhD, DVM Demir Akin, PhD Mehmet Ozgun Ozen

OF14.O05 Nanoscale Flow Cytometry Quantification of Blood-based Extracellular Vesicle Biomarkers Distinguishes MCI and Alzheimer's Disease.

Dr. Thamara Dayarathna, Dr. Austyn Roseborough, Dr. Janice Gomes, Dr. Reza Khazaee, Dr. Shaun Whitehead, Dr. Hon Sing Leong, Professor Stephen Pasternak

OF14.O06 Comparative analysis of plasma-derived small extracellular vesicles and whole plasma-derived miRNAs as biomarker targets for Parkinson's disease

Ms. Sanskriti Rai, Mr. Rishabh Singh, Dr. Prahalad Singh Bharti, Dr. Roopa Rajan, Dr. Saroj Kumar

OF15.O02 Encapsulate the components of CRISPR/Cas9 into extracellular vesicles by protein palmitoylation

Ph.d Yaoyao Lu, research assistant Nathalie Majeau, Ph.D Gabriel Lamothe, Research assistant Joel Rousseau, Professor Jacques-P Tremblay

OF15.O03 Nanofluidic platform with ultrahigh-throughput for versatile loading of small extracellular vesicles

Hui Yang, PhD Rui Hao, PhD Candidate Zitong Yu, PhD Candidate Shi Hu, PhD Yanhang Hong, Professor Yi Zhang, Sihui Chen

OF15.O05 Affinity-based bategorization of antimicrobial and cell-penetrating peptides in vesicle interactions

Phd Tamas Beke-Somfai, Tasvilla Sonallya, PhD Imola Cs. Szigyarto, PhD Tunde Juhasz, Kinga Ilyes, Priyanka Singh, Delaram Khamari, DSc Edit Buzas, PhD Zoltan Varga

OF15.O06 Tonicity-driven osmotic cargo loading for engineering extracellular vesicles

Professor Yoon-Kyoung Cho, Chaeeun Lee, Sumit Kumar

OF16.O02 Cancer-derived small extracellular vesicles reprogram the DNA methylome of normal epithelial cells adjacent to the primary cancer

Hanguo Jiang, Professor Zhijie Chang

OF16.O03 ULK1 enhances biogenesis of oncogenic small extracellular vesicles (sEV) to induce tumorigenesis and metastasis in hepatocellular carcinoma (HCC)

Mr Samuel Wan Ki Wong, Miss Claudia Wing Lam Tam, Mr Nicolas Cheuk Hang Lau, Dr Xiaowen Mao, Prof Judy Wai Ping Yam

OF16.O04 Extracellular vesicle encapsulated miR-1307-5p confers chemoresistance by modulating cancer stem cells in oral cancer

Mrs. Aditi Patel^{Ahmedabad University, Ahmedabad, Gujarat, India}, Dr. Shanaya Patel^{Ahmedabad University, Ahmedabad, Gujarat, India}, Ms. Vaishnavi Patel^{Ahmedabad University, Ahmedabad, Gujarat, India}, Dr. Vivek Tanavde^{Ahmedabad University, Ahmedabad, Gujarat, India}

OF16.O05 PTPN23 Downregulation by WDR4 Determines the Exosome Secretion Fate of MVB to Promote Cancer Metastasis and Immune Evasion

Phd Candidate Nai Yang Yeat, Li-Heng Liu, Yu-Hsuan Chang, PhD Kui-Thong Tan, PhD Ruey-Hwa Chen

OF16.O06 The elevated ECM1 protein in circulating sEVs is associated with integrin-β2, and it mediates the enhanced breast cancer growth and metastasis under obesity conditions

Mr Keyang Xu

OS17.O02 Extracellular vesicles carrying tenascin-C: a

Highly sensitive & specific, multi-omics compatible, pan-tumor liquid biopsy platform

Dr. Yanan Zhang, Dr. Alexander Koepp, Adela Brzobohata, Dr. Emanuele Puca, Dr. Roberto De Luca, Dr. Cesare Di Nitto, Dr. Teresa Hemmerle, Dr. Yingchao Meng, Dr. Stavros Stavrakis, Dr. Alexander Ring, Prof. Dr. Andreas Wicki, Prof. Dr. Julia Furtner, Dr. Caroline Hertler, Dr. Marcel Buehler, Prof. Dr. Michael Weller, Dr. Emilie Le Rhun, Prof. Dr. Dario Neri, Dr. Tobias Weiss

OS17.O03 Lectin microarray profiling of plasma EV glycosylation for gastric cancer diagnosis, prognosis, and prediction of immunotherapy response

Dr Fanqin Bu, Dr Guangyu Ding, Dr Yunzi Wu, Dr Chenjie Xu, Dr Liyi Bai, Professor Xintao Qiu, Professor Pengfei Yu, Professor Yibin Xie, Professor Li Min

OS17.O04 Higher concentration of small extracellular vesicles-GCC2 in the pulmonary veins as a prognostic biomarker for patients with surgically resected lung adenocarcinoma

Dr Byeong Hyeon Choi, MD Jun Hee Lee, Dr Ok Hwa Jeon, Mr Chang Gun Kim, Professor Yeonho Choi, Professor Yong Park, Professor Ji-Ho Park, Professor Sunghoi Hong, Professor Hyun Koo Kim

OS17.O05 Single EV protein and RNA expression detection via an in-situ concurrent technology: sEV-PREDICT for PD-L1 positive extracellular vesicles analysis in plasma

Student Tong Liao, PhD Weilun Pan, Professor Lei Zheng, Professor Bo Li

OS18.O02 Deletion of P2RX7 ameliorates cognitive dysfunction and neurodegeneration in PS19 mice via suppression of extracellular vesicle mediated tau transfer

Seiko Ikezu, Post doctoral fellow Victor Santos, Postdoctoral fellow Mohammad Abdullah, Technician Justice Ellison, Research associate Zhi Ruan, Professor Tsuneya Ikezu

OS18.O03 Circulatory extracellular vesicles transport complement C1q for promoting neuronal amyloid-beta production in alzheimer's disease

Dr Yang Yu, Dr Wenjun Xiao, Associate Professor Zhigang Li

OS18.O04 APOE genotype alters lipidomic and proteomic profiling of Alzheimer's disease brain-derived extracellular vesicles reflecting inflammation and lipid dysbiosis

Dr. Zhengrong Zhang, Dr. Kaiwen Yu, Dr. Hanmei Bao, Dr. Michael DeTure, Ms. Clara Scholes, Dr. Yang You, Dr. Seiko Ikezu, Dr. Dennis Dickson, Dr. Xianlin Han, Dr. Junmin Peng, Dr. Tsuneya Ikezu

OS18.O05 Extracellular vesicle remodeling in response to mutant huntingtin

Natayme Rocha Tartaglia, Francesca Farina, Morgane Fontaine, Johanna Cormenier, Damarys Loew, Florent Dingli, Heike Rohweder, Chantal Bazenet, Emmanuel Brouillet, Lorena Martin-Jaular, Frédéric Saudou, Clotilde Théry, Christian Neri

OS19.O02 Simultaneous tracking of big and small extracellular vesicles via multiplexed bioluminescence resonance energy transfer reporters

Dr. Anthony Yan-Tang Wu, Ms. Wendy Wan-Ting Wong, Ms. Shannon Yu-Hsuan Yeh, Ms. Angela Yun-Fei Zhang, Dr. Charles Pin-Kuang Lai

OS19.O03 Nanoscale visualization and tracking of small extracellular vesicles and their DNA-associated cargo in the recipient cells using single-molecule localization microscopy

Dr Basant Kumar Thakur, Prof. Dr. Cremer Christoph, Dr Jamal Ghanam, Prof. Dr. Dirk Reinhardt, Dr. Xiaomin Liu, Xingfu Zhu, Dr. Venkatesh Kumar Chetty

OS19.O04 Endovesiclosis: a novel technology for quantum dot-based extracellular vesicles labeling

Dr. Koushik Debnath^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Sadiq Umar^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Kasey Leung^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Chun-Chieh Huang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Miya Kang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Yu Lu^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Prof. Praveen Kumar Gajendrareddy^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Prof. Sriram Ravindran^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}

OS19.O05 Tracing extracellular vesicle subpopulations using HaloTag fusion proteins

Ms. Willemijn De Voogt, Dr. Sander Kooijmans, Mr. Kevin Harrijvan, Ms. Soultana Karakyriakou, Dr. Richard Wubbolts, Dr. Pieter Vader

OS20.O02 Bottom-up assembly of synthetic extracellular vesicles for the regulation of immune activity in atopic dermatitis

Phd Student Amelie Chane, PhD Student Meline Macher, PhD Student Sarada Muduli, Doctor (Dr.) Ilia Platzman, Professor (Prof.) Joachim Spatz

OS20.O03 Precise and robust cell-free synthesis of artificial extracellular vesicles

Mr. Tanner Henson, Alessandra Arizzi, Hyehyun Kim, David Wang, Neona Lowe, Conary Meyer, Keerthana Ananda, Dr. Erkin Seker, Dr. Randy Carney, Dr. Aijun Wang, Dr. Cheemeng Tan

OS20.O04 Extracellular vesicle-iron oxide nanoparticle hybrid system: ExoFeR to induce ferroptosis and reverse therapeutic resistance in cancer

Assistant Professor Akhil Srivastava, Anjugam Paramanantham, Yariswamy Manjunath, Rahmat Asfiya, Siddharth Das, Grace McCully, Assistant Professor Jussuf Kaifi

OS20.O05 Novel noncoding RNA drugs bioinspired by therapeutic EV cargo

Professor And Executive Director Eduardo Marban, Dr Ahmed Ibrahim, Dr Russell Rogers, Dr Alessandra Ciullo, Dr Ke Liao

OS21.O02 Synergistic combination of extracellular vesicle formulations for the treatment of KRAS-driven cancer

Dr. Cao Dai Phung, Thi Tuyet Trinh Tran, Brendon Zhi Jie Yeo, Gao Chang, Rebecca Carissa Prajogo, Migara Kavishka Jayasinghe, Thi Thanh Xuan Dang, Yuan Ju, Mai Trinh Nguyen, Boya Peng, Hong Anh Le, Eric Yew Meng Yeo, Bonney Glenn, Boon Cher Goh, Dahai Luo, Wai Leong Tam, Minh TN Le

OS21.O03 Fecal derivatives and extracellular vesicles enhance response to immune checkpoint blockade

Postdoctoral Fellow Golnaz Morad, Brenda Melendez, Sarah Johnson, Manoj Chelvanambi, Matthew Wong, Ashish Damania, Nadim Ajami, Jennifer Wargo

OS21.O04 Designed extracellular vesicles for therapeutic applications in neurofibromatosis type 1 (NF1)

Miss Maria Angelica Rincon-Benavides, Miss Aarti Patel, Mrs. Tatiana Cuellar-Gaviria, Mr. Ethan Stamas, Mr. Jad Hussein, Mr. Diego Alzate-Correa, Miss Yuyan Yu, Miss Cintia Gomez, Mrs. Heather Powell, Mr. Daniel Gallego-Perez, Mrs. Natalia Higuita-Castro

OS21.O05 Extracellular vesicles-mediated targeting of the glioma microenvironment

Miss Jacqueline YT Yeo, Dr Yuganthini Vijayanathan, Miss Janice HY Tan, Mr Fikri Mohamad, Ms Rachel LY Ho, Miss Nurashikin Abdul Halim, Dr Tatsuya Kozaki, Mr Zhi Wei Zhang, Dr Hai Tao Tu, Dr Jann Sarkaria, Dr Florent Ginhoux, Dr Li Zeng, Dr Ivy Ho

OS21.O06 Therapeutic exosomes targeting neuroendocrine prostate cancer

Associate Professor Sharanjot Saini, Dr Sandip Nathani, Ms Diana Asante, Ms Amritha Sreekumar, Dr. Matthew Simmons

OS23.O02 EXO-CD24-is a revolutionary immunomodulator that is smarter than steroids: the road from an idea to a ground-breaking reality

Shiran Shapira, Prof., MD, MHA, CMO Nadir Arber

OS23.O03 In situ production of engineered extracellular vesicles for efficient delivery of protein biotherapeutics

Samantha Roudi, Post-Doc Dhanu Gupta, Professor Samir El Andaloussi

OS23.O04 Treatment of NASH utilizing engineered extracellular vesicles with surface-displayed FGF21 and encapsulated miR-223

Associate Professor Kyungmoo Yea, Professor Moon-Chang Baek

OS23.O05 Engineered red blood cells extracellular vesicles as therapeutic strategy for the treatment of renal diseases

Dr Alessia Brossa¹, Dr Michela Arena¹, Dr Alessandro Gori², Dr Marina Cretich², Dr Ilaria Giusti³, Prof Vincenza Dolo³, Benedetta Bussolati¹

¹Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy, ²Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Milano, Italy, ³Pathology Unit, Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy

OS23.O06 Engineered let-7a-5p-enriched extracellular vesicles

To alleviate inflammation for acute lung injury

Mr. Sin-Yu Chen, Mr. Po-Chen Li, Dr. Tai-Shan Cheng, Ms. Hsin-Tung Chen, Ms. Wei-Ni Tsai, Dr. Hsiu-Jung Liao, Professor Ly James Lee, Professor Chi-Ying F. Huang

OS24.O02 Microfluidic isolation of complete platelet-free plasma for enhanced detection of blood extracellular vesicles (EV) microRNAs and surface proteins

Sheng Yuan Leong, Ms. Wan Wei Lok, Ms Hui Min Tay, Mr. Hong Boon Ong, Dr. Poh Loong Soong, Dr. Roger Sik Yin Foo, Dr. Rinkoo Dalan, Dr. Han Wei Hou

OS24.O03 Optimized isolation of fecal bacterial extracellular vesicles and its application in colorectal cancer diagnostics

Student Yicong Xue, doctor Zihao Ou, Professor Bo Situ, Professor Lei Zheng

OS24.O04 A low-cost kit for gentle, effective and timely extracellular vesicle (GET EV) isolation: accelerating development of RNA-based liquid biopsies for neuroendocrine neoplasms

Mr. Boyang Su, Dr. Morteza Jeyhani, Dr. Xiaojing Yang, Jina Nanayakkara, Reese Wunsche, Dr. Neil Renwick, Dr. Scott Tsai, Dr. Hon Leong

OS24.O05 Light-induced extracellular vesicle adsorption

Colin Hisey, Xilal Rima, Colin Hisey, Chiranth Nagaraj, Sophia Mayone, Kim Nguyen, Sydney Wiggins, Chunyu Hu, Divya Patel, David Wood, Zachary Schultz, Derek Hansford, Eduardo Reategui

OS24.O06 Hybrid microfluidic tangential flow filtration and herringbone microstructures for rapid extracellular vesicles isolation from blood plasma

Mr. Jia Yi Voo, Dr. Sheng Yuan Leong, Dr. Rinkoo Dalan, Prof. Han Wei Hou

OT01.O02 miR-151a-5p cargo in neuron-derived extracellular vesicles is a biomarker and mediator of antidepressant treatment response

PhD Dariusz Żurawek, PhD Alice Morgunova, PhD Laura Fiori, M.S. Jennie Yang, PhD Claudia Belliveau, M.S. Pascale Ibrahim, M.S. Jean Francois Théroux, M.S. Ryan Denniston, Prof. Sidney H. Kennedy, Prof. Raymond W. Lam, Prof. Roumen Milev, PhD Susan Rotzinger, MD Claudio N. Soares, MD Valerie H. Taylor, MD Rudolf Uher, PhD Jane A. Foster, MD Benicio N. Frey, PhD Cecilia Flores, PhD Corina Nagy, MD Gustavo Turecki

OT01.O03 First-in-human clinical trial of allogeneic platelet extracellular vesicles as a potential therapeutic for chronic wound healing

Dr. Jancy Johnson, Dr. Gregor Lichtfuss

OT01.O04 Navigating the regulatory and commercial challenges of translating extracellular vesicle-based biomarkers into clinical practice

Dr Olasehinde Olusanya

OT01.OWP01 Serum extracellular vesicle profiling to determine extracorporeal photopheresis response in graft versus host disease

Miss Kimberly Schell, Doctor Aisling Flinn, Professor Matthew Collin, Professor Andrew Gennery, Doctor Rachel Crossland

OT01.OWP02 Cracking the code: Understanding oncogenic small EVs in pancreatic cancer diagnostic landscape

Ms Arunima Panda, Mrs Ilaria Casari, Dr Abir Halder, Dr Walid Abu Shawish, Dr Danielle Dye, Prof Krish Ragunath, A/Prof David Greening, Prof Marco Falasca

OT01.OWP03 Hydrogel loaded with microalgae-derived extracellular vesicles for preventing skin ultraviolet damage

Miss Jiarong Cui, Prof. Min Zhou

OT02.O02 Multi-omic insights into extracellular vesicles mediating drug resistance in leishmania parasites

Associate Professor Christopher Fernandez-Prada

OT02.O03 Common mechanisms of protection against pathogenic gram-negative bacteria by host-derived sEV

Dr. Adam Fleming, Mr. Graham Matulis, Ms. Heather Hobbs, Dr. Valentin Giroux, Mr. Hunter Mason, Dr. Weidong Zhou, Dr. Valerie Calvert, Dr. Nitin Agrawal, Professor Emanuel Petricoin, Dr. Rekha Panchal, Professor Igor Almeida, Dr. Sina Bavari, Professor Ramin Hakami

OT02.O04 Parasite extracellular vesicles selectively target human monocytes to induce T-cell anergy and amelioration of DSS-induced colitis in mice

Dr Anne Borup, Dr Farouq Mohammad Sharifpour, Dr Litten Sørensen Rossen, Dr Bradley Whitehead, MSc Anders Toftegaard Boysen, Dr Paul Giacomin, Mrs Kim Miles, Ms Maggie Veitch, Dr Andrea Ridolfi, Dr Marco Brucale, Dr Francesco Valle, Dr Lucia Paolini, Dr Paolo Bergese, Dr Alex Loukas, Professor Peter Nejsum

OT02.O05 Legionella pneumophila outer membrane vesicles promote macrophage survival while Legionella pneumophila induce inflammatory cell death pathways

Ms. - Ayesha, Dr Franklin Wang Ngai Chow, Prof. Polly Hang-mei LEUNG

OT03.O02 Extracellular vesicles as mediators of retinal homeostasis and immune modulation

Dr Yvette Wooff, Dr Adrian Cioanca, Miss Rakshanya Sekar, Associate Professor Riccardo Natoli

OT03.O03 Matrimeres: Cell-secreted nanoscale mediators enabling systemic maintenance of tissue integrity and function

Dr. Koushik Debnath, Dr. Irfan Qayoom, Mr. Steven O'Donnell, Ms. Julia Ekiert, Ms. Can Wang, Mr. Mark Sanborn, Mr. Chang Liu, Ms. Ambar Rivera, Dr. Ik Sung Cho, Ms. Saiumamaheswari Saichellappa, Dr. Peter Toth, Prof. Dolly Mehta, Prof. Jalees Rehman, Prof. Xiaoping Du, Prof. Yu Gao, Jae-Won Shin

OT03.O04 Elucidation of the mechanisms of participation of mesenchymal stromal cells extracellular vesicles in the regulation of myofibroblasts differentiation on 2D and 3D models of fibrosis

Ms Anastasiya Tolstoluzhinskaya, Ms Natalia Basalova, Ms Anastasiya Efimenko

OT03.O05 Mechanical overload-induced extracellular mitochondria and particles release from tendon cells leads to inflammation in tendinopathy

Dr. Ziming Chen, Mengyuan Li, Peilin Chen, Andrew Tai, Jiayue Li, Euphemie Bassonga, Junjie Gao, Delin Liu, David Wood, Brendan Kennedy, Qiujian Zheng, Professor Minghao Zheng

OT05.O02 Interferon induced isoform of ADAR1 aids in Ewing sarcoma metastasis by fueling the pro-inflammatory response in tumor microenvironment

Mr. Manideep Pachva, Dr. Peter Ruzanov, Dr. Valentina Evdokimova, Dr. Melanie Rouleau, Dr. Laszlo Radvanyi, Dr. Poul Sorensen

OT05.O03 Tumor -derived extracellular vesicles endogenously released by tumors are captured by resident and non-resident cells in the pre-metastatic niche and activate the inflammasome in macrophages

Dr. Laurence Blavier-Sarte, Dr. Irina Matei, Dr. David Lyden, Professor Yves DeClerck

OT05.O04 Extracellular vesicles derived from plasma of exercise mice attenuated aggressive breast cancer tumour growth and metastasis

Dr Pamali Fonseka, Dr Sanjay Shahi, Prof Mark Febbraio, Prof Suresh Mathivanan

OT05.O05 RalA enhances hepatocellular carcinoma metastasis via upregulating protein cargos of small extracellular vesicles

Dr Lu Tian, Miss Jingyi Lu, Dr Karen Man-Fong Sze, Dr Goofy Yu-Man Tsui, Dr Daniel Wai-Hung Ho, Prof Irene Oi-Lin Ng

OT05.O06 CRISPR/Cas9-based deletion of cortactin reduces the secretion of small extracellular vesicles, blocks cancer-associated cachexia, and prolongs survival

Dr Sai Vara Prasad Chitti¹, Mrs Akbar L Marzan¹, Professor Suresh Mathivanan¹

¹La Trobe Institute For Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia, Australia

OT06.04 Spirulina EVs induce pro-inflammatory response by targeting innate immune cells, demonstrating potential as a vaccine adjuvant

Dr. Mohammad Farouq Sharifpour, Dr. Suchandan Sikder, Dr. Yide Wong, Dr. Na'ama Koifman, Dr. Matthias Floetenmeyer, Dr. Robert Courtney, Prof. Jamie Seymour, Prof. Alex Loukas

OT06.05 Targeting PD-L1 in cholangiocarcinoma using milk-derived nanovesicle-based immunotherapy

Dr. Piyushkumar Gondaliya, Mr. Adil Ali Sayyed, Julia Driscoll, Irene K Yan, Dr. Tushar Patel

OT06.O02 Engineered extracellular vesicles mediate the in situ propagation of antibacterial signaling enabling biofilm eradication: in vitro and in vivo studies

Postdoctoral Scholar Tatiana Cuellar-Gaviria, Maria Rincon-Benavides, Hatice Topsakal, Ana Salazar-Puerta, Mia Kordowski, Pranav Rana, Orlando Combita-Heredia, Daniel Wozniak, Daniel Gallego-Perez, Natalia Higuita-Castro

OT06.O03 Celery exosome-like nanovesicles as dual function anti-cancer nanomedicine

Xin Lu, Qing Han, Professor Weiliang Xia¹

¹Shanghai Jiao Tong University, Shanghai, China

OT06.OWP01 Reshaping the landscape of prostate cancer treatment: FeS-Functionalized OMVs as a promising nanodrug for immunotherapy

Doctor Xinxing Du, Doctor Huan Chen, Doctor Cong Hu, Doctor Yanhao Dong, Doctor Xinrui Wu, Doctor Jinyao Liu, Doctor Liang Dong, Doctor Wei Xue

OT06.OWP02 Bovine milk extracellular vesicles (mEVs)-liposomes hybrid systems: a potential strategy for oral delivery of siRNA

Dr. Yunyue Zhang

OT06.OWP03 Turmeric-derived extracellular vesicles laden polyphenol-based hydrogel synergistically restores skin barrier in atopic dermatitis

Mingzhen Zhong, PhD Weilun Pan, Professor Lei Zheng

OT06.OWP1 Oral and rectal administration of bovine milk derived EVs in a colitis mouse model

Miss Nidhi Seegobin¹, Miss Marissa Taub¹, Dr Atheer Awad^1,2, Dr Sudax Murdan¹, Prof Abdul Basit¹

¹University College London, London, United Kingdom, ²University of Hertfordshire, Hatfield, United Kingdom

OT07.O02 EHD4 cooperates with Ral GTPase to drive multi-vesicular body maturation and exosome secretion

Dr Vincent Hyenne, Dr Kuang-Jin Huang, Dr Jacky G. Goetz

OT07.O03 Exogenous bacterial Cas9 expression alters small EV secretion and their protein cargo in p53 dependent manner

Professor Suresh Mathivanan¹

¹La Trobe University, Melbourne, Australia

OT07.O04 Screening for an inhibitor of EV secretion in ovarian cancer cells using a small molecule library

Dr. Yusuke Yoshioka, Dr. Akira Yokoi, Prof. Takahiro Ochiya

OT07.O05 Piezo1 activation increases release of therapeutic extracellular vesicles after mechanical stimulation in bioreactors

Phd André Cronemberger Andrade, Sarah Razafindrakoto, Lea Jabbour, Florence Gazeau, Amanda Silva Brun

OT07.O06 Regulation of EV biogenesis by ubiquitination and deubiquitination

Professor Sharad Kumar, Dr Ammara Farooq, Dr Natalie Foot, Dr Yoon Lim

OT08.O02 Detection of multi-cancer signatures from extracellular vesicles using automated high-performance liquid chromatography

Dr Andrew Lai, Dr Dominic Guanzon, Dr Carlos Palma, Dr Flavio Carrion, Dr Ryan Cohen, Prof Andreas Obermair, Prof Andreas Moller, Prof Carlos Salomon

OT08.O03 Double digital assay for single extracellular vesicle and single molecule detection

Dr. Jina Ko

OT08.O04 High-sensitive rapid detection of urinary EVs with upconverting nanoparticle-based lateral flow immunoassay

PhD Md Khirul Islam, Professor Urpo Lamminmäki, Adjunct professor Janne Leivo

OT08.O05 Optofluidic lab-on-a-chip for point-of-need diagnostics and monitoring of treatment effectiveness by detection and quantification of EV subpopulations

Dr. Vasiliy Chernyshev, Mr. Alexey Kuzin, Dr. Vadim Kovalyuk, Dr. Pavel An, Mr. Alexandr Golikov, Mr. Sergey Svyatodukh, Mr. Stanislav Perevoschikov, Dr. Irina Florya, Dr. Alexey Schulga, Dr. Sergey Deyev, Dr. Gregory Goltsman, Dr. Dmitry Gorin

OT08.OWP01 Isolation and molecular characterization of exosomes from glioblastoma patients using a microfluidic device after ultrasound-based opening of the blood brain barrier

Ms Abha Kumari, Dr Mark Youngblood, Andrew Gould, Dr Yoon-Tae Kang, Li Chen, Karl Habashy, Thiago Reis, Dr Chris Amidei, Dr Rachel Ward, Cristal Gomez, Guillaume Bouchoux, Michael Canney, Dr Roger Stupp, Prof. Adam Sonabend, Prof. Sunitha Nagrath

OT08.OWP02 EV biomarker discovery for ultra-early differential diagnosis of stroke

Lee-Ann Clegg, MD, PhD Rolf A. Blauenfeldt, Bioinformatician, PhD Jesper Just, MSc in Engineering Rikke Bæk, Professor Peter Kristensen, Professor, MD Grethe Andersen, MSc, PhD Kim R. Drasbek, MSc, Ph.D Malene M. Jørgensen

PF01.01 Biomarkers for diagnosis of abdominal aortic aneurysm using small extracellular vesicle-associated microRNA in human serum

Dr. Kazuki Takahashi^{Department of Molecular Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shinjuku-ku, Japan}, Dr. Yusuke Yoshioka^{Department of Molecular Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shinjuku-ku, Japan}, Dr. Naoya Kuriyama^{Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japan}, Dr. Shinsuke Kikuchi^{Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japan}, Professor Nobuyoshi Azuma^{Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japan}, Professor Takahiro Ochiya^{Department of Molecular Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shinjuku-ku, Japan}

PF01.04 Enhancing concurrent chemoradiation outcome prediction for locally advanced cervical cancer patients through plasma extracellular vesicle proteomics analysis

Mr. Kittinun Leetanaporn, Mr. Jitti Hanprasetpong, Miss Wararat Chiangjoing, Mr. Sitthiruk Roytrakul, Miss Piyatida Molika, Mrs. Raphatphorn Navakanitworakul

PF01.05 Extracellular vesicles as dual messengers: Deciphering microbial and host interaction for periodontitis

Miss Chun Liu, Dr Chaminda Jayampath Seneviratne, Prof Sašo Ivanovski, Dr Pingping Han

PF01.06 Glioblastoma-derived salivary proteins in small extracellular vesicles as prognostic biomarkers

Dr Juliana Müller Bark, Dr Lucas Trevisan França de Lima, Dr Xi Zhang, Dr Daniel Broszczak, Dr Paul J. Leo, Dr Rosalind L. Jeffree, Dr Benjamin Chua, Dr Bryan W. Day, Professor Chamindie Punyadeera

PF01.08 Multicenter, prospective, observational study for urinary extracellular vesicle biomarkers of kidney allograft fibrosis

Professor Sung Shin, Dr. Hye Eun Kwon, Dr. Mi Joung Kim, Professor Heungman Jun, Professor Sang Jun Park, Professor Jun Gyo Gwon

PF01.09 Placental EVs enriched with Chromosome 19 and 14 cluster miRNAs as predictive biomarkers for Idiopathic Recurrent Pregnancy Loss

Ms. Chitra Bhardwaj, Dr. Priyanka Srivastava, Dr. Minakshi Rohilla, Dr. Seema Chopra, Dr. Anupriya Kaur, Dr. Inusha Panigrahi

PF01.10 Plasma-derived EVs as biomarkers of sepsis in burn patients via label-free Raman spectral analysis

Ms. Hannah O'Toole, Ms. Neona Lowe, Ms. Visha Arun, Ms. Anna Kolesov, Prof. Tina Palmieri, Prof. Nam Tran, Prof. Randy Carney

PF01.11 Proteome signature in serum extracellular vesicles reflects bronchial asthma pathophysiology

Md.PhD Yoshito Takeda, MD Hanako Yoshimura, MD.PhD Yuya Shirai, MD Takahito Enomoto, PhD Jun Adachi, MD.PhD Atsushi Kumanogoh

PF01.13 Proteomic study of small extracellular vesicle protein biomarker profiles for breast cancer liquid biopsy

Ms Yu Jin Lee, Dr Jie Ni, Dr Valerie Wasinger, Mr Qi Wang, Dr Joanna Biazik, A/Prof Peter Graham, Prof Yong Li

PF01.14 Rapid and non-invasive diagnosis of high PI-RADS prostate cancer by high-performance serum extracellular vesicles key metabolites

Postgraduate Zehong Peng, Postgraduate Yuning Wang, Postgraduate Xinrui Wu, Postgraduate Xingxing Du, Postgraduate Cong Hu, Postgraduate Yanhao Dong, Postgraduate Qi Chen, Postgraduate Yang Ge, Professor Kun Qian, Associate Research Fellow Liang Dong, Professor Wei Xue

PF01.16 Small extracellular vesicles in body fluids: promising prognostic biomarkers for head and neck cancer

Mr Abolfazl Jangholi, Dr Sarju Vasani, Prof Liz Kenny, Prof Sudha Rao, Prof Riccardo Dolcetti, Prof Chamindie Punyadeera

PF01.17 Spatiotemporal characteristics of tissue derived small extracellular vesicles is associated with tumor relapse and anti-PD-1 response

Doctor Qiu-Yun Fu, Professor Gang Chen

PF01.18 Urinary exosomal miRNA biomarkers for antibody-mediated

Rejection after kidney transplantation

Professor Sung Shin, Dr. Mi Joung Kim

PF01.20 Use of small RNAs from follicular fluid-derived extracellular vesicles as biomarkers for predicting success rates of fertility treatment with assisted reproductive technology

Dr. Ayako Muraoka, Dr. Akira Yokoi, Dr. Kosuke Yoshida, Mrs. Masami Kitagawa, Dr. Hiroaki Kajiyama

PF01.21 A Pilot Study on Intra-Articular Injection of Umbilical Cord-derived Mesenchymal Stem Cell (UC-MSC) Secretome in Temporomandibular Joint Dysfunction

drg. Dhanni Gustiana¹, Dr. Cynthia Retna Sartika², Mrs. Rima Haifa³, Ms Marsya Nilam Kirana³, Mrs Nisa Zulfani³, Ms Karina Kalasuba³, Mrs Ditta Kalyani Devi³, Mrs Vinessa Dwi Pertiwi³

¹RSUD Tangerang Selatan, Tangerang, Indonesia, ²Faculty of Pharmacy Universitas Padjadjaran, Jatinangor, Indonesia, ³Prodia StemCell Indonesia, Central Jakarta, Indonesia

PF01.22 Investigating the Therapeutic Effects of Umbilical Cord-derived Mesenchymal Stem Cell and Umbilical Cord Mesenchymal Stem Cell-derived Secretome in Chronic Ulcer Treatment: A Case Report

Dr Lisa Hasibuan¹, Dr Cynthia Retna Sartika^2,3, Mrs. Rima Haifa², Miss Atikah Anwar Hasibuan², Mrs. Ditta Kalyani Devi², Mrs. Adina Novia Permata Putri²

¹Immanuel Hospital, Bandung, Indonesia, ²Prodia StemCell Indonesia, Central Jakarta, Indonesia, ³Faculty of Pharmacy, Universitas Padjajaran, Jatinangor, Indonesia

PF01.23 International standardization concept to promote the technological development of extracellular vesicles

Senior Expert Ikuo Kawauchi¹

¹Fujifilm Holdings, Tokyo, Japan

PF01.23 A microfluidic device for isolation and quantitation of hepatocyte-secreted extracellular vesicles and monitoring their exosomal cytochrome P450 activities on-chip

Doctoral Researcher Ehsanollah Moradi, Ph.D. Päivi Järvinen, Ph.D. Markus Haapala, Associate Professor Tiina Sikanen

PF01.24 Fostering Consistency in EV-Based Vaccine Development and Clinical Trials: Advancing Towards Standardization

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, France

PF01.24 A single-particle-level detection of miRNA in extracellular vesicles using gold particle molecular beacons

Parvez MD SORWER ALAM, Takahiro Kochi, Prof Shin-ichi Kano, Prof Atsuo Sasaki, Prof Kazuhiko Tabata, Dr. Eisuke Dohi

PF01.25 Aptasensor detection of infectious viral disease by targeting extracellular vesicles

Ms Harleen Kaur, Professor Nathan Bartlett, Doctor Renee V Goreham

PF01.26 Characterization of prostate-specific antigen (PSA) associated with extracellular vesicles (EVs) from prostate cancer patients to develop a lateral flow diagnostic test

Kimberly Luke, Casey Scott-Weathers

PF01.28 Detection of extracellular vesicles from bacteria or mammalian cells using aptasensor technology

Dr Renee Goreham

PF01.29 Detection of human immunodeficiency virus (HIV) proteins in extracellular vesicles (EVs) by immunocapture lateral flow method

Mr. Casey Scott-Weathers, Ms. Kaitlyn King, Kimberly Luke

PF01.30 Fluorescent Nanoparticle-Based Glycoprofiling of Colorectal and Pancreatic Cancer-Derived Extracellular Vesicles for Early Detection

Mr. Rufus Vinod, Ms. Priyadharshini Parimelazhagan Santhi, Mrs. Erica Routila, Ms. Marina Alexeeva, Dr. Kjetil Søreide, Dr. Kim Pettersson, Dr. Janne Leivo

PF01.33 Optimizing diagnostic accuracy: a comprehensive standardization approach for CL-ELISA with extracellular vesicles isolated from toxoplasma gondii

Master Letícia Pedrini, Master Paula Meneghetti, Doctor Vera Lúcia Chiocolla, Doctor Ana Claudia Torrecilhas, Doctor Blima Fux

PF01.34 Rapid assessment of single extracellular vesicles using ultrathin nanoporous membranes for ‘catch and display’ of surface biomarkers

Samuel Walker^{Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA}, PhD James McGrath^{Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA}, MD, MBA Jonathan Flax^{Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA; Department of Urology, University of Rochester Medical Center, Rochester, NY, USA}

PF01.36 Single-particle analysis of circulating bacterial extracellular vesicles reveals their biogenesis, changes in blood and links to intestinal barrier

Ph.d Zihao Ou

PF01.38 Surface modification of cellulose acetate membrane for fabrication of microfluidic platforms for express extracellular vesicle-based liquid biopsy

Ms. Ekaterina Moiseeva, Dr. Vasiliy Chernyshev

PF01.39 Transferrin-conjugated magnetic nanoparticles for the isolation of brain-derived blood exosomal microRNAs: a novel approach for parkinson's disease biomarker

Associate professor Eun-jae Lee, Professor Yong Shin

PF01.42 Mapping the Multi-omics of Small Extracellular Vesicles in Diffuse Intrinsic Pontine Gliomas Reveals Biomarker Composition with Diagnostic Impact

Mr. Gaoge Sun¹, M.D. Tian Li³, Ying Zhang¹, Hang Yin^1,2

¹School of Pharmaceutical Sciences, Tsinghua University, Beijing, China, ²Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China, ³Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, China.

PF01.43 Impact of diabetes in proteomic profile of serum extracellular vesicles in obese patients after bariatric surgery

Dr. Jae-a Han¹, M.D. Haekyung Lee², Hee-Sung Ahn³, Dr. Soon Hyo Kwon³, Dr. Kyunggon Kim³, Dr. Seongho Ryu¹

¹Soonchunhyang Institute of Med-Bio Science (SIMS), Soonchunhyang University, Cheonan-si, South Korea, ²Division of Nephrology, Department of Internal Medicine, Seoul, South Korea, ³Asan Institute for Life Sciences, Asan Medical Center, Seoul, South Korea

PF01.44 Exploring small extracellular vesicles as a new biomarker to monitor duodeno-pancreatic neuroendocrine tumors (DPNET) in the PRODIGE 31- REMINET cohort

Valentin Vautrot¹, Isen Naiken¹, Carmen Garrido¹, Pr Côme Lepage², Dr Jessica Gobbo³

¹INSERM 1231, Label “Ligue National contre le Cancer” and Label d'Excellence LipSTIC, DIJON, France, ²Federation Francophone de Cancérologie Digestive (FFCD), EPICAD INSERM 1231, DIJON, France, ³INSERM 1231, Label “Ligue National contre le Cancer” and Label d'Excellence LipSTIC, Department of Medical Oncology, Early phase unit INCa CLIP²; Center Georges-François Leclerc, DIJON, FRANCE

PF01.46 Development of RNA sequencing platform for extracellular vesicles for identification of RNA markers for pancreatic cancer diagnosis

Visiting Researcher Yuta Shimizu^1,2, Researcher Fumi Asai², Researcher Keidai Miyakawa², Assistant Professor Kenji Takahashi³, Director Tatsutoshi Inuzuka²

¹Baylor Genetics, Houston, United States, ²H.U. Group Research Institute, Akiruno, Japan, ³Asahikawa Medical University, Asahikawa, Japan

PF01.47 Spectral flow cytometry of plasma EVs for detection of endometrial stromal cell markers CD10, CD90 and CD140b for endometriosis biomarkers

Ms Emily Paterson¹, Dr Simon Scheck^1,2, Dr Simon McDowell², Dr Nick Bedford², Associate Professor Jane Girling³, Dr Claire Henry¹

¹University of Otago, Wellington, New Zealand, ²Te Whatu Ora - Capital and Coast, Wellington, New Zealand, ³University of Otago, Dunedin, New Zealand

PF01.48 miR-15a from tear-derived EVs in diabetic retinopathy

Professor Tengku Ain Fathlun Kamalden¹, 2 Nur Musfirah Mahmud¹, 3 Ying Jie Liows¹, 4 Sujaya Singh¹, 5 Samarjit Das²

¹UM Eye Research Centre, Department of Ophthalmoogy, Universiti Malaya, Kuala Lumpur, Malaysia, ²Department of Anaesthesiology and Department of Pathology, Johns Hopkins School of 37 Medicine, Baltimore, United States of America

PF01.49 Proteomics discovered differential extracellular vesicle enriched protein cargo for hepatocellular carcinoma early diagnosis.

Zhenxun Wang¹, Ph.D Bodeng Wu², Qiaoting Wu¹, Jiawei Li¹, Jiaming Chen¹, Quan Zhong¹, Phd Xin Zhang², Prof. Lei Zheng², Prof. Yu Wang¹

¹Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China, ²Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, China

PF01.50 EV-derived circular RNAs as biomarkers for pleural mesothelioma

Dr Ben Johnson¹, Mr Winston Lay¹, Dr Tamkin Ahmadzada², Mr Richard Zelei¹, Dr Anthony Linton¹, Dr Elham Hosseini-Beheshti¹

¹Asbestos And Dust Diseases Research Institute, Concord, Sydney, Australia, ²The University of Sydney, Camperdown, Sydney, Australia

PF01.53 Circadian Changes in mouse plasma miRNAs

Dr. Eisuke Dohi¹

¹National Center Of Neurology And Psychiatry, Kodaira city, Japan

PF01.54 Exploring the utility of exosome subpopulations for biomarker discovery

CEO, R&D Se-Hwan Paek¹, Associate Research Engineer Taekmin Kim¹, Research Engineer Dayeon Choi¹, Research Director Seung-Cheol Choi¹

¹SOL Bio Corporation, Seoul, South Korea

PF01.55 Radiation-induced miR-126-5p in extracellular vesicles suppresses cholesterol efflux by targeting ABCG5

Min Eon Park¹, You Yeon Choi¹, Ki Moon Seong¹

¹Korea Institute of Radiological & Medical Sciences (KIRAMS), Seoul, KOREA

PF01.56 Screening of Exosomal miRNAs in Radiation-induced AKR/J leukemia mice model

Min Eon Park¹, You Yeon Choi¹, Ki Moon Seong¹

¹Korea Institute of Radiological & Medical Sciences (KIRAMS), seoul, KOREA

PF01.57 Enhanced characterization of extracellular vesicles using oni nanoimager: a comparative analysis of isolation techniques for jurkat cell-derived extracellular vesicles

Diane Nelson¹, Mahir Mohiuddin, Investigator Jennifer Jones, Jeffrey Fagan, Jerilyn Izac, Sumeet Poudel, Bryant Nelson, Lili Wang

¹Nist, United States

PF01.58 Harnessing extracellular vesicles for precise drug delivery across CNS barriers

Dr. Marie Pauwels¹, Dr. Nele Plehiers¹, Dr. Charysse Vandendriessche¹, Prof. Matthew JA Wood², Dr Lien Van Hoecke¹, Prof Roosmarijn E Vandenbroucke¹

¹VIB-UGent, Gent (Zwijnaarde), Belgium, ²University of Oxford, Oxford, UK

PF01.59 Benzo[a]pyrene exposure detection by Raman spectroscopy of large extracellular vesicles

Ms. Geetika Raizada, Dr. Benjamin Brunel, Mr. Joan Guillouzouic, Dr. Eric Le Ferrec, Dr. Eric Lesniewska, Dr. Wilfrid Boireau, Dr. Céline Elie-Caille

¹FEMTO-ST Institute, CNRS, University of Franche-Comté, Besançon, France

PF01.61 Label-free biomarker detection in advanced colorectal cancer plasma exosomes

Dr Rana Rahmani¹, Dr. Sanduru Thamarai Krishnan^1,2, Dr. David Rudd^1,2, Ehud Hauben^4,5, Prof. Nicolas H. Voelcker^1,2,3

¹Monash Institute of Pharmaceutical Sciences, Monash University, Australia, ²Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, Clayton 3168, Australia, ³Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia, ⁴The Basil Hetzel Institute for Translational Health Research, Australia, ⁵Discipline of Surgery, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Australia

PF01.64 Profiling single extracellular vesicle phenotypes in cancer using advanced nanotechnologies

Dr Richard Lobb¹, Dr Alain Wuethrich¹, Associate Professor David Fielding², Professor Andreas Möller³, Professor Matt Trau¹

¹University of Queensland, Brisbane, Australia, ²Department of Thoracic Medicine, Royal Brisbane and Women's Hospital, Brisbane, Australia, ³5JC STEM Lab, Li Ka Shing Institute of Health Sciences, Department of Otorhinolaryngology, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, China

PF01.65 Title: Proteomics-based Molecular Signatures of Alzheimer's Disease – From Blood to Extracellular Vesicles

Associate Prof. Shona Pedersen¹

¹College of Medicine, Qatar University, Doha, Qatar

PF02.01 A novel circulating extracellular vesicle miRNA panel regulates tumour cell migration, chemoresistance and patient survival outcomes in ovarian cancer

Dr Soumyalekshmi Nair, Anas Emerizal, Dominic Guanzon, Andrew Lai, Flavio Carrion, Yaowu He, Aase Handberg, Lewis Perrin, Gregory Rice, John Hooper, Carlos Salomon

PF02.03 Acquisition of cancer stem cell-like characteristics in tumor cells by application of EVs derived from 5-FU-treated human gastric cancer cells

Ms. Akane Sato, Mr. Kyo Okita, Dr. Etsuro Ito

PF02.05 Alteration of bone marrow-derived mesenchymal stem cell exosomes on cervical cancer spheroids

Miss Piyatida Molika, Assoc. Prof. Dr. Raphatphorn Navakanitworakul

PF02.06 Anti-cancer effects of extracellular vesicles from a Lacticaseibacillus paracasei strain in triple negative breast cancer cells

Professor Wen-wei Chang, Miss Hui-Yu Jiang, Dr. Wan-Hua Tsai, Professor Hsueh-Te Lee

PF02.07 Application of extracellular vesicles in 3D cell culture model of primary hepatocyte and liver tumoral cells

PhD Student Silvia López-Sarrió, PhD student Clara Garcia-Vallicrosa, PhD Student Guillermo Bordanaba-Florit, PhD Maria Azparren-Angulo, Postdoctoral researcher Félix Royo, Principal investigator Juan Manuel Falcón-Pérez

PF02.09 Blocking the secretion of small extracellular vesicles prevents muscle atrophy, lipolysis and cancer-induced cachexia

Dr Sai Vara Prasad Chitti, Akbar Marzan, Prof Suresh Mathivanan

PF02.10 Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition

Xue Liu

PF02.11 Clathrin light chain A-enriched small extracellular vesicles remodel microvascular niche to induce hepatocellular carcinoma metastasis

Dr. Yi Xu

PF02.12 Comparative proteomics analysis of small EVs derived from mouse oral cancer cells

Research Fellow Adnan Shafiq, Shinya Sato, Alissa Weaver

PF02.13 CXCR4-mediated ciliogenesis controls cancer cell death

Mr Tae-Kyu Jang, Dr Eunyi Moon

PF02.14 Devising integrin β4-enriched small extracellular vesicle as drug delivery vehicle for targeting pulmonary metastasis of hepatocellular carcinoma

Dr Tung Him Ng, Ms Aijun Liang, Prof Judy Wai Ping Yam

PF02.15 Endocytosis of EV into HNSCC cancer cells is required for increased sensitization to tyrosine kinase inhibitor

Dr Darren Toh, Ms Hui Sun Leong, Ms Fui Teen Chong, Ms Mengjie Ren, Dr Gopalakrishna Iyer

PF02.16 Exosomal G6PD drives metabolic reprogramming and reshape the pre-metastastic niche to facilitate metastasis in hepatocellular carcinoma.

Dr. Xiaoxin Zhang

PF02.20 Exploring exosome-mediated mechanisms in gefitinib-resistant lung adenocarcinoma

Mr. Chun-Fan Lung, Ph. D Student Chun Fan Lung

PF02.21 Exploring the role of obesity-induced extracellular vesicles secretion and associated oncogenic proteins in endometrial cancer pathogenesis

Dr Takahiko Sakaue, Kalpana Deepa Priya Dorayappan, Dr Wafa Khadraoui, Dr Muralidharan Anbalagan, Dr Adrian Suarez, Dr Casey Cosgrove, Dr Larry J. Maxwell, Dr Hironori Koga, Dr David O'Malley, Dr David Cohn, Dr Selvendiran Karuppaiyah

PF02.22 Extracellular vesicle-dependent inhibition effects of EGR1 on HCC metastasis

Phd Xin Zhang, Quan Zhong, Jiaming Chen, Zhenxun Wang, Bin Xu, Boyan Boyan Huang, Jinsheng Zheng, Tianyu Wu, Yu Wang, Lei Zheng

PF02.25 Extracellular vesicles secreted during oncolytic viral therapy with hmgb1 promotes melanoma outgrowth

PhD Darshak Bhatt, Msc Annemarie Boema, PhD Silvina Bustos, PhD Andreia Otake, PhD Alexis Carrasco, Professor Patricia Reis, Professor Roger Chammas, Professor Toos Daemen, PhD Luciana Andrade

PF02.26 Functional assessments of extracellular vesicles coronated with human epidermal growth factor receptor 2 (HER2) protein

Extracellular vesicles and breast cancer Mina Mobin Rahni, Immunology Marzieh Ebrahimi, Extracellular Vesicles Faezeh Shekari

PF02.27 GRP78-rich extracellular vesicles derived from gastric cancer cells promote gastric cancer stemness and chemoresistance

Dr. Jen-Lung Chen, Ms. Hsin-Yi Tsai, Assistant Professor Ming-Wei Lin

PF02.30 Impeding the secretion of tumor cell-derived small extracellular vesicles attenuates breast cancer progression and metastasis

Dr. SANJAY SHAHI, Prof. Suresh Mathivanan

PF02.31 Interrogation of the spatial tissue architecture and miRNA sequencing of extracellular vesicles in matched epithelial ovarian cancers

Dr Andrew Lai, Dr Priyakshi Kalita-de Croft, Dr Dominic Guanzon, Dr Soumyalekshmi Nair, Mr Nihar Godbole, Dr Flavio Carrion, Dr Shayna Sharma, A/Prof Margaret Cummings, Prof Lewis Perrin, Prof John Hooper, Prof Ken O'Byrne, Prof Sunil Lakhani, A/Prof Fernando Guimaraes, Dr Arutha Kulasinghe, Prof Andreas Moller, Prof Carlos Salomon

PF02.33 Macrophage function is modulated by EVs derived from plasma of HNSCC patients through the NF-κB signaling pathway

Ms Diana Huber, Mrs Tsima Abou Kors, PhD Linda Hofmann, Prof Monika Pietrowska, PhD Marta Gawin, Prof Ramin Lotfi, Prof Thomas K Hoffmann, Prof Cornelia Brunner, Prof Marie-Nicole Theodoraki

PF02.34 Melanoma secreted melanosomes induce immune tolerance in lymphatic endothelial cells through overexpression of CEACAM-1

Ms Daniela Likonen

PF02.35 Metabolic reprogramming into a glycolysis phenotype induced by extracellular vesicles derived from prostate cancer cells

Professor Yoon-Jin Lee, Ms. Shinwon Chae, Ms. Haekang Yang, Mr. Chul Won Seo, Mr. Chang Yeol Lee, Professor Sang-Han Lee, Dongsic Choi

PF02.36 MiR-195-5p-loaded tumor-derived extracellular vesicles restrains melanoma spheres growth and radiotherapy-induced resistant phenotype

Ms Nathalia Leal Santos, Roger Chammas, Luciana Andrade

PF02.37 Monitoring cancer extracellular vesicle transfer within tumor tissue context

Nao Nishida-Aoki

PF02.40 Pancreatic cancer cell derived extracellular vesicles enriched microRNAs play a critical role in macrophage reprograming

Dr. Baldev Singh, Dr. Pankaj Gaur, Dr. Jeyalakshmi Kandhavelu, Mr. Yanjun Zhang, Mr. Zihao Zhang, Dr. Shivani Bansal, Mr. Meth Jayatilake, Mr. Yaoxiang Li, Dr. Pritha Bose, Dr. Seema Gupta, Dr. Partha Banerjee, Dr. Vivek Verma, Dr. Baldev Singh

PF02.41 Pancreatic cancer cell-derived EVs promote monocyte differentiation towards immunosuppressive tumor-associated macrophages

Mr. Yanjun Zhang, Dr. Baldev Singh, Dr. Pritha Bose, Dr. Jeyalakshmi Kandhavelu, Mr. Zihao Zhang, Dr. Shivani Bansal, Dr. Sunil Bansal, Mr. Meth Jayatilake, Mr. Yaoxiang Li, Dr. Shu Wang, Dr. Baldev Singh

PF02.44 Proteomic analysis of Butyrate-resistant colorectal cancer-derived exosomes reveals potential resistance to anti-cancer drugs

Kesara Nittayaboon, Kittinun Leetanaporn, Prof. Surasak Sangkhathat, Prof. Sittiruk Roytrakul, Assoc. Prof. Raphatphorn Navakanitworakul

PF02.46 Proteomics analysis of the small extracellular vesicles and soluble secretory proteins from cachexia-inducing cells and their effect on C2C12 myotubes

Mrs Akbar Marzan, Dr. Sai Chitti, Prof Suresh Mathivanan

PF02.47 Quantification and characterization of circulating extracellular vesicles in cervical cancer patients before, during, and after treatment

Prof Muriel Meiring, Ms Noluthando Gasa

PF02.49 Small but mighty: CD-sEV cargos promote pancreatic cancer metastasis and stem cell reprogramming

Mr Harrison Rudd, Dr Geeta Upadhyay

PF02.50 Small extracellular vesicle PD-1 leads to senescence-initiated epithelial-mesenchymal transition in oral cancer through intrinsic PD-L1-p38 MAPK signaling

Doctor Lin-Zhou Zhang, Professor Gang Chen

PF02.51 Small extracellular vesicles derived from cancer cells modulate breast cancer patients’ immune system via affecting Th1/Th2 and T-reg cells

Mr Abdulwahab Teflischi Gharavi, Ms Raheleh Tahmasvand, Dr Amirabbas Rahimi, Dr Saeed Irian, Prof Mona Salimi

PF02.52 The enrichment of death associated miRNAs in placental explant culture promoted cervical tumor tissue undergoing necrosis

Lin Wang

PF02.53 The role of extracellular vesicle-contained CD155 during cancer progression

Li-Ying Wu, Dr. Luize Lima, Dr. Sunyoung Ham, Student Mina Lim, Dr. Edna Chai, Prof. Yong-Soo Choi, Prof. Andreas Möller

PF02.54 Three-dimensional matrix stiffness drives piezo1 activation in cancer spheroid-derived small extracellular vesicles

Maulee Sheth, Dr Manju Sharma, Maulee Sheth

PF02.55 Title: Osteoclasts educated by prostate cancer cells promote bone destruction via EV-mediated communication networks in bone metastatic site

M.D. Takaaki Tamura, Ph.D. Tomofumi Yamamoto, Ph.D. Akiko Kogure, Ph.D. Yusuke Yoshioka, M.D., Ph.D. Shinichi Sakamoto, M.D., Ph.D. Tomohiko Ichikawa, M.D. Takahiro Ochiya

PF02.56 Tracking the EMT-like phenotype switching during targeted therapy in melanoma by analyzing extracellular vesicle phenotypes

Mr Quan Zhou, Prof. Jing Wang, Dr. Zhen Zhang, Dr. Alain Wuethrich, Dr. Richard Lobb, Prof. Matt Trau

PF02.57 Tumor cell-derived extracellular vesicles promote ROS-induced DNA damage in hepatocellular carcinoma

Mr. Zhixian Chen, Prof. Judy Yam

PF02.58 Uncovering extracellular vesicle microRNA and protein cargo from chemoresistant osteosarcoma: Shedding light on the potential transfer of therapy resistance

Mr. Joaquín Jurado-Maqueda, Alessandra De Feo, Prof. Katia Scotlandi

PF02.59 Understanding the role of mesothelioma cell-derived extracellular vesicles in modulating fibroblast functions

Mr Vivek Dharwal, Dr Vivek Dharwal, Mr Jiawei Chang, Dr Zaklina Kovacevic, Dr Elham Hosseini-Beheshti

PF02.60 Unraveling the role of CD133 in Breast cancer-Extracellular Vesicles in invasion and metastasis

Mireia Gomez

PF02.61 Y-box binding protein 1 in small extracellular vesicles reduces the osteogenic differentiation of bone marrow-derived mesenchymal stem cells – significance in acute myeloid leukemia

Dr Venkatesh Kumar Chetty, Dr Jamal Ghanam, Prof. Dr Dirk Reinhardt, Dr Basant Kumar Thakur

PF02.62 Comparison of profile and functional activities of EVs from fresh tumor biopsies and decellularized tumor tissue in colorectal cancer

Dr Sarah Tassinari¹, Dr Federica Collino², Benedetta Bussolati¹

¹Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy, ²8 Department of Clinical Sciences and Community Health, University of Milano, Milano, Italy

PF02.63 Adipocyte-origin exosomes induce metastasis in models of TNBC

Graduate Student Yuhan Qiu¹, Undergraduate Student Rebecca Yu¹, Graduate Student Andrew Chen¹, Postdoc Matt Lawton¹, Graduate Student Pablo Llevenes¹, Lab Manager Manohar Kolla¹, Postdoc Naser Jafari¹, Kiana Mahdaviani², Naomi Ko², PI Stefano Monti¹, PI Gerald Denis¹

¹Boston University School Of Medicine, Boston, United States, ²Boston Medical Center, Boston, United States

PF03.02 Are mouse blood EV-derived miRNA profiles consistent across studies? systematic reviews

Parvez MD　SORWER　ALAM, Dr. Eisuke Dohi

PF03.03 Characterization of human follicular fluid extracellular vesicle subtypes and their impact on human granulosa-like tumor cell line KGN

Ms Inge Varik, Ms Katariina Johanna Saretok, Dr Ileana Quintero, Dr Maija Puhka, Dr Aleksander Trošin, Ms Kristine Roos, Dr Paolo Guazzi, Dr Agne Velthut-Meikas

PF03.04 Comprehensive and specific analysis of surface glycans on extracellular vesicles (EVs) for understanding EV diversity

Ph.D. Asako Shimoda, Professor Emeritus Kazunari Akiyoshi

PF03.05 Extracellular particles are present in platelet concentrates

Lauren Litchfield, Dr Rebecca Wellburn, Dr Sarah Bajan, A/Prof Yoke Lin Fung, A/Prof John-Paul Tung

PF03.06 Extracellular vesicle associated markers present on lipid droplets

Miss Irumi Amarasinghe, Dr Ebony Monson, Dr Eduard Wilms, Mr William Phillips, Dr Shuai Nie, Miss Abbey Milligan, Dr Donna Whelan, Prof Andrew Hill, Prof Karla Helbig

PF03.07 Extracellular vesicles in fresh frozen plasma and cryoprecipitate

Ji Hui Hwang, A/Prof John-Paul Tung, Prof Damien Harkin, Prof Robert Flower, Dr Natalie Pecheniuk

PF03.08 Large extracellular vesicles subsets and contents discrimination: the potential of morpho mechanical approaches at single EV level

Ms. Geetika Raizada, Mr. Joan Guillouzouic, Dr. Eric Le Ferrec, Dr. Eric Lesniewska, Dr. Wilfrid Boireau, Dr. Céline Elie-Caille

PF03.09 MBsomes and other EVs intercellular communication in skin wound healing

Phd Student Mariane Shouky, Graca Raposo

PF03.10 Nano-flow cytometry-based discrimination of extracellular vesicles and non-vesicular particles: insights into extracellular carriers of specific biomolecules

Xiaomei Yan, Yunyun Hu, Haonan Di, Dr. Ye Tian

PF03.11 Quantitative profiling of single exosome heterogeneity using single-molecule binding assay

Ms Jiyoung Goo, Ms Somi Park, Ms Hyeyeong Ku, Ms Jeongmin Lee, Mrs Jeong Hee Kim, Mr In-San Kim, Mr Cherlhyun Jeong

PF03.12 Quantitively mapping the EV field and its trajectory through scientometrics

Mr Liam Hourigan, Mr William Phillips, Mr Chaomei Chen, Mr Amirmohammad Nasiri Kenari, Mr Krishna Chaitanya Pavani, Mrs Lesley Cheng, An Hendrix, Mr Andrew Hill

PF03.13 Spatial diversity of intraperitoneal extracellular vesicles and potential tumor-suppressive roles of liver-surface extracellular vesicles in the development of high-grade serous ovarian carcinoma

Dr Kosuke Yoshida, Dr Akira Yokoi, Dr Kazuhiro Suzuki, Dr Yukari Nagao, Dr Ryosuke Uekusa, Ms Masami Kitagawa, Dr Eri Inami, Dr Takao Yasui, Dr Hiroaki Kajiyama

PF03.14 The effects of acute and chronic hypoxia on EV production and phenotype in cancer cells

Dr Chris Pridgeon, Ms Julia Monola, Ms Kerttu Airavaara, Dr Daniel Palmer, Prof. Marjo Yliperttula, Dr Riina Harjumäki

PF03.15 Matrix-bound nanovesicles: biogenesis and ties to the ECM

Marley Dewey, Assistant Professor George Hussey, Professor Stephen Badylak

PF03.17 The impact of follicular fluid small and large extracellular vesicles on the gene expression of human granulosa-like tumor cell line KGN

Dr Agne Velthut-Meikas¹, Inge Varik¹, Katariina Johanna Saretok¹, Kristine Rosenberg^1,2, Aleksander Trošin³, Maija Puhka⁴, Ileana Quintero⁴, Paolo Guazzi⁵

¹Tallinn University Of Technology, Tallinn, Estonia, ²Nova Vita Clinic, Tallinn, Estonia, ³East Tallinn Central Hospital, Tallinn, Estonia, ⁴University of Helsinki, Helsinki, Finland, ⁵HansaBioMed Life Sciences Ltd, Tallinn, Estonia

PF03.18 Towards development of detergent-based strategy for the enrichment of extracellular particle subpopulations and subdomains

Dr. Igor V Kurochkin¹, Lausonia Ramaswamy

¹Central Research Laboratory, Sysmex Co., Kobe, Japan

PF03.22 Exosomes Isolation by Ultracentrifugation: Novel Subpopulations reveal Extracellular Vesicle Heterogeneity and Diverse Functional Signatures

Director, Computational Oncology Unit Ahmed Fadiel², Process Development Lead Shuaizhen Yuan¹, Associate Scientist Eileah Loda¹, Ceo Adam Koster¹, Chair, Medical Scientific Advisory Board Frederick Naftolin¹, Director, Medical Affairs Matthew Peterson

¹Interactome Biotherapeutics, Grand Rapids, United States, ²University of Chicago, Chicago, USA

PF04 Cell-derived nanovesicles as a scalable production of extracellular vesicles-mimetics for therapeutic applications

Dr Wei Heng Chng¹, Mr Ram Pravin Kumar Muthuramalingam¹, Dr Yub Raj Neupane¹, Dr Chenyuan Huang¹, Dr Wei Jiang Goh¹, Dr Choon Keong Lee¹, Bertrand Czarny², Assistant Professor Jiong-Wei Wang¹, Associate Professor Giorgia Pastorin¹

¹National University of Singapore, Singapore, ²Nanyang Technological University, Singapore

PF04.03 Advancements in lung cancer immunotherapy using engineered exosome to deliver PD-L1 siRNA

Dr. Farrukh Aqil, Raghuram Kandimalla, Disha Moholkar, Margaret Wallen, Chuanlin Ding, Ramesh Gupta

PF04.05 Engineered exosomes for HLA-G-targeted co-delivery of MSI1 siRNA and chemotherapeutics to reduce the tumor progression

PhD Chih-Ming Pan, MS Yu-Ting Liao, PhD Shao-Chih Chiu

PF04.07 Extracellular vesicle-mediated delivery of customized ASOs targeting driver mutants for personalized Non-Small Cell Lung Cancer treatment

PhD student Trinh Tran, Doctor Dai Phung, Brendon Yeo, Rebecca Prajogo, Migara Jayasinghe, Yuan Ju, Eric Yeo, Doctor Boon Cher Goh, Doctor Wai Leong Tam, Doctor Minh Le

PF04.08 FDA-approved ETA antagonist regulates cellular and exosomal B7-H4 through N-glycosylation inhibition

Ms Sua Kim, Dr. Dokyung Jung, Professor Moon-Chang Baek

PF04.09 Hydrogel-encapsulated exosome vaccine as a novel immunotherapeutic approach and its role in enhancing immunotherapy for prostate cancer

Liang Dong, QI Chen

PF04.10 In vivo CAR-T generated by CD3ɛ nanobody-engineered exosomes eliminates solid tumors and promotes the immunological memory formation

Dr. Shi-Wei Huang, Dr. Mei-chih Chen, Dr. Yu-Chuan Lin, Dr. Chih-Ming Pan, Dr. Chung-Chun Wu, Miss Chen-Yu Lin, Miss Pei-Ying Lin, Miss Yu-Ting Chiang, Miss Yu-Han Huang, Miss Wan-Yu Mao, Miss Steffany Rusli, Professor Shao-Chih Chiu, Professor Der-Yang Cho

PF04.11 Novel personalized cancer vaccine using attenuated tumor extracellular vesicles with enhanced immunogenicity

Graduate student Jihoon Han, Graduate student Yeongha Hwang

PF04.12 Redirecting pre-existing noncancer immunity to cancer cells using tumor-targeting extracellular vesicles for delivery of MHC-I-compatible peptides for cancer immunotherapy

Yang Lu, Songbo Qiu, Professor Zhen Fan

PF04.16 Surface-engineered NK cell-derived small extracellular vesicles induce potent anti-tumor effects in lung cancer cells

Dr. Sung-Min Kang, Dr. Dokyung Jung, Ms Soojeong Noh, Ms Sanghee Shin, Ms Minju Kim, Professor Byungheon Lee, Professor Kyungmoo Yea, Professor Moon-Chang Baek

PF04.17 Synthetic immunogenicity-induced DNA accumulation in colorectal cancer extracellular vesicles enhances T cell stemness

Ms Seong A Kim, Ms Yeji Lee, Dr. In-San Kim

PF04.18 Therapeutic plasma exchange as a method to combat extracellular vesicle-mediated immunotherapy resistance in melanoma

Dr. Jacob Orme, Henan Zhang, Prashanth Lingamaneni, Yohan Kim, Roxane Lavoie, Jacob Hirdler, Elizabeth Bering, Joanina Gicobi, Heather Dale, Lisa A Kotschade, Matthew S. Block, Svetomir N. Markovic, Haidong Dong, Fabrice Lucien, Annie T. Packard, Jeffrey L. Winters, Sean S. Park

PF04.21 Vitamin B enhances anti-tumor immunity by inhibiting CD47 on cellular and extracellular vesicles

MS course Na-Eun Kim, Dr. Dokyung Jung, Professor Moon-Chang Baek

PF04.24 Synthetic biology-based bacterial extracellular vesicles displaying BMP-2 and CXCR4 to ameliorate postmenopausal osteoporosis

Associate Professor Han Liu

PF04.25 TNFα-bearing small extracellular vesicles synergize with SMAC mimetics to eradicate tumor cells

Dr. Rostyslav Horbay, Daniel Panting, Michaela van der Meerwe, Maria Dimancheva, Dr Eric LaCasse, Dylan Burger, Dr Shawn Beug

¹Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and University of Ottawa, 401 Smyth Rd, Ottawa, Ontario, K1H 8L1, Canada, Ottawa, Canada, ²Kidney Research Centre, The Ottawa Hospital Research Institute and University of Ottawa, 401 Smyth Rd, Ottawa, ON, K1H 8L1, Canada,

PF04.26 Therapeutic poxviruses trigger the secretion of anti-tumor extracellular vesicles with immunomodulatory potential

Lucas Walther³, Jacky Goetz¹, Karola Rittner², Dr Vincent Hyenne¹

¹INSERM U1109, Strasbourg, France, ²Transgene SA, Illkirch-Graffenstaden, France, ³INSERM U1109 and Transgene SA, Strasbourg, France

PF04.27 CAR-T derived extracellular vesicles demonstrate in vitro therapeutic efficacy in breast and blood cancer cells

Dr Kartini Asari¹, Siena Barton¹, Sadman Bhuiyan¹, Kol Thida Mom¹, Amirah Fitri¹, Dr Mozhgan Shojaee¹, Dr Carlos Palma¹, Dr Sara Nikseresht¹, Dr Ramin Khanabdali¹, Professor Gregory Rice^1,2

¹INOVIQ Ltd, Notting Hill, Australia, ²UQ Centre for Clinical Research, Herston, Brisbane City, Australia

PF05.02 Watermelon alleviates IBD by modulating intestinal lactobacillus plantarum colonisation and the release of bacterial extracellular vesicles

Phd Qianbei Li, Professor Lei Zheng

PF05.03 L. amazonensis amastigotes release unique extracellular vesicles in a calcium and pH dependent manner

Deborah Brandt Almeida, Ms Jenicer Kazumi Umada Yokoyama Yasunaka, Doctor Verônica Feijoli Santiago, Doctor Simon Ngao Mule, Ms Paula Menegheti, Doctor Giuseppe Palmisano, Doctor Ana Claudia Torrecilhas, Doctor Mauro Cortez

PF05.05 A human host-defense peptide LL-37 ameliorates mouse sepsis by orchestrating the chemotaxis of neutrophils and secretion of anti-inflammatory extracellular vesicles

Assistant Professor Yumi Kumagai^1,2, Special Appointed Professor Isao Nagaoka^1,3, Professor Etsuo Susaki¹

¹Dept. of Biochemistry and Systems Biomedicine, Graduate School of Medicine, Juntendo University, Bunkyo-ku, Japan, ²Biomedicine Research Core Facility, Graduate School of Medicine, Juntendo University, Bunkyo-ku, Japan, ³Faculty of Medical Science, Urayasu, Japan

PF05.06 Induction of proinflammatory response in bystander macrophages by extracellular vesicle-delivered SARS-CoV-2 accessory protein ORF3a

Dr Sin-Yee Fung¹, Kam-Leung Siu¹, Man Lung Yeung², Prof Judy Wai Ping Yam³, Prof Dong-Yan Jin¹

¹School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong, ²Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong, ³Department of Pathology, The University of Hong Kong, Pokfulam, Hong Kong

PF05.07 Redox-active outer-membrane vesicles boost extracellular electron uptake in marine sedimentary bacteria under energy starvation conditions

Dr. Xiao Deng¹

¹National Institute For Materials Science, Tsukuba, Japan

PF05.08 “Strain-Based Comparison and Pharmacological Investigation of Bacterial Extracellular Vesicles”

Master Seoah Park¹, Jongsoo Mok², Junghoon Choi¹, Hye-Min Yu³, Hye-Jin An³, Ga-Hyun Choi³, Yeon-Seon Lee³, Ki-Jin Kwon³, Sung-Jun Choi³, Soo-Jin Kim³, Joonghoon Park^1,2

¹Graduate School of International Agricultural Technology, Seoul National University, Korea, ²Institute of Green Bio Science & Technology, Seoul National University, Korea, ³Schofield Biome Research Lab, HK inno.N, Korea

PF05.09 Cracking the egg: probing Schistosoma mansoni eggs for tolerogenic products

Mx Madeleine Rogers¹, Dr Athena Andreosso¹, Dr Jagan Billakanti², Dr Sandip Kamath³, Prof Donald McManus¹, Prof Malcolm Jones¹, Dr Catherine Gordon¹, A/Prof Severine Navarro¹, A/Prof Severine Navarro⁴

¹QIMR Berghofer Medical Research Institute, Herston, Australia, ²Cytiva, Brisbane, Australia, ³Medical University of Vienna, Vienna, Austria, ⁴Centre for Childhood Nutrition Research, Brisbane, Australia

PF06.01 Evaluating the immunogenicity of circulating extracellular vesicles from gestational diabetes patients: insights for therapeutic use

Professor Flavio Carrion, Dr Soumyalekshmi Nair, Katherin Scholz-Romero, Dr Carlos Palma, Dr Andrew Lai, Dr Dominic Guanzon, Professor Bernardo Morales, Associate Professor Martha Lappas, Professor Carlos Salomon

PF06.03 Pancreatic cancer-derived small extracellular vesicles alter immune cell behaviour via the sphingosine-1-phosphate signalling pathway

Miss Jordan Fyfe, Dr Danielle Dye, Dr Pat Metharom, Professor Marco Falasca

PF06.04 The HLA-I immunopeptidome of platelet-derived extracellular vesicles

Dr Caitlin Boyne, Mr Jordan Marsh, Dr Sally Shirran, Dr Alan Stewart, Dr Simon Powis

PF06.05 Therapeutic rescue of sepsis induced liver damage by immune-regenerative HIF1α enriched extracellular vesicles

Miss YEJI LEE, Miss Jiyoung Goo, Mr In-San Kim

PF06.06 Unfolding the role of placental small extracellular vesicles in preeclampsia in the development of the fetal immune system

Ms Michaela Klaczynski, Ms Birgit Hirschmugl, Ms Barbara Darnhofer, Ms Katharina Eberhard, Mr Harald Köfeler, Mr Karl Kashofer, Mr Christian Wadsack

PF06.07 Aerobic fitness levels can alter the secretion of circulating extracellular vesicles during moderate intensity exercise

Dr Mee Chee Chong, Dr Anup D. Shah, Associate Professor Ralf B. Schittenhelm, Dr Anabel Silva, Dr Patrick F. James, Professor Jason Howitt

PF06.08 Akkermansia muciniphila alleviates lipid metabolism disorders in mice via delivery of Amuc_1100-Containing vesicles

Phd Qianbei Li, Professor Lei Zheng

PF06.10 Calpeptin alters insulin-mediated glucose uptake and extracellular vesicle secretion in human adipocytes

Msc Johanna Matilainen, Viivi Berg, Maija Vaittinen, Janne Tampio, Ville Männistö, Jussi Pihlajamäki, Tanja Turunen, Marjo Malinen, Pirjo Käkelä, Dorota Kaminska, Veera Luukkonen, Anne-Mari Mustonen, Uma Thanigai-Arasu, Kristiina Huttunen, Reijo Käkelä, Sanna Sihvo, Petteri Nieminen, Kirsi Rilla

PF06.11 Changes in insulin sensitivity across gestation is associated with changes in the profile of maternal circulating extracellular vesicle protein and miRNA: A Longitudinal study.

Dr Soumyalekshmi Nair, Lilian Kessling, Dominic Guanzon, Andrew Lai, Flavio Carrion, David Simmons, Mireille Van Poppel, Harold David McIntyre, The Dali Core Investigator Group, Gernot Desoye, Carlos Salomon

PF06.12 Early pregnancy serum maternal and placenta-derived exosomes miRNAs vary based on pancreatic β-cell function in gestational diabetes

MD, PhD Melissa Razo-Azamar, PhD Rafael Nambo-Venegas, PhD Iván Rafael Quevedo, PhD Gregorio Juárez-Luna, PhD Carlos Salomon, MD, PhD Martha Guevara-Cruz, PhD Berenice Palacios-González

PF06.13 Functional implications of hepatic EV alteration in NAFLD and T2DM

Pin Hsuan Chu, Dr. Han-Yi E. Chou, Dr. Tien-Jyun Chang, Dr. Shiau-Mei Chen

PF06.14 Induction of renal damage by methylglyoxal-lysine dimer (MOLD) through exosome-mediated miR-130a-3p

Principal Researcher Eun Hee Han, Ms. Hye Min Kim, Dr. Jin Young Min, Mr. Min Sung Park1

PF06.15 Multifaceted action of stem cell-derived extracellular vesicles for nonalcoholic steatohepatitis

Ph.D. Jimin Kim, M.S. Seul Ki Lee, M.S. Haedeun You, M.S. Sang-Deok Han, Ph.D. Tae Min Kim, Ph.D. Soo Kim

PF06.17 Podocyte-derived urinary extracellular vesicles in membranous nephropathy

PhD student Karen Lahme, PhD Wiebke Sachs, PhD Desiree Loreth, Stephanie Zielinski, Johannes Brand, PhD Kristin Surmann, Professor Uwe Völker, Thorsten Wiech, Professor Tobias N. Meyer, Lars Fester, Professor Catherine Meyer-Schwesinger

PF06.18 Primary pancreatic ductal cells from normal cadaveric donors are responsive to type 1 diabetes-mimicking proinflammatory cytokines in vitro and secrete extracellular vesicles

Neslihan Erdem, Nathaniel Hansen, Min Talley, Heather Zook, Kevin Jou, Jose Ortiz, Nagesha Guthalu Kondegowda, David Arribas-Layton, Fouad Kandeel, Enrique Montero, Helena Reijonen, Rupangi Vasavada, Patrick Pirrotte, Tijana Jovanovic-Talisman, Hsun Teresa Ku

PF06.19 Small extracellular vesicles inhibit NLRP3 inflammasome activation in diabetic retinopathy

Henry Louie, Ilva D. Rupenthal, Odunayo O. Mugisho, Lawrence W. Chamley

PF06.22 Investigating the impact of extracellular vesicles in obese pregnancies: Are EVs from obese

Pregnant dams during early pregnancy sufficient to cause obesity in offspring?

Phd Student Taylor Hollingsworth¹, Pharm.D., Ph.D. Thea Golden¹, M.D. Rebecca Simmons¹

¹University Of Pennsylvania, Philadelphia, United States

PF06.23 Characterizing plasma-derived EVs from pregnant Black cis-women as a potential tool to predict adverse pregnancy outcomes

Ms. Kobe Abney¹, Pharm.D., Ph.D. Thea Golden¹, Dr. Yu-Chin Lien¹, Dr. Nadav Schwartz¹, Dr. Rebecca Simmons¹

¹University Of Pennsylvania, United States

PF06.24 Human seminal fluid extracellular vesicles induce immune responses in female cervical cells in vitro

Miss Cottrell Tamessar^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Miss Chishan Burch^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Miss Piper Miller^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Miss Jane Durbidge^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Miss Tegan Bryde^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Miss Shanu Parameswaran^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Associate Professor Geoffry De Iuliis^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Doctor Judith Weidenhofer^{3 Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 4 School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, The University of Newcastle, Ourimbah, NSW 2258, Australia.}, Dr Hui-ming Zhang^{5 Central Analytical Facility, Research and Innovation Division, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Professor Sarah Robertson^{7 Robinson Research Institute and School of Biomedicine, The University of Adelaide, SA 5005, Australia.}, Doctor Elizabeth Bromfield^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia. 6 Bio21 Institute, School of BioSciences, The University of Melbourne, VIC 3010, Australia.}, Doctor David Sharkey^{7 Robinson Research Institute and School of Biomedicine, The University of Adelaide, SA 5005, Australia.}, Professor Brett Nixon^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Doctor John Schjenken^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}

¹Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, Newcastle, Australia, ²School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, Callaghan, Newcastle, Australia, ³Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, Newcastle, Australia, ⁴School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, The University of Newcastle, Ourimbah, Central Coast, Australia, ⁵Central Analytical Facility, Research and Innovation Division, The University of Newcastle, Callaghan, Newcastle, Australia, ⁶Bio21 Institute, School of BioSciences, The University of Melbourne, Parkville, Melbourne, Australia, ⁷Robinson Research Institute and School of Biomedicine, The University of Adelaide, North Adelaide, Adelaide, Australia

PF06.25 Local treatment of inflammatory bowel disease by EV drug carriers

Miss Nidhi Seegobin¹, Miss Victoria Chris^1,2, Miss Marissa Taub¹, Dr Sudaxshina Murdan¹, Prof Abdul Basit¹

¹University College London, London, United Kingdom, ²University of Oxford, Oxford, United Kingdom

PF06.27 The induction of pro-inflammatory extracellular vesicles in the progression of metabolic-associated fatty liver disease (MAFLD)

Dr. Allen Wei-Lun Huang¹, Ms. Tzu-Ching Kao², Dr. Sin-Tian Wang², Ms. Yi-Wen Chiu², Dr./Prof. Pin-Nan Cheng³, Dr./Prof. Chi-Yi Chen⁴, Prof. Kung-Chia Young²

¹Center of Applied Nanomedicine, National Cheng Kung University, Tainan, Taiwan, ²Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ³Department of Internal Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ⁴Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chia-Yi Christian hospital, Chiay, Taiwan

PF06.28 Stem Cell-Derived Nano Vesicles' Impact on Muscle Regeneration in Cachexia Models

Postgraduate student Keren Esther Kristina Mantik^1,2, Researcher Sohee Moon¹, Sujin Kim¹, Researcher, MS Bon-Sang Gu¹, Postgraduate student Jubi Lee^1,2, Postgraduate student Chan-Young So^1,2, CEO Shingyu Bae⁴, Professor, MD, PhD Ju-Hee Kang^1,2,3

¹Department of Pharmacology and Research Center for Controlling Intercellular Communication, College of Medicine, Inha University, 22212, South Korea, ²Program in Biomedical Science and Engineering, Inha University, 22212, South Korea, ³Institute of Sports & Arts Convergence (ISAC), Inha University, 22212, South Korea, ⁴BioDrone Research Institute, MDimune Inc, South Korea

PF07.01 19F-MRI-labeled extracellular vesicle mimetics for specific monitoring of targeted drug delivery

Dr. Andrea Galisova, Dominik Havlicek, Ayca Tunca, Ondrej Sedlacek, Daniel Jirak

PF07.02 A dynamic, label-free, and efficient small EVs sensing platform for assessment of EV based drugs —- case study of MSC-sEVs for Cardiac Oxidative Stress Injury Therapy

Dr Chunlian Qin, Dr Danyang Li, Dr Ning Hu, Dr Lizhou Xu

PF07.04 A novel pathogen sensing platform for detection of Escherichia Coli

Miss Shiana Malhotra, Dr Renee Goreham, Dr Thomas Nann

PF07.05 Analysis of tumor-derived small extracellular vesicles with spectral flow cytometry

Dr Linda Hofmann, Dr Annika Betzler, Prof Thomas Hoffmann, Prof Cornelia Brunner, Prof Marie-Nicole Theodoraki

PF07.10 Fueling strategy-based self-sacrificed MOF@DNAzyme integrated chip for the isolation and detection of tumor-derived extracellular vesicles

Student Zehan Zeng, PhD Weilun Pan, Professor Jinxiang Chen

PF07.11 Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system

Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Yulin Cao, Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Yuxuan Jiang, Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Qiubai Li, Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Yong Deng

PF07.12 Improvement of extracellular vesicle detection sensitivityon a surface-functionalized power-free microchip

Associate Professor Ryo Ishihara, Hinako Yokohari, Ren Ogata, Kotomi Katori, Kentaro Doi, Kurumi Omiya, Tadaaki Nakajima, Eri Shimura, Takeshi Baba

PF07.14 Multiparametric analysis of single small extracellular vesicles using nanoflow cytometry (nFCM): optimized experimental design and implementation

Research Scientist Prashant Kumar, Brian Dobosh, Rabindra Tirouvanziam

PF07.19 Robotic fluidic force microscopy (robotic FluidFM)-based nanoinjection of extracellular vesicles into individual living cells

Dr. Tamás Visnovitz, Ms Kinga Dóra Kovács, Dr. Tamás Gerecsei, Dr. Beatrix Péter, Dr. Sándor Kurunczi, Ms Anna Koncz, Dr. Krisztina Németh, Ms Dorina Lenzinger, Dr. Krisztina V Vukman, Ms Anna Balogh, Ms Imola Rajmon, Dr. Péter Lőrincz, Dr. Inna Székács, Prof. Edit I Buzás, Dr. Róbert Horváth

PF07.20 Single vesicle flow cytometry with enhanced small particle detection using spectral flow cytometry

Dr. Maria Gracia Garcia Mendoza, Dr. John Nolan, Erika Duggan, Patrick Nolan, Kate Pilkington, Dr. Haley Pugsley

PF07.22 Tracking system of CD63-positive extracellular vesicles under in vitro coculture conditions

Lecturer Yutaka Naito, Professor Kazufumi Honda

PF07.24 Visualizing intercellular transfer of extracellular vesicle RNA cargo using an improved metabolic labelling approach

Ms. Willemijn de Voogt, Dr. Richard Wubbolts, Dr. Pieter Vader

PF07.25 The comparison and optimalization of sEV s staining protocols for visualization of cellular uptake in super-resolution microscopy

Mr Jakub Tomaszewski¹, Bsc Wiktoria Klimek², PhD Hanna Kozłowska³, PhD Małgorzata Czystowska-Kuźmicz¹

¹Department of Biochemistry, Medical University of Warsaw, Warsaw, Poland, ²Faculty of Biology and Biotechnology, Warsaw University of Life Sciences, Warsaw, Poland, ³Laboratory of Advanced Microscopy Techniques, Mossakowski Medical Research Institute Polish Academy of Sciences, Warsaw, Poland

PF07.26 Fluorescence Polarization Utilizing Aptamers for Targeted Sensing of sEVs

Mr Satendra Jaysawal^1,2, Dr. Rocky Chowdhury^1,2, Mr. Rajindra Napit^1,2, Ms. Jasmine Catague^1,2, Mr. Haben Melke^1,2, Dr Cuong Pham³, Dr. Wei Duan^1,2

¹School of Medicine, Deakin University, Geelong, Australia, ²Institute of Mental and Physical Health and Clinical Translation (IMPACT), Deakin University, Geelong, Australia, ³Molecular Imaging and Theranostics Laboratory, Baker Heart and Diabetes Institute, Melbourne, Australia

PF07.27 A flow cytometry approach for the characterization and isolation of extracellular vesicles

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, Lyon, France

PS01.02 Apple-derived nanovesicles influence bone regeneration by acting on THP-1-derived macrophage polarization and mesenchymal stem cell osteogenic differentiation

Martina Trentini, Dr. Luca Lovatti, Prof. Dr. Kathrin Becker, Dr. Giulia Brunello, Prof. Dr. Barbara Zavan

PS01.03 Atractylodes macrocephala derived EV-like particles alleviate ulcerative colitis by modulating intestinal flora and TH17 signaling pathway

Professor Kewei Zhao, Xuejun Tan, Bowen Gao, Yukun Xu, Yue Cao, Qing Zhao, Tianxin Qiu, Mingzhen Zhang

PS01.04 Bacteria Gram+ derived nanovesicles and mimetics as new vaccine for Streptococcus pneumoniae (Pn)

Bertrand Czarny

PS01.05 Bacteria-derived mimetic vesicles: their role in the invitro immune response against Streptococcus pneumoniae infection

Dr Dinesh Kesavan

PS01.08 Delineating the pro-osteogenic potential of Lactobacillus rhamnosus derived extracellular vesicles in ameliorating glucocorticoid induced osteoporosis ex vivo

Ms. Megha Sharma^{All India Institute of Medical Sciences (AIIMS), New Delhi, India}, Dr. Rupesh K. Srivastava^{All India Institute of Medical Sciences (AIIMS), New Delhi, India}

PS01.09 Delivery of nucleic acids using red blood cell-derived extracellular vesicles to the central nervous system

Ms Melissa Tan, Dr Brenda Wan Shing Lam, Dr Waqas Muhammad Usman, Dr Thach Tuan Pham, Dr Chang Gao, Dr Harwin Sidik, Ms Rachel Tan, Dr Minh TN Le

PS01.100 Therapeutic potential of IL-1β-primed mesenchymal stromal cells-derived soluble factors and extracellular vesicles in wound healing

PhD Marina Trouillas, Mrs Marine De Taddeo, Mr Pierre Maincourt, Mrs Muriel Nivet, MD Guillaume Valade, Mrs Claire Langle, Mrs Marion Grosbot, Mrs Sylvie Goulinet, PhD Philippe Mauduit, MD, PhD Sébastien Banzet, PhD Juliette Peltzer

PS01.102 Therapeutic role of MSC exosomes in rabbit temporomandibular joint model of osteoarthritis

Dr Yuanyuan Jiang, Dr Shipin Zhang, Dr Sai Kiang Lim, Dr Wei Seong Toh

PS01.105 Umbilical cord mesenchymal stromal cells-derived small extracellular vesicles: advancing knee osteoarthritis therapeutics

Mr. Aliosha I. Figueroa-Valdés¹, Mr. Nicolás Georges², Ms. Catalina Adasme-Vidal¹, Ms. Yeimi Herrera-Luna³, Ms. Patricia Luz-Crawford^{1, 3}, Mr. Maroun Khoury^1,2,4,5,6, Ms. Francisca Alcayaga-Miranda^1,2,4,5,6

¹IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Santiago, Chile, ²Universidad de los Andes, Centro de Investigación e Innovación Biomédica (CiiB), Laboratory of Nano-Regenerative Medicine, Santiago, Chile, ³Universidad de los Andes, Centro de Investigación e Innovación Biomédica (CiiB), Laboratory of Molecular and Cellular Immunology, Santiago, Chile, ⁴Consorcio Regenero, Chilean Consortium for Regenerative Medicine, Santiago, Chile, ⁵Universidad de los Andes, Faculty of Medicine, School of Medicine, Santiago, Chile, ⁶Cells for Cells, Santiago, Chile,

PS01.107 Unveiling the power of adipose tissue stem cell-derived peptide-engineered CD81+/Tsg101+ extracellular vesicles for precision targeting and neural stem cell rejuvenation

Mr. Satyajit Ghosh, Dr. Surajit Ghosh

PS01.108 Urine derived stem cells: A unique robust production platform for autologous immunomodulatory EVs

Anders Boysen, Doctor Bradley Whitehead, Doctor Anne Louise S. Revenfeld, Doctor Anna Karina Juhl, Doctor Reza Yarani, Doctor Yonglun Luo, Doctor Thor Petersen, Doctor Peter Nejsum

PS01.108 An acellular targeted therapeutic approach using extracellular vesicles from human endometrial mesenchymal stem cells

Dr Shanti Gurung^1,2, Ms Diem-Mai Pham Diem-Mai Pham¹, Ms Molly McLaughlin^1,2, Dr Jill C. Danne⁴, Dr Joel R. Steele³, Professor Ralf B. Schittenhelm³, Professor Jerome A. Werkmeister^1,2, Professor Caroline E. Gargett^1,2

¹The Ritchie Centre/Hudson Institute Of Medical Research, Clayton, Australia, ²Obstetrics and Gynaecology, Monash University, Clayton, Australia, ³Proteomics and Metabolomics Platform, Monash University, Clayton, Australia, ⁴Monash Ramaciotti Centre for Cryo-Electron Microscopy, Clayton, Australia

PS01.109 Extracellular vesicles as a treatment for metabolic dysfunction-associated steatohepatitis (MASH) and hepatocellular carcinoma (HCC)

Miss Mihiri Goonetilleke^1,2, Ms Jeanne Correia¹, Dr Yuan Chen¹, Ms Hannah McDonald¹, Dr Siow Teng Chan¹, Mr Ian Simpson⁵, Dr Ishmael Inocencio¹, Prof. William Sievert^3,4, A/Prof Rebecca Lim¹

¹Hudson Institute Of Medical Research, Clayton, Australia, ²Obstetrics and Gynaecology, Monash University, Clayton, Australia, ³Gastroenterology and Hepatology Unit, Monash Health, Clayton, Australia, ⁴Centre for Inflammatory Disease, Monash University, Clayton, Australia, ⁵Monash Health, Clayton, Australia

PS01.11 Development of microbial nanovesicle-based (OMVs) multivalent Vaccine formulation against human/animal diseases

Duvvada Srinivas

PS01.114 Effect of extracellular vesicles isolated from osteoblast differentiation medium of dedifferentiated adipocytes on osteogenic differentiation.

4th grade in Ph.D Yusuke Nishiguchi¹, Ph.D Mamoru Ueda², Ph.D Hirohito Kubo², Ph.D Junichiro Jo³, Ph.D Yoshiya Hashimoto³, Ph.D Toshihiko Takenobu²

¹Graduate school of dentistry department of oral and maxillofacial surgery, Osaka Dental University, Osaka/Chuoku Otemachi, Japan, ²Second Department of Oral and Maxillofacial Surgery, Osaka Dental University, Osaka/Chuoku Otemachi, Japan, ³Department of Biomaterials Osaka Dental University, Hirakatashi/Kuzuhahanazonocho, Japan

PS01.115 Acute Toxicity Effect of UC-MSC Secretome in Different Route of Administration

Dr. Angliana Chouw^1,2, Dr. Cynthia Retna Sartika^1,2, Miss Geofanny Facicilia¹, Miss Annisa Nur Arofah¹, Miss Riska Agustina¹, Miss Zulfa Maulidah¹

¹Prodia Stemcell Indonesia, Jakarta, Indonesia, ²Universitas Padjajaran, Sumedang, Indonesia

PS01.116 Therapeutic Potential of Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles on Atopic Dermatitis: A Comparative Study

Mrs Maimonah Al-Masawa¹, Associate Professor Dr Angela Min Hwei Ng¹, Dr Jhi Biau Foo², Dr Chee Wun How³, Dr. Jia Xian Law¹

¹Centre For Tissue Engineering and Regenerative Medicine, Faculty of Medicine, National University Malaysia, Kuala Lumpur, Malaysia, ²School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, Malaysia, ³School of Pharmacy, Monash University, Malaysia

PS01.118 Intravenous Administration of Extracellular Vesicles Derived from Mesenchymal Stem Cells (MSC-EVs) Mitigates Tendon and Cartilage Degeneration in Type II Diabetes (T2D) Rats

Dr Sik Loo Tan¹, Dr Zahrah Shamim¹, Omar Maged¹, Nik Aizah¹, Dr Qi Hao Daniel Looi², Dr JhiBiau Foo³, Professor Tunku Kamarul^1,4

¹Department of Orthopaedic Surgery, National Orthopaedic Center for Excellence in Research and Learning (NOCERAL), Faculty of Medicine, Universiti Malaya, 50603, Kuala Lumpur, Malaysia., Malaysia, ²My CytoHealth Sdn. Bhd., Lab 6, DMC Level 2, Hive 5, Taman Teknologi MRANTI, Bukit Jalil, Kuala Lumpur 57000, Malaysia, Bukit Jalil, Malaysia, ³School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, Subang Jaya 47500, Selangor, Malaysia, Subang Jaya, Malaysia, ⁴Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, Bertam 13200 Kepala Batas, Pulau Pinang, Malaysia., Bertam, Kepala Batas, Malaysia

PS01.12 Edible plant-derived nanovesicles to systemic deliver nucleic acid medicine for oral administration

Dr. Tomohiro Umezu, Mamoru Yanagimachi, Ph.D. Masakatsu Takanashi, MD, Ph.D. Yoshiki Murakami, MD, Ph.D. Masahiko Kuroda

PS01.13 Effects of ginger-derived extracellual vesicles on preadipocyte differentiation: implications for obesity

Ph.d Student Diksha Choudhary

PS01.130 Comprehensive characterization of olive-derived nanoparticles (ODNPs) as a new drug delivery system.

Graduate Student Zhu Zhao¹, Dr. Jerome Lacombe¹, Dr. Frederic Zenhausern¹

¹Univ Of Arizona, Phoenix, United States

PS01.14 Engineering plant-derived extracellular vesicles for targeted inflammatory therapy in colitis

Su Jin Kang, Ph.D Won Jong Rhee

PS01.15 Enhanced chemo-resistance in liver and breast cancer cells: synergistic interplay of seaberry and garlic-derived extracellular vesicles with cisplatin

Mr. Yasir Mohamed Riza, Dr. Faisal Alzahrani, Dr. Rami Mosaoa

PS01.16 Exosome-delivered curcuminoids to target the brain: Modulation of key markers of Alzheimer's disease (AD)

Dr. Ramesh Gupta, Dr. Raghuram Kandimalla, Ms. Disha Moholkar, Dr. Margaret Wallen, Mr. Jeyaprakash Jeyabalan, Dr. Wendy Spencer, Dr. Neetu Tyagi, Dr. Farrukh Aqil

PS01.18 Extracellular vesicles from bovine milk loaded miR146a-5p prevented group 2 innate lymphoid cells-dominant allergic airway inflammation

Li Chan-gu, Professor Fu Qing-Ling

PS01.19 Extracellular vesicles-derived from VGBR induces cell cycle arrest and apoptosis in human colon cancer cells through p53- pathway

PhD Student Abhinay Kumar Singh, Dr. Win-Ping Deng

PS01.20 Gut commensal bacteria derived outer membrane vesicles tamp down skin inflammatory in psoriasis

Dandan Su, Manchun Li, Professor Hongbo Chen, Fang Cheng

PS01.22 Investigating the therapeutic potential of human amniotic epithelial cell derived extracellular vesicles for inflammatory fetal brain and lung injury in a large animal model

Dr Ishmael Inocencio, Mr Naveen Kumar, A/Prof Rebecca Lim, Dr Tamara Yawno

PS01.23 Isolation, characterization, and functional studies of the Gardenia-derived EVs for potential Parkinson's disease treatment

Haobo Wang, Dr Wen Chen, Dr Yan Wang, Dr Danyang Li, Dr Lizhou Xu

PS01.25 Morinda officinalis-derived extracellular vesicle-like particles anti-osteoporosis by regulating MAPK signaling pathway

Professor Kewei Zhao, Doctor Yue Cao, Master Xuejun Tan

PS01.26 Oral delivery of dihydroartemisinin for the treatment of melanoma via bovine milk-derived small extracellular vesicles (sEVs)

Mr. Dulla Naveen Kumar, Ms. Aiswarya Chaudhuri, Ms Deepa Dehari, Dr. Dinesh Kumar, Dr. Ashish Kumar Agrawal

PS01.27 Parabacteroides goldsteinii-derived exosomes alleviate acute lung injury by regulating gut microbiota

Ms Wensi Zhu, Ms Linxiao Han, Ms Ludan He, Dr Chih-Jung Chang, Jian Zhou

PS01.28 Polygonum cuspidatum derived nanoparticles and acupuncture combined to alleviate rheumatoid arthritis via immunomodulation

Dr Ningcen Li, Prof Bo Li, Prof Lei Zheng

PS01.29 Polygonum cuspidatum derived nanovesicles accelerate wound healing of deep second-degree burn by Nrf 2-Keap pathway

Qi Xiu, Prof. Bo Li, Prof. Lei Zheng

PS01.30 Polyphenol-rich cocoa supplementation elevates neuroactive compounds in escherichia coli nissle 1917 membrane vesicles

PhD Student Amelie Legare, Miss Michele Iskandar, Andre Marette, Stan Kubow

PS01.32 Potential of milk-derived extracellular vesicles for oral drug delivery

HoChung Jang, Dr. Yoosoo Yang

PS01.33 Recombinant extracellular vesicles as vaccines within animal health

Professor Hanne Winther-Larsen

PS01.34 Rhizoma Drynariae-derived nanovesicles reverse osteoporosis by potentiating osteogenic differentiation of human bone marrow mesenchymal stem cells via targeting ERα signaling

Ph.d Qing Zhao, Ph.D Junjie Feng, Ph.D Lei Zheng, Ph.D Kewei Zhao

PS01.37 Turmeric-derived nanoparticles functionalized aerogel regulates multicellular networks to promote diabetic wound healing

Ph.D Bodeng Wu, Ph.D Weilun Pan, Ph.D Shihua Luo, MD Mingzhen Zhong, Professor Bo Li, Professor Lei Zheng

PS01.38 Two-photon responsive microneedles loaded with engineered turmeric-derived extracellular vesicles for detection and treatment of subcutaneous infections

Dr Weilun Pan, Master Mingzhen Zhong, Prof Lei Zheng

PS01.40 Harnessing the delivery potential of milk extracellular vesicles as innovative therapeutic tools for treating bacterial infections

Jitendra Kumar¹

¹ICAR-National Dairy Research Institute, Karnal-132001, India

PS01.40 Amplifying the regenerative and immunomodulatory potential of mesenchymal stem cell-derived small extracellular vesicles via apoptotic induction

Professor Sujata Mohanty, Ms Meenakshi Mendiratta, Ms Mohini Mendiratta, Dr Suchi Gupta

PS01.41 Assessing the cellular effects of ASC-EVs in the context of autologous fat grafting

Dr Emma Symonds, Rachelle Smith, Mr Alexander Brown, Associate Professor Margaret Currie, Dr Kathryn Hally, Dr Kirsty Danielson

PS01.42 Large-scale preparation of milk-derived extracellular vesicles for medical cosmetics application

Xue Wu, Jiuheng Shen, Youxiu Zhong, Xian Zhao, Peifen Gao, Wantong Zhou, Xudong Wang, Professor Wenlin An

¹National Vaccine & Serum Institute (NVSI), China National Biotech Group (CNBG), Sinopharm Group, Beijing, China

PS01.44 Characterization of extracellular vesicles in mesenchymal stem cell co-cultures derived from different tissue origins and potential therapeutic applications

Tsuyoshi Kawaharada, Daisuke Watanabe, Shuhei Iida, Amaka Watanabe, Akio Mizushima

PS01.44 Strain-Based Comparison and Pharmacological Investigation of Bacterial Extracellular Vesicles

Extracellular Vesicles Seoah Park¹

¹Seoul National University, South Korea

PS01.45 Counter-selection of EVs using Kupffer cells alters protein corona and EV biodistribution in vivo

Dr Chintan Bhavsar¹, Dr Rui Chen¹, Ms Elaina Coleborn¹, Ms Shuying Li¹, Ms Sarah Wilkey¹, Mr Trent Neilson¹, Dr Katharine Irvine², Dr James Cuffe¹, Dr Sherry Wu¹

¹The University Of Queensland, Brisbane, Australia, ²Mater Research, Brisbane, Australia

PS01.45 Charting cardiac cell repair: dual therapy combining nanovesicles and biomaterials

Phd Student Auriane Drack, Doctor Alin AR Rai, Hien A Tran, Associate professor Jelena Rnjak-Kovacina, Associate professor David Greening

PS01.46 Characterization and biological activity potential of extracellular vesicles from industrial bovine milk: a comparative analysis of multiple sources

Mr. Aliosha I. Figueroa-Valdés¹, Ms. Catalina Adasme-Vidal¹, Dr. Maroun Khoury¹, Dra. Francisca Alcayaga-Miranda¹

¹IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Santiago, Chile

PS01.46 Comparative analysis of the therapeutic potential of extracellular vesicles from aged and young bone marrow-derived mesenchymal stem cells in osteoarthritis pathogenesis

Ms Shital Wakale, Dr Antonia Sun, Dr Yang Chan, Dr Jennifer Gunter, Dr Chamikara Liyanage, Prof Ross Crawford, Dr Song Wu, Dr Hai Hu, Dr Indira Prasadam

PS01.47 Comparison of the therapeutic effect of fetal and perinatal MSCs derived EVs on inflamed chondrocytes in vitro

Karyna Tarasova, MSc Belen Arteaga, PhD Harini Nivarthi, MSc Johanna Gamauf, MSc Angkana Kidtiwong, PhD Sinan Gültekin, PhD Mathias Hackl, PhD Regina Grillari, Prof. PhD Christopher Gerner, Ass. Prof. Dr. Iris Gerner, Prof. Dr. Florien Jenner

PS01.49 Effects of bone marrow mesenchymal stem cell-derived small extracellular vesicles (BM-MSC-sEVs) on H2O2-induced oxidative damage in human retinal pigment epithelial cells

Dr. Nithikan Suthumchai, Miss Panjaree Siwaponanan, Miss Payalak Sudcharee, Dr. Siripakorn Sangkitporn, Miss Acharaporn Dambua, Miss Patcharaporn Boonchu, Miss Phatcharaphon Nopprang, Prof.Dr. Kovit Pattanapanyasat, Prof. La-ongsri Atchaneeyasakul

PS01.50 Effects of hypoxia precondition on enhancing the anti-inflammatory effects of mesenchymal stem cells derived exosomes may involve microRNA-21-5p

MD Kuan-Wen Chen, PhD Chao-Yuan Chang, MD,PhD Chun-Jen Huang

PS01.51 Engineering adipose-derived stem cell-derived extracellular vesicles by calcium silicates activated for chronic wound healing

Associate Professor Jian-Jr Lee, Dr En-Wei Liu, Dr Yen-Hong Lin, Ms Min-Hua Yu, Associate Professor Ming-You Shie

PS01.52 Enhancing extracellular vesicles yields and functionality for cardiac repair through scalable bioreactor production of human-induced pluripotent stem cells

PhD Student Ana Meliciano, Pedro Vicente, Ana Filipa Louro, Cláudia Diniz, João Jacinto, Paula Marques Alves, Margarida Serra

PS01.53 EVs from hiPSC-derived NSCs are proficient in inhibiting traumatic brain injury-induced NLRP3-p38/MAPK, cGAS-STING activation, and IFN-I signaling

Ashok Shetty, Dr Maheedhar Kodali, Dr Leelavathi N Madhu, Dr Shama Rao, Dr. Raghavendra Upadhya, Ms Sahithi Attaluri, Dr Bing Shuai

PS01.54 Exosome derived from 3D-cultured hADSCs exhibited enhanced osteogenesis capacity via intravenous injection

Dr. Ruijing Chen, Dr. Taojin Feng, Dr. Ming Chen, Dr. Ruijing Chen

PS01.56 Exploring induced pluripotent stem cell-derived exosomes as a potent antimicrobial and immunomodulatory agent against vibrio vulnificus

Phd Pei-Ling Chi

PS01.57 Exploring the immunomodulatory and wound-healing potential of Extracellular vesicles derived from iMSCs

Denise Zujur, MSc William Theoputra, PhD Makoto Ikeya

PS01.58 Exploring the therapeutic potential of extracellular vesicles derived from human mesenchymal stem cells in tumor-bearing mice

Dr. Prapatsorn Charoenyingpaisal, Dr. Hsien-Hen Lin, Dr. Toru Okubo, Mr. Hayato Kurata, Mr. Tetsuo Koike, Mr. Yoichi Honma

PS01.59 Extracellular vesicles derived from iMSC primed with hyaluronic acid enhance cardiac function.

Ph.D. Seon-Yeong Jeong, Ph.D. Jimin Kim, M.S. Seul Ki Lee, M.S. Haedeun You, Ph.D. Soo Kim

PS01.60 Extracellular vesicles derived from mesenchymal stem cells and cartilage tissue to promote cartilage regeneration

Dr. Jia Xian Law, Ms. Chiew Yong Ng, Assoc. Prof. Min Hwei Ng, Prof. Ying Yang, Assoc. Prof. Jhi Biau Foo, Dr. Chee Wun How, Assoc. Prof. Kien Hui Chua, Assoc. Prof. Kok Yong Chin, Dr. Rizal Abdul Rani, Prof. Nor Hamdan bin Mohamad Yahaya

PS01.61 Extracellular vesicles derived from mesenchymal stem cells reduce inflammation and restore intestinal barrier integrity in a new in vitro co-culture model of intestinal inflammation

Miss Mona Belaid, Giorgia Pastorin, Driton Vllasaliu

PS01.63 Extracellular vesicles of senescent mesenchymal stromal cells lose their antifibrotic potential both in vitro and in vivo

Mr. Maksim Vigovskii, Ms. Nataliya Basalova, Ms. Olga Grigorieva, Ms. Uliana Dyachkova, Mr. Vladimir Popov, Ms. Anastasia Tolstoluzhinskaya, Ms. Anastasia Efimenko

PS01.65 First European Medicines Agency approved study with Umbilical Cord Mesenchymal Stromal Cell Extracellular Vesicles in the prevention of Bronchopulmonary Dysplasia: merit of a scalable GMP production platform

Ms. Sofia Baptista, Ms. Cristina Manfredi, Dr. Marcin Jurga, Mr. Gabrielis Kundrotas, Mr. Dimitri Stevens, Mr. Domenico Mancuso, Ms. Elisabetta Gramegna, Mr. Rudra Kashyap, Ms. Sandrine Mores, Prof Eugenio Baraldi, Prof Maurizio Muraca, Dr. Beatrice De Vos

PS01.66 Human platelet lysate enhanced angiogenic potential of extracellular vesicles derived from mesenchymal stem cells

Dr Yue Zhang, Professor Tao-tao Tang, Professor Lin-li Lv, Professor Bi-cheng Liu

PS01.67 Hypoxia primed WJ-MSCs-sEVs exhibit enhanced immunomodulatory & regenerative activity in wound milieu primarily via miR125b-5p/IL-6R axis

Ms Yashvi Sharma, Dr Sujata Mohanty

PS01.68 Immunomodulatory potential of IL-1β-primed mesenchymal stromal cells-derived extracellular vesicles and soluble factors to prevent organ dysfunction after a traumatic hemorrhagic shock

PHD Student Guillaume Valade, MD Clément DEVAUTOUR, Mrs Marion GROSBOT, Mrs Muriel NIVET, Phd Student Marine DE TADDEO, PHD Ahmad HAIDAR, PHD Patrice DECKER, Mrs Sylvie GOULINET, PHD Philippe MAUDUIT, MD, PHD Sébastien BANZET, PHD Marina TROUILLAS, PHD Juliette PELTZER

PS01.69 Immunomodulatory properties of dental pulp stem cell derived EVs

Dr Sadiq Umar^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Koushik Debnath^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Chun-Chieh Huang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Kasey Leung^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Miya Kang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Yu Lu^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Praveen Gajendrareddy^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Sriram Ravindran^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}

PS01.70 Intranasal delivery of NAMPT-MSC-sEV improves cognitive function in mice with repeated mild traumatic brain injury by reducing acetylated tau

Prof. Qing-Ling Fu

PS01.71 Locoregional intra-arterial delivery of MSC-derived extracellular vesicles directly into the pancreas maintains glycemic regulation in diabetic rats

Dr. Reza Yarani, Dr. Rosita Primavera, Dr. Shashank Chetty, Dr. Jing Wang, Prof. Flemming Pociot, Dr. Avnesh Thakor

PS01.72 Lyophilization of engineered EVs for regenerative medicine

Dr. Chun-chieh Huang, Dr Miya Kang, Dr Koushik Debnath, Ms Yu Lu, Dr Sriram Ravindran

PS01.73 Mechanism of miR-155-5p in hiMSC-sEV in treating androgenic alopecia by activating AKT/β-catenin/GSK3β signal pathway

Post-doctor Ruiyun Tian, Professor Furong Li

PS01.74 Mensenchymal stem cell exosomes and its effect on facial skin regeneration and rejuvenation

Ms. Ching-fen Yang, Ms. Hoei Ser Chong, Dr. Takaaki Matsuoka

PS01.75 Mesenchymal stem cells activate cellular autophagy by delivering exosomes to alleviate LPS-induced endothelial inflammatory injury in sepsis

Shiyue Lu, Zhe Li, Yuqing Xu, Yuxiao Deng

PS01.76 Mesenchymal stromal cells-derived small extracellular vesicles For corneal wound healing

Seyedmohammad Moosavizadeh, PhD Student Jiemin Wang, Dr. Ellen Donohoe, PhD Student Aoife Canning, Dr. Aideen Ryan, Professor Thomas Ritter

PS01.77 MicroRNA-100-5p mediates the therapeutic efficacy of mesenchymal stem cell-derived exosomes in a murine psoriasis model

Dr Yu Chen Huang, Dr. Chao Yuan Chang, Dr. Chun Jen Huang

PS01.79 MSC-small extracellular vesicles alleviated Th2-airway inflammation by regulating the metabolism of DCs in mice

Prof. Qing-Ling Fu, Lifen Wen, Longxin Huang

PS01.81 pcMSCs-derived exosome promoting stem cell reprogramming and suppressing inflammatory condition in LPS-induced ARDS/ALI model

Phd Student Kajal Singh, Mr. Abhinay Kumar Singh, Dr. Yen-Hua Huang

PS01.83 Potential therapeutic effect of human dental stem cells-derived exosomes enhances neurological function and cerebral blood flow after ischemic stroke in rats

Assistant Professor Sukonthar Ngampramuan, Doctor Anyapat Atipimonpat, Associate Professor Hathaitip Sritanaudomchai, Assistant Professor Paranee Yatmark

PS01.84 Purification and characterization of extracellular vesicles derived from induced pluripotent mesenchymal stem cells for treatment of vaginal prolapse

Dr. Olivia Cardenas- Trowers, Ralph Perkerson, Tammee Parsons, Nabanita Halder, Nisha Durand, Abba Zubair, Jing Zhao, Takahisa Kanekiyo

PS01.86 Rejuvenation by OCT4/SOX2 in extracellular vesicles through regulation of the toll-like receptor 4 (TLR4) pathway

Professor Jisook Moon

PS01.87 Safety evaluation of Wharton's Jelly Mesenchymal Stem Cells (WJMSCs) derived small extracellular vesicles (sEVs) on healthy Sprague Dawley rats

Mr Illayaraja Krishnan, Associate Professor Dr Min Hwei Ng, Dr. Jia Xian Law, Ms Shathiya Rajamanickam, Dr Baskar Subramani, Associate Professor Dr Yogeswaran Lokanathan

PS01.88 Secretome derived from wharton jelly-mesenchymal stem cells mitigate acute graft-versus-host-disease: impact of hypoxia and apoptosis

Ms Mohini Mendiratta, Ms Meenakshi Mendiratta, Dr. Sandeep Rai, Professor Ritu Gupta, Dr. Sabyasachi Bandyopadhyay, Dr. Hariprasad GuruRao, Professor Sujata Mohanty, Dr. Ranjit Sahoo

PS01.89 Senolytic CD38 antigen receptor-modified mesenchymal stem cell-derived extracellular vesicles slowing age-associated degeneration

Dr Yaoying Long, Dr. Bianlei Yang, Prof. Zhichao Chen, Prof. Qiubai Li

PS01.93 Small extracellular vesicles derived from human chemically induced liver progenitors (hCLiPs) improve liver fibrosis via inactivation of hepatic stellate cells.

Ms Tomoko Yamaguchi, Dr Juntaro Matsuzaki, Dr Takeshi Katsuda, Ms Noi Tokuda, Mr Yuzhi Tan, Dr Masaki Kimura, Dr Takahiro Ochiya, Dr Yoshimasa Saito

PS01.94 Small extracellular vesicles derived from human mesenchymal stem cells prevent Th17-dominant neutrophilic airway inflammation via immunoregulation on Th17 cells

PhD. Bi-Xin He, Prof. Qing-Ling Fu, PhD. Shu-Bing Fang, PhD. Chan-Gu Li

PS01.95 Stem Cell-Derived Extracellular Vesicles: a potential therapy for premature lung disease

Dr Hala Saneh, Heather Wanczyk, Joanne Walker, Dr Christine Finck

PS02.01 3D culture of human adipose stem cells in physiological oxygen for therapeutic extracellular vesicle production

Doctoral Researcher Julia Monola, Postdoctoral Researcher Chris Pridgeon, Alisa Jokela, Principal investigator Riina Harjumäki

PS02.02 Biomanufacturing of immature cardiomyocytes derived extracellular vesicles in 2L stirred tank bioreactor

MSc João Jacinto, MSc Ana Meliciano, MSc Lara Inocêncio, MSc Pedro Vicente, PhD Margarida Serra

PS02.03 Bioprocess optimization for extracellular vesicles derived from mesenchymal stem cells

Aslan (mehdi) Dehghani, Senior Scientist Eric Black, Senior Scientist Zheng Zhao, Senior Scientist Namitha Haridas, Senior Manager of Process Development Sunandan Saha, Senior Manager of Process Development David Splan, Head of Process Development Services Mark Szczypka, Head of Advanced Bioprocessing David Pollard

PS02.04 Development of extracellular vesicles collect media for naïve and engineered HEK293 cells

Dr Kartini Asari, Kol Thida Mom, Amirah Fitri, Sadman Bhuiyan, Dr Ramin Khanabdali, Professor Gregory Rice

PS02.05 Extracellular vesicles bioprocess design and economic modeling

Aslan (mehdi) Dehghani

PS02.06 High-yield exosome production from cells-laden 3D auxetic scaffolds with cyclic mechanical stimulation for an effective drug delivery

Associate Professor Ming-You Shie

PS02.07 Impact of donor variability on the therapeutic potential of platelet-derived extracellular vesicles in regulating endothelial cell permeability

Malvika Gupta, Dr. Mandeep Kaur, Dr. Sowmya Shree Gopal, Dr. Jessica Cardenas, Dr. Amit Srivastava

PS02.10 Scalable production of modified HEK293T extracellular vesicles using adherent packed bed bioreactor culture system

Dr Choon Keong Lee^{Esco Aster Pte Ltd}, Ms Claudine Ming Hui Lim^{Esco Aster Pte Ltd}, Ms Winnie Faustinelie^{Esco Aster Pte Ltd}, Dr Desy Silviana^{Esco Aster Pte Ltd}, Mr Xiangliang Lin^{Esco Aster Pte Ltd}

PS02.11 A decision-making tool to navigate through extracellular vesicle research and product development

Francesca Loria, Sabrina Picciotto, Giorgia Adamo, Andrea Zendrini, Samuele Raccosta, Lucia Paolini, Mauro Manno, Paolo Bergese, Giovanna L. Liguori, Paolo Guazzi, Antonella Bongiovanni, Nataša Zarovni

PS02.12 Analytical toolbox for reliable characterization of extracellular vesicles

Aslan (mehdi) Dehghani, Paul Keselman, Prabuddha Mukherjee, Meng Chai, michael Olszowy, Jordan Speidel, Thomas Gaborski, Nick Luey

PS02.13 In vivo and in vitro studies on the role of sEVs as a drug delivery system in breast cancer; a systematic review

Mr Abdulwahab Teflischi Gharavi, Prof Keykavous Parang, Dr Saeed Irian, Prof Mona Salimi

PS02.14 Orthogonal measurement of number concentration standards for NTA calibration

Julie Chen, Product Line Manager - Particle Characterization Jeffrey Bodycomb, Ph.D.

PS03.01 Effects of exosomes derived from skeletal muscle of senescent mice on bone metabolism

Dr Mingming Zhang, Dr Ran Li, Dr Zhongqi Wang

PS03.02 Elucidating the role of extracellular vesicles in mediating reprogramming processes: connecting in vitro insights to in vivo applications for enhanced peripheral nerve tissue repair

PhD Ana Salazar Puerta, Neil Ott, Sara Kheirkhah, Jon Stranan, Grant Barringer, Samuel Cortes, Roxanne Vermette, Emily Moser, MS Hallie Harris, PhD William Lawrence, PhD Devleena Das, MD PhD Mana Saffari, MD Amy Moore, PhD Daniel Gallego-Perez

PS03.04 Extracellular vesicles secreted by mesenchymal stromal cells may regulate the pool of activated stromal cells during the development of fibrosis

Ms NATALIYA BASALOVA, Ms Olga Grigorieva, Ms Anastasiya Tolstoluzhinskaya, Ms Uliana Dyachkova, Mr Maxim Vigovsky, Ms Maria Kulebyakina, Mr Vladimir Popov, Ms Natalia Kalinina, Ms Zhanna Akopyan, Ms Anastasia Efimenko

PS03.05 Extracellular vesicles secreted by multipotent mesenchymal cells contribute to the suppression of macrophage proinflammatory phenotype and reduce their profibrotic properties

A. E. Tolstoluzhinskaya, Ms Uliana Dyachkova, Mr Maksim Vigovskiy, Dr Nataliya Basalova, Ms Anna Gardzhuk, Dr Anastasia Efimenko, Dr Olga Grigorieva

PS03.06 Facilitating muscle formation via bone-derived extracellular vesicles induced by HDAC-inhibition

Dr. Ming Chen, Dr. Taojin Feng, Dr. Mingming Zhang, Dr. Ruijing Chen, Prof. Yi Li, Prof. Licheng Zhang, Prof. Pengbin Yin, Prof. Peifu Tang

PS03.08 Plasma EV-miR-887-3p levels reflect the therapeutic effect of the antifibrotic agent in patients with liver cirrhosis

Dr. Juntaro Matsuzaki, Ms. Mayu Yoshida, Dr. Koji Fujita, Dr. Masamichi Kimura, Ms. Noi Tokuda, Ms. Tomoko Yamaguchi, Dr. Masahiko Kuroda, Dr. Takahiro Ochiya, Dr. Yoshimasa Saito, Dr. Kiminori Kimura

PS04.01 Cardiomyocytes-derived EVs for the treatment of COVID-19-induced cardiac damage

Dr Marta Prieto-Vila, Dr Yusuke Yoshioka, Professor Takahiro Ochiya

PS04.03 Elucidating pathophysiology of hypertrophic cardiomyopathy (HCM): Proteomics in extracellular vesicles (EVs) of HCM patient tissue reveals altered metabolic state and increased cardiac EV release

Msc Sarah Hilderink^{Physiology, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1118, Amsterdam, the Netherlands}, Rita Najor^{Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA}, Richard Goeij-de Haas^{Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Berend Gagestein, Jaco Knol^{Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Thang V Pham^{Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Kenneth C Bedi Jr^{Cardiovascular Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA}, Kenneth B Marguiles^{Cardiovascular Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA}, Michelle Michels^{Department of Cardiology, Erasmus MC, Rotterdam, The Netherlands}, Connie R Jimenez^{Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Asa Gustafsson^{Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA}, Jolanda van der Velden^{Physiology, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1118, Amsterdam, the Netherlands}, Diederik WD Kuster^{Physiology, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1118, Amsterdam, the Netherlands}

PS04.04 Endothelial cell derived extracellular vesicles contribute to laminar shear stress adaptation

Research Specialist II Amber Eliason, Graduate Student Santiago Moreno, Assistant Professor David Marciano

PS04.05 Epithelial cell-derived extracellular vesicle mediated inflammation, infection, and cellular senescence in chronic obstructive pulmonary disease

Miss Georgia Bateman, Professor Cliff Taggart

PS04.06 Inflammatory endothelial cell-derived apoptotic bodies modulate innate and adaptive immune processes

Dr Amy Baxter, Ms Caitlin Vella, Dr Pamali Fonseka, Dr Tien Nguyen, Dr Emma Grant, Prof Suresh Mathivanan, Prof Stephanie Gras, Prof Mark Hulett, A/Prof Ivan Poon

PS04.06 Extracellular Vesicles mediated communication between fat and heart during heart failure

Achala Moncy, Assistant Professor Sam Das

PS04.08 Lipogenic lung fibroblast-derived extracellular vesicles attenuate cigarette smoke-induced COPD pathology by enhancing alveolar type II cell stemness

Dr Yu Fujita, Dr Shota Fujimoto, Dr Reika Kaneko, Dr Jun Araya

PS04.11 Reduced CD63+ extracellular vesicle levels associate with atherosclerosis in hypercholesteraemic mice and humans

Mr Brachyahu Kestecher

PS04.14 Small EV-associated miR-145 is a driver in mitral valvular interstitial cell transition in mitral valve prolapse

Associate Professor Vicky Yang, Dawn Meola, Nicole Moyer, Runzi Zhou, Sally Carnevale, Guoping Li, Saumya Das

PS04.15 VCAM-1+ endothelial cell derived extracellular vesicles mediate the acute phase response following myocardial infarction

Naveed Akbar, Mr Daan Paget, Mr Lewis Timms, Dr Daniel Radford Smith, Ms Rebecca Rooney, Ms Heleah Soulati, Ms Carla De Villiers, Professor Paul Riley, Professor Robin Choudhury, Professor Daniel Anthony

PS04.17 Hypoxia induced extracellular vesicles of the neurovascular unit in a model of blood-brain barrier disruption.

Miss Rebecca Raven¹, Doctor Jessica Williams¹, Professor Keith Morris¹, Professor Philip James¹

¹Centre for Cardiovascular Health and Aging, Cardiff Metropolitan University, Cardiff, United Kingdom

PS04.18 Extracellular vesicles in aging cold-stored whole blood do not seem to compensate for the decreasing hemostatic function

Petra Ilvonen¹, Sanna Susila^1,2, Reetta Pusa¹, Ulla Impola¹, Tuukka Helin³, Lotta Joutsi-Korhonen³, Saara Laitinen¹, Jouni Lauronen¹, Minna Ilmankunnas^1,4,5

¹Finnish Red Cross Blood Service, Helsinki, Finland, ²Emergency Medical Service and Emergency Department, Päijät-Häme wellbeing services county, Lahti, Finland, ³Department of Clinical Chemistry, HUS Diagnostic Center, Helsinki University Hospital and University of Helsinki, Helsinki, Finland, ⁴Department of Anesthesiology and Intensive Care Medicine, Helsinki University Hospital and University of Helsinki, Helsinki, Finland, ⁵Meilahti Hospital Blood Bank, Department of Clinical Chemistry, HUS Diagnostic Center, Helsinki University Hospital and University of Helsinki, Helsinki, Finland

PS05.01 A novel approach of T cell engineering by targeted exosomes delivering CRISPR/Cas9 system for PD-1 knock-out

Ms, Ph.D. candidate Mahboubeh Shahrabi Farahani, Ph.D. Elham Hosseini-Beheshti, Ph.D. Mehdi Forouzandeh Moghadam, Ms, Ph.D. candidate Leila Darzi

PS05.01 Production of a targeted delivery system for T cell modifications by engineering exosomes to express ICAM-1

Ms, Ph.D. candidate Mahboubeh Shahrabi Farahani, Ph.D. Elham Hosseini-Beheshti, Ph.D. Mehdi Forouzandeh Moghadam, Prof Seyed Mohammad Moazzeni, Ms Leila Darzi

PS05.02 Advanced SIRPα-enhanced extracellular vesicles: a novel approach in fibrosis treatment

Advanced Sirpα-enhanced Extracellular Vesicles: A Novel Approach In Fibrosis Treatment Minjeong Kwon, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Min Kyoung Jo, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Seohyun Kim, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Dong-U Shin, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Gi Beom Kim, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Gi-Hoon Nam, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment In-San Kim

PS05.03 Alleviating lung inflammation via effective miRNA delivery to alveolar macrophages using extracellular vesicles conjugated surfactant protein A

Student Miji Kim, Student Sujeong Park, Student Nayeong Lee, Student Dohyun Kim, Student Dongwoo Kim, Ph.D Seon-Jin Lee, Ph.D DVM Jung Joo Hong, Ph.D (Professor) Heedoo Lee

PS05.04 Antigen binding extracellular vesicles for targeted drug delivery

Mr Madhusudhan Bobbili, Nuria Gimeno, Mr Stefan Vogt, Florian Rüker, Gordana Wozniak-Knopp, Johannes Grillari

PS05.05 Cassette-like modification of biofunctional peptides on extracellular vesicles (PepEVs) for on-demand intracellular delivery

Dr. Ikuhiko Nakase

PS05.07 Development of an EV-based siRNA delivery platform for targeting metastatic cancers

Chia-Ling Hsieh, Doctor Anh Duy Do, Miss Mafewu Olga Raboshakga, Professor Shian-Ying Sung

PS05.09 Development of DC-targeting exosome-based drug delivery platform

Ph.D. Student Sheng-Yun Hsu, Undergraduate Hsi-Ming Chiang-Hsieh, M.S. Chen-Guang Zhang, Ph.D. Chen-Yun Yeh, Ph.D. Pi-Hui Liang, Ph.D. Han-Chung Wu, Ph.D. Yungling Leo Lee

PS05.10 Development of Dendritic cells derived exosomes based novel vaccine formulation against Latent tuberculosis

Dr Saima Naz, Dharani bandi, farhan ahmed

PS05.11 Development of exosome-based antibiotic transport for enhanced intracellular efficacy

Miss Ayaulym Nurgozhina, Shynggys Sergazy, Madiyar Nurgaziyev, Laura Chulenbayeva, Mohamad Aljofan

PS05.12 Development of lung-directed siRNA-carrier using autologous serum-derived small EVs for lung metastases of melanoma

Dr. Mai Hazekawa, Dr. Dasuke Watase, Dr. Takuya Nishinakagawa, Dr. Masato Hosokawa, Dr. Daisuke Ishibashi

PS05.13 Development of targeted exosome as plasmid delivery vehicles to HER2-expressing breast cancer cells

Miss Leila Darzi, Dr Mehdi Forouzandeh Moghadam, Dr Mehdi Shamsara, Dr Elham Hosseini-Beheshti

PS05.14 Development of targeted exosomes as CRISPR/Cas9 delivery platforms to HER2-expressing breast cancer cells

Miss Leila Darzi, Dr Mehdi Forouzandeh Moghadam, Dr Mehdi Shamsara, Dr Elham Hosseini-Beheshti

PS05.15 Doxorubicin-loaded therapeutic EVs as effective drug delivery vehicles to neuroblastoma cells

Doctoral Candidate Marc Liébana, Doctoral Candidate Silvia López, PhD Esperanza González, PhD Juan Manuel Falcón

PS05.16 Endogenous protease mediated delivery of engineered immunomodulatory extracellular vesicles

Ms. Kasey Leung^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Miya Kang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Koushik Debnath^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Chun-Chieh Huang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Sadiq Umar^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Mrs. Yu Lu^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Sriram Ravindran^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}

PS05.17 Engineered exosomes loaded with let-7i-5p microRNA allay acute lung injury in mice with gastric content aspiration

Professor Chun-Jen Huang, Doctor Chao-Yuan Chang, Doctor Ching-Wei Chuang

PS05.18 Engineered extracellular vesicles for targeting and activation of lymphatic VEGFR-3

Dr. Wolf Holnthoner

PS05.20 Engineered MSC-derived exosomes alleviate radiation-induced lung injury via transferring mitochondrial component to improve homeostasis of lung epithelial cells

Distinguished professor KS Clifford Chao, Attending Physician Chi-Hsien Huang, Research Assistant Shi-Xuan Yan, Research Assistant Hsin-Yu Chang, Research Assistant Pei-Chen Yang, Associate Professor Kevin Chih-Yang Huang

PS05.21 Engineered MSC-EVs scavenge self-antigen for alleviating psoriasis via modulating metabolic and immunological disorders

Mr Xin Zhou, Dr Jiancheng Wang, Dr Danyang Li

PS05.22 Engineered targeting extracellular vesicles as nano-carriers loaded with chemo-drug for cancer therapeutics

Professor Yiwen Chen, Seiner Engineer Kai-Wen Kan, Professor Ming-You Shie, Professor Shao-Chih Chiu, Superintendent Der-Yang Cho

PS05.24 Enhancing the targeting and regenerative efficacy of mesenchymal stem cell-derived small extracellular vesicles via dual modification strategies

Ms Meenakshi Mendiratta, Dr Sujata Mohanty

PS05.25 Enveloped protein nanocages (EPN) as a versatile and controllable engineered EV platform

Dr Daniel Humphrys

PS05.26 Ex vivo T cell editing as a therapeutically relevant model to evaluate EV cargo delivery

Juliette Suermondt, PhD Xiuming Liang, Guannan Zhou, Houze Zhou, Oskar Gustafsson, PhD H. Yesid Estupiñan, PhD Yang Liu, Professor Samir EL Andaloussi, Assistant professor Joel Nordin

PS05.27 Exploring HER2 isoform in secreted EV as a co-treatment for HNSCC cells to tyrosine kinase inhibitors

Ms Fui Teen Chong, Ms Hui Sun Leong, Ms Mengjie Ren, Dr Shen Yon Toh, Prof N Gopalakrishna Iyer

PS05.28 Exploring the loading of cell penetrating peptides (CPPs) into extracellular vesicles (EVs) for therapeutic applications

Ms. Neona Lowe, Rachel Mizenko, Dr. Alyssa Panitch, Dr. Randy Carney

PS05.31 Generalizable anchor aptamer strategy for loading and targeted delivery of nucleic acid therapeutics on exosomes

PhD Gang Han

PS05.32 Generating engineered EVs with targeting properties against EGFR+ triple-negative breast cancers

Ragnar Axel Adolfsson^{Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland}, Erna Jonsdottir^{Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland; Biomedical Center, University of Iceland, Iceland}, Dr. Jens Guðmundur Hjörleifsson^{Department of Biochemistry, Science Institue, University of Iceland}, Dr. Berglind Eva Benediktsdóttir^{Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland; Biomedical Center, University of Iceland, Iceland}

PS05.34 In silico protein design with cyclization facilitates efficient delivery into cells and extracellular vesicles

Ms Yeonju Lee, Mr Kyung-Min Kim, Mr Young-Pil Kim

PS05.35 Intraarticularly delivered mRNA-encapsulating extracellular vesicles for osteoarthritis therapy

Researcher Hsiu-Jung Liao, Dr. Tai-Shan Cheng, Miss Yi-Shan Shen, Mr. Sin-Yu Chen, Professor Chih-Hung Chang, Professor Ly Lee, Professor Chi-Ying Huang

PS05.37 mRNA loading of extracellular vesicles for the treatment of neurological disorders

Miss Patricia Wongsodirdjo, Dr Ya Hui Hung, Dr Fazel Shabanpoor, Dr Laura Vella, Dr Rebecca Nisbet

PS05.38 Nanofluidic constriction enables encapsulation of biomacromolecule in small extracellular vesicles for efficient intracellular delivery

Zitong Yu, Huitao Zhang, PhD Rui Hao, PhD Candidate Shi Hu, Sihui Chen, Professor Hui Yang

PS05.39 Optimization of mRNA loading into extracellular vesicles for in vivo therapeutic delivery

Ph.D Candidate Liouba Le Roux, Ph.D. Adityas Purnianto, Ph.D. Laura Vella, Ph.D. Ya Hui Hung

PS05.42 RBCEVs: A promising platform for safe and efficient gene therapy, mitigating risks and enhancing expression

Ms Melissa Tan, Dr Brenda Wan Shing Lam, Dr Harwin Sidik, Dr Tenzin Gocha, Dr Ronne Wee Yeh Yeo, Dr Minh TN Le, Dr Waqas Muhammad Usman

PS05.43 Reactive oxygen species responsive multifunctional fusion extracellular nanovesicles: prospective treatments for acute heart transplant rejection

Professor Hongbo Chen, Xingyu Lu, Dr Fang Cheng

PS05.44 Reprogramming of T cell-derived small extracellular vesicles using IL2 surface engineering induces potent anti-cancer effects through miRNA delivery

Dr. Dokyung Jung, Sanghee Shin, Dr. Sung-Min Kang, Inseong Jung, Suyeon Ryu, Soojeong Noh, Dr. Sung-Jin Choi, Jongwon Jeong, Beom Yong Lee, Kwang-Soo Kim, Dr. Christine Seulki Kim, Dr. Jong Hyuk Yoon, Dr. Chan-Hyeong Lee, Dr. Felicitas Bucher, Dr. Yong-Nyun Kim, Prof. Sin-Hyeog Im, Dr. Byoung-Joon Song, Prof. Kyungmoo Yea, Prof. Moon-Chang Baek

PS05.45 Research on the treatment of knee osteoarthritis with CXCR7 delivered by engineered extracellular vesicles

Bin Zeng, Duan Li

PS05.46 Sensitisation of EGFR-driven cancers to EGFR tyrosine kinase inhibitors by application of exosomal EGFR isoform D as a co-drug

Ms Hui Sun Leong, Dr Shen Yon Toh, Ms Fui Teen Chong, Ms Mengjie Ren, Prof N. Gopalakrishna Iyer

PS05.48 Slow controlled release of extracellular vesicles with hydrogel based nanoparticles

Ms. Reese Wunsche, Dr. Morteza Jeyhani, Mr. Boyang Su, Dr. Hon Sing Leong, Dr. Scott Tsai

PS05.49 Small extracellular vesicles as a superior targeted drug delivery system compared to liposomes

Dr. Diem Nguyen, Thieu Nguyen, Nhan Vo, Dr. Lan N Tu

PS05.50 Strategic loading of epitopes onto EV subtypes using Craftgen@EV for vaccine of cellular immunity

Ph.D. student Shota Shinagawa, Technical Staff Tamiko Minamisawa, Technical Staff Saki Matsumoto, Project Leader Kazuma Kiyotani, Kiyotaka Shiba

PS05.51 Targeted cargo delivery to mouse lower limb by exosome carrying a muscle targeting moiety with intravenous injection

Mr. Minghao Sun, Associate Director Mafalda Cacciottolo, Principal Scientist Yujia Li, Senior Scientist Mahrou Sadri, Senior Scientist Michael LeClaire, Research Associate David Tran, Chief Scientific Officer Kristi Elliott

PS05.53 The development of engineered exosome-conjugated nanobody for nuclei acids/drug delivery in glioblastoma treatment

Dr. Shao-chih Chiu, Dr. Ming-You Shie, Dr. Shi-Wei Huang, Dr. Chih-Ming Pan, Dr. Der-Yang Cho

PS05.54 Using autologous blood-derived extracellular vesicles as precision therapeutics for retinal degenerations

Ms Rakshanya Sekar, Dr Yvette Wooff, Dr Adrian Cioanca, Associate Professor Riccardo Natoli

PS05.55 Utilising engineered stem cell-derived nanovesicles (scNVs) as a scalable, cell reprogramming therapeutic for cardiac repair

Phd Candidate Jonathan Lozano, Dr. Jarmon G Lees, Dr. Alin Rai, Dr. Kyah Grigolon, Dr. Helen Kiriazis, Ren Jie Phang, Jonathon Cross, Haoyun Fang, Dr. Daniel Donner, Shiang Y Lim, Dr. David W. Greening

PS05.56 Engineered serum extracellular vesicles deliver CRISPR-Cas9 ribonucleoproteins to modify the dystrophin gene

Ph.d Yaoyao Lu, Research assistant Nathalie Majeau, Ph.D Camile Bouchard, Professor Jacques-P Tremblay

PS05.58 Engineered extracellular vesicles for delivery of therapeutic small RNA

PhD student Julia Anna Rädler, Giulia Corso, Antje Zickler, Noriyasu Kamei, Wenyi Zheng, Dhanu Gupta, Samir El Andaloussi

PS05.63 Multifunctional aggregation-induced emission-based extracellular vesicles to remodel microenvironment for infected wound healing

Doc. Pingping Wang¹, Professor Hang Zou¹, Prof. Lei Zheng¹

¹Department Of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China

PS05.64 Arginine-rich cell-penetrating peptide-modified microvesicles for macropinocytosis induction and enhanced intracellular delivery

Dr. Ikuhiko Nakase¹, Kenta Morimoto¹, Jojiro Ishitobi¹, Dr. Kosuke Noguchi¹, Ryoichi Kira¹, Dr. Tomoka Takatani-Nakase², Dr. Ikuo Fujii¹, Dr. Shiroh Futaki³, Dr. Masamitsu Kanada⁴

¹Graduate school of Science, Osaka Metropolitan University, Sakai, Japan, ²School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan, ³Institute for Chemical Research, Kyoto University, Uji, Japan, ⁴Department of Pharmacology and Toxicology, Michigan State University, East Lansing, USA

PS05.65 Extracellular Vesicle Sorting Motif Platform for Natural Multiplex Cargo Delivery

Laboratory Director Gibeom Kim¹, Senior Researcher Inkyu Lee^1,3, CEO Gi-hoon Nam^1,2, Professor In-San Kim^3,4

¹Department of Research and Development, SHIFTBIO INC., Seoul, South Korea, ²Department of Biochemistry & Molecular Biology, Korea University College of Medicine, Seoul, South Korea, ³KU-KIST Graduate School of Converging Science and Technology. Korea University, Seoul, South Korea, ⁴Chemical and Biological Integrative Research Center, Korea Institute of Science and Technology, Seoul, South Korea

PS06.01 Airway basal stem cell-derived extracellular vesicles: a promising strategy for fibroblasts regulation

Dr. Lisi Luo, Dr. Huijie Yang, Dr. Junfeng Huang, Dr. Shiyue Li

PS06.06 Evaluating the role of GM-1 ganglioside in neuronal uptake

Miss Thitikan Jirakittisonthon, Dr. Orman Snyder, Dr. Hong He, Dr. Mark Weiss

PS06.08 Hyaluronan coat enhances the targeting of extracellular vesicles into CD44 overexpressing cells

MSc Heikki Kyykallio, BSc Kirsti Härkönen, PhD Martina Hanzlíková, PhD Tatu Lajunen, Professor Tapani Viitala, PhD Kirsi Rilla

PS06.09 Identifying proteins that impact differential uptake of extracellular vesicles from ovarian follicular fluids collected at early and late in follicle growth phases

Assistant Professor Wei-Ting Hung, Professor John S. Davis, Professor Lane Christenson

PS06.10 In vivo differential kinetic distribution of extracellular vesicles affected by oncogenic RAS and RAF transformation

Ms Shinwon Chae, Mr Chul Won Seo, Ms Haekang Yang, Professor Yoon-Jin Lee, Professor Dongsic Choi

PS06.11 NaTaLi: Nanobody-Tag Ligand click strategy for targeted multicolor EVs

Dr. Andrea Galisova, Dr. Jiri Zahradnik, Dr. Daniel Jirak

PS06.12 NHE7 upregulation potentiates the uptake of small extracellular vesicles by enhancing maturation of macropinosome in hepatocellular carcinoma

Dr Yao Yue, Dr Xu Yi, Dr Judy Wai Ping Yam

PS06.13 Nodal flow transfers polycystin to determine mouse left-right asymmetry

Dr. Yosuke Tanaka, Dr. Ai Morozumi, Dr. Nobutaka Hirokawa

PS06.16 Study on the bio-distribution of autologous serum-derived small EVs in a melanoma spontaneous metastasis mice model for the development of nucleic acid carriers for cancer metastasis

Dr. Daisuke Watase, Mai Hazekawa, Ayano Yamada, Mitsuhisa Koga

PS06.17 Using single molecule microscopy to measure EV uptake, sub-cellular localization and dynamics

Dr James Rhodes, Dr Stefan Balint, Mr Andras Miklosi, Dr Nina Jajcanin-Jozic, Mr Andrei Traista, Dr Pradeep Kumar, Dr Grace DeSantis

PS06.18 Cancerous Extracellular Vesicles Induced Platelet mRNA Degradation by RNaseL Activation

Mr. Gaoge Sun¹, Zihan Liu¹, Ying Zhang¹, Hang Yin^1,2

¹School of Pharmaceutical Sciences, Tsinghua University, Beijing, China, ²Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China

PS06.19 Forward-Thinking Insights: Exploration of future therapeutics through HNF4-AS1 regulation of HNF4a promoter selection

Dr Pevindu Abeysinghe¹, Ms Breanna Humber², Mr Riccardo Cecchin², Prof. Kevin Morris¹

¹Centre for Genomics and Personalised Health, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia, ²Menzies Health Institute Queensland, School of Pharmacy and Medical Science, Griffith University, Gold Coast, Australia

PT01.01 A High-efficiency isolation system combined with proteomics in studying urinary small extracellular vesicles proteins for improving prostate cancer diagnosis

Dr. Cheng Zhou, Ms. Jie Gong, Mr. Baokun Fan, Ms. Xuan Ding, Dr. Bairen Pang, Prof. Yong Li, Dr. Junhui Jiang, Dr. Zejun Yan, Dr. Yue Cheng, Mr. Yingzhi Chen, Dr. Zhaohui Jiang, Mr. Tiannan Guo

PT01.02 A novel bladder cancer liquid biopsy using mutated proteins in urinary extracellular vesicles

M.D., Ph.D. Yuji Hakozaki, M.D., Ph.D. Yuta Yamada, M.D., Ph.D. Haruki Kume, Ph.D. Koji Ueda

PT01.03 A validated workflow and bioinformatic analysis pipeline for extracellular vesicle-based RNA biomarker signature discovery in molecular diagnostics

Christian Grätz, Dr. Benedikt Kirchner, PD Dr. Marlene Reithmair, Dr. Florian Brandes, Dr. Agnes S. Meidert, Prof. Dr. Gustav Schelling, Prof. Dr. Michael W. Pfaffl

PT01.04 Advancements in biomarker development for toxicology and safety assessment studies

Tasvilla Sonallya, Annamária Minus, Ferenc Fekete, Dr. Anikó Gaál, Kinga Ilyés, Dr. Tamás Beke-Somfai, Dr. Zoltán Varga, Dr. Katalin Monostory

PT01.05 Advancing precision: Development of extracellular vesicles protein-based panel for validation of endometrial cancer biomarkers

Dr Anastasiia Artuyants, Martin Middleditch, Deanna Shea, Bianca Nijmeijer, Sophia Bebelman, Dr Cherie Blenkiron

PT01.06 Analysis of secreted small extracellular vesicles from activated human microglial cell line reveals distinct pro- and anti-inflammatory proteomic profiles

Miss Xueming Niu, Dr Zhen Zhang, Mr Quan Zhou, Dr Alain Wuethrich, Dr Richard Lobb, Professor Matt Trau

PT01.07 Aquaporin 3 detection in placental extracellular vesicles in normal human pregnancy and preeclampsia

PhD Natalia Szpilbarg, Matías Nicolás Sierra, MD Juan Sebastián Sar, PhD Alicia Ermelinda Damiano

PT01.08 Automated high-throughput isolation of extracellular vesicles (EVs) and small RNA sequencing profile in serum of breast cancer patients

Dr Ramin Khanabdali, Dr Scott Zhu, Dr Mathew Moore, Dr Gregory Rice

PT01.09 Cargo content in extracellular vesicles from a murine cell model of organotropic metastatic breast cancer

Graduate Student Amélie Nadeau, Graduate Student Thupten Tsering, PhD Kyle Dickinson, PhD Daniela Quail, PhD Peter Siegel, PhD Julia V Burnier

PT01.10 Changes to small and large urinary extracellular vesicles in glioblastoma

Dr Susannah Hallal, Mr Liam Sida, Dr Agota Tűzesi, Dr Elissa Xian, Dr Daniel Madani, Dr Krishna Muralidharan, Dr Brindha Shivalingam, Associate Professor Michael Buckland, Dr Laveniya Satgunaseelan, Dr Kimberley Alexander

PT01.11 Circulating EVs as diagnostic biomarkers of indeterminate thyroid nodules

Dr Nada Ahmed, Dr Kevin Beatson, Dr Jigisha Patel, Dr Mohammad Eddama, Dr Tarek Abdel-Aziz, Professor Lucie Clapp

PT01.13 Detection of MTA1 in plasma sEVs derived from cancer patients

Graduate Research Assistant Kritisha Bhandari, Laboratory Technician Jeng Shi Kong, Physician Scientist Haoyao Sun, Professor Jinchang Wu, Assistant Professor Bethany Hannafon, Professor William Dooley, Professor Wei-Qun Ding

PT01.14 Developing metabolomic approach in profiling extracellular vesicle biomarkers for prostate cancer diagnosis and progression risk stratification

Mr Mahmoud Hamed, Dr Valerie Wasinger, Mr Qi Wang, Associate Professor Peter Graham, Dr David Malouf, Dr Joseph Bucci, Professor Yong Li

PT01.15 Early cancer detection made easy: liquid biopsy analysis of low-concentration EGFR mutations in NSCLC using large-volume plasma and urine

Young-Hye Seo, Sung-Kyung Bong, Beomhee Ahn, Hanna Kim, Hwanghee Ryu, Myunghee Jang, Ph.D Seung-Hak Choi, Ph.D Vijaya Sunkara, Juhee Park, Ph.D Yoon-Kyoung Cho, Ph.D Kyusang Lee, Ph.D Beomseok Lee

PT01.16 Effect of X-ray irradiation on quantity and tetraspanin markers expression of extracellular vesicles (EVs) derived from peripheral blood mononuclear cells (PBMCs) and plasma from patients undergoing total-body irradiation (TBI)

Zi Huai Chew, Senior Research Scientist Christelle Chua

PT01.17 Establishing the capacity of liver derived extracellular vesicle cargo to reflect variability in drug exposure and response

Ms Lauren Newman, Dr Zivile Useckaite, Associate Professor Andrew Rowland

PT01.21 Exploring plasma-derived small extracellular vesicles as novel biomarkers for early-stage detection of pancreatic neuroendocrine tumors

Ms Priya Kumari Gorai, Ms Simran Rastogi, Dr Surabhi AS, Dr Seema Singh, Dr Shipra Agarwal, Dr Sujoy Pal, Dr Tapas Chandra Nag, Prof Renu Dhingra, Prof Mehar Chand Sharma, Prof Rakesh Kumar, Dr Saroj Kumar, Dr Neerja Rani

PT01.22 Extracellular vesicle-derived RNA profiling predicts melanoma and non-small cell lung cancer (NSCLC) response to immune checkpoint inhibitors

Ms Lidia Medhin, Doctor Lydia Warburton, Professor Benhur Amanuel, Doctor Leslie Beasley, Professor Elin Gray

PT01.23 Extracellular vesicles are diagnostic and predictive of blood pressure before and during exercise in people with hypertension

Samantha Upson, Dr. Sabrina LaSalvia, Eric Trillaud, Dr. Emily Heiston, Nathan Stewart, Dr. Steven Malin, Dr. Uta Erdbrügger

PT01.24 Extracellular vesicles as potential biomarkers for non-alcoholic fatty liver disease (NAFLD)

Malene Joergensen, Anders Askeland, Rikke Bæk, Charlotte Sten, Rikke Wehner Rasmussen, Morten Hjuler Nielsen, Nahuel Garcia, Maiken Mellergaard, Aase Handberg

PT01.25 Forecasting post-COVID syndrome: leveraging molecular signatures of extracellular vesicles for pedictive analysis

Dr Edina Gyukity-Sebestyen, Gabriella Dobra, Matyas Bukva, Dr Maria Harmati, Timea Boroczky, Dr Szabolcs Nyiraty, Dr Barbara Bordács, Dr Margareta Korsos, Dr Zoltan Szabo, Dr Gabor Kecskemeti, Prof. Dr Tamas Varkonyi, Prof. Dr Zoltan Konya, Prof. Dr Marta Szell, Dr Peter Horvath, Dr Krisztina Buzás

PT01.26 Glioblastoma biomarkers in urinary extracellular vesicles reveal the potential for a ‘liquid gold’ biopsy

Dr Susannah Hallal, Dr Agota Tuzesi, Mr Liam Sida, Dr Elissa Xian, Dr Daniel Madani, Dr Krishna Muralidharan, Associate Professor Brindha Shivalingam, Associate Professor Michael Buckland, Dr Laveniya Satgunaseelan, Dr Kimberley Alexander

PT01.27 Glycosignatures of small extracellular vesicles secreted by breast cancer cells

Lifang Yang, Benjamin Johnson, Caleb Smack, Professor Eric Feliberti

PT01.28 High-throughput and automated isolation of plasma derived extracellular vesicles to identify microRNAs with diagnostic potential for ovarian cancer

Dr Ramin Khanabdali, Dr Carlos Palma, Miss Siena Barton, Professor Greg Rice

PT01.30 Identification of extracellular vesicles and particles derived proteins as novel biomarkers for prostate cancer diagnosis, risk stratification and monitoring metastasis

Mr Qi Wang, Dr Bairen Pang, Dr Cheng Zhou, Dr Meng Han, Jie Ni, David Malouf, Joseph Bucci, Peter Graham, Tiannan Guo, Junhui Jiang, Yong Li

PT01.32 Investigation of the immunopeptidome carried by MHC class I molecules on extracellular vesicles (EV) released from lung cancer cells

Miss Debra Lennox, Dr Caitlin Boyne, Dr Sally Shirran, Dr Simon Powis

PT01.33 Isocitrate dehydrogenase 1 is increased in urinary extracellular vesicles from type 2 diabetic model rats

Ph.D. Student Haruka Sei, M.S. Naoya Hirade, Ph.D. Fumie Nakashima, Ph.D. Takahiro Shibata

PT01.34 Isolation and characterisation of extracellular vesicles from tumour and non-tumour lung tissues for next generation sequencing

Edward Stephens, Dr Tian Mun Chee, Mr Vihanga Dharmasena, Professor Kwun Fong, Professor Ian Yang

PT01.36 Leveraging extracellular vesicle glycan signatures for prostate cancer detection

MS Trevor Enright, PhD Kai Tao, PhD Sinan Sabuncu, PhD Emek Demir, MD Mark Garzotto, BS Randall Armstrong, PhD Michelle Gomes

PT01.37 Lipidomic and proteomic approaches revealed glycerophospholipids as a signatures of hypoxic small extracellular vesicles from head and neck squamous cell carcinoma

Dr Alicja Głuszko, dr. hab. Mirosław Szczepański, dr. Andrzej Ciechanowicz, Prof. Theresa Whiteside, dr. Nils Ludwig

PT01.38 Lipidomic identification of novel small extracellular vesicle biomarkers for prostate cancer early diagnosis and risk progression stratification

PhD Meng Han, PhD Jie Gong, Professor Qi Wang, PhD Bairen Pang, PhD Cheng Zhou, PhD Zhihan Liu, Professor Junhui Jiang, Professor Yong Li

PT01.39 Multiplex profiling of endometriosis-derived extracellular vesicles reveals novel potential biomarkers for endometriosis

MSc Karolina Soroczyńska, Tobias Tertel, Bernd Giebel, Małgorzata Czystowska-Kuźmicz

PT01.40 Novel set of extracellular vesicle proteins as biomarkers for early detection of high grade serous ovarian cancer

Kalpana Deepa Priya Dorayappan, Dr. Michelle Lightfoot, Dr. Lianbo Yu, Dr. Colin Hisey, Dr. Takahiko Sakaue, Dr Muralidharan Anbalagan, Dr Casey Cosgrove, Dr Larry Maxwell, Dr Premal Thaker, Dr Beth Y. Karlan, Dr David O'Malley, Dr Raphael E. Pollock, Dr David E. Cohn, Dr Rajan Gogna, Dr Selvendiran Karuppaiyah

PT01.41 Proteomic analysis of cerebrospinal fluid in medulloblastoma and associated extracellular vesicle protein - TKT as a potential biomarker

Research Professor Seung Ah Choi, Professor Seung-Ki Kim, Professor Ji Hoon Phi

PT01.41 Plasma extracellular vesicle miR-512-3p modulates the GTPase activity and the angiogenic function of endothelial colony-forming cells by targeting ARHGEF3 in pediatric Moyamoya disease

Research Professor Seung Ah Choi, Professor Eun Jung Koh, Professor Seung-Ki Kim

PT01.42 Proteomic profiling of extracellular vesicles from lymphatic drainage fluid after optimized isolation reveals enriched tumor-associated markers compared to plasma

Dr XINYU QU, Dr Leanne Leung, Dr Bojie Chen, Professor Zigui Chen, Professor Katie Meehan, Professor Jason Chan

PT01.45 Raman spectroscopy-based profiling of plasma-derived extracellular vesicles: a novel approach for differentiating cancerous diseases

Timea Boroczky, Matyas Bukva, Gabriella Dobra, Maria Harmati, Edina Sebestyen-Gyukity, Yasmin Ranjous, Laszlo Szivos, Katalin Hideghety, Krisztina Budai, Judit Olah, Peter Horvath, Gyorgy Lazar, Zoltan Konya, Pal Barzo, Almos Klekner, Krisztina Buzas

PT01.46 Revealing urinary exosomal eiomarkers in progressive NAFLD: proteomic analysis in a rat model

Chao-Yuan Chang, Visiting Staff Chun-Jen Huang, Visiting Staff Syuan-Hao Syu, Visiting Staff Tze-Sian Chan

PT01.48 Small extracellular vesicle (sEV) proteins as a potential biomarker for endometriosis

Dr Hannah Nazri, Dr Raphael Heilig, Associate Professor Roman Fischer, Professor Benedikt Kessler, Dr Kavita S Subramaniam, Professor Christian Becker, Dr Thomas Tapmeier

PT01.49 Storage stability study of human urinary extracellular vesicles

MD Cahyani Gita Ambarsari, Professor MW Taal, MRCPCH MD(res) JJ Kim, Assistant Professor Dong-Hyun Kim, Assistant Professor AM Piccinini

PT01.51 SWATH-MS identified differentially expressed proteins in extracellular vesicles isolated from pleural effusions of Malignant Pleural Mesothelioma

Dr. Kelly Tian Mun Chee, Prof. Kwun M Fong, Prof. Ian A Yang, Assoc. Prof. Rayleen V Bowman

PT01.52 Systemic changes in Immune System-Related Plasma Extracellular Vesicles During Healthy Aging

Dr. Xin Zhang, Dr. Sisi Ma, Syeda Iffat Naz, Janet Huebner, Dr. Erik Soderblom, Noor Alnemer, Dr. Constantin Aliferis, Dr. Virginia Kraus

PT01.53 The altered levels of urinary extracellular vesicles pre- and post-surgery relative to proteomics change in breast cancer patients

Miss Nilobon Jeanmard, Dr. Rassanee Bissanum, Mr. Kittinun Leetanaporn, Mr. Pongsakorn Choochuen, Assoc.Prof. Hutcha Sriplun, Miss Sawanya Charoenlappanit, Dr. Sittiruk Roytrakul, Assoc.Prof. Raphatphorn Navakanitworakul

PT01.54 The circulating extracellular vesicles in ovarian cancer study

Dr Andrew Lai, Dr Dominic Guanzon, Dr Shayna Sharma, Mrs Katherin Scholz-Romero, Dr Yaowu He, Mr Weitong Huang, Dr Tanja Pejovic, Dr Carmen Winters, Professor Terry Morgan, Professor Jermaine Coward, Associate Professor Amy McCart Reed, Professor Sunil Lakhani, Professor Andreas Obermair, Professor Amanda Barnard, Professor Anna deFazio, Professor Lewis Perrin, Professor John Hooper, Professor Gregory Rice, Professor Carlos Salomon

PT01.58 Unique lipidomic profile sets Extracellular vesicles apart from other cellular fractions in ovarian cancer

Ms Shikha Rani, Dr Andrew Lai, Dr Dominic Guanzon, Mr Kaltin Ferguson, A/Prof Lewis C. Perrin, Prof John D. Hooper, Prof Carlos Salomon

PT01.59 Unlocking ovarian cancer detection: Long-read sequencing reveals promising biomarkers using extracellular vesicle DNA methylation and mutation patterns

Dr Dominic Guanzon, Dr Subash Rai, Mr Rakesh Sankar, Ms Pragati Lodha, Ms Vidya Gummagatta, Dr Andrew Lai, Professor Lewis Perrin, Professor John Hooper, Professor Carlos Salomon

PT02.01 Altered protein nitrosylation patterns in extracellular vesicles isolated from activated microglia

Dr Natasha Vassileff, Dr Jereme Spiers, Miss Sarah Bamford, Dr Rohan Lowe, Dr Keshava Data, Professor Paul Pigram, Professor Andrew Hill

PT02.02 Antidepressant effects of aerobic exercise: are circulating EVs responsible?

Reine Khoury, Dr. Dariusz Zurawek, Gabriella Frosi, Assistant Professor Corina Nagy

PT02.03 BDNF/TrkB system dysregulation at the cell environment: extracellular vesicles as carriers of TrkB-ICD in Alzheimer's disease

Mr. Tiago Costa-Coelho, João Fonseca-Gomes, Gonçalo Garcia, Mafalda Ferreira-Manso, Catarina B. Ferreira, Carolina de Almeida-Borlido, Juzoh Umemori, Mikko Hiltunen, Eero Castrén, Ana M. Sebastião, Alexandre de Mendonça, Dora Brites, Maria José Diógenes

PT02.04 Brain region-specific changes in extracellular vesicles release and composition in tau R406W human organoid tauopathy model

Dr. Tina Bilousova, Nina Knitowski, Dr. Qing Cao, Shengkai Zhao, Swetha Atluri, Mikhail Melnik, Achyutha Kodavatikanti, Dr. Ranmal Samarasinghe, Dr. Jessica Rexach, Dr. Karen Gylys

PT02.05 Cerebrospinal fluid extracellular vesicle miRNAs identify synaptic transmission alterations in Alzheimer's disease

PhD Ursula Sandau, Trevor McFarland, PhD Sierra Smith, MD Douglas Galasko, MD Joseph Quinn, MD, PhD Randy Woltjer, PhD Julie Saugstad

PT02.06 Eukaryotic and microbiota-derived extracellular vesicles in Parkinson's disease.

Tiana Koukoulis, Purnianto Adityas, David Finkelstein, Leah Beauchamp, Kevin Barnham, Dr Laura Vella

PT02.07 Exploitation of vitreous-derived extracellular vesicles to study the central nervous system dynamics

Lien Cools, Dr. Cristiano Lucci, Sam Noppen, Dr. Charysse Vandendriessche, Drs. Kaat Verleye, Drs. Laura Raes, Elien Van Wonterghem, Prof. Inge Mertens, Prof. Dominique Schols, Prof. Roosmarijn E Vandenbroucke, Prof. Lies De Groef

PT02.09 How do tumour derived EVs interact with the maturing nervous system and lead to altered pain processing in cancer survivors?

Dr Hannah Jackson, Dr Anna Grabowska, Dr Victoria James, Dr Federico Dajas-Bailador, Dr Beth Coyle, Dr Gareth Hathway

PT02.10 Inflammation-associated microglial EVs exhibit morphological differences and enrichment for ribosomes

Mr William Phillips, Ms Irumi Amarasinghe, Dr Ebony Monson, Dr Nicholas Reynolds, Prof Karla Helbig, Dr Lesley Cheng, Prof Andrew F Hill

PT02.11 Isolation of spontaneously-released brain extracellular vesicles: implications for stress-driven brain pathologies

Dr Ioannis Sotiropoulos, Dr Patricia Gomes, Dr Cristian Bodo, Dr Carlos Noguera-Ortiz, Dr Martina Samiotaki, Dr Minghao Chen, Dr Carina Soares-Cunha, Dr Joana M. Silva, Dr Bárbara Coimbra, Dr George Stamatakis, Dr Liliana Santos, Dr George Panayotou, Professor Clarissa L. Waites, Proffessor Christos Gatsogiannis, Professor Nuno Sousa, Professor Dimitrios Kapogiannis, Dr Bruno Costa-Silva

PT02.12 Metabolic analysis of extracellular vesicles isolated from human brain tissue in Alzheimer's disease context

PhD student Patricia Hernández-López, Dr. Elisabeth Rackles, Dr. Oihane E. Albóniga, Dr. Juan Manuel Falcon-Pérez

PT02.13 Mitochondrial proteins are exported from cells via sEVs in Parkinson's disease

Mr Adityas Purnianto, Ms Mitali Kulkarni, Professor Scott Ayton, Professor Catriona McLean, Professor Ashley Bush, Professor David Finkelstein, Professor Kevin Barnham, Dr Laura Vella

PT02.14 Plasma-derived small extracellular vesicles in alzheimer's disease progression: insights into synaptic dysfunction and neuroinflammation

Mr Rishabh Singh, Ms Sanskriti Rai, Dr Prahalad Singh Bharti, Dr Prasun Chatterjee, Dr Saroj Kumar

PT02.15 Primary rat cortical tri-culture to study cellular response to cancer EVs

Mrs. Rachel Rachel, Dr. Randy Carney, Hyehyun Kim, Dr. Erkin Seker

PT02.16 Protein nitrosothiol patterns altered in extracellular vesicles from Alzheimer's disease brain cortex

Dr Natasha Vassileff, Dr Rohan Lowe, Dr Keshava Datta, Professor Catriona McLean, Professor Andrew Hill, Dr Jereme Spiers

PT02.17 Proteomic analysis of brain-dervied extracellular vesicles in Huntington's disease

Miss Mitali Manish Kulkarni, Mr. Adityas Purnianto, Miss Tiana Koukoulis, Miss Huaqi Su, Miss Geraldine Kong, Professor Anthony Hannan, Dr. Laura.J Vella

PT02.19 Uncovering the composition of extracellular vesicles (EVs) regulated by Translin-Associated Factor X (TRAX) that modulates microglial identity

Dr. Yu-Ting Weng, Ph.D. Yijuang Chern

PT02.22 Extracellular vesicles in mood disorders: a systematic review

Dr Cristian-Daniel Llach¹, Ms Gia Han Le¹, Dr Gerard Anmella², Dr Joshua Rosenblat¹, Dr Anna Gimenez-Palomo², Dr Isabella Pacchiarotti², Dr Eduard Vieta², Dr Roger McIntyre¹, Dr Rodrigo Mansur¹

¹University Of Toronto, Toronto, Canada, ²Bipolar and Depressive Disorders Unit, Hospital Clinic de Barcelona, Barcelona, Spain

PT03.01 Analysis of the phenotypical changes of plasma EVs over time in healthy donors

Rikke Bæk, Maiken Mellergaard, Rikke Wehner Rasmussen, Rikke Bülow Eschen, Evo Lindersson Søndergaard, Aase Handberg, Malene Møller Jørgensen

PT03.02 Comparison of primed mesenchymal stromal cells secretome following different methods of purification with a large panel of characterization tools

PHD Student Guillaume Valade, PHD Student Marine DE TADDEO, Mrs Muriel NIVET, Mrs Marion GROSBOT, Mrs Claire LANGLE, Mrs Sylvie GOULINET, PHD Philippe MAUDUIT, Mr Vincent JUNG JUNG, PHD Chiara GUERRERA, MD, PHD Sébastien BANZET, PHD Juliette PELTZER, PHD Marina TROUILLAS

PT03.03 Evaluation of the physical properties and pharmacokinetics of EVs purified by the microfiltration membrane with ion exchange function

Ms. Ayano Higaki, Mr. Keita Inoue, Ms. Mizuki Kobayashi, Ms. Makiko Hiraoka, Mr. Yoshitaka Kawakami, Ph.D. Naohiro Seo

PT03.04 Is it feasible to distinguish extracellular vesicles by their biophysical properties?

Mr Fredrik Stridfeldt, MSc Hanna Kylhammar, Dr Vipin Agrawal, MSc VIkash Pandey, Dr André Görgens, Professor Samir El Andaloussi, Professor Dhrubaditya Mitra, Professor Apurba Dev

PT03.06 Characterization of human MSC-derived extracellular vesicle preparations using size-exclusion HPLC and ion-exchange HPLC coupled with multi-angle light scattering detection

Dr. Hirotaka Nishimura, Dr. Tomofumi Yamamoto, Dr. Noritaka Hashii, Dr. Akiko Ishii-Watabe

PT03.07 EVs during zebrafish larvae development

Dr.med. Linda-marie Mulzer¹, Tim Felger¹, PD Dr. med. habil. Dr. rer. nat. Luis Muñoz², Gesa Engl¹, Prof. Dr. med. Heiko Reutter¹, Leila Pourtalebijahromi³, Prof. Dr. Gregor Fuhrmann³, Philipp Arnold⁴, Dr. med. Alina Hilger¹

¹Department of Pediatrics and Adolescent Medicine, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany, ²Department of Rheumatology and Immunology, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany, ³Friedrich-Alexander University Erlangen-Nürnberg, Department of Biology, Chair of Pharmaceutical Biology, Erlangen, Germany, ⁴Friedrich-Alexander University Erlangen-Nürnberg, Institute for Functional and Clinical Anatomy, Erlangen, Germany

PT03.08 Activated human mast cells produce extracellular vesicles that change the metabolic function of target cells

Senior Investigator Marianna Kulka, Dr. Marcelo Marcet-Palacios, Sabrina Rodrigues Meira

PT03.09 Advancing ultra-low, ultra-deep extracellular vesicle proteomics

Prof David Greening, Mr Alin Rai, Ms Haoyun Fang, Ms Bethany Claridge, Mr David Greening

PT03.10 Amniotic fluid EV proteome is a clear representation of gestational age-dependent fetal development

Dr Ishara Atukorala, Dr Ching-Seng Ang, Ms Sally Beard, Ms Bianca Fato, Dr Natasha de Alwis, Dr Hamish Brown, Professor Natalie Hannan, Professor Lisa Hui

PT03.11 Assessing the compartmentalisation of small non-coding RNAs in the circulation

Dr I-Jou Teng, Dr Kaloyan Takov, Dr Clemens Gutmann, Prof. Manuel Mayr

PT03.12 Astrocyte-enriched extracellular vesicle protein concentrations after proteinase K treatment

Dr. Leandra Figueroa-Hall, Dr. Kaiping Burrows, Dr. Ahlam Alarbi, Dr. Chibing Tan, Dr. Bethany Hannafon, Dr. Rajagopal Ramesh, Dr. Victoria Risbrougn, Dr. T. Kent Teague, Dr. Martin Paulus

PT03.13 Characterization of extracellular vesicles with capillary electrophoresis

Aleksandra Steć, Ph.D. Joanna Jońca, Ph.D. Agata Płoska, Prof. Leszek Kalinowski, Assoc. Prof. Bartosz Wielgomas, Ph.D. Krzysztof Waleron, Prof. Bogdan Lewczuk, Mr. Grzegorz Czyrski, Ph.D. Andrea Heinz, Ph.D. Szymon Dziomba

PT03.14 Circadian mass spectrometry-based proteome profiling of salivary extracellular vesicles

Dr Carlos Andres Palma Henriquez, Ms Siena Barton, Dr Sara Nikseresht, Mr Sadman Bhuiyan, Dr Mozhgan Shojaee, Dr Kartini Asari, Dr Pingping Han, Dr Ramin Khanabdali, Dr Gregory Rice

PT03.15 FunRich enables enrichment analysis of extracellular vesicles OMICs datasets

Mr Sriram Gummadi

PT03.16 Modulating nonspecific uptake of engineered extracellular vesicles

Beth DiBiase^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Roxana Mitrut^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Taylor Gunnels^{Biomedical Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Dr. Neha Kamat^{Biomedical Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Dr. Joshua Leonard^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}

PT03.17 Multi-omics characterization of highly enriched human plasma small extracellular vesicles

Ms Huaqi Su, Assoc. Prof. Kevin Barnham, Prof. Gavin Reid, Dr. Laura Vella

PT03.20 Proteomic analysis of extracellular vesicles secreted by human umbilical cord mesenchymal stem/stromal cells under stimulated conditions

Dr. Chaiyong Koaykul, Dr. Kunthika Mokdarta, Dr. Poorichaya Somparn, Dr. Jiradej Makjaroen, Dr. Chatikorn Boonkrai, Dr. Trairak Pisitkun

PT03.22 Sizing and visualization of single EVs using a super-resolution based workflow to characterize EV populations

Dr James Rhodes, Dr Kathleen M Lennon, Dr Colbie Chinowsky, Dr Abigail Neininger-Castro, Ms Chloe Celingant-Copie, Dr Daniel Zollinger, Dr Grace DeSantis

PT03.23 The modulating effect of short, cationic peptides on EV's protein corona

PhD Imola Cs. Szigyarto, Priyanka Singh, Tasvilla Sonallya, PhD Aniko Gaal, PhD Lilla Turiak, PhD Laszlo Drahos, PhD Zoltan Varga, PhD Tamas Beke-Somfai

PT03.24 Vesiclepedia and ExoCarta: A web-based compendiums of extracellular vesicles cargo and extracellular particles

Mr Sriram Gummadi

PT03.26 Establishment of an immunocapture method for the separation of a rheumatoid arthritis-related CD90+ subpopulation of extracellular vesicles

M.Sc. Stefanie Kurth, PhD André Tiaden, M.Sc. Edveena Hanser, Ute Heider, PhD Stefan Wild, Professor Diego Kyburz

PT03.27 ExoPAS: numerous and pure isolation of exosomes using cationic material and PEG

Wonjae Kim, Student Kangmin Lee

PT03.28 A biomimetic vortex tangential flow filtration (VTFF) system for efficient isolation and purification of extracellular vesicles

Ph.D. Candidate Yuxin Qu^{School of Biomedical Engineering, Tsinghua University, Beijing, China}, Assistant professor Han Wang^{School of Biomedical Engineering, Tsinghua University, Beijing, China}, Lan Xie^{School of Basic Medical Sciences, Tsinghua University, Beijing, China}

PT03.29 A protocol to differentiate the chondrogenic ATDC5 cell-line for the collection of chondrocyte-derived extracellular vesicles

Mr Jose Marchan-Alvarez, Miss Loes Teeuwen, Mr Doste Mamand, Prof Susanne Gabrielsson, Prof Klas Blomgren, Dr Oscar Wiklander, Dr Phillip Newton

PT03.30 A quick, cost-free, and user-friendly cleanup protocol for dye and drug removal from small extracellular vesicle solution

Ioannis Isaioglou, Gloria Lopez-Madrigal, Jasmeen Merzaban

PT03.31 A standardized multi-stage purification process and comprehensive characterization of extracellular vesicles derived from HEK293F cells

Research associate Nhan Vo, Research associate Chau Tran, Research associate HB Nam Tran, Scientist T Nhat Nguyen, Research associate Thieu Nguyen, Scientist DN Diem Nguyen, Research associate Tran Pham, R&D lead Hoai-Nghia Nguyen, R&D specialist Lan-N Tu

PT03.32 A survey study on the status of extracellular vesicle (EV) research in malaysia: current updates

Ts. Dr. Norhayati Liaqat Ali Khan, Dr. Nadiah Abu, Dr. Wai Leng Lee, Dr. Muhammad Farid Nazer Muhammad Faruqu, Dr. Jia Xian Law, Associate Professor Dr. Norshariza Nordin, Dr. Maryam Azlan, Associate Professor Dr. Rajesh Ramasamy, Dr. Sik Loo Tan, Associate Professor Dr. Wan Nazatul Shima Shahidan, Mr. See Nguan Ng, Dr. Kok Lun Pang, Dr. Vijayendran Govindasamy, Mr. Benson Koh, Dr. Pan Pan Chong, Miss Yoong Yi Chong, Mrs. Nur Hidayah Hassan, Mr. Nazmul Huda Syed, Mrs. Maimonah Eissa Sheikh Al-Masawa

PT03.33 Advancing scalable production of purified adipose-derived stem cell extracellular vesicles

Jing Zhou, Ph.D candidate Jiajia Dai, Ph.D candidate Haonan Di, Ph.D candidate Yunyun Hu, Ph.D candidate Niangui Cai, professor Xiaomei Yan

PT03.35 Benchmarking surface functionalization strategies for marker independent EV capture and profiling

Mr. Hugues Martin, Dr. Andreas Wallucks, Dr. Andy Ng, Ms. Molly Shen, Dr. David Juncker

PT03.36 Beyond the boundaries of conventional isolation techniques: Functional self-assembled coordination polymer nanoparticles for instant one-step selective and efficient enrichment of exosomes – ExoFlocs™

Mr Mohamed Sallam, Mr Cong-Minh Nguyen, Dr Amandeep Singh Pannu, Dr Indira Prasadam, Mr Yezhou Yu, Professor Serge Muyldermans, Dr Frank Sainsbury, Professor Nam-Trung Nguyen, Professor Nobuo Kimizuka

PT03.38 Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy

Jie Gong, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Meng Han, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Bairen Pang, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Qi Wang, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Haotian Chen, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Zhihan Liu, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Cheng Zhou, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Yong Li, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Junhui Jiang

PT03.39 Comparison of asymmetric depth filtration and ultrafiltration combined with size-exclusion chromatography for EV isolation from cell culture media

Dr. Vasiliy Chernyshev, Dr. Elena Svirshchevskaya, Mr. Mikhail Ivanov, Dr. Denis Silachev

PT03.40 Comprehensive evaluation of extracellular vesicle markers through diverse isolation strategies

Dr. Kaiping Burrows, Dr. Leandra Figueroa-Hall, Dr. Ahlam Alarbi, Dr. Bethany Hannafon, Cole Hladik, Dr. Rajagopal Ramesh, Dr. Victoria Risbrough, Dr. T. Kent Teague, Dr. Martin Paulus

PT03.41 Confident isolation and proteomics of bacterial extracellular vesicles by size exclusion chromatography

Ms Haekang Yang, Ms Shinwon Chae, Mr Chul Won Seo, Ms Seoyeon Kim, Professor Yoon-Jin Lee, Professor Dongsic Choi

PT03.42 Development of a method for large-scale purification of extracellular vesicles using the PS affinity method

Dr. Afshin Iram, Shotaro Masuda, Hana Onizuka, PhD. Ryo Ukekawa, PhD. Takahiro Nishibu

PT03.43 Development of an applicable method for bacterial extracellular vesicle isolation from mouse stool supernatant

Shujin Wei, Professor Wanli Xing

PT03.44 Does EV purity affect downstream functionality?

Research Officer Janice Tan, Principal Investigator Ivy Ho

PT03.46 Evolution of an EV enrichment protocol: from minimal information to proteomics

Dr Felicity Dunlop, Dr Shaun Mason, Dr Taeyoung Kang, Professor Suresh Mathivanan, Professor Aaron Russell

PT03.47 ExoCAS-2: rapid and pure isolation of exosomes by anionic exchange using magnetic beads

Student Jaeeun Lee

PT03.48 ExoFilter: large capacity extraction of EVs using a positive charge mesh filter in continuous flow

Student Yongwoo Kim

PT03.49 miRQuick: An innovative charge-based EV isolation method for highly efficient extraction of EV-miRNAs from liquid samples

Student Lee Kangmin

PT03.50 High-throughput isolation and sorting of nanoparticle loaded exosomes

Dr. Hye Sun Park, Taewoong Son, Mi Young Cho, Hyunseung Lee, Eun Hee Han, Dr. Kwan Soo Hong

PT03.52 Impact of hyaluronidase on tetraspanin expression of extracellular vesicles (EVs) in synovial fluid from patients with rheumatoid arthritis and osteoarthritis using the Exoview platform.

Mrs. Edveena Hanser, Prof. Dr. Diego Kyburz

PT03.54 Isolation and enrichment of extracellular vesicles with double-positive membrane protein for subsequent biological studies

Dr. Huixian Lin, Dr. Chunchen Liu, Prof. Bo Li, Prof. Lei Zheng

PT03.55 Molecular imprinted polymer-based artificial peptide (MIPap) enables isolation of astrocyte-specific extracellular vesicles (asEV) in serum

Yong Shin, Professor Eun Jae Lee

PT03.56 Novel strategy for affinity capture and release sEV

Professor Wei Duan, Mr Rajindra Napit, Dr Rocky Chowdhury, Mr Satendra Jaysawal

PT03.57 Optimization of separation methodologies for obtaining high yield-high purity urinary extracellular vesicles

Beatriz Martín-Gracia, Optimization of separation methodologies for obtaining high yield-high purity urinary extracellular vesicles Håkon Flaten, Optimization of separation methodologies for obtaining high yield-high purity urinary extracellular vesicles Krizia Sagini, Optimization of separation methodologies for obtaining high yield-high purity urinary extracellular vesicles Alicia Llorente

PT03.58 Optimizing high-throughput isolation of extracellular vesicles from primary cells in small to medium-scale 3-dementional bioreactors with serial purification methods

Dr. Zheng Zhao

PT03.59 Pillared interdigitated electrodes for small extracellular vesicle capture

Miss Emma Morris, Associate Professor Karl Hassan, Professor Craig Priest, Dr Bin Guan, Dr Renee Goreham

PT03.60 Protocol optimisation for extracellular vesicle isolation and characterisation: evaluation of ultracentrifugation, size exclusion chromatography and charged core bead chromatography methods

Dr Farha Ramzan, Hui Hui Phua, Dr Vidit Satokar, Dr Shikha Pundir, Dr Anastasia Artuyants, Dr Cherie Blenkiron, Dr Chris Pook, Prof Mark Vickers, Dr Ben Albert

PT03.62 Rapid and efficient isolation platform for plasma extracellular

vesicles: EV-FISHER

Dr Weilun Pan, Prof Lei Zheng, Prof Jinxiang Chen, Prof Bo Li

PT03.63 Salivary extracellular vesicles isolation methods impact the robustness of biomarkers detection

Dr Jérémy Boulestreau^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Dr Laurence Molina^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Alimata Ouedraogo^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Louen Laramy^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Ines Grich^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Dr Thi Nhu Ngoc Van^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France; SkillCell, Montpellier, France}, Dr Franck Molina^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Dr Malik Kahli^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}

PT03.64 Single-particle multiplex analysis of EV-biophysical properties of fractionated particle populations by ion exchange chromatography

Professsor Takanori Ichiki, Chiharu Mizoi, Kento Toyoda, Professor Naohiro Seo

PT03.65 Tailored cellulose nanofiber sheets capture and preserve small extracellular vesicles from micro-volume body fluids and reveal the unknown profiles of extracellular vesicles

M.D., Ph.D. Akira Yokoi, M.D., Ph.D. Kosuke Yoshida, B.Sc. Masami Kitagawa, Ph.D. Takao Yasui, M.D., Ph.D. Hiroaki Kajiyama

PT03.66 Targeting EV enriched lipids for non-biased capture and analysis

Dr Bradley Whitehead, PhD Litten S Sørensen, Anders T Boysen, Prof Peter Nejsum

PT03.68 Xeno-free human platelet lysate depleted of exosomes for enhanced extracellular vesicle yield from stem cells, immune cells, and cancer cells

Mr. Yee-Hsien Lin, Mr. Han-Tse Lin, Mr. William Milligan, Dr. Min-Chang Huang

PT03.70 Real-time Label-free platforms for size determination and cell interaction studies of extracellular vesicles

Msc In Medicinal Chemistry, doctoral researcher in Pharmacy Elena Scurti¹, PhD Martina Hànzlikova¹, MSc Johanna Puutio², PhD Fadak Howaili³, PhD Kai Härkönen⁴, Professor Pia Siljander², PhD Saara Laitinen⁴, Professor Tapani Viitala^1,3

¹Division of Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland, ²EVcore facility, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland, ³Åbo Akademy University, Turku, Finland, ⁴Finnish Red Cross Blood Service, Helsinki, Finland

PT03.71 How Centrifugation Can Improve Your EV Workflow

Ms. Amy Henrickson¹, Dr. Lutz Ehrhardt, Dr. Shawn sternisha

¹Beckman Coulter, Indianapolis, United States

PT03.72 Optimizing a workflow for the analysis of extracellular vesicles

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, France

PT03.73 MISEV 2023: the Beckman Coulter Life Sciences approach for Extracellular Particles

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, France

PT03.75 EV Quant: A quantitative web-based compendium of extracellular vesicles cargo for studies in vesiclepedia

Mr SRIRAM GUMMADI¹

¹Latrobe University, Australia

PT03.76 Optimized protocol for isolation of extracellular vesicles (EV) - carried microRNAs from platelet-free plasma using size-exclusion chromatography (SEC) and phenol – guanidine extraction.

MD Miłosz Majka¹, PhD Katarzyna Czarzasta², MD, PhD Małgorzata Wojciechowska², PhD Małgorzata Czystowska-Kuźmicz¹

¹Medical University of Warsaw, Chair and Department of Biochemisrty, Warsaw, Poland, ²Medical University of Warsaw, Laboratory of Centre for Preclinical Research, Chair and Department of Experimental and Clinical Physiology, Warsaw, Poland

PT03.77 Comparative Analysis of Plasma and Serum Exosomal Small RNA Sequencing Profiles

Dr. Alex Chauhan¹, Hinal Zala¹, Simone Yamasaki¹, Enaam Merchant¹, Dr. Mohamed El-Mogy¹, Dr. Songsong Geng¹, Dr. Taha Haj-Ahmad¹, Dr. Yousef Haj-Ahmad¹

¹Norgen Biotek Corp., Thorold, Canada

PT03.80 Innovative Ultrapure Exosome Extraction Using Hybrid Charge-Based Filtration and Tangential Flow Filtration

Mr. Yoing-woo Kim¹, Mr. Kang-Min Lee¹, Professor Sehyun Shin¹

¹Korea University, Seoul, South Korea

PT03.81 Use of advanced aptamer technology in EV research

Mr. Rajindra Napit¹, Mr. Satendra Jyasawal¹, Ms. Jasmine Catague¹, Mr. Haben Melke¹, Dr. Rocky Chowdhury¹, Dr. Lingxue Kong¹, Dr. Wei Duan¹

¹Deakin University, Warun Ponds, Geelong, Australia

PT03.82 Purifying exosomes to meet manufacturing demand using a gentle, size-based, and scalable purification solution

Dr Jagan Billakanti¹, Dr Jon Lundqvist, Dr Peter Guterstam

¹Cytiva, Brisbane, Australia

PT03.83 Isolation of extracellular vesicles in aqueous two-phase systems for cancer diagnosis

Ph.D. Candidate Minyeob Lim¹

¹POSTECH, Pohang, South Korea

PT04.01 25HC depleted accessible cholesterol to restrict SFTSV infection and infectious-EVs mediated tramsmission

Postdoctor Rui Zhang

PT04.04 Bacterial extracellular vesicles contain metabolites that could contribute to the pathological hallmarks of Alzheimer's disease

Samuel Wachamo^{Department of Neuroscience, Neuroscience Graduate Program, Center for Brain Immunology and Glia, Medical Scientist Training Program, University of Virginia, Charlottesville, VA, USA}, Alisha Thakur, Mallarie Broadway^{Department of Neuroscience, Neuroscience Graduate Program, Center for Brain Immunology and Glia, Medical Scientist Training Program, University of Virginia, Charlottesville, VA, USA}, Dr. Alban Gaultier^{Department of Neuroscience, Neuroscience Graduate Program, Center for Brain Immunology and Glia, Medical Scientist Training Program, University of Virginia, Charlottesville, VA, USA}

PT04.05 Bacterial outer membrane vesicles trigger mitochondrial stress in macrophages

Ms Chantelle Blyth, Dr Michael Lazarou, Dr Thomas Naderer

PT04.06 Comparative analysis of intestinal microbiota-derived extracellular vesicles in newborns from vaginal and cesarean section delivery: implications for modulation of immune system cells

Ms. Catalina Adasme-Vidal, Mr. Aliosha I. Figueroa-Valdés, Ms. Camila Fuentes, Ms. Patricia Valdebenito, Mr. Sebastián Illanes, Ms. Francisca Alcayaga-Miranda

PT04.08 Exosomal miRNAs as markers of the biological effects of plant extracts

Doctor Alisa Petkevich, Doctor Aleksandr Abramov, Professor Vadim Pospelov

PT04.09 Exploration of Purified Extracellular Vesicles (EVs) from Trypanosoma cruzi Y and G Strains on Host Cell Interaction

PhD student Paula Meneghetti, Ana Claudia Torrecilhas

PT04.10 Explore food microbes with preservative tolerance to spread of antimicrobial resistance from perspective of extracellular vesicles

Dr. Bao-Hong Lee, MS. Yi-Tsen Chang, Mr. You-Zuo Chen, Mr. Hui-Chun Lin, Dr. Wei-hsuan Hsu

PT04.11 Exploring the extracellular vesicles derived from food spoilage microorganisms in the transmission of antibiotic resistance and potential impact on the gut environment

Dr. Bao-Hong Lee, MS. Yi-Tsen Chang, Mr. Hui-Chun Lin, Mr. You-Zuo Chen, Dr. Tang-Long Shen, Dr. Wei-hsuan Hsu

PT04.13 Extracellular vesicles derived from Akkermansia muciniphila

Promote placentation and mitigate preeclampsia

Ph.d Zihao Ou

PT04.14 Extracellular vesicles derived from Candida albicans promote lung injury through inducing ferroptosis of macrophages

Miss Yiyi Huang, Doctor Kening Zhao, Miss Yuneng Hua, Miss Mei Huang, Doctor Ruyi Zhang, Doctor Jingyu Wang, Mr Fan Bu, Miss Junhui Wang, Professor Lei Zheng, Professor Qian Wang, Professor Xiumei Hu

PT04.15 Extracellular vesicles derived from Naegleria fowleri stimulate cytokine production by innate immune cells

Asst. Prof. Sakaorat Lertjuthaporn, Ms Narinee Srimark, Mrs Hathai Sawasdipokin, Ms Kasama Sukapirom, Ms Jinjuta Somkird, Prof. Kovit Pattanapanyasat, Ladawan Khowawisetsut

PT04.16 Extracellular Vesicles isolated from Virulent and Non-Virulent trypomastigotes forms from Trypanosoma cruzi in Host Cell Modulation

Ana Claudia Torrecilhas, Master Nicholy Lozano, Full Professor Sergio Schenkman

PT04.17 Extracellular vesicles release from Aeromonas hydrophila: proteomic analysis and immunomodulatory activity

Professor MAHANAMA DE ZOYSA, Mr. Mawalle Kankanamge Hasitha Madhawa Dias

PT04.18 Gram-positive bacterial extracellular vesicles released by Streptococcus parauberis: Proteomic profiling and anti-inflammatory activity

Professor MAHANAMA DE ZOYSA, Mr. E.H.T. Thuslahn E.H.T. Thuslahn Jayathilaka, Mr. Mawalle Kankanamge Hasitha Madhawa Dias, Dr. Chamilani Nikapitiya Nikapitiya

PT04.20 Helicobacter pylori cytotoxin, VacA, hijacks dendritic cell extracellular vesicles

Miss Ruby Gorman-batt¹, Associate Professor Meredith O'Keeffe, Doctor Terry Kwok-Schuelein

¹Monash University, Clayton, Australia

PT04.20 Helicobacter pylori cytotoxin, VacA, hijacks dendritic cell extracellular vesicles to dysregulate immune cell functions

Miss Ruby Gorman-batt, Meredith O'Keeffe, Terry Kwok

PT04.21 Helicobacter pylori extracellular vesicles contain functionally active enzymes promoting bacterial survival

Miss Nina Colon, Mr Liam Gubbels, Professor Richard L. Ferrero

PT04.23 Infective forms of Leishmania show different biological aspects and differ in the extracellular vesicle target mechanisms: Looking at the fundamental bases to understand differences

Dr Mauro Javier Cortez Veliz, Miss Deborah Brandt-Almeida, Mrs Jenicer Kazumi Umada Yokoyama Yasunaka, Dr Simon Ngao Mule, Dr Giuseppe Palmisano, Dr Ana Claudia Torrecilhas

PT04.23 Host-Parasite Interface: Exploring the Interaction of Trypanosoma cruzi trypomastigotes forms Y strain Extracellular Vesicles (EVs) with Human Monocytes and Macrophage

PhD student Juliana Fortes, Master student Nathani Negreiros, Ana Claudia Torrecilhas

PT04.24 Investigating the impact of Pseudomonas aeruginosa outer membrane vesicles on alveolar macrophage responses

Miss Isabella Stuart, Mr Joshua Nickson, Dr Seong Hoong-Chow, Associate professor Thomas Naderer

PT04.25 Isolation and physiological characterisation of Ascochyta rabiei small extracellular vesicles

Ms Matin Ghaheri, Dr Ido Bar, Dr Prabhakaran T. Sambasivam, Dr Muhammad J. A. Shiddiky, Mr Abolfazl Jangholi, Prof Chamindie Punyadeera, Prof Rebecca Ford

PT04.26 Leishmania extracellular vesicles genomic cargo: sharing is caring

Associate Professor David Langlais, MSc Atia Amin, PhD Ana Victoria Ibarra Meneses, Associate Professor Christopher Fernandez-Prada

PT04.27 Microbiome derived EVs regenerate intestinal stem cells against radiation injury

Dr. Payel Bhanja, Dr. Rishi Man Chugh, Dr. Kafayat Yusuf, Dr. Badal Roy, Dr. Shahid Umar, Dr. Subhrajit Saha

PT04.29 Microbiota-derived extracellular vesicles regulate host liver gluconeogenesis

Dr Jian Tan, Ms Jemma Taitz, Dr Duan Ni, Ms Camille Potier, Prof Ralph Nanan, Prof Laurence Macia

PT04.30 Presence of viral particles and origin of extracelluar vesicles isolated from patients with COVID-19 and their association with clinical outcome

Msc Jaques Franco Novaes De Carvalho, Msc. Gabriela Rodrigues Barbosa, Msc Marina Malheiros Araújo Silvestrini, Dr. Sidneia Sousa Santos, Dr. Flávio Freitas, Dr. Nancy Cristina Junqueira Bellei, Dr. Andréa Teixeira de Carvalho, Dr. Ana Claudia Torrecilhas, Dr. Reinaldo Salomão

PT04.31 Probiotic extracellular vesicles: Characterisation and unravelling the proteomic cargo of extracellular vesicles derived from Lactobacillus delbrueckii

Mr Kyle Bramich, Dr Rahul Sanwlani, Prof Suresh Mathivanan

PT04.32 Proteomic analysis of Olive flounder (Paralichthys olivaceus) plasma derived exosomes responses to Edwardsiella piscicida infection

Professor MAHANAMA DE ZOYSA, Mr. E.H.T. Thuslahn Jayathilaka, Mr. Mawalle Kankanamge Hasitha Madhawa Dias, Dr. Chamilani Nikapitiya Nikapitiya

PT04.33 Role of HIV-associated extracellular vesicles in human papillomavirus (HPV) infection

Professor Ge Jin, Dr. Zhimin Feng

PT04.33 Role of extracellular vesicles in the pathogensis of Citrus exocortis viroid infection tomato plants

Professor Tang-long Shen, Hao-Yuan Chien, Ta-Hsin Ku

PT04.35 TcVPS23: A component of ESCRT-I complex is a key factor in secretion of extracellular vesicles, endocytosis of Transferrin and act as important virulence factor in Trypanosoma cruzi experimental infection

Pos-doctoral Nadjania Saraiva de Lira Silva, Ana Claudia Torrecilhas, Full Professor Sergio Schenkman

PT04.37 The Helicobacter pylori autotransporter ImaA associates with extracellular vesicles to promote host inflammatory responses in gastric epithelial cells

Mr Angus Cramond, Ms Nina Colon, Mr Jack Emery, Dr Dongmei Tong, Dr Caroline Skene, Professor Richard L. Ferrero

PT04.38 The Helicobacter pylori virulence factor, Tipa, is carried by bacterial extracellular vesicles to the nuclear compartment of host cells

Mr Jack Emery, Doctor Variya (Way) Nemidkanam, Ms Nina Colon, Ms Kate Friesen, Ms Georgie-Wray McCann, Associate Professor David McGee, Doctor Natalia Castaño-Rodríguez, Doctor Dongmei Tong, Doctor Caroline Skene, Doctor Laurent Terradot, Professor Richard L. Ferrero

PT04.39 The impact of maternally-derived gut bacterial extracellular vesicles on the offspring's developing immune system

Miss Jemma Taitz, Mr Jian Tan, Mr Duan Ni, Mr Georges Grau, Mr Nicholas King, Mr Ralph Nanan, Ms Laurence Macia

PT04.40 The role of Neisseria gonorrhoeae outer membrane vesicles in inducing trained immunity in Macrophages

Dr Jiaru Yang, Dr. Seong Hoong Chow, Dr. Pankaj Deo, Associate Professor Thomas Naderer

PT04.43 Using circulating bacterial outer membrane vesicles to diagnose bacterial infections

Phd Qianbei Li, Professor Lei Zheng

PT04.45 Foam cell-derived extracellular vesicles regulate the environment surrounding atherosclerotic plaques

Foam cell-derived extracellular vesicles regulate the environment surrounding atherosclerotic plaques Akihiko Okamura¹, Dr. Yusuke Yoshioka¹, Shungo Hikoso², Takahiro Ochiya¹

¹Department of Molecular and Cellular Medicine, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Japan, ²Department of Cardiovascular Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Japan

PT04.46 Hypericum perforatum-derived exosomes-like nanovesicles-based photosensitizer for photodynamic therapy in adipose tissues

Professor Jianbo Wu¹, Dr. Ziyu Li

¹Southwest Medical University, Luzhou, China

PT05.03 Characterising soluble TREM2 in extracellular vesicles derived from cultured human macrophages

Miss Drishya Mainali, Mr Anjie Ge, Dr Monokesh Sen, Miss Yvonne A. Candia, Dr. Claire Goldsbury, A/Prof. Laura Piccio

PT05.04 CRISPR/Cas9, genome editing and EVs: Exogenous bacterial Cas9 expression alters small EV secretion and their protein cargo in p53 dependent manner

Professor Suresh Mathivanan

PT05.05 Cytoplasmic DNA accumulation upon the inhibition of small extracellular vesicles secretion induces cGAS and ULBP1 activation in acute myeloid leukemia blasts

Dr. Jamal Ghanam, Dr. Venkatesh Kumar Chetty, Prof. Dr. Dirk Reinhardt, PD. Dr. Basant Kumar Thakur

PT05.06 Defining the parameters for sorting of RNA cargo into extracellular vesicles

Associate Professor Mona Batish, Graduate Student Ahmed Abdelgawad, Assistant Professor Vijay Parashar

PT05.07 Effects of electromagnetic fields on the release and content of extracellular vesicles

Hailong Wang, Research Associate YiHua Wang, Professor Gregory Worrell

PT05.10 Establishment of a method for mass production of extracellular vesicles based on microRNA regulation

Dr. Tomofumi Yamamoto, Dr. Hirotaka NIshimura, Dr. Noritaka Hashii, Dr. Akiko Ishii-Watabe, Dr. Yusuke Yamamoto, Prof. Takahiro Ochiya

PT05.12 Exploiting the human peptidome for discovery of EV release-modulating agents

Dr. Ruediger Gross, Ms. Hanna Reßin, Mr. Nico Preißing, Dr. Ludger Ständker, Prof. Dr. Jan Münch

PT05.13 Gene copy numbers dictate extracellular vesicle cargo

Sumeet Poudel, Jerilyn Izac, Zhiyong He, Lili Wang

PT05.16 Identification of cis-regulatory elements involved in exosomal cargo sorting

Phd Student Gizaw Gebeyehu, Research Professor Tibor Rauch, Associate Professor Marianna Pap, Dr Geza Makkai, Dr Tibor Janosi

PT05.17 Lipid nanoparticles (LNPs) alter transcriptomic contents of extracellular vesicles (EVs) leading to functional LNP-mRNA repackaging into EVs

Benyapa Tangruksa^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41346, Sweden 2. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde SE-54128, Sweden}, Doctor Muhammad Nawaz^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41346, Sweden}, Adjunct Professor Sepideh Heydarkhan-Hagvall^{2. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde SE-54128, Sweden}, Professor Jane Synnergren^{2. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde SE-54128, Sweden 3. Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41345, Sweden}, Associate Professor Hadi Valadi^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41346, Sweden}

PT05.18 Metalloprotease ADAM10 in secretion, composition, and targeting of extracellular vesicles

MSc Christopher C. Reimann, MSc Feizhi Song, Dr. rer. nat. Dipl. Hum.-Biol. Hermann C. Altmeppen, Dr. Lesley Cheng, Prof. Markus Glatzel, Prof. Marina Mikhaylova, Prof. Andrew F. Hill

PT05.20 The “torn bag mechanism” of small extracellular vesicle release through rupture of the limiting membrane of en bloc released large EVs

Dr. Tamás Visnovitz, Ms Dorina Lenzinger, Ms Anna Koncz, Ms Tünde Bárkai, Dr. Krisztina V Vukman, Ms Alicia Galinsoga, Dr. Krisztina Németh, Ms Kelsey Fletcher, Dr. Péter Lőrincz, Dr. Gábor Valcz, Prof. Edit I Buzás

PT05.23 β-catenin regulates the biogenesis and secretion of small extracellular vesicles by modulating senescence

Dr Taeyoung Kang

PT05.24 The RNA-Binding Protein NPM1 Is a Component of Exosomal Machinery Controlling mRNA Sorting through Binding to Specific RNA Motif

Student Kaixiang Zhang^1,2, Ying Zhang¹, Hang Yin^1,2

¹School of Pharmaceutical Sciences, Tsinghua University, Beijing, China, ²Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China

PT05.25 Glycosylphosphatidylinositol-anchored Proteins Promote Cellular Membrane Trafficking and Enhance Extracellular Vesicles Production

Scientist Tong Zhao¹, Associate Scientist Wei Zhao¹, Associate Researcher Shengya Xu¹, Associate Researcher Moxuan Yang¹

¹TheraXyte Bioscience, Beijing, China

PT05.26 Immunomodulatory effect of AFSC conditioned media generated using Micregen's cell-free secretome technology platform linked with neural protection

Dr Robert Mitchell¹, Mr Andrew Parnell¹, Dr Ben Mellows¹, Professor Ketan Patel¹, Dr Steve Ray¹

¹Micregen Limited, Reading, United Kingdom

Dr. Ruediger Gross, Hanna Reßin, Pascal von Maltitz, Dan Albers, Laura Schneider, Hanna Bley, Markus Hoffmann, Mirco Cortese, Dhanu Gupta, Miriam Deniz, Jae-Yeon Choi, Jenny Jansen, Christian Preußer, Kai Seehafer, Stefan Pöhlmann, Dennis R Voelker, Christiane Goffiniet, Elke Pogge-von Strandmann, Uwe Bunz, Ralf Bartenschlager, Samir El Andaloussi, Konstantin MJ Sparrer, Eva Herker, Stephan Becker, Frank Kirchhoff, Jan Münch, Janis A Müller

Plenary Session 1 and Featured Abstract, Plenary 1, May 9, 2024, 9:00 AM - 10:15 AM

Introduction

Several human viral pathogens, including flaviviruses Zika and Dengue virus, are present in human body fluids, yet rarely transmitted sexually or orally. A possible explanation are extracellular vesicles (EVs) in semen and saliva which have been shown to inhibit flavivirus infection. The exact mechanism, whether this activity occurs in EVs from different sources, and which other viruses are affected are unclear.

We here propose that the antiviral activity of these EVs involves competitive binding of phosphatidylserine (PS) receptors. PS-receptors are commonly used as EV attachment factors and are sensors of apoptosis, a phenomenon exploited by several virus families in a process termed “viral apoptotic mimicry”, leading to uptake of virions and subsequent infection.

Methods

To study the molecular mechanism of antiviral EVs, we purified EVs from human semen, saliva, urine, breast milk, and blood using tangential flow filtration and size-exclusion chromatography. We characterized them biophysically by NTA and quantified the abundance (shotgun lipidomics) and exposure (bead-assisted and nano-flow cytometry) of PS. The antiviral activity was tested against a diverse panel of apoptotic mimicry viruses and viruses known to use other modes of attachment. To elucidate the specificity of PS on antiviral activity, we modified the exposed lipid leaflet enzymatically and imaged their interaction with PS receptors using confocal microscopy.

Results

Using Zika virus as a model for viral apoptotic mimicry, we show that EVs from all used sources inhibit infection of cell lines and primary tissues, while synthetic control vesicles lacking PS are inactive. Lipidomics and flow cytometry using lactadherin revealed that antiviral EVs are rich in surface-exposed PS. PS-exposing EVs prevented virion attachment and infection by competing with virions for cellular PS-receptors. This antiviral activity was reduced upon enzymatic removal of lipid headgroups and restored by cyclodextrin-mediated PS-donation. The antiviral activity extended to other viruses known to use PS receptors, including Dengue, West-Nile-, Chikungunya-, Ebola and vesicular stomatitis viruses, but not viruses using other receptors such as SARS-CoV-2, HIV-1 or herpesviruses.

Conclusions

Overall, our results identify PS-exposure as an EV-based innate defense mechanism that may play a key role in restricting viral transmission via body fluids.

Dr Susannah Hallal, Dr Ágota Tűzesi, Dr Abhishek Vijayan, Dr Laveniya Satgunaseelan, Associate Professor Hao-Wen Sim, Associate Professor Brindha Shivalingam, Associate Professor Michael Buckland, Associate Professor Fatemeh Vafaee, Dr Kimberley Alexander

Plenary Session 2 and Featured Abstract, Plenary 1, May 10, 2024, 9:00 AM - 10:10 AM

Introduction: Glioblastoma (GBM) is the most common and aggressive adult primary brain tumour, and patients face distressingly short survival outcomes of only 14 months. GBM tumours often recur quickly, acquiring more aggressive and treatment-resistant features that cannot be accurately detected with currently available monitoring methods. The development of circulating biomarkers that offer early and precise indications of GBM recurrence holds immense potential for enhancing patient care. To address this critical need, we have developed a novel, extracellular vesicle (EV)-based multianalyte liquid biopsy strategy that detects sensitive biomarker signatures or ‘blood fingerprints’ for routine assessment of GBM tumour activity and treatment response.

Methods: Using size-exclusion chromatography, EVs were isolated from 104 serial plasma specimens (1 mL) from 50 patients diagnosed with GBM IDH-wildtype. The serial plasma samples were collected at three clinical timepoints: before (Pre-OP, n = 27) and after (Post-OP, n = 49) first surgeries, and upon pathologically-confirmed recurrence (REC, n = 28). Captured plasma-EV populations were characterised by nanoparticle tracking analysis, cryo-transmission electron microscopy and mass spectrometry. The plasma-EVs were analysed using established, complementary EV proteomics and small-RNA (sRNA) sequencing platforms. Filtered and normalised proteomics and sRNA datasets were passed through a cross-validation pipeline (30 iterations; 80%-train:20%-test) using multiple feature selection methods and classification models. The best-performing protein and sRNA candidate biomarkers were identified separately. Multianalyte models were generated using an ensemble stacking machine learning method that combined the performance power of the best performing protein and sRNA biomarkers.

Results: Our pipeline generated 4117 proteins and 272 sRNA transcripts common to all plasma specimens. We described three multianalyte (protein and sRNA) blood ‘fingerprints’ that comprised the best-performing biomarkers for classifying patients according to GBM tumour burden (Pre-OP vs Post-OP), recurrence (Post-OP vs REC) and treatment resistance (Pre-OP vs REC). All models had a training performance of 100% and cross-validation test accuracy rates that ranged from 89.9-93.4%.

Conclusions: Our EV-based liquid biopsy strategy holds promise for accurately monitoring GBM patients and distinguishing recurrence from treatment-effects. Independent validation of our blood fingerprints is underway utilising plasma specimens and clinicopathologic data captured from two external GBM cohorts, the VERTU study (trial ACTRN12615000407594) and GlioNET observational study.

Prof. Dr. Ryosuke Kojima, Mr. Koki Kunitake, Professor Tadahaya Mizuno, Professor Yasuteru Urano

Plenary Session 3 and Featured Abstract, Plenary 1, May 11, 2024, 9:00 AM - 10:05 AM

Small Extracellular Vesicles (sEVs) are important mediators of cell-to-cell communication in both physiological and pathological contexts, including cancer metastasis. These facts highlight the potential of sEV biogenesis and release processes (“release” processes hereafter) as novel therapeutic targets. Furthermore, sEVs are also attracting attention as highly biocompatible delivery vesicles, and therefore methods to control/enhance their production are of great interest for biotechnological applications. Despite the importance of sEV release processes, a comprehensive understanding of their regulation has been difficult with conventional low-throughput assays using small-molecule inhibitors or siRNAs in separate wells.

Here we report a novel high-throughput pooled screening system to identify key players of sEV release processes. We actively incorporated guide RNA (gRNA) for Cas9 into sEVs through the interaction of gRNA and dead Cas9 (dCas9) fused with an sEV marker in a pooled CRISPR screening format. This allows sEV-loaded gRNA to work as a “barcode” linking each sEV to the perturbation of gene expression in its originating cell. Quantification of the composition of barcode gRNA in both sEVs and cells allows high-throughput, genome-wide exploration of genes involved in sEV release while canceling out the effects on cellular activities (e.g., proliferation, barcode transcription). We call this assay platform CRISPR-assisted individually barcoded sEV-based release regulator (CIBER) screening. CIBER screening using multiple sEV markers in combination with bioinformatic analyses revealed both known and previously unknown factors controlling sEV release processes, uncovering different effects of V-type ATPases, mitochondrial electron transport, and the cell cycle on the release of CD63+ and CD9+ sEVs. We believe this work provides a basis for detailed studies on the biogenesis, release, and heterogeneity of sEVs. (Kunitake, Kojima* et al, bioRxiv 2023, doi.org/10.1101/2023.09.28.559700)

Dr Georgia Atkin-Smith, Jascinta Santavanond, Amanda Light, Joel Rimes, Andre Samson, Jeremy Er, Joy Liu, Darryl Johnson, Melanie Le Page, Pradeep Rajasekhar, Raymond Yip, Niall Geoghegan, Kelly Rogers, Catherine Chang, Vanessa Bryant, Mai Margetts, Cristina Keightley, Trevor Kilpatrick, Michele Binder, Sharon Tran, Erinna Lee, Doug Fairlie, Dilara Ozkocak, Andrew Wei, Edwin Hawkins, Ivan Poon

Plenary Session 4 and Featured Abstract, Plenary 1, May 12, 2024, 9:00 AM - 10:00 AM

Introduction:

Endothelial cells are integral components of all vasculature within complex organisms and are vital in diverse processes such as embryonic development, angiogenesis and wound healing. Despite their low turnover, endothelial cells undergo rapid growth and remodelling under both homeostatic and stressed conditions. Understanding how these processes are regulated, and how endothelial cells mediate communication throughout the organism remains a fundamental question in cell biology. The generation of extracellular vesicles (EVs) has emerged as a key mechanism for cell-to-cell communication, as well as for the removal of cellular waste. However, the formation, function and clearance of large, endothelial cell-derived EVs in vivo is yet to be fully defined.

Methods:

We used a series of world class imaging techniques to study the formation of large endothelial cell-derived EVs in model organisms. This includes 4D intravital microscopy of the bone marrow calvarium of live, endothelial cell reporter mice, as well as dual confocal multiphoton microscopy of cleared tissue. To complement our mouse studies, we also performed extensive time lapse microscopy on endothelial reporter zebrafish. We paired our elegant imaging approaches with comprehensive traditional and imaging flow cytometry analysis to examine the formation, contents and clearance of EVs under homeostatic settings and under the stress of various blood cancer models.

Results:

For the first time, we captured the formation of large endothelial cell-derived EVs in vivo, and acquired 4D information of this process including spatial and temporal insights. We identified these EVs to be a population of large, mitochondria-rich EVs present under homeostatic settings, and identified in murine, zebrafish and human samples. We demonstrate that these EVs can interact with and be efferocytosed by various immune cell populations as a potential clearance mechanism. Excitingly, we show that elevated levels of circulating large, endothelial cell-derived EVs correlated with the degradation of the bone marrow vasculature caused by acute myeloid leukemia (AML).

Summary/Conclusions:

Collectively, our study provides in vivo spatio-temporal characterization of EV formation in the murine vasculature, and suggests that circulating, large endothelial cell-derived EVs may provide a snapshot of endothelial cell health that reflects the extent of tissue damage at distal sites.

PhD Dariusz Żurawek, PhD Alice Morgunova, PhD Laura Fiori, M.S. Jennie Yang, PhD Claudia Belliveau, M.S. Pascale Ibrahim, M.S. Jean Francois Théroux, M.S. Ryan Denniston, Prof. Sidney H. Kennedy, Prof. Raymond W. Lam, Prof. Roumen Milev, PhD Susan Rotzinger, MD Claudio N. Soares, MD Valerie H. Taylor, MD Rudolf Uher, PhD Jane A. Foster, MD Benicio N. Frey, PhD Cecilia Flores, PhD Corina Nagy, MD Gustavo Turecki

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM - 12:00 PM

Introduction

The difficulty of studying the biochemical processes of the living brain has been a limitation in advancing our understanding of the molecular mechanisms of antidepressant treatment response. Recently, it has been reported that various brain cell types, including neurons, release extracellular vesicles with specific molecular cargo that can cross the blood-brain barrier. Detecting these vesicles in the periphery may serve as non-invasive biomarkers, providing insights into the true molecular state of the brain and aiding our understanding of antidepressant treatment mediators.

Methods

We analyzed 430 human plasma samples from the CAN-BIND-1 clinical trial (clinicaltrials.gov, NCT01655706). Informed consent was obtained from participants and the trial has an ethical approval. The samples were collected before and after an 8-week escitalopram treatment from depressed patients, distinguishing between responders and non-responders to the treatment, and untreated healthy controls. Neuron-derived extracellular vesicles (NEVs) were isolated from plasma using size exclusion chromatography following by immunoprecipitation against SNAP25, a protein exclusively expressed in neurons and found on the surface of NEVs. Presence of EV markers and potential contamination in NEV samples were tested by Western blot. Transmission electron microscopy and nanoparticle tracking analysis were used to determine the morphology and size distribution of NEVs. Small RNA-seq profiling followed by RT-qPCR validation were used to determine miRNA cargo in NEVs.

Results

Before treatment, depressed patients had lower miR-151a-5p levels in NEVs than controls. However, these levels significantly increased over time only in the group of depressed patients who responded to antidepressant treatment. miR-151a-5p levels in NEVs demonstrated good predictive value in discriminating between responders and non-responders. In vitro and in silico experiments revealed that miR-151a-5p negatively regulates a gene set enriched in the prefrontal cortex, responsible for regulating glutamatergic signaling. Engineered NEVs enriched with miR-151a-5p and delivered to prefrontal cortex displayed antidepressant properties in vivo and effectively ameliorated the behavioral deficits induced by chronic social defeat stress in mice. Animal studies were approved by an appropriate ethical committee.

Conclusion

miR-151a-5p cargo in NEVs may be a molecular mediator of effective antidepressant treatment response.

Dr. Jancy Johnson, Dr. Gregor Lichtfuss

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM - 12:00 PM

Introduction:

Chronic wounds are damaged tissue that stay unhealed for longer than 4 weeks and are currently lacking effective treatment methods. In vitro and animal studies have shown that the release of growth factors and nucleic acids by activated platelets through extracellular vesicles (EVs) are pivotal for healthy wound healing. Hence, platelet-derived EVs (pEVs) could represent a next-generation therapeutic for the treatment of chronic wounds.

However clinical assessment of pEV-based therapeutics has been hampered to-date by challenges in scalable and reproducible EV manufacturing. This study aimed to address these gaps by isolating clinical-grade pEVs using a scalable, proprietary chromatography-based method, evaluating their functional efficacy in cell-based assays and lastly, assessing their safety upon administration in humans in a Phase I clinical trial.

Methods:

pEVs were isolated using Ligand Exosome Affinity Purification (Exopharm) and characterized for particle size, count and morphology. In vitro assays were performed to assess the effect of pEVs on cellular processes important for wound healing. Lastly, a Phase I Clinical trial approved by the Australian Red Cross Lifeblood was conducted in healthy volunteer adults to assess safety of pEV administration following a skin punch biopsy (n = 11).

Results:

Clinical-grade pEVs were found to adhere to MISEV guidelines, ranging between 65-200nm in size, possessing a lipid bilayer and positive for EV proteins such as ALIX and Syntenin. pEVs significantly improved cellular functions such as dermal fibroblast proliferation and migration in vitro suggesting a potential regenerative function of pEVs after injury. Sterility and endotoxin testing revealed that the pEVs were suitable for administration for clinical trials.

In humans, a single dose of pEVs (300 µg mL-1) was injected, following a skin punch biopsy. A mean healing time of 22.8 ± 8.7 days was recorded for pEV and placebo-treated groups. Importantly, pEV treatment was found to be safe and well-tolerated with no adverse events recorded.

Conclusion:

These results demonstrated for the first time, that allogeneic pEVs can be manufactured under clean-room conditions, at clinical scale, and are safe when administered to humans. These findings also support future studies that assess efficacy of pEV-therapeutics in patients with disrupted wound healing.

Dr Olasehinde Olusanya

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM - 12:00 PM

Method: This project involved the formation of a team including academic experts with varied backgrounds in the field of extracellular vesicles (EVs). The primary objective of the team was to investigate the potential of biomarkers derived from EVs for the purpose of medicine development. The research team engaged in a comprehensive investigation of the available literature and executed a series of tests to ascertain and assess the prevailing obstacles and prospects associated with the regulation and commercialization of biomarkers derived from extracellular vesicles (EVs).

Results: Based on the results of our research, we suggest that the extracellular vesicle (EV) community should prioritize the advancement of therapeutic applications grounded on empirical evidence. Additionally, efforts should be made to modify current regulatory criteria to encompass biomarkers derived from EVs. Furthermore, it is imperative to make endeavours in the identification of commercially feasible uses of extracellular vesicle (EV)-based biomarkers and to produce comprehensive, integrated data at the systems level to enhance their utilization in clinical decision-making processes. To comprehensively comprehend the practical consequences of utilizing EV-based biomarkers in the realms of diagnosis and therapy, it is imperative to engage in collaborative endeavours with clinical organizations. These measures have the potential to facilitate the integration of EV-based biomarker development with their application in clinical settings.

Summary/Conclusion:

In summary, by the resolution of regulatory and commercialization obstacles, as well as the promotion of cooperation between the extracellular vesicle (EV) community and clinical institutions, we can facilitate the effective incorporation of EV-derived biomarkers into clinical settings. This would not only improve disease diagnosis and therapeutic evaluation, but also expedite the advancement of novel treatment modalities. Further investigation is warranted in this domain to effectively harness the potential of extracellular vesicle (EV)-based biomarkers in the context of pharmaceutical development, with the ultimate goal of enhancing patient outcomes.

Keywords: Extracellular vesicles (EVs), EV-based biomarkers, Regulation, Clinical applications Biomarker development, Diagnostic tools

Miss Kimberly Schell, Doctor Aisling Flinn, Professor Matthew Collin, Professor Andrew Gennery, Doctor Rachel Crossland

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM - 12:00 PM

Introduction

Graft versus Host disease (GvHD) is a serious complication of allogeneic haematopoietic stem cell transplantation (HSCT), affecting 40-70% of patients and leading to mortality in 40-60%. Standard steroid treatment can cause adverse effects, and lead to steroid refractory disease. Extracorporeal photopheresis (ECP) therapy is a safe alternative, though the mechanism of action is unknown, and biomarkers to determine ECP response are lacking. This study aims to investigate the role of serum extracellular vesicles (EVs) during ECP therapy for GvHD.

Methods

Serum and peripheral blood mononuclear cell (PBMC) samples were acquired from n = 12 paediatric ECP patients before each treatment cycle with appropriate ethical approval and informed consent. EVs were isolated and characterised from four sequential timepoints (TP) by precipitation for n = 6 complete (CR) and n = 6 partial/non-responders (PR). EV microRNA cargo and surface marker expression profiling was performed using NanoString, MACSPlex and ImageStream, respectively. T Cell Receptors (TCRs) will be assessed via the TCR diversity nCounter panel, and serum cytokines via MSD assays in PBMC and serum, respectively.

Results

EV modal size was significantly larger for patients who reached CR vs. PR, and this was most pronounced after commencement of ECP. Analysing EV microRNA composition showed 3 principal component analysis (PCA) clusters; PR and two separate clusters of mostly CR. There was no treatment cycle-based clustering. Eight microRNAs were significantly differentially expressed in CR vs. PR patients; hsa-miR-23a-3p was downregulated while hsa-miR-144-3p, hsa-miR-548ar-3p, hsa-miR-378e, hsa-miR-21-5p, hsa-let-7i-5p, hsa-miR-579-3p, and hsa-miR-1283 were upregulated (p<0.05). MicroRNA target analysis showed enrichment of GvHD and/or ECP-related pathways, such as allograft rejection, interleukin, and TLR-signalling. The most highly expressed EV-microRNA post-ECP in all patients was anti-inflammatory miR-451a. Focusing on EV-markers, there was a significant interaction between ECP cycle and response for CD40, CD42a, CD56, CD41b, CD31, and CD49e, while HLA-DPDQDR, CD14, CD20 and CD44 were significantly downregulated post-ECP, and CD8, CD44, CD142 and CD146 were expressed at a significantly higher level in PR vs. CR.

Conclusions

EVs show potential for biomarkers of ECP response, and their molecular cargo and surface markers may help elucidate the mechanisms of ECP action.

Ms Arunima Panda, Mrs Ilaria Casari, Dr Abir Halder, Dr Walid Abu Shawish, Dr Danielle Dye, Prof Krish Ragunath, A/Prof David Greening, Prof Marco Falasca

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM - 12:00 PM

Introduction

Pancreatic cancer (PC) is one the most aggressive malignancies marked by rising incidence and high fatality rates. PC is characterised by a lack of early symptoms, delayed diagnosis, and resistance to conventional chemotherapy, making it challenging. The PC marker, Ca19.9, lacks efficiency and efficacy. Consequently, early detection and effective treatment options are crucial in confronting this challenge. Small extracellular vesicles (sEVs) are employed by cancer cells for intracellular communication and to promote cancer progression. Identification and molecular analysis of the distinctive molecules transported by sEVs associated with PC hold the potential to serve as biomarkers, offering molecular signatures for PC diagnosis.

Methods

We performed characterisation of sEVs from PC and patient-derived xenograft (PDX) cell lines based on phenotypical, physiological, and functional characteristics. To support our functional results, we performed a CRISPR knockdown. Plasma samples were collected from 30 suspected PC patients and 14 disease-free participants. Ethical approval was obtained for the use of human blood samples from PC patients and age-matched healthy participants (Royal Perth Hospital RGS4208).

Results

Our research has identified specific oncoproteins only found in sEVs derived from PC cells. Interestingly, sEVs derived from human PC cell lines and PDX cells contain unique cargo, including metastatic regulatory factors and pivotal signalling molecules crucial for PC progression. Furthermore, our proteomic analysis of sEVs from human PC cell lines and PDX cell lines has shown higher expression of critical enzymes involved in inositol synthesis and metabolism, including the transporter SLC5A3. SLC5A3 plays a role in inositol transport and the generation of essential signalling molecules like phosphatidylinositol 4,5-bisphosphate. SLC5A3 can play the role of a prognostic factor for PC. Our findings have been validated in sEVs from PC, PDX cell lines, knockdown of PDX cell lines and plasma samples from PC and healthy participants.

Conclusion

Oncoproteins such as SLC5A3 can be potential PC biomarkers and improve the diagnostic landscape of PC diagnosis.

Funding:

This research is funded by PanKind, The Australian Pancreatic Cancer Foundation (www.pankind.org.au) and the Royal Perth Springboard Plus Grant.

Miss Jiarong Cui, Prof. Min Zhou

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM - 12:00 PM

Introduction

Spirulina platensis (S. platensis, SP), a naturally occurring edible microalgae, has been noticed for its numerous beneficial properties, including antioxidant, anti-inflammation, and immunomodulatory effects. However, the challenges associated with its micron-scale size resulting in difficult skin absorption, and potential for skin allergies hinder its application in the treatment of skin diseases. As nanoscale phospholipid bilayer vesicles, SP extracellular vesicles (SP-EVs) contain components from the original cells, which is expected to inherit the application value of SP and be used in the skin diseases. Consequently, this research aims to isolate extracellular vesicles from the culture supernatants of SP and examine their potential to prevent skin damage caused by ultraviolet radiation.

Methods

In this study, SP-EVs were isolated using differential ultracentrifugation, and their structure and size were determined through the utilization of a transmission electron microscope, atomic force microscope, and dynamic light scattering. To enhance the adhesion of this system, SP-EVs were incorporated into a mixture of carboxymethyl chitosan (CMCS) and sodium alginate (SA), and subsequently cross-linked with genipin to form a hydrogel complex. To ensure optimal biosafety and adhesion properties, hydrogels with a final concentration of 0.04% genipin were selected from various concentration ratios for subsequent cell and animal experiments.

Results

The results of our study indicate that treatment with SP-EVs effectively mitigated the generation of reactive oxygen species, DNA damage, and mitochondrial alterations in keratinocytes stimulated with H2O2 or exposed to UV irradiation. Furthermore, SP-EVs therapy demonstrated enhanced antioxidant capacities, as evidenced by increased activity of glutathione peroxidase and superoxide dismutase, along with a decrease in malondialdehyde concentration, thus protecting against oxidative stress-induced skin damage. Additionally, in both cellular and animal models, SP-EVs treatment exhibited anti-inflammatory properties in cells and tissues.

Conclusion

In brief, we have successfully fabricated a hydrogel with exceptional adhesion properties by incorporating SP-EVs into the biocompatible CMCS/SA substrates and subsequently cross-linking them with genipin. Considering the remarkable stability, absence of adverse toxicity, and absence of anaphylactic reactions, SP-EVs exhibit promising potential as a dermatological nanotherapeutic agent for mitigating skin UV damage via their antioxidant, anti-inflammatory, and other therapeutic effects.

Associate Professor Christopher Fernandez-Prada

Introductory Talk and Oral Session: OT02 Pathogen Host Response, Eureka, May 9, 2024, 10:40 AM - 12:00 PM

Introduction: Leishmaniasis, a significant tropical disease caused by Leishmania protozoan parasites, presents challenges due to emerging drug-resistant strains. Understanding the role of extracellular vesicles (EVs) in propagating drug resistance is crucial for the development of effective treatments and potential vaccines.

Methods: Our comprehensive study combined proteomics, lipidomics, and genomics to analyze both Leishmania parasites and their EVs. The EVs were isolated from Leishmania infantum promastigotes using a protocol applicable to various Leishmania spp. strains. This involved multiple centrifugation steps, filtering, and ultracentrifugation to retrieve and purify small EVs. DNA was extracted from these EVs using the DNeasy Blood and Tissue Kit and sequenced using an Illumina sequencer. Proteomic analysis involved mass spectrometry to identify and quantify proteins in both parasites and EVs. Lipidomic profiling was conducted using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS), focusing on the lipid composition of EVs from drug-resistant strains. Genomic analysis included Next-Generation Sequencing (NGS) and PCR assays to examine the DNA content of EVs and assess their role in horizontal gene transfer (HGT).

Results: Our findings revealed significant changes in the EVs of drug-resistant parasites, including variations in morphology, size, and distribution. The proteomic analysis identified a diverse array of proteins, including virulence factors and proteins encoded by drug-resistance genes. Lipidomics revealed a notable shift in the lipid composition of EVs from drug-resistant strains. Genomic studies confirmed the enrichment of circular amplicons carrying drug-resistance genes in EVs. These EVs were shown to transfer drug-resistance traits to naïve parasites, altering their drug sensitivity, enhancing their growth, and improving their control of reactive oxygen species.

Summary/Conclusion: This research provides the first evidence of EVs as an efficient platform for HGT in eukaryotic parasites, facilitating the rapid transmission of drug-resistance genes and enhancing the global fitness of recipient parasites. Our findings highlight the critical role of EVs in the dissemination of drug resistance in Leishmania, offering novel insights for therapeutic strategies targeting these vesicles to combat drug-resistant strains.

Dr. Adam Fleming, Mr. Graham Matulis, Ms. Heather Hobbs, Dr. Valentin Giroux, Mr. Hunter Mason, Dr. Weidong Zhou, Dr. Valerie Calvert, Dr. Nitin Agrawal, Professor Emanuel Petricoin, Dr. Rekha Panchal, Professor Igor Almeida, Dr. Sina Bavari, Professor Ramin Hakami

Introductory Talk and Oral Session: OT02 Pathogen Host Response, Eureka, May 9, 2024, 10:40 AM - 12:00 PM

Introduction:

Gram-negative bacteria (GNB) have significant clinical importance in hospitals and are among the world's most significant public health problems due to their high resistance to antibiotics. We have used two well-established model human pathogens, Yersinia pestis (Yp), and Burkholderia pseudomallei (Bp), to address the strong gap in knowledge regarding the molecular mechanisms by which host-derived sEV help protect against pathogenic gram-negative bacteria.

Methods:

sEV were purified from naïve U937 monocytes (EXu) and infected U937 (EXi) by differential centrifugation followed by density gradient purification, and characterized by SEM, confocal microscopy, NTA, marker analysis (CD63, TSG101, Flotillin-1), and LC-MS/MS analysis to profile sEV content and check for presence of LPS. Immune responses of naïve U937 cells and response mechanisms were analyzed following treatment with equivalent amounts of EXi or EXu (as control). These included macrophage differentiation assays, multiplex measurements of inflammatory cytokines both in vitro and in vivo, bacterial clearance assays, quantitative reverse protein microarray (RPMA) analysis of 173 host signaling proteins, and siRNA knockdown of signaling proteins identified by RPMA and of the EXi-induced cytokines in recipient cells. For all assays, at least four biological replicates were performed.

Results: Our results demonstrate that EXi induce activation of p38 kinase in recipient naïve monocytes that leads to their differentiation to macrophages and significant release of IL-6, IL-8 and IL-10 cytokines, phenotypes that are intriguingly similar to when the cells are infected with the bacteria. The IL-6 cytokine release in turn primes the recipient immune cells, leading to a dramatic increase in their capacity to clear the bacteria if they become infected. MS analysis showed lack of LPS in EXi, and also demonstrated the presence of specific bacterial proteins that have antigenic properties.

Summary/Conclusion: We have identified some of the main molecular mechanisms by which host-derived EXi assist the host in clearing infection caused by gram-negative pathogens. EXi prime distant naïve monocytes through modulation of distinct pathways such as p38 to mount IL-6 dependent immune responses, protecting the cells from a possible subsequent infection. These results provide a basis for development of novel prevention strategies against infection with gram-negative bacterial pathogens.

Dr Anne Borup, Dr Farouq Mohammad Sharifpour, Dr Litten Sørensen Rossen, Dr Bradley Whitehead, MSc Anders Toftegaard Boysen, Dr Paul Giacomin, Mrs Kim Miles, Ms Maggie Veitch, Dr Andrea Ridolfi, Dr Marco Brucale, Dr Francesco Valle, Dr Lucia Paolini, Dr Paolo Bergese, Dr Alex Loukas, Professor Peter Nejsum

Introductory Talk and Oral Session: OT02 Pathogen Host Response, Eureka, May 9, 2024, 10:40 AM - 12:00 PM

Introduction: Helminths cause chronic infection in the host through modulation of the host immune response. They suppress the pro-inflammatory type 1 response in favour of a modulated type 2 response by releasing excretory/secretory products (ESPs). Extracellular vesicles (EVs) have been shown to be released with the ESPs; however, their immunomodulatory mechanism is not fully understood.

Methods: We isolated EVs from Ascaris ESPs using size exclusion chromatography (SEC) and characterized them using nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM). Labelled EVs were generated in vivo, via incorporation of fluorescent lipid analogues during EV biogenesis, followed by flow cytometry and imaging flow cytometry to evaluate EV uptake in human PBMCs. Protease inhibition and PNGase were used further to understand the interaction between EVs and immune cells, while co-cultures and cytokine release were used to evaluate T-cell activation. A mouse model of inducible chemical colitis (dextran sodium sulfate) was used to assess the in vivo suppressive effects of Ascaris EVs.

Results: Using flow cytometry, we demonstrate that Ascaris EVs are primarily internalized in monocytes of human PBMCs. EVs induced a unique phenotype in the monocyte which was partly due to enzymatic activity by metalloproteinases. Enzymatic removal of N-linked glycans from the surface of EVs did not alter their effect on the monocytes' expression profile. Ascaris EVs attenuated T-cell activation in a monocyte dependent manner, and we demonstrate for the first time that these immunomodulatory effects are predominantly caused by EVs and not the EV-depleted fractions. Lastly, we find that Ascaris EVs partly recover DSS induced colitis in mice as measured by weight gain/loss and clinical score.

Summary/Conclusion: We show that EVs from intestinal helminths can suppress the immune response in circulating human immune cells and ameliorate colitis in mice.

Ms. - Ayesha, Dr Franklin Wang Ngai Chow, Prof. Polly Hang-mei LEUNG

Introductory Talk and Oral Session: OT02 Pathogen Host Response, Eureka, May 9, 2024, 10:40 AM - 12:00 PM

Introduction: Legionella pneumophila is a gram-negative bacterium and a respiratory pathogen associated with Legionellosis. Recent research has revealed that these bacteria release extracellular vesicles called outer membrane vesicles (OMVs) that contain effector molecules capable of triggering inflammatory responses in the host. However, there are insufficient comprehensive studies on the immunomodulatory capabilities of L. pneumophila OMVs, particularly when compared to the effect of L. pneumophila infection. To address this gap, we purified and characterized the L. pneumophila OMVs and explored their immunostimulatory effect on THP-1-derived macrophages in comparison with L. pneumophila infection

Methods: Highly purified OMVs were obtained from L. pneumophila using ultracentrifugation followed by density gradient ultracentrifugation. Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were employed to prove the purity of vesicles. A human proteome profiler kit which contains 105 cytokines, growth factors, and chemokines was used for the analysis. The intensities of the resulting blots were measured using quick spot software and the resulting protein expression data was further analysed by IPA (Ingenuity pathway analysis).

Results: NTA and TEM revealed that L. pneumophila OMVs have diameter ranging between 100 and 250 nm. Human cytokine array analysis revealed that these OMVs greatly induced the expression of various cytokines, growth factors, and enzymes, such as IL-1β, GM-CSF, IL-5, IL-4, IL-10, Growth hormone, FGF, DPPIV, and VCAM-1 in THP-1 derived macrophages when compared to L. pneumophila infection. Further IPA revealed that the immunostimulatory effects of L. pneumophila OMVs promote cell survival pathways while L. pneumophila infection induces macrophage cell death and inflammation pathways.

Conclusion: Our finding provides valuable insights into the role of OMVs as the key modulators of the immune system however, further research is necessary to understand their exact function in the pathogenesis of Legionnaires disease.

K E Y W O R D S

Cytokines, gram-negative bacteria, inflammation, Legionella pneumophila.

Dr Yvette Wooff, Dr Adrian Cioanca, Miss Rakshanya Sekar, Associate Professor Riccardo Natoli

Introductory Talk and Oral Session: OT03 EVs in Tissue Function, Room 105-106, May 9, 2024, 10:40 AM - 12:00 PM

Introduction: Extracellular vesicles (EV) are membrane-enclosed delivery vehicles, that function in cellular communication through the selective transfer of molecular cargo between cells. Since cell-to-cell communication is critical for tissue survival, dysregulation in EV communication is linked to the development of inflammatory and neurodegenerative diseases. Currently, little is known about retinal EV, due to technical difficulties in EV isolation. We therefore developed a novel protocol for the isolation of retinal EV, and quantified, characterised, and profiled the molecular contents of retinal EV in health and across degeneration.

Methods: Retinal EV were isolated from mouse and human retinas using papain digestion and ultracentrifugation. Retinal EV were characterised using nanotracking analysis, electron microscopy and western blot. Small RNA-seq and tandem mass spectrometry was performed on retinal EV. To investigate the role of EV or EV-miRNA on retinal homestasis, EV from healthy mouse retinas or EV-abundant microRNA were supplemented into the degenerating retina via intravitreal injection. Electroretinography and optical coherence tomography were used to measure retinal function and morphology respectively following EV/miRNA administration, while TUNEL and IBA-1+ immunohistochemistry was conducted on retinal cryosections to determine levels of cell death and inflammation.

Results: EV were found to alter in their concentration, size and molecular contents in degeneration compared to healthy controls. The top ten EV-miRNA were found to make up ∼67% of the total retinal miRNA concentration, and along with upregulated differentially expressed EV proteins were identified to control inflammatory and cell survival pathways known to be heavily involved in retinal degenerations. Compared to controls, mice injected with healthy retinal EV had significantly higher retinal function, reduced inflammation and decreased photoreceptor cell death. Further, intravitreal administration of key EV miRNA protected the retina against photo-oxidative damage-induced retinal degeneration, ameliorating inflammation and cell death, and preserving retinal function.

Conclusions: Taken together, our data suggests that a loss of EV-miRNA bioavailability is correlated to progressive retinal degeneration, while supplementation of healthy retinal EV or highly abundant EV-miRNA can restore homeostatic communication pathways and slow the progression of retinal degeneration.

Dr. Koushik Debnath, Dr. Irfan Qayoom, Mr. Steven O'Donnell, Ms. Julia Ekiert, Ms. Can Wang, Mr. Mark Sanborn, Mr. Chang Liu, Ms. Ambar Rivera, Dr. Ik Sung Cho, Ms. Saiumamaheswari Saichellappa, Dr. Peter Toth, Prof. Dolly Mehta, Prof. Jalees Rehman, Prof. Xiaoping Du, Prof. Yu Gao, Jae-Won Shin

Introductory Talk and Oral Session: OT03 EVs in Tissue Function, Room 105-106, May 9, 2024, 10:40 AM - 12:00 PM

Introduction: Rapid restoration of tissue barriers is essential for promoting regeneration post-injury. However, the mechanism involved, especially when the extracellular matrix (ECM) remains compromised, is not well understood. Recent studies propose that nanoscale mediators secreted by cells play a role in transporting ECM molecules. However, these mediators consist of diverse subpopulations, including lipid membrane-bound extracellular vesicles (EVs) and non-vesicular extracellular particles (NVEPs). The specific subpopulation responsible for competently activating ECM signaling over a distance remains unclear. Here, we present the discovery of matrimeres as constitutive nanoscale mediators of tissue integrity and function.

Methods: We utilized the standard differential ultracentrifugation technique (EV-TRACK ID EV150007) to isolate the crude nanoscale fraction secreted from mesenchymal stromal cells (MSCs). Subsequently, we employed immunoaffinity-based approaches, nanoparticle tracking analysis, and conditional probability calculations in order to quantify nanoscale subpopulations based on CD63, fibronectin (FN), and Triton-X sensitivity. Characterization of different nanoscale subpopulations involved various assays, including transmission electron microscopy and mass spec proteomics analysis. Following the isolation and characterization, we administered the fractionated or reconstituted nanoscale fractions to mice after inducing infectious or sterile inflammatory injury. Subsequent evaluations included the assessment of edema, vascular permeability, and tissue function.

Results: We define matrimeres as non-vesicular nanoparticles released by cells, identifiable by a primary composition consisting of at least one matrix protein and DNA molecules serving as scaffolds. MSCs form matrimeres from FN and DNA within acidic intracellular compartments. Drawing inspiration from this natural process, we successfully reconstituted matrimeres using purified matrix proteins and DNA molecules. Matrimeres containing plasma FN circulate in the blood under normal conditions, but their levels significantly decrease during systemic inflammation in vivo. Administering exogenous matrimeres leads to a rapid restoration of vascular integrity and tissue function by actively reannealing endothelial cells post-injury, and these matrimeres persist in the host tissue matrix.

Conclusions: We show that cells utilize a mechanism involving the assembly and systemic circulation of matrimeres to constitutively maintain tissue integrity. The ability to produce matrimeres at scale shows potential as a biologically inspired platform for tissue regeneration.

Ms Anastasiya Tolstoluzhinskaya, Ms Natalia Basalova, Ms Anastasiya Efimenko

Introductory Talk and Oral Session: OT03 EVs in Tissue Function, Room 105-106, May 9, 2024, 10:40 AM - 12:00 PM

Introduction. The main effectors cells of fibrogenesis are myofibroblasts, that participate in formation and remodeling of the fibrotic unit named fibroblastic focus, consisting of specific extracellular matrix (ECM). Accumulating evidence indicates that mesenchymal stromal cells could inhibit the fibrosis development mainly by regulating the myofibroblast pool through secreted extracellular vesicles (EV-MSCs) and specific microRNAs within EV-MSCs. We explored the molecular mechanisms of the effect of EV-MSCs and selected microRNAs as their cargo on myofibroblast transdifferentiation during fibrogenesis.

Methods. EV-MSC fraction were isolated by ultrafiltration from MSC conditioned medium and characterized according to MISEV-2018 recommendations. We added EV-MSCs to the previously developed 2D and 3D in vitro models of fibrosis. A 2D model was a culture of myofibroblasts differentiated under the influence of the transforming growth factor β (TGFb). 3D model was created using a decellularized spheroid ECM surrounded by myofibroblasts, which imitated the fibroblastic focus. We studied the contribution of selected microRNAs transferred within EV-MSCs and associated with fibrosis by transfection of EV-MSCs with synthetic microRNA inhibitors (Qiagen).

Results. The addition of EV-MSCs to 2D fibrosis model decreased the acquisition of myofibroblasts’ features and fibrotic ECM markers such as collagen type I and EDA-fibronectin. Using 3D model, we showed that addition of EV-MSCs resulted in a reduction of myofibroblasts features and the destruction of the structure of fibroblastic focus. We conducted an inhibitory analysis and showed that the antifibrotic effect of EV-MSCs was associated with the presence of microRNA-129, -29c and -92a in its composition.

Conclusions. Using 2D and 3D in vitro model simulating fibroblastic focus, we revealed that microRNAs-129, -29c and -92a secreted by MSC within EV-MSCs could significantly contribute to the regulation of myofibroblast transdifferentiation. These data further can be used to consider EV-MSCs and specific antifibrotic microRNAs as promising candidates for the treatment of fibrosis. The study was supported by the State Assignment of Lomonosov MSU.

Dr. Ziming Chen, Mengyuan Li, Peilin Chen, Andrew Tai, Jiayue Li, Euphemie Bassonga, Junjie Gao, Delin Liu, David Wood, Brendan Kennedy, Qiujian Zheng, Professor Minghao Zheng

Introductory Talk and Oral Session: OT03 EVs in Tissue Function, Room 105-106, May 9, 2024, 10:40 AM - 12:00 PM

INTRODUCTION: Tendinopathy, the most prevalent musculoskeletal disease, is typically caused by mechanical overload. While its underlying pathology is associated with inflammation, it is not clear how overload induces the pathological process. The aim of the present study is to explore the link between the overload and inflammatory reactions in tendon.

METHODS: We performed RNA sequencing on human tendinopathic tissues to explore the cellular response in tendinopathy. We then generated mouse tendon organoid by performing three-dimensional uniaxial stretching in bioreactors. Cyclic strain of uniaxial loadings included underload with 0% or 3% strain, normal load with 6% strain, and overload with 9% strain. Functional tests were performed by RT-qPCR. Medium extracellular particles (mEPs) were isolated from culture medium by differential centrifugation. Flow cytometry, dynamic light scattering, electron microscopy, and immunoblotting were performed to characterize mEPs. MitoTracker-labelled intracellular mitochondria of tendon organoid after loaded by different strain were observed through confocal live-cell imaging. Raw 264.7 mouse macrophage cell line was used for chemotaxis assay in a Boyden Chamber System with Magnetic-Activated Cell Sorting Technology to elucidate the role of ExtraMito in macrophage chemotaxis. Cytokines secretion by macrophages was analyzed by a bead-based multiplex assay panel. N-Acetyl-L-cysteine (NAC) was used as the antioxidant to tendon organoid to regulate mitochondrial fitness.

RESULTS: RNA sequencing showed that cellular activities including oxygen-related reactions, extracellular particles, and inflammation were activated in human tendinopathic tissues. Compared to underload, normal load induced better formation of mitochondrial network, while overload fragmented mitochondrial network and induced tendon organoid to release mEPs including ExtraMito. High-resolution confocal microscopy identified two forms of ExtraMito, including mitochondria-encapsulated mEPs and free extracellular mitochondria. Overload led to the degeneration of the organoid and induced mEPs release containing ExtraMito. Chemotaxis assay showed that ExtraMito from overloaded tendon organoid induced macrophages chemotaxis. In addition, mEPs from overloaded tendon organoid induced the production of proinflammatory cytokines including IL-6, CXCL1 and IL-18. NAC treatment attenuated overload-induced macrophage chemotaxis.

Conclusion: We identify for the first time that tendon cells release ExtraMito as a subtype of mEPs to the extracellular environment and mediate the inflammation, a process regulated by mechanical loading.

Dr. Zach Troyer, Sarah Marquez, PhD Olesia Gololobova, PhD Kenneth Witwer

Introductory Talk and Oral Session: OT04 EV Communication and Uptake, Room 109-110, May 9, 2024, 10:40 AM - 12:00 PM

Introduction: Extracellular vesicles (EVs) have been reported to fuse with cellular membranes pre- or post-endocytosis and thereby to deliver cargo to the recipient cell cytosol; however, the efficiency of this process appears to be low and may depend on producer:recipient cell identities. One approach to boosting fusion-mediated cargo delivery is to engineer EVs to display viral fusion proteins such as the vesicular stomatitis virus glycoprotein (VSV-G) envelope. While potentiating delivery, proteins of pathogenic viruses may also be immunogenic, limiting clinical applications. Human endogenous retroviruses (HERVs) are found in the human genome and, although no longer infectious, encode proteins that may be less immunogenic than foreign viral proteins. Here, we engineered EV display of several HERV envelope (Env) proteins (Syncytin-1, Syncytin-2, and HERV-K-108-Env) and assessed how they affect EV/cell interaction for two cell types (HEK293T and HeLa) that are commonly used for in vitro experiments.

Methods: Expi293F cells were used to produce EVs loaded with PalmGRET (an EV-labelling EGFP-Nanoluciferase (NLuc) fusion) and displaying one of several selected HERV Env proteins. EVs were separated by combined ultrafiltration and size exclusion chromatography. EVs were characterized per MISEV by electron microscopy, Western blot, and nanoflow cytometry and assessed for incorporation of PalmGRET and HERV Env. EVs were incubated with HEK293T or HeLa cells, and cell/EV association was measured by measuring cellular NLuc activity after washing away unbound EVs and treating cells with trypsin to remove surface-bound EVs.

Results: EV enrichment of HERV Envs and PalmGRET was confirmed by immunoblot and nanoflow. For recipient HEK293T cells, EVs displaying Syncytin-1 (HERV-W Env) had increased cellular association (2.027-fold/p<0.0001/n = 6), but not uptake (1.121-fold/p = 0.838/n = 3), compared with non-surface-modified control EVs. For HeLa cells, Syncytin-1+ EVs had both increased association (5.701-fold/p = 0.0038/n = 3) and uptake (2.857-fold/p = 0.01/n = 3). Syncytin-2+ and HERV-K-108-Env+ EVs did not have increased HEK293T/HeLa interactions.

Conclusions: HERV Envs that are expressed in EV-producing cells are also displayed on EVs. Certain HERV Env+ EVs have enhanced levels of interaction with target cells. However, the extent of this enhancement may differ by recipient cell type. These findings emphasize the importance of understanding the interactions of native and engineered EVs with different recipient cells.

Ms Mia Kordowski, Dr Ana Salazar-Puerta, Ms María Rincon-Benavides, Mr Justin Richards, Dr Nina Tang, Dr Safdar Khan, Dr Elizabeth Yu, Dr Judith Hoyland, Dr Devina Purmessur, Dr Natalia Higuita-Castro

Introductory Talk and Oral Session: OT04 EV Communication and Uptake, Room 109-110, May 9, 2024, 10:40 AM - 12:00 PM

Introduction

Chronic low back pain is a leading cause of disability worldwide and often results from intervertebral disc (IVD) degeneration¹. Our innovative approach involves using engineered extracellular vesicles (eEVs) loaded with transcription factors (TFs) and functionalized with cell specific transmembrane ligands to target nucleus pulposus (NP) and annulus fibrosus (AF) cells within the diseased IVD. This strategy offers a potential treatment for chronic low back pain.

Materials and Methods

In vitro, eEVs loaded with key developmental TFs FOXF1 and T to target NP and MKX and SCX to target AF, were derived from adult human dermal fibroblasts after nanoelectroporation with expression plasmids for each factor²-⁴. Characterization involved nanoparticle tracking analysis, Western Blot, ELISA, and qRT-PCR to confirm therapeutic TF packing and ligand presence. Fluorescently labeled eEVs were used to assess preferential uptake by human degenerate IVD cells (IRB 2015H038). Therapeutic efficacy of the functionalized TF loaded eEVs was evaluated via qRT-PCR analysis of catabolic, inflammatory, and pain markers in degenerate cells before and after exposure to eEVs. Excitingly, we developed a method of high-resolution microscopy to visualize efficient loading of protein cargo inside the eEVs and ligand presence on the membrane.

Results

Our research emphasizes the potential of using eEVs loaded with developmental TFs and functionalized with ligands for targeted delivery to NP or AF. Characterization validated successful loading of developmental TFs and ligands. Functionalized eEVs exhibited preferential internalization by NP or AF, leading to robust upregulation of TF expression. In contrast, non-functionalized EVs were captured nonspecifically. Additionally, our findings demonstrated successful reprogramming of NP and AF cells towards a healthier, pro-anabolic phenotype following exposure to eEVs, as confirmed by qRT-PCR and enhanced collagen production.

Conclusions

Our research demonstrates the potential of functionalized eEVs for efficient and selective delivery of developmental TFs to degenerate cells within the IVD, resulting in reprogramming of both cell types towards a healthier phenotype. These findings underscore the importance of further exploring eEV-based nanocarriers for targeting specific cell types and employing various techniques to confirm proper protein loading inside or on the membrane of eEVs. This research opens possibilities for tailored interventions in regenerative medicine.

Ms Akbar Marzan, Ms Monika Petrovska, Professor Suresh Mathivanan, Sarah Stewart

Introductory Talk and Oral Session: OT04 EV Communication and Uptake, Room 109-110, May 9, 2024, 10:40 AM - 12:00 PM

Introduction

Most mammalian proteins are secreted through the conventional secretory pathway, however a subset of cytosolic proteins are also secreted through unconventional protein secretion (UPS) pathways. We previously described a novel mechanism for UPS of non-vesicular protein cargo, involving bi-directional phospholipid scrambling at the plasma membrane. Extracellular vesicles (EVs) embody a major pathway for UPS. Therefore, we are now investigating whether phospholipid scrambling is also important for vesicular UPS, and its impact on EV biogenesis, packaging and function.

Methods

To investigate a potential role for phospholipid scrambling in EV biology, we investigated whether scramblases were expressed in EVs. EVs were isolated using ultracentrifugation and analysed using proteomics. Candidates were then investigated using CRISPR/Cas9 to generate scramblase knockout mammalian cell lines. EV biogenesis, secretion and function were assessed using biochemical approaches, confocal microscopy and proliferation assays.

Results

Proteomic analyses showed that EVs isolated from multiple mammalian cell lines contain phospholipid scramblases. Upon knockout of at least one scramblase, the protein content of EVs is altered. Loss of scramblase activity also reduces the number of EVs released per cell without affecting EV morphology. Confocal microscopy revealed that there may be perturbations both in the plasma membrane and endocytic system, leading to altered EV biogenesis and secretion. Finally, we showed that the pro-proliferative function of cancer-cell derived EVs is abolished when purified from scramblase knockout parent cells.

Summary/Conclusion

Together these results suggest that phospholipid scrambling and membrane homeostasis is important for the UPS of both soluble and vesicular cargo. EV biogenesis/secretion is decreased when scrambling activity is abolished. Equally, the protein cargo carried by scramblase-deficient EVs is altered, and this leads to functional impairments. Together this data demonstrates phospholipid scramblase activity is an important regulator of EV biogenesis and function.

Dr. Maria Harmati, Akos Diosdi, Ferenc Kovács, Ede Migh, Gabriella Dobra, Timea Boroczky, Matyas Bukva, Edina Gyukity-Sebestyen, Peter Horvath, Krisztina Buzas

Introductory Talk and Oral Session: OT04 EV Communication and Uptake, Room 109-110, May 9, 2024, 10:40 AM - 12:00 PM

Introduction. There are strong evidences that extracellular vesicle (EV) -mediated crosstalk between tumor and stroma cells has an essential role in tumor evolution and determines the outcome of the disease. However, there is a lack of quantitative data on the EV network of the tumor microenvironment (TME). Here, we aimed to establish 3D tumor model systems for live tracking of EV crosstalk. Our goal was to identify the primary communication axes and quantititatively analyse the EV transfer in different tumor types under physiological conditions and chemotherapeutic stress.

Methods. To monitor the EV transfer between cells, we used in-cell EV-labelling using CellTracker dyes. We generated three different 3D tumor models co-culturing one type of tumor cells (T-47D ductal carcinoma, A375 melanoma, or MG-63 osteosarcoma) and various stroma cells (MRC-5 fibroblasts, EA.hy926 endothelial cells and THP-1 monocytes) in hydrogel matrix. To mimic chemotherapeutic stress, low dose doxorubicin was applied. We analyzed the quantitative features of transfered EVs between different cell types using our patented high-content screening plates, a Leica SP8 Digital LightSheet microscope and the image processing and machine learning software called BIAS (Biological Image Analysis).

Results. By co-culturing four different cell types, we have optimized the 3D tumor model sytems for quantitative monitoring of EV crosstalk and mapping the EV network of the TME. The three types of tumor models showed significant differences in their EV crosstalk activity, the primary communication axes and drug-induced effects as well.

Conclusions. In this study, we identified potential target cells for EV-blocking drugs, which may be integrated to the medicine as a complementary therapy in tumor diseases. Also, the established 3D model systems and experimental pipeline may help to develop a high-throughput personalized medicine platform supporting clinical decision-making.

Funding. TKP2021-EGA09, OTKA-K143255, ÚNKP-23-4-SZTE-639, INKP_2024-11.

Dr. Cheng Zhou, Ms. Jie Gong, Mr. Baokun Fan, Ms. Xuan Ding, Dr. Bairen Pang, Prof. Yong Li, Dr. Junhui Jiang, Dr. Zejun Yan, Dr. Yue Cheng, Mr. Yingzhi Chen, Dr. Zhaohui Jiang, Mr. Tiannan Guo

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

Prostate cancer (PCa) is the most common cancer among men globally, particularly in developed countries. The current diagnostic tools such as prostate specific antigen (PSA) test, magnetic resonance imaging and tissue biopsy lack sensitivity and specificity. Extracellular vesicles (EVs) are nano-sized vesicles and play an important role in cancer communications and metastasis. The advancements in a novel EV separation system such as EXODUS and proteomics are providing an excellent chance in EV protein biomarker discovery The aim of this study was to leverage a robust EV isolation method- EXODUS, coupled with data-independent acquisition mass spectrometry (DIA-MS), to profile the urinary small EV (sEVs) protein biomarkers for accurate PCa diagnosis.

Methods:

Urinary sEVs were isolated using a novel EXODUS technology, and sEVproteins were identified by DIA-MS to enhance coverage and quantitation. A comprehensive landscape of urinary sEV-associated proteins was developed based on samples (n = 168), including low-intermediate risk PCa (n = 58), high-risk PCa (n = 58), metastatic PCa (n = 22), and controls (n = 30). Machine learning models were established and validated in an independent cohort consisting of PCa patients (n = 60)and controls (n = 20). This cohort also includes both grey zone PCa (n = 34) and grey zone controls (n = 11). The independent validation set was also performed with ELISA.

Results:

Total 5409 sEV proteins were identified in all samples. The key identified pathways in sEVs associated with PCa incidence, progression, and metastasis. include lysosome, proteasome, and cholesterol metabolism. was employed for classification evaluation, Two machine learning diagnostic models including sEV CD177, PEPD, DNASE1, CTNNB1, SEMA3D, SCGB1A1 proteins were established, demonstrating enhanced diagnostic efficiency for PCa diagnosis, especially in the grey zone PCa.

Summary/Conclusions:

In conclusion, this study presents a translational workflow focusing on identifying urine sEV proteins as molecular markers to advance the clinical diagnosis of PCa. The enriched pathways shed light on the mechanistic aspects of PCa development. Our established diagnostic models hold promise in improving PCa early diagnosis, especially in the grey zone PCa, contributing to the best personalized treatment choice.

M.D., Ph.D. Yuji Hakozaki, M.D., Ph.D. Yuta Yamada, M.D., Ph.D. Haruki Kume, Ph.D. Koji Ueda

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

We have previously identified a group of proteins with quantitatively cancer-specific high expression in plasma EVs of cancer patients for the purpose of enabling liquid biopsy of renal or colorectal cancer (Int J Cancer (2018);142:607)(Mol Cancer Res (2021);19:834). However, in many cases, these biomarker candidates were also found to be expressed in several normal organs, and their cancer specificity was insufficient.

Therefore in this study, we established an original proteogenomics analysis method and developed a cancer liquid biopsy technology targeting proteins with cancer-specific somatic mutations, which are expected to have 100% cancer specificity.

Bladder cancer is a cancer with a high risk of recurrence and requires regular follow-up using cystoscopy and contrast-enhanced CT after endoscopic resection. In this study, we aimed to develop a new liquid biopsy technique by detecting mutated proteins in urinary extracellular vesicles as a new, minimally invasive method of diagnosing recurrence.

Seven patients with urothelial carcinoma who underwent transurethral resection of bladder tumor at the University of Tokyo Hospital were included in the study. Genomic DNA was extracted from the resected tumors and blood samples from the patients, and somatic mutations were identified by whole exon sequencing. As the result, 55-444 nonsynonymous mutations and 5-16 frameshift mutations were identified.

Based on this information, the personalized proteogenomic analysis of bladder cancer tissue was performed, resulting in identification of 4-20 mutated proteins. More importantly, analysis of the patients’ urinary EVs revealed 1-3 mutated proteins in common with tissues.

An absolute quantification system using stable isotope-labeled peptide standards was established for these urinary EV mutated proteins, and the quantitative changes in the patient's treatment prognosis with disease progression were also monitored.

In conclusion, we introduce the potential of EV mutant proteins as a new cancer liquid biopsy modality.

Christian Grätz, Dr. Benedikt Kirchner, PD Dr. Marlene Reithmair, Dr. Florian Brandes, Dr. Agnes S. Meidert, Prof. Dr. Gustav Schelling, Prof. Dr. Michael W. Pfaffl

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Extracellular vesicles (EV) are shed by almost every cell type and can be found in any biofluid. They contain a variety of molecules, including several RNA species, which are protected from degradation. Over the last decade, we developed a validated workflow and analysis pipeline for the discovery and characterisation of EV-associated transcriptomic biomarkers in molecular diagnostics. Transcriptomic disease indicators offer several advantages over other types of biomarkers used in liquid biopsies. However, EV-associated RNA from liquid biopsy is limited in amount and quality, making it even more critical to ensure validity, transparency and repeatability throughout the process. In the biomarker analysis pipeline we highlight the key steps and give particular emphasis to the necessary quality control checkpoints, which are linked to numerous international guidelines that should be considered along the workflow, e.g. GMP, MIQE, MISEV, or GLP. Our discovery workflow starts with patient cohort recruitment and continues with liquid biopsy sampling and processing. The purification and characterization of EVs is explained in detail, as well as the isolation and quality control of EV-associated RNA species. We point out the possible pitfalls during next generation sequencing library preparation and subsequent RNA sequencing. Advanced bioinformatics assist to identify and correct for unwanted bias possibly introduced in an earlier step of the biomarker workflow. Applied multivariate methods, HCA, PCA and PLA-DS, are useful visualization tools to identify powerful biomarker candidates from the sequencing data set. For EV small-RNA biomarker studies, isomiRROR and caRNAge are in-house developed software routines, which were successfully applied for miRNA isoform analysis, batch correction, in-silico functional validation, and the overall process of small RNA sequencing data evaluation, respectively. Validation of the biomarker signature obtained this way is mandatory using RT-qPCR / RT-dPCR and miREV, following the recommendations according to the MIQE guidelines. Numerous studies have demonstrated the validity and diagnostic potential of EVs, and it is only a matter of time before EV-associated transcriptomic biomarkers will find their way into clinical routine.

Tasvilla Sonallya, Annamária Minus, Ferenc Fekete, Dr. Anikó Gaál, Kinga Ilyés, Dr. Tamás Beke-Somfai, Dr. Zoltán Varga, Dr. Katalin Monostory

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

INTRODUCTION

Toxicity testing is a crucial aspect of evaluating the potential harm of substances on living organisms. Currently, routine assessments often involve measuring enzymes such as GPT and GOT. However, the quest for more effective methodologies is ongoing, with a focus on developing specific biomarkers that can detect toxicity at earlier stages. Extracellular vesicles (EVs) could be exploited as biomarkers providing more accurate and timely indications of adverse effects, enhancing our ability to assess and mitigate potential risks. Their enhanced potential is owing to their varied contents, stability, and ease of access to different bodily fluids allowing them to be used as potential biomarkers. The microRNAs (miRNAs) in EVs are non-coding RNA molecules and in extracellular miRNAs can be organ-specific markers of deleterious lesions affecting a particular organ. EVs may be expressed in different sizes and concentrations, and their miRNA content may vary, which can be an early predictor of adverse effects.

METHODS

EVs were isolated from rat liver cell culture medium and rat blood plasma. They were characterized using MRPS, FACS, and FT-IR. In in vitro hepatotoxicity studies, various drugs such as paracetamol were used to check the hepatotoxicity at different concentrations, and toxicity was characterized by the miRNA concentration in EVs. We tested the applicability of reference miRNA((let-7-a, miR-103, miR23a, miR-16, miR-92, U6)) expression at constant concentrations independent of experimental design, and the expression of miRNAs(miR-122, miR-151, miR-155, miR-192, miR-194, miR-193a, miR-21, miR1, miR-375, miR133a, miR-146a) predictive of potential liver toxicity.

RESULTS

The extracellular vesicle isolation protocol, extracellular vesicle characterization method, and liver-specific miRNA profiling and concentration determination protocols were standardised to follow the hepatotoxicity. Paracetamol was used as a model compound with a well-established hepatotoxicity at high concentrations. Paracetam ol toxicity was characterized by the miRNA(miR-122 & miR146a) concentration in EVs that increased with treatment. We found that the labelling ability of glycoproteins on the surface of EVs is significantly reduced by paracetamol treatment.

SUMMARY

EVs can be employed as toxicity biomarker and the selective expression of miRNAs for hepatic damage and the change in their concentration are the criteria for their applicability as biomarkers.

Dr Anastasiia Artuyants, Martin Middleditch, Deanna Shea, Bianca Nijmeijer, Sophia Bebelman, Dr Cherie Blenkiron

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Endometrial cancer (EC) is the most prevalent gynaecologic malignancy, originating in the lining of the uterus. Existing diagnostic methods, such as ultrasound, hysteroscopy, and endometrial biopsy, while effective, are invasive, affecting patient acceptance and quality of life. This underscores the imperative for a non-invasive, easily accessible biomarker-based assay for early EC detection. Extracellular vesicles (EVs) emerge as promising entities in cancer research, offering a less intrusive means of testing due to their unique molecular cargo and systemic presence. Our goal is to develop a multiplex Multiple Reaction Monitoring (MRM)-based targeted panel for validation of our previously identified EV protein candidates (identified by tissue-derived EV profiling) that can be translated into routine clinical practice.

Methods

Building on our prior findings, we shortlisted peptides of interest representing diagnostic (EC vs non-cancer), prognostic (discriminating between EC histotypes endometrioid and serous), and biological (obesity, co-morbidities) purposes. Rigorous exclusion criteria, such as avoiding oxidizable amino acids and peptides with multiple adjacent cleavage sites, ensure assay specificity. Our approach involves optimising the relative quantitation of the targets using existing datasets of peptide identifications, followed by absolute quantitation using stable isotope-labelled peptides. Evaluation of the multiplex method includes analytical selectivity, carryover, matrix effects, and linearity. Analytes will be verified on various sample types (SEC isolated EVs from tissue, biofluids, and cell lines) before patient biofluid testing.

Summary

The envisioned multi-biomarker panel, designed to differentiate between cancer and benign samples and categorise histological subtypes, promises not only improved diagnostic accuracy to complement or replace current EC clinical tools, thereby revolutionising early diagnostic outcomes for people diagnosed with endometrial cancer.

Miss Xueming Niu, Dr Zhen Zhang, Mr Quan Zhou, Dr Alain Wuethrich, Dr Richard Lobb, Professor Matt Trau

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Microglia are a specialized population of innate immune cells located in the central nervous system, CNS. In response to physiological and pathological changes in their microenvironment, there are two classical inflammation-related phenotypes based on in vitro studies: pro-inflammatory or anti-inflammatory phenotype microglia. The balance between microglia pro- and anti-inflammatory polarization can be a potential biomarker for the various brain pathologies in the CNS. Here, we perform proteomic analysis of small extracellular vesicles (sEVs) derived from microglia cells to identify sEVs biomarkers indicative of pro-inflammatory and anti-inflammatory phenotypic changes.

Method

We have utilized nanoparticle tracking analysis, nanoflow cytometry, western blot and mass spectrometry to characterize sEVs and investigated the alterations in the proteome of sEVs purified from human microglia cell line following lipopolysaccharides, or a cytokine cocktail of IL-4, IL-10 and TGFβ stimulation. Cellular morphological changes and inflammatory gene expression validated the activated microglia cell models. Gene Ontology (GO) analysis was performed to determine the biological and functional properties of enriched proteins.

Result

Our analysis showed a distinct protein profile that influences specific biological pathways involved in inflammation found in sEVs released from LPS and IL-4/10/TGFβ treated cells compared to control. 188 proteins were identified in LPS-sEVs and 242 proteins were identified in IL-4/10/TGFβ-sEVs via mass spectrometry. According to GO analysis, we found a few proteins in LPS-sEVs that are related to immune response. Additionally, the enrichment of anti-inflammatory and tumor-associated proteins was identified in IL-4/10/TGFβ-sEVs. GO enrichment analysis revealed the downregulation of numerous pathways involved with antigen presentation and signaling cascades involved in neuroinflammation.

Conclusion

These results indicate that the activation of microglia through LPS and IL-4/10/TGFβ can lead to the secretion of sEVs reflective of the pro- and anti-inflammatory phenotype of the cells they were derived from. Although beyond the scope of this investigation, clinical analysis of proteins enriched in LPS- and IL-4/10/TGFβ-sEVs could provide important information when using advanced approaches to detect trace levels of sEVs. This can provide critical insight into biomarker discovery to monitor the pathogenesis of CNS disorders.

PhD Natalia Szpilbarg, Matías Nicolás Sierra, MD Juan Sebastián Sar, PhD Alicia Ermelinda Damiano

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Preeclampsia (PE) is a human gestational syndrome associated with placental insufficiency. In the most severe cases, trophoblast migration is impaired and the placenta is not adequately formed. The syncytiotrophoblast releases extracellular vesicles (EVs) into maternal circulation from the sixth week of gestation until term. In addition, placental EVs have been reported to increase in number and change their content in PE. Therefore, it has been proposed that they could function as biomarkers. It has been reported that aquaporin 3 (AQP3) is involved in trophoblast migration and its expression is decreased in placentas with PE. The aim of this work was to validate a method to detect AQP3 in EVs from (i) maternal plasma and (ii) supernatant of cultured placental explants to evaluate the potential utility of AQP3 as a PE biomarker.

Methods: This study was approved by the Ethics Committee of the Hospital Naval Pedro Mallo, Ciudad de Buenos Aires, Argentina. EDTA-anticoagulated maternal blood and placentas from normal and PE pregnancies were collected under informed consent. Placentas were obtained immediately and processed within one hour after cesarean section. Explants from normal and PE placentas were cultured 18 h at 37°C and supernatants were collected. Plasma and explant EVs were obtained by differential centrifugation, filtration and ultracentrifugation. Samples enriched in EVs were characterized by DLS, NTA, transmission electron microscopy and western blot to analyze the presence of CD63 and HSP70. AQP3 protein expression was also determined. Placental alkaline phosphatase (PLAP), a syncytiotrophoblast marker, was analyzed to confirm the presence of EVs of placental origin in plasma EVs samples.

Results: Preliminary results show that samples enriched in EVs were obtained and EVs of placental origin were present in plasma EVs fraction. AQP3 was detectable in both plasma and explant EVs samples.

Conclusion: This work lays the foundations to evaluate whether AQP3 is differentially expressed in placental-released EVs under normal and pathological conditions. Further studies are needed to confirm if the results obtained in placental explant cultures are reflected in maternal plasma in order to consider the potential evaluation of AQP3 as a PE biomarker.

Dr Ramin Khanabdali, Dr Scott Zhu, Dr Mathew Moore, Dr Gregory Rice

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) and their molecular profiling have been regarded as ideal candidates for biomarker developments for the earlier detection of different diseases for diagnostic applications. The current major obstacle for clinical translation is the lack of efficient, robust and high-throughput EV isolation and downstream analysis method that can be easily integrated into clinical laboratory workflows. In this study, we used a high-throughput automated bead-based immunoaffinity system to isolate EVs and Identify differentially expressed miRNAs from serum samples obtained from women diagnosed with breast cancer to identify EV-associated biomarkers for earlier detection of breast to improve disease prognosis.

Methods: EVs were isolated from 500 µL of serum obtained from women diagnosed with breast cancer (I, II, III, IV n = 12 per stage) and age matched normal healthy women (n = 48) using EXO-NET EV isolation kit on high-throughput automated KingFisher Apex system. Small RNAs were isolated on the same system using Promega Maxwell® HT miRNA Plasma and Serum kit for small RNA sequencing and RT-PCR analysis.

Results: High-throughput EVs isolation and their associated miRNAs from 96 serum samples was conducted in less than 2 hours. Small RNA seq analysis identified top 50 significantly differentially expressed miRNAs (log2 = 2, p < 0.01) between normal healthy women and women diagnosed with breast cancer. Data modelling including GO and KEGG pathway analysis identified molecular pathways associated with breast cancer and EV compartments.

Conclusion: We have established an efficient, robust, and fit-for- purpose high-throughput and automated system for EVs and their associated RNA and protein isolation on the same system for downstream analysis that holds great promise for facilitating the translation of EV diagnostics into routine clinical practice.

Graduate Student Amélie Nadeau, Graduate Student Thupten Tsering, PhD Kyle Dickinson, PhD Daniela Quail, PhD Peter Siegel, PhD Julia V Burnier

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: While primary disease is often well controlled, metastatic breast cancer (mBC) is fatal and the mechanisms underlying organotropism remain largely unknown. Extracellular vesicles (EVs) carry various biomolecules and are involved in organ-specific metastasis, but their role as biomarkers is still being explored. Our aim is to study EV-associated cargo of murine mBC cell lines that possess different metastatic potentials and organotropism to the lung, the bone, the liver, and the brain. This will allow us to identify genomic and proteomic drivers that can potentially be used as informative biomarkers of metastatic disease.

Methods: EVs were isolated from the cell culture media of metastatic 4T1 and non-metastatic 67NR parental cell lines, as well as lung-metastatic (533, 537), bone-metastatic (592, 593), liver-metastatic (2776, 2792), brain-metastatic (BP, LM) and normal (NMuMg) cell lines using centrifugal filtration followed by ultracentrifugation. The presence of EVs was confirmed and characterized by nanoparticle tracking analysis, transmission electron microscopy, and western blot. EV-DNA and cell free (cf)DNA were isolated using the DNA Purification kit and the EZ2 ccfDNA kit, respectively. Both were quantified using Qubit. Digital PCR was used to detect DNA mutations in EV-DNA and cfDNA. Proteomics analysis of EV proteins from all cell lines was conducted with label-free mass spectrometry.

Results: Varying levels of EV-DNA and cfDNA were measured in all parental and metastatic cell-derived EVs. The genomic alterations of Trp53 P31X and Kit A942S were detected in the EV-DNA and cfDNA of all cancer cells and absent from the normal cells. When compared to NMuMg, untargeted proteomics revealed that parental and mBC cell-derived EVs are enriched in several proteins that play important roles in cancer growth and metastasis, including HTRA1, PDCD6, Annexin A11, NT5E, TSPAN14, CPNE8, H2-L, H2-D1, and SH3GL1. Moreover, four variants of histones (H2AX, H2A.Z, H1.4, H1.0) were found only in site-specific cell-derived EVs and were especially enriched in brain-metastatic derived EVs.

Summary/Conclusion: We found mutated EV-DNA, cfDNA and differentially expressed protein cargo in mBC cell-derived EVs compared to normal cells. Further investigation of EV cargo will help in understanding metastatic properties in organotropism, allowing us to identify potential biomarkers for mBC.

Dr Susannah Hallal, Mr Liam Sida, Dr Agota Tűzesi, Dr Elissa Xian, Dr Daniel Madani, Dr Krishna Muralidharan, Dr Brindha Shivalingam, Associate Professor Michael Buckland, Dr Laveniya Satgunaseelan, Dr Kimberley Alexander

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Patients with glioblastoma face major obstacles to effective treatment, partly due to unreliable methods for monitoring tumour progression and recurrence. The availability of a liquid biopsy that can accurately monitor glioblastoma biomarkers from patient urine has great potential enhance patient care. The urine contains diverse extracellular vesicle populations (EV; 30-1000 nm membrane particles) which undergo alterations in tumour states and mirror tumour pathophysiological changes. As urinary-EV populations may offer clinically-relevant diagnostic endpoints for glioblastoma, we have investigated the biochemical differences between small urinary-EV (< 200 nm) and large urinary-EV (200-1000 nm) subtypes, and molecular changes associated with glioblastoma recurrence.

Methods: Urine (15-60 mL) was collected from 24 catherterised glioblastoma IDH-wildtype patients at three clinical timepoints; before (Pre-OP; n = 9) and after (Post-OP; n = 7) first surgeries, prior to recurrence surgery (REC; n = 6), and from age/gender matched healthy controls (HC; n = 13). Urinary-EVs were isolated by differential ultracentrifugation, separating large and small EVs at 17,000 xg and 100 000 xg, respectively. The size, morphology and integrity of urinary-EVs was characterised by nanoparticle tracking and cryo-electron microscopy, while molecular differences were assessed via fourier-transform infrared (FT-IR) spectroscopy. Urinary-EV proteomes were analysed with data independent acquisition mass spectrometry (DIA-MS) and aligned to a custom glioma protein library comprised of spectral coordinates for 8662-protein species.

Results: Small and large urinary-EVs had distinct FT-IR spectral profiles for regions associated with functional groups of nucleic acids, proteins and lipids. DIA-MS confidently identified 2294 and 928 proteins in at least 80% of small and large urinary-EV samples, respectively. Across the three glioblastoma timepoints, there were significant alterations in protein distribution between small and large urinary-EVs, reflecting changes associated with tumour burden (Pre-OP vs Post-OP), treatment resistance (Pre-OP vs REC) and recurrence (Post-OP vs REC). Notably, significant large urinary-EV proteins were highly accurate for glioblastoma (classification accuracy = 96.7%) and displayed consistent trends across the three clinical stages, shifting at Post-OP and returning to Pre-OP levels at REC.

Conclusions: Our findings demonstrate significant alterations between small and large urinary-EVs in glioblastoma. Putative large urinary-EV biomarkers described here merit further validation with longitudinal cohorts of glioblastoma urine.

Dr Nada Ahmed, Dr Kevin Beatson, Dr Jigisha Patel, Dr Mohammad Eddama, Dr Tarek Abdel-Aziz, Professor Lucie Clapp

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Preoperative differentiation of benign from cancerous indeterminate thyroid nodules (ITN) remains one of the most challenging issues in endocrine oncology. Most patients with an ITN will undergo diagnostic surgical resection with around 80% receiving a benign diagnosis on final histology. Liquid biopsy-based analysis of circulating extracellular vesicles (EVs) presents a promising diagnostic modality. The aims of this study were to measure the levels of circulating total and subpopulations of EVs and identifying differentially expressed EV miRNAs in patients with benign versus cancerous ITNs. Methods: For EV quantification, EVs where isolated by differential centrifugation from plasma of 28 patients with ITN (14 benign and 14 cancers on final histology) and 16 matched healthy controls (HCs). The total phosphatidylserine+ and subpopulations of cancer related EVs (CXCR7+, CD147+, SDC4+, EpCAM+) were measured in the plasma using a scatter calibrated flow cytometer with a limit of detection of ∼ 210 nm EV diameter. All controls were performed according to MiFlowCyt-EV guidelines. For EV miRNA profiling, EVs were isolated by membrane affinity spin columns, and total EV-RNA extracted from 6 HCs, 6 benign and 9 cancer patient plasma samples. miRNA sequencing was performed using DNBseq-G400 sequencing platform. Results: The concentration of the phosphatidylserine+ plasma EVs was significantly higher in patients with ITN (both cancer and benign) (p<0.0001) compared to HCs. Furthermore, the plasma concentrations of CXCR7+, CD147+, SDC4+, EpCAM+ EVs were all significantly higher in patients with ITN in comparison to HCs (p<0.0001 for all). A total of 650 miRNAs were identified. Two miRNAs (hsa-miR-195-3p, hsa-miR-619-5p) were significantly upregulated (p<0.0001) and 4 were downregulated: hsa-miR-3176, hsa-miR-205-5p, a novel miRNA: hsa-miR208-3p (p<0.0001 for all 3), and hsa-let-7i-3p (p = 0.03) in patients with malignant versus benign ITNs. KEGG pathway analysis of each of the differentially expressed miRNA targets revealed genes involved in cancer pathways. Conclusions: Circulating EVs, especially EV miRNAs, have a high diagnostic value for ITNs and may improve the diagnostic strategy for patients with ITNs.

Graduate Research Assistant Kritisha Bhandari, Laboratory Technician Jeng Shi Kong, Physician Scientist Haoyao Sun, Professor Jinchang Wu, Assistant Professor Bethany Hannafon, Professor William Dooley, Professor Wei-Qun Ding

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Metastasis-associated protein 1 (MTA1) is overexpressed in different types of human cancer and has been suggested as a cancer therapeutic target and diagnostic biomarker. We have recently reported that MTA1 is contained in small extracellular vesicles (sEVs) released from human cancer cells.

Objective: The objective of this study is to evaluate the potential of the MTA1 levels in plasma sEVs as indicators of human cancer.

Methods: Plasma from the patients with breast (n = 10) and pancreatic cancer (n = 30) were collected by the Cooperative Human Tissue Network. Nude mice implanted with the breast cancer cell line MDA-MB-231 (n = 6) were utilized to determine whether sEV-associated MTA1 is released into the circulation. A Cancer Biomarker Antibody Array was applied to plasma sEVs isolated from cancer patients, and MTA1 level in plasma sEVs was further determined using an ELISA kit. Statistical analysis was performed with the GraphPad Prism 10.

Results: Plasma sEV MTA1 level was significantly elevated in mice implanted with MDA-MB-231 cells for 6-10 weeks compared with mice without implantation, indicating the release of sEV-associated MTA1 from cancer cells to the circulation. Consistently, MTA1 level was found to be significantly higher in plasma sEVs derived from patients with breast cancer compared with matched healthy controls. The Cancer Biomarker Antibody Array showed that the level of MTA1 is three times higher in plasma sEVs derived from patients with early stage pancreatic cancer compared with that in matched healthy subjects. This was further confirmed by ELISA showing that the level of MTA1 is significantly elevated in plasma sEVs derived from patients with early stage pancreatic cancer.

Summary: We observed that the level of MTA1 in plasma sEVs is significantly elevated in nude mice implanted with MDA-MB-231 cells and in patients with breast and pancreatic cancer. These observations indicate that sEV-associated MTA1 is released from tumor cells to the circulation and that plasma sEV MTA1 may serve as a biomarker indicative of breast and pancreatic cancer.

Mr Mahmoud Hamed, Dr Valerie Wasinger, Mr Qi Wang, Associate Professor Peter Graham, Dr David Malouf, Dr Joseph Bucci, Professor Yong Li

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Background

Current prostate cancer (PCa) diagnosis tools such as serum prostatic-specific antigens (PSA) test, magnetic resonance imaging and tissue biopsy are not specific and inaccurate. Extracellular vesicles (EVs) are nano-sized vesicles and secreted by all living cells into the extracellular milieu and contain cellular components encapsulated by lipid membranes. Liquid biopsy is a fast-growing research area and can provide an alternative solution that is less invasive and more reliable. Advances in metabolomics show a comprehensive analysis of EVs’ cargo could lead to the discovery of more accurate biomarkers. In this study, we hypothesise that EVs’ metabolic profiles are different between PCa cell lines and normal prostate cell line or between small EVs (sEVs) and large EVs (lEVs). Our objective is to isolate sEVs and lEVs from a panel of PCa cell lines and a normal prostate cell line to identify differences in key metabolites using global approaches.

Methods

Using ultracentrifugation (UC), EVs were isolated from different PCa cell lines (PC3, DU145, LNCaP and 22RV1) and a normal prostate epithelial cell line (PNT2). Isolated EVs were characterised into lEVs and sEVs. Confirmation tests including nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blotting (WB) were conducted to check the characteristics of isolated EVs. Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) is employed to investigate the metabolomic profiles of EVs isolated from different cell lines.

Results

The characterisation of both sEVs and lEVs is confirmed by NTA, TEM and WB. We found that EVs concentration of at least 108 vesicles is required to achieve reliable metabolomics outcomes. We have preliminarily identified differences in sEVs and lEVs metabolite content between PCa cell lines and a control cell line.

Conclusion

Our established protocol in EVs isolation using UC is confirmed to yield EVs with high purity and quality for metabolomic analysis. We have established the metabolomic analysis in EVs using PC3 cell line and a control cell line. In our following study, additional experiments will be run to test more PCa cell lines and normal control prostate cell line and further validate metabolite candidates identified for PCa diagnosis and risk stratification.

Young-Hye Seo, Sung-Kyung Bong, Beomhee Ahn, Hanna Kim, Hwanghee Ryu, Myunghee Jang, Ph.D Seung-Hak Choi, Ph.D Vijaya Sunkara, Juhee Park, Ph.D Yoon-Kyoung Cho, Ph.D Kyusang Lee, Ph.D Beomseok Lee

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Lung cancer, a leading cause of cancer mortality, finally improved overall survival thanks to targeted therapies in recent years. This relies on identifying somatic mutations in genes such as EGFR, through tissue biopsies, which is not feasible to early stage patients who will benefit the most. Thus, liquid biopsy, utilizing blood samples, has been gaining interest, and extracellular vesicles (EVs) emerged promising improved sensitivity over cell-free DNA.

This study explores the utility of large-volume clinical samples (e.g., plasma exceeding 3 mL and urine reaching 30 mL) for detecting EGFR mutations at low concentration in early-stage non-small-cell lung cancer (NSCLC) patients. We focus on RNA-based EGFR mutation detection qPCR assays using both plasma and urine-derived EVs.

The enrichment of extracellular vesicles from sizable plasma and urine volumes preserves scarce nucleic acid in the liquid samples. We employed a quick and high yield EV concentration method that uses ionic strength modulation technique (ExoPRISM) for selective EV precipitation followed by high performance TFF (tangential flow filtration) on a lab-on-a-disc. The low-molecular-weight electrolytes used for immediate EV aggregation are washed on the lab-on-a-disc along with soluble proteins.

For validation, we obtained EVs from the cell culture of H1975 and HCC827 cell lines which have EGFR genes with specific mutations (L858R, T790M, Exon19del). The EVs were spiked into plasma and urine samples from normal human donors for EV enrichment and RNA preparation. The RNA samples undergo reverse transcription, pre-amplification of cDNA, and multiplex quantitative real-time PCR to determine the limit of detection (LOD), which is consistently below 30 copies (less than 0.01%) for all mutations.

The ongoing clinical assessment of EGFR mutation detection sensitivity across 50 plasma and urine specimens from individual non-small cell lung cancer patients will establish the clinical potential of this method. Due to the higher concentration of RNAs with mutations than DNA counterparts in the sample, our assay will be more useful for early stage detection, as well as treatment monitoring. The comparative analyses of plasma, urine, and tissue samples aim to provide improved insights into precise management, encompassing treatment response assessment, and minimal residual disease in lung cancer patients.

Zi Huai Chew, Senior Research Scientist Christelle Chua

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

EVs are believed to be involved in cell-to-cell communication after exposure to ionising radiation (IR), but while PBMCs are known to be particularly sensitive to IR,it is only lately that researchers have begun to characterise PBMC-derived EVs Previous studies focused mainly on the impact of EV cargo after high doses of radiation typically applied for radiotherapy, and frequently at later time points of >24h.We sought to explore the impact of TBI on EVs profiles derived from plasma of cancer patients approximately 24h after undergoing their first fraction of TBI treatment (2Gy),and compare this with EVs derived from PBMCs after 2Gy ex vivo irradiation as it is not well understood.

(1) Whole blood or PBMCs collected from healthy donors were exposed to X-ray irradiation and cultured in plasma for 24 hours.(2) Plasma was collected from cancer patients before and 24 hours after their first 2Gy fraction of TBI.EVs were isolated using size-exclusion chromatography and analysed by interferometric imaging.

The overall quantity of EVs does not appear to increase significantly, 24 hours after exposure for (1) and (2).This is contrary to expectations that EV populations always increases in response to IR,and suggests that at earlier time points this may be otherwise.We observed that CD9 is generally highly expressed in EVs derived from PBMCs of healthy donors and from plasma of cancer patients. However,while CD81 expression is also highly expressed for EVs derived from plasma of cancer patients before and after exposure,this was not the case for EVs derived from PBMCs of healthy donors,in agreement with other published data reported.CD63 expression increases with exposure for EVs derived from PBMCs of healthy donors while expression of all 3 tetraspanin markers (CD9,CD63,CD81) decreases with exposure for EVs derived from plasma of cancer patients.

Our results suggest that in vivo (TBI for radiotherapy) compared to ex vivo (IR of whole blood) exposure of ionizing radiation have differing extent of effects on the quantity of EVs and expression of various tetraspanin markers.Hence,care must be applied when interpreting results derived from in vivo experiments and comparing it into ex vivo experiments.

Ms Lauren Newman, Dr Zivile Useckaite, Associate Professor Andrew Rowland

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: An individual's exposure and response to a drug is determined by the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of the drug. Critically, between subject differences in abundance of drug metabolizing enzymes (DMEs) and drug target proteins underpin variability in PK and PD and impact treatment efficacy and tolerability. Differences may result from normal physiology, genetics, environment or chronic liver disease including metabolic associated fatty liver disease (MAFLD), but have historically been challenging to define on a molecular level in vivo. This study sought to (i) establish the fundamental capacity of liver derived EVs to define between-subject variability in abundance of DME proteins and MAFLD drug targets and (ii) explore the concordance between absolute abundance in tissue and tissue derived EVs.

Methods: EVs were recovered from human liver tissue samples (LT-EVs, n = 11) using our reported protocol of enzymatic digestion followed by size exclusion chromatography. The presence of EVs in the extracellular space of hepatic cells was observed by TEM. EVs were subject to particle analysis by NTA and targeted liquid chromatography mass spectrometry (LC-MS) assays to assess EV markers and contaminants and quantify a panel of DMEs and drug targets for MAFLD.

Results: LT-EVs were enriched in CD81, CD9 and TSG101 and significantly depleted of albumin relative to whole tissue. Of the 26 proteins evaluated in the DME and MAFLD panel, 23 (88%) were detected and quantified in LT-EV. Moderate-strong positive correlations (Pearson r > 0.6) between tissue and LT-EVs were observed for 15 of 23 targets, with greater concordance for proteins with reported liver specificity or enrichment relative to other tissues.

Conclusions: This study developed a workflow to robustly quantify proteins in LT-EVs associated with interactions of drugs with the liver. Accounting for possible contribution from extra-hepatic EVs in the tissue, LT-EVs could report on between-subject variability in tissue protein abundance. Further work in liver-derived plasma EVs will address the intriguing potential to track markers of drug exposure and treatment response in vivo by liquid biopsy.

Ms Priya Kumari Gorai, Ms Simran Rastogi, Dr Surabhi AS, Dr Seema Singh, Dr Shipra Agarwal, Dr Sujoy Pal, Dr Tapas Chandra Nag, Prof Renu Dhingra, Prof Mehar Chand Sharma, Prof Rakesh Kumar, Dr Saroj Kumar, Dr Neerja Rani

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

The five-year survival rate of rare Pancreatic Neuroendocrine Tumors (PanNET) is the lowest among neuroendocrine tumors because initial detection is being done at late stages due to the unavailability of appropriate diagnostic markers; therefore, there is an urgent need for an early-stage biomarker for PanNETs. The small extracellular vesicles (sEV) play a key role in tumor growth and metastasis, owing to their remarkable capacity to induce metastatic behavior and proliferation. For the first time, as proof of concept, this study pioneers the investigation of the relationship between sEV concentration and PanNET grades, as well as the presence of BIRC2/cIAP and the autophagy marker Beclin-1 as cargo within sEVs derived from plasma. By examining plasma-derived small extracellular vesicles, this research sheds new light on the potential role of these vesicles as diagnostic biomarkers for PanNETs, offering a promising avenue for early detection and improved patient outcomes.

Materials and methods:

Plasma-derived sEVs were examined via TEM to characterize their morphology and western blot of CD63 and TSG101 confirmed the presence of sEVs, with calnexin serving as the negative control. Nano Tracking Analysis was employed to quantify their abundance and size. Western blot analysis was used to probe the presence of BIRC2 and Beclin-1 in sEVs. Additionally, IHC assessed the expression of BIRC2 and Beclin-1 in both PanNET and control tissues.

Results:

This study presents compelling evidence of elevated plasma secretion of sEVs in PanNETs (Grade I & II) individuals compared to healthy controls (HCs) (p<0.0001) with a sensitivity of 100%. Moreover, higher protein expression levels of cancer marker BIRC2/cIAP1 (p = 0.002) and autophagy marker Beclin-1 (p = 0.02) were observed in PanNETs compared to HCs. Notably, the immunohistochemistry (IHC) analysis of PanNET tissue revealed a parallel expression pattern for both proteins.

Conclusion:

The findings unveil a potential correlation between elevated plasma secretion of sEVs and PanNET pathogenesis. Heightened expression of BIRC2 and Beclin-1 proteins further highlights their potential as important PanNET biomarkers. This study provides valuable insights into PanNET biology, paving the way for new diagnostic and therapeutic approaches.

Ms Lidia Medhin, Doctor Lydia Warburton, Professor Benhur Amanuel, Doctor Leslie Beasley, Professor Elin Gray

Poster Pitches (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:45 PM - 1:00 PM

Introduction: Melanoma's aggressiveness underscores the need for a reliable and accessible biomarker. Our preliminary analysis identified a circulating extracellular vesicle-derived gene signature (cEVsig) from plasma and linked to response to immune checkpoint inhibitors. This study aims to validate the predictive role of the EV signature in an independent cohort of melanoma and NSCLC patients undergoing immunotherapy.

Methods: Samples were collected before treatment and EVs were isolated from plasma. EV-RNA was extracted for qRT-PCR analysis of a 21-gene set signature (previously identified). To evaluate differences in gene expression between responders and non-responders to treatment, Mann-Whitney U-test was conducted. Logistic regression models were assessed including all 21 genes or a subset thereof, and their Receiver Operating Characteristic- Area Under the Curve (ROC-AUC) values were determined. Cox proportional hazard models and log-rank tests were used to assess the predictive value of the gene signature on patient survival.

Results: We analyzed a total of 50 eligible metastatic melanoma patient samples, comprising 37 patients commencing treatment with ipilimumab/nivolumab, 11 pembrolizumab, and 2 nivolumab. Notably, there was a significant difference in gene expression between responders and non-responders using Mann-Whitney U-test analysis. Our predictive model exhibited strong performance with a model quality score of 0.82, AUC of 0.902, sensitivity of 71%, specificity of 93%, PPV of 96%, and NPV of 58%. Survival analysis indicated that the presence of the gene signature was linked to improved PFS (HR:0.13 (95%CI: 0.04-0.39), p = 0.0019) and OS (HR:0.16 (95%CI: 0.05-0.55), p = 0.0093). Analysis of a cohort of 54 NSCLC treated with pembrolizumab monotherapy is ongoing to ascertain the biomarker's predictive potential across diverse cancers.

Conclusion: Our validation study underscores the predictive potential of the identified EV-derived RNA signature in categorizing melanoma patients as responders or non-responders to immune checkpoint inhibitors. Determining the likelihood of response before commencing therapy can enable personalizing treatment selection, minimizing unnecessary toxicity and expediting patients' alternative therapies and participation in clinical trials.

Disclosure note: The Abstract outlines EV-derived genes with pending patent, restricting disclosure of their specific names.

Samantha Upson, Dr. Sabrina LaSalvia, Eric Trillaud, Dr. Emily Heiston, Nathan Stewart, Dr. Steven Malin, Dr. Uta Erdbrügger

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Hypertension (HTN) is a leading modifiable risk factor for cardiovascular disease. Although plasma extracellular vesicles (EVs) have been investigated for diagnostic purposes, limitations in methodology have raised uncertainty for EV's role in HTN. Therefore, we utilized advanced EV flow cytometry to characterize a range of EVs in patients across HTN stages as well as analyze their relation to exercise blood pressure responses.

Using a cross-sectional design, adults with obesity (n = 62, 83% F, 53.2±1.2yr, BMI:36.0±0.7kg/m2, VO2max: 22.0±0.5mg/kg/min) underwent clinical testing. HTN was defined based on systolic blood pressure (SBP): Normotensive (n = 12, 92% F, <120mmHg), Elevated (n = 15, 80% F, 120-129mmHg), and HTN (n = 35, 80% F, >129mmHg). Blood work was done after an overnight fast to assess tetraspanin EVs+, platelet (CD41+), endothelial (CD105, CD31+/CD41-), platelet endothelial cell adhesion molecule (CD31), and leukocyte derived EVs (CD45+) using nanoflow cytometry (Northern Light, Cytek) following MyFlowCyt guidelines. A subgroup of 34 individuals underwent a VO2max (maximal oxygen consumption aerobic fitness) test at self-selected speeds and treadmill incline rising every 2 minutes until exhaustion to assess SBP responses (VO2max-0min, ΔSBP). Individuals were characterized as having normo- (n = 12, 92% F) and hyper-tensive (n = 22, 73% F; men: >60 and women: >50mmHg) ΔSBP responses to exercise.

There were no group differences in age, BMI, or VO2max (all p>0.05). However, fasting total EV+ counts varied between BP groups (p = 0.0037). EVs were elevated in people with HTN compared to those with elevated (p = 0.015) and normotensive (p = 0.022) SBP. CD105+ EVs varied among BP groups (p = 0.0144) and were higher in those with HTN compared to with Elevated SBP(p = 0.042). As expected, ΔSBP to exercise was greater in those with hypertensive compared to normotensive (75.0±19.9 vs. 43.8±5.7mmHg, p<0.001) responses. Interestingly, platelet and endothelial EVs+ only correlated with ΔSBP for those with hypertensive (r = 0.53, r = 0.56, r = 0.68 and r = 0.50 respectively, all p<0.02), but not normotensive (r = 0.00, r = 0.01, r = 0.01 and r = 0.20, all p>0.05) responses.

EVs were related to HTN status, exercise, and blood pressure responses. While this work supports EVs as possible diagnostic markers of vascular health, additional work in plasma and urine is warranted to determine how EVs impact vascular physiology for optimization of chronic disease risk reduction.

Malene Joergensen, Anders Askeland, Rikke Bæk, Charlotte Sten, Rikke Wehner Rasmussen, Morten Hjuler Nielsen, Nahuel Garcia, Maiken Mellergaard, Aase Handberg

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction:

Non-alcoholic fatty liver disease (NAFLD) is a common obesity-related metabolic disorder lacking non-invasive diagnostic biomarkers. Extracellular vesicles (EVs) have emerged as potential sources of tissue- and disease-specific biomarkers, for which EV Array (Jørgensen et al. 2013, JEV) provides a high-throughput method for screening of candidate biomarkers. Here, we present preliminary findings for identifying EV-based biomarkers for NAFLD utilizing the EV Array platform.

2) Methods:

Plasma was collected from obese individuals with NAFLD and the metabolic syndrome (obese NAFLD-MetS; n = 33), obese without these conditions (obese controls; n = 28), and lean controls (n = 35). Obese individuals with NAFLD-MetS were followed and samples prepared during weight loss intervention (baseline, visit 1 (1 month), visit 2 (5-6 month).

Microarray technology (EV Array) was used to screen the plasma samples for their content of small EVs against a panel of 90 surface- or surface-associated markers. The investigated markers can be categorized as general EV markers, tissue-specific markers (liver), fatty acid transporters, chemokine receptors (CXCR), Notch receptors, inflammatory (TNF), and immune markers (Garcia et al., 2023, Int. J. Mol).

3) Result:

Data from the EV Array analysis was analyzed both in univariate and multivariate manners and in comparison with Proton Density Fat Fraction (PDFF) obtained by MRI scanning as well as several other clinical and metabolic characteristics (Askeland et al, submitted). Comparing the three groups at baseline showed that the EV phenotypes can indeed distinguish the groups. In addition, several markers showed tendency to decrease during weight loss in the obese NAFLD-MetS group. Specific results are provided to the extent it is allowed according to a non-disclosure agreement as a biomarker patent is pending.

4) Summary/Conclusion:

Our findings suggest that EV Array analysis could facilitate novel NAFLD biomarker discovery. Several markers demonstrated potential as biomarkers for NAFLD progression. However, analysis of the full dataset and subsequent validation are required to confirm the clinical utility of EVs as non-invasive biomarkers to screen for NAFLD.

Dr Edina Gyukity-Sebestyen, Gabriella Dobra, Matyas Bukva, Dr Maria Harmati, Timea Boroczky, Dr Szabolcs Nyiraty, Dr Barbara Bordács, Dr Margareta Korsos, Dr Zoltan Szabo, Dr Gabor Kecskemeti, Prof. Dr Tamas Varkonyi, Prof. Dr Zoltan Konya, Prof. Dr Marta Szell, Dr Peter Horvath, Dr Krisztina Buzás

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: The post-COVID-19 condition (also known as long COVID) refers to long-term symptoms that some people experience more than 12 weeks after recovering from COVID-19. These symptoms may persist from the onset of their initial illness, or may develop after their recovery, and may disappear over time and then recur. We hypothesized that the molecular pattern of small extracellular vesicles (sEV) found in the plasma of patients during acute SARS-CoV-2 infection could predict the course of COVID-19 disease, the risk of developing post-COVID syndrome, and its symptoms.

Methods: We conducted a detailed investigation involving 60 volunteers, from whom we isolated - by size exclusion chromatography - small extracellular vesicles (sEVs) from serum samples collected during their acute phase of COVID-19 infection. Based on the symptoms occurred after COVID-19, we perform hierarchical clustering classifying the patients into 3 groups. Subsequent analyses are carried out on the 3 groups: i) a group exhibiting a wide range of symptoms in a cumulative manner, ii) a group manifesting a limited set of symptoms, and iii) a group showing no residual symptoms post-infection. Following a characterization of sEV samples, we employed advanced Raman spectroscopic measurement, liquid chromatography–mass spectrometry (LC-MS) analysis and enzyme–linked immunosorbent assay (ELISA) to examine their properties.

Results: The Raman spectra of the 3 patients group showed different characteristics allowing us to draw predictions over the potential complications. According to a proteomic analysis of sEVs, more than 25 proteins were significantly enriched in the two patient groups exhibiting post-COVID symptoms compared to the symptom-free control group. These proteins include some members of the complement system, such as C2, C4, C5, C1 inhibitors.

Conlusion: As the protein composition of sEVs isolated from serum collected during acute SARS-CoV-2 infection shows a significant difference between patients exhibiting post-COVID symptoms and those who are asymptomatic, plasma EV analysis might be suitable for forecasting the post-COVID syndrome and may help the patient care.

Dr Susannah Hallal, Dr Agota Tuzesi, Mr Liam Sida, Dr Elissa Xian, Dr Daniel Madani, Dr Krishna Muralidharan, Associate Professor Brindha Shivalingam, Associate Professor Michael Buckland, Dr Laveniya Satgunaseelan, Dr Kimberley Alexander

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Readily accessible biomarkers that reflect tumour activity and treatment response are urgently needed to improve the clinical management of the aggressive primary brain tumour glioblastoma, especially for recurrent cases. Given that the urinary system is a major clearance route for circulating extracellular vesicles (EVs; 30-1000 nm membranous particles), we investigated whether sampling urinary-EVs could offer a reliable non-invasive liquid biopsy strategy for diagnosing and monitoring glioblastoma.

Methods: Fifty urine specimens (15-60 mL) were collected from 24 catheterised glioblastoma IDH-wildtype patients at three clinical timepoints; before (Pre-OP, n = 17) and after (Post-OP, n = 9) gross-total-resection of a de novo glioblastoma tumour, and prior to recurrence surgery (REC, n = 7). We also collected urine samples from age/gender-matched healthy controls (HC, n = 14). The urinary-EVs were isolated by differential ultracentrifugation, and characterised by nanoparticle tracking analysis and cryo-transmission electron microscopy. The urinary-EV proteomes were analysed by high-resolution data-independent acquisition mass spectrometry (DIA-MS), and the data was extracted by alignment to our custom glioma protein library comprised of 8662-protein species.

Results: Overall, 6857 proteins were confidently identified in urinary-EVs (q-value≤0.01). A stepwise logistic regression identified five urinary-EV biomarker proteins with significantly elevated levels in Pre-OP compared to HCs, with an excellent cumulative diagnostic performance of 95.8% (AUC = 0.958). Strikingly, urinary-EV protein levels effectively distinguished glioblastoma patients at the three clinical stages (FC≥|2|, adj.p-val≤0.05, AUC>0.9). Many significant urinary-EV proteins aligned with previously defined EV biomarkers from GBM cell culture, neurosurgical fluids and plasma, and showed consistent trends across the three clinical timepoints, altering at Post-OP and reverting to Pre-OP levels at REC. Notably, we identified three urinary-EV proteins, GGH, GRN and ITM2B, with excellent sensitivity and specificity for glioblastoma recurrence (AUC>0.92), and known links to glioblastoma progression and/or treatment-resistance.

Summary/Conclusion: Comprehensive DIA-MS characterisation confirms the urine as a viable source of EV-associated biomarkers for glioblastoma diagnostics and monitoring. The urinary-EV biomarkers discovered here warrant further investigation using large longitudinal cohorts of glioblastoma urine specimens.

Lifang Yang, Benjamin Johnson, Caleb Smack, Professor Eric Feliberti

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction: Breast cancer (BC) is a highly heterogenous disease with many subtypes that differ in clinical behavior. However, current tissue-based diagnostic routines are simplification of the inherent biology of BC and provide limited information for personalized diagnosis. Extracellular vesicles (EVs) have been recognized as a mode of intercellular communication. Like their parent cell, EVs are heavily glycosylated. Glycoconjugates of EVs has been shown to play a role in EV protein sorting, cell targeting, and recognition. As aberrant glycosylation is a hallmark of cancer cells, little is known about the molecular basis of glycosylated EV cargos as well as their presentiveness of known cancer characteristics. Here, we characterized the EV glycoproteins released by different BC cell lines to define subtype-specific signatures.

2) Methods: Breast cell lines representing normal breast cells and 4 BC subtypes (luminal A, luminal B, HER2 enriched, and triple negative) were cultured in appropriate cell media. Small extracellular vesicles (sEVs) were isolated via a differential ultracentrifugation approach. The morphology and size of isolated sEVs were determined by transmission Electron Microscopy and NanoSight. The sEV purity was assessed by Western blotting with a panel of markers. Lectin blots were performed to examine the glycosylation patterns of specific carbohydrate moieties in the protein lysates from sEVs relative to their parent cells. In addition, metabolic labeling and click chemistry were employed to further characterize cellular and sEV surface sialoglycoproteins.

3) Results: Collected particles with nanoscale size (50–150 nm) harbored membrane-encapsulated vesicular structure and presented typical small EV markers. Lectin blot analysis showed distinct glycosylation patterns in various BC subtypes. Comparison of the glycosignatures of sEVs with their parent cells revealed both enrichment and depletion of specific glycosproteins in these vesicles. Furthermore, abundant sialylated glycoproteins were detected on the surface of cells and sEVs. Sialylation patterns and levels on the sEV surface, which reflect the cell of origin, were correlated with the aggressiveness of BC subtypes.

4) Conclusions: sEVs from BC cells display specific glycosignatures and are enriched in subtype-associated sialoglycoproteins on their surface. The identification of these glycoproteins could provide novel biomarkers to improve BC stratification and diagnosis.

Dr Ramin Khanabdali, Dr Carlos Palma, Miss Siena Barton, Professor Greg Rice

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Despite the growing number of disease-associated extracellular vesicle (EV) biomarkers being identified, the translation of EV diagnostics into routine clinical laboratory tests remains limited. To effectively leverage EVs as diagnostics requires identification of disease-associated biomarkers in specific EV subpopulations; and rapid, reproducible, and scalable sample processing. Here, we have developed a high-throughput bead-based immunoaffinity system that captures a highly enriched subpopulation of EVs to Identify biomolecular profile of any biofluids including plasma, serum, urine and saliva samples for biomarker discovery and clinical translation for diagnostic applications.

Methods: EVs were isolated from 500 µL of plasma obtained from women diagnosed with ovarian cancer (I, II, III n = 10 each stage) and age matched normal healthy women (n = 30) using EXO-NET EV isolation kit on high-throughput automated KingFisher system. Following EXO-NET EV isolation, small RNAs were isolated on the same system using Promega Maxwell® HT miRNA Plasma and Serum kit for microRNAs (miRNAs) sequencing and RT-PCR analysis.

Results: High-throughput EXO-NET EVs isolation and their associated miRNAs from 60 clinical plasma samples was performed on the KingFisher system in less than 2 hours. Small RNA sequencing analysis identified top 27 significantly differentially expressed miRNAs (log2 = 2, p <0.01) between normal healthy women and women diagnosed with ovarian cancer across different stages. Data modelling identified top GO molecular pathways associated with ovarian cancer and EV compartments.

Conclusion: We have established an efficient, robust, and fit-for-purpose high-throughput and automated system for EVs and their associated RNA and protein isolation for downstream analysis which holds great promise for facilitating the integration of EV diagnostics into routine clinical practice.

Mr Qi Wang, Dr Bairen Pang, Dr Cheng Zhou, Dr Meng Han, Jie Ni, David Malouf, Joseph Bucci, Peter Graham, Tiannan Guo, Junhui Jiang, Yong Li

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1. Introduction

Prostate cancer (PCa) is the second leading cause of death in men in Australia. Current diagnostic tool blood prostate-specific antigen test is insufficient in PCa diagnosis and risk stratification. There is an unmet need to develop novel approaches for more cost-effective and accurate biomarkers for PCa early diagnosis and treatment stratification to enable personalised medicine. PCa-derived extracellular vesicles have garnered attention in recent years due to their important role in communications in tumour microenvironment as well as in invasion, progression, metastasis, therapeutic resistance, and immune escape. Our objective in this study was to identify different protein profiles in four different extracellular vesicles and particles (EVPs) subpopulations, including large extracellular vesicles, small extracellular vesicles, exomeres and supermeres, from a panel of PCa cell lines and PCa patients’ plasma using proteomic analysis for PCa diagnosis and progression risk stratification.

2. Methods

PC3, DU145, LNCaP and 22Rv1 PCa cell lines as well as a normal prostate epithelial cell line RWPE-1 were cultured, and the supernatants were collected for EVPs isolation. PCa patients were selected based on a series of criteria and divided into four groups (control, low-risk, intermediate-risk, high-risk, metastasis, n = 3 in each group) following the NCCN guideline 2023. All EVPs were isolated by ultracentrifugation and then characterised by transmission electron microscopy, nanoparticle tracking analysis and western blotting, atomic force microscopy, zeta potential analyzer and nano-flowcytometry. LC-MS/MS proteomics and bioinformation analysis was applied for the pathway analysis of PCa derived EVPs proteins.

3. Results

A series of gradient ultracentrifugation was applied for isolating PCa derived EVPs. The EVPs isolated were characterised by morphology, size, classical protein biomarkers and some new physical parameters for confirmation. A group of potential EVP proteins were identified and need to be further validated in PCa cell lines and an independent set of PCa clinical samples.

4. Summary/Conclusion

We have successfully established a reliable and reproducible method for isolating distinct EVP populations from PCa cells and human plasma samples and comprehensively characterised their properties. Investigation of the details of protein profiles from EVPs is ongoing and holds promise for PCa early diagnosis, risk stratification and metastasis monitoring.

Miss Debra Lennox, Dr Caitlin Boyne, Dr Sally Shirran, Dr Simon Powis

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

As cancer cells produce extracellular vesicles (EV) more abundantly than normal cells it is possible that EVs from solid tumours could be detected in the patient bloodstream, which has already been shown in breast cancer and melanoma. Characterisation of the peptides from HLA-I molecules present on the surface of these EVs could provide information on treatment efficacy and gain more insight into the molecular mechanism and more accurate prognostic indicators in a minimally invasive procedure compared to biopsy. Within the scope of EV immunopeptidome derived for lung cancer (cell line or patient blood) we will seek the presence of tumour associated and tumour specific antigens, which can be the target for immune attack of cancer cells.

Methods

Isolation of HLA-I molecules from EVs via size exclusion chromatography and immunoprecipitation before eluting bound peptides and performing LC/MS/MS as described by Synowsky et al., 2017. Then bioinformatic analysis through various software programmes including but not limited to the TAA database and Pather.

Results

So far, we have determined from 4 different cells lines that we can detect Tumour Associated Antigens (TAAs) from the peptides found on EVs and that the is an overlap in what is presented. 7 distinct peptides and many TAAs such as NCOR, EEF2, SON were found in more than one cell line and had similar prominent pathways detected. We have also began the analysis on 17 patient blood samples which will be compared to donor blood and is currently suggesting similar TAAs.

Summary/Conclusion

EV released by lung cancer cells do indeed have HLA-I molecules on the surface for antigen presentation that is detectable in both cell models and in patient blood samples. The HLA-I bound peptides present relevant TAAs can also give some indication of the molecular processes likely occurring in the cells of origin in both cell lines and patient samples which with further investigation could allude to possible future biomarkers for diagnosis/prognosis the possibility of a non-invasive method for determining prognosis in the future.

Ph.D. Student Haruka Sei, M.S. Naoya Hirade, Ph.D. Fumie Nakashima, Ph.D. Takahiro Shibata

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Diabetic nephropathy (DN) is one of the complications of diabetes and is the main cause of dialysis. In this study, we attempted to discover the candidate(s) of the DN markers from urinary extracellular vesicles (uEVs).

Method: The uEVs isolated from type 2 diabetic (T2D) model rats were subjected to proteomic analysis. We also investigated the effects of calorie restriction (CR) on the levels of a potential marker candidate in uEVs from T2D model rats since it was reported that calorie restriction effectively attenuated the promotion of diabetes in the T2D model rats. The candidate expression in the renal cortex was assessed by Western blotting and real-time PCR. To identify the origin of uEVs containing the candidate, we focused on the differences in the renal cell surface sugar chains between renal segments. We performed lectin affinity enrichment of uEVs and immunohistochemical staining.

Results: Proteomic analysis revealed that isocitrate dehydrogenase 1 (IDH1) in uEVs from T2D model rats was significantly upregulated compared to control rats. The upregulation was significantly suppressed by CR. Western blotting and real-time PCR revealed that IDH1 was significantly upregulated in the renal cortex of T2D model rats. Using immunohistochemical staining, we found that IDH1 was highly expressed in proximal tubular cells of the renal cortex and collecting duct cells of the inner medulla. To confirm that IDH1-containing uEVs came from proximal tubular cells, we developed the lectin enrichment method using biotinylated Lentil culinaris lectin (LCA). Biotinylated LCA enabled to enrich IDH1-containing uEVs and proximal tubular cells-derived uEVs. Furthermore, immunohistochemical staining showed that IDH1-positive cells and LCA-positive cells were colocalized in the renal cortex.

Summary/Conclusion: IDH1 was significantly upregulated in uEVs from T2D model rats. The upregulation was suppressed by CR. We also identified proximal tubular cells as the main host cells of IDH1-containing uEVs. These findings indicate that IDH1 might be one of the candidates of the DN markers in uEVs.

Edward Stephens, Dr Tian Mun Chee, Mr Vihanga Dharmasena, Professor Kwun Fong, Professor Ian Yang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Lung cancer is the leading cause of cancer death worldwide, largely due to late-stage diagnoses. Biomarkers of disease detectable using minimally invasive methods, such as blood tests, could improve lung cancer detection and screening capabilities. Extracellular vesicles (EVs) have emerged as leading candidates for biomarker studies as EVs contain clinically relevant bioactive cargo that bears notable resemblance to their parent cells, and therefore make them attractive targets for cancer biomarker studies. In this study, we aimed to isolate and characterise EVs derived from tumour and non-tumour lung tissue to demonstrate their utility as a novel bioresource for an EV-specific signature of lung cancer tumour biomarkers.

Methods: Thirty milligrams of frozen lung tissue was incubated in a collagenase/dispase-based digestion buffer for one hour at 37°C and then filtered through a 40µm cell strainer. Digested tissue was then centrifuged at low speeds to remove cells, large vesicles and other debris, followed by ultracentrifugation for 100 minutes at 100,000xg at 4°C. The resulting EV pellet was then purified using size exclusion chromatography, and concentrated further using ultrafiltration. Prior to ultrafiltration, a small volume of sample was reserved for EV characterisation using the IZON Exoid instrument to measure size and concentration, western blotting to qualitatively assess the EVs present, and electron microscopy to visualise the EVs. RNA and DNA were extracted from EVs using QIAGEN AllPrep Mini kits. The resulting RNA and DNA fractions were quantified using a Qubit 4 Fluorometer and Agilent Tapestation.

Results: EV concentration and size analyses revealed an average particle diameter of 121nm and a concentration of 4.59x10¹⁰ particles/mL. Western blotting indicated the presence of EVs with positive staining of CD9 and FLOT1 antibodies. Preliminary concentration analyses of EV-extracted DNA and RNA yielded DNA concentrations of 37.4ng/µL and 39.4ng/µL, and RNA concentrations of 2.9ng/µL and 8.5ng/µL for tumour and non-tumour tissues, respectively.

Conclusion: Here, we demonstrate a lung tissue digestion technique to isolate EVs from tumour and non-tumour lung tissues. Ample concentrations of DNA and RNA were obtained from lung tissue EVs, enabling downstream sequencing experiments to interrogate EV-specific signatures of lung cancer tumour biomarkers.

MS Trevor Enright, PhD Kai Tao, PhD Sinan Sabuncu, PhD Emek Demir, MD Mark Garzotto, BS Randall Armstrong, PhD Michelle Gomes

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Levels of Prostate-specific antigen (PSA) in the blood used to screen patients for prostate cancer, but lacks specificity. Of the patients referred for diagnostic biopsy a third are cancer-negative. Minimally invasive fluid-based biomarkers distinguishing between low-risk, high-risk, or benign prostatic conditions are needed to reduce the incidence of unnecessary biopsies. Tumor-derived extracellular vesicles (EVs) are a fingerprint of biomarkers on cancer cells. Since cancer cells exhibit glycosylation aberrancies, we hypothesize that the glycan landscape of prostate-derived EVs from expressed prostatic fluid (DRE-urine) may provide unique markers for early detection and disease progression.

Methods: EVs were enriched from DRE-urine samples of low-risk (Gleason <7; n = 25), high-risk (Gleason >7; n = 25) prostate cancer patients and benign controls (n = 25) using density gradient ultracentrifugation and characterized by Nanoparticle Tracking Analysis (NTA), and Transmission Electron Microscopy (TEM). Immunoblotting confirmed EV markers. To test our hypothesis, we profiled and compared the glycosylation patterns of prostate-derived DRE urine-enriched EVs from prostate cancer patients and controls by multi-parametric flow cytometry using panels of fluorescently conjugated lectins and an antibody to the prostate cancer marker, PSMA.

Results: All 75 patient samples had >1x 10¹⁰ EV particles. Patient EVs were in the typical size ranges of 50-150nm and showed characteristic cup-shaped morphology. Computational analysis of lectin/antibody intensities on individual prostate-derived EVs showed glycan patterns associated with prostate cancer. Using glycan biomarkers in a Random Forest Classifier, we could classify patients into the relevant cohort with high sensitivity and specificity. Furthermore, Shapley Analysis quantifies the contribution of each glycan biomarker and shows distinct glycan signatures for prostate cancer patients and controls.

Conclusion: Taken together, our data indicates that EV glycan signatures can distinguish high-risk, and low-risk prostate cancer patients from benign/screen-negative disease status suggesting EV glycosylation is a promising biomarker for prostate cancer detection.

Limited Information Disclosure: The lectin panels are retained by OHSU as a patent candidate, preventing detailed disclosure of the lectin and glycan signature identities.

Ethical Statement: Human subject samples were collected from consenting individuals for the CEDAR Biorepository under OHSU and VA clinic Institutional Review boards.

(IRB#18048, VAIRB#4214)

Funding: CEDAR- Full 2020-1251, Full 2023-1688

Dr Alicja Głuszko, dr. hab. Mirosław Szczepański, dr. Andrzej Ciechanowicz, Prof. Theresa Whiteside, dr. Nils Ludwig

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Hypoxia, a hallmark of solid tumors, including head and neck squamous cell carcinomas (HNSCCs), molecularly and functionally modulates activity of cancer cells. In our prior experiments, we demonstrated that HNSCC cell lines exposed to hypoxia release an increased number of small extracellular vesicles (sEVs). This study aims to analyze metabolic shift by combining lipidomic and proteomic profiles of sEVs released from HNSCC cell cultures under normoxic and hypoxic conditions, comparing them with plasma samples from HNSCC patients and normal donors.

Materials and Methods: We collected sEVs from plasma of HNSCC and normal donors, as well as from the supernatants of HNSCC cells (PCI-30) and normal control cells (HaCaT keratinocytes) exposed to 21 % (normoxia) and 1 % (hypoxia) oxygen supply. sEVs were isolated from plasma and supernatants using size exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis, electron microscopy, immunoblotting, and, for cell line-derived sEVs, high-resolution mass spectrometry lipidomic and proteomic analysis.

Results: The analyzed sEVs had an average diameter of 125–135 nm and carried CD63 and CD9 but not Grp94. The analysis of hypoxia-derived sEVs in tumor demonstrated 815 and 735 significantly dysregulated lipids compared to normal counterparts exposed to hypoxia and to tumor-derived under normoxic conditions, respectively. Among the 263 lipids in common, the most abundant were glycerophospholipids (GPLs). Hypoxia determined the distribution of 281 unique lipids in tumor-derived sEVs compared to normal keratinocytes. The adaptation of HNSCC cells to hypoxia was also associated with an enrichment in the increased number of unique GPLs.

This data was overlaid on the proteomic level. The expression of proteins involved in the catabolism of GPLs to lysophosphatidic acid increased in both hypoxic cell line-derived sEVs and HNSCC plasma-derived sEVs but not in those from normal donors. Choline transporter was characteristic feature of hypoxic HNSCC-derived sEVs.

Conclusion: Adaptation to environmental stress such as hypoxia, unveils the plasticity of the lipid profile in HNSCC-derived sEVs, marked by the upregulation of GPLs. This lipid signature correlates on the proteomic level, suggesting that sEV-associated lipids may serve as a signature for tissue hypoxia in HNSCC and act as signaling molecules in tumor progression.

PhD Meng Han, PhD Jie Gong, Professor Qi Wang, PhD Bairen Pang, PhD Cheng Zhou, PhD Zhihan Liu, Professor Junhui Jiang, Professor Yong Li

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1. Introduction:

Current diagnosis and risk progression stratification using blood prostate specific antigen (PSA) test for prostate cancer (PCa) is inaccurate and unreliable. Tissue biopsy is harmful and cannot cover tumor heterogeneity. Therefore, developing innovative approaches for accurate PCa diagnosis and risk stratification is critically important in choosing the best treatment and personalized medicine. Extracellular vesicles (sEVs) play an important role in regulating cell-to-cell communication and tumor initiation, progression, and metastasis positioning them as an important source of biomarkers for liquid biopsy. Lipidomic analysis of EVs for cancer biomarker discovery is a new developing research area and holds promise for PCa diagnosis and personalized therapy. Our Aim in this study was to identify novel lipid biomarkers from plasma sEVs of different stages of PCa patients for personalized treatment choice.in.

2. Methods: sEVs from plasma and urine samples of 6 controls and PCa patients (n = 6. low-risk: 3 and high-risk: 3) and control subjects (n = 6) were isolated. Employing nanoflow and liquid chromatography-mass spectrometry (LC-MS/MS) were applied for lipid biomarker discovery and targeted parallel reaction monitoring (PRM) was use for lipid biomarker validation using urine samples including control, low-risk, and high-risk groups (n = 15 each group).

3. Results: Total 727 distinct lipids were identified form sEVs of urine and plasma samples by LC-MS/MS. The lipid markers identified was found more variable in urinary sEVs than in plasma sEVs. A significant upregulation of most lipid types was observed in the urine and plasma sEVs of PCa patients compared to control subjects. In addition, lipid significant reduction of sEV lipids expression in thigh-risk PCa group was found compared to low-risk PCa group.

4. Conclusion: Our findings indicate that a panel of sEV lipid biomarkers identified such as carnitine C14:1, carnitine C16:1-OH, hold potential for PCa early diagnosis and risk stratification. Validation of these biomarkers are ongoing in an independent set of urine samples for clinical value confirmation.

Keywords: Prostate cancer, extracellular vesicles, lipidomics, liquid biopsy, diagnosis, risk stratification

MSc Karolina Soroczyńska, Tobias Tertel, Bernd Giebel, Małgorzata Czystowska-Kuźmicz

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Endometriosis, a prevalent and challenging gynecological disorder, poses therapeutic challenges with a lack of effective treatments and early diagnostic biomarkers. The multifaceted nature of this condition calls for innovative approaches, highlighting the pivotal role of exploring a diverse panel of biomarkers for accurate diagnostics and tailored therapeutic interventions. In this context, extracellular vesicles (EVs), present in various body fluids, emerge as promising candidates for liquid biopsy biomarkers, given their accessibility and potential diagnostic value. Implementing a comprehensive strategy that integrates state-of-the-art EV analysis techniques, including high-resolution molecular profiling of EVs using proteomics and single-vesicle imaging flow cytometry (IFCM) analysis, is crucial for identifying novel EV-based biomarkers or therapeutic targets for this complex and heterogeneous condition.

Methods

EVs were isolated from plasma and peritoneal fluid (PF) of endometriosis and control patients using SEC and were verified by WB, fluorescent mode NTA (F-NTA), imaging flow cytometry, and TMT-based quantitative proteomics analysis. Molecular profiling of EVs directly in plasma and PF samples was conducted using high-throughput IFCM with a specialized antibody panel. This panel included detection of antigens elevated during chronic inflammatory states (CD152), associated with early endometriotic lesions (CD82; CD44), immune suppression (CD16; CD206), and endometrial receptivity leading to infertility (CD227).

Results

A heterogeneous collection of EVs was identified in plasma and PF samples from both endometriosis patients and controls. These vesicles exhibited typical characteristics of small EVs and contained bona fide EV markers. Single EV analyses on the IFCM platform, along with EV proteomics analysis, revealed that EV populations derived from endometriosis patients contain a wide range of molecules, with some associated with the pathogenesis of endometriosis.

Summary/Conclusion

In summary, our study highlights the potential of EVs as promising liquid biopsy biomarkers for endometriosis. The presence of diverse EV populations, along with identified endometriosis-specific signatures, suggests promising applications in diagnostics, prognostics, and therapeutics. Further evaluation of these EV signatures is crucial for advancing non-invasive approaches in managing this complex gynecological disorder, providing valuable insights into potential diagnostic and therapeutic avenues for improved clinical outcomes.

Kalpana Deepa Priya Dorayappan, Dr. Michelle Lightfoot, Dr. Lianbo Yu, Dr. Colin Hisey, Dr. Takahiko Sakaue, Dr Muralidharan Anbalagan, Dr Casey Cosgrove, Dr Larry Maxwell, Dr Premal Thaker, Dr Beth Y. Karlan, Dr David O'Malley, Dr Raphael E. Pollock, Dr David E. Cohn, Dr Rajan Gogna, Dr Selvendiran Karuppaiyah

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

High-grade serous ovarian cancer (HGSOC) accounts for over 75% of all epithelial ovarian cancer and has a high mortality rate due to a lack of early detection methods. Many biomarkers have been proposed; however, none have been shown to improve detection at an early stage in this patient population. The purpose of this study is to assess whether there are unique extracellular vesicle (EV) protein signatures of early-stage HGSOC that could serve as early detection biomarkers.

Methods:

250 serum samples were obtained from a multi-institutional retrospective cohort of patients with all stages of HGSOC and healthy controls. LC-MS/MS and PEA of serum samples from patients with early-stage HGSOC identified differentially expressed EVs proteins in HGSOC versus controls. Tob candidate proteins are validated by ELISA.

Results:

We have identified the top 10 EVs candidate proteins based on their fold change and statistical significance. When comparing early-stage HGSOC and controls using ELISA, CFH, PCP, and CCNE1 exhibited the highest Area Under the Curve (AUC) values of 0.94, 0.91, and 0.83, respectively. In contrast, MUC16 (also known as CA-125) exhibited an AUC of 0.78 in EVs but only 0.42 in whole serum. Furthermore, MUC16 distinguished between controls and both early and advanced-stage HGSOC in EVs, while in whole serum, it was only differentially expressed between controls and advanced-stage disease. Our results revealed generally weak correlations between pairs of biomarkers. In this study, we integrated the identified ten biomarkers, deriving the optimal combination biomarker by maximizing the corresponding Sum of Harmonic Means (SHUM). The overall true positive rate (TPR) of 0.943, false positive rate (FPR) of 0.000, and Mathew's correlation coefficient (MCC) were presented for both the combined biomarker approach and individual biomarkers. The results demonstrated that the predictive performance of the combined biomarkers surpassed that of any individual biomarker.

Conclusion:

Expression of EV-based protein biomarkers is significantly different in early-stage HGSOC compared to both control and late-stage HGSOC. This work can be readily translated to clinical use and potentially improve the early detection of HGSOC substantially.

Research Professor Seung Ah Choi, Professor Seung-Ki Kim, Professor Ji Hoon Phi

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Cerebrospinal fluid (CSF) is a crucial factor in brain tumor studies, particularly medulloblastoma (MBL). Previous CSF proteomics research has predominantly concentrated on extracellular vesicle (EV) proteins rather than the comprehensive CSF proteome. Recent progress in mass spectrometry systems and ‘Omics’ data analysis techniques has opened the door to unbiased and in-depth exploration of the proteome. Our goal was to discover specific diagnostic biomarkers associated with EV proteins through proteomic profiling of the comprehensive CSF. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of CSF samples from MBL (n = 31) and hydrocephalus (HC, control, n = 19) patients, we identified 1,112 quantified proteins per CSF sample. Enrichment analysis and differential expression profiling revealed 273 differentially expressed proteins (DEPs). Feature selection highlighted four upregulated soluble proteins (SPTBN1, HSP90AA1, TKT, and NME1-NME2) in MBL CSF. Validation with ELISA confirmed elevated levels of TKT in MBL compared to HC, particularly in Group 4 MBL and leptomeningeal seeding (LMS). Moreover, TKT-positive EVs were significantly enriched in MBL CSF compared to HC CSF and correlated with LMS burden. These results provide significant insights into the proteomic profile of the total CSF in MBL patients, underscoring the potential of TKT as a meaningful biomarker for MBL, particularly in the diagnosis of LMS.

Research Professor Seung Ah Choi, Professor Eun Jung Koh, Professor Seung-Ki Kim

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Background

Moyamoya disease (MMD) is a chronic occlusive cerebrovascular disease known to be a major cause of pediatric stroke. Emerging evidence suggests that circulating extracellular vesicle (EV) containing miRNAs in cerebrovascular disease plays a significant role in intercellular communication by delivering RNA cargo involved in biological processes. This study aimed to investigate the specific miRNAs loaded in EVs from MMD plasma, followed by identification of their roles and mechanisms.

Methods

EVs were isolated from plasma of normal group and MMD patients. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, ExoView, RT-qPCR and western blot. Profiling of miRNAs in EVs were determined using NanoString nCounter miRNAs analysis. Guanosine triphosphatase (GTPase) activity, tubule formation and cell viability were observed by transfection of miR-512-3p inhibitor in MMD endothelial colony forming cells (ECFCs).

Results

EVs were successfully isolated from normal and MMD plasma. Compared with normal EVs, the number of MMD EVs were small, but there was no difference in size. miRNA profiling demonstrated that miR-512-3p was significantly upregulated in MMD EVs. The target prediction analysis of EV-miR-512-3p suggested that rho guanine nucleotide exchange factor 3 (ARHGEF3) was down-regulated and could be targeted in MMD ECFCs. Inhibition of miR-512-3p in MMD ECFCs resulted in increased expression of ARHGEF3 and its downstream effector RHOA, enhanced GTPase activity, and restoration of tubule formation.

Conclusion

This study suggests the potential of miR-512-3p in EVs from plasma as a diagnostic biomarker of MMD. Downregulation of ARHGEF3, a target gene of miR-512-3p, may be involved in the aberrant angiogenesis of MMD through downstream RHOA signaling.

Dr XINYU QU, Dr Leanne Leung, Dr Bojie Chen, Professor Zigui Chen, Professor Katie Meehan, Professor Jason Chan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Oral cancers tend to metastasize to locoregional, tumor-draining lymph nodes other than distant sites, and lymph node metastasis is the well-known prognostic factor. Therefore, it is assumed that lymph-rich drainage fluid is the proximal biofluid to the surgically resected tumor, and tumor-related markers are more highly enriched in lymphatic fluid than in blood.

Methods: Matched plasma and lymphatic drainage fluid (LDF) samples were collected from oral cancer patients (n = 9). Extracellular vesicles (EVs) were isolated by iodixanol density gradient ultracentrifugation followed by size-exclusion chromatography, and then purified EVs were treated with trypsin. All EV samples were characterized and validated based on guidelines. The protein cargo of EVs was determined using a TMT-based high-resolution quantitative proteomic approach.

Results: Through an optimized isolation strategy, most lipoproteins and albumin were eliminated from EV samples. In LDF samples, a total of 3,771 EV proteins were identified and 3,596 of them were quantified, while in plasma samples, only 1,256 EV proteins were identified and 822 of them were quantified. More importantly, the plasma EV proteome is completely contained in the LDF EV proteome, and LDF EVs contain more head and neck cancer-associated protein markers compared with plasma.

Summary/Conclusion: LDF is a better source of EV protein biomarkers and is superior to plasma. By combining iodixanol density gradient ultracentrifugation and size-exclusion chromatography, we successfully isolated and purified EVs from LDF for proteomics analysis. Lymph liquid biopsy will enable earlier and more precise care following surgery and aid in post-surgical treatment decisions.

Timea Boroczky, Matyas Bukva, Gabriella Dobra, Maria Harmati, Edina Sebestyen-Gyukity, Yasmin Ranjous, Laszlo Szivos, Katalin Hideghety, Krisztina Budai, Judit Olah, Peter Horvath, Gyorgy Lazar, Zoltan Konya, Pal Barzo, Almos Klekner, Krisztina Buzas

Poster Pitches (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:45 PM - 1:00 PM

Introduction

Investigation of the molecular composition of small extracellular vesicles (sEVs) for tumor diagnostic purposes is gaining popularity, particularly for diseases with difficult diagnosis, such as central nervous system malignancies. Using spectroscopy analysis for diagnosing cancerous diseases is promising, but yet an underexplored method. Therefore our aim is to elucidate the potential role of plasma-derived sEVs in diagnosing seven distinct patient groups using a sufficient number of clinical samples and Raman spectroscopy.

Methods

The study was conducted in accordance with the Declaration of Helsinki, informed consent forms were collected and the study was approved by national ethics committee. Up to 532 plasma samples from seven patient groups (glioblastoma multiforme, meningioma, melanoma and non-melanoma brain metastasis, colorectal tumors, melanoma and lumbal disc herniation patients as a control group) were collected. SEV isolation was performed through differential centrifugation. The isolates were characterized by Western Blot, transmission electron microscopy and nanoparticle tracking analysis. For Raman spectra classification Principal Component Analysis–Support Vector Machine algorithm was used. Classification accuracy, sensitivity, specificity and the Area Under the Curve (AUC) value were used to evaluate the performance of classification.

Results

According to our results, there are no significant differences in the particle numbers belonging to the 7 patient groups. Raman measurements indicate that the patient groups are distinguishable with 80–95% sensitivity and 61–100% specificity. AUC scores of 0.63–1 suggest excellent classification performance. The highest sensitivity and specificity can be reached with the comparison of the malignant brain tumors and the control group.

Summary/Conclusion

Our findings indicate that Raman spectroscopic analysis of sEV-enriched plasma isolates is a promising strategy for the development of noninvasive and cost-effective methods supporting the clinical diagnosis of various cancers.

Chao-Yuan Chang, Visiting Staff Chun-Jen Huang, Visiting Staff Syuan-Hao Syu, Visiting Staff Tze-Sian Chan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

Disease progress of non-alcoholic fatty liver disease (NAFLD) to liver fibrosis is often asymptomatic, challenging the effectiveness of blood-based biomarker tests. The use of urine samples for biomarker measurement presents a promising alternative. Exosomes, nanosized double-membrane particles containing nucleic acids, microRNAs, and proteins, hold potential as clinical biomarkers. However, the cargo of urine exosomes in NAFLD remains largely unexplored. This study aims to elucidate established biomarkers in urine exosomes across three stages of NAFLD, employing a high-fat diet/fructose-induced NAFLD and liver fibrosis rat model.

Methods:

Male Sprague-Dawley rats were randomly assigned to receive a normal diet (ND group) or high-fat diet/fructose feeding for 6, 12, or 18 weeks (HFr/HFD_E, HFr/HFD_M, or HFr/HFD_L groups, respectively). Following euthanasia, liver tissues and urine samples were collected. Liver histopathological assessment included hematoxylin & eosin, Oil Red, and Masson's trichrome staining for NAFLD activity score, fat accumulation, and fibrosis. Urine exosomes were isolated through ultra-centrifugation. Proteomic analysis of urinary exosomes utilized liquid chromatography–tandem mass spectrometry, followed by MetaboAnalyst 5.0 and Ingenuity Pathway Analysis (IPA).

Results:

Histopathological analysis revealed a significant increase in NAFLD activity score in the HFr/HFD_E, HFr/HFD_M, and HFr/HFD_L groups compared to the ND group as the disease progressed with longer feeding duration (all p<0.05). Oil Red staining showed significantly higher levels of fat accumulation in the HFr/HFD_M and HFr/HFD_L groups compared to both the HFr/HFD_E and ND groups (all p<0.05). Masson's trichrome staining depicted significantly higher fibrosis levels in the HFr/HFD_L group than the other 3 groups (all p<0.05). These data confirmed that a high-fat diet/fructose diet induces NAFLD in rats, progressing to liver fibrosis in 16 weeks. Proteomic profiling identified 675 proteins with expression variations among the groups. MetaboAnalyst 5.0 demonstrated distinct clusters, and IPA revealed altered upstream regulators in urinary exosomes. Notably, hepatocyte nuclear factor 4 alpha, angiotensin, tumor necrosis factor-α, interleukin-1β, and interleukin-6 displayed increased expression with disease progression.

Conclusion:

This study explores potential NAFLD biomarkers in urine exosomes across disease stages using proteomic analysis, offering insights for non-invasive detection and monitoring of NAFLD progression.

Dr Hannah Nazri, Dr Raphael Heilig, Associate Professor Roman Fischer, Professor Benedikt Kessler, Dr Kavita S Subramaniam, Professor Christian Becker, Dr Thomas Tapmeier

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

Endometriosis, defined as ectopic endometrial-like tissue, causes pain and/or subfertility in 10% of reproductive-age women. The cause is unknown, resulting in inadequate diagnostic methods (no clinically relevant biomarker) and treatment. The sEV-protein cargo, described in cancer, diabetes and pre-eclampsia, could similarly serve as an endometriosis biomarker.

Methods:

Peritoneal fluid (PF) samples were obtained from 18-49-year-old women (n = 63) who were investigated for abdominal/pelvic pain and/or subfertility via diagnostic laparoscopy within the ENDOX study at the Endometriosis CaRe Centre, Nuffield Department of Women's and Reproductive Health, University of Oxford (REC 09/H0604/58). PF was collected and classified according to cycle phase (proliferative/secretory/menstrual) and endometriosis severity (ASRM stages I+II/III+IV) according to WERF EPHect standards. Exclusion criteria were hormonal treatment, malignancy, pregnancy, breastfeeding, and an inability to understand the consent form. PF was centrifuged to remove cells, debris, and microvesicles. sEVs were isolated using size exclusion chromatography (SEC) and analysed by nanoparticle tracking analysis (NTA), immunoblotting, and mass spectrometry.

Results:

We confirmed the presence of sEVs in PF of women at different endometriosis stages and from disease-free women in different menstrual cycle phases by NTA, immunoblotting and mass spectrometry. Enriched sEVs were positive for ALIX, CD9, and syntenin. The endometriosis PF-derived sEV mode size was 130±8.7 nm (n = 44), compared to 134±2.12 nm in controls (n = 19). Irrespective of the menstrual cycle phase, sEV concentrations were highest in stage III-IV endometriosis (n = 19), followed by stage I-II endometriosis (n = 25) and controls (P = 0.0210) (n = 19). sEV concentrations in stage I-II endometriosis were highest in proliferative compared to secretory cycle phases. Proteomic analysis found CD44, in sEVs a protein unique to endometriosis, contributing most to the separation of endometriosis and control samples (high variable importance projection, VIP score).

Conclusions:

PF-derived sEV concentrations vary regardless of cycle phase and disease stage, and this difference appears to be reflected in the protein cargo. Correlating these findings to other, easily accessible biological fluids such as blood or urine will aid our understanding of endometriosis and in endometriosis biomarker identification.

MD Cahyani Gita Ambarsari, Professor MW Taal, MRCPCH MD(res) JJ Kim, Assistant Professor Dong-Hyun Kim, Assistant Professor AM Piccinini

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Urinary extracellular vesicles (uEVs) are potentially clinically valuable biomarkers, particularly in patients with kidney and urinary tract problems. uEVs encapsulate their cargoes, including nucleic acids, proteins and metabolites, protecting them from enzymatic degradation. We implemented methods reported by previous studies for collecting, storing, and preserving uEVs to examine particle concentration stability in random morning urine samples stored for 6 months.

Methods

Fresh urine samples were collected from seven healthy volunteers, three females and four males, 100 mL each. Two tablets of cOmpleteä Mini Protease Inhibitor Cocktail tablet (Product No: 11836153001 Roche) were added to each urine container. Pre-processing to remove cells and cell debris was done within 4 hours from sample collection by centrifugation at 800xg at 4°C for 10 minutes with a swing-bucket rotor. Samples were divided into aliquots of 10 mLs each for uEV isolation at 0 and 6 months post collection and stored at -80°C. We isolated uEVs using three different commercial kits, which use precipitation- (Total Exosome Isolation (from urine) Reagent (TEIR; Invitrogen)), pH and precipitation (Urine Exosome Purification Kit (UEPK; Norgen)), and size-exclusion chromatography (SEC)-based methods (IZON), respectively. Cell-free urine samples were defrosted overnight at 4°C on ice for uEV isolation and quantification. uEV isolation was conducted following manufacturers’ instructions. uEV characterization, including quantification and size distribution assessment, was done using nanoparticle tracking analysis (NTA) with Zetaview PMX-120.

Results

Performing NTA analysis, we found that particle concentration after 6 months of storage was not different from baseline. uEV particle size distributions at 0-month and 6-month post collection were also similar. Among the three kits utilized, Total Exosome Isolation (from urine) Reagent (TEIR; Invitrogen) resulted in the highest uEV concentration, followed by SEC (IZON) and Urine Exosome Purification Kit (UEPK; Norgen).

Summary/Conclusion

Protease inhibitors for preservation, pre-processing done within 4 hours after sample collection, and storage at -80°C may be useful for future long-term uEV-derived biomarker studies.

Dr. Kelly Tian Mun Chee, Prof. Kwun M Fong, Prof. Ian A Yang, Assoc. Prof. Rayleen V Bowman

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Malignant pleural mesothelioma (MPM) is a rare type of cancer affecting serosa that lines body cavities, most commonly pleura. The extracellular vesicle (EV) compartment of pleural fluid could be selectively enriched with protein cargo specific for pathologic entities responsible for pleural effusions. The protein content of the EV contained within pleural fluid has not been extensively studied. Therefore, the protein cargo of pleural fluid EV was studied with the aim of identifying specific protein candidates of MPM capable of distinguishing it from other causes of pleural effusion.

Methods

This study was approved by The Prince Charles Hospital ethics committee (HREC/18/QPCH/312). Pleural fluid was centrifuged at 800 x g for 10 minutes at 4 ⁰C, followed by ultracentrifugation at 100,000 x g (w2t = 5.46e10) for 1 hour 40 minutes at 4⁰C to isolate EV. Sequential Window Acquisition of all Theoretical fragment-ion spectra - Mass Spectrometry (SWATH-MS) was performed on 19 pleural effusion cases of MPM (n = 6), lung adenocarcinoma metastatic to the pleura (LUAD; n = 5), breast cancer metastatic to the pleura (BRCA; n = 4) and non-malignant conditions (NM; n = 4). R package LIMMA algorithm based on empirical Bayesian methods was used for differential expression analysis.

Results

Analysis of all 19 PFEV samples collectively showed a total of 830 proteins, 10477 peptides, and 76042 spectra detected at 5% local FDR. The number of proteins detected in each disease state were 630 (MPM), 439 (LUAD), 406 (BRCA) and 625 (NM), respectively. Four proteins (LG3BP, FCGBP, MXRA5 and STOM) demonstrated greatest potential as protein marker candidate for MPM. The level of LG3BP was higher in MPM than in any other cause of pleural effusion: p-values of 0.0087 (MPM-LUAD) and 0.0095 (MPM-BRCA), and 0.0095 (MPM-NM). FCGBP was higher in MPM than in LUAD and BRCA (p-values of 0.03 and 0.038, respectively), while MXRA5 and STOM levels were reduced in MPM PFEV compared with BRCA (p = 0.019 and p = 0.038, respectively) and NM (p = 0.038 and p = 0.038, respectively).

Conclusions

The protein candidates warrant further testing to determine whether they could be useful components of a panel of protein biomarkers with diagnostic utility for patients with pleural effusions.

Dr. Xin Zhang, Dr. Sisi Ma, Syeda Iffat Naz, Janet Huebner, Dr. Erik Soderblom, Noor Alnemer, Dr. Constantin Aliferis, Dr. Virginia Kraus

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: We previously reported that multiple immune cell-associated circulating extracellular vesicle (EV) subpopulations declined with aging in healthy humans and mice. In this study, we aimed to identify systemic changes in immune system-related plasma EVs during healthy aging.

Methods: Plasma was isolated from blood of younger (18-31 years, n = 6) and older (47-83 years, n = 22) healthy donors by centrifugation at 3000 rpm for 15 minutes at 4°C to remove cells and debris, and stored at −80°C. Frozen plasma was completely thawed and centrifuged at 2000 g for 10 minutes at 4°C to remove remaining debris; EVs and EV-depleted supernatants were separated by polymer-based precipitation. EVs were validated to have a bilayer structure and size diversity, and carry mitochondria, traditional EV markers (CD81, CD9, CD63, CD29), and hematopoietic cell-related markers (CD4, CD8, CD56, CD15, CD14, CD68, CD19, CD235a, CD41a, CD31, CD34, HLA-ABC, HLA-G, HLA-DRDPDQ) with various frequencies. We measured concentrations of endo-EV (in EVs) and exo-EV (in EV-depleted supernatants) cytokines by ELISA. The endo-EV proteome was quantified by high-resolution mass spectrometry. High-resolution flow cytometry was used to quantify the frequency of EV subpopulations carrying the tested surface markers, and internalization of PKH67 pre-labeled EVs by WI-38 fibroblasts and their change in proliferation.

Results: Compared with matched exo-EV supernatants, endo-EV contents had higher TNF-α and IL-27, lower IL-6, IL-11, IFN-γ, and IL-17A/F, and similar concentrations of IL-1β, IL-21, and IL-22. There were significant correlations of endo-EV and exo-EV TNF-α, IL-27, IL-6, IL-1β, and IFN-γ. Endo-EV IFN-γ and exo-EV IL-17A/F and IL-21 significantly declined with age. Age was significantly associated with EV peptides, positively (n = 37) and negatively (n = 257); the corresponding age-related EV proteins were predominately enriched in liver and innate immune system. WI-38 fibroblasts (>95%) internalized similar amounts of both young and old EVs. Compared to cells that did not take up PKH67-EVs, the cells with PKH67-EVs (that internalized EVs), particularly young EVs, underwent greater cell proliferation.

Conclusions: Our results identify EV phenotypes reflecting immunosenescence during healthy aging and the ability of plasma EVs of young donors to stimulate proliferation of recipient cells in vitro. These EV biomarkers are candidates for future aging studies.

Miss Nilobon Jeanmard, Dr. Rassanee Bissanum, Mr. Kittinun Leetanaporn, Mr. Pongsakorn Choochuen, Assoc.Prof. Hutcha Sriplun, Miss Sawanya Charoenlappanit, Dr. Sittiruk Roytrakul, Assoc.Prof. Raphatphorn Navakanitworakul

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Urinary extracellular vesicles (uEVs) have shown great promise as non-invasive biomarkers for various cancerous diseases due to their ability to reflect pathological states along the course of the disease and treatment. Thus, this study aimed to evaluate the uEVs concentration and uEVs proteomes in BC patients before and after surgery.

Methods: Urine samples were collected from BC patients (n = 30) at two time points: before surgery and after surgery. The uEVs were isolated using differential ultracentrifugation and were then characterized by western blot and TEM. The concentration of uEVs was measured by nanoparticle tracking analysis (NTA). LC-MS/MS was utilized for proteomic analysis of uEVs.

Results: The uEVs concentrations of individual BC patients were compared before and after surgery. In comparison to pre-surgery, we observed two trends in the uEVs level after surgery: an increase and a decrease from pre-surgery. The eight BC patients had an elevated uEV level, whereas the twenty-two BC patients had a decrease of uEVs concentration after surgery. Proteomic analysis of uEVs clarified the differentially expressed proteins (DEPs) between pre- and post-surgery samples, which were grouped into an increasing trend (sig-up 26, sig-down 35) and a decreasing trend (sig-down 121, sig-up 121). The heatmap between the pre- and post-surgery samples revealed remarkable patterns of protein expression. It's interesting to note that while cancer-associated proteins were down-regulated in post-surgery samples, which showed a declining trend of uEVs concentration, the significantly up-regulated proteins in post-surgery samples were mapped to proteins in cancer-related pathways annotated by the KEGG database that might be related to the increase of uEVs after surgery.

Conclusion: The alteration of uEVs concentration pre- and post-surgery revealed the dissimilarity of uEVs proteomes and might be relative to pathological changes in breast cancer patients.

Dr Andrew Lai, Dr Dominic Guanzon, Dr Shayna Sharma, Mrs Katherin Scholz-Romero, Dr Yaowu He, Mr Weitong Huang, Dr Tanja Pejovic, Dr Carmen Winters, Professor Terry Morgan, Professor Jermaine Coward, Associate Professor Amy McCart Reed, Professor Sunil Lakhani, Professor Andreas Obermair, Professor Amanda Barnard, Professor Anna deFazio, Professor Lewis Perrin, Professor John Hooper, Professor Gregory Rice, Professor Carlos Salomon

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) contain bioactive molecules, making them strong candidates as disease biomarkers. Ovarian cancer (OVCA) survival rates have remained stagnant over the past two decades due to the absence of clinically useful biomarkers for early detection. This study aims to evaluate their sensitivity and specificity in accurately classifying women with OVCA using Machine Learning (ML).

Methods: A total of 926 patients were analysed between 2016 and 2022. Plasma samples were collected, and patients were randomly assigned into three groups: 1) Training set with cross-validated models (n = 220), 2) Validation set (n = 235), and 3) Assay refinement (n = 471). EVs were isolated using size exclusion chromatography and a capture immunoaffinity magnetic bead-based system compatible with routine pathology testing and characterised in compliance with MISEV2018. The EV content (miRNAs, proteins, lncRNA, miscRNA, MtrRNA, MttRNA, rRNA, scaRNA, snRNA, and tRNA) was used as ML classifiers. EV biomarker stability was evaluated in a heterogeneous population of OVCA cell lines in the absence or presence of RNase/proteinaseK. All procedures were conducted in compliance with ISO17025.

Results: Ten classifiers were trained in group 1 to detect cancer, benign, and healthy patients. Across all groups, a sensitivity of 98% specificity (2% false-positive rate) was reported, with statistical comparisons made for each of the 10 classifiers independently and in combination. The most sensitive classifiers at 98% specificity for detecting cancer were miRNAs (training: 78%, validation: 82%), proteins (training: 85%, validation: 90%), and the pan-feature classifier (training: 90%, validation: 95%). Notably, identifying healthy controls exhibited high sensitivity (95% CI 84%-100%) compared to cancer and benign conditions. An Early Identification Multivariate Index Assay was developed using data from stages I and II OVCA patients and healthy controls, achieving consistent performance in the training set (95%CI, 88%-100%), validation set (95%CI, 90%-100%), and assay refinement (95%CI, 80%-95%). Hierarchical clustering identified two distinct clusters based on OVCA cell line stability: A) CAOV-3, TOV-112D; B) HEY, OVCA-420, OVCAR-3, SKOV-3, OVCA-429, OVTOKO, OV90.

Conclusions: This study is the most comprehensive investigation of EVs in ovarian cancer. Given the potential value of early detection in OVCA, further evaluation of this test is warranted in prospective population-level studies.

Ms Shikha Rani, Dr Andrew Lai, Dr Dominic Guanzon, Mr Kaltin Ferguson, A/Prof Lewis C. Perrin, Prof John D. Hooper, Prof Carlos Salomon

Poster Pitches (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:45 PM - 1:00 PM

Introduction:

Lipids serve a dual role, acting as fundamental structural components of extracellular vesicles (EVs) and vital messengers in intercellular communication. They potentially influence the reprogramming of recipient cells during cancer progression. This study aimed to optimize a lipidomic approach for identifying and quantifying a broad range of plasma-derived EV-associated lipid classes, including phospholipids, sphingolipids, glycerolipids, and cholesterols. It aimed to distinguish EVs from other cellular reservoirs and evaluate the EV-lipid signature across the progression of ovarian cancer.

Methods:

EV and non-EV fractions were isolated from plasma samples of healthy donors using size exclusion chromatography (SEC). Characterization involved size analysis, the abundance of classical protein markers, and morphology assessment using nanoparticle tracking analysis (NTA), immunoblotting, and transmission electron microscopy. Samples were spiked with SPLASH® II LIPIDOMIX® Mass Spec Standard, and lipid extraction used solid-phase extraction (SPE) cartridges. Targeted lipidomic analysis was performed on a Shimadzu Nexera UHPLC/5500QTRP tandem mass spectrometer system. Clinical relevance was assessed in a high-risk ovarian cancer population (n = 97).

Results:

Comparative lipidomic mass spectrometry (LC-MS) analysis revealed distinct enrichment of phospholipids, sphingolipids, and glycerolipids within plasma-derived EVs compared to non-EV fractions. Abundant phospholipids such as phosphatidylcholine, lyso-phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine were identified in EV fractions. Additionally, EVs exhibited notable enrichment of sphingomyelins, ceramides, and diacylglycerol, distinguishing them from other vesicular pools. Specific changes in EV lipid composition associated with disease stage and ovarian cancer histotype were identified.

Summary:

Our findings highlight the unique abundance of molecular lipid species within plasma-derived EVs. Characterizing the EV lipidome holds potential for the development of improved diagnostic and prognostic biomarkers for ovarian and various gynaecological cancers.

Dr Dominic Guanzon, Dr Subash Rai, Mr Rakesh Sankar, Ms Pragati Lodha, Ms Vidya Gummagatta, Dr Andrew Lai, Professor Lewis Perrin, Professor John Hooper, Professor Carlos Salomon

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

Extracellular vesicles (EVs) have emerged as significant carriers of molecular biomarkers and mediators of intercellular communication. While research has concentrated on understanding the protein and RNA content of these EVs, the role of EV DNA remains inadequately explored. The potential functional aspects of EV DNA have been proposed in pathological conditions such as cancer, highlighting its relevance as a promising biomarker. Moreover, increasing utilization of cell-free DNA as a diagnostic tool in liquid biopsy applications underscores the potential importance of EV DNA as a novel and noteworthy biomarker for ovarian cancer (OVCA) detection.

Methods:

For initial optimisation, the EV and cell-free components were isolated from normal (n = 3) and ovarian cancer (n = 3) plasma using size-exclusion chromatography, and characterized using Nanoparticle Tracking Analysis and Bicinchoninic acid assay. The DNA was extracted, quantified and sizing of DNA fragments performed using Qubit dsDNA and DNA ScreenTape assays. Long-read Oxford Nanopore sequencing was used to comprehensively characterize and compare EV DNA with cell-free DNA. A larger independent cohort (n = 30) with different OVCA histotypes will be used to refine DNA methylation and mutation signatures in circulating EVs.

Results:

The average particle concentration within the EV fraction was 1.96e+09 particles/mL for normal and 5.4e+09 particles/mL for cancer patients. Among normal patients, the average DNA concentration was 1.68 ng/µL and 2.29 ng/µL for EV DNA and cell-free DNA, respectively. Conversely for cancer patients, the average DNA concentration was 1.35 ng/µL and 1.63 ng/µL for EV DNA and cell-free DNA, respectively. Nanopore long-read sequencing revealed a median read length of 200 base pairs for cell-free DNA, and 350 base pairs for EV DNA. Notably, methylation signatures were observed in EV DNA of cancer patients, contrasted with the absence of this signature in normal patients, for specific genomic loci.

Conclusion:

This research marks the first effort to utilize long-read sequencing for EV DNA analysis to investigate methylation and mutation patterns in ovarian cancer. The results indicate that EV DNA holds significant promise as a biomarker for diagnosing ovarian cancer. This study lays a foundation for further exploration and validation of EV-based DNA biomarkers in cancer diagnostics and treatment monitoring.

Dr Natasha Vassileff, Dr Jereme Spiers, Miss Sarah Bamford, Dr Rohan Lowe, Dr Keshava Data, Professor Paul Pigram, Professor Andrew Hill

Poster Pitches (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:45 PM - 1:00 PM

Introduction:

Neuroinflammation is a common feature of neurodegenerative conditions, often appearing very early in disease pathogenesis. Microglial activation is known to be a prominent initiator of neuroinflammation and can be induced through lipopolysaccharide treatment (LPS). This activation leads to the expression of the inducible form of nitric oxide synthase (iNOS), resulting in the production of nitric oxide (NO). NO targets cysteine thiols resulting in post-translational S-nitrosylation, which can alter the target protein's function. Furthermore, packaging of these NO-modified proteins into Extracellular Vesicles (EVs) propagates the neuroinflammatory phenotype by allowing the exertion of NO signalling in distant locations. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. Therefore, this study aimed to identify the effect NO signalling exerts through protein post translational modifications in neuroinflammation.

Methods:

Extracellular Vesicles (EVs) were isolated from LPS-treated microglia using differential ultracentrifugation with a sucrose density gradient. This was followed by characterisation to meet the Minimal Information for Studies of EVs 2018, including nanoparticle tracking analysis, electron microscopy and western blot analysis. The EVs subsequently underwent novel advanced surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS) in addition to, iodolabelling and comparative proteomic analysis to identify post-translation nitrosylation changes.

Results:

ToF-SIMS imaging successfully identified NO modified cysteine thiol side chains in the EV proteins isolated from LPS treated microglia. Additionally, the iodolabelling proteomic analysis revealed the EVs from LPS-treated microglia carried nitrosylated proteins indicative of neuroinflammation, microglial activation, and protein clearance pathway regulation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an important role in neuroinflammatory communication.

Conclusions:

Together, these results show activated microglia are capable of exerting broad signalling changes through the selective packaging of EVs during neuroinflammation.

Reine Khoury, Dr. Dariusz Zurawek, Gabriella Frosi, Assistant Professor Corina Nagy

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Exercise, a potent non-drug therapy, maintains mental health and has been used in the treatment of depression, anxiety, and schizophrenia, among others. Exercise is acknowledged for promoting brain plasticity, memory, and delaying neurodegeneration. Aerobic exercise's positive effects are thought to be driven by “exerkines” (exercise factors) including metabolites, proteins, nucleic acids, and hormones. The precise mechanisms behind exercise's benefits for MDD are not fully understood. The literature indicates that exercise might directly or indirectly affect the brain by releasing small extracellular vesicles (sEVs) into the bloodstream. However, studies on exercise-induced sEVs often lack control in timepoints, sex, exercise duration, and sEV collection timing.

Methods: Mice underwent a light/dark switch for two weeks before starting a treadmill exercise regimen. A week of low-intensity training preceded a 2-week treadmill program at 10m/min x 60min/d x 5d/week, followed by 15m/min x 60min/d x 5d/week. Sedentary mice spent an hour daily on a stationary treadmill for two weeks. Daily body weight was recorded. Mice were sacrificed pre-exercise, immediately post-exercise, and 3h, 9h, and 18h post-exercise. Trunk blood was collected, centrifuged, and plasma obtained. Skeletal muscle, liver, and brain tissues were snap-frozen on dry ice. Plasma was clarified, and EVs were isolated using 70nm qEV. EVs were isolated directly from muscle using collagenase D, centrifugation, and 70nm qEV columns. Western blotting, TEM, and NanoSight assessed EV quality. Small RNA was assessed using sequencing adaptors, and protein was quantified using LC-MS/MS.

Results: We anticipate EVs from exercise will differ from sedentary conditions, primarily in cargo rather than size or concentration. Given the sex specific effects of exercise on health and behaviour, we expect sex-specific effects of exercise on EV cargo, including an initial increase in inflammatory markers in females and a later increase in males.

Summary: Examining EVs and their contents within controlled exercise paradigms in vivo is an essential initial exploration into how context-dependent EV release may contribute to exercise's beneficial effects. The crucial next phase involves exploring whether myocyte-released EVs can cross the blood-brain barrier, potentially exerting therapeutic effects.

Mr. Tiago Costa-Coelho, João Fonseca-Gomes, Gonçalo Garcia, Mafalda Ferreira-Manso, Catarina B. Ferreira, Carolina de Almeida-Borlido, Juzoh Umemori, Mikko Hiltunen, Eero Castrén, Ana M. Sebastião, Alexandre de Mendonça, Dora Brites, Maria José Diógenes

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: The neuroprotective BDNF/TrkB-FL system is compromised in Alzheimer's disease (AD). Amyloid-beta triggers calpain-mediated TrkB-FL receptor cleavage, giving rise to the formation of TrkB-ICD, a novel intracellular toxic fragment. Biological samples are used to pinpoint potential disease biomarkers and within these, extracellular vesicles (EVs) are cell-specific carriers of promising pathological hallmarks. Thus, this work aims to investigate the presence of extracellular TrkB-ICD in the post-mortem brain and CSF samples from AD patients, as well as in EVs from plasma samples of AD patients and neuroblastoma cells.

Methods: Human AD patient post-mortem brain samples were collected for RNA and protein extraction and classified according to the Braak staging – stages 0-II, III-IV. For CSF and plasma samples, patients fulfilled the criteria for Mild Cognitive Impairment (MCI, controls) or MCI due to AD (MCI/AD). Such controls reported cognitive complaints despite showing no Aβ deposition nor neuronal injury. CSF AD biomarkers, neuropsychological analysis, and brain imaging were used for patient characterization. Plasma-derived EVs (pdEVs) from the same MCI/AD cohort were isolated using the ExoQuick reagent and characterized. In parallel, EVs from 48-hour conditioned medium of control, GFP- and TrkB-ICD-V5-transduced (ICD-V5) differentiated SH-SY5Y cells were isolated through differential ultracentrifugation. Large (lEVs) and small (sEVs) EVs were characterized and, together with the concentrated EV-depleted secretome, probed for ICD-V5 and the endogenous TrkB-ICD fragments.

Results: Human post-mortem samples revealed a decrease in TrkB-FL protein levels concomitant with an increase in the TrkB-ICD fragment levels (p = 7.3 x10-3 and 3.9 x10-3, n = 7-11). CSF analysis showed increased TrkB-ICD immunoreactivity in MCI/AD patients (p = 7.55x10-3, n = 23-47), and a negative correlation between the levels of Aβ1-42 and TrkB-ICD (ρ=-0.47, n = 47). Regarding EV presence, plasma of MCI/AD patients contained higher levels of TrkB-ICD (p = 0.010, n = 17-18). TrkB-ICD and ICD-V5 were detected in both SH-SY5Y EV subpopulations equally (p>0.05, n = 3). Importantly only the endogenous TrkB-ICD fragment was detected in the EV-depleted secretome.

Conclusions: Altogether, these data demonstrate TrkB-ICD extracellular secretion, alluding for its potential toxicity dissemination. TrkB-ICD as a proxy of BDNF/TrkB-FL dysregulation may prove itself as a relevant hallmark of AD in the future.

Dr. Tina Bilousova, Nina Knitowski, Dr. Qing Cao, Shengkai Zhao, Swetha Atluri, Mikhail Melnik, Achyutha Kodavatikanti, Dr. Ranmal Samarasinghe, Dr. Jessica Rexach, Dr. Karen Gylys

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Progress in the development of organoid culture models opens up a new avenue for investigating extracellular vesicle (EV) biogenesis, cargos, and their role in intercellular communications in health and diseases allowing a more natural 3D environment without a risk of EV sample contamination with intracellular vesicles from tissue dissociation. Utilizing an organoid tauopathy model, our overall goal is to evaluate the role of EVs in immune cell reprogramming, leading to the establishment of a permissive environment for tau pathology spread and neuronal circuit disruption.

Methods: We compared four different methods of EV isolation from brain organoid culture media, including ultracentrifugation, polymer-based precipitation, size exclusive chromatography, and immunoprecipitation. Levels of exosomal markers and immune checkpoint molecules were evaluated in EVs isolated from cortical (CTX), ganglionic eminence (GE), and fused CTX+GE organoid cultures derived from tau R406W (frontotemporal dementia mutation) and isogenic control human iPSCs.

Results: Pan-exosomal immunoprecipitation, utilizing three tetraspanins enriched in exosomes (CD63, CD9, and CD81), was chosen for analysis as the most successful method of organoid EV isolation. We observed region- and genotype-specific changes in CD63 and CD47 expression in EVs isolated from the organoid cultures. There was a significant upregulation of CD63 in EV fractions from mutant CTX and CTX+GE organoid cultures compared to isogenic control, coinciding with a decrease in CD63 levels in mutant GE-derived EVs. These results indicate a differential effect of the tau R406W mutation on EV biogenesis and/or cell viability of excitatory (CTX) and inhibitory (GE) neurons and/or a region-specific effect on glial cells. An upregulation of CD47, but not another immune checkpoint molecule, PDL-1, in EVs isolated from mutant CTX+GE fused organoid cultures (Day 120), suggests a potential role of exosomal CD47 in immune cell regulation through interactions with its cognate receptor, signal regulatory protein alpha (SIRPα). Further experiments are required to evaluate this hypothesis.

Summary: Most neurodegenerative disorders, including tauopathies, are characterized by endo-lysosomal dysfunctions and dysregulation of intercellular communications. The accompanied changes in EV biogenesis and composition are part of the disease mechanism, and a better understanding of their role can provide potential therapeutic targets and biomarkers for disease progression.

PhD Ursula Sandau, Trevor McFarland, PhD Sierra Smith, MD Douglas Galasko, MD Joseph Quinn, MD, PhD Randy Woltjer, PhD Julie Saugstad

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Alzheimer's disease (AD) is the most common form of dementia and is the fifth leading cause of death for individuals aged 65 and older. With only six FDA approved therapies, none of which cure the disease, there is an urgent need to identify new therapeutic targets. Cerebrospinal fluid (CSF) contains brain derived EVs with cargo that reflects disease alterations. Thus, we performed a target prediction analysis with human CSF EV miRNA data from AD and control (CTL) donors, to identify relevant gene targets that we show are altered in human AD brain and may contribute to pathogenesis.

Methods: CSF from living donors (AD n = 28; CTL n = 28) was fractionated by size exclusion chromatography and characterized by transmission electron microscopy; immunoblot for EV and non-EV markers (e.g., flotillin, CD81, apolipoproteins); and tunable resistive pulse sensing. 190 miRNAs were assessed by qPCR using primer-probe arrays and those significantly changed in AD (FDR corrected) were used for target prediction and Ingenuity Pathway Analysis. Human postmortem frontal cortex (FC) and hippocampus from AD and CTL was used for miR-16-5p staining and immunoblot analysis of SNAP-25 and MUNC18-1.

Results: In AD CSF EVs, four miRNAs (miR-16-5p, -331-3p, -409-3p, -454-3p) were significantly increased compared to CTLs. CSF EV miR-16-5 expression was also found to be sex and genotype dependent, with significant increases specific to APOE4 positive AD females. We also found the miR-16-5p was expressed in human brain and trends towards increased expression in AD (n = 4) white matter, compared with CTLs (n = 3). Relevant to synaptic dysfunction in AD, miR-16-5p predicted targets included mRNAs integral to synaptic transmission (SNAP-25, MUNC18-1). In AD females both SNAP-25 and MUNC18-1 were significantly decreased in the hippocampus (n = 17) and FC (n = 12) compared with control females (hippocampus and FC, n = 11). While in AD males there was only a significant decrease in FC (n = 10) SNAP-25 compared with CTL males (n = 12) and no other differences (hippocampus: CTL n = 11, AD n = 17).

Summary/Conclusion: We demonstrate that miRNAs altered in CSF EVs are informative to changes that occur in brain and have the potential to identify targets that may serve as novel therapeutics to treat AD.

Tiana Koukoulis, Purnianto Adityas, David Finkelstein, Leah Beauchamp, Kevin Barnham, Dr Laura Vella

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Parkinson's disease (PD) is an increasingly common neurodegenerative disease. The etiology of idiopathic PD is complex and multifactorial involving environmental contributions, such as viral or bacterial infections and microbial dysbiosis, in genetically predisposed individuals.

A limited understanding of the molecular events that drive neuroinflammation and neurodegeneration causes a major hindrance in the development of efficacious treatments for PD. Understanding these systems and triggers of disease may provide novel biological drug targets for the development of neuroprotective treatments as well the discovery of biochemical biomarkers of preclinical disease.

Here, we will report on our preclinical Parkinson's disease research program that uses cell (bacterial, human and mouse) and animal models to elucidate the contribution of eukaryotic and microbiota-derived extracellular vesicles to disease progression.

Lien Cools, Dr. Cristiano Lucci, Sam Noppen, Dr. Charysse Vandendriessche, Drs. Kaat Verleye, Drs. Laura Raes, Elien Van Wonterghem, Prof. Inge Mertens, Prof. Dominique Schols, Prof. Roosmarijn E Vandenbroucke, Prof. Lies De Groef

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

The evolving role of extracellular vesicles (EVs) in intercellular communication within the central nervous systems (CNS) has sparked interest in using EVs as biomarkers, drug delivery tools and therapeutic targets for neurodegenerative diseases. Since established protocols to isolate EVs from CNS tissue are lacking, current research mainly uses cerebrospinal fluid or plasma, albeit both having drawbacks. Here, we propose using vitreous-derived EVs to investigate CNS EVs. Being an integral part of the CNS, the retina has a similar cellular composition as the brain and (patho)physiological processes are conserved. Only separated by the inner limiting membrane, the vitreous humor is suggested to be a liquid biopsy of the retina, and is known to be rich in EVs. Although vitreous-derived EVs are being studied in ophthalmological diseases, their application to study neurodegenerative processes has remained unexplored thus far.

Methods

Mouse vitreous-derived EVs were isolated via size exclusion chromatography, and their concentration and size were characterized with nanoparticle tracking analysis and electron microscopy. Western blot for tetraspanin markers, TSG101 and calnexin was performed on retinal lysates and EV fractions. Lastly, glial and neuronal cell markers were assessed via a surface plasmon resonance bioassay. In a second step, retinal neuroinflammation and -degeneration was induced via lipopolysaccharide injections or optic nerve crush, and changes in EV dynamics were evaluated. An ongoing proteomic analysis will assess the EV cargo in all experimental conditions.

Results

EV concentrations isolated from 30µL vitreous comprised on average 10E10 particles/mL, with diverse morphologies and sizes ranging between 50-350nm. EV fractions displayed several tetraspanin markers and TSG101, but not calnexin. Moreover, neuronal and glial EVs were identified in the vitreous humor. Lastly, neuroinflammatory or neurodegenerative stimuli impacted the EV release/uptake ratio, yet no discernible changes in EV size were found.

Summary

This study demonstrates the potential of the vitreous humor as a liquid biopsy to obtain and study retinal EVs. The presence of neuronal and glial EVs suggests that this biofluid provides unique opportunities to study CNS-derived EVs, including their involvement in neuroinflammatory and neurodegenerative processes. Ongoing proteomics analysis will help to further unravel their cargo and functions.

Dr Hannah Jackson, Dr Anna Grabowska, Dr Victoria James, Dr Federico Dajas-Bailador, Dr Beth Coyle, Dr Gareth Hathway

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

Childhood cancer-related pain and its treatment lack sufficient clinical attention due to limited understanding of the underlying mechanisms. While adult literature has extensively studied chemotherapy-induced peripheral neuropathic pain (CIPN), mechanisms in early life remain unexplored. In the CIPN literature there is a growing body of evidence for tumour derived factors altering pain processing. Our study aims to understand how tumour derived EVs interact with the developing nervous system and impact pain processing in childhood brain tumour survivors.

Methods:

We determined dose-response curves and IC50 values for several medulloblastoma (MB) cell lines using standard-of-care chemotherapy drugs: vincristine, etoposide, cisplatin, and lomustine. EVs were isolated via size exclusion chromatography and characterised through western blotting, flow cytometry and transmission electron microscopy (TEM). We quantified the effects of chemotherapy on EV release using ZetaView analysis. We then tested whether EVs from relevant MB cell lines could influence axon development in primary mouse embryonic day 16.5 dorsal root ganglion (DRG) neurons in vitro after pre-treatment with chemotherapy-treated EVs.

Results:

EVs from chemotherapy-treated and untreated cells exhibited similar size and shape. However, a combination of vincristine, etoposide, and cisplatin at low concentrations significantly increased EV secretion by MB cell lines. We are currently assessing the impact of these EVs on axonal length and growth in early-phase neuronal cultures of primary DRG neurons.

Conclusion:

Standard chemotherapy drugs substantially enhanced EV release from MB cells, and co-culture of embryonic DRG neurons with chemotherapy exposed EVs resulted in neuronal death. Our next step is to investigate whether administering these selected EVs in healthy animals alters pain responses and pain maturation, and to study the biodistribution of these EVs within the nervous system and body, after infusion into the cerebrospinal fluid as well as how EV cargo is altered by chemotherapeutics.

Mr William Phillips, Ms Irumi Amarasinghe, Dr Ebony Monson, Dr Nicholas Reynolds, Prof Karla Helbig, Dr Lesley Cheng, Prof Andrew F Hill

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Neuroinflammation is a fundamental aspect of multiple neurological conditions, including neurodegenerative conditions such as Alzheimer's disease and chronic traumatic encephalopathy (CTE). Microglia are the brain's primary immune cells involved with inflammation. Extracellular vesicles (EVs) are bilayered lipid nanoparticles that reflect a cell's state, making them a rich source of biomarkers. Omics methods, including proteomics, lipidomics, and small RNAseq, were used to evaluate changes in EV populations from LPS-treated microglia. Small-angle X-ray scattering (SAXS) and cryo transmission electron microscopy (Cryo-TEM) were used to comprehensively assess EVs from activated microglia and any associated morphology changes.

Methods

EVs were isolated from 200 mL of serum-free conditioned media from either control or 50 ng/mL LPS-treated SIM-A9 murine microglial cells by tangential flow filtration and size-exclusion chromatography. EV physical characteristics were examined using Zetaview nanoparticle tracking analysis and SAXS at the Australian Synchrotron. 30 Cryo-TEM images were taken per replicate, and vesicles were quantified. LC/MS proteomics and lipidomics were conducted at the Bio21 Melbourne Mass Spectrometry and Proteomics Facility. Small RNA isolation was performed using established protocols, and RNAseq conducted an Ion GeneStudio S5.

Results

NTA measurements from treated and untreated cells did not reveal any significant changes in the size profile of the EVs. A lower particle count but larger size in LPS versus control was detected in both SAXS and Cryo-TEM. Additionally, Cryo-TEM saw more multi-membraned vesicles compared to control. Demonstrating the utility of SAXS and Cryo-TEM for evaluating EV morphology.

Proteomic analysis revealed enrichment for ribosomal components in EVs isolated from LPS-treated cells and immune response components such as IL-1β and the inflammasome component NLRP3. Comparing lipid composition between LPS treated and control also provided further insight into compositional EV changes during inflammation. Finally, small RNA analysis found multiple enriched small RNA, including mir-146a, upregulated in LPS EVs compared to control.

Conclusions

This study reveals the dynamic and heterogeneous nature of EVs, both compositionally and morphologically, while highlighting their potential as valuable biomarkers for evaluating inflammation in the brain. These findings contribute significantly to understanding the role of microglial EVs during neuroinflammation and their potential role in a greater systemic context.

Dr Ioannis Sotiropoulos, Dr Patricia Gomes, Dr Cristian Bodo, Dr Carlos Noguera-Ortiz, Dr Martina Samiotaki, Dr Minghao Chen, Dr Carina Soares-Cunha, Dr Joana M. Silva, Dr Bárbara Coimbra, Dr George Stamatakis, Dr Liliana Santos, Dr George Panayotou, Professor Clarissa L. Waites, Proffessor Christos Gatsogiannis, Professor Nuno Sousa, Professor Dimitrios Kapogiannis, Dr Bruno Costa-Silva

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: In the era of Precision Medicine, extracellular vesicles (EVs) exhibit great potential for the theragnostic of brain disorders such as Alzheimer's disease (AD), a complex disease with no effective treatment and poorly understood risk factors, where pathological heterogeneity and diverse clinical presentations complicate the development of precise patient-tailored therapies. Thus, the collection and characterization of physiologically relevant EVs, as well as the study of its precipitating/risk factors, are of the utmost importance. However, standard brain EV isolation approaches rely on tissue dissociation, which can contaminate EV preparations with intracellular vesicles.

Methods: Based on a multiscale analysis, including cryo-EM, label-free proteomics, and ExoView, we hereby present a novel isolation method of small EVs (sEVs), named “release method”, based on their spontaneous release from the human and mouse brain tissue. Moreover, we have also tested the release method under conditions of chronic psychological stress, a known risk factor of AD.

Results: Our advanced EVs analysis demonstrated that the release method represents an efficient method that captures a small EV-enriched population spontaneously released by brain tissue. In addition, we tested the significance of the release method under conditions where biogenesis/secretion of sEVs was pharmacologically manipulated and under exposure to chronic stress, a clinically-relevant precipitant of AD. Here we found that the release method monitors the drug-evoked inhibition or enhancement of sEVs secretion in a very sensitive manner, while chronic stress induced the secretion of Tau-carrying brain-derived EVs accompanied by memory loss and mood deficits suggesting a potential role of sEVs in the brain response to stress and progression of related stress-driven brain pathologies, such as AD.

Summary/Conclusion: This spontaneous release method may contribute to the characterization and biomarker profile of physiologically relevant brain-derived exosomes in brain function and pathology. Also, the clarification of the “footprints” of chronic stress on brain EVs may contribute to the identification of stress biomarkers helping towards early detection of stress-related disorders. Given that the modern lifestyle increasingly exposes individuals to high stress loads, the understanding of the mechanistic link(s) between chronic stress and AD pathogenesis may facilitate both AD treatment and prevention.

PhD student Patricia Hernández-López, Dr. Elisabeth Rackles, Dr. Oihane E. Albóniga, Dr. Juan Manuel Falcon-Pérez

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Alzheimer's disease (AD) is the most common form of dementia, which affects more than 55 million people worldwide (according to WHO in 2023). It is characterized by loss of cognitive functions through the accumulation of insoluble deposits (hyperphosphorylated-tau and amyloid-β-peptide-42) in brain. In the context of metabolism, pathological changes are also observed consistently during the progression of the disease. Recently, a link between the presence of specific metabolic pathway intermediates in the cell and the development of AD has been described (González-Domínguez et al. 2021). Extracellular vesicles (EVs) secreted by Central Nervous System (CNS)-derived cells are considered to be involved in AD pathogenesis as they carry a cargo including AD intermediators. The exosomal cargo, which varies on the biological-pathological state of the secretory cell, is considered as a potential source of reliable biomarkers for different diseases. So, we hypothesize that comparing the metabolic cargo of CNS-derived EVs between AD patients and healthy controls could be a tool to understand the metabolic changes in AD.

Methods: EVs were isolated from post-mortem human temporal cortex tissues using in-house size exclusion chromatography. Two groups of samples were included: AD patients, displaying Braak stages III-V (n = 14); and healthy controls with no evidence of dementia (n = 10). Lipid and metabolite profiling of EVs were analysed by ultra-high performance liquid chromatography coupled to a time of flight mass spectrometer (UHPLC-ToF-MS) using positive and negative ionization modes to find those compounds that were carried in these EVs and were involved in group separation.

Results: In total, we found 8 metabolic features from polar positive and 43 from lipidomics negative analysis that are responsible of the separation observed between the two EV groups (AD patients and healthy controls).

Summary/Conclusion: Untargeted metabolomics and lipidomics analysis of EVs showed some features that are different between AD patients and healthy control.

Keywords: extracellular vesicles, Alzheimer's disease, metabolomics, lipidomics

Mr Adityas Purnianto, Ms Mitali Kulkarni, Professor Scott Ayton, Professor Catriona McLean, Professor Ashley Bush, Professor David Finkelstein, Professor Kevin Barnham, Dr Laura Vella

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Under oxidative stress, cells release EVs with an altered cargo composition, potentially as a protective response against oxidative stress. This phenomenon has been reported in various diseases, especially those associated with inflammation and aging. However, this has not been thoroughly investigated in Parkinson's disease (PD), despite the prominence of oxidative stress and mitochondrial dysfunction as features of the disease. Therefore, this study has investigated the impact of oxidative stress on EV composition within the context of PD.

Methods

Small EVs (sEVs) were isolated using size exclusion chromatography from two in vitro models of oxidative stress relevant to PD (rotenone, a mitochondrial toxin, and erastin, an inducer of iron-dependent cell death) as well as post-mortem caudate tissue of PD subjects. sEVs were validated by immunoblotting, nanoparticle tracking analysis, and transmission electron microscopy. The EVs’ content of proteins with lipid peroxidation adducts and mitochondrial proteins were examined using LC-MS-MS bottom up proteomic and immunoblotting. Density gradient ultracentrifugation (DGUC) was used to isolate a subpopulation of sEV with a mitochondrial origin.

Results

Rotenone and erastin-induced oxidative stress significantly increased mitochondrial proteins and proteins with lipid peroxidation adducts in sEVs while minimal changes were observed in the cells. The change in mitochondrial protein content in sEVs, particularly prominent in the rotenone model, suggests a phenomenon highly sensitive to oxidative stress originating from mitochondria. These findings were supported by identification of increased mitochondrial oxidative phosphorylation complexes in the sEVs isolated from post-mortem caudate tissue of PD subjects. A subpopulation of sEVs, separated using DGUC, contained the mitochondrial proteins and proteins with lipid peroxidation adducts. This suggests a common origin for these types of protein cargo within sEVs.

Conclusion

This study demonstrates that sEVs can serve as sensitive indicators of oxidative stress and mitochondrial dysfunction in PD, owing to their ability to export cellular components damaged by oxidative stress, such as mitochondrial components. Hence, EVs may offer a promising avenue as a potential source of biomarker for early stages of PD when cellular changes are relatively minimal and for investigating early neuropathological changes linked to oxidative stress and mitochondrial dysfunction in PD.

Mr Rishabh Singh, Ms Sanskriti Rai, Dr Prahalad Singh Bharti, Dr Prasun Chatterjee, Dr Saroj Kumar

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Alzheimer's disease (AD) is a neurodegenerative disease characterized by Aβ plaques and neurofibrillary tangles, with chronic inflammation and synaptic dysfunction playing a significant contributor to disease progression and cognitive decline. Small extracellular vesicles (sEVs) are implicated in AD progression by facilitating the spread of pathological proteins and inflammatory cytokines. This study investigates the role of plasma-derived sEVs (PsEVs) in synaptic dysfunction and neuroinflammation and their association with amyloid-β and tau pathologies in AD progression.

Method: A total of 45 [15 each in AD, mild cognitive impairment (MCI), and age-matched healthy control (AMC)] subjects were recruited, and written informed consent was obtained (Ethics Ref No: IECPG-670/25.08.2022). PsEVs were isolated using a chemical precipitation method, and their morphology was characterized by transmission electron microscopy. The size and concentration of PsEVs were determined using nanoparticle tracking analysis (NTA). Antibody-based validation of PsEVs was done using CD63, CD81, TSG101, and L1CAM antibodies. Synaptic dysfunction and neuroinflammation were evaluated with Synaptophysin, Glial Fibrillary Acidic Protein (GFAP), IL-1β, and TNF-α antibodies. AD-specific markers, Aβ-42, and pTau were examined using Western blot and ELISA.

Results: Our findings reveal higher concentrations of PsEVs in AD and MCI compared to AMC (p<0.0001). Aβ42 and pTau expression are significantly elevated in MCI (AUC = 0.9711, sn = 100%, sp = 80%, p<0.0001; AUC = 0.9689, sn = 100%, sp = 93.3%, p<0.0001, respectively); AD (AUC = 1, sn = 100%, sp = 100%, p = p<0.0001; AUC = 1, sn = 100%, sp = 100%, p<0.0001, respectively). Synaptophysin exhibits decreased expression from AMC to MCI to AD (p = 0.047), whereas, IL-1β, TNF-α, and GFAP showed increased expression in MCI and AD compared to AMC (p = 0.002, 0.0006, 0.0184, respectively). The increased levels of PsEVs correlate with synaptic dysfunction and neuroinflammation.

Conclusion: Elevated PsEVs and upregulated Aβ42 and pTau expression show high diagnostic accuracy in AD. The decreasing synaptophysin expression and increased neuroinflammatory markers in AD and MCI patients suggest synaptic degeneration and neuroinflammation as potential indicators. These findings support the potential of sEV-associated biomarkers for AD diagnosis and highlight synaptic dysfunction and neuroinflammation in disease progression.

Funding: CSIR [09/006(0533)/2021-EMR-I], ICMR (2020-1194), DHR (YSS/2020/000158).

Keywords: Alzheimer's disease, mild cognitive impairment, small extracellular vesicles, synaptic dysfunction, neuroinflammation.

Mrs. Rachel Rachel, Dr. Randy Carney, Hyehyun Kim, Dr. Erkin Seker

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Understanding interaction of EVs with CNS cells will be important for identifying mechanisms of disease, as well as for developing EV-based therapies for them. However, models to study this often use isolated single cell types that lack the intercellular signaling that may mediate EV function and fate, such as the complex processes in neuroinflammation. Critically, metastatic niche formation in the brain is hypothesized to be mediated in part by cancer-derived EVs, though details are poorly understood. Here, we employ primary rat cortical tri-culture (neurons, astrocytes, microglia) and contrasting co-culture (neurons, astrocytes) to compare interactions of EVs from breast cancer cells (MDA-MB-231) to their brain-tropic variant (231-Br), and a non-cancer control (HEK293T), to identify EV fate and neuroinflammatory response.

Methods

EVs were isolated via differential ultracentrifugation and characterized via nanoparticle tracking analysis, flow cytometry, resistive pulse sensing, and interferometric imaging. Cells from rat-pup neocortices were dissociated and grown in chambered coverslips. For uptake, EVs were fluorescently labeled, separated by size exclusion chromatography, and incubated at 7.5*109 EVs/mL in cultures for 4-24 hrs. Cells were immunostained and imaged by confocal microscopy to assess uptake, while cell culture media was collected and probed for cytokines to assess inflammatory response.

Results

231-Br and HEK293T EVs were taken up at similar levels while MDA-MB-231 had lower overall uptake in co- and tri-culture. EVs mainly associated with neurons and astrocytes. Despite low uptake, MDA-MB-231 EVs resulted in the highest increase in inflammatory cytokines (e.g., MCP-1 and MIP-1α) in tri-culture. Though 231-Br EVs had similar cytokine trends, increases were not significant compared to controls. In contrast, HEK293T EVs decreased VEGF, MCP-1, and IP-10 expression. Cytokine expression (except of fractalkine, produced by neurons) did not change in co-culture.

Discussion

These results suggest that functional effect and broad uptake rate, rather than cell targeting, could be an important contributor to EV-mediated metastasis, with EVs from brain-metastatic cells causing less inflammation yet having higher overall uptake. More broadly, the interaction of EVs with each cell type suggests that EV function may be multifaceted, highlighting the importance of complex in vitro systems to better understand EV function in the CNS.

Dr Natasha Vassileff, Dr Rohan Lowe, Dr Keshava Datta, Professor Catriona McLean, Professor Andrew Hill, Dr Jereme Spiers

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: The cargo of extracellular vesicles (EVs) is known to be altered in neurodegenerative conditions like Alzheimer's disease (AD) and may facilitate disease pathology. Neuroinflammation is an underlying feature of neurodegenerative conditions including AD and is characterised by the production of numerous cytokines and free radicals such as nitric oxide (NO). NO exerts its effect through post-translational S-nitrosylation of target cysteine thiols (nitrosothiols), altering the protein's function and subcellular localisation, which may include packaging into EVs. Despite this, the NO-modified proteome of EVs in AD has not been investigated. Therefore, this study aimed to determine the NO signalling changes occurring through post-translational modifications on EV protein cargo in AD.

Methods: Brain derived EVs (BDEVs) isolated from the frontal cortex of human post-mortem AD brain tissue samples using enzymatic digestion and sucrose density gradient separation were thoroughly characterised, to meet the Minimal Information for Studies of Extracellular Vesicles guidelines, before undergoing iodolabelling and comparative proteomic analysis to identify NO-induced post-translational changes.

Results: The iodolabelling proteomic analysis revealed a protein profile heavily representative of microglial and antioxidant proteins indicative of neuroinflammation in the AD BDEVs. These included inflammatory, membrane surface, and mitochondrial proteins, suggesting protein nitrosothiols may play an important role in neuroinflammatory communication via EVs in AD.

Conclusions: Together, this study is the first to show NO-induced S-nitrosothiol post-translational changes in BDEVs, demonstrating the effect NO signalling exerts on the protein cargo of EVs during AD. Further work should investigate the functional consequence of protein nitrosothiolation of EV cargo and how this may affect cellular processes in AD.

Miss Mitali Manish Kulkarni, Mr. Adityas Purnianto, Miss Tiana Koukoulis, Miss Huaqi Su, Miss Geraldine Kong, Professor Anthony Hannan, Dr. Laura.J Vella

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Huntington's disease (HD) is a genetic neurological disorder that is characterised by decline in movement, cognition, and behavior. It is imperative to understand the progression of HD due to the absence of treatment and therapeutic targets. Despite the genetic cause of HD being known, there is still considerable progress to be made to understand the cellular and molecular mechanism associated with the disease pathogenesis. In the past decade, research has demonstrated that extracellular vesicles (EVs) play a role in the pathogenesis of neurodegenerative diseases and that the protein content of EVs can provide insights into underlying disease mechanisms. To elucidate the potential role of EVs in HD, we investigated the protein cargo of EVs isolated from human tissue and a mouse model of HD.

Methods: EVs were isolated from the extracellular matrix of human HD and neurological control parietal brain tissue (n = 8) or the cortex, striatum, and hippocampus of the R6/1 transgenic mouse model of HD and wild-type controls (n = 10) by differential centrifugation, size exclusion chromatography and ultrafiltration. EVs were characterised by immunoblotting, transmission electron microscopy and liquid chromatography mass spectrometry-based proteomics.

Results: Our studies highlighted the enrichment of EV markers in immunoblotting and proteomics demonstrating successful isolation of EVs. We identified an overrepresentation of proteins associated with mRNA splicing, via the spliceosome in human HD EVs. These novel findings demonstrated that spliceosome and spliceosomal components are exported by the cells via EVs in HD. In the R6/1 mice model, we identified overrepresentation of proteins associated with the MAPK signaling cascade revealing that EVs export proteins associated with the MAPK signaling pathway.

Summary/Conclusions: This is the first study to comprehensively characterise the cargo of EVs in HD human and mouse brain. Our findings illustrate the potential of EVs to offer insights into both early and late stage disease mechanisms in HD.

Dr. Yu-Ting Weng, Ph.D. Yijuang Chern

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

Translin-Associated Factor X (TRAX) controls the expression of a specific set of miRNAs. Ablation of TRAX in striatum leads to the disruption of miRNA-mRNA networks, accelerating the progression of Huntington's disease (HD), an inherited neurodegenerative disease linked to the elongated glutamine-containing mutant huntingtin protein. Unexpectedly, reducing TRAX expression, primarily expressed in neurons, decreases several signature genes in microglia that lack TRAX expression, suggesting the role of TRAX in neuron-microglia communication. Our investigation focuses on the potential of TRAX to modify the composition of extracellular vesicles (EVs), thereby impacting the identity of microglia receiving these EVs.

Methods:

To examine the effect of TRAX on EV composition, we established TRAX knockout WT and HD striatal cell lines using CRISPR-Cas9. EVs were isolated from control and TRAX knockout cell culture medium containing 10% of EV-depleted FBS by differential centrifugation followed by total exosome isolation kit (Invitrogen) and characterized by nanoparticle tracking (NTA), western blotting and miRNA sequencing. To assess the effect of EVs secreted by TRAX knockout cells on microglial identity, WT primary microglia were treated with EVs secreted by control and TRAX knockout cells and the expression of microglial signature genes was evaluated by qRT-PCR.

Results:

The isolated EVs exhibited enrichment of EV markers, CD63 and TSG101, in comparison to the total cell lysate. Surprisingly, TRAX was detected in EVs secreted by both WT and HD striatal cells, suggesting the potential function of TRAX within EVs. Treatment of WT primary microglia with EVs secreted by TRAX knockout HD striatal cells resulted in the loss of microglial identity. We identified 53 differentially expressed (DE) miRNAs, with a log2 fold change > 1 or < -1 and p < 0.05, between EVs secreted by control and TRAX knockout HD striatal cell lines. Further analysis of the predicted miRNA-targeted genes suggested the involvement of several these genes in regulating microglial signature genes. The characterization of the TRAX-regulated EV miRNA-mRNA axis in modulation of microglia identity will be discussed.

Summary/Conclusions:

We demonstrated that TRAX affects the identity of microglia that uptake neuron-secreted EVs by potentially regulating the miRNA content within the EVs.

Dr Cristian-Daniel Llach¹, Ms Gia Han Le¹, Dr Gerard Anmella², Dr Joshua Rosenblat¹, Dr Anna Gimenez-Palomo², Dr Isabella Pacchiarotti², Dr Eduard Vieta², Dr Roger McIntyre¹, Dr Rodrigo Mansur¹

¹University Of Toronto, Toronto, Canada, ²Bipolar and Depressive Disorders Unit, Hospital Clinic de Barcelona, Barcelona, Spain

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: EVs hold promise as CNS biomarkers, traversing the blood-brain barrier with diverse cargo and offering non-invasive “liquid biopsies” for brain pathology assessment. This systematic review consolidates EV research in mood disorders, highlighting their biomarker and therapeutic potential.

Methods: Studies of extracellular vesicles in both pre-clinical and clinical research on mood disorders were identified from PubMed, Scopus, Web of Science and Psycinfo from 2010 to December 2023. Risk of bias was assessed, and results are presented as a systematic review following PRISMA guidelines.

Results: Limited research has explored extracellular vesicles in mood disorders, with the existing evidence lacking reproducibility. Besides, heterogeneity in materials and methods and overall lack of adherence to MISEV guidelines makes it difficult to synthetize all results. Most studies focused on exosome mi-RNA cargo, reporting interesting molecules such as miR-139-5p, miR-26a, miR-335-5p or miR-1292-3p, that could help differentiate between healthy subjects and mood disorders, as well as among diverse subtypes of mood disorders. Some of them may be useful to identify subthreshold depression or predict antidepressant response, especially those regulating TLR4. Noteworthy proteins and metabolites in EVs, including SERPINF1 and various metabolites, exhibited potential as biomarkers for MDD and BD. Several studies linked their findings to synaptic plasticity and neurogenesis, to the theory of neuroinflammation, insulin resistance, mitochondrial or circadian rhythms dysfunction, as well as to the functioning of key molecules like GSK-3, BDNF, VEGF or second messenger systems such as MAPK, Ras and PI3K-AKT. A work under the umbrella of the gut-brain axis paradigm also focused on bacteria-derived exosomes to differentiate MDD from non-depressive patients. On the other hand, engineered EVs, like RVG-circDYM EVs, hold promise for targeted gene therapy in MDD, exhibiting alleviation of depressive symptoms in mouse models.

Summary: EVs, and neural EVs in particular, exhibit significant potential in aiding the diagnosis and treatment of intricate mood disorders. Enhanced EV-based methodologies are gaining traction, paving the way for personalized treatment strategies. However, technical limitations remain and regulatory approval for EV-based therapies is pending. Integration of various data sources could enhance precision in biomarker discovery, heralding a potential era of EV-based brain diagnostics and therapeutics.

Rikke Bæk, Maiken Mellergaard, Rikke Wehner Rasmussen, Rikke Bülow Eschen, Evo Lindersson Søndergaard, Aase Handberg, Malene Møller Jørgensen

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction

Extracellular vesicles (EVs) in plasma have great diagnostic potential as biomarkers for several diseases. To use EVs in clinical settings, it is of great importance to establish whether EV phenotypes and numbers change over time in a healthy cohort. The aim of this study was to determine both the long-term (week-to-week) as well as the short-term (day-to-day) variation of EV concentration and composition in plasma from healthy donors and to compare the findings with blood cell counts.

2) Methods

Venous peripheral blood (citrate and EDTA) was obtained from 4 fasting healthy donors once a week over a period of 6 weeks. Furthermore, blood samples were drawn daily for one week. Blood cell counts were measured using a Sysmex XN-1000. Small EV concentration and composition were analyzed by the EV Array (Jørgensen et al., 2013, JEV) using antibodies against 23 selected surface-markers for capture in combination with detection by antibodies against CD9, CD63, CD81 or a cocktail of these. Capture antibodies included antibodies against EVs in general (CD9, CD63, CD81, Alix, Flotilin-1 etc.), and immunological and inflammatory markers (CD4, CD8, CD80, HLA ABC, HLA DR/DP/DQ, TNF RI and RII). In comparison, high resolution flow cytometry (hfcm, Sanden et al., 2018) was used for analyzing EV populations based on the expression of CD9, CD63, and CD81 as well as colocalization of these markers. Nanoparticle Tracing analysis was performed to determine size and numbers of EVs.

3) Results

In total, 40 plasma samples were collected and analyzed. Large inter-individual variation was found in EVs analyzed by EV Array and hFCM. For certain blood cell types, the long-term intra-individual variation varied significantly over time, which was not seen in the context of small EVs. Minor short-term and intra-individual variation (day-to-day) was observed in cellular composition but was not reflected in the obtained phenotypes of small EVs.

4) Summary/Conclusion:

A few of the selected EV surface markers from circulating blood cells showed minor changes over time, although this did not reflect the significant changes identified at the cellular level.

PHD Student Guillaume Valade, PHD Student Marine DE TADDEO, Mrs Muriel NIVET, Mrs Marion GROSBOT, Mrs Claire LANGLE, Mrs Sylvie GOULINET, PHD Philippe MAUDUIT, Mr Vincent JUNG JUNG, PHD Chiara GUERRERA, MD, PHD Sébastien BANZET, PHD Juliette PELTZER, PHD Marina TROUILLAS

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Acellular therapies have recently emerged as a new therapeutic option in the field of tissue repair. Extracellular Vesicles (EV) and, more generally, the secretome derived from Mesenchymal Stromal Cells (MSC) possess immunomodulatory and pro-healing properties similar to those of MSC themselves. Moreover, MSC are able to adapt their secretion profile in response to stimuli of their environment and we have previously shown that IL-1β priming of MSC is effective to improve the properties of their secretome. This study aims to characterize the impact of IL-1β priming doses and different secretome purifications containing EV more and less enriched in soluble factors (EV+SF), with a wide range of analytical methods, and to better define our products.

Methods

MSC are primed with 1 or 5ng/mL of IL-1β for 24h. After 72 hours of secretion in serum-free medium, the conditioned medium is collected. Tangential flow filtration is used to isolate and concentrate EV or EV+FS, using filters with respective cut-off sizes of 500 or 10kDa. Particles concentration and size are measured using nanoparticle tracking analysis and interferometric light microscopy technology. Cryo-electron microscopy is used to confirm EV structure. Total protein is quantified by BCA technic. Phenotype is analyzed by mass spectrometry and flow cytometry for nanoparticle analysis.

Results

Our results indicate that neither IL-1β priming doses nor concentration methods induce major variations in particle concentrations and sizes. However, significant differences are observed between NTA, Videodrop and NanoFCM methods. As expected, our products express CD9, CD63, CD81 but also MSC specific markers. Similar mass spectrometry profiles are obtained between conditions with the different doses of priming. However, major differences are observed between EV and EV+FS. EV+FS are more enriched in growth factors, interleukins, chemokines, proteins linked to matrix remodeling and also metabolic enzyme. The EV fraction contains more proteins implicated in vesicles trafficking.

Summary/Conclusion

This study, using a broad spectrum of characterization methods, allowed to improve our understanding of the impact of production/purification processes on the characteristics of our acellular products. This knowledge will allow us to develop scalable and efficient manufacture of medicinal products for tissue repair.

Ms. Ayano Higaki, Mr. Keita Inoue, Ms. Mizuki Kobayashi, Ms. Makiko Hiraoka, Mr. Yoshitaka Kawakami, Ph.D. Naohiro Seo

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Among extracellular vesicles (EVs), exosomes (EXO) derived from multivesicular bodies of late endosome are highly expected to be used in clinical treatment and diagnosis. However, there is no existing technology to easily purify EXO from biological samples. Since it has been reported that EXO and other EVs can be separated by differences in surface negative charge [1], we developed a new microfiltration membrane (developed purification membrane) with ion-exchange groups and micropores through which EXO can pass, and evaluated its accuracy in EXO purification.

Methods: EVs were prepared by passing HEK293 culture supernatant through the developed purification membrane followed by eluting with buffer solutions containing high salt. The obtained EVs were characterized by measurement of physical properties such as zeta potential, proteome analysis, western blot analysis, and DNA content. In addition, we conducted administration experiments of the obtained EVs in mice to investigate their pharmacokinetics.

Results: By using the developed membrane, EVs that contained the EXO markers such as Tsg101, and were free of actin and DNA, could be prepared in a short time with a high unnecessary protein removal rate. In a pharmacokinetic study, it was confirmed that the EVs purified by the developed membrane circulated systemically and localized in various organs, whereas most of the EVs prepared by ultracentrifugation were localized mainly in the liver via macrophages.

Summary/Conclusion: This method has enabled us to purify EXO-rich EVs that have the ability to circulate systemically. Our technology can be applied not only to basic research areas but also to clinical applications by incorporating it into GMP-manufacturing processes, and has the potential to become indispensable for future EV research.

[1] N. Seo, et al., J. Extracell Vesicles, (2022) 11(3): e12205.

Mr Fredrik Stridfeldt, MSc Hanna Kylhammar, Dr Vipin Agrawal, MSc VIkash Pandey, Dr André Görgens, Professor Samir El Andaloussi, Professor Dhrubaditya Mitra, Professor Apurba Dev

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Studies have shown that the stiffness properties of extracellular vesicles might be important in disease and drug monitoring as the mechanical properties may change as a response to treatment or because of disease progression. However, multiple reasons including their small size, liquid-like membrane, and inhomogeneous composition complicate the application of traditional and well-established methods. Atomic Force Microscopy (AFM) is a high-resolution technique that enables precise single EV analysis. It can extract both physical properties such as height and radius but also biomechanical properties such as linear stiffness K and bending modulus κ. However, there exists no satisfiable model explaining the vesicle's response to indentation and earlier studies report a large spread of stiffness values.

Methods

4 EV samples (WT, WT-SEC, CD63-KO, and Pan-KO) were adhered to poly-l-lysine-coated substrates and imaged in AFM. Individual EVs were scanned at a resolution of 4 nm/pixel. 150 indentations were carried out up to a set force of 800 pN. From there, linear stiffness was extracted from all approach curves and the bending modulus was extracted from the retract curves showing membrane tether formations to the AFM tip. A liquid shell theory was developed to describe indentation experiments.

Results

45 WT, 36 WT-SEC, 24 CD63-KO, and 18 Pan-KO EVs were investigated. T-testing reveals an increase in mean linear stiffness in the CD63-KO vesicles (11.7 mN/m) compared to the WT vesicles (7.2 mN/m). Analysis of one individual EV reveals a spread of stiffness values. By comparing the possible linear stiffness distributions, we discover a larger uncertainty within the samples than across different samples. This complexity and heterogeneity on a single EV level would have been missed without repeated measurements. No difference could be found in the bending modulus (27-34 kBT).

Summary/Conclusion

This study highlights the importance of repeated measurements on a single vesicle when analyzing biomechanical properties. Our data suggest that both K and κ can vary significantly on both individual and ensemble levels. We present a theoretical model that explains most of the indentation behavior. We also suggest an alternative statistical method to better handle the low throughput AFM data.

Dr. Hirotaka Nishimura, Dr. Tomofumi Yamamoto, Dr. Noritaka Hashii, Dr. Akiko Ishii-Watabe

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Clinical applications of human mesenchymal stem cell (MSC)-derived EVs are being actively investigated. EV preparations often contain a variety of extracellular vesicles, lipoproteins, and other particulate matter. Furthermore, there is heterogeneity in the physical properties and contents of EVs of the same size range. These variabilities may impact their pharmacological activity and safety, necessitating robust quality control, which is not established yet. This study aimed to evaluate the heterogeneity of particle size and charge in MSC-derived EV samples using high-performance liquid chromatography (HPLC) and multi-angle light scattering (MALS).

Methods: Several batches of EV preparations purified and condensed from MSC culture supernatants using 100k MWCO tangential flow filtration (TFF) were used. Size exclusion HPLC (SEC)-UV-MALS was employed to analyze soluble protein impurities and particle size variation across batches and enrichment rates. Fractions of SEC-MALS were analyzed by CD9/CD63 ELISA to identify particle types in each fraction. Anion exchange HPLC (AEX)-MALS was utilized for the assessment of surface charge heterogeneity of EV preparations. Fractions of AEX-MALS were analyzed by SEC-MALS, CD9/CD63, and phosphatidylserine (PS)/CD63 ELISA.

Results: SEC-UV-MALS revealed that the EV samples contained soluble proteins and three particle populations with different sizes, and the particle composition and the amount of protein impurities varied with the production batch and concentration rate. CD9/CD63 ELISA revealed that exosomes were mainly contained in the main peak in the SEC-MALS chromatogram, with very few in the other peaks. The analysis by AEX-MALS revealed the presence of several types of particles with different charges in the EV samples, and that their composition varied with the enrichment rate in TFF. SEC-MALS analysis of each AEX-fraction revealed that the fractions contained the same size particles with different charges. These fractions were assessed by ELISA and found to contain exosomes and to differ in PS presentation, which suggested that PS contributes to the charge diversity of exosomes.

Conclusion: SEC-MALS and AEX-MALS effectively characterized size and charge heterogeneity in EV samples. Variances due to production batches and enrichment rates were readily discernible. These methodologies would help quality control of EV-based therapies.

Funding: This work was supported by AMED under Grant No.JP23mk0101218.

Dr.med. Linda-marie Mulzer¹, Tim Felger¹, PD Dr. med. habil. Dr. rer. nat. Luis Muñoz², Gesa Engl¹, Prof. Dr. med. Heiko Reutter¹, Leila Pourtalebijahromi³, Prof. Dr. Gregor Fuhrmann³, Philipp Arnold⁴, Dr. med. Alina Hilger¹

¹Department of Pediatrics and Adolescent Medicine, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany, ²Department of Rheumatology and Immunology, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany, ³Friedrich-Alexander University Erlangen-Nürnberg, Department of Biology, Chair of Pharmaceutical Biology, Erlangen, Germany, ⁴Friedrich-Alexander University Erlangen-Nürnberg, Institute for Functional and Clinical Anatomy, Erlangen, Germany

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

EVs play a crucial role in cell migration and differentiation. Despite zebrafish being a well-established vertebrate model, limited knowledge exists about EVs in zebrafish embryogenesis. This pioneering study investigates large (lEVs) and small (sEVs) EVs throughout the first 96 hours of zebrafish larvae (zfl) development, analysing size and number changes over time.

Methods:

Wild-type AB strain zfl were collected at 24, 48, 72 and 96 hours post fertilization (hpf) and mechanically homogenized. LEVs and sEVs were isolated by differential centrifugation and characterized using transmission electron microscopy (TEM), flow cytometry, and nanoparticle tracking analysis (NTA). AxV-FITC and FM4-64 were used during flow cytometry to detect phosphatidylserine and a lipid bilayer. Statistics were conducted using Prism 6.0. Results were considered significant at p<0.05.

Results:

TEM showed a clear population of lEVs and sEVs. NTA revealed a significant separation of the lEV and sEV fractions based on their size at every observation point and showed a significantly increasing number of lEVs and sEVs over time with a peak at 72hpf. No correlation of the increase in numbers of EVs with the length development of the zfl was detected. Further NTA indicated that sEVs become significantly larger over time. Flow cytometry showed high positivity of lEVs for AxV-FITC (99,8%) and FM4-64 (99,0%).

Conclusion:

The significant size difference observed via NTA indicates the quality of the isolation method. Although the number of EVs increase over time, no correlation with length growth of the zfl was detected. With 72hpf the highest number of EVs was observes, at this time most organs have formed, mainly maturation and growth occur; the decreasing number of EVs afterwards could be due to a reduced need for EV-mediated cross talk. EVs did not only increase in number but also in size. Since EVs serve as cargo deliveries, larger EVs may reflect a higher transport capacity. Flow cytometry showed a high purity of the lEV samples with a strong expression of phosphatidylserine and the presence of a lipid bilayer. We suggest that an increase in EV number and EV size is necessary to orchestrate the maturing zfl.

Senior Investigator Marianna Kulka, Dr. Marcelo Marcet-Palacios, Sabrina Rodrigues Meira

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Small extracellular vesicles (EV), or exosomes, are 30-150 nm phospholipid-encased nanoparticles that carry information between cells. Mast cells are myeloid-derived immune cells that mediate innate/adaptive immunity, tissue remodeling, allergic inflammation and clearance of parasitic infection. Methods: Human mast cells (HMC-1 and LAD2) were activated and their production of exosomes was analyzed by proteomic profiling, transmission electron microscopy (TEM), nuclear magnetic resonance (NMR) metabolomic profiling and flow cytometry. Results: Activated mast cells produced at least three distinct subpopulations of EVs with unique structures observed by EM. Atom scale modeling indicated that each EV size could potentially carry specific protein configurations. Proteomic profiling showed unique protein content, and a comparison of EV proteomes from activated and resting EVs showed that 16 proteins were significantly more abundant and 32 less abundant in the activated EVs. A STRING analysis of the more abundant proteins revealed a correlation with mitochondria biology (COX6A1 – Cytochrome c oxidase subunit 6A1), catalytic activity (MAP4K4 – Mitogen-activated protein kinase 4), calcium mediated signaling (PPP3CB - Serine/threonine-protein phosphatase), as well as regulators of intracellular membrane trafficking (RAB31 – Ras-related protein) and exon junction complex components (CCDC9 – Coiled-coil domain containing protein 9). On the other hand, the STRING analysis of the less abundant proteins revealed strong correlation with the protein kinase domain, microtubule turnover regulation (KIF2C – Kinesin-like protein), and chromatin assembly mediator (CHAF1B – Chromatin assembly factor 1 subunit B). The proteomic profiling also revealed the presence of the markers CD9, CD81, CD63, ALIX, and TSG101 in both activated and resting samples, which indicates the presence of characteristic EVs and that the cell activation process does not significantly affect EV marker expression. EVs from activated cells induced an increase in the metabolic rate of target cells, suggesting that the EV contents modified the mitochondrial function of target cells; the proliferation and viability of the target cells remained unchanged. Conclusions: This data suggests that activated cells produce different populations of EVs that differ in size and cargo that activates the metabolomic profile of target cells.

Prof David Greening, Mr Alin Rai, Ms Haoyun Fang, Ms Bethany Claridge, Mr David Greening

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

In recent decades the study of extracellular vesicles (EV) has gained attention due to their function and role in intercellular communication and cargo transfer. Advancements in liquid chromatography and mass spectrometry (LC-MS/MS) has demonstrated the ability to comprehensively define EV proteomes. However, these studies are often limited by sample availability, requiring upscaled EV production from cell culture or biofluids, limiting its applicability to lower yield EV sources.

Methods:

Here, we establish a workflow to enable precise and comprehensive proteomic characterisation of small EVs (sEVs) from ultralow starting quantities. This pipeline is defined by its optimised sample preparation methods, short chromatography lengths, and high-resolution quantitative proteomics using data-independent acquisition (DIA). This refined DIA approach combined robust single-pot, solid-phase-enhanced sample preparation with temporally optimised enzymatic digestion and short chromatography gradients using ultra-low input loads of 0.5 to 50 ng of EV peptide across each LC gradient.

Results:

For 50 ng loading, more than 3730 proteins for all gradient lengths were observed, with 4599 identifications in our 44 min workflow. The short gradient lengths favoured low peptide loads, with a 15 min gradient consistently quantifying >1100 protein groups from 500 pg of EV peptide, and >3800 protein groups from 50 ng, including the robust quantification of 22 core EV marker proteins. Furthermore, we optimised bead-based sample preparation for ultra-low quantities of EV (0.5 to 1 µg) to obtain sufficient peptide source for MS quantification. Our easy to apply approach enables the generation of meaningful proteome insights from <1 µg starting EV protein, encompassing the identification of >1900 protein groups and capturing sufficient proteomic diversity of EV from different cell sources to determine known EV biology.

Summary/Conclusion:

This optimised workflow addresses the pressing need to capture precise and comprehensive proteomes of EVs from ultralow sample quantities, without compromising depth and accuracy. Our workflow pipeline is straightforward and can be implemented to suit various laboratory conditions. This adaptability facilitates the characterisation of EVs, particularly where sample availability is constrained.

Dr Ishara Atukorala, Dr Ching-Seng Ang, Ms Sally Beard, Ms Bianca Fato, Dr Natasha de Alwis, Dr Hamish Brown, Professor Natalie Hannan, Professor Lisa Hui

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Amniotic fluid (AF) surrounds the developing fetus, providing nutrients and protecting the fetus from biological and mechanical dangers. In addition to a myriad of proteins, immunoglobulins, electrolytes and growth factors, the AF is a rich source of extracellular vesicles (EVs). As the keratinization of the fetal skin begins around 20 weeks of gestation, fetal urination and lung secretions become the main sources of increasing AF volume. This makes the amniotic fluid a rich source of fetal biological material including EVs.

Methods

Second trimester and term amniotic fluid samples (6 each) were obtained from routine amniocentesis and Caesarean section, consecutively, with written informed consent from patients. EVs were isolated using differential centrifugation coupled with filtration and ultracentrifugation. EVs were characterized using nano-particle tracking analysis, cryo-electron microscopy and Western blotting for Alix, CD9 and CD63. EVs were subjected to label-free proteomics and data analysis was performed using Spectronaut® and MaxQuant-Perseus.

Results

The number of EVs per 1 mL of amniotic fluid and the protein amount per EV did not significantly change according to gestation. Cryo-electron microscopy revealed the presence of unilamellar, multilamellar, multicompartmental and granular-centred EVs in each sample. The analysis of 220 EVs using ImageJ revealed that there is no significant gestation-dependent difference in the ratio of these diverse types of EVs. The proteomic signature showed a stark difference according to the gestation of the amniotic fluid EVs. Moreover, majority of the proteins uniquely expressed in the second trimester was involved in the development of the central nervous system, with cardiac development scoring the second place. The proteins upregulated in the term amniotic fluid EVs corresponded to the impending newborn functions such as breathing and eating.

Conclusions

The EV proteome can be considered as an accurate representation of the fetal developmental processes according to gestation. Therefore, amniotic fluid derived EVs can be used as a liquid biopsy for fetal physiology and pathology.

Dr I-Jou Teng, Dr Kaloyan Takov, Dr Clemens Gutmann, Prof. Manuel Mayr

Poster Pitches (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:45 PM - 1:00 PM

Extracellular vesicles (EVs) have attracted attention as carriers of proteins and nucleic acids that are important for intercellular communication and as biomarkers in the circulation. Small non-coding RNAs (sncRNAs), such as microRNAs (miRNAs) and YRNAs, have been described as part of the functional cargo of the EVs. However, lipoproteins and RNA-binding proteins have also been demonstrated as putative carriers of sncRNAs. The lack of reliable methods to resolve these carriers from human blood hampers the assignment of sncRNA signatures to different carriers.

In this study, automatised high-performance size-exclusion chromatography (HP-SEC) workflow was established to improve the separation of nanoparticles from plasma and minimise the co-isolation of lipoproteins with small EVs. Immunoblotting for small EV markers (CD9 tetraspanin) and apolipoproteins (ApoA1 and ApoB for high- and low-density lipoproteins, respectively) was used to characterise the isolates. Quantitative real-time PCR of HP-SEC fractions showed that miRNAs were mainly detected in the late-eluting fractions enriched in ApoA1 and small, soluble proteins. Contrarily, YRNAs were predominantly found in the albumin-rich and ApoB-containing fractions. To further investigate whether lipoproteins are the main carriers, HDL and ApoB immunodepletion methods were employed. Both failed to substantially deplete sncRNAs from plasma indicating that lipoproteins are minor carriers of circulating sncRNAs. To identify other potential carriers, conditioned medium from human hepatoma cells was subjected to HP-SEC. In agreement with plasma experiments, sncRNAs were primarily found in fractions that did not contain EV markers (CD63, CD81, CD9, syntenin-1). Instead, most of the sncRNAs were identified in the late-eluting fractions containing smaller, soluble protein complexes, including numerous RNA-binding proteins.

In summary, we observed only a minor role for lipoproteins and EVs as carriers of circulating sncRNAs. Instead, soluble RNA-binding proteins, co-eluted with sncRNAs from plasma suggestive of a role in the secretion and protection of sncRNAs. The current study established an automatic and reproducible HP-SEC procedure for a better understanding of the compartmentalisation of circulating sncRNAs and EV cargo. In the future, methods established here can determine changes in sncRNA distribution across different carriers and help delineate the function of circulating sncRNAs in the intercellular communication.

Dr. Leandra Figueroa-Hall, Dr. Kaiping Burrows, Dr. Ahlam Alarbi, Dr. Chibing Tan, Dr. Bethany Hannafon, Dr. Rajagopal Ramesh, Dr. Victoria Risbrougn, Dr. T. Kent Teague, Dr. Martin Paulus

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) are nanoparticles whose cargo is highly enriched with microribonucleic acids, and other biomolecules like lipids, carbohydrates, and proteins. EVs diverse cargo allows for cell-to-cell communication, and their ability to cross the blood brain barrier bi-directionally enables enrichment of EVs from brain cells to examine central nervous system mechanisms. Here, we investigated the feasibility of measuring proteins expressed in AEEVs with high-sensitivity multiplex assays.

Methods: A human serum sample (one subject) was treated with or without proteinase K (PK; 0.5 mg/mL-final concentration) and EVs were isolated with various isolation methods (precipitation, precipitation + purification, resin, and size exclusion). Astrocyte-enriched (AE)EVs were enriched with an astrocyte-specific, glutamate transporter (GLAST). AEEVs on beads were lysed and protein used for detection of tetraspanins (CD9, CD81, CD63), apolipoprotein E (APOE), and neural cell adhesion molecule (NCAM) protein concentrations. All samples were run in duplicate, and leftover human serum was used as a positive control for all proteins.

Results: AEEV protein concentrations were reduced with PK treatment, with Norgen at below detectable limit (BDL). All proteins were detectable in AEEVs except for NCAM, which was BDL. CD9 concentrations were detectable in all methods, CD81 concentrations were above the lower limit of detection (LLOD) for ExoQuick, ExoQuick Ultra, and SmartSEC, and CD63 concentrations were above standard 7 for all methods. All methods were comparable for APOE concentrations after PK treatment. Interassay coefficients of variation for samples not treated and treated with PK: CD9 (2.04, 10.8), CD81 (7.02, 24.02), CD63 (2.85, 20.55), APOE (5.19, 5.44) and NCAM (2.16, NA).

Summary: Tetraspanins were detectable in AEEVs for all methods indicating the presence of EVs. APOE, a protein mainly produced in astrocytes, was highly expressed in AEEVs confirming the origin of our EV-enriched population. NCAM, a neuronal marker, was not expressed in AEEVs, confirming the absence of neuronal type. Treatment with PK is necessary to remove lipoproteins, but care must be taken when measuring proteins of interest including tetraspanins, as PK treatment decreased these in AEEVs. Our preliminary results indicate that all methods are appropriate for APOE detection and ExoQuick and ExoQuick Ultra for tetraspanins.

Aleksandra Steć, Ph.D. Joanna Jońca, Ph.D. Agata Płoska, Prof. Leszek Kalinowski, Assoc. Prof. Bartosz Wielgomas, Ph.D. Krzysztof Waleron, Prof. Bogdan Lewczuk, Mr. Grzegorz Czyrski, Ph.D. Andrea Heinz, Ph.D. Szymon Dziomba

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Most of the techniques currently used for the characterization of extracellular vesicles (EVs) are low selective and ineffective in the assessment of vesicles’ heterogeneity. Impurities profiling is also strongly limited and usually requires the application of additional technique like Western Blotting or mass spectrometry. To address these issues, a novel approach to EVs characterization was developed with capillary electrophoresis (CE) technique. The advantage of CE lies in its simultaneous separation of various analytes such as small molecules, polysaccharides, proteins and particles, which provides complete information on the presence of contaminants in the analyzed isolates [1,2]. Moreover, good agreement between the EVs quantitation results obtained by CE, nanoparticles tracking analysis (NTA) and bicinchoninic acid assay (BCA) has already been proved [1,3].

Methods

EVs were isolated from bacteria culturing media (Pectobacterium sp., E.coli) [1,2], plant material (Citrus limon) [3], and mammalian cell cultures (CHO cells) [4] using different method – ultracentrifugation (bacterial vesicles), and combination of size exclusion chromatography with ultrafiltration (for plant material and mammalian samples). The obtained isolates were characterized using NTA, BCA, microscopy (Cryo-EM or TEM), and CE equipped in UV and LIF detection.

Results

CE was able to separate subpopulations of EVs in the isolates obtained from various sources. The difference in electrophoretic mobility of certain EVs was related to their cargo composition. It has also been shown that staining of selected vesicle components with fluorescent dyes might be utilized for the identity confirmation of EVs with CE.

Conclusion

CE was proven to be able to solve numerous problems encountered in today's EVs-focused research. It might be used for the monitoring of the isolation process [3,4] or for the quality control of the isolates.

[1] Steć A, et al. Int J Mol Sci. 23: 4347 (2022).

[2] Jońca J, et al. Int J Mol Sci. 22: 12574 (2021).

[3] Steć A, et al. Food Chem. 424, 136333 (2023).

[4] Steć A, et al. Anal Bioanal Chem. 415, 3167–3176 (2023).

Dr Carlos Andres Palma Henriquez, Ms Siena Barton, Dr Sara Nikseresht, Mr Sadman Bhuiyan, Dr Mozhgan Shojaee, Dr Kartini Asari, Dr Pingping Han, Dr Ramin Khanabdali, Dr Gregory Rice

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Saliva is an easily accessible and non-invasive biofluid with huge potential for biomarker discovery and diagnostic applications. However, using whole saliva requires considering a few factors, including sample collection time, methods, food contamination, individual variability, and relatively lower concentrations of specific biomolecules. Salivary extracellular vesicles (EVs) have gained significant attention recently to overcome these barriers. However, collection time may play an important role in the quantity, quality, and compositions of saliva derived EVs.

Methods: Saliva samples were collected from 3 volunteers using spitting method at 07:00 (A), 13:00 (B), 19:00 (C) and 01:00 (D). Saliva (1mL) was diluted with 1 mL of PBS and centrifuged at 10,000 x g for 5 min to remove cell/membrane and food debris. EVs were isolated using 30 µL of EXO-NET® according to the manufacturer's instructions. EXO-NET captured salivary EVs were then lysed in 1% SDS before processing for mass spectrometry and western blot analysis.

Results: Western blot analysis confirmed canonical EV markers including CD9, CD81 and CD63 across all time points. Mass spec analysis identified around 903 proteins in (A), 813 in (B), 803 in (C) and 871 in (D). Out of that, 718 proteins were present in all 4 times, with 16 proteins identified only in (A), 3 in (B), 2 in (C) and nothing uniquely expressed in (D). Data modelling identified 62 EV-associated proteins out of the top 100 proteins including Alix, CD63, ENO1, Annexins, HSP90, Rab7a, Actin, and GAPDH. In terms of differential expression of proteins among the 4 times, RAB10 and ALDH4A1 showed to be significantly high during the morning and reduced by the end of day, while GOLM1, FUBP1 and TFF3 showed significantly low in the morning compared to the evening time.

Summary/Conclusion: Immunoaffinity bead-based method represent a simple and rapid method for isolating salivary EVs for proteomic profiling and downstream analysis. This study identified diurnal variation in the number and composition of salivary EV proteins identified. Time of sample collection thus should be considered an important covariate when analyzing salivary EV cargo.

Mr Sriram Gummadi

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: High-throughput methods to profile the genome, transcriptome, proteome and metabolome of various systems has become a routine in multiple research laboratories around the world. Hence, user-friendly software tools to analyse, integrate and interpret multi-OMICs datasets are essential.

Methodology: FunRich tool was developed in C# language using Microsoft.NET library and is compatible with Microsoft Windows. Hypergeometric distribution test was performed to check the statistical significance of enriched and depleted terms. In addition, Bonferroni and Benjamini–Hochberg (BH) aka FDR (false discovery rate) method is also implemented to correct for multiple testing.

Results: We report FunRich tool that enables biologists to perform functional enrichment analysis on the generated datasets.

Users can perform enrichment analysis with a variety of background databases and have complete control in updating or modifying the content in most of the databases. Specifically, users can download and update the background database from UniProt at any time thereby allowing a robust background database that can support annotations from >18 taxonomies.

The new additional features implemented in FunRich include miRNA enrichment analysis, plugin to analyse extracellular vesicle datasets from Vesiclepedia database, customisable heatmaps, comparison of oncogenes using COSMIC database, ID conversion and customisable colour for all the publication quality graphs.

Importantly, since 2015, FunRich website has been visited by >82,173 users around the world with >248,000 page views. FunRich has also been widely used for analysis extracellular vesicles dataset and users have compared their data with Vesiclepedia.

Conclusion: Overall, FunRich (http://www.funrich.org) tool is user-friendly and enables users to perform various analysis on their datasets with minimal or no aid from bioinformaticians.

Beth DiBiase^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Roxana Mitrut^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Taylor Gunnels^{Biomedical Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Dr. Neha Kamat^{Biomedical Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Dr. Joshua Leonard^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Engineered extracellular vesicle (EV) therapies are emerging treatment modalities for many diseases. An open challenge for clinical use is the susceptibility of EVs to nonspecific uptake by the mononuclear phagocytic system, leading to fast clearance times. Extending circulation time and decreasing nonspecific uptake would provide therapeutics with more opportunities to reach their targets while maintaining the biological advantages of EVs as delivery vehicles. We hypothesized that engineering EVs with biopolymers, specifically mucins, would reduce their nonspecific uptake and increase their circulation time. We envision this technique may represent a tunable handle for modulating EV uptake to fit a desired application.

Methods

In vitro, we isolated EVs from mScarlet-expressing FreeStyle 293-F cells, stably engineered to display varying densities of GFP-tagged mucin on their plasma membranes, using differential centrifugation. Measurement of mucin loading on EV surfaces was determined via GFP fluorescence, and mucin EV morphology was evaluated using cryo-EM. Changes in nonspecific uptake conferred by mucins were determined by flow cytometry using an in vitro uptake assay. RAW 264.7 mouse macrophage-like cells were utilized in this assay, as they provide an indication of the potential effects on nonspecific uptake by the MPS in vivo.

Results

Cryo-EM images confirm the presence of mucin-like projections from the EV surface, as well as changes in EV morphology leading to non-spherical structures. Fluorescent protein gels indicate that EVs isolated from cells that express different amounts of mucin display different levels of mucin on the EV. Preliminary uptake study results indicate a reduction in non-specific uptake compared to control conditions when EVs are loaded with high amounts of mucin biopolymer.

Summary/Conclusions

We theorize that EV nonspecific uptake can be modulated by tuning the amount of mucin biopolymer displayed on the EV surface. Further investigation into balancing mucin-conferred EV shielding with targeting is ongoing. Studies investigating membrane property changes due to mucin loading which affect uptake will elucidate the underlying mechanisms, and preclinical studies investigating circulation time will advance therapeutic treatments. We anticipate that this study will expand the field of engineered EV therapeutics and accelerate their development to combat diseases.

Ms Huaqi Su, Assoc. Prof. Kevin Barnham, Prof. Gavin Reid, Dr. Laura Vella

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Cellular processes such as endocytosis, autophagy, phagocytosis and mitochondrial OXPHOS, among others, influence small extracellular vesicles (sEVs) biogenesis and cargo packaging, and these processes have been implicated in various diseases. The ability to measure alterations in these pathways in humans would be valuable for understanding disease mechanisms and potentially offer biomarkers for the early detection of disease. However, directly measuring changes in these pathways is challenging. The biogenesis of sEVs, including cargo sorting, happens at the convergence of these pathways, offering an opportunity to examine plasma sEVs, which can provide insight of cellular conditions in diseases. Moreover, sEVs are enriched in lipids, providing a window into diseases involving lipid dysregulation. To explore potential of sEVs-based biomarkers in blood, it is essential to enrich and characterize blood sEVs with minimal blood contaminants (blood proteins and lipoprotein particles), that are easily co-isolated. The co-isolated blood contaminants pose challenges for mass spectrometry in determining sEVs molecular composition (proteins and lipids), making the value of plasma sEVs to report on cellular pathway changes or lipid alteration unclear.

Methods: Human plasma sEVs were isolated by density fractionation and size exclusion chromatography. The highly enriched sEVs were characterised by western blot, transmission electron microscopy, and quantitative mass spectrometry-based proteomics and lipidomics.

Results: Lipidomic analysis showed that sEVs are depleted of cholesteryl esters but rich in sphingomyelin (SM), glycerophosphoethanoamine (PE), and glycerophosphoserine (PS), which have been implicated in various diseases. Proteomic analysis revealed that plasma sEVs harbour protein networks associated with endosomal, autophagic and lysosomal (EAL) pathways and mitochondria that have not been reported previously.

Conclusion: Highly enriched plasma sEVs harbour protein networks associated with EAL and mitochondrial pathways, and subsequently, can provide a platform for identifying disease associated changes in these pathways outside the cell. The functional lipids, such as SM, PE, and PS, that enriched in plasma sEVs, provide the opportunity to investigate diseases associated with lipid dyshomeostasis.

Dr. Chaiyong Koaykul, Dr. Kunthika Mokdarta, Dr. Poorichaya Somparn, Dr. Jiradej Makjaroen, Dr. Chatikorn Boonkrai, Dr. Trairak Pisitkun

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Protein cargos inside the extracellular vesicles (EVs) are signaling molecules that affect several functions in the receiving cells. Mesenchymal Stem/Stromal Cell (MSC)-derived Extracellular Vesicles have been growing interest in drug development for many clinical applications. MSC is the cell that produces EVs by responding to the surrounding environment. However, little is known about the cargo content of MSC-EVs secreted by MSCs from different culture conditions. Therefore, this study focuses on analyzing the protein cargo profile in EVs from MSCs cultured under normal and stimulating conditions.

Methods

Human umbilical cord-derived MSCs were grown under xeno-free culture media, and the cells at cumulative population doubling levels (PDLs) between 16 – 20 were used in this study. MSCs at 70-80 % confluence were cultured under several EV harvesting conditions: (1) condition media (control condition), (2) condition media with inflammatory cytokines (cytokine condition), and (3) condition media with hypoxia condition (hypoxia condition). After that, EVs from each condition were isolated by ultrafiltration and size-exclusion chromatography process. Then, the protein cargo in EVs was investigated by mass spectrometry technique.

Results

The size and physical characteristics of EVs from all harvesting conditions showed no significant difference. Based on mass spectrometry analysis, we found a total of 700-900 proteins in EV cargo in each condition. The EVs from cytokines stimulating condition showed the upregulation of anti-inflammatory function-related proteins, and the EVs from hypoxia stimulating condition presented upregulation of redox proteins and angiogenic function-related proteins than control conditions.

Conclusion

This study provides the proteomic information of EV cargos from MSCs collected from controlled and stimulated culture conditions.

Dr James Rhodes, Dr Kathleen M Lennon, Dr Colbie Chinowsky, Dr Abigail Neininger-Castro, Ms Chloe Celingant-Copie, Dr Daniel Zollinger, Dr Grace DeSantis

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Extracellular vesicles (EVs) are a group of heterogeneous membranous particles ranging in biogenesis, size, and biomarker content. The characterization of EVs remains challenging due to the heterogeneity of EV samples and the limited sensitivity and specificity of current EV detection methods. Single-molecule localization microscopy, such as dSTORM imaging, enables researchers to visualize and characterize EV populations with 20 nm resolution.

Methods

Purified EVs from three different cell lines were immobilized on the surface of functionalized coverslips using ONI's EV Profiler 2, either by individual or by pooled antibodies against CD9, CD63 and CD81. Affinity-isolated EVs were then fluorescently labeled with a pan-EV marker and selected antibodies against CD9, CD63, and CD81. EVs were imaged with dSTORM on a Nanoimager, and vesicle characterization was performed using cluster-based analysis to extract key features, including circularity, size, density, and biomarker content inside and outside of the EV. EV size from each sample was compared to data obtained using Transmission Electron Microscopy (TEM).

Results

EV Profiler 2 showed high efficiency of capturing purified EVs using either biotinylated antibodies or ONI's PS capture method. EV profiling with an anti-CD9, CD63 and CD81 antibody cocktail or 2 tetraspanin antibodies and the pan-EV stain enabled precise characterization of EV size across 3 different populations. In addition, differences in biomarker positivity fractions were observed across the 3 populations, showing the power of dSTORM imaging at quantifying biomarkers at a single EV level.

Summary/Conclusions

Single-molecule localization microscopy, such as dSTORM imaging, allows researchers to determine EV size across populations with a higher throughput than TEM. Additionally, multicolor dSTORM imaging allows for the detection of biomarkers on single EVs, opening up a range of possibilities for EV research.

PhD Imola Cs. Szigyarto, Priyanka Singh, Tasvilla Sonallya, PhD Aniko Gaal, PhD Lilla Turiak, PhD Laszlo Drahos, PhD Zoltan Varga, PhD Tamas Beke-Somfai

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Identifying the spontaneously formed protein corona on the surface of nanoscale materials is of critical importance for the therapeutic and diagnostic development of nanoparticles. In order to explore the potential role in manipulating the surface of nanoparticles and also to progress towards surface engineering, a few short, cationic peptides with well-characterized action mechanism were selected. The interactions of selected peptides with EV protein corona create new opportunities in wound healing, tissue repair or biofilm inhibition, as both could be present in the complex microenvironment.

Methods

Nanoscale liquid chromatography coupled to tandem mass spectrometry was performed for characterizing protein corona composition. Red blood cell-derived extracellular vesicles (RBCEVs) were isolated, as they are easily produced in high concentrations, and have several unique advantages, such as the lack of DNA, prevention of immune clearance and extended circulation due to the presence of several integral membrane proteins. As a first step, the effective peptide concentration for treatment of RBCEVs was determined using spectroscopy, flow cytometry and microfluidic resistive pulse sensing techniques. Size exclusion chromatography measurements were performed to separate intact vesicles from protein rich soluble fractions after peptide treatment.

Results

Based on results a total of 125 proteins were identified in the control sample, consisting of membrane-bound and cytosolic proteins and metabolic enzymes. The protein content of control and peptide-treated samples were compared, which resulted in high number of common proteins, supporting the good selection strategy of the peptides. Furthermore, the relative abundance of characteristic membrane and subcellular proteins remained mainly constant after treatment. The list of proteins found in treated sample was subtracted from the control protein list, which resulted in seventeen proteins, identified as protein corona. Interestingly, some applied peptides also caused the appearance of several new proteins, not identified in the control sample. These may originate from the electrostatic attraction of partially negatively charged EV surfaces and cationic peptides, which acts as membrane anchors in the attachment of these proteins to EV surfaces.

Conclusions

Our results envisage the applicability of cationic peptides in modulating protein corona content, as a possible tool in nanoparticles surface engineering.

Mr Sriram Gummadi

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) are nanosized membrane bound vesicles that are shed by cells into extracellular space. In addition to EVs, some of the mammalian cells are also reported to secrete extracellular particles (EPs). EVs have a key role in multiple pathophysiological processes due to their signalling capabilities and are classified into various subtypes based on their biogenesis and origin. Exosomes are small EVs that range in 30–150 nm diameter and they originate due to the fusion of multivesicular bodies with the plasma membrane.

In terms of the molecular composition, EVs sequester a diverse and rich cargo of proteins, lipids, and nucleic acids, and the ensuing cargo is altered according to the pathophysiological conditions of the host cells that secrete them. Hence, a catalogue of the EV cargo in various conditions could aid in identifying an EV fingerprint that are specific to a tissue, cell type, and/or pathology.

Methodology: Vesiclepedia and ExoCarta are built using a combination of MySQL for the background database, Zope content management system and Python as the programming language that connects the database to the content management system and the data is manually annotated and curated from published and unpublished studies.

Results: We report an update of Vesiclepedia (http://www.microvesicles.org) an online database that contains a list of RNA, proteins, lipids and metabolites that are identified in EVs and EPs and ExoCarta (http://www.exocarta.org), a manually curated web-based compendium of exosomal cargo and it features dynamic protein-protein interaction networks and biological pathways of exosomal proteins.

Currently, Vesiclepedia contains data obtained from 3,533 EV studies, 50,550 RNA entries, 566,911 protein entries, 3,839 lipid entries, 192 metabolite and 167 DNA entries. In the latest update, a new feature EV-QUANT is added where it allows for relative quantification between EV protein/RNA/lipid cargo samples within one specific study. Furthermore, Vesiclepedia also contains EV and EP-associated DNA data that were obtained from 52 studies including 167 data entries for oncogenes and mutated genes.

Conclusion: A catalogue of EV cargo will immensely benefit the research community in identifying an EV fingerprint that are specific to a tissue, cell type, and/or pathology.

M.Sc. Stefanie Kurth, PhD André Tiaden, M.Sc. Edveena Hanser, Ute Heider, PhD Stefan Wild, Professor Diego Kyburz

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) were previously reported to contribute to the pathogenesis of various diseases including rheumatoid arthritis (RA) – a chronic inflammatory autoimmune disease with high global socioeconomic burden. However, most studies are based on bulk analysis of EVs, neglecting the complexity of different EV subsets present in biofluids of patients. To investigate and characterize distinct EV subpopulations within a heterogeneous mix of EVs in RA we established an immunocapture separation method based on magnetic nano-sized beads targeting a relevant marker in RA synovial tissue. In this context CD90/THY1 was selected due to the reported contribution of CD90+ fibroblasts in RA synovitis.

Methods: EVs secreted from cultured synovial fibroblasts of arthritis patients (RA and Osteoarthritis) were isolated via size exclusion chromatography (SEC) and subsequently characterized via Transmission Electron Microscopy (TEM), Nano Tracking Analyzer (NTA), NanoFCM and WesternBlot (WB). The separation of a CD90+ population from the heterogenous EV population was performed with anti-CD90 EV Isolation MicroBeads (Miltenyi). WB and mass-spectrometry based proteomics were applied to identify characteristics and differences between the two populations. Spiking of fibroblast-derived EVs in RA patient-derived synovial fluid was used to test the separation approach in more complex source material.

Results: WB and NanoFCM results confirmed the presence of CD90 on in vitro fibroblast-derived EVs. The magnetic bead-based separation of heterogenous fibroblast-derived EVs resulted in a CD90+ and a CD90- EV subpopulation. WB and proteomics confirmed the enrichment of CD90 in the CD90+ population. Furthermore, both populations exhibited distinct EV marker patterns with enriched Annexin-1 in CD90+ EVs, and higher expression of CD63 and Flotillin-1 in the CD90- population. Additional EV markers like CD9 and Syntenin were similarly expressed. Finally, spiked CD90+ EVs were successfully retrieved from synovial fluid demonstrating the feasibility of the separation approach in complex biofluids.

Summary/Conclusion: Using a magnetic immunocapture approach we were able to separate a CD90+ population from a heterogeneous mixture of in vitro fibroblast-derived EVs and also from synovial fluid spiked with CD90+ EVs. The ability to investigate distinct EV subsets present in biofluids of patients with various diseases may offer novel insights into their role in pathogenesis.

Wonjae Kim, Student Kangmin Lee

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction

Extracellular vesicle-derived microRNAs (EV-miRNAs) are promising biomarkers for early cancer diagnosis. Nevertheless, existing EV-miRNA extraction technologies involve a complex two-step process that results in low extraction efficiency and inconsistent outcomes. This study introduces miRQuick, a novel single-step extraction method designed for the efficient and high-recovery extraction of EV-miRNAs from various samples.

2) Methods

The miRQuick method involves adding positively charged substances to the sample, causing negatively charged EVs to quickly aggregate and precipitate. Subsequently, EV sediment is then lysed, followed by miRNA extraction utilizing the spin column extraction method. This entire process, achievable with standard laboratory equipment, can be completed within an hour.

3) Results

We validated the miRQuick method using diverse analytical techniques with a comparative assessment against existing methods for plasma, urine, and saliva samples. The miRQuick method demonstrated significantly higher performance than other methods, not only for blood plasma but also for urine and saliva samples. Furthermore, we successfully extracted and detected 9 biomarker candidate miRNAs in the plasma of breast cancer patients using miRQuick.

4) Summary/Conclusion

Our results demonstrate that miRQuick stands out as a revolutionary single-step method, addressing the limitations of current extracellular vesicle-derived microRNA (EV-miRNA) extraction techniques across diverse sample types with excellent repeatability. Its implementation holds promise for groundbreaking early cancer diagnosis, opening avenues for further research and clinical applications in the evolving landscape of biomarker discovery and diagnostics.

Ph.D. Candidate Yuxin Qu^{School of Biomedical Engineering, Tsinghua University, Beijing, China}, Assistant professor Han Wang^{School of Biomedical Engineering, Tsinghua University, Beijing, China}, Lan Xie^{School of Basic Medical Sciences, Tsinghua University, Beijing, China}

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

According to MISEV2018, EV isolation protocols with both high recovery and high purity have not been achievable yet. This is mainly attributed to the dilemma of effectively retaining EVs while simultaneously removing contaminants. To address this issue, we drew inspiration from nature and took cues from the unique gill arch structure of suspension-feeding fishes. These fishes (e.g., paddlefishes) benefit from rib-and-groove gill rakers that allow them to efficiently filtrate zooplanktons for food without clogging. Mimicking this process, we designed a vortex tangential flow filtration (VTFF) microfluidic system for effectively recovering and purifying of EVs.

Methods

Inspired by the suspension-feeding mechanism, we have developed microfluidic chips and auxiliary systems for efficient filtration of EVs. First, the generation of vortex profile was evaluated through numerical simulations and fluidic experiments. Subsequently, scanning electron microscopy (SEM) was employed to verify the prevention of clotting. After optimizing experimental parameters, the VTFF system was utilized for the isolation of EVs from human plasma and urine, with a comparative analysis against ultracentrifugation and PEG-based approaches. The isolated EVs were characterized using transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), enzyme-linked immunosorbent assay (ELISA), and nanoparticle flow cytometry (NanoFCM). Moreover, this system was utilized to isolate EVs from urine of mice with and without myocardial infarction for miRNA sequencing.

Results

During chip operation, EVs undergo continuous flow and separate without adhering to the filter membranes. Baffles in the microchannels induce vortex where samples continuously scour the membranes and prevents clogging during continuous tangential flow through the membranes. The recovery of EVs from human plasma was >88% while the removal rate of free proteins was >99%, which outperformed ultracentrifugation and PEG-based approaches. TEM images showed typical round and cup-shaped structures, suggesting high integrity of isolated EVs. Besides, miRNA sequencing from isolated urine EVs has discovered novel differentially expressed miRNAs for myocardial infarction with biological significance.

Summary

In summary, The VTFF systems can generate vortex streaming during tangential flow filtration, and effectively purify and recover EVs from complex body fluids. Based on this system, novel urinary EV markers for myocardial infarction diagnosis were discovered.

Mr Jose Marchan-Alvarez, Miss Loes Teeuwen, Mr Doste Mamand, Prof Susanne Gabrielsson, Prof Klas Blomgren, Dr Oscar Wiklander, Dr Phillip Newton

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Endochondral ossification leads to longitudinal bone growth and fracture healing through the actions of chondrocytes, which synthesize and modify cartilage by the release of a variety of particles including extracellular vesicles (EVs). However, our understanding of the role of EVs in endochondral ossification is yet to be fully uncovered. Particularly, it remains a challenge to isolate typical populations of chondrocyte-derived EVs due to the difficulties both in preserving the morphology and functional capability of primary chondrocytes in culture as well as in applying the serum-free conditions required for EV production. Here, we used the ATDC5 cell line - one of the most well-characterized tools used to study chondrocytes in vitro - to recover chondrocyte-derived EVs from early- and late-differentiation stages, representing chondrocytes before and during cartilage mineralization.

Methods: To select a suitable EV-collection medium, we compared custom EV-depleted media with normal growth conditions, at an early (day 10) and a late (day 18) differentiation stage, in terms of phenotype, conditioned media pH, histochemical staining, gene expression and cell viability. Using the selected medium, we then scaled-up culture volumes and studied the functionality and viability of the chondrocytes at a cellular and ultrastructural level. Thereafter, EVs were isolated from the conditioned medium using size-exclusion chromatography and EV characteristics, including particle size, ultrastructure and protein: particle ratio, were assessed.

Results: After screening different culture conditions, our data indicate that a serum-free Opti-MEM-based culture medium preserves chondrocyte identity (expression of Sox9, Col2a1 and Col10a1 genes) as well as proteoglycan synthesis, matrix mineralization, and cell viability at both time-points. A scaled-up production was used to obtain EVs from early- and late-stage chondrocytes. The chondrocyte-derived EVs had typical cup-shaped ultrastructure and expression of classical EV markers (CD63 and ALIX), at quantities suitable (3.27E+7 particles/cm2 at day 10 and 1.12E+8 particles/cm2) for downstream experiments.

Conclusion: We established a method to obtain chondrocyte-derived EVs before and during cartilage mineralization that may aid the further understanding of their roles in endochondral bone growth and fracture healing.

Funding: This work was supported financially by the Novo Nordisk foundation, Swedish Research Council, Region Stockholm and Karolinska Institutet.

Ioannis Isaioglou, Gloria Lopez-Madrigal, Jasmeen Merzaban

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Labeling of extracellular vesicles, such as exosomes, is a key component in studies detecting their uptake rate and their organotropic biodistribution. The most common way of labeling is through lipophilic dyes, such as DIO. Removal of the excess dye from the exosome solution is necessary to proceed in downstream assays. However, current cleaning-up methods require either a time-consuming, extra ultracentrifugation step, usage of specific equipment, such as size exclusion chromatography system, or single-use cleaning-up columns.

Here, we present a novel, rapid, user-friendly, and free-of-cost protocol for eliminating the excess dye from the exosome solution. Introducing the exosome parental cells into the solution containing the excess lipophilic dye leads to the trapping of the latter into the cells. Afterward, by using a simple centrifugation step, the parental cells form a pellet, resulting in a supernatant containing labeled exosomes free of excess dye. By performing the necessary controls using the Nanoparticle Analysis System and Dynamic Light Scattering assays, we confirmed that the introduction of the parental cells into the exosome solution did not cause any qualitative or quantitative alterations in the isolated exosomes.

In addition, given the role of the exosomes as a next-generation drug delivery system, we evaluated the efficiency of the proposed protocol in eliminating the excess daunorubicin, an anti-tumor drug. Indeed, the suggested protocol successfully removed the excess drug from the exosome solution.

In conclusion, here we present a novel, fast, user-friendly, and free-of-cost methodology to clean up the excess dye or drug from an exosome solution, without affecting the quality or the quantity of the isolated particles.

Research associate Nhan Vo, Research associate Chau Tran, Research associate HB Nam Tran, Scientist T Nhat Nguyen, Research associate Thieu Nguyen, Scientist DN Diem Nguyen, Research associate Tran Pham, R&D lead Hoai-Nghia Nguyen, R&D specialist Lan-N Tu

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Background: Small extracellular vesicles (sEVs) with the size of less than 200 nm are gaining attention as a promising drug nanocarrier. However, the clinical translation of sEVs remains challenging due to the lack of robust manufacturing protocols to produce sEVs with high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns.

Methods: We established a quality-controlled multi-stage procedure to produce and isolate sEVs from human embryonic kidney (HEK293F) cells. We compared several 2-step and 3-step purification procedures for the particle yield and purity. Subsequently, the purified sEVs were fully characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and preclinical safety profile in both in-vitro and in-vivo testing.

Results: Our findings demonstrated that culturing HEK293F cells at a high-density saturatory phase in a semi-continuous culture provided the optimal yield of sEVs. A novel 3-step purification procedure combining tangential flow filtration, sucrose-cushion ultracentrifugation, and bind-elute size-exclusion chromatography outperformed all other procedures in terms of sEV purity without affecting particle yield and integrity. Upon characterization, about 50% of the HEK293F-derived sEVs were positive for all three tetraspanin markers CD81, CD9, CD63. The highly abudant proteins and miRNAs in the sEVs were mostly involved in cell cycle, protein folding and organelle organization processes. Purified sEVs further demonstrated excellent preclinical safety profile after 6 repeated intravenous injections in mice.

Conclusions: Our developed multi-stage workflow allowed robust and reproducible production of ultrapure sEVs with stable characteristics and highly adaptable to mass-scale production. The well-characterized sEVs derived from HEK293F cells could be safe and reliable drug carriers for future therapeutic applications.

Ts. Dr. Norhayati Liaqat Ali Khan, Dr. Nadiah Abu, Dr. Wai Leng Lee, Dr. Muhammad Farid Nazer Muhammad Faruqu, Dr. Jia Xian Law, Associate Professor Dr. Norshariza Nordin, Dr. Maryam Azlan, Associate Professor Dr. Rajesh Ramasamy, Dr. Sik Loo Tan, Associate Professor Dr. Wan Nazatul Shima Shahidan, Mr. See Nguan Ng, Dr. Kok Lun Pang, Dr. Vijayendran Govindasamy, Mr. Benson Koh, Dr. Pan Pan Chong, Miss Yoong Yi Chong, Mrs. Nur Hidayah Hassan, Mr. Nazmul Huda Syed, Mrs. Maimonah Eissa Sheikh Al-Masawa

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Malaysia is witnessing a surge in the study of extracellular vesicles (EVs) providing valuable insights into intercellular communication and potential applications in diagnostics and therapeutics. Recognizing the pivotal role of collaborative efforts in advancing the field, The Malaysian Society for Extracellular Vesicles (MySEV) was established. This study aims to provide a comprehensive overview of the current status of EV research in Malaysia, including assessing the depth of understanding, the stage of research development, the encountered challenges, the awareness and adherence to existing guidelines (MISEV2018).

Methods: A targeted participant approach was done by disseminating a link to questionnaires (Google-form) through close contacts of the MySEV Committee members to government and private institutions as well as industrial partners.

Results: 54 complete responses were gathered from participants from public and private higher education institutions and industrial partners. The majority (85.2%) of survey respondents were actively involved in EV research, reporting 1 to 3 years of experience. The remaining respondents refrained from involvement, citing challenges related to grant applications, inadequate facilities, and lack of collaboration. The primary focus was on exploring EVs as therapeutics (48.1%), with 46.3% of respondents working on EVs from stem cells. Other areas of focus include targeting EVs as biomarkers and drug carriers. The majority were at the stage of experimenting the role/application of EVs in their field of interest (vaccine/diagnostic/therapeutic). Ultracentrifugation was the most commonly used isolation process, followed by ultrafiltration and Tangential Flow Filtration. The participants showed high awareness about the MISEV2018 guidelines whereby 90% of respondents were informed about and adhered to it. The key challenges in EV research in Malaysia included the lack of analysis instruments and incomplete availability of protocols.

Conclusion: This study sheds light on the dynamic landscape of EV research in Malaysia, evidenced through respondents active engagement, primarily on its therapeutic potential. The establishment of MySEV signifies a collaborative effort to advance this field. Challenges such as securing grants, inadequate facilities and limited collaborations need to be addressed. We believe, EV research in Malaysia is heading in the right direction and has great potential to contribute to the overall EV ecosystem internationally.

Jing Zhou, Ph.D candidate Jiajia Dai, Ph.D candidate Haonan Di, Ph.D candidate Yunyun Hu, Ph.D candidate Niangui Cai, professor Xiaomei Yan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Extracellular vesicles derived from adipose-derived stem cells (ADSC-EVs) demonstrate remarkable therapeutic potential in degenerative and inflammatory disease. However, prevailing methods for ADSC-EV generation are resource-intensive, time-consuming, and often lack the necessary assurance of high purity and mass production capability. In this study, we introduce a scalable workflow designed for the efficient cultivation, isolation, enrichment, and purification of ADSC-EVs. This innovative approach integrates three-dimensional (3D) cultivation using microcarriers with a custom-built tangential flow filtration (TFF) system linked to chromatography. Employing a laboratory-built nano-flow cytometer (nFCM) enables the real-time assessment of particle concentration and size distribution of ADSC-EVs at various stages, providing immediate insights into their yield. Additionally, nFCM enhances the efficiency and precision of evaluating ADSC-EV purity by integrating Gaussian Mixture Model (GMM) analysis with the size distribution data.

Methods

ADSCs underwent cultivation in a microcarrier-based three-dimensional (3D-M) culture system to promote growth. A specialized tangential flow filtration (TFF) system, uniquely designed for highly-efficiency isolation and enrichment of EVs from culture supernatants, was constructed and employed. This was followed by chromatography separation to further remove impurity components. Three chromatography columns with different resins were examined for their performance increasing ADSC-EV purifty. The nFCM was pivotal in assessing particle concentrations, purity, and size distribution of ADSC-EVs at each stage to optimize the preparation method. GMM analysis integrated into nFCM data provided a rapid purity assessment.

Results

The TFF system significantly boosted EV yield by approximately 10-fold compared to classical ultracentrifugation (UC) while maintaining EV integrity. However, the purity of EVs upon TFF separation (TFF-EVs) was approximately 45%, half that of UC-EVs. Incorporating a preferred chromatography column elevated ADSC-EV purity to 90%, increasing total yield 4-fold compared to UC-EVs. Validation against Triton-X100 lysis data affirmed the accuracy of GMM analysis in the rapid determination of ADSC-EV purity.

Conclusions

The as-developed scalable workflow achieves high-quality production of ADSC-EVs suitable for potential large-scale clinical therapy. Its integration of 3D cultivation, TFF, chromatography, and nFCM analysis offers a promising avenue for efficient, high-purity ADSC-EV generation at scale, while acknowledging the cost-intensive nature of traditional methods.

Mr. Hugues Martin, Dr. Andreas Wallucks, Dr. Andy Ng, Ms. Molly Shen, Dr. David Juncker

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Various extracellular vesicle (EV) analysis techniques often rely on surfaces to capture the EVs, notably planar surfaces in immunofluorescence imaging and beads in flow cytometry. Commonly, affinity binders enrich EVs positive for one protein and measure other targets’ presence with detection antibodies. This inherently restricts analysis and fails to establish a protein expression baseline in the total population, convoluting data interpretation. Marker independent capture surfaces can be used to establish an unbiased expression profile or to classify subpopulations in single EV analysis. Most marker independent surfaces capture EVs by their physical properties such as surface charge or hydrophobicity. The resulting biases in EV proteomic composition, however, are so far poorly characterized. Here, we provide a systematic comparison between common and newly proposed marker independent surfaces.

Methods: Glass coverslips were functionalized with silanes terminated with various functional groups including aldehyde, amino, and methoxy. These surfaces were studied for physisorption, electrostatic capture, or hydrophobic capture. Furthermore, aldehyde functionalized coverslips were functionalized with either membrane curvature-sensing peptides, lactadherin, an affinity binder of common EV lipids, or a tetraspanin antibody cocktail. We used size photometry and fluorescence imaging (SPFI), an in-house single EV imaging platform that allows characterization of both size and protein expression, to characterize EVs captured on the different surfaces. The expression profiles were compared with flow cytometry, and the size distribution, with nanoparticle tracking analysis, both capture-free methods. We used SEC purified EVs from HT29 human adenocarcinoma and 293T human embryonic kidney cells.

Results: Electrostatic capture using silanes with an aldehyde functional group had similar protein expression profile, while electrostatic capture-based amine-functionalized surfaces had higher expression of CD9 but lower expression of CD63 and CD81. Hydrophobic capture based methoxy-functionalized surfaces had lower EV protein expression of all markers and, furthermore, tend to capture smaller EVs. Peptide, lactadherin and tetraspanins capture surfaces will be presented at the conference.

Conclusion: Target-independent EV capture mechanisms impact the observed EV populations. While surfaces that capture based on physisorption, electrostatic capture or hydrophobic capture are marker independent, they aren't bias-free. These results should be considered when used with capture surface techniques.

Mr Mohamed Sallam, Mr Cong-Minh Nguyen, Dr Amandeep Singh Pannu, Dr Indira Prasadam, Mr Yezhou Yu, Professor Serge Muyldermans, Dr Frank Sainsbury, Professor Nam-Trung Nguyen, Professor Nobuo Kimizuka

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

The advancement of innovative isolation techniques for extracellular vesicles (EVs) has garnered significant attention owing to their pivotal roles in intercellular communication and potential as disease biomarkers. This study introduces an original approach utilising self-assembled functional coordination polymer nanoparticles (CPNs) for the rapid and efficient isolation and enrichment of small extracellular vesicles (sEVs), particularly exosomes, from complex biological fluids. Our pioneering nanostructured material called ExoFlocs™ is employed in conjunction with anti-PLAP monoclonal antibodies for selective sEVs enrichment, followed by characterisation and biomarker analysis. The investigation employs a one-pot synthesis method with adaptive antibody inclusion, yielding ExoFlocs™ nanostructures with adjustable size and composition. Comprehensive characterisations, encompassing dynamic light scattering, zeta potential analysis, high-resolution scanning electron microscopy, and energy-dispersive X-ray spectroscopy, validate the stability and attributes of ExoFlocs™. The study successfully isolates placental sEVs from cell culture supernatant using anti-PLAP@ExoFlocs particles, achieving a notable capturing efficiency of 97.5%. These isolated sEVs are characterised by their expression of endosomal markers. Notably, a distinctive approach is introduced to rescue sEVs from ExoFlocs™ nanoparticles, utilising inorganic phosphate ions and simplifying downstream processes. Furthermore, the utility of isolated sEVs for subsequent research is validated through RT-qPCR analysis of a specific mRNA biomarker, KiSS1. The study demonstrates that the isolated sEVs maintain their RNA content and integrity. The innovative one-step, chelation-free adaptive inclusion technique holds promise for diverse applications in the realms of targeted drug delivery, biomarker discovery, and enhanced diagnostic tools.

Jie Gong, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Meng Han, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Bairen Pang, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Qi Wang, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Haotian Chen, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Zhihan Liu, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Cheng Zhou, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Yong Li, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Junhui Jiang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Prostate cancer (PCa) is a major man's health challenge. Current diagnostic tools in PCa such as PSA test and biopsy are inaccurate and not specific. Liquid biopsy is a non-invasive approach and can overview all pictures of tumor development. Urine is an excellent source for PCa liquid biopsy using extracellular vehicles (EVs), which play important roles in cancer communications. However, a standardized and reliable method for urine EVs separation remains elusive. In this study, we aimed to compare EXODUS (a novel automated exosome separation system), with ultracentrifugation (UC) and two commercial kits to find which approach is the best one for small EVs (sEVs) isolation applied for PCa liquid biopsy isolation

Methods: Urine sEVs were isolated from the urine samples of six healthy individuals (n = 6) using four distinct methods including EXODUS, UC and two commercial kits (exoEasy™ Maxi Kit and Total Exosome Isolation-TEI from urine). sEVs isolated were characterized by, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting (WB). The purity indexes of sEV isolated were evaluated using particle number and protein concentration ratio. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) was utilized to profile urine sEVs isolated from 4 different separation techniques.

Results: We demonstrate that UC and EXODUS isolations show more consistent repeatability and the particle concentration of EXODUS isolates was significantly higher compared to UC and exoEasy, but not significantly different compared to TEI. The purity of UC and EXODUS isolates was significantly higher than that exoEasy. Downstream proteomic analysis is ongoing.

Conclusion: This study provides a comprehensive evaluation of four urine sEVs isolation techniques, laying the groundwork for selecting appropriate methods tailored to specific research objectives. EXODUS demonstrates a notable enhancement in urine sEVs extraction efficiency and purity compared to other methods, showing promise for its integration into clinical practice for the rapid isolation of urine EVs from human urine and the exploration of potential protein markers in PCa patients. This advancement holds potential in revolutionizing PCa early diagnosis and risk stratification in liquid biopsy.

Dr. Vasiliy Chernyshev, Dr. Elena Svirshchevskaya, Mr. Mikhail Ivanov, Dr. Denis Silachev

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction

It is challenging to isolate EVs from complex biofluids such as plasma, urine, saliva, amniotic fluid, and the growth medium of cultured cells. A complex biomolecular milieu of biofluids, such as protein agglomerates and lipid nanoparticles, which overlap with EVs in size and other physicochemical features, are frequently contaminants of EV samples. In a recent study we developed and presented an innovative method of EV isolation that is based on asymmetric depth filtration (DF). The procedure is low-cost and straightforward. In 3-4 h it reproducibly allows to isolate EVs with a high yield and purity from complicated biological fluids using just simple equipment, e.g. traditional centrifuge. In this study we performed a comparison between DF and ultrafiltration coupled with size-exclusion chromatography (UF-SEC) method to isolate EVs from cell culture media which was not done previously but has significance in future studies where isolation may be critical in the experimental pipeline.

2) Methods

HCT116 and HT29 colorectal cancer cell lines were used in this study. Cell culture growth medium was split into two equal volumes (50 mL each), one used for DF and the other for UF-SEC to isolate EVs. Obtained EV samples underwent complete characterization which included nanoparticle tracking analysis, western-blotting, electron microscopy and proteomics. Data was then compared between the two types of EV isolation techniques.

3) Results

Hydrodynamic and geometric size distribution of EVs was comparable between the two isolation methods. The number of EVs isolated from equal sample volume and purity which was determined by evaluating the amount of EVs present per microgram of protein was nearly the same. Proteomics of EVs isolated by DF from both cell lines showed more protein types that were identified when compared to UF-SEC.

4) Summary/Conclusion

In this side-by-side comparison of two techniques of EV isolation from cell culture media, DF and UF-SEC, it was determined that both provide high purity samples. When taking into account the initial sample volume it was also found that both provide nearly the same yield from cell culture media. However, DF showed advantage over UF-SEC in proteomics which should be taken into account.

Dr. Kaiping Burrows, Dr. Leandra Figueroa-Hall, Dr. Ahlam Alarbi, Dr. Bethany Hannafon, Cole Hladik, Dr. Rajagopal Ramesh, Dr. Victoria Risbrough, Dr. T. Kent Teague, Dr. Martin Paulus

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1)Introduction. Extracellular vesicles (EVs) play pivotal roles in intercellular communication, influencing various physiological and pathological processes. With increasing interest in EVs for therapeutic applications, establishing standardized methodologies for their isolation is crucial. The heterogeneous nature of EVs and contaminants in biological fluids present challenges, necessitating a comprehensive comparison of isolation methods and their impact on well-established EV markers—CD9, CD63, and CD81. Understanding the variability in isolation methods is vital for enhancing the reproducibility and reliability of EV research.

2)Methods. Seven isolation methods were employed: 1) polymer-based precipitation, 2) low-molecular-weight electrolytes-based precipitation, 3) Size Exclusion Chromatography (SEC), 4) SEC with different fraction collections, 5) Precipitation followed by SEC, 6) Silicon-Carbide methods, and 7) Differential Ultracentrifugation. EVs were isolated from human plasma, plasma with proteinase K (PK) treatment, human serum, and serum with PK treatment. Blood collected in BD Vacutainer tubes was processed, and plasma and serum aliquots were stored at -80°C until EV isolation. EV isolation was performed from 250µL - 500µL of each sample type using the seven methods. EV specificity, size, and concentration were assessed through western blot, immunoassays, and microfluidic resistive pulse sensing. EV markers (CD9, CD63, and CD81) concentrations were evaluated using immunoassays.

3)Results. The SEC method on serum samples yielded the highest CD9 concentrations(6.4AU/mL), while both SEC and polymer-based precipitation from serum+PK showed the highest CD63 yield(0.55AU/mL). Polymer-based precipitation from plasma or serum yielded the highest CD81 concentrations (0.27AU/mL). Evaluating purity (EV markers to total protein ratio) revealed the SEC method on serum EVs had the best CD9 purity, and the SEC method on serum+PK EVs had the best CD63 purity. Both SEC methods and Precipitation + SEC demonstrated good CD81 purity in plasma and serum.

4)Summary/conclusion. This comprehensive study highlights the superior performance of the Size Exclusion Chromatography (SEC) method among seven isolation techniques for extracellular vesicles across various sample types. The method consistently demonstrated high yields and purity for CD9, CD63, and CD81, emphasizing the importance of selecting appropriate isolation methods for specific EV markers. These findings contribute valuable insights toward standardizing EV research methodologies, ultimately enhancing reproducibility and reliability in the field.

Ms Haekang Yang, Ms Shinwon Chae, Mr Chul Won Seo, Ms Seoyeon Kim, Professor Yoon-Jin Lee, Professor Dongsic Choi

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Bacteria release the extracellular vesicles (EVs), known as outer membrane vesicles from Gram negative and membrane vesicles from Gram positive. These lipid bilayer particles are ranged from 20 nm to 100 nm which play diverse roles in bacteria pathogenesis such as antibiotics resistance, transfer of materials, and quorum sensing. Bacterial EVs have a unique density with 1.15 g/mL and thus ultracentrifuge or density gradient ultracentrifuge have been widely used to enrich the EVs. However, these methods are time-consuming and often suffer from contamination issues. Therefore, we established the confident procedure of bacteria EV isolation using optimized size exclusion chromatography (SEC) method while minimizing contamination and optimizing time utilization.

Methods: Limosilactobacillus reuteri and Lactiplantibacillus paraplantarum were obtained from Korean Collection for Type Cultures (KCTC). Bacteria were inoculated into the MRS broth and anaerobic cultured at 37°C and 30°C upto O.D. 1.2 for L. reuteri and L. paraplantarum, respectively. After preclearing of bacteria, the supernatant was concentrated by 100 kDa centrifugal filter. For ultracentrifuge procedure, the concentrated medium (CM) was 50-fold diluted and then subjected to ultracentrifugation at 150,000g. For SEC procedure, CM was loaded on the column and fractionated. We compared the yield and purity using NTA, microBCA assay, SDS-PAGE, Coomassie blue staining, TEM and Western blotting for each of the experimental steps. Mass spectrometry-based quantitative proteomics were conducted to reveal unique proteome of bacterial EVs.

Results: Coomassie blue staining, TEM and Western blotting showed the enriched bacterial EVs from SEC fractions. To compare the purity of each methods, particles (by NTA) per protein amount was applied and SEC method showed the better purity of EV isolations than ultracentrifuge method. Furthermore, proteomic analyses represented that the proteins from membrane and cytosol are enriched in EVs implying unique sorting of selective proteins into EVs form parental bacteria.

Summary/Conclusion: Taken together, we established the efficient isolation method of bacterial EVs by SEC with validation of their proteome by mass spectrometry. This procedure would provide the information of more reliable EV cargos and practical protocol for large scale isolation of bacterial EVs.

Dr. Afshin Iram, Shotaro Masuda, Hana Onizuka, PhD. Ryo Ukekawa, PhD. Takahiro Nishibu

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction

Extracellular vesicles (EVs) contain various bioactive substances such as lipids, nucleic acids, and proteins. Since EVs derived from mesenchymal stem cells (MSCs), have therapeutic potential for various diseases, the use of EVs as therapeutic agents has been thought to be promising. Consequently, multiple methods, such as tangential flow filtration (TFF), size-exclusion chromatography (SEC), anion exchange chromatography (AEX), and their combination have been developed for a larger scale EV purification. Here, we have developed a scalable and reproducible method for affinity purification of EVs by using Tim4 protein, which specifically binds phosphatidylserine displayed on the surface of EVs. Because the binding is Ca²+-dependent, intact EVs can be easily released from Tim4 by adding Ca²+ chelators. In this study, we demonstrated purification of EVs from bone marrow-derived MSC culture supernatant.

2) Methods

Recombinant Tim4 protein was covalently immobilized on the surface of agarose regin and the Tim4-resin was packed into a 1 mL column. 1000 mL of bone marrow-derived MSC culture supernatant was filtrated through ⌀0.22 µm filter to remove cell debris and 200 mL of the resulted supernatant was directly applied into the Tim4 column. After washing the column, 4 mL of PBS containing Ca²+ chelators was applied to elute the captured EVs. The resulted EVs were analyzed by using NTA, ELISA and BCA methods. In parallel, EVs were also purified from the 200 mL of the same culture supernatant by using TFF, TFF + SEC, or TFF + AEX, and the resulted EVs were analyzed for comparison.

3) Results

From 200 mL of MSC supernatant, Tim4 column, TFF, TFF + SEC, and TFF + AEX methods gave 1.68 x 10¹¹, 2,74 x 10¹¹, 1,08 x 10¹¹, and 0.7 x 10¹¹ particles, respectively. The highest purity of the purified EVs was obtained by the Tim4 column method (0.5 x 10¹⁰ particles/µg protein) compared to the other methods (0.10 - 0.14 particles/µg protein). ELISA assays using anti-tetraspanin antibodies also showed a similar trend.

4) Summary/Conclusion

We propose that our affinity purification method has the potential for use in the manufacturing process of therapeutic EVs in fewer purification steps.

Shujin Wei, Professor Wanli Xing

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

The animal intestinal tract is colonized by a huge number of microorganisms, which establish a dynamic and bidirectional interaction with the host. Bacterial extracellular vesicles (BEVs), which contain bioactive substances from their parental cells, can undertake diverse biological functions. Therefore, analyzing BEVs in stools may provide more information for disease research. However, the isolation of BEVs is still facing challenges, particularly in distinguishing BEVs from EVs derived from the host. There is an urgent need to develop approaches to separate BEVs conveniently. We aim to develop a new method based on epsilon-poly-L-lysine (ϵ-PL), which is a broad-spectrum antimicrobial peptide targeting the surface of bacteria, to enrich BEVs from mouse stool supernatant rapidly.

Methods

We first precipitated BEVs from bacterial culture media using our in-house ϵ-PL-based method (PL). The isolated BEVs were identified by transmission electron microscopy, nanoparticle tracking analysis, and LC-MS/MS analysis. The results were compared with those of the commonly used ultracentrifugation method (UC). Next, the cell selectivity of ϵ-PL was validated in a simulated sample containing EVs derived from both mammalian cells and bacteria using western blotting analysis. Thirdly, a protocol to enrich BEVs from mouse stool supernatants was established. Using 16S rRNA gene sequencing, the microbial compositions of BEVs enriched by UC and PL were analyzed and compared with stool samples.

Results

By binding to the surface of BEVs, ϵ-PL can facilitate precipitation of BEVs at a relatively low centrifugal speed (10,000 × g). BEVs isolated by PL from bacterial culture media were comparable to those isolated by UC in size distribution, morphology, and protein profile. Moreover, ϵ-PL shows a higher affinity for BEVs relative to EVs derived from mammalian cells, which makes it possible to selectively enrich BEVs from stool supernatants in a single step. The microbial composition analysis of BEVs isolated from mouse stools conveyed unique profiles and may identify novel biomarkers for disease.

Summary/Conclusion

This study provides a new method to enrich BEVs from mouse stool supernatant. As a cost-effective, simple, and scalable method, the PL method could be used as a powerful tool for therapeutic or diagnostic purposes requiring BEV enrichment.

Research Officer Janice Tan, Principal Investigator Ivy Ho

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

Extracellular vesicles (EVs) are small membranous structures released by all cell types into the extracellular environment. EVs play a pivotal role in intercellular communication and have garnered interest as biomarkers of various diseases. In addition, transfer of EVs between cell types was shown to affect the functionality of the recipient cells. The purity of EV is crucial in their function and potential clinical application. In this study, we employed two different methods to isolate EVs from conditioned media (CM) and compared their size, purity and functionality on microglia/macrophage.

Methods:

EVs were isolated from the same batch of human glioma cells U251MG cultured in serum-free DMEM. The CM was divided equally and subjected to ultracentrifugation (UC) or size exclusion chromatography (SEC). Isolated EVs were characterised based on MISEV2018 guidelines (western blot, transmission electron microscopy, nano-flow cytometry). The immune-modulation effect of the EVs was tested in microglia/macrophages (migration, phagocytosis and cytokine expression).

Results:

To avoid deleterious effect from contaminating proteins co-purified during UC or SEC, the purity of the EVs were assessed. Protein measurement against particles number evaluation showed higher protein concentration in UC-isolated EVs compared with SEC. Using immunoblotting, both UC and SEC-EVs were found to express CD81, CD63, and TSG101. CD81 was significantly increased in UC-isolated EVs. Interestingly, Syntenin-1 was only present in SEC-EVs. Data from Nano-flow showed minimal size difference despite a difference in concentration. EV-exposed microglial cells were evaluated and the effect on migration, phagocytosis, filopodia formation and cytokine expression was tested. Net migration of microglial cells was quantified after 24 hours. The data showed that both EVs isolated using UC and SEC exert similar effects on the microglia/macrophages in vitro.

Summary/conclusion:

Both methods isolated EVs with minimal variations which may indicate a difference in cargo. This difference could potentially influence downstream functions. The absence of syntenin-1 in UC-isolated EVs, despite their similar size, suggests that the two isolation methods may have captured distinct EV populations of the same size. Additional investigations will be conducted to elucidate the reasons for the exclusive presence of syntenin-1 in SEC-EVs and to explore its potential involvement in subsequent cellular functions.

Dr Felicity Dunlop, Dr Shaun Mason, Dr Taeyoung Kang, Professor Suresh Mathivanan, Professor Aaron Russell

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Extracellular vesicles (EVs) and major plasma protein constituents have several overlapping biophysical properties. This provides considerable challenges for EV enrichment, accurate quantification and downstream analysis. The objective of this project was to combine multiple purification approaches, including size exclusion chromatography (SEC), strong anion exchange (SAX) magnetic bead enrichment and single-pot, solid-phase-enhanced sample preparation (SP3) to develop a method to enrich EVs from 500 µL of human plasma for proteomics analysis.

Methods

Blood was collected in EDTA tubes and centrifuged to remove red blood cells and platelets. Plasma stored at -80°C was thawed and centrifuged (precleared plasma). Precleared plasma was subjected to SEC. Fractions were collected for SAX magnetic bead enrichment, with or without concentration by centrifugal filtration. After overnight binding with SAX magnetic beads and washing, EVs were lysed and EV proteins solubilised using an SDS-based lysis buffer and heating at 95⁰C. Reduction and alkylation was conducted simultaneously with TCEP and IAA. Proteins were then prepared using a standard SP3 protocol with STAGE tip desalting prior to injection on an LC-MS (Orbitrap). To establish a more robust reproducible approach, 500 µL plasma samples were split into three 150 µL aliquots after preclearing. Each aliquot was individually processed using SEC/SAX then analysed via LC/MS. Triplicate proteomics data was combined.

Results

The minimal information for EV studies were met using electron microscopy, TRPS and Western blotting; the latter indicating the presence of EV markers CD63 and syntenin, absence of calnexin and a reduction in albumin and ApoA1 in SEC-enriched plasma EVs, compared with precleared plasma. Syntenin, CD9 and CD81 were detected by proteomics analysis in SEC-enriched plasma. However, ApoB100 lipoprotein was also enriched. Therefore, the SEC output was subsequently subjected to SAX to further enrich for EVs based on surface charge. The addition of the SAX purification step doubled the number of quantifiable EV-associated markers, measure by LC/MS. This protocol consistently detected ∼500 proteins with 80-100 of these, quantifiable EV markers.

Summary/Conclusion

Our SEC/SAX enrichment protocol doubles the amount of quantifiable EV markers, compared with SEC alone, as measure by LC/MS from 150 µL of human plasma analysed in triplicate.

Student Jaeeun Lee

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction

Extracellular vesicles (EVs) are emerging as crucial biomarkers in liquid biopsies. However, despite extensive efforts to translate EVs into clinical practice, the use of EVs is currently hindered by the limitations of existing EV-isolation technologies, which remain in their early stages of development.

2) Methods

This study introduces a novel magnetic bead-based ion exchange platform, termed ExoCAS-2 (exosome clustering and scattering), for efficient EV isolation. This platform leverages the inherent negative charge of exosomes to facilitate their binding to magnetic beads coated with a polycationic polymer. The magnetic properties of the beads enable facile manipulation through washing and elution steps, allowing for the isolation of highly pure and high-yield exosomes within 40 minutes.

3) Results

The proposed method successfully isolates exosomes, as evidenced by analyses of their size distribution, morphology, surface and internal protein markers, and exosomal RNA. Compared to commercially available methods, ExoCAS-2 demonstrates superior performance in terms of key aspects such as operation time, purity, and recovery rate.

4) Summary/Conclusion

ExoCAS-2 highlights the significant potential of the magnetic bead-based ion exchange platform, ExoCAS-2, for efficient and high-quality isolation of exosomes from blood plasma, paving the way for their further translation into clinical applications.

Student Yongwoo Kim

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction

The clinical potential of extracellular vesicles (EVs) as therapeutic agents and drug delivery vehicles becomes more and more evident. So, many techniques for EVs isolation have been developed. However, most of the exosome isolation technologies developed so far have been customized in small quantities in a laboratory environment. Therefore, large-scale processing technology is required.

2) Methods

We have proposed a novel method (ExoFilter) for isolating EVs with a charge-based filter in continuous flow mode. The cationic materials were bound to a porous nylon mesh (pore size 1 µm) installed on a spin column (d = 6 mm) and then passed through the plasma 1mL. NTA, RT-qPCR, and Western blot analyses were used to evaluate the efficiency of exosome isolation. SEM image analysis confirmed EVs captured on the mesh surface.

3) Results

The Exofilter method successfully extracted EVs from plasma and serum. EV isolation efficiency increased exponentially as the number of stacked meshes increased. These results demonstrated that the positively charged mesh filter can rapidly and effectively capture and extract exosomes from continuously flowing samples.

4) Summary/Conclusion

Based on the experimental results of this study, we scaled up the ExoFilter, which can accommodate 1 mL of sample, to a large-capacity ExoFilter which can accommodate 1 L of sample. We also demonstrated that the current continuous ExoFilter method can be used to effectively isolate exosomes from large-scale samples (up to 1 L) such as cell culture media.

Student Lee Kangmin

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction

Extracellular vesicles (EVs) are minute membrane-bound particles released by cells, capable of carrying crucial biological information. While scientists recognize their potential as biomarkers for various diseases, the technology to isolate and purify them remains in its early stages.

2) Methods

This study presents a novel method for isolating EVs, termed ExoPAS (exosome precipitation and scattering), which utilizes a cationic material and polyethylene glycol (PEG). The cationic material attracts the negative charge of EVs in biofluids, resulting in the formation of EV clusters. Subsequently, PEG's mesh-like structure binds to these clusters, offering stabilization and preventing undesired aggregation with other molecules. This study meticulously analyzes the isolated exosomes from blood plasma, examining their size, morphology, protein content, and RNA composition.

3) Results

The ExoPAS method successfully extracted EVs from diverse biofluids, including plasma, serum, saliva, and urine. Notably, it yielded significantly higher quantities and purity of EVs compared to existing precipitation-based extraction techniques.

4) Summary/Conclusion

The proposed ExoPAS method outperformed commercially available methods in terms of both purity and recovery rate, indicating that the combination of the cationic material and PEG holds significant promise for robust EV isolation from various biofluids.

Dr. Hye Sun Park, Taewoong Son, Mi Young Cho, Hyunseung Lee, Eun Hee Han, Dr. Kwan Soo Hong

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1. Introduction

Extracellular vesicles (EVs) have been known to play an important role in intercellular communication. Depending on their origin, EVs can be classified exosomes, which are produced by the endosomal pathway, and microvesicles, which are originated from the plasma membrane. Although various technologies for isolating and purifying exosomes from EVs have been continuously reported, the diversity of characteristics depending on the methods and the difficulty in standardization still remain challenging issues.

2. Methods

Here, we propose a method to isolate and sort exosomes from various cell lines, such as cancer, immune, and stem cells, using nanoparticles. The optimal purification methods were applied according to the characteristics of the nanoparticles used, the purified exosomes were qualitatively analyzed using exosome markers, and the purification process was optimized through quantitative analysis according to unique properties of the nanoparticles.

3. Results

The morphological analysis of nanoparticle containing exosomes were confirmed through TEM images. And Western blot and protein quantitative analysis were conducted to confirm exosome characteristics. As the concentration of nanoparticles increased, the amount of purified exosomes increased, and the luminescence properties of the nanoparticles were quantitatively analyzed with various parameters. Also, it was observed that the isolation of exosomes using nanoparticles could give different yield depending on the type of the cells.

4. Summary

We showed that exosomes containing nanoparticles can be isolated from cell culture media via particle characteristics. We demonstrate collection of exosomes derived from various types of cells sorted from different EVs without differential centrifugation. We suggest that this method opens a new way to investigate EVs based on nanomaterials.

Mrs. Edveena Hanser, Prof. Dr. Diego Kyburz

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Synovial fluid (SF) is a complex biofluid that is produced in excess in patients with joint diseases and is used for diagnostic purposes. SF is highly viscous due to its content of hyaluronic acid, which contributes to a poor inter-study reproducibility of EV analysis in SF. In the present study we have assessed the impact of hyaluronidase treatment on tetraspanins expression profile in SF and in enriched EVs using a single-particle interferometric reflectance imaging sensor (SP-IRIS)-based ExoView R200 platform.

Method: SF was obtained from knee joints of 4 rheumatoid arthritis (RA) and 4 osteoarthritis (OA) patients, with informed consent. SF was centrifuged, aliquoted and kept at -80° until further use. SF aliquots were thawed and treated with 30U/ml of hyaluronidase (Sigma) at 37°C for 45 minutes, treated and untreated aliquots were centrifuged and diluted with PBS. EVs were isolated from treated SF using size exclusion chromatography (SEC). The characterization was done by nanoparticle tracking analyzer and transmission electron microscopy. The concentration of treated, untreated SF and of isolated EVs was adjusted to 1.00E+09 particles/ml for Exoview R200, NanoView Biosciences.

Result: The expression profile of tetraspanins in terms of the number of events on capture spots CD63, CD81 and CD9 was higher in hyaluronidase treated SF from patients with RA compared to untreated SF. In contrast, the tetraspanin expression of treated and untreated OA SF samples was similar. This may be attributed to the fact that OA SF is more viscous and might need higher concentrations of hyaluronidase. The colocalization profile of tetraspanins differed between EVs from treated and non-treated SF. When EVs were measured directly in SF by Exoview, a higher number of EVs was detected as compared to measurement after EV isolation by SEC.

Conclusion: Hyaluronidase treatment of SF in RA resulted in a higher number of events compared to untreated SF, suggesting removal of hyaluronic acid increased EV yield. Thus, hyaluronic acid treatment of SF should be considered to optimize EV analysis. In addition, enrichment of EVs by SEC resulted in a lower count, suggesting that SF can be used directly without prior EV isolation.

Dr. Huixian Lin, Dr. Chunchen Liu, Prof. Bo Li, Prof. Lei Zheng

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: The isolation and enrichment of specific extracellular vesicle (EV) subpopulations are essential in the context of precision medicine. However, the current methods predominantly rely on a single-positive marker and are susceptible to interference from soluble proteins or impurities. This limitation represents a significant obstacle to the widespread application of EVs in biological research.

Methods: Herein, we propose a novel approach that utilizes proximity ligation assay (PLA) and DNA-RNA hybridization to facilitate the binding of two proteins on the EV membrane in advance enabling the isolation and enrichment of intact EVs with double-positive membrane proteins followed by using functionalized magnetic beads for capture and enzymatic cleavage for isolated EVs release. The TEM, SEM, NTA were used for the isolated EVs characterization and the NanoFCM was used for capture efficiency assessment. For further exploring its application, RNA sequencing was conducted and a breast cancer cohort was collected.

Results: The capture efficiency of this method reaches approximately 83.87%, with each magnetic bead providing an average of 1425 EVs captured. The method we constructed facilitates the isolation and enrichment of large-scale subpopulations of EVs with a double-positive membrane protein. Additionally, this method demonstrates excellent specificity, avoiding interference from soluble proteins to a certain extent. Furthermore, our study highlights the potential application of isolating and enriching EVs with a double-positive membrane protein in biological function studies and biomarker screening. Notably, this technology shows promise in isolating and enriching EVs from heterogeneous plasma-derived EVs for cancer diagnosis, thus promoting the clinical application of specific EV subpopulations.

Conclusion: In conclusion, our study presents a simple, effective, and novel strategy for isolating and enriching EVs with a double-positive membrane protein without complex equipment. This strategy has the potential to drive forward EVs research.

Yong Shin, Professor Eun Jae Lee

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction

Exosomes are small vesicles enclosed by a membrane that serve as essential mediators of intercellular communication and are also valuable biomarkers for various diseases, including cancer. However, traditional methods for isolation exosomes, including cell type specific exosomes, are labor-intensive and require complicated pretreatment techniques.

2) Methods

In this study, molecular imprinted polymers-based articial peptide (MIPaps) for exosome isolation utilizing protein and peptide from the tetraspanin protein family (CD63/CD9 and CD81) were developed. The technique was confirmed by size distribution by NTA, morphology by SEM/TEM, miRNA level by RT-qPCR, and protein content of exosomes. Additionally, the capture percentage and recovery rate were calculated.

3) Results

Our findings suggest that peptide templates can be used as an alternative to protein templates for synthesizing MIP-based MNPs. Lastly, MIP-based MNPs made with GLAST templates were employed to capture astrocyte-derived exosomes. The results indicate that MIP-based MNPs created with GLAST peptides as templates can be utilized to isolate serum exosomes and analyze astrocyte protein markers and miRNA, which may be useful in the diagnosis and monitoring of Neuromyelitis Optica Spectrum Disorder (NMOSD).

4) Summary/Conclusion

The MIP-based MNPs demonstrate the potential to capture exosomes from biofluids and provide a rapid, simple, and high-yield approach for clinical applications of human excreta analysis.

Professor Wei Duan, Mr Rajindra Napit, Dr Rocky Chowdhury, Mr Satendra Jaysawal

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

CD63/CD81 and CD9 are well-established sEV biomarkers. However, the existing methods for isolating sEVs have several significant limitations that hinder their widespread adoption in research laboratories and clinical settings. Aptamers are single-stranded DNA/RNA molecules that adopt a specific three-dimensional structure, enabling them to bind with high affinity and specificity to target molecules. Through a peptide-based SELEX approach, we isolated DNA aptamers against a specific epitope on one of extracellular domains of human CD9. With several rounds of molecular engineering, we have evolved our CD9 aptamer to a ligand that binds to native CD9 proteins on the plasma membrane of both human cells and sEVs with high specificity. Utilising this newly developed CD9 aptamer and CD81/CD83 aptamers, we developed a novel tetraspanin aptamer-based affinity isolation methods for the scalable isolation of cancer biomarker-positive sEVs. Both the EV capture and release are carried out in physiological buffer and pH. Further refinement of our novel aptamer-guided EV isolation strategy will pave the way for future personalized precision oncology.

Beatriz Martín-Gracia, Optimization of separation methodologies for obtaining high yield-high purity urinary extracellular vesicles Håkon Flaten, Optimization of separation methodologies for obtaining high yield-high purity urinary extracellular vesicles Krizia Sagini, Optimization of separation methodologies for obtaining high yield-high purity urinary extracellular vesicles Alicia Llorente

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction

According to the Global Cancer Observatory, approximately 1.4 million new cases of prostate cancer (PCa) and 375,000 related-deaths were registered worldwide in 2020. Even if the detection PSA is a widely used tool for the diagnosis of PCa, many studies agree that its use leads to overdiagnosis and overtreatment because this protein is not cancer-specific. For this reason, the search for new biomarkers represents an urgent need in the management of PCa. In the last decade, extracellular vesicles (EVs) have emerged as a very promising source of biomarkers for PCa in liquid biopsies because they are a molecular fingerprint of the cells of origin and are found in biofluids. Particularly, several studies have focused on urinary EVs because urine is easily collected and is in close contact with the prostate. However, as with other biofluids, it is important to optimize the methodology around urinary EV separation and analysis to promote the use of EV-associated molecules as PCa biomarkers.

2) Methods

In this study two EV separation methods, differential centrifugation (DC) and ultrafiltration coupled to size exclusion chromatography (SEC), were optimized and compared to each other in terms of small EV yield and purity by analyzing both the number of EVs by nanoparticle tracking analysis and the expression of EV markers by Western blot.

3) Results

When performing DC, results showed that the addition of a 200 nm filtration step largely removed from the EV pellet uromodulin, a very abundant protein in urine that co-isolates with EVs. On the other hand, concentration of urine samples with a 10 kDa ultrafiltration device prior to SEC gave a higher EV yield compared to 30 kDa and 100 kDa devices. After concentration, the isolation of EVs by SEC also avoided the contamination with uromodulin.

4) Summary

Preliminary studies resulted in SEC providing a slightly higher yield of EV recovery with a similar purity to DC. Considering that DC is a more laborious and time-consuming technique than SEC, the use of ultrafiltration coupled to SEC is a good EV isolation alternative that can help in the implementation of EV-based biomarkers in the clinic.

Dr. Zheng Zhao

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction: In the burgeoning field of extracellular vesicle (EV) research, efficient isolation from primary cells at small to medium scales (15 mL to 2 L) remains a critical challenge. Addressing this, our study explores a novel approach utilizing Mesenchymal stem cells (MSCs) with 3-demtional bioreactors. Tangential Flow Filtration (TFF), diafiltration, and ion-exchange purification are applied to achieve high-throughput isolation. The isolated EV were validated by Nanoparticle Tracking Analysis (NTA), western blot, uptaking assay, wound healing assay, to show the therapeutic potential of MSC EVs.

2) Methods: MSCs were sustained in 2 L bioreactor, and EV depleted media was exchanged in day 5, and conditioned media was collected on day 8. A 0.8 um filter was used for clarification, and 750 kDa hallow fiber was used for TFF and diafiltration. A 4 mL volume of ion exchange column was used to removal rest of protein impurities. The isolated EV were store under 4C and validated by western blot, uptaking assay, wound healing assay, and proteomic analysis to show the therapeutic potential of MSC EVs

3) Result: Particle concentration analysis revealed consistently robust concentrations throughout the entire process, ranging from 1.2E9 particles/mL to 3.02E10 particles/mL, with a final volume reduction from 2 L to 45 mL. The overall particle yield reached approximately 1.36E12 particles, demonstrating a commendable total recovery rate of 85%. Western blot results corroborated the presence of essential EV markers, including CD9, CD63, ALEX, and TSG101. Cellular uptake studies demonstrated rapid internalization within the first hour, while wound healing assays underscored the therapeutic potential of isolated MSC EVs by significantly enhancing the cell healing process compared to the control.

4) Summary/Conclusion: By focusing on this innovative method, we enhanced the EV purification processes from MSC with small to medium scales range up to 2 L and also validated the integrity and therapeutic potentials of these EV. With proper upstream equipment's, the process can be easily upscaled to 50 L.

Miss Emma Morris, Associate Professor Karl Hassan, Professor Craig Priest, Dr Bin Guan, Dr Renee Goreham

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths annually. Currently, the only way to diagnose lung cancer is to undertake a biopsy, which is usually done after symptoms are present. At this point, the patient survival rate is low. For this reason, a screening process that is non-invasive and portable is needed to increase patient survival rates. With each person exhaling 10,000 L of breath each day, breath offers an alternative sampling source that is accessible and does not cause the patient discomfort. This project aims to develop a breathalyser to target biomarkers found within breath, namely extracellular vesicles. Extracellular vesicles, released by various cell types, have recently been discovered in exhaled breath. Since exhaled breath condensate holds a spectrum of molecules and biomarkers, it's crucial to employ a detection method that can isolate small extracellular vesicles from impurities. The integration of pillared interdigitated electrodes facilitates the organic isolation of these nanoparticles. Leveraging thiol-modified DNA aptamers with high specificity and affinity to surface proteins, such as the common biomarker CD63 and CD44 associated with lung cancer, the micropillar device adeptly captures small extracellular vesicles on its gold surface. The thiol group of the modified aptamer will interact with the gold surface creating a stable bond, following incubation withextracellular vesicles derived from A549 lung adenocarcinoma cell line and exhaled breath condensate from healthy patients within a concentration curve of 3.10 x 10⁷ – 7.94 x 10⁷ particles per mL, a monolayer will form upon the gold surface. The monolayer can be characterised, as the gold surface acts as an electrode, by electrochemical impedance spectroscopy. This response serves as a measurable indicator of successful small extracellular vesicle capture. Future adaptation of this work presents the incorporation of a polyclonal aptamer library to offer increase specificity to an extracellular vesicle target. The envisioned outcome is the creation of a portable screening device for lung cancer, promising advancements in prognosis and facilitating effective monitoring of cancer progression.

Dr Farha Ramzan, Hui Hui Phua, Dr Vidit Satokar, Dr Shikha Pundir, Dr Anastasia Artuyants, Dr Cherie Blenkiron, Dr Chris Pook, Prof Mark Vickers, Dr Ben Albert

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) are reported to play a key role in intercellular communication and are present in abundance across a range of body fluids. Emerging evidence has suggested that EVs in breast milk may impact the health and well-being of infants. Various methodologies have effectively isolated EVs from breastmilk, including ultracentrifugation, size exclusion chromatography (SEC), and density-based separation. However, each approach has strengths and limitations, often involving complex and time-consuming approaches. Therefore, there is an urgent need to establish an efficient isolation technique that optimally balances ease of use and affordability while maximising yield and purity. This is key for identifying the functional components within EVs that may mediate infant health outcomes.

Methods: Using banked breastmilk samples from the Fish Oil in Pregnancy Trial (ACTRN12617001078347; 1mL), a comprehensive evaluation of EV isolation methods, including ultracentrifugation, SEC, and charged core bead chromatography using Capto™ Core (CC) was examined. Isolated EVs from different methods were compared by nanoparticle tracking analysis (NTA) to determine particle size and quantity, transmission electron microscopy (TEM) to visualise EV integrity and morphology, and western blot to investigate selected EV-associated protein cargo.

Results: Following TEM analysis, all EV isolation methods were shown to contain 40-150 nm sized EVs. However, CC isolation visibly enhanced EV integrity with less contaminant debris and lipid-type droplets. As evidenced by western blotting, all isolates were positive for small EV markers (CD9, CD81 and CD63). However, CC isolation resulted in cleaner EV isolates with less lactoferrin (milk contaminant) and Calnexin (cellular marker). Interestingly, both UC (9.04*1010 particles/ml) and SEC (1.16*1011 particles/ml) resulted in higher concentrations of particles as compared with CC isolation (2.45*109 particles/ml).

Conclusion: Based on our findings, the CC isolation method (Capto™ Core) serves as a viable option for isolating an EV-enriched subset, with the removal of larger quantities of contaminant material than alternatives whilst maintaining the integrity of the EVs. The next step involves conducting a comprehensive molecular characterisation of the isolated subset and evaluating the functionality of the EV cargo in cell models.

vesicles: EV-FISHER

Dr Weilun Pan, Prof Lei Zheng, Prof Jinxiang Chen, Prof Bo Li

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Extracellular vesicles (EVs) have found diverse applications in clinical theranostics. However, the current techniques to isolate plasma EVs suffer from burdensome procedures and limited yield. Herein, we report a rapid and efficient EV isolation platform, namely EV-FISHER, constructed from the metal-organic framework featuring cleavable lipid probes (PO43−-spacer-DNA-cholesterol, PSDC). The EV-FISHER baits EVs from plasma by cholesterol and separates them with an ordinary centrifuge. The captured EVs could be released and collected upon subsequent cleavage of PSDC by deoxyribonuclease I. We conclude that EV-FISHER dramatically outperforms the ultracentrifugation (UC) in terms of time (∼40 min vs. 240 min), isolation efficiency (74.2% vs. 18.1%), and isolation requirement (12 800 g vs. 135 000 g). In addition to the stable performance in plasma, EV-FISHER also exhibited excellent compatibility with downstream single-EV flow cytometry, enabling the identification of glypican-1 (GPC-1) EVs for early diagnosis, clinical stages differentiation, and therapeutic efficacy evaluation in breast cancer cohorts. This work portrays an efficient strategy to isolate EVs from complicated biological fluids with promising potential to facilitate EVs-based theranostics.

Dr Jérémy Boulestreau^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Dr Laurence Molina^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Alimata Ouedraogo^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Louen Laramy^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Ines Grich^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Dr Thi Nhu Ngoc Van^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France; SkillCell, Montpellier, France}, Dr Franck Molina^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Dr Malik Kahli^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) are lipid bound nanoparticles known to play a key role in cell-to-cell communication, including homeostatic and pathological processes. Their presence in the peripheral fluids including saliva, a biofluid easy to collect in a non-invasive way, makes EVs potent tools for discovery of diagnosis or prognosis biomarkers. Several methods to isolate and characterize salivary EVs (sEVs) have been described but require further optimization and standardization to foster the translation to clinical application. The aim of this research was to rigorously characterize and directly compare salivary EVs isolated from two different fast and scalable technics (co-precipitation and immuno-affinity) to the current time-consuming ultracentrifugation method.

Methods: In this work, we collected saliva samples from nine healthy volunteers. sEVs were isolated using three different methods: ultracentrifugation (UC), co-precipitation (Q) and immunoaffinity (M). The EVs size and distribution were analyzed using NTA and their morphology by cryo-EM. Protein and miRNA cargos were also determined by western blot, proteomics and RT-qPCR. We also systematically assessed the impact of saliva filtration on 0.22 and 0.45 µm filters before EV isolation as this was never clearly investigated.

Results: Our findings reveal that: sEVs are abundant and can be isolated from small volumes (1ml) of saliva. UC and Q EVs have the same size distribution, but twice less particles for Q samples. M EVs are significantly smaller (84 nm vs 264 and 227 nm for UC and Q respectively). Protein and small RNA cargos greatly varies depending on the isolation method chosen and we show that EVs isolation allows detecting specific biomarkers undetected in the whole saliva. We confirm that miRNAs are principally contained in EVs and not in free circulating form in saliva. We also have determined that filtration is detrimental for EVs isolation. Finally, our results suggest that co-precipitation method is compliant, suitable for biomarker discovery, and diagnostic.

Summary/Conclusion: This work characterizes a neglected source of EVs and provide evidence that co-precipitation method is efficient, suitable for analysis of clinical samples and cost-effective for isolation of salivary EVs from small volumes of saliva.

Professsor Takanori Ichiki, Chiharu Mizoi, Kento Toyoda, Professor Naohiro Seo

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

Recent efforts have been directed towards the development of methods for isolating and purifying exosomes from extracellular vesicles (EVs). While techniques utilizing surface molecules such as tetraspanins and phospholipids have been considered highly specific, there is emerging evidence suggesting that leveraging physical properties like size and charge differences, in combination with appropriate separation methods, may offer a highly efficient means of exosome isolation. Looking ahead to clinical applications of exosomes, it is imperative to explore platform technologies for the scalable and quality-assured preparation of exosomes.

Methods:

Culture supernatant of human dental pulp stem cells (DPSC) was subjected to ultrafiltration to concentrate EVs. Subsequently, ion exchange column chromatography was employed to fractionate EVs based on their charge differences. This method, following the high-efficiency exosome recovery protocol developed by Seo et al., utilized a DEAE Sepharose column with a linear gradient of NaCl aqueous solution [N. Seo et al., J. Extracellular Vesicles 11, e12205 (2022)]. The isolated particle populations were then subjected to single-particle multiplex measurements using a microfluidic device developed by Ichiki‘s group, evaluating the size and zeta potential of each particle [T. Ichiki et al., Microcapillary chip-based extracellular vesicle profiling system. Extracellular vesicles: methods and protocols, 209-217 (2017)].

Results:

Fractions extracted in NaCl solutions at specific salt concentrations (200 mM) and (400 mM) exhibited maximal particle number density. Multiplex measurements using the microfluidic device revealed distinct particle populations: one showing a size distribution in the range of 60-300 nm and a relatively narrow distributions of zeta potential peak at approximately -16 mV, and the other displaying a size distribution of 30-450 nm and zeta potential peak at approximately -20 mV, respectively. These results support the successful recovery of particle populations resembling exosomes and microvesicles.

Summary:

By employing ion exchange chromatography, distinct particle populations resembling exosomes and microvesicles were obtained. The results of single-particle multiplex measurements using the microchip-based nano-particle analysis system clarified the biophysical differences in isolated EV particles. This achievement is anticipated to contribute to the development of a technological platform for the convenient and quality-assured preparation of exosomes in large quantities, fostering their potential clinical applications.

M.D., Ph.D. Akira Yokoi, M.D., Ph.D. Kosuke Yoshida, B.Sc. Masami Kitagawa, Ph.D. Takao Yasui, M.D., Ph.D. Hiroaki Kajiyama

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

Extracellular vesicle (EV) isolation is the fundamental and critical factor for EV research, and one of the limitations of EV recovery is the requirement for a certain volume of biofluids. We recently invented tailored cellulose nanofiber (CNF) sheets (Yokoi A et al., Nat Commun. 2023), and here, we aimed to test the CNF sheets' performance toward EV isolation and explore their utility in clinical applications.

Methods:

To test the quality of EVs, which were captured by CNF sheets, we performed nanoparticle tracking (NTA) assays, western blotting (WB) analyses for EV markers, and a cryo-electron microscope. We applied small RNA sequencings to assess the profiles of EVs. For functional analyses, we use an ovarian cancer mouse model using ID8 cells, a murine epithelial ovarian cancer cell line. To validate the utility of CNF sheets in the human body, we obtained EVs from 210 samples of seven perioperative patients.

Results:

Using 10 µL of human serum, small-size EVs were successfully isolated by CNF sheets, and purity and yield were significantly higher than with serial-ultracentrifugation methods. Moreover, sEVs were preserved in the sheet for a week in a dry condition. By attaching the EV sheet to the moistened organs in vivo, the sEVs from trace ascites were collected. In the mouse model, CNF sheets revealed that cancer-related miRNAs were detected in the very early phase. In cancer patients, the direct EV sheet attaching method during the surgery identified the location-based unique sEV miRNA profile, and the tumor surface sEVs had distinct profiles from tissues or whole ascites. Trajectory analyses revealed that the connection pattern differed in patients with localized or advanced disease. Tumor-associated sEV-miRNAs on tumor surfaces were also detectable in serum, urine, or saliva and decreased in post-operation.

Summary/Conclusion:

CNF sheets provide a whole new concept of EV analyses, including EV isolation from micro-volume body fluids and EV preservation in dry conditions for a week. Furthermore, CNF sheet attachment methods enable the capture of ascites sEVs on organ surfaces, revealing the location-based EV heterogeneity of cancer patients. This tool has broad potential applications contributing to basic and translational EV research.

Dr Bradley Whitehead, PhD Litten S Sørensen, Anders T Boysen, Prof Peter Nejsum

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction

Single vesicle imaging has advanced significantly, allowing multiplex analysis of EV markers to determine EV heterogeneity and phenotyping of vesicles. However, many systems require EV immobilisation and rely upon antibodies targeting EV markers, such as CD63 or CD81 for capture that introduces positive selection bias that can obscure true phenotypic analysis. Therefore, the aim of this study was to assess a mechanism of capture via targeting ubiquitously expressed EV lipids and to determine the level of sample pre-processing required to enable selective capture to enable a “clinic friendly” method of EV isolation and characterisation

2) Methods

Cholera toxin subunit B (CTxB) with known binding to GM1 sphingomyelin was selected for assessment. Extracellular vesicles were harvested from the cell culture supernatant of RAW macrophage cell lines and human plasma from anonymous donors from Danish blood bank, Aarhus Hospital. EVs were isolated using ultracentrifugation (UC), size exclusion chromatography (SEC) or a combination of SEC and UC. Binding of biotinylated CTxB to EVs and non-EV fractions was determined using dot-blot arrays and contrasted to known markers of EVs (CD63) and markers of LDL (ApoB) and HDL (ApoA). Binding of CTxB to purified LDL and HDL was assessed. Removal of LDL and HDL by immunoprecipitation, anionic polymer precipitation, UC, SEC or SEC plus UC was assessed using dot blot. Finally, EV capture was mediated by immobilisation of biotinylated CTxB capture ligand on streptavidin coated slides and binding of fluorescently labelled EVs was assessed.

3) Results

Lipid mediated detection of EVs from conditioned cell culture media was selective and efficient, however, EVs isolated from human plasma using UC or SEC alone showed significant contamination with HDL or LDL, respectively. Furthermore, the CTxB showed cross-reactivity with non-vesicle SEC fractions and it was determined the ligand also bound with high affinity to LDL/HDL. The removal of lipoprotein contamination was assessed and a combination of SEC followed by UC was sufficient to remove lipoprotein and enable selective EV capture by GM1 lipid binding ligand.

4) Conclusion

EVs can be selectively analysed using unbiased capture via lipids, however this is dependent on efficient removal of lipoprotein contamination.

Mr. Yee-Hsien Lin, Mr. Han-Tse Lin, Mr. William Milligan, Dr. Min-Chang Huang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Background and Aim

The therapeutic potential of extracellular vesicles (EVs) has emerged as a prominent focus in biomedical research. EVs play a crucial role in cell-to-cell communication, influencing various disease processes, cancer development, and tissue regeneration. Exosomes, a subcategory distinguished by their size, contain a plethora of bioactive molecules and genetic information, sparking interest in novel therapeutic and diagnostic applications. Leveraging their distinctive biological properties, lipid bilayer exosomes can be engineered as an effective drug delivery system for tissue regeneration and cancer treatment. Therefore, meticulous methodology is imperative to generate high-quality cell derived EVs, particularly in the context of cancer research and therapeutic applications.

However, conventional cell culture supplements like FBS or human platelet lysate (hPL) pose challenges, potentially influencing target cell physiology due to supplement-derived EVs. This not only raises concerns of misinterpreted results but also complicates downstream isolation and analysis. Hence, the use of qualified ancillary materials and a controlled culture environment is paramount to ensure consistent EV expression from cells, both in research and clinical settings.

In this context, a novel gamma-irradiated hPL (ED hPL Gi), characterized by its xeno-free nature and high EV depletion, is employed in the culture system for producing cell-derived EVs. ED hPL Gi creates an effective environment, supporting the long-term secretion of EVs from MSC, immune cells, as well as cancer cells.

Methods, Results & Conclusion

When cells reached 30–50% confluency in a culture plate or reached a cell density of 1x106/ml, the culture medium was changed to 0-5% ED hPL Gi medium to initiate EV production. Our results indicate that ED hPL Gi is viable for all the target cells in the study, producing a substantial amount of EVs while extending cell activity. Moreover, cells exhibited continuous growth throughout the experiment, maintaining over 85% cell viability. Therefore, ED hPL Gi emerges as a promising supplement for the production of MSC-, immune cell- and cancer cell-derived EVs, suitable for both exosome research and GMP manufacturing for clinical applications.

Msc In Medicinal Chemistry, doctoral researcher in Pharmacy Elena Scurti¹, PhD Martina Hànzlikova¹, MSc Johanna Puutio², PhD Fadak Howaili³, PhD Kai Härkönen⁴, Professor Pia Siljander², PhD Saara Laitinen⁴, Professor Tapani Viitala^1,3

¹Division of Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland, ²EVcore facility, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland, ³Åbo Akademy University, Turku, Finland, ⁴Finnish Red Cross Blood Service, Helsinki, Finland

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Proper characterization of extracellular vesicles (EVs), specifically subpopulations of EVs, is a challenging task. Also, cell assays for assessing EV cell uptake are mainly static, which omits the dynamic nature of biological interactions, and require labels, which may alter EV properties and cell behavior. The aim of this work was to develop unique real-time label-free surface plasmon resonance (SPR)-based analysis platforms for EV size determination, subpopulation identification and EV-cell interaction studies.

Methods

Red blood cell (RBC)- and platelet (PLT)-derived EVs, and commercial reference EVs were captured by general EV (i.e., CD9, CD63, CD81) and EV specific antibodies (i.e., CD41 (PLT EVs), CD235ab (RBC EVs)) immobilized on the SPR sensor surface. The ratio between the SPR signal responses measured at two different wavelengths (i.e., 670nm and 785nm) was used to determine the size of the EVs through mathematical calculations. DLS and NTA were used as comparative size values. Different concentrations of EVs were allowed to interact with HeLa and PC3 cell lines seeded on fibronectin coated SPR sensor, and kinetic data for the cell interactions were extrapolated from the SPR signal responses.

Results

EV antibodies were successfully immobilized on the SPR sensor and demonstrated effective capturing of EVs, which enabled size determination of EVs. The EV sizes obtained from the SPR analyses ranged from 150 nm for RBC EVs, between 30-200 for PLT EVs and their subpopulations, and 150-200 nm for commercial reference EVs, largely agreeing with DLS and NTA data. EV cell interactions with HeLa cells showed concentration dependent SPR responses. In case of PC3 cells the interaction kinetics measured with the cell-based SPR platform revealed that PLT EVs are taken up by the cells more readily than RBC EVs.

Conclusions

Real-time label-free SPR analysis platforms for EV characterization were successfully developed. The size of EVs determined with the SPR analysis platform proved to be accurate. The cell-based SPR analysis platform proved the selectivity of cell layers towards a specific type of EV, and the cellular responses, which were dependent on the EV concentration, provided information about the interaction kinetics between EVs and cells under dynamic conditions.

Ms. Amy Henrickson¹, Dr. Lutz Ehrhardt, Dr. Shawn sternisha

¹Beckman Coulter, Indianapolis, United States

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: With the progress in EV research, an understanding of the different workflows is essential. As different production and purification methods typically lead to different purities, while different characterization techniques highlight different perspectives of the purified vesicles, it is essential that an in-depth understanding of these methods is available. Here, we will discuss several different workflows that center on centrifugation purification and analysis to help researchers understand the benefits and limitations of the different methods.

Methods: Preparative ultracentrifugation offers many different purification options. The current need to improve EV purification might require re-thinking the currently used purification strategy to further remove contaminations, which might interfere with your workflow. Centrifugation offers multiple purification strategies, including density gradient ultracentrifugation, rate-zonal centrifugation, and differential centrifugation. Each method has its benefits and limitations, which will be discussed.

Centrifugation can be used for both the preparation of EVs and the characterization of samples using the analytical ultracentrifuge (AUC). AUC works by monitoring the sedimentation and diffusion patterns of particles under centrifugal force. From this data, several sample properties can be determined, including sample purity and size distribution, both of which are important aspects of EV research. Additional biophysical properties, including sedimentation and diffusion coefficients, anisotropy, hydrodynamic radius, and partial specific volume, can also be determined by AUC.

Results: Here, we demonstrate several different preparation methods and discuss their benefits and limitations in regard to use and contaminant removal. While demonstrating the characterization that can be achieved with AUC.

Summary/Conclusion: Understanding the different purification methods will enable researchers to choose the best method for their EVs. AUC has long been established as a strong characterization of viral vectors; it is a first principal characterization technique that enables researchers to study their samples in their final formulation. Collectively, these findings will help researchers with sample purification and highlight the advantages of adding AUC as an orthogonal characterization technique for EVs.

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, France

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Several methods are employed for extracellular vesicles (EVs) analysis. Isolation methods typically involve ultracentrifugation, size-exclusion chromatography, or precipitation-based approaches. Characterization techniques, such as electron microscopy, nanoparticle tracking analysis, flow cytometry, and Western blotting, provide information about EV size, morphology, surface markers, and protein content. Here we sought to present a typical workflow for the integration of flow cytometry in EV characterization.

Methods

A comprehensive workflow for the characterization of EVs is proposed, with two distinct models depending on the purpose: biomarker/heterogeneity analysis (commonly used in R&D) and specific EV production (for manufacturing purposes). The workflow includes several key steps such as EV preparation, purification, quality control, and characterization. Flow cytometry is a widely used method in this process and is often complemented with orthogonal methods for comparison.

Results

We highlight the significant benefits of automation in EV preparation, which effectively reduces variability introduced by human operators. This automation ensures consistent and reliable results throughout the process. Additionally, flow cytometry stands out as a superior method for EV characterization due to its ability to sensitively detect and count single EVs using fluorescence. The ability to analyze EVs individually is particularly important in biomarker/heterogeneity analysis. Moreover, the user-friendly nature and robustness of the tested cytometer offer significant advantages, especially in a manufacturing setting. Lastly, the seamless integration of the flow cytometry into an EV workflow analysis further proves its advantages.

Conclusion

In this work, we demonstrate the seamless integration of flow cytometry into EV research. The utilization of the flow cytometry allows for the acquisition of high-quality and reproducible data, owing to its exceptional sensitivity. Collectively, these findings strongly suggest the advantages of incorporating flow cytometry as a complement to, or even a replacement for, orthogonal methods in EV analysis.

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, France

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

The recent release of MISEV2023 marks a significant milestone for the EV research community, aiming to establish standardized guidelines and recommendations for the analysis and reporting of extracellular vesicle studies. The pursuit of knowledge in this field involves utilizing various solutions to generate data, while ensuring quality and reproducibility. This report focuses on how current practices in centrifugation and flow cytometry align with MISEV2023, along with potential gaps. Detailed protocols are provided for centrifugation methods like differential ultracentrifugation and analytical ultracentrifugation, as well as flow cytometry protocols for particle counting, characterization, and sorting. Specifically, the ability to detect and characterize small EVs by flow cytometry is highlighted for its exceptional fluorescence sensitivity (scatters and fluorescences). We also delved into the analysis and reporting aspects of the different techniques, offering a critical review of the current status quo. By examining the needs for proper analysis and reporting of EV-derived data scientists can gain valuable insights to enhance their EV research endeavors.

Mr SRIRAM GUMMADI¹

¹Latrobe University, Australia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

EV Quant: A quantitative web-based compendium of extracellular vesicles cargo for studies in vesiclepedia

Introduction: Extracellular vesicles (EVs) are nanosized membrane bound vesicles that are shed by cells into extracellular space. EVs have a key role in multiple pathophysiological processes due to their signalling capabilities and are classified into various subtypes based on their biogenesis and origin. Exosomes are small EVs that range in 30–150 nm diameter and they originate due to the fusion of multivesicular bodies with the plasma membrane.

In terms of the molecular composition, EVs sequester a diverse and rich cargo of proteins, lipids, and nucleic acids, and the ensuing cargo is altered according to the pathophysiological conditions of the host cells that secrete them. Hence, a catalogue of the EV cargo in various conditions could aid in identifying an EV fingerprint that are specific to a tissue, cell type, and/or pathology.

Methodology: EV Quant is a quantitative plugin added to Vesiclepedia and are built using a combination of MySQL for the background database, Zope content management system and Python as the programming language that connects the database to the content management system and the data is from both published and unpublished studies.

Results: We describe an update of Vesiclepedia (http://www.microvesicles.org) an online database that contains RNA, proteins, lipids and metabolites that are identified in EVs and Eps.

EV Quant, a plugin added to vesiclepedia which contains the quantified EV cargo for the experiments in vesiclepedia. Users can search for a biomolecule and compare the quantification data between treatments in a specific experiment and in addition to that, all of the EV cargo identified in that experiment is summarised as a data table and a heatmap showcasing the top 100 differentially regulated candidates will be plotted in real time.

Currently, Vesiclepedia contains data obtained from 3,533 EV studies, 50,550 RNA entries, 566,911 protein entries, 3,839 lipid entries, 192 metabolites and EV Quant has 62,822 quantification entries.

Conclusion: A catalogue of EV cargo will immensely benefit the research community in identifying an EV fingerprint that are specific to a tissue, cell type, and/or pathology.

MD Miłosz Majka¹, PhD Katarzyna Czarzasta², MD, PhD Małgorzata Wojciechowska², PhD Małgorzata Czystowska-Kuźmicz¹

¹Medical University of Warsaw, Chair and Department of Biochemisrty, Warsaw, Poland, ²Medical University of Warsaw, Laboratory of Centre for Preclinical Research, Chair and Department of Experimental and Clinical Physiology, Warsaw, Poland

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

MicroRNAs (miRNAs) have been identified as potent EV-derived molecules with promising clinical implementation. Packaging of miRNAs into vesicles increases their stability but also complicates their specific isolation. There are several methods for the extraction of circulating miRNAs from plasma with following quantification by RT-qPCR. We optimized a protocol for RNA isolation from EV-rich SEC fractions and determined how it influences EV-miRNA characteristics in a set of platelet-free plasma (PFP) from clinically characterised individuals.

Methods:

EVs were isolated from 1ml of human PFP using SEC and nanoparticle tracking analysis (NTA, ZetaView) was used to analyze the particle size and number. The EV-protein markers were then characterized by Western blotting. MiRNeasy Serum/Plasma Kit (Qiagen) was used to isolate RNA from PFP and EV-rich samples. RNase and detergent sensitivity assay was performed prior to isolation. A Nanodrop spectrophotometer was used to evaluate the total RNA concentration and purity and Agilent Bioanalyzer small RNA chips were used to evaluate the size range of isolated RNA. Extraction efficiency was evaluated by Taqman advanced miRNA assay in RT-qPCR using array cards and singe-tube assay.

Results:

EV-rich fractions isolated by SEC consisted of RNA of higher purity than the protein-rich fractions. RNA content of those fractions was well protected against RNAse-proteinase treatment while RNA concentration significantly decreased after addition of detergent. The modified protocol rendered significantly more RNA of higher purity compared to the standard protocol. Analysis of clinical samples showed that the modified protocol enables to isolate microRNAs from EV-rich fractions of different profile compared to PFP.

Summary:

This study succeeded in providing conditions to extract a good-quality EV-RNA from PFP for the RT-PCR miRNA assays. The optimized protocol increases yield and purity of obtained small RNAs allowing to more sensitive and reproducible analysis of microRNA content of extracellular vesicles.

Dr. Alex Chauhan¹, Hinal Zala¹, Simone Yamasaki¹, Enaam Merchant¹, Dr. Mohamed El-Mogy¹, Dr. Songsong Geng¹, Dr. Taha Haj-Ahmad¹, Dr. Yousef Haj-Ahmad¹

¹Norgen Biotek Corp., Thorold, Canada

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Peripheral blood is commonly collected using various devices, with potassium EDTA and serum tubes being prevalent. Plasma and serum serve as primary samples for monitoring disease progression. Exosomes in these samples offer real-time surveillance of prevalent diseases. Extensively studied, exosomal RNA extracted from extracellular vesicles plays a role in regulating transcriptional expression. Due to their low abundance, next-generation sequencing is optimal for characterizing these RNAs. Despite being primary in exosome studies, there's limited data comparing exosomal small RNA sequencing profiles in plasma and serum. This study highlights differences in these profiles from the same donors. Blood was collected from 4 donors in EDTA and Serum tubes. Plasma and Serum were separated and were stored at -80°C until further use. Intact Exosomes were purified from 0.2, 0.5 and 1.0 mL plasma and serum volumes. Extracted exosomes were further processed to extract exosomal RNA. Small RNA library was constructed from all the purified exosomal RNA and sequenced using Illumina's NextSeq 550 platform. A pattern showing an increase in the reads mapped to genome was observed with the increase in plasma (p = 0.0122) and serum (p = 0.0141) volumes. Similar trend was observed in total small RNA species for plasma (p = 0.0037) and serum (p = 0.0396) samples. This trend was further reflected in the percentage of reads assigned to miRNA (p = 0.0031), piRNA (p = 0.042) and rest of the small RNA species combined (gencode; p = 0.0473) in plasma samples. Serum samples showed higher percentage of reads mapped to genome as compared to plasma sample, however this was significant only for 1.0 mL serum volume. Among the reads that were mapped to the genome, there was a significant difference in the percentage of miRNA, piRNA and circularRNA reads, between plasma and serum samples. This study reveals a significant impact of sample volume on exosomal small RNA sequencing profiles in plasma and serum. Both plasma and serum exhibited comparable proportions of miRNA, piRNA and circularRNA reads for a 1.0 mL sample volume. At volumes below 1.0 mL (0.2 mL and 0.5 mL), serum demonstrates a higher percentage of small RNA species compared to plasma, suggesting its preference in such cases.

Mr. Yoing-woo Kim¹, Mr. Kang-Min Lee¹, Professor Sehyun Shin¹

¹Korea University, Seoul, South Korea

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: To utilize extracellular vesicles (EVs) as therapeutic agents and drug delivery vehicles, it is crucial to address the issue of minimizing impurities present in the extracts of extracellular vesicles. Unfortunately, most of the currently developed exosome isolation techniques have failed to address this purity issue, demanding urgent development of innovative technologies.

Methods: Here, we propose a novel method for isolating EVs using a recently developed charge-based filter by our research group, along with hybridizing the conventional tangential flow filtration (TFF) method. The ExoFilter consists of a multi-layered structure composed of a positively charged porous filter (pore size 1 µm). In this study, three methods were employed: 1) TFF followed by ExoFilter, 2) ExoFilter followed by TFF, and 3) repeated application of ExoFilter-TFF. The results were analyzed using techniques such as NTA, BCA, RT-PCR, etc., to evaluate the efficiency of exosome separation

Results:All three methods, which involved the combination of ExoFilter and TFF, yielded unexpected outcomes by significantly decreasing impurities while maintaining a consistent exosome extraction yield. Particularly, the pre-ExoFilter followed by TFF method, which initially captures positively charged nanoparticles and subsequently removes particles smaller than 50nm, exhibited the highest purity, achieving a performance level approximately one-tenth of the impurity level of TFF. The results of the TFF followed by ExoFilter and repeated ExoFilter-TFF processes showed impurity levels approximately one-fourth of those of TFF with comparable yields.

Summary/Conclusion: These findings present a novel method that combines two different extraction techniques, resulting in innovative reduction of impurity content without compromising extraction yield. It is deemed as a combination of charge-based extraction and size-based filtration techniques, which complement each other's drawbacks without conflict. Moreover, the proposed method offers practicality for large-scale implementation, achieving over a tenfold increase in purity without compromising yield.

Mr. Rajindra Napit¹, Mr. Satendra Jyasawal¹, Ms. Jasmine Catague¹, Mr. Haben Melke¹, Dr. Rocky Chowdhury¹, Dr. Lingxue Kong¹, Dr. Wei Duan¹

¹Deakin University, Warun Ponds, Geelong, Australia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

Extracellular Vesicles (EVs) play a vital role in intercellular communication by transporting genetic material and proteins. Despite their significance, current purification methods such as ultracentrifugation and chromatography are time-consuming and can damage EVs, hindering their potential clinical applications. Given their importance in diagnostics and therapeutics, there's a pressing need for alternative purification techniques. This project aims to address this need by exploring new methods to isolate and enrich EVs effectively.

Methodology:

The method involves utilizing predictive tools aid in optimizing aptamer design, ensuring spacer arm addition minimally impacts aptamer affinity. Followed by use of denaturing agents to disrupt the 3D structure of aptamers, enabling effective capture of extracellular vesicles (EVs) without the need for harsh reagents like detergent, high salt, or heat. Aptamers targeting universal EV biomarkers like CD63, CD81, and CD9 are employed for small EV capture, with a focus on the CD63 aptamer due to its commonality in small EVs. Various matrix immoboolization strategies for the conjugation of aptamers will be explored.

Results:

The CD63 aptamer was modified with the help of computational prediction tools to enable its immobilization without apparent affecting its affinity. The aptamer affinity was then tested on antibody captured EVs to assess the binding capacity of aptamer. The result showed modified aptamer is capable of binding to EVs in PBS buffer. Next the aptamer will be immobilized on the solid surface to capture and purify the EVs.

Conclusion:

By leveraging predictive tools for aptamer design and employing denaturing agents, this research demonstrates a promising approach for efficient EV capture under mild or physiological conditions. The successful modification of the CD63 aptamer and its demonstrated binding capacity to EVs pave the way for further development of novel purification strategies with potential clinical applications.

Dr Jagan Billakanti¹, Dr Jon Lundqvist, Dr Peter Guterstam

¹Cytiva, Brisbane, Australia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

A scalable workflow for the purification of exosomes, a type of extracellular vesicle (EV), is a major challenge for therapeutic-grade exosome manufacture. Exosomes are large ― between 40 and 150 nanometres (nm) in diameter ― and downstream processing includes the removal of much smaller size contaminants such as host cell proteins and DNA. Established technologies for research, R&D, and diagnostic purposes include ultracentrifugation, density gradient separation, gravity separation, or a combination of these methods. Although these technologies generate enriched exosomes suitable for characterization, they can damage the exosome structure, suffer from poor yields and scalability, and require long preparation times, which could reduce biological function. Therefore, these methods are not suitable for manufacturing large quantities of therapeutic exosomes.

Methods:

We describe a workflow for both research and clinical scales of manufacturing. The EV enrichment includes depth filtration to remove cells, Benzonase treatment to degrade DNA/RNA, tangential flow filtration using 750 kDa to concentrate exosomes, followed by either gentle size-exclusion chromatography (SEC) with Cytiva™ superSEC resin or chromatography (MMC) with Capto™ Core 700 resin; the latter combines size exclusion and binding mode for separation different species from the load material.

Results:

During the primary tangential flow filtration (TFF), a 750 kDa hollow fiber produced 100% recovery of EV while different chromatography steps produced different recovery profiles. For example, superSEC resin showed 7% more EV recovery and 3.3 times faster separation over Sepharose™ CL-2B. superSEC resin demonstrated similar performance at two different scales with three different feed materials.

Summary:

In this presentation, we will demonstrate a start-to-finish exosome production process suitable for clinal scale manufacturing of exosomes harvested from three different cell lines. Both chromatography options mentioned above are amenable to scale-up and packing in large-scale columns for clinical-grade EV manufacturing. Depending on the EV dose requirements, a secondary TFF with 750 kDa is proposed for further concentration of EV before sterile filtration.

Ph.D. Candidate Minyeob Lim¹

¹POSTECH, Pohang, South Korea

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction

Extracellular vesicles (EVs) have significant diagnostic potential, yet their efficient isolation remains challenging. The aqueous two-phase systems (ATPSs) offer a high-purity and high-yield method for EV separation, but the underlying mechanism needs to be better understood, limiting its application. In order to elucidate the principle of separation, we apply Kramers’ theory to the system. Our results show that the liquid-liquid interface acts as a size-selective filter, improving the efficiency of EV isolation and providing a pathway to effective diagnostics.

2) Methods

A theoretical and simulation model for nanoparticle transport in ATPS was validated using beads of various sizes. Plasma samples from prostate cancer patients with metastasis (n = 23) and without metastasis (n = 31) were separated using ATPS. The efficiency of separating EVs, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and albumin was evaluated using total internal reflection fluorescence microscopy, western blot, and transmission electron microscopy. EV markers (CD9, CD63) and cancer markers (PD-L1, PSMA) were quantified via enzyme-linked immunosorbent assay.

3) Results

The study discovered a negative correlation between interfacial tension and the diameter of particles that can pass through the interface. Using this mechanism, over 80% of EVs were isolated from plasma, with only 10% of impurities (LDL, HDL, and albumin) retrieved. Applying this technique to prostate cancer diagnosis, PD-L1 and PSMA levels in exosomes turned out to have a strong correlation with metastasis. Metastasis detection through EV showed high efficiency (AUC = 0.88), in contrast to the traditional liquid biopsy marker, serum PSA, which demonstrated poor detection capability (AUC = 0.62).

4) Summary/Conclusion

This study demonstrated that a potential barrier developed by low interfacial tension (10 ∼ 60 µJ/m²) acts as a filter with pore sizes of 40 ∼ 100 nm, enabling efficient isolation of EVs from plasma. We expect that the potential barrier will facilitate further research using nanoparticles.

Postdoctor Rui Zhang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a novel tick-borne phlebitis virus of Bunyaviridae, characterized by high fever, thrombocytopenia and leukopenia with high case fatality. Currently, no specific antiviral drugs available to treat patients. Extracellular vesicles are lipid bilayer vesicles that carry various contents (protein, genetic information,), communicate and regulate neighboring cells. We found that EVs derived from SFTSV-infected cells contained infectious virions that were efficiently transported by these secreted vesicles into neighboring cells. Cholesterol in the PMs plays an important role in regulating the entry of viruses into cells. 25HC is an endogenous oxidized sterol involved in various metabolic pathways, and it is generally considered to be a soluble factor that involves in antiviral activity.

Methords: The large extracellular vesicles secreted by SFTSV infected cells were isolated and purified by gradient centrifugation, and the virions were found in MVs-SFTSV by NTA analysis, electron microscopy analysis and content identification.

Results: We reported a natural lipid metabolite 25HC that inhibited SFTSV entry by activating the activity of lipid metabolism enzyme-ACAT, affecting the cholesterol translocation from the cytoplasm to plasma membranes. At the same time, through this way, 25HC also inhibit the extracellular vesicles carried virions into target cells, prevented the MVs-SFTSV mediated virus-transmission and spread.

Summary: Our data showed the large vesicles form SFTSV infected cells contained viral particles and mediated virus transmission. 25HC simultaneously inhibited the entry of SFTSV and infectious MVs-SFTSV, which could be regarded as an antiviral strategy to kill two birds with one stone.

Samuel Wachamo^{Department of Neuroscience, Neuroscience Graduate Program, Center for Brain Immunology and Glia, Medical Scientist Training Program, University of Virginia, Charlottesville, VA, USA}, Alisha Thakur, Mallarie Broadway^{Department of Neuroscience, Neuroscience Graduate Program, Center for Brain Immunology and Glia, Medical Scientist Training Program, University of Virginia, Charlottesville, VA, USA}, Dr. Alban Gaultier^{Department of Neuroscience, Neuroscience Graduate Program, Center for Brain Immunology and Glia, Medical Scientist Training Program, University of Virginia, Charlottesville, VA, USA}

Poster Pitches (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:45 PM - 1:00 PM

Introduction: Alzheimer's disease (AD) is the most common form of dementia affecting 50 million people worldwide, and this number is projected to be 152 million by 2050. AD is characterized by a progressive decline in cognitive function associated with the formation of amyloid beta (Aβ) plaques, neurofibrillary tangles (NFTs), and neuroinflammation resulting in neuronal dysfunction and death. It is still not well understood what triggers the pathological hallmarks of AD, and thus there are currently few clinically efficacious disease modifying therapies for AD. However, emerging evidence confirms a crucial role of the gut microbiota through microbially produced metabolites, but the precise mechanisms remain to be elucidated. Based on previous studies that showed altered microbiota profile in AD, we hypothesized that the gut microbiota regulates microbiota-gut-brain axis and affects the pathological hallmarks of AD through release of bacterial extracellular vesicles (BEVs) that contain microbial metabolites that have been implicated in AD.

Methods: BEVs from intestinal contents of C57BL/6J mice were isolated by ultracentrifugation followed by size exclusion chromatography. BEVs were characterized by BCA assay, nanoparticle tracking analysis, transmission electron microscopy, ELISA, western blot, and metabolomics.

Results: Our preliminary results, for the first time, show a significant presence of metabolites, that have been associated with AD, within BEVs derived from the intestinal contents of mice. These metabolites are among the most biologically active bacterial metabolites, and their functional relevance in health and disease, especially in AD, will be investigated using 5XFAD mice, specific strains of bacteria that are known to produce the specific metabolites, and Cre-LoxP system in future studies.

Summary/Conclusion: We discovered that BEVs contain metabolites, which can potentially regulate local and systemic immune response in AD. Ongoing studies in our lab will determine if BEVs play a functional role in the brain and mediate onset and progression of AD. The new insights into mechanisms in these studies may lead to new, effective therapeutic strategies for AD.

Funding: National Institutes of Health (T32 GM007267), Owens Family Foundation, and the Miller Family.

Keywords: Bacterial Extracellular Vesicles (BEVs), Alzheimer's disease (AD)

Ms Chantelle Blyth, Dr Michael Lazarou, Dr Thomas Naderer

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Neisseria gonorrhoeae-derived outer membrane vesicles (OMVs) target mitochondria and cause mitochondrial dysfunction and apoptosis [1, 2]. How mitochondria sense OMV-mediated damage to activate apoptosis remains unclear. Here, we focused on the role of mitochondrial quality control systems, including organelle-specific stress pathways and PINK1/Parkin mitophagy, to determine their role in regulating OMV levels, macrophage survival and immune responses.

Methods: Neisseria gonorrhoeae OMVs were isolated from Neisseria gonorrhoeae (MS11A) grown to mid-log phase (OD600 < 1) in gonococcal-specific broth media and then harvested via ultracentrifugation. Protein and particle concentrations were assessed using BCA and NTA, respectively. Primary bone marrow-derived macrophages (BMDMs) from wild-type and Parkin-/- mice were differentiated and exposed to N. gonorrhoeae OMVs at 20 µg/ml. Cell viability was assessed over a 48 hours via live cell imaging with mitochondrial stress confirmed via immunoblot. The cytokine profile of OMV-treated cells asses assessed via flow cytometry using the legend plex assay.

Results: Preliminary data show a reduction in cell death at 48 hours after OMV treatment in the absence of Parkin and a delayed mitochondrial stress response, indicated by PGAM5 cleavage assessed by immunoblot. Parkin-/- BMDMs also produced an altered cytokine profile, with reduced expression of proinflammatory cytokines.

Summary: This data highlights key differences between wild-type and knockout genotypes in response to N. gonorrhoeae OMV exposure, suggesting a potential role of mitochondrial quality control systems in regulating macrophage survival and innate immune responses. 

References:

1. Deo, P., et al., Mitochondrial dysfunction caused by outer membrane vesicles from Gram-negative bacteria activates intrinsic apoptosis and inflammation. Nat Microbiol, 2020. 5(11): p. 1418-1427. 

2. Deo, P., et al., Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis. PLoS Pathog, 2018. 14(3): p. e1006945. 

Ms. Catalina Adasme-Vidal, Mr. Aliosha I. Figueroa-Valdés, Ms. Camila Fuentes, Ms. Patricia Valdebenito, Mr. Sebastián Illanes, Ms. Francisca Alcayaga-Miranda

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

The type of birth -vaginal delivery (VD) or cesarean section (CS)- determines the composition of commensal intestinal microbiota in newborns (CIMN). CIMN has been associated with pediatric diseases, including those related to the immune system. The interaction between gut microbiota and the immune system remains poorly understood, yet it may be mediated by bacterial extracellular vesicles (bEVs) derived from the gut microbiota. This study aims to determine whether VD/CS influences the characteristics and immunomodulatory potential of microbiota-derived bEVs in newborns.

Methods:

bEVs were isolated from fecal samples collected from VD/CS newborns and characterized in terms of concentration and size using nanoparticle tracking analysis, morphology via transmission electron microscopy, and gram-negative/gram-positive bacterial markers via western blot. The immunomodulatory potential of bEVs was assessed in vitro through a peripheral blood mononuclear cell immunosuppression assay using human and mouse cells, along with phytohemagglutinin and anti-CD3/CD28 activating beads and determining lymphocyte subpopulations by flow cytometry.

Results:

A protocol for bEVs isolation was established. No differences were observed in the concentration and size of bEVs isolated from CIMN. However, bEVs' shapes varied depending on their VD/CS origin. Likewise, bEVs derived from VD exhibited greater immunomodulation capacity than those obtained from CS, inhibiting lymphocyte proliferation.

Summary/Conclusions:

The established protocol allowed for obtaining a concentrate enriched in bEVs, whose morphology and immunosuppressive effect depend on their VD/CS origin, perhaps influenced by the Gram-positive/negative bacterial composition of CIMN.

Doctor Alisa Petkevich, Doctor Aleksandr Abramov, Professor Vadim Pospelov

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction. According to the data of NCHS among U.S. adults aged 20 and over, 57.6% used any dietary supplement in the past 30 days. Although among the most used dietary supplements are vitamin D3 and omega-3 fatty acids, there is a huge variety of plant extracts based dietary supplements. Different extract forms of the same plant may have completely different biological effects. With growing interest to the dietary supplements, these plant extracts become one of the important environmental epigenetics factors. Exosomes, as one of the main tools of the cell messenger system, may provide a valuable data regarding epigenetic changes in the cells being exposed to these plant extracts.

Methods. Several breast cancer cell lines (including MCF7, MCF10F, SK-BR-3, MDA-MB-231 from ATCC) were incubated for 24 hours with different plant extracts including water and isobutanol extracts of Laminaria Angustata. After incubation exosomal miRNAs (exoEasy Maxi kit, Qiagen, Germany) and supernatant miRNAs were isolated with following miRNA sequencing (Illumina 5 Mio read pairs 2*150).

Results. Exosomal miRNAs showed more significant difference in the levels of expression compare to supernatant miRNAs, what may be partly explained by their specificity to the changes in regulation of signal pathways. There were miRNAs with mostly significant changes in expression levels, which are associated with regulation of the genes, associated with cytoskeletal proteins, proliferation and apoptosis. It is interesting to note, that many miRNAs with most significant changes in expression level bind directly to DNA as the main mechanism of gene regulation.

Conclusion. Exosomes and exosomal miRNAs in regard to study of epigenetic influence of different environmental factors, including food, may be a valuable tool, providing the relevant information on the changes in the gene regulation.

PhD student Paula Meneghetti, Ana Claudia Torrecilhas

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction Extracellular vesicles (EVs) are naturally released into the culture medium by Trypanosoma cruzi, the causative agent of Chagas disease is a Neglected Tropical Diseases (NTDs). Previous research has shown that EVs release is important in facilitating communication between host cells and the protozoan parasite. The characterization of EVs released during metacyclogenesis in different strains of T. cruzi (Y and G) metacyclic trypomastigotes, as well as their role in interactions with human host cells will be the focus of our research.

Methods: T. cruzi metacyclic forms were cultured in Liver infusion tryptose (LIT) medium to isolated EVs (Y and G strains), then purified using ultracentrifugation and size exclusion chromatography (SEC). THP-1 cells were used in uptake assays with EVs labeled with PHK26 and was analyzed using flow cytometry and confocal microscopy at 30 and 60 minutes, 24 and 48 hours. In parallel, qRT-PCR was also used to perform TNF-alpha, CCL5, IL-1, IL-6, IP-10 and IL-10 detection assays in the THP-1 cells incubation with EVs. Simultaneously, EVs isolated from Y and G strains were characterized using mass spectrometry (MS).

Results: We observed the interaction of EVs with THP-1 cells within the first 30 minutes and maximum internalization occurring after 48 hs. qRT-PCR analyses revealed the presence of cytokines and chemokines in both strains, with higher concentrations observed in the Y strain and a significant increase in the G strain, particularly in the detection of IL-6. The MS revealed 167 proteins, 37 of which were found exclusively in the Y strain and 6 in the G strain. Among the proteins highlighted in this study were tropomyosin and guanylate binding proteins, which are known for their roles in cellular regulation and pathogen replication within host cells.

Summary/Conclusion: The active interaction observed between host and EVs derived from T. cruzi strongly suggests that these EVs may play a mediating role in facilitating interactions with host. This discovery highlights the dynamic interaction between T cruzi and its host vector and human environment, shedding light on the intricate mechanisms involved in communication as well as the potential influence of EVs during the interaction process.

Dr. Bao-Hong Lee, MS. Yi-Tsen Chang, Mr. You-Zuo Chen, Mr. Hui-Chun Lin, Dr. Wei-hsuan Hsu

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: The widespread and inappropriate use of antibiotics lead to the generation and spread of antimicrobial resistance (AMR) microorganisms, limiting the therapeutic benefits of medicine. Preservative food additives are used to prevent the growth of microorganisms in food products, which may also lead to the emergence and spread of AMR microorganisms. However, their risks and mechanisms of action have not been paid attention to and explored. The aim of this study is to investigate the spread of AMR microorganisms to preservatives in fermented food product. Methods: This study focuses on understanding benzoic acid (BA) resistance in microorganisms from pickled vegetables. Microbes were isolated from pickled cucumbers and made resistant to BA. BA-resistant microorganisms were identified through Sanger sequencing. EVs were isolated using ultracentrifugation and characterized for shape and size via transmission electron microscopy (TEM). EVs concentrations were measured using nanoparticle tracking analysis (NTA). Results: EVs from BA-resistant strains were co-cultured with other microorganisms to assess potential BA resistance transmission. Proteomic analysis identified the protein responsible for generating and spreading BA resistance, while fluorescence staining confirmed EVs uptake by microbes. The isolated microorganism from pickled cucumbers, identified as Pichia krudriavzevii (PK), produced round-shaped EVs with lipid bilayers observed by TEM. Co-culturing PK with these EVs allowed it to adapt to high BA concentrations. The protein SNQ2, an ABC transporter known for drug resistance, was highly expressed in BA-resistant EVs, likely contributing to BA resistance development. Summary: Taken together, microorganisms in fermented products can develop BA resistance, undergo vertical transmission, and potentially spread resistance through secreted EVs.

Dr. Bao-Hong Lee, MS. Yi-Tsen Chang, Mr. Hui-Chun Lin, Mr. You-Zuo Chen, Dr. Tang-Long Shen, Dr. Wei-hsuan Hsu

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Antibiotics are frequently employed to treat bacterial infections, contributing to the rise of antibiotic resistance. The exchange and transfer of microbial DNA through extracellular vesicles (EVs) facilitates the horizontal dissemination of these resistant genes. These nano-scale vesicles, containing DNA, RNA, and proteins, play a crucial role in intercellular communication. However, a limited body of research exists exploring the correlation between microbial EVs, drug resistance, and food safety. Pickled vegetables, such as cucumbers preserved with salt, organic acid, or sugar, often incorporate benzoic acid (BA) as a food preservative, effectively inhibiting the growth of yeast and mold. This study seeks to examine whether microorganisms within pickled vegetables can develop resistance to BA, elucidate the underlying mechanisms, and assess if this phenomenon adversely affects the gut environment. Methods: Microorganisms were isolated from pickled cucumbers, and BA was employed to induce resistance, with microbial species identified through Sanger sequencing. The transmission of BA resistance through EVs was evaluated by co-culturing EVs with microorganisms, gut microbiota, and gut cells separately. Proteomic analysis aimed to identify proteins responsible for generating and spreading BA resistance, while flow cytometry was employed to identify microorganisms capable of uptaking EVs. Results: Pichia krudriavzevii (PK) was successfully isolated from pickled cucumbers, demonstrating an ability to adapt to higher concentrations of BA when co-cultured with EVs. Furthermore, candidate proteins linked to the spread of BA resistance were analyzed. Flow cytometry was used to determine the proportion of gut microbiota that uptakes EVs, and sorted bacteria were identified through sequencing. Fluorescence staining confirmed the absorption of EVs by PK and gut microbes, as well as gut cells. Conclusion: Microorganisms in fermented products can develop resistance to BA, undergo vertical transmission, and potentially spread resistance through secreted EVs. This process may impact gut environment, posing risks to human health.

Promote placentation and mitigate preeclampsia

Ph.d Zihao Ou

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Preeclampsia (PE) is a multisystem disorder with high maternal morbidity and mortality rates. Currently, no practical therapeutic approach is available to prevent PE progression, except for early delivery. Gut dysbiosis is associated with PE development. Previous data showed that the abundance of Akkermansia muciniphila (Am) was lower in patients with PE than in normotensive pregnant women. Here, in this study, decreased abundance of Am was observed in a PE mouse model. Also, we found that administration with Am could significantly attenuate systolic blood pressure, promote foetal growth and improve the placental pathology in mice with PE. Moreover, Am-derived extracellular vesicles (AmEVs) were transferred from the gastrointestinal (GI) tract to the placenta and mitigated pre-eclamptic symptoms in PE mice. These beneficial effects of AmEVs were mediated by enhanced trophoblast invasion of the spiral artery (SpA) and SpA remodelling through activation of the epidermal growth factor receptor (EGFR)–phosphatidylinositol-3-kinase (PI3K)–protein kinase B (AKT) signalling pathway. Collectively, our findings revealed the potential benefit of using AmEVs for PE treatment and highlighted important host–microbiota interactions.

Miss Yiyi Huang, Doctor Kening Zhao, Miss Yuneng Hua, Miss Mei Huang, Doctor Ruyi Zhang, Doctor Jingyu Wang, Mr Fan Bu, Miss Junhui Wang, Professor Lei Zheng, Professor Qian Wang, Professor Xiumei Hu

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Candida albicans-induced lung injury is one of the most prevalent diseases in patients with invasive candidiasis. However, the mechanism by which C. albicans causes invasion and lung injury in the host remains unclear. Recently, studies have reported that C. albicans secrete extracellular vesicles (EVs), a group of nanostructures carried bioactive components and participated in the pathogenic processes. In the present study, we aim to explore the pathogenic mechanism of C. albicans EVs damage to the host lung, and to provide theoretical basis and new therapeutic strategies for the clinical treatment of severe invasive candidiasis.

Methods

1. To explore whether C. albicans promote host lung injury through EVs, GW4869 was used to inhibit the EVs secretion of Candida albicans. Subsequently, C. albicans /GW4869 pretreated C. albicans infection models were constructed respectively.

2. To explore whether EVs derived from C. albicans induce host lung injury, EVs secreted by Candida albicans were extracted by ultracentrifuge, and then C. albicans EVs/ S. cerevisiae EVs infection models were constructed respectively.

3. To explore whether C. albicans EVs promote lung injury through inducing ferroptosis of macrophages, the expression of ferroptosis related indicators and molecules were detected.

Results

1. In vivo experiments showed that C. albicans and C. albicans EVs both induce acute lung injury in mice, while GW4869 pretreated C. albicans has a weakened damage effect. C. albicans and C. albicans EVs triggered decreased frequency of lung macrophages, increased MDA level and PTGS2 expression in lung.

2. In vitro experiments showed that C. albicans and C. albicans EVs induce decreased viability and increased cytotoxicity of macrophages.

3. In vitro experiments showed that C. albicans EVs increase the intracellular total iron level, MDA level and mitochondrial superoxide in macrophages, while decrease GSH level. C. albicans EVs also increased the expression of PTGS2 while decreased the expression of GPX4, which are consistent with the main features of ferroptosis in cells.

Conclusion

In the present study, we found that C. albicans was able to induce ferroptosis in macrophages by secreting EVs, thus promoting acute lung injury in host.

Asst. Prof. Sakaorat Lertjuthaporn, Ms Narinee Srimark, Mrs Hathai Sawasdipokin, Ms Kasama Sukapirom, Ms Jinjuta Somkird, Prof. Kovit Pattanapanyasat, Ladawan Khowawisetsut

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis, a severe central nervous system infection with a high mortality rate. The disease's pathogenesis is linked to both the pathogenicity of the amoeba and the robust inflammatory response elicited by host immune cells, particularly recruited blood-borne innate leukocytes. As extracellular vesicles (EVs) have been recognized as key mediators of intercellular communication between pathogenic amoebas and the hosts, the activation of the host immune response by EVs might significantly influence the outcome of infection. Therefore, this study seeks to investigate the influence of N. fowleri-derived extracellular vesicles (Nf-EVs) on gene expression and cytokine production in innate immune cells, with a specific focus on neutrophils and monocytes.

Methods: The pathogenic N. fowleri trophozoites were cultivated axenically in Nelson's medium with supplementation. After replacing the culture medium with buffer saline, the culture supernatant was collected following a 24-hour incubation period. Nf-EVs were isolated from the N. fowleri trophozoite culture supernatant using a multi-step differential centrifugation and characterized through electron microscopy and nanoparticle tracking analysis. Primary neutrophils and peripheral blood mononuclear cells were purified via Ficoll-Hypaque gradient density centrifugation. Subsequently, primary monocytes were further isolated using immunomagnetic bead separation. These primary cells were then incubated with Nf-EVs, and the study explored the effects of Nf-EVs on gene expression and cytokine production (TNF-α, IL-1β, IL-6, and IL-8) by these cells.

Results: Nf-EVs exhibited the potential to elevate the expression levels of pro-inflammatory molecules, specifically inducing heightened production of TNF-α by neutrophils and IL-6 and IL-8 by monocytes. Notably, the activation of primary neutrophils and monocytes by Nf-EVs varied in the timing of response.

Summary/Conclusion: These findings demonstrated the immunostimulatory properties of Nf-EVs, emphasizing their role in orchestrating the activation of host immune cells and the subsequent induction of inflammation.

Ana Claudia Torrecilhas, Master Nicholy Lozano, Full Professor Sergio Schenkman

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) derived from trypomastigote forms of Trypanosoma cruzi modulating host cells invasion and immune responses. Although the parasite can survive in tissue culture-infected cells, it decreases virulence in mice. In our study, we successfully restored virulence in mice upon 20 consecutive passages. As a result, we performed a comparison study, comparing the impact of EVs released by non-virulent trypomastigotes maintained in vitro (P2) to virulent trypomastigotes maintained in vivo (P20). Our findings suggest a new link between T. cruzi virulence and EV release. Surprisingly, these EVs increase parasitism in the heart first, then the bladder, intestine, and lungs.

Methods: P2 and P20 EVs were isolated using a combination of ultracentrifugation and SEC column. THP-1 cells were subjected to uptake assays using P2 and P20 EVs, labeled with PHK26, and then examined using the Image Streams system microscopy at 15-, 30, and 60-minutes intervals, as well as 24 and 48 hours. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to examine TNF-alpha, CCL5, IL-1, IL-6, IP-10, and IL-10 expression in THP-1 cells after EVs P2 and P20 incubation.

Results: We investigated the dynamic cellular uptake of PHK26-labeled EVs using ImageStreams system microscopy. Both P2 and P20 EVs interacted immediately with human monocytes. Concurrently, we employed qRT-PCR to examine cytokine and chemokine expression in THP-1 cells upon incubation with P2 and P20 EVs. Notably, cytokine production increased in P20 EVs when compared to P2 EVs.

Conclusion: This comprehensive approach allowed us to investigate the properties of EVs released by virulent (P20) and non-virulent (P2) parasites during their interactions with host cells and assess the impact on immune response communication, providing a detailed understanding of the complex interactions between Trypanosoma cruzi and host cells. This allows us to understand how virulence factors influence different stages of Chagas disease.

Funding: FAPESP, CAPES, CNPq

Professor MAHANAMA DE ZOYSA, Mr. Mawalle Kankanamge Hasitha Madhawa Dias

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Bacterial EVs (BEVs) are known for harbor various virulence factors and signaling molecules that facilitate cell communication and host-pathogen interactions. The present study focuses on isolation, characterization of BEVs from Aeromonas hydrophila (AhEVs), and understand their immunomodulatory role in vitro and in vivo experiments.

Methods: BEVs were isolated using the ultracentrifugation method. Characterization was carried out using transmission electron microscopy (TEM), nanoparticle tracking (NTA), and analyzing bacterial EV markers. The proteomie profile was analyzed using LC-MS/MS. Internalization of AhEVs was tested using fathead minnow (FHM) cells, followed by analysis of toxicity and reactive oxygen species (ROS) production. In vitro and in vivo immunomodulatory effects were investigated using FHM cells and adult zebrafish.

Results: From TEM imaging, ultrastructural morphology was confirmed to have a spherical shape, and NTA determined the mean particle size as 105.5 ± 2.0 nm. Proteomic analysis revealed 4825 unique peptides and 1284 proteins, associated with antioxidant function, transduction regulation, and transportation activity. No cytotoxicity was observed in AhEVs treated FHM cells and zebrafish larvae up to 50 µg/mL. Furthermore, fluorescent-labeled AhEVs internalized into FHM cells. Cellular pro-inflammatory cytokines, nuclear factor (NF)-κB, interferon (Ifn), interferon regulatory factor (Irf) 7, interleukin (il) 8, and il11 were upregulated in AhEVs treated FHM cells. In vivo gene expression analysis revealed that AhEVs treated adult zebrafish-induced toll-like receptor (tlr) 2 and 4, tumor necrosis factor α (tnfα), heat shock protein (hsp) 70, il6, il10, and il1β. In the spleen of AhEVs treated adult zebrafish showed induced NF-κB and TNF-α proteins.

Summary/Conclusion: Overall results exhibit AhEVs have morphological and physicochemical characteristics of Gram (-ve) bacteria. Furthermore, AhEVs display immunomodulatory activity in FHM and zebrafish. In-depth analysis of the proteome of AhEV may provide its mechanisms of action associated with immunomodulatory function.

Professor MAHANAMA DE ZOYSA, Mr. E.H.T. Thuslahn E.H.T. Thuslahn Jayathilaka, Mr. Mawalle Kankanamge Hasitha Madhawa Dias, Dr. Chamilani Nikapitiya Nikapitiya

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Streptococcus parauberis is a Gram-positive pathogenic bacterium that causes Streptococcosis in fish and bovine mastitis. This study was focused on isolation, characterization, proteomie profiling, and anti-inflammatory activity of S. parauberis derived EVs (SpEVs).

Methodology: SpEVs were obtained from S. parauberis free culture media using the ultracentrifugation method. The isolated SpEVs were characterized based on morphology through transmission electron microscopy, size determination via nano tracking analysis, identification of bacterial EV markers, and protein profile. Additionally, the proteomie profile was analyzed using LC-MS/MS. The anti-inflammatory activity of SpEVs was assessed in murine macrophages (Raw 264.7) cells treated with lipopolysaccharide (LPS).

Results: The isolated SpEVs exhibited oval-shaped clear membrane-bound particles with an average diameter of 168.3 ± 6.5 nm. Flagellin, a bacterial EV marker, was detected, and SDS-PAGE confirmed the presence of three significant protein bands in SpEVs. Proteomic analysis identified 6209 unique peptides and 1039 proteins in SpEVs, with a predominant protein mass distribution in the range of 10–50 kDa. Gene ontology analysis revealed that expressed proteins in SpEVs were enriched for catalytic activity, with other notable terms including toxin activity and antioxidant activity. Functionally, SpEVs illustrated anti-inflammatory activity in Raw264.7 cells upon LPS treatment.

Summary/Conclusion: SpEVs were successfully isolated from S. parauberis and they showed the characteristics of BEVs. Proteomic analysis affirmed the expression of distinctive proteins in SpEVs, associated with diverse physiological and immunological functions. In addition, the study confirmed the potent anti-inflammatory activity of SpEVs.

Miss Ruby Gorman-batt¹, Associate Professor Meredith O'Keeffe, Doctor Terry Kwok-Schuelein

¹Monash University, Clayton, Australia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Dendritic cell (DC)-derived extracellular vesicles (EVs) are known to regulate host immunity, however, their role during bacterial pathogenesis remains unclear. We observed that a critical Helicobacter pylori (Hp) virulence factor, the vacuolating cytotoxin A (VacA), specifically targets DC-EVs. Hp is a gram-negative bacterium that persistently infects 50 % of the world's population, and is the greatest risk factor for the development of gastric cancer (GC). VacA has been shown to assist in bacterial persistence by limiting dendritic cell (DC), T-cell and macrophage functions. However, how the toxin interacts with these cell types in vivo remains poorly understood, but may be explained by VacA dissemination to distant immune centres by DC-EVs. Here, we report VacA's influence on the physical properties of DC, DC-EV and the interactions between VacA+-EVs and recipient immune cells.

Methods

The impact of Hp 60190 VacA on the maturation state of murine DC cell line, MutuDC, was evaluated by flow cytometry and immunofluorescence microscopy. VacA localisation during MutuDC and human primary DC intoxication was assessed by correlative light electron microscopy. Small EVs (sEVs) were isolated by multi-step ultracentrifugation for analysis by immunoblot, transmission electron microscopy, nanotracking analysis and mass spectrometry. Finally, the impact of VacA+-sEVs on recipient immune cells was assessed by flow cytometry and immunofluorescence microscopy.

Results

VacA modulated the expression of cell surface markers, without being phagocytosed by the DCs. Instead, VacA localised exclusively to EVs of MutuDC and human primary DCs. VacA did not impact DC-EV size, however VacA+-sEVs were enriched in key markers of immune cell functions and possessed the ability to deliver VacA to naïve immune cells. Analysis concerning the functional impacts of VacA+-sEVs is ongoing.

Conclusions

This study reveals that VacA specifically binds EV at the DC surface, altering DC and DC-EV immune marker composition. We propose that VacA perturbs DC maturation and hijacks DC-EVs to dysregulate immune cell signalling, assisting in the chronic persistence of Hp. Our findings highlight a previously unrecognised role of EVs in bacterial pathogenesis.

Miss Ruby Gorman-batt, Meredith O'Keeffe, Terry Kwok

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Dendritic cell (DC)-derived extracellular vesicles (EVs) are known to regulate host immunity, however, their role during bacterial pathogenesis remains unclear. We observed that a critical Helicobacter pylori (Hp) virulence factor, the vacuolating cytotoxin A (VacA), specifically targets DC-EVs. Hp is a gram-negative bacterium that persistently infects 50 % of the world's population, and is the greatest risk factor for the development of gastric cancer (GC). VacA has been shown to assist in bacterial persistence by limiting dendritic cell (DC), T-cell and macrophage functions. However, how the toxin interacts with these cell types in vivo remains poorly understood, but may be explained by VacA dissemination to distant immune centres by DC-EVs. Here, we report VacA's influence on the physical properties of DC, DC-EV and the interactions between VacA-EVs and recipient immune cells.

Methods

The impact of Hp 60190 VacA on the maturation state of murine DC cell line, MutuDC, was evaluated by flow cytometry and immunofluorescence microscopy. VacA localisation during MutuDC and human primary DC intoxication was assessed by correlative light electron microscopy. Small EVs (sEVs) were isolated by multi-step ultracentrifugation for analysis by immunoblot, transmission electron microscopy, nanotracking analysis and mass spectrometry. Finally, the impact of VacA-sEVs on recipient immune cells was assessed by flow cytometry and immunofluorescence microscopy.

Results

VacA modulated MutuDC expression of the costimulatory molecule, CD86, without being phagocytosed by the DCs. Instead, VacA localised exclusively to EVs of MutuDC and human primary DCs. This interaction was dependant on cholesterol-rich lipid rafts. VacA did not impact DC-EV size, however VacA-sEVs were enriched in key markers of immune cell functions and possessed the ability to deliver VacA to naïve immune cells. Analysis concerning the functional impacts of VacA-sEVs is ongoing.

Conclusions

This study reveals that VacA specifically binds EV at the DC surface, altering DC and DC-EV immune marker composition. We propose that VacA perturbs DC maturation and hijacks DC-EVs to dysregulate immune cell signalling, assisting in the chronic persistence of Hp. Our findings highlight a previously unrecognised role of EVs in bacterial pathogenesis.

Miss Nina Colon, Mr Liam Gubbels, Professor Richard L. Ferrero

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) that are released during bacterial growth contain various cargo involved in pathogenesis. Proteomic studies showed that the gastric pathogen Helicobacter pylori produces EVs containing urease (UreA, UreB) and catalase (KatA), known to be essential for bacterial colonisation in the stomach. Nevertheless, it was not known whether urease and catalase in EVs retain functional activity. As these enzymes are colonisation factors for the bacterium, we hypothesised that functionally active urease and catalase within H. pylori EVs may promote bacterial survival against environmental stresses. Methods: EVs from H. pylori wild-type (WT) bacteria and ureA, ureB and katA isogenic mutants were isolated by ultracentrifugation and characterised by Western blotting. Enzymatic activities of the EVs and sonicated preparations of the whole bacteria were determined using quantitative assays. The impact of EV-associated enzymes on bacterial survival and host cell responses was assessed in in vitro assays. Results: Western blotting confirmed the presence of UreA, UreB and KatA subunits within EVs and whole cell preparations. EVs had similar levels of urease and catalase activity as the whole bacteria (1.31 ± 0.321 vs. 1.21 ± 0.504 µmol/min/mg protein, p = 0.8741; and 784.4 ± 30.543 8 vs. 817.3 ± 27.8 µmol/min/mg protein, p = 0.6979). EV-associated urease had no effect on bacteria survival at acid pH. Conversely, WT EVs (40 µg/mL) protected katA mutant bacteria from exposure to 10 mM H₂O₂, similar to the WT bacteria, whereas katA mutant bacteria were killed. As bacterial EVs are highly adept at entering host cells, we next determined the localisation of EV-delivered H. pylori catalase within human gastric epithelial cells. KatA was detected by Western blotting analyses on the nuclear fractions from cells exposed to WT, ΔureA or ΔureB but not ΔkatA EVs. Further, catalase activity was detected in the nuclear fractions from cells incubated with either WT, ΔureA or ΔureB EVs. Conclusions: Together, these data highlight that H. pylori EVs contain functionally active urease and catalase. Moreover, EV-associated catalase was shown to protect the bacterium against environmental stresses and to be delivered to the nucleus of host cells, potentially leading to DNA damage.

Dr Mauro Javier Cortez Veliz, Miss Deborah Brandt-Almeida, Mrs Jenicer Kazumi Umada Yokoyama Yasunaka, Dr Simon Ngao Mule, Dr Giuseppe Palmisano, Dr Ana Claudia Torrecilhas

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

IINTRODUCTION The regulatory mechanism of CD200R/CD200 participates in several cellular processes mediated by the host's immune response. Cells expressing CD200 ligand regulate macrophages, which present the CD200R on their membrane. During infection with virulent Leishmania, the induction of CD200 in macrophages by extracellular vesicles (Evs) of infective amastigotes is essential to inhibit the iNOS/NO mechanism, which leads to parasite resistance and proliferation. However, little is known whether infective metacyclic promastigotes can induce CD200. OBJECTIVE Therefore, the present work evaluates the ability of the two infective forms of Leishmania to release Evs and induce CD200.OBJECTIVE Therefore, the present work evaluates the ability of the two infective forms of Leishmania to release Evs and induce CD200. MATERIALS AND METHODS Leishmania amastigotes and metacyclic promastigotes were incubated in their own medium for vesiculation by 1h, and the supernatants were filtered through a 0.45-mm sterile cell strainer. Isolated Evs from the different infective forms were concentrated using amicon, and different assays were performed to validate the quantity and biological effect of Evs in macrophages by immunofluorescence and Immunoprecipitation/western blot (WB), both using polyclonal antibodies against Leishmania-Evs. RESULTS Concentrated Evs from the different forms were incubated in macrophages for 1h, and then fixed for IF assays to detect Evs signals. Representative images showed that stationary-phase promastigotes (enriched with metacyclic forms) and amastigotes presented a similar pattern of Evs-immunofluorescence signal in macrophages, confirmed by quantifying Evs signal per cell. When the same approach for Evs detection was used to detect CD200, amastigote-Evs induced a strong signal of the inhibitory ligand in macrophages. To confirm the specificity of amastigotes in inducing CD200, the different infective forms were incubated with macrophages, and the expression of CD200 was detected by IP/WB. Amastigotes induce CD200 in macrophages, but this molecule is not detected during infection of metacyclic forms. CONCLUSION Leishmania infective forms induce similar Evs-quantities, but only amastigote-Evs can induce CD200 in macrophages, suggesting a different Evs-content from both infective forms. Further experiments will be prepared to understand better the difference between both infective forms to highlight new aspects of biogenesis and the releasing mechanism of Evs.

PhD student Juliana Fortes, Master student Nathani Negreiros, Ana Claudia Torrecilhas

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: The protozoan Trypanosoma cruzi causes Chagas disease (CD). CD is classified as a neglected tropical disease (NTD) and is estimated to kill 14,000 people across Latin America every year. The disease's treatment, diagnosis, and epidemiological control remain challenging. T. cruzi delivers EVs at various stages of its life cycle, which aid in communication with cells and modulation of the host's immune response. EVs may be useful as CD biomarkers and targets for new therapeutic strategies. The goal is to investigate to how T. cruzi-released EVs influence the profile and behavior of primary monocytes and macrophages, with a focus on activation markers.

Methods: Nanoparticle Tracking Analysis (NTA) was used to determine the concentration and size of T.cruzi EVs. Following that, the EVs were labeled with PKH26 Dye Solution (SIGMA). Flow cytometry (FC) and confocal microscopy (CM) were used to assess the interaction of the EVs with THP-1 and differentiated THP-1 after 5, 15, 30 minutes, 1 h, 24 h, and 48 h. After isolating mononuclear cells from whole blood using Ficoll-Paque (SIGMA), human monocytes were isolated using a negative selection strategy according to the commercial Pan Monocyte Isolation Kit, human (Miltenyi Biotec). Monocytes were incubated with EVs that contained the following markers: CD14-V450 (BD Biosciences), CD16-PE-CY7 (ExBio), CD80-FITC (BD Biosciences), CD86-APC (BD Biosciences), CD69-PE (ExBio), HLA-DR-V500 (BD Biosciences), TexasRed (BD Biosciences), and FC.

Results: Our results show that internalization of T. cruzi EVs was uptake using confocal microscopy after only 5 minutes of incubation with human THP-1 cells. Flow cytometry (FC) also confirmed this uptakeand cells interactions. Furthermore, after 24 hours of incubation, significant changes in monocyte morphology were observed. Furthermore, we found a significant increase in the expression of key surface markers on monocytes, which could be important in their differentiation into macrophages.

Summary/Conclusion: The T.cruzi EVs interact with host cells, resulting in observable changes in cellular morphology and suggesting the potential to alter the host's immune response profile. These findings provide insight on the quick internalization dynamics of T. cruzi EVs, their effect on cellular morphology, and the implications for monocyte differentiation.

Miss Isabella Stuart, Mr Joshua Nickson, Dr Seong Hoong-Chow, Associate professor Thomas Naderer

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Pseudomonas aeruginosa is an opportunistic pathogen, that can cause life threatening infections, particularly those who are immunocompromised. While conventional treatments target infection with antimicrobials, our research explores an alternative approach by focusing on host and virulence factors. To develop new treatment strategies, a comprehensive understanding of host factors and responses are needed. For P. aeruginosa infections we have turned to focusing on their outer membrane derived vesicles and how they affect alveolar macrophage functions.

Methods: Bone marrow-derived alveolar macrophages (BMDAMs) were exposed to P. aeruginosa derived outer membrane vesicles (POMVs) with cytokine responses assessed by multiplex assays, immunoblotting, and flow cytometry. Host responses of cell-death, apoptotic caspase activation, and mitochondrial health were examined through real-time analysis using live cell imaging. To investigate the role of BCL-2 family members in AMs, A1-deficient AMs and BH3 mimetics, such as ABT-737, and S63845, were used. Additionally, A1-deficient mice were infected with intranasally administered P. aeruginosa. Colony-forming units (CFUs) were enumerated from the lung, liver, and blood, and flow cytometry was used to assess cytokine secretion in the serum using a multiplex assay.

Results: POMVs induce pro-inflammatory cytokine secretion in BMDAMs, however cell death levels remain low. Treatment with POMVs led to increased expression of anti-apoptotic proteins, A1 and MCL-1 in BMDAMs. Live cell imagining of A1-deficient cells, combined with MCL-1 inhibitor S63845, resulted in higher levels of cell death compared to WT. Infected A1-deficient mice lost more weight than WT across the duration of infection. However, CFUs from both lung and liver returned equivalent numbers.

Conclusions: BMDAMs show increased survival rates and secretion of pro-inflammatory cytokines in responses to POMVs. Anti-apoptotic proteins A1, and MCL-1 play crucial roles in alveolar macrophages by promoting macrophage survival when challenged with POMVs. Additionally, A1 appears to play an important role in P. aeruginosa lung infections. The absence of A1 does not impact the replication of P. aeruginosa, possibly suggesting there is another factor at play. These findings highlight the intricate interplay between macrophages and bacterial vesicles, shedding light on their role in modulating inflammation during infections.

Ms Matin Ghaheri, Dr Ido Bar, Dr Prabhakaran T. Sambasivam, Dr Muhammad J. A. Shiddiky, Mr Abolfazl Jangholi, Prof Chamindie Punyadeera, Prof Rebecca Ford

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Across kingdoms, small extracellular vesicles (sEVs) are produced by pathogenic organisms that are associated with signalling for host recognition and invasion, including by fungi and when in contact with their plant hosts. Very little is known about plant pathogenic fungal sEVs, with data limited to a few early studies on Fusarium oxysporum, Botrytis cinerea and Penicillium digitatum. Meanwhile, Ascochyta rabiei is an endemic and widespread necrotrophic ascomycete fungus that causes significant impact on chickpea production. The presence of sEVs in A. rabiei invasion of chickpea have not yet been established which is the focus of this study.

Methods: A. rabiei sEVs were isolated from liquid broth cultures (with and without presence of host tissue) through optimised ultracentrifugation method. The isolated sEVs were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Mass Spectrometry.

Results: In this study, TEM and NTA analyses confirmed that our optimised ultracentrifugation method, successfully isolated sEVs from A. rabiei with an acceptable size range and morphology, revealing typical cup-shaped structures with double membranes of 30-150 nm in size with greater frequencies produced in response to the presence of powdered and concentrated host tissues (AP) compared to whole detached leaves (AFL). sEVs isolated from AFL media showed larger mean and mode sizes than the other. Protein concentration of sEVs isolated from medias with the presence of host, AFL, and AP, were higher than the isolated sEVs without the host. The mass spectrometry analysis led to the identification of proteins involved in cell wall synthesis, biological processes, ribosomal structure, oxidation/reduction-related proteins, and stress responses.

Conclusions: This is the first time that the sEVs have been identified and characterised in an Ascochyta species. Bioinformatics analysis of sEV proteomics revealed a variety of pathogenic-related proteins, like oxidation/reduction-related proteins and stress response proteins. Our results will help better understanding the biological function of the sEVs during plant and A. rabiei interaction and providing the evidence for the role of sEVs in pathogenesis of the A. rabiei.

Associate Professor David Langlais, MSc Atia Amin, PhD Ana Victoria Ibarra Meneses, Associate Professor Christopher Fernandez-Prada

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Leishmaniasis is a neglected tropical disease causing about one million new cases and over twenty thousand deaths every year worldwide. The disease is caused by the parasite Leishmania spp. transmitted to its host by a sandfly and affecting some of the poorest populations, already afflicted by poor housing, displacement, and malnutrition. Despite its endemicity and important health and social consequences, the treatment arsenal is limited, and drug resistance is rising. We have recently shown that Leishmania can share and propagate drug resistance genes through extracellular vesicles (EVs). However, the genomic events leading to this generous propagation are still unclear. The objective of this project is to understand how Leishmania genomic plasticity support their capacity to develop drug resistance to antimony and what genomic cargo is packaged in EVs. Methods: Of the >20 species causing Leishmaniasis in humans, most still have incomplete reference nuclear and mitochondrial (kinetoplast) genome assemblies and annotations, which complicates their study. Firstly, we have performed a hybrid genome assembly for L. infantum using a combination of long-read Nanopore sequencing (ONT) and short-read Illumina sequencing. Secondly, to study the genomic event leading to antimony resistance, we have cultivated L. infantum and two other species under increasing drug pressure. Genomic DNA was extracted at every incremental step for ten independent clonal populations for each species and subjected to ONT sequencing. Clinical isolates of different resistance level to antimony were also sequenced. Thirdly, EVs were purified from antimony-resistant clones, their DNA extracted and submitted to sequencing. Results: The hybrid assembly approach allowed us to obtain a high-quality reference genome, resolving large telomeric repeats and gaps, and solving the complete kinetoplast maxicircle sequence. The ONT sequencing of lab and clinical antimony-resistant isolates allowed the identification of new tricks used by the parasites to develop resistance, including various intra- and extra-chromosomal amplifications. Moreover, the parasites can release these newly amplified regions into EVs to spread resistance. Summary/Conclusion: The newly assembled genome will be a resource for future studies and our results will help understand the drug resistance mechanisms of the parasite and develop new surveillance and/or therapeutics against this deadly disease.

Dr. Payel Bhanja, Dr. Rishi Man Chugh, Dr. Kafayat Yusuf, Dr. Badal Roy, Dr. Shahid Umar, Dr. Subhrajit Saha

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Gut microbiome plays an important role in gastrointestinal homeostasis and repair. Akkermansia muciniphila (AM) is the dominant human bacterium that abundantly colonizes in nutrient-rich mucus layer and protects against gastrointestinal damage and inflammation. Clinical trials based on anti-inflammatory or anti-neoplastic effect of AM are promising. Moreover, AM derived extracellular vesicles (EVs) carry important paracrine signals for intestinal homeostasis. Previous reports demonstrated that therapeutic use of bacteria derived EV to mitigate injury and inflammation. Our study suggested that anaerobic commensal microbiome such as AM are sensitive to ROS and absence of such species due to irradiation exposure contribute in intestinal injury. We hypothesized that oral supplementation of A. muciniphila, derived EV (AM-EV) can mitigate radiation induced intestinal injury. In mice model of radiation induced intestinal injury we have demonstrated that oral supplementation of AM-EV (50mg/mice for 3 times) at 24 hours after whole or partial body irradiation can mitigate radiation induced intestinal injury with restitution of crypt villus structure. To mitigate intestinal epithelial injury it is important to rescue intestinal stem cells (ISCs) and activate regenerative function of ISCs. AM-EV treatment activated the regenerative function Lgr5+ve ISCs both in ex-vivo intestinal organoid system and in Lgr5-EGFP-ires-CreERT2-TdT mice exposed to irradiation. Proteomics study of these EVs identified several key proteins including activator of folate pathway, HIF1alpha signaling pathway and TRAF6 which are important for ISC regeneration. In summary, present study demonstrated that AM-EV based therapy can be considered as a potential mitigator of Gastrointestinal acute radiation syndrome.

Dr Jian Tan, Ms Jemma Taitz, Dr Duan Ni, Ms Camille Potier, Prof Ralph Nanan, Prof Laurence Macia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: The gut microbiota regulates many host processes including immunity and metabolism. Bacteria-derived extracellular vesicles are involved in a variety of biological processes such as quorum sensing, material exchange, antibiotics resistance and host immunomodulation. However, much less is known about how extracellular vesicles produced by the gut microbiota influence host physiology.

Methods: Whole microbiota extracellular vesicles (MEV) were isolated from faeces of control diet (AIN93G) fed animals. Faeces were resuspended in 0.02µm filtered PBS and supernatant centrifuged at 10,000g for 10min, then 18,000g for 45min before filtration through a 0.22µm filter and centrifuged at 100,000g for 2 hours. MEV were characterized by Nanoparticle Tracking Analysis using the ZetaView and visualized using transmission electron microscopy. For biodistribution study, MEV was stained with the lipophilic dye DiD and 60µg of stained MEV orally administered to mice and organs imaged using the IVIS 6 hours later. For chronic exposure study, 1x10¹⁰ MEV were administered orally by gavage to mice daily for at least 4 weeks. For in vitro studies, HEPG2 cell line was cultured in complete DMEM media and stimulated with 3x10⁹ MEV per well in a 6 well plate.

Results: Administration of MEV is biodistributed predominantly to the liver and led to upregulation of glucogenic genes Pepck and G6p in mice livers. MEV upregulated hepatic gluconeogenesis directly, as its addition to the HEPG2 human hepatocyte cell line led to an increase in PEPCK and G6P expression in vitro. This impact of MEV on gluconeogenesis was confirmed by pyruvate tolerance testing, with MEV-treated animals having significantly elevated glucose levels compared to control mice. Sustained gluconeogenesis in MEV treated mice led to metabolic impairment with the development of mild insulin resistance and glucose intolerance. Interestingly, we also found that fasting, which induces gluconeogenesis, led to a significant increase in the production of extracellular vesicles by the gut microbiota.

Summary/Conclusion: While the gut microbiota can affect host metabolism, this work highlights a novel role for MEV as inducer of liver gluconeogenesis. Our work suggests the evolutionary role of the gut microbiota in supporting host glucose demands via their production of extracellular vesicles.

Msc Jaques Franco Novaes De Carvalho, Msc. Gabriela Rodrigues Barbosa, Msc Marina Malheiros Araújo Silvestrini, Dr. Sidneia Sousa Santos, Dr. Flávio Freitas, Dr. Nancy Cristina Junqueira Bellei, Dr. Andréa Teixeira de Carvalho, Dr. Ana Claudia Torrecilhas, Dr. Reinaldo Salomão

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

INTRODUCTION. Extracellular Vesicles (EVs) isolated from cells infected with the SARS-CoV-2 virus are still unknown, but they are known to be used as EVs in the transfer of viral components in other viruses. Our goal is to use flow cytometry to characterize EVs isolated from plasma samples from patients with COVID-19 sepsis and confirm the presence of viral particles as well as SARS-CoV-2 variants. METHODS. Plasma samples were collected from patients with COVID-19 and sepsis (n = 42) admitted to Hospital Sepaco in So Paulo between 03/2021 and 08/2021, as well as from healthy individuals (n = 09). We isolated EVs by UC at 100,000 x g for 16 hours and used NTA to select 20 patient samples with the highest concentrations of EVs (particles/mL) to evaluate the presence of SARS-CoV-2 viral particles using the RT-qPCR kit targeting the RdRp, Envelope (E), and Nucleocapsid (N) genes. EVs, phosphatidylserine (Annexin V), and tetraspanins (CD9 and CD81) were identified using flow cytometry (CytoFlex S), as well as the cellular origins of neutrophils (CD66b), endothelial cells (CD144), and T lymphocytes (CD3), and platelets (CD42a).RESULTS. RT-qPCR analysis revealed gene amplification in 14 of 20 patient EV samples. Two samples revealed a Gamma variant. Flow cytometry revealed that the average percentage expression of EVs labeled with Annexin V and platelet origin markers was (17.94% 6,357), followed by CD3 T lymphocytes (17.62% 5,834), CD66b neutrophils (10.42% 4,832), and CD144 endothelial cell (8.60% 2,151). There were no significant differences in each marker when comparing gender groups, differences in clinical stage, days of hospital stay, and EVs with PCR (+) versus PCR (-).CONCLUSION. EVs have been identified as possible carriers of SARS-CoV-2 viral particles. Flow cytometry analysis of patients revealed a higher proportion originating from platelets. However, the evidence for the definitive role of EVs in differentiating between patient groups remains inconclusive. The images of EVs isolated by electron microscopy will be analyzed in the following steps. Financial Support: CAPES, FAPESP, CNPq.

Mr Kyle Bramich, Dr Rahul Sanwlani, Prof Suresh Mathivanan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Probiotic species of bacteria are typically introduced into the body through various food sources and can confer a beneficial effect on the host. Over the past decade, extensive research has revealed the therapeutic potential of probiotic extracellular vesicles (EVs) in several disease contexts including cancer, ulcerative colitis, viral infections, and obesity. However, the current limitations in biogenesis knowledge, universal isolation methods, and understanding of intestinal epithelium uptake have prevented realisation of their clinical utility. To further understand how probiotic EVs can affect specific mechanisms, the proteomic contents of EV isolated from a probiotic strain was investigated.

Methods: Using combinational isolation techniques and omics approaches, we aimed to isolate and purify EVs from a probiotic bacterial strain and identify enriched protein cargo. Isolation was achieved using tangential flow filtration and differential centrifugation coupled with ultracentrifugation. Characterisation of isolated L. delbrueckii EVs was further confirmed by microscopy and proteomic analysis of EV samples was performed using mass spectrometry (LC-MS/MS).

Results: LC-MS/MS hits identified a total of 301 proteins in the EV samples and comparison to whole cell lysate revealed 285 common proteins between samples. Interestingly, gene ontology biological processes implicated these proteins in several biological processes such as biosynthetic processes (52.4%), nucleotide binding (34.1%), and metabolism (70.7%). Gene ontology molecular function further validated these findings, suggesting that proteins from L. delbrueckii EVs have functions as ribosome structural constituents and binding to RNA, metal ions, and nucleotides.

Conclusions: Proteomic analysis revealed the multiplicity of L. delbrueckii EV protein cargo and the associated diverse roles in various important biological processes. These findings also indicate that cargo from probiotic EVs have roles in a range of metabolic processes including, peptide and cellular amide metabolism, tRNA metabolic processes, and carboxylic acid metabolism.

Professor MAHANAMA DE ZOYSA, Mr. E.H.T. Thuslahn Jayathilaka, Mr. Mawalle Kankanamge Hasitha Madhawa Dias, Dr. Chamilani Nikapitiya Nikapitiya

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Etiological agent of the fish disease Edwardsiellosis, Edwardsiella piscicida is an important pathogen in various marine and fresh water animals and can transmitted via food chain to humans and other animals. Overuse of antimicrobials in the agriculture and aquaculture arouse multi-drug resistance (MDR), thus prioritizing various types of vaccines against edwardsiellosis. To date, few commercial fish vaccines were successful, hence, searching of new control strategies are essential. This study aimed to investigate alterations of plasma-derived exosomes proteins in olive flounder with experimentally challenged E. piscicida and to investigate the differentially expressed proteins (DEPs) that may responsible for diagnosing the infection.

Methods: We isolated and characterized exosomes from phosphate buffered saline (PBS) injected (PBS-Exo) and E. piscicida challenged (Ep-Exo) olive flounder plasma. For quantitatively identify the DEPs between two, isobaric tags for relative and absolute quantitation (iTRAQ)-based high performance liquid chromatography-tandem mass spectrometry analysis was conducted. The DEPs were subjected to Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analyses.

Results: Nanoparticle tracking analysis suggested higher plasma exosome concentration and lower diameter in length of exosomes are circulating in fish plasma that infected with E. piscicida compared to PBS-injected controls, but was statistically non-significant. Out of expressed 407 abundant proteins, up or downregulated 321 DEPs were identified. Among those, 75 were significantly upregulated while 24 were significantly downregulated (fold change >1.2 or < 0.83; q-value <0.05) and 76 were at basal (fold change <1.2 and >0.83). Enrichment analysis indicated that E. piscicida mainly modulated the complement activation proteins and most DEPs were in complement activation pathways. STRING network analysis further confirmed most DEPs are mainly involved in innate immune responses.

Summary/Conclusion: This study revealed that E. piscicida infection possesses number of DEPs that could consider for promising biomarkers for diagnosis of E. piscicida infection in fish. Further, our findings give clues to understand host-pathogen interaction mechanism, which may assist development of new therapeutics and drug delivery carriers in future.

Professor Ge Jin, Dr. Zhimin Feng

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Role of HIV-associated extracellular vesicles in human papillomavirus (HPV) infection

Zhimin Feng and Ge Jin

Case Western Reserve University School of Medicine, Cleveland, OH, USA

Introduction: People living with HIV (PLWH) are more susceptible to co-infections, including high-risk human papillomavirus (HPV) in the oral cavity. This co-infection increases the risk of HPV-related oropharyngeal cancers. HIV-associated extracellular vesicles (EVs) are implicated in various HIV comorbidities, including co-infection and cancer. However, their role in promoting high-risk HPV co-infection in the oral cavity remains unclear. This study investigates the potential role of HIV-associated EVs in facilitating HPV infection in the oral cavity.

Methods: EVs were isolated using the differential ultracentrifugation method from culture supernatants of HIV-positive J1.1 and HIV-negative Jurkat T cells grown in media supplemented with EV-depleted FBS. Western blotting confirmed the presence of tetraspanin proteins. The ZetaView Twin (Particle Metrix Inc.) was used to quantify the EVs. HPV16 pseudoviruses (PsV) were prepared from HEK293TT cells transfected with the mCherry-HPV16-L1 and HPV16-1 constructs. The 3-dimentional (3-D) oral mucosal cultures (MatTek Inc.) were used for HPV infection experiments.

Results: EVs isolated from culture supernatants of J1.1 T cells and the saliva of HIV-infected individuals contained viral transcripts spanning the trans-activation response element (TAR), Tat, and Nef regions of the HIV genome. We observed that HIV-associated J1.1 EVs significantly enhanced HPV16 PsV infection in the 3D oral mucosal cultures compared to EVs from HIV-negative T cells. HPV16 PsV only infected oral mucosal cultures with simulated microwounds, suggesting its dependence on the basal layer cells exposed through epithelial disruptions. Importantly, cetuximab, a monoclonal antibody to epidermal growth factor receptor (EGFR) for cancer treatment, blocked the pro-infection effect of HIV-associated EVs, suggesting EGFR's role in mediating HPV infection in the presence of HIV-associated EVs in the oral cavity.

Summary/Conclusion: Our findings demonstrate that HIV-associated EVs play a crucial role in promoting HPV acquisition and infection in the oral cavity of PLWH. Further research on HIV-associated EVs and their interplay with HPV infection may lead to novel therapeutic strategies to prevent HPV infection in this vulnerable population.

Funding: This work was funded by NIH/NCI R01CA264910.

Professor Tang-long Shen, Hao-Yuan Chien, Ta-Hsin Ku

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Viroids are naked, circular single-stranded RNA molecules, about 250–400 nucleic acids in size, and do not translate any proteins. Citrus exocortis viroid (CEVd) can infect not only citrus but also other crops such as tomatoes (S. lycopersicum L. cv. Rutgers) and cause epinasty, necrosis, and stunning. Currently, known pathogenic mechanisms are mainly viroid-derived small interfering RNAs (vd-siRNAs) that target and cleave host messenger RNA and cause host mRNA silencing to produce symptoms. Extracellular vesicles (EVs) are lipid-bilayer particles that play a role in communicating between cells or tissues by transporting substances such as proteins, lipids, or nucleic acids. Previous studies indicated that the host would transport small RNAs to the pathogen through EVs and cause low expression of Botrytis cinerea-related pathogenic genes, thereby inhibiting its pathogenicity. Since viroids move through the plant and cause systemic diseases, it is important to study how naked and lacking protein capsids protect viroids in the plant. For this, we assume that EVs can act as a vector for the transport of viroids within the plant to cause systemic diseases. First, we inoculated CEVd onto tomatoes (S. lycopersicum L. cv. Rutgers), then purified the stem and leaf EVs six weeks post-inoculation and confirmed the particle number, diameter, and structure by NTA (nanoparticle tracking analysis) and TEM. Second, we detected the viral content contained in EVs by RT-qPCR and found that the CEVd amount of the inoculation group (CEVd-EVs) was significantly higher than the mock group (mock-EVs). In addition, the proteomic analysis revealed that CEVd-EVs contain several proteins involved in metabolic and catabolic process. In order to understand whether the viroids could be transported by EVs or not and cause symptoms or stress on the plants, we re-inoculated CEVd-EVs and mock-EVs on healthy tomato plants. After four weeks post-inoculation, the biomass and leaf area decreased in the CEVd-EVs inoculation group. These findings provide evidence that EVs could serve as a mode of transport for viroids, potentially contributing to the spread of infection. Additionally, the alterations in plant phenotype suggest that the interaction between viroids and EVs may have broader implications for plant health and disease resistance.

Pos-doctoral Nadjania Saraiva de Lira Silva, Ana Claudia Torrecilhas, Full Professor Sergio Schenkman

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: The ESCRT machinery (Endosomal Sorting Complex Required for Transport) is made up of four multi-subunit complexes: ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. These complexes play a role in the transport of ubiquitinated cargoes to intraluminal vesicles that form from multivesicular bodies derived from endosomal compartments. These multivesicular bodies eventually fuse with lysosomes to degrade endocytosed cargoes or with the plasma membrane to secret intraluminal vesicles as a heterogeneous mixture of exosomes. Trypanosomatids are protozoan parasites that secrete extracellular vesicles and rely on endocytosis for survival. However, the EV biogenetic pathways in Trypanosoma cruzi are almost completely unknown, and no ESCRT proteins have been identified, despite their predicted existence.

Methods: We used CRISPR/Cas9 to investigate the ESCRT-I complex, specifically the TcVPS23 protein, for its role in extracellular vesicle (EV) formation in T. cruzi trypomastigotes and epimastigotes. TcVPS23 emerged as a critical virulence factor in T. cruzi, correlating with other pathogen findings. We also performed Nanoparticle Tracking Analysis (NTA), detected the specific markers such as trans-sialidase (TS), and functional assays (in vitro and in vivo).

Results: TcVPS23 mRNA was found to be more abundant in trypomastigotes than in epimastigotes, indicating that this system is fully functional in Kinetoplastida and can act as a virulence factor in T. cruzi. The partial deletion of the TcVPS23 gene reduced Transferrin uptake but not BSA endocytosis. TcVPS23 has also been shown to be important for the secretion of epimastigotes and trypomastigotes-derived EV, as well as the expression of trans-sialidase on the surface of trypomastigotes. Lower TcVPS23 expression reduced the parasite's ability to invade cells and increased the intracellular amastigotes in L6 cells at 48, 72, and 96h. TcVPS23 mutant was inoculated in vivo showed decreased parasitemia and mast cell recruitment in the heart, and it increased the percentage of infected mice that survived.

Summary/Conclusion In conclusion, these findings demonstrate the importance of TcVPS23 in EV secretion, endocytic modulation, and performance as an important virulence factor in T. cruzi, highlighting the ESCRT protein as a promising candidate for vaccine development against Chagas disease

Mr Angus Cramond, Ms Nina Colon, Mr Jack Emery, Dr Dongmei Tong, Dr Caroline Skene, Professor Richard L. Ferrero

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Helicobacter pylori produces several autotransporter proteins that use a conserved beta barrel to transport passenger domains through the bacterial membrane. Each H. pylori autotransporter protein has a distinct passenger domain; except for the vacuolating cytotoxin, VacA, these autotransporters remain largely unstudied. Previous work on the H. pylori autotransporter ImaA found that imaA knockout bacteria induce stronger IL 8 responses in gastric epithelial cells (AGS), suggesting that ImaA may be an immunomodulatory protein. Proteomic studies showed that H. pylori ImaA associates with extracellular vesicles (EVs). Thus, we hypothesised that EVs containing ImaA may be able to modulate host responses. Methods: We generated H. pylori imaA mutant bacteria, then isolated EVs from these and the corresponding wild-type (WT) bacteria by ultracentrifugation. EVs were characterised by Nanoparticle tracking analysis (NTA). Interleukin-8 (IL-8) responses of AGS gastric epithelial cells to EVs were determined by ELISA. To investigate the immunomodulatory effects of ImaA in vivo, we stimulated splenocytes from mice that had been vaccinated on days 0 and 28 with H. pylori WT or imaA EVs, or PBS, then euthanised on day 56. Results: NTA showed that H. pylori imaA and WT EVs have similar particle sizes (median sizes 95 and 100 nm, respectively). imaA EVs induced weaker IL 8 responses than WT EVs (3.7- and 1.1-fold change from unstimulated for WT and imaA EVs, respectively, p = 0.0043). Splenocytes from mice that were vaccinated with either H. pylori WT or imaA EVs ex vivo showed reduced IFN γ and IL-17 responses to ConA stimulation ex vivo, when compared with splenocytes from unvaccinated mice, suggesting EVs suppress Th1 and Th17 responses. Summary/Conclusions: Taken together, we propose that H. pylori ImaA contributes to the pro-inflammatory effects of EVs on epithelial cells. Further work is required to understand the role of EV-associated ImaA in vivo.

Mr Jack Emery, Doctor Variya (Way) Nemidkanam, Ms Nina Colon, Ms Kate Friesen, Ms Georgie-Wray McCann, Associate Professor David McGee, Doctor Natalia Castaño-Rodríguez, Doctor Dongmei Tong, Doctor Caroline Skene, Doctor Laurent Terradot, Professor Richard L. Ferrero

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: The bacterium Helicobacter pylori tightly regulates the host immune response to modulate inflammation, but if dysregulated, this leads to cancer. H. pylori tumour necrosis factor-α-inducing protein (Tipα) was reported to promote tumour necrosis factor (TNF) responses and carcinogenesis. Although Tipα is secreted in a soluble form by the bacterium, it is also packaged with bacterial extracellular vesicles (EVs). We hypothesised that H. pylori EVs carry Tipα to the nucleus, resulting in perturbed nuclear processes that promote carcinogenesis.

Methods: H. pylori isogenic tipA and tipA/tipA+ mutant strains were generated and characterised by NTA and Western blotting. EVs isolated from H. pylori wild-type (WT) and mutant bacteria were incubated with gastric epithelial cells (AGS) and THP-1-derived macrophages. Intracellular localisation of EV-associated Tipα was determined by cell fraction and confocal microscopy. Proinflammatory cytokine responses were determined by ELISA.

Results: We confirmed H. pylori EVs harbour Tipα and that WT and mutant EVs shared similar particle numbers and median sizes. Also, the amounts of Tipα secreted into culture supernatants varied between H. pylori strains but was not associated with disease outcome. EV-associated Tipα was present within the cytoplasm of AGS cells at 4-hours post-treatment and accumulated in the nuclear compartment by 18 hours. Consistent with previous work, the recombinant form of Tipα induced TNF production in THP-1 cells. Similarly, tipA EVs induced significantly more TNF (p = 0.0007) and interleukin-8 (IL-8; p = 0.0127) than WT or tipA/tipA+ EVs in THP-1 cells. Interestingly, however, tipA EVs induced significantly lower levels of IL-8 than those from WT or tipA/tipA+ EVs in AGS cells (p = 0.0402).

Summary/conclusion: Taken together, we propose that EV-associated Tipα is an immunoregulatory virulence factor which targets the nuclear compartment of host cells to promote gastric carcinogenesis.

Miss Jemma Taitz, Mr Jian Tan, Mr Duan Ni, Mr Georges Grau, Mr Nicholas King, Mr Ralph Nanan, Ms Laurence Macia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

The gut microbiota is established at birth and has a profound impact on host immunity. The stage before weaning presents a critical window where exposure to microbiota components is essential for proper immune development. It is less known, however, how exposure to maternal gut microbiota components may affect immune development at an earlier stage in utero. Bacterial extracellular vesicles (BEVs) produced by gut microbiota are novel players in the microbiota-host relationship, however, their role, particularly during pregnancy, remains largely unexplored.

Methods

C57BL6 mice were orally administered 60ug of gut-derived BEVs during pregnancy from E0 to E19 (PBS administered as control). Offspring were transferred to untreated foster dams at birth and culled prior to weaning at D21.

Results

Bodyweight and liver weight of BEV-exposed offspring was significantly increased compared to offspring of PBS mice, suggesting a potential impact on offspring's metabolism. BEV-exposed offspring had increased spleen and mesenteric lymph node cellularity, while thymus and bone marrow cellularity were unaffected, suggesting BEV exposure in utero may affect secondary lymphoid organ development. By spectral cytometry, we observed significantly increased proportions of activated CD8 and CD4 T cells in secondary lymphoid organs in the BEV-exposed mice but did not detect changes in thymocyte profiles, suggesting BEV exposure in pregnancy does not impact offspring lymphopoiesis but rather may affect lymphocytes in the periphery. We identified that in vitro, BEVs induce induced T cell proliferation equally in both groups, suggesting BEV exposure in utero does not induce-antigen specific proliferation.

Summary:

Our findings suggest that in utero exposure to gut derived-BEVs alters T cells located in secondary lymphoid tissues. While the mechanism and receptors involved remain to be elucidated, this work highlights the role of MEV on the developing immune system in utero.

Dr Jiaru Yang, Dr. Seong Hoong Chow, Dr. Pankaj Deo, Associate Professor Thomas Naderer

Poster Pitches (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:45 PM - 1:00 PM

Introduction

Trained immunity refers to innate immune cells showing increased immune response to pathogens after stimulation with nonlethal concentration of bacterial molecules or pathogens. Gonorrhoea is sexually transmitted disease caused by Neisseria gonorrhoeae. In the absence of new antibiotics, developing a gonorrhoea vaccine is a pressing need. Outer membrane vesicles (OMVs) are a promising vaccine platform. The meningococcal vaccine (4CMenB) that is based on OMVs elicits some protection (∼30%) against Neisseria gonorrhoeae infections in humans and animal models using vaginal challenge. Current efforts are aiming at identifying the proteins that confer cross-protection against N. gonorrhoeae to develop a more specific OMV vaccine. How OMVs activate the innate immune system that leads to protective immunity and whether OMVs in general induce trained immunity remains unclear. We hypothesise that N. gonorrhoea OMVs induce trained immunity in monocytes and macrophages, which can be explored to develop a N. gonorrhoea vaccine.

Methods

We isolated OMVs from N. gonorrhoeae and trained human monocytes for 24 hours. Trained monocytes were differentiated to macrophages and challenged with N. gonorrhoeae, Candida albicans, E. coli or with purified molecules, such as LPS. We then measured immune responses, such as TNF-α and IL-6 secretion, and programmed cell death signalling in trained and control macrophages. Inhibitors that target histone deacetylases (TSA, Butyrate sodium, LMK235, RGFP966 and Tubastatin), pro-apoptotic factors (BCL-2 and BCL-xL inhibitor), NLRP3 inflammasome (MCC950) and caspases (QVD) were included during the training to identify host pathways that promote immunity.

Results

OMV-trained monocytes and macrophages produced higher concentrations of IL-6 and TNF-α when restimulated with N. gonorrhoeae, E. coli and LPS compared to the untrained cells. Trained cells display higher survival rate when they encounter restimulations compare to the untrained cells. Inhibitors targeting histone deacetylases, cell death signalling and NLRP3, impaired trained immunity as determined by IL-6 and TNF-α secretion and cells survival rate.

Conclusions

OMVs derived from N. gonorrhoeae activate trained immunity in human monocytes. OMV-dependent training depends on histone acetylation and is regulated by the apoptosis, NLRP3 and caspase activity. We will discuss how OMV-dependent training of innate immune cells confers protection against various pathogens.

Phd Qianbei Li, Professor Lei Zheng

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Bacterial infections pose a significant global public health concern, leading to sepsis and lethal organ dysfunctions. Traditional diagnostic approaches, however, are time-consuming and suffer from low positivity rates, thus impeding timely treatment for bacterial infections. In essence, this stems from our limited understanding of the pathogenic mechanisms behind bacterial infections and the absence of more precise markers. Bacterial outer membrane vesicles (OMVs) serve as critical intermediaries for bacterial-host communications, yet their roles and clinical significance in bacterial infections remain largely uncharted. Herein, we introduce a novel OMV-specific labeling technique predicated on the selective recognition of bacteria by antibiotics, based on witch enable to distinguish and quantify OMVs from other sources of EVs when coupled with a Flow Nanoanalyzer. Leveraging this methodology, we discerned markedly elevated OMV levels in the blood samples of bacterial infection patients, particularly those with bloodstream infections (BSI). Moreover, we found that bacteria released OMVs from infection foci into the bloodstream, which subsequently instigated mitochondrial damage and triggered apoptosis in mouse macrophages. This study furnishes a pioneering quantitative approach for the specificity analysis of OMVs and unveils their diagnostic and pathogenic potential in bacterial infections.

Foam cell-derived extracellular vesicles regulate the environment surrounding atherosclerotic plaques Akihiko Okamura¹, Dr. Yusuke Yoshioka¹, Shungo Hikoso², Takahiro Ochiya¹

¹Department of Molecular and Cellular Medicine, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Japan, ²Department of Cardiovascular Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Japan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Ischemic heart disease (IHD) is a leading cause of death worldwide, and it is essential to understand the molecular mechanisms of plaque progression and rupture to prevent the incidence of IHD. This study aims to investigate the effect of extracellular vesicles (EVs) derived from foam cells (FCs), which are considered to be the main component of plaque, on the cells surrounding the plaque.

Methods

Human monocyte-derived cell lines differentiated into macrophage-like cells and smooth muscle cells (SMCs) were loaded with enzyme-modified non-oxidizing LDL for 96 hours to form FCs. Each FC was cultured in serum-free medium for 48 hours, and EVs were purified from the culture supernatant by ultracentrifugation. FC-derived EVs (FC-EVs) were analyzed for particle size and number by NTA, and their components were also analyzed for lipidome. FC-EVs were added to macrophages, SMCs, and endothelial cells (ECs), and the expression levels of atherosclerosis-related genes were analyzed by qPCR. Cell migration and proliferation were also measured by the live cell imaging system. Macrophages and SMCs treated with FC-EVs were loaded with LDL, and the viability of the cells and the amount of lipid uptake were evaluated. In addition, FC-EV-treated ECs and macrophages were co-cultured to evaluate the adhesive ability of macrophages to ECs.

Results

FC-EV-treated ECs upregulated the expression of genes related to intercellular adhesion ability and increased the number of macrophages adhering to ECs. FC-EVs internalized more lipids than non-FC-EVs, and their lipid composition was influenced by the lipid profile of LDL. FC-EV-treated SMCs increased cell migration capacity and enhanced lipid uptake. Furthermore, they were less resistant to the cytotoxic effects of LDL and induced more cell death compared to EV-untreated SMCs. FC-EV-treated macrophages increased migration capacity. All effects were more significant in the group of macrophage-derived FC-EVs added.

Conclusions

This study suggests that FC-EVs may increase plaque volume by accumulating macrophages and SMCs in the surrounding plaque. Furthermore, increased lipid uptake and induction of cell death were observed, suggesting that FC-EVs may accelerate atherosclerosis by functioning surrounding the plaque. We are investigating the effects of FC-EV treatment on atherosclerosis model mice.

Professor Jianbo Wu¹, Dr. Ziyu Li

¹Southwest Medical University, Luzhou, China

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Recent investigations underscore the capacity of photodynamic therapy (PDT) to induce adipocyte apoptosis, thereby mitigating obesity. Nonetheless, extant synthetic photosensitizers manifest limitations that hinder their clinical viability.

Methods: In the current study, we used Hypericum perforatum-derived exosomes-like nanovesicles (HPExos) as a novel photosensitizer, and investigated its PDT effects in adipose tissue during obesity. HPExos-were administered to high fat diet mice via intraperitoneal injection, followed by targeted irradiation with specialized LED lights.

Resutls: We here show that HPExos combined with PDT accumulated in visceral white adipose tissues results in a reduced body weight and improved insulin sensitivity. HPExos combined with PDT induced apoptosis by driving high levels of ROS. In addition, HPExos combined with PDT significantly downregulated the expression of transcription factors, PPARγ, C/EBPα, and SREBP and lipogenesis protein FABP4 both in vitro and in vivo, associated with a decreased FFA levels. Conclusion: These findings suggest that HPExos could act as an effective photosensitizer in the regulation of glucose hemostasis through inhibiting adipocyte differentiation and lipogenesis in the treatment of obesity.

Miss Drishya Mainali, Mr Anjie Ge, Dr Monokesh Sen, Miss Yvonne A. Candia, Dr. Claire Goldsbury, A/Prof. Laura Piccio

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Triggering receptor expressed on myeloid cells 2 (TREM2) is an important signalling mediator for central nervous system immune modulation and repair. Ectodomain shedding of TREM2 results in the formation of soluble TREM2. sTREM2 is found elevated in the cerebrospinal fluid (CSF) of people with neuroinflammatory conditions in comparison to healthy controls (Piccio et al., 2008). However, the function of soluble TREM2 beyond its biomarker potential remains unclear to date. Extracellular vesicles (EVs) play a vital role in cell-to-cell communication, potentially modulating pathological events in recipient cells. The overall hypothesis of this study is that TREM2/sTREM2 is localised to myeloid cell derived extracellular vesicles (EVs) and could mediate its function.

Methods: PBMC from healthy controls were cultured for seven days with 5% autologous human serum. The cultured supernatant was spun serially at 1000g-force and at 20,000 g-force for 20mins at 4C. The supernatant was ultracentrifuged at 18,000 g-force to isolate large EVs and again at 100, 000 g-force to isolate small EVs. ELISA measured sTREM2 concentrations in the supernatant after the 18k and 100k runs. The EVs pellets were digested with 1:30 trypsin to protein ration and analysed with LC/TMS under data-independent acquisition (DIA). The EVs were characterised via Nanoparticle Tracking Analysis (Zetaview) after cell-mask green (CMG) labelling of the veiscles and negative stain transmission electron microscopy (TEM).

Results: We observed a significant depletion of sTREM2 by ELISA in the cell culture supernatant post EV isolation, indicating the presence of sTREM2 in the EV pellet. sTREM2 fragment (VLVEVLADPLDHR) was found in both large and small EVs as well as the supernatant. CMG labelling showed less concentration of EV in the sample (almost by a factor 1000x) in comparison to unlabelled particle analysis. Negative stain TEM confirmed the presence of EVs.

Summary: This study indicates a potential association between sTREM2 and extracellular vesicles for the first time. However, the presence of sTREM2 in the EVs needs further investigation to confirm this association. The next step will be to examine sTREM2 abundance in the plasma of individuals with Multiple Sclerosis compared to healthy controls.

Professor Suresh Mathivanan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

CRISPR-Cas9 is a widely utilised genome editing technique that has provided an efficient means to study the function of genes in various cells and tissues. Despite this utility, there is currently a limited understanding of the impact of Cas9 expression in mammalian cells, particularly in the biogenesis and secretion of small extracellular vesicles (sEVs). Here, we investigated the changes in the protein cargo and secretion of sEVs upon expression of SpCas9 in human colorectal carcinoma HCT116 and human embryonic kidney HEK293 cells.

Methods:

A panel of cells with varying P53 status were utilised. sEVs were isolated by ultracentrifugation and characterised by Western blotting, EM, NTA and quantitative proteomics. Human and murine cancer cells releasing EVs were implanted in immune-compromised (nude) and immune-competent (C57BL/6) mice, respectively.

Results:

Expression of Cas9 impeded the proliferation of HCT116 cells and increased the abundance of p53 and its downstream target p21. Cas9 expression also increased the abundance of EV-enriched proteins CD63 and CD9 in HCT116 cells and further accelerated the secretion of sEVs. Subsequent label-free quantitative proteomics analysis of whole cell lysates highlighted small but noticeable alterations in the abundance of the few proteins upon expression of Cas9. In contrast, proteomic analysis of sEVs revealed a cell type-dependent differential abundance of several proteins. Knockout of P53 impacted the phenotype of sEVs.

Conclusion:

Overall, the study highlighted the cell-type dependent alterations in the protein cargo and secretion of sEVs upon expression of Cas9.

Dr. Jamal Ghanam, Dr. Venkatesh Kumar Chetty, Prof. Dr. Dirk Reinhardt, PD. Dr. Basant Kumar Thakur

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Cancer cells secrete small extracellular DNA (sEVs) with higher DNA (EV-DNA) content than their normal counterparts. However, the question remains: how is EV-DNA selected for secretion in sEVs, and under which circumstances? Here, we studied the implication of inhibiting sEVs secretion on cytoplasmic DNA (cyDNA) accumulation, DNA sensing activation, and cancer immunogenicity in acute myeloid leukemia (AML).

Methods

To inhibit sEVs secretion, 2D and 3D AML cultures were treated with the farnesyltransferase (FT) and sphingomyelinase (SP) inhibitors Manomycin A and GW4869 in the presence or absence of DNA damage-inducing agents Cytarabine (Ara-c) or hydroxyurea (HU). Rab27a-knockout cells were also cultured under the same conditions. sEVs isolation was done based on size exclusion chromatography, and characterized according to MISEV2018 guidelines. cyDNA was characterized by dsDNA staining and quantification after subcellular fractionation.

Results

sEVs secretion was considerably suppressed by using the combination of FT and SP inhibitors similar to Rab27a-KO, as seen by western blot and beads-assisted FACS analyses of sEVs markers. The relative EV-DNA concentration has significantly increased in sEVs isolated from cells treated with Ara-c and HU. Next, we observed a substantial accumulation of cyDNA with concomitant activation of DNA sensor cGAS and a significant increase in IRF3-S386 phosphorylation in cells. A subtle increase in pIRF3 (S396) nuclear foci has been observed when combining FT and SP inhibitors and Ara-c treatment. Analysis of DNA damage markers revealed a specific increase in ɣH2A.X nuclear foci and a high expression of the exonuclease TREX1. In AML cells, expression of surface ligands for natural killer (NK) cells receptor (NKG2D-L) such as ULBPs family is upregulated upon cellular stress, including DNA damage. AML cells were stained for ULBP1 expression, which was significantly higher under FT and SP inhibitors and Ara-c treatment. This has significantly increased NK cells toxicity against AML blasts.

Conclusion

The inhibition of sEVs secretion has resulted in a significant accumulation of cyDNA, enhancing the anti-tumor activity of NK cells against AML blasts. EV secretion appears to be another mechanism evolved by cells to prevent cyDNA accumulation. Therefore, targeting cytoplasmic DNA excretion via sEVs opens up new possibilities for cancer treatment.

Associate Professor Mona Batish, Graduate Student Ahmed Abdelgawad, Assistant Professor Vijay Parashar

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: There is mounting evidence that different RNA species are packaged into extracellular vesicles (EVs). Recent work has shown that not all RNAs are equally likely to be packaged into EVs but the process of sorting and selection of RNA cargo to be preferentially packaged into EVs is not completely understood. Here, we explored the role of inherent features including RNA length, GC content, secondary structure, number of exons and presence of consensus motifs that can be attributed for preferential enrichment of RNAs into EVs.

Methods: We utilized RNA sequencing on RNA isolated from DLD-1 cells to determine the RNAs enriched in EVs. Breifly, EVs were isolated and characterized as per MISEV guidelines. Total RNA was isolated from these EVs and cells and sent for RNA sequencing. The RNA candidates were identified by mapping to human reference genome. The raw data was analyzed for differential enrichment of different RNA sub types into EVs.

Results: We found a significant correlation between the length of RNA, GC content and structuredness to its enrichment into EVs while the number of exons did not show any effect on its enrichment. We also found presence of GC-rich novel motif specifically in some EV bound RNA species.

Conclusions: These results indicate that the RNAs physical attributes govern its eventual fate for packaging into EVs and extracellular transport. Thoroughly validation of these features could pave way for better engineering of RNA cargo into EVs.

Hailong Wang, Research Associate YiHua Wang, Professor Gregory Worrell

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction:

Extracellular vesicles (EVs) are small membrane-bound structures that originate from various cell types and carry molecular cargo to influence the behavior of recipient cells. The use of EVs as biomarkers and delivery vehicles for diagnosis and treatment in a wide range of human disease is a rapidly growing field of research and clinical practice. Four years ago, we postulated the hypothesis that electromagnetic field (EMF) can influence the release and content of EVs.

Since then, we have optimized several technical aspects of our experimental setup.

2) Methods:

We used a bioreactor system that allows cells to grow in a three-dimensional environment mimicking in-vivo conditions. We designed a custom-made EMF stimulation device that encompasses the bioreactor and delivers uniform EMFs. We established a three-step EV purification protocol that enables high-density production of EVs. We then performed mass spectrometry-based proteomics analysis on EV-related proteins and used high-resolution nanoparticle flowcytometry for single-vesicle analysis.

3) Results:

We demonstrated that electrical stimulations of current amplitudes at physiological level that are currently applied in therapeutic deep brain stimulation can modulate EV content in a frequency-dependent manner, which may have important implications for basic biology and medical applications.

4) Conclusion:

First, it raises intriguing questions about how the endogenous electrical activity of neuronal and other cellular assemblies influence the production and composition of EVs. Second, it reveals an additional underlying mechanism of how therapeutic electrical stimulations can modulate EVs and treat human brain disorders. Third, it provides a novel approach of utilizing electrical stimulations in generating specific EV cargos.

Dr. Tomofumi Yamamoto, Dr. Hirotaka NIshimura, Dr. Noritaka Hashii, Dr. Akiko Ishii-Watabe, Dr. Yusuke Yamamoto, Prof. Takahiro Ochiya

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

In recent years, therapeutic application of extracellular vesicles (EVs) has become active in the fields of cancer, inflammatory diseases, and regenerative medicine, and clinical trials on EVs derived from mesenchymal stem cells (MSCs) are also being conducted around the world. However, one of the issues that must be resolved in applying EVs as biological medicines for more general indications is establishing a manufacturing method that can stably produce EVs. Therefore, we attempted to establish a new method that highly promotes the secretion of high-quality EVs from mesenchymal stem cells.

Methods

As reported previously (Urabe F, et al., Sci. Adv., 2020), miRNAs regulating EV were screened. ExoScreen method, a high-sensitive EV detection system, was used for this study. Combined with miRNA-mimic library, we identified miRNAs, which enhanced the EV secretion in many types of normal cells including MSCs. The microarray and in silico analysis were performed to examine the direct targets of the specific miRNA. Anti-inflammatory effect of EVs was also investigated in a LPS-stimulating inflammatory model.

Results

The screening results showed that the production of EVs from miR-3202 transfected cells was found to be 1.5- to 4-fold higher than that of the control. Nanoparticle tracking analysis showed no significant difference in particle size. In addition, ABCA1 was identified as a direct target of miR-3202. Attenuating the ABCA1 with siRNAs and the treatment of the ABCA1 inhibitors promoted EV secretion 1.5- to 3.5-fold higher than each control. These results suggested that an miR-3202/ABCA1 axis facilitated the EV secretion in MSCs. Next, we performed functional assays of miR-3202-induced MSC-derived EVs. MSC-derived EVs usually have anti-inflammatory effects, so we used LPS-stimulating inflammatory model to investigate the biological function of EVs produced from miR-3202 treated MSCs. As a result, the effect of miR-3202-induced MSC-derived EVs on anti-inflammation was comparable to that of control MSC-derived EVs as long as the same number of particles were treated. This data indicates that the EVs induced by miR-3202 are functional.

Conclusions

We found that miR-3202 enhanced EV secretion by reduction of ABCA1 expression in MSCs, and miR-3202-induced MSC-derived EVs had anti-inflammatory effects.

Dr. Ruediger Gross, Ms. Hanna Reßin, Mr. Nico Preißing, Dr. Ludger Ständker, Prof. Dr. Jan Münch

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Despite advances in the understanding of the molecular biology of EV biogenesis and release, details on how these processes are regulated are limited. Consequently, few agents with potent/selective EV-release modulating activity are known. Considering known functions of EVs in numerous infectious, oncological and neurodegenerative conditions but also the emerging use of EV biotechnology for drug delivery, agents inhibiting or boosting EV release, respectively, would be of great interest. We here explored the human peptidome, containing millions of compounds with enormous structural and functional diversity, to identify such agents endogenously present in humans.

Methods

1000 liters of human hemofiltrate (HF) was used to prepare a peptide library by cation-exchange chromatography followed by reverse-phase chromatography. The resulting ∼ 500 fractions contain the entire human blood peptidome and were screened for modulating EV release using a HEK293T CD63-Tluc reporter cell line. 48h after addition of fractions, luciferase activity was measured in the supernatant (EV release) and in lysates (retained EVs/EV biogenesis) and the quotient calculated as the EV release factor. Cytotoxic effects were evaluated by ATP levels. Fractions were ranked for magnitude of release/biogenesis modulation and compared to controls and hit fractions evaluated for dose-dependency.

Results

In the initial screening, EV release-boosting activity was observed for several neighboring fractions. In fractions with most pronounced effects, quantification suggested stimulation of EV release without changes in biogenesis (4.2-fold increase in supernatant-, 5-fold decrease cellular luciferase activity, EV release factor 20). Distinct eluates also exerted inhibitory effects on both cellular and release EV simultaneously, hinting at a general biogenesis blockade (4-fold reduced supernatant-, 2-fold reduced cellular luciferase levels, release factor 0.5). For these primary hit fractions, effects were dose-dependent and exerted at non-cytotoxic concentrations.

Summary/Conclusion

These results offer first insights into the diversity of EV-modulating agents to be discovered in the human peptidome. Following further rounds of chromatography, the responsible peptide(s) will be identified using mass spectrometry and chemically synthesized to confirm the activity. In addition to potential therapeutic and biotechnological applications of such EV-release modulating peptides, identifying their endogenous role will offer new insights into how EV-release is physiologically fine-tuned.

Sumeet Poudel, Jerilyn Izac, Zhiyong He, Lili Wang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) are nanometer-sized vesicles that carry heterogeneous cellular components including proteins, nucleic acids, lipids, and metabolites. The intravesicular and surface contents of EVs are a goldmine of biomarkers and have both physiological and pathological importance. Because of the heterogeneous cargo, EVs can act as mediators of local and distal cell-cell communication that could potentially trigger cancer metastasis, for example. However, how the genome signature regulates EV cargo and an understanding of the function is not well studied. This study aims to understand how genetics impact EV cargo loading.

Methods: EVs were extracted from cultures of the vector copy number cell lines with a fluorescent reporter (green fluorescent protein, GFP) ranging from 0 – 4 copies. Extracted EVs were characterized by Spectradyne nCS1 for count and size distribution. Imaging flow cytometry was used to analyze the fluorescent intensity for GFP+ EVs and their counts from the total EVs (CD9+ EVs). RNA content of the EVs was also compared among the copy number cells along with the total RNA by ddPCR. In addition, naturally occurring copy number cells are also being analyzed to understand the role of copy numbers for EV cargo loading.

Results: Preliminary results suggest that the gene copy number dictates the protein cargo of the EVs but not the RNA content. Increasing copies of reporter GFP in genetic loci correlated with increasing count of GFP+ EV intensity and count with flow cytometry.

Conclusion: It is important to understand the role of copy numbers in EV cargo. It is especially important to understand its role in cell and gene therapy space because as an effective measure to reduce the genotoxic and tumorigenic potential caused by uncontrolled integration, the US Food and Drug Administration recommends the integrated vector copy number (VCN) must be less than 5 copies per cell. Other studies are also being conducted to understand if gene copy number plays a role in cargo loading in naturally occurring or pathological states.

Phd Student Gizaw Gebeyehu, Research Professor Tibor Rauch, Associate Professor Marianna Pap, Dr Geza Makkai, Dr Tibor Janosi

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Sepsis remains one of the most serious diseases with the greatest health and economic impact on societies around the world. In the context of sepsis, circulating exosome-transported mRNAs and miRNAs can regulate the expression of immune response genes of the recipient cells. The details of exosome-based cell-to-cell communication are not well known including how mRNAs are recruited and packaged into the exosomes and other extracellular vesicles we aimed to explore molecules and mechanisms related to exosomal cargo (e.g. mRNA and miRNA) loading under well-defined conditions in cell cultures. We studied exosomal LPS-treatment-related RNA profile changes of THP-1 cells and identified RNA sequences that might be implicated in cargo sorting and loading.

THP-1 (human monocyte) cell line was treated with LPS and then exosomes were purified from the supernatant by multiple-step ultra-centrifugation. Total RNA was isolated from exosomal fractions using a TRISOL-based method and subsequently sequenced by next-generation sequencing (RNA-Seq). The MEME program analyzed the most abundant exosomal mRNAs (i.e., the top 50 mRNAs) at their UTRs. Conventional and NEBuilder-based cloning methodologies were employed to generate reporter gene constructs to verify the functional relevance of the MEME program-indicated conserved sequences in UTRs. Cells were transfected by these reporter gene constructs and RNA content of exosomal fractions was monitored by qPCR.

We identified a highly conserved sequence element among the most abundant exosomal mRNAs. We have constructed Reporter genes that are suitable for investigating the function of this exosome-associated RNA motif. Transfection studies with the reporter gene constructs are ongoing. These experiments may lead to insights into the RNA motif, protein factors, and mechanisms involved in exosomal packaging in the context of sepsis.

Benyapa Tangruksa^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41346, Sweden 2. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde SE-54128, Sweden}, Doctor Muhammad Nawaz^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41346, Sweden}, Adjunct Professor Sepideh Heydarkhan-Hagvall^{2. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde SE-54128, Sweden}, Professor Jane Synnergren^{2. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde SE-54128, Sweden 3. Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41345, Sweden}, Associate Professor Hadi Valadi^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41346, Sweden}

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction:

Lipid nanoparticles (LNPs) are widely used as effective vehicles for delivering functional mRNA, for example, in COVID-19 mRNA vaccines. Previously, it was shown that after LNPs and its mRNA (e.g. EPO mRNA) are uptaken by cells, a fraction of the LNP-mRNA is repacked into the cells’ own vehicles, extracellular vesicles (EVs). This process facilitates the transport of the LNP-mRNA between cells through EVs. One emerging strategy for cardiovascular disease treatment is delivering angiogenic molecules, such as VEGF-A mRNA, to stimulate blood vessel formation. In this study, we investigated how LNP containing VEGF-A mRNA affects the transcriptomic content and functions of EVs.

Methods:

LNPs with encapsulated VEGF-A mRNA were administered to lung epithelial, umbilical cord endothelial, and cardiac progenitor cells. The EVs from these cells were isolated, and their transcriptomic profiles were defined using RNA-Seq analysis. Using either LNPs or the three EV types, equal amount of VEGF mRNA was administered to recipient cells in vitro, or in vivo via intracardiac injection. The heart tissues at and around the injected area were examined.

Results:

Following LNP administration to cells, secreted EVs contained a fraction of internalised VEGF-A mRNA. A significant amount of the VEGF-A mRNA was detectable in EVs from all three cell types. RNA-Seq results demonstrate that cells respond to LNP treatment, and the RNA contents of EVs are altered, resulting in over 1000 dysregulated genes. Several pro-angiogenic transcripts were overexpressed in EVs after LNP treatment. Additionally, EVs deliver functional VEGF-A mRNA in vitro and in vivo, leading to protein production. Cardiac progenitor EVs promote the most angiogenesis during in vitro VEGF-A delivery compared to other EVs. Intracardiac injections of VEGF-A mRNA into mouse hearts produce local VEGF-A protein without spillover to other organs. EVs from cardiac progenitor cells demonstrate the least inflammatory cytokine production in cardiac tissue compared to other EVs and LNPs.

Conclusions:

LNP treatment alters the transcriptomic contents of secreted EVs. LNPs use EVs as functional extensions to transport therapeutic mRNA between cells, but EVs do it differently by delivering a mixture of mRNAs, including the mRNA from LNPs.

MSc Christopher C. Reimann, MSc Feizhi Song, Dr. rer. nat. Dipl. Hum.-Biol. Hermann C. Altmeppen, Dr. Lesley Cheng, Prof. Markus Glatzel, Prof. Marina Mikhaylova, Prof. Andrew F. Hill

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Proteases are a mostly unexplored factor in the biology of extracellular vesicles (EVs). Their cellular presence may determine protein sorting into and release via EVs, while their continuous activity at the EV surface towards membrane proteins may alter the specificity of EV-to-cell interaction and cargo delivery. Here, we focus on the metalloprotease ADAM10 as a master regulator of signaling and adhesion proteins and a key player in brain cell communication, embryonic development, and cancer. Studying the effects of ADAM10's presence and activity on EV composition and targeting will shed new light on the factors driving EV function and could help model the consequences of ADAM10 dysregulation.

Methods: Human A549 lung carcinoma cells with wildtype (WT) or ADAM10 KO (A10KO) genotype, as well as ADAM10 inhibitor-treated WT cells (A10inh.), were cultured in serum-free medium. Small EVs (sEVs) were isolated from the cell supernatant by ultracentrifugation and compared by NTA, EM, western blot, and mass spectrometry. The isolated sEVs were then used for treatment of WT and A10KO cells, and cells were fixed and imaged for EV-mediated ADAM10 transfer by confocal microscopy. ADAM10 activity in WT EV-treated A10KO cells was determined by analyzing the conditioned media for shed fragments of the cellular prion protein (PrPc), as this substrate is solely shed by ADAM10 (i.e., no involvement of related proteases such as ADAM17/TACE) and cleavage site-specific antibodies were available.

Results: Genetic depletion of ADAM10 (A10KO), but not inhibition, decreased the number of released sEV particles compared to WT. Focusing on PrPc as a major ADAM10 substrate, we found minor expression in WT sEVs but strong signal in sEVs from A10KO and A10inh. cells, suggesting changes in its sorting or processing. Treatment of A10KO cells with ADAM10-positive WT sEVs caused the reappearance of mature ADAM10 in A10KO cells and partially restored PrPc shedding.

Summary/Conclusion: Our results provide new insights into ADAM10 as modulator of EV trafficking and function and suggest EVs as potential vectors for the extracellular transfer of active ADAM10, paving the way for future studies to address the functional relevance of this mechanism in health and disease.

Dr. Tamás Visnovitz, Ms Dorina Lenzinger, Ms Anna Koncz, Ms Tünde Bárkai, Dr. Krisztina V Vukman, Ms Alicia Galinsoga, Dr. Krisztina Németh, Ms Kelsey Fletcher, Dr. Péter Lőrincz, Dr. Gábor Valcz, Prof. Edit I Buzás

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Recent studies have revealed that extracellular vesicle (EV) biogenesis is more complex than predicted previously. Our recent findings highlighted that colon cancer cells release large, membrane-enclosed structures with small inner vesicles. This process is distinct from canonical exosome release via exocytosis and small ectosome discharge by plasma membrane shedding. The current study aims at investigating whether this unique large, multivesicular EV subtype was exclusively characteristic for colorectal cancer cells.

Methods

We used confocal microscopy (of both fixed and live-cells), transmission electron microscopy (EPON-embedded and immunogold techniques), super-resolution live-cell imaging and Western blot analysis. We also tested the effect of inhibiting different cellular pathways. The detection of the large EVs (lEVs) was made possible through a novel in situ fixation approach, which prevented disintegration during centrifugation and pipetting. Comprehensive characterization of the large multivesicular extracellular structures included assessment of classical EV markers such as CD63, CD81, ALIX, TSG101, as well as non-conventional markers like TSPAN4 and LC3B.

Results

Our findings indicate that all tested cell types, including different cell lines and cells within their tissue environment, release lEVs containing intraluminal vesicles. We demonstrated that upon spontaneous rupture of the limiting membrane of these lEVs, internal small vesicles escaped into the extracellular environment through a “torn bag mechanism” and we could monitor this process by super-resolution live-cell imaging. Our studies suggest that the multivesicular lEVs may have an unusual intracellular origin, distinguishing them from any conventionally secreted EVs.

Conclusion

We propose that the “torn bag release” of small EVs represents a previously undescribed release mechanism of EVs of endosomal origin, potentially having roles in both homeostatic functions and cell-cell communication. The observed “torn bag mechanism” prevalent in both in vitro and in fixed tissue samples, differ from exocytosis. It involves plasma membrane budding, ectocytosis, and subsequent extracellular rupture of the limiting membrane of the large multivesicular EVs. This novel mechanism offers valuable insights into the origin of the small EV release.

Dr Taeyoung Kang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Wnt signalling pathway has been attributed in pathogenesis of cancer and is known to regulate cellular senescence. The functional role of Wnt signalling pathway in the biogenesis and secretion of small extracellular vesicles (sEVs) is poorly understood.

Methods

sEVs secreted from cells in culture were isolated via the conventional differential-ultracentrifugation and further characterised using Western blotting, nanoparticle tracking analysis, transmission electron microscopy. To investigate the implications cellular senescence and Wnt signalling pathway in the biogenesis and secretion of sEVs, CRISPR/Cas9-based knockout, luciferase assay, quantitative proteomic analysis, immunofluorescence, and siRNA-based knockdown and cellular survivability assays were conducted in isogenic cells, whose survival was either dependent or independent on the canonical Wnt signalling pathway.

Results

Knockout of CTNNB1 (β-catenin) using CRISPR/Cas9 genome editing technique highlighted significant attenuation of Wnt signalling activity as well as reduced proliferation and induction of cellular senescence via the activation of AKT signalling pathway in human colorectal cancer cells. The induction of cellular senescence resulted in increased secretion of sEVs with altered protein cargo. Furthermore, immunofluorescence microscopy, proteomic analysis, and Western blotting collectively suggested alterations in the abundance of several regulators of sEV biogenesis and secretion, such as CD63, Cortactin and Rab27a upon induction of cellular senescence via the loss of β-catenin. siRNA-mediated knockdown of sEV regulators resulted in rescuing the secretion of sEVs in human colorectal cancer cells that lacked β-catenin. Moreover, p53 and p21 knockout cells as well as exogenous expression of p21 or p16 further highlighted the implications of cellular senescence in the biogenesis and secretion of sEVs in human colorectal cancer cells that exhibit Wnt-dependency.

Conclusion

In conclusion, the current findings of the study highlight a novel mechanism by which cells undergoing cellular senescence regulate the biogenesis and secretion of sEVs in a Wnt-dependent manner.

Student Kaixiang Zhang^1,2, Ying Zhang¹, Hang Yin^1,2

¹School of Pharmaceutical Sciences, Tsinghua University, Beijing, China, ²Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) carry genetic materials such as miRNA and mRNA to modulate gene expression in the cells that receive these vesicles, playing crucial roles in both physiological state homeostasis and disease development. EV mRNAs are not only promising candidates for non-invasive diagnostic tests but also can be used for therapeutic purposes across various diseases. However, the mechanism for sorting mRNA into EVs, an important question in EV biogenesis, remains unclear. Here we have identified the typical mRNA binding protein NPM1, which facilitates the transportation process of mRNAs into EVs. This process relies on the involvement of both NPM1 can specific motifs on mRNA.

Methods: EVs from both cultured cells and serum samples were collected with ultracentrifugation. RNA binding proteins in EVs were identified by the oligo d(T) capture method. Next-Generation Sequencing was employed to identify total mRNAs in EV. Sequence data was analyzed with MEME to discover specific motifs.

Results: In this work, we have identified NPM1 as an RNA-binding protein that accounts for sorting of mRNA into EVs. In addition, we found that in NPM1-depleted EVs, the mRNA level of cancerous genes decreased significantly, despite some non-cancerous genes remaining unchanged. Utilizing computational analysis, we identified motifs in mRNA that are specifically recognized by NPM1 protein and facilitate the sorting into EVs. Moreover, when conjugating our identified motifs at 3’UTR of mRNA, enhanced level of mRNA in EVs was observed. Importantly, increased functional protein level was also found in cells taken up these EVs. Furthermore, in non-small cancer patient serum, we found that NPM1 is highly expressed in serum EVs, also the level of EGFR mRNA in EVs, in agreement with our observation from EVs isolated from cell culture.

Summary: In conclusion, our findings that NPM1 is a binding protein for EVs RNA, which selectively transports mRNA, not only help elucidate the mechanism for mRNA sorting into EVs but also offer hints for the development of specific mRNA transport tools using EVs. These findings also offer clinical evidence supporting the association of NPM1 with cancerous mRNA shuttling via EVs.

Scientist Tong Zhao¹, Associate Scientist Wei Zhao¹, Associate Researcher Shengya Xu¹, Associate Researcher Moxuan Yang¹

¹TheraXyte Bioscience, Beijing, China

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) are naturally derived from all types of cells and have broadly categorized into ectosomes and exosomes according to the different plasma membrane budding orientations. EVs provide numerous advantages as drug carriers, however, large-scale manufacturing EVs is challenging due to the limited yield in cells. Multiple Glycosylphosphotidylinositol-anchored proteins (GPI-APs) are organized into cholesterol-dependent nanoscale clusters at the plasma membrane and delivered to endosomes via the CLIC/GEEC pathway. Notably, endocytosis of the cells is critical for the biogenesis of EVs, especially for exosomes. In our study, two GPI-APs, CD55 and CD59 can increase the EVs yields in HEK293 cells significantly. Moreover, this function is highly regulated by the GPI-anchor part, which is confirmed by various truncations and engineering.

Methods: HEK293 cells were transfected with plasmids and stably expressed target proteins after selection with antibiotics such as puromycin/hygromycin. Cells were cultured in DMEM containing 10% EV-depleted FBS for adherent culturing or in a serum-free medium for suspension culturing. The culture supernatant was treated with the 0.2 um filter after removing the cells and cell-debris. EVs were then isolated by ultra-centrifugation at 100,000 g for 85 min, followed by an SEC column. The particle number and size distribution were detected with both NTA (ZetaView) and nanoFCM (ApogeeFlow). The EV biomarkers such as ALIX, TSG101, CD9, CD81, and CD63 were detected with Western Blot or nanoFCM. The morphology of EVs was obtained by TEM or CyroEM.

Results: We initially performed the proteomics analysis, comparing EV with its cell counterpart, and found multiple GPI-APs were sorted into EVs such as CD55 and CD59. After overexpression of CD55 or CD59, HEK293 cells increased its EVs production significantly. Various truncations of CD55 also can boost EVs production except for the GPI-anchor deficiency. Furthermore, mCherry fusion with CD55 GPI-anchor still increased the EVs yield. More importantly, mCherry can be loaded into EVs with high enrichment, which reveals the great potential of programmable engineering EVs for drug loading and targeting.

Summary: Here we found that GPI-anchor can be the booster of EVs production, and simultaneously used as a scaffold for drug loading and targeting.

Dr Robert Mitchell¹, Mr Andrew Parnell¹, Dr Ben Mellows¹, Professor Ketan Patel¹, Dr Steve Ray¹

¹Micregen Limited, Reading, United Kingdom

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction

Micregen, a UK-based biotech company investigating the regenerative capabilities of stem cell paracrine factors has developed a platform technology to manipulate stem cells to produce secretome products for use in regenerative medicine.

Here we characterise an amniotic fluid-derived stem cell secretome (AFSC CM). This secretome has been quantified to contain over 2,000 components: miRNA, cytokines, anti-inflammatory factors and proteins including growth factors and a major extracellular vesicle (EV) component.

This study investigated the AFSC CM immunomodulatory effects. We demonstrate the EV component to be uptaken by various cell types at a cellular and organ-wide level and demonstrate its role in retaining neuronal plasticity in an Alzheimer's disease model.

Methods

EVs were characterised in accordance with published ISEV guidelines, size measured via TEM and NTA and purity via western blot. Immunomodulatory effects of AFSC CM were measured in vitro via ELISA and luciferase activity and the quantification of inflammatory cytokines in vivo via a Luminex panel.

EV biodistribution was quantified by fluorescent staining of isolated EV and detected via 3D confocal imaging in vitro and In Vivo Imaging System (IVIS) in vivo.

Neuronal efficacy was measured using a murine model of Alzheimer's, quantifying inflammatory markers via immunohistochemistry and behavioural effects via novel object recognition.

Results

AFSC CM improved endogenous stem cell activity, reduced stress-induced senescence and pro-inflammatory signalling in vitro.

The secretome contained a major EV component and these were delivered systemically to a wide range of organs in vivo and taken up by different cell types in vitro.

Systemic immunomodulation was detected by the reduction of pro-inflammatory cytokines in two inflammatory in vivo systems. Neuronal plasticity was quantified in an Alzheimer's disease model that was linked to the recruitment of inflammatory astrocytes or microglia in the hippocampus.

Summary/Conclusions

A secretome generated from an amniotic fluid-derived stem cell (AFSC CM) under proprietary conditions improve multiple key cellular measures that have been associated with regeneration with immunomodulation a key pathway.

EVs within AFSC CM are a key component and can be delivered systemically, reaching and effecting inflammation in a variety of organs such as the gut and brain.

Mr. Manideep Pachva, Dr. Peter Ruzanov, Dr. Valentina Evdokimova, Dr. Melanie Rouleau, Dr. Laszlo Radvanyi, Dr. Poul Sorensen

Introductory Talk and Oral Session: OT05 Cancer Metastasis, Plenary 1, May 9, 2024, 4:00 PM - 5:35 PM

Ewing sarcoma(EwS) is a malignant bone cancer of childhood and adolescence, where 40% cases are present with metastasis. Our recent whole transcriptomic sequencing unveiled that EwS cells express RNA derived from various classes of repetitive elements of DNA, including but not limited to HSATs, HERVs, LINEs and SINEs. It was also unraveled that these repetitive RNAs are predominantly secreted in small bag like structures called extracellular vesicles (EVs). Interestingly, sequencing analysis revealed that the repetitive RNA secreted in EVs are highly A-to-I (Adenosine to Inosine) edited, compared to the cellular repetitive RNA. We hypothesized that this A-to-I editing is necessary for their secretion and subsequent actions in recipient tumor microenvironment cells.

We performed the knockdown of ADAR1 enzyme responsible for A-to-I editing using lentivirus based transduction of short-hairpin RNA. Later, small EVs were purified from EwS ADAR1 +/- cells using ultrafiltration followed by ultracentrifugation. Purified small EVs were analyzed for size and small EV markers by Nanoparticle tracking analysis and Immunoblotting analysis, respectively. RNA purified from EwS ADAR1 +/- cells and EVs was used for whole transcriptomic sequencing. Immunofluorescence, immunoblotting, ELISA and droplet digital PCR techniques were used to monitor various functional read outs from the recipient cells (monocytes and fibroblasts) treated with EwS ADAR1 +/- EVs.

ADAR1 knockdown in EwS cells caused significantly reduced secretion of various types of SINE-ALUs RNA in EVs. These various types of SINE-ALU RNA, including but not limited to AluSz, AluJo, Alujb, AluSx, are present in mRNAs of genes belonging to stress granule assembly, anti-viral immune response and NF-Kβ pathway. Notably, these SINE-ALUs are specifically edited by interferon induced isoform of ADAR1 (p150 isoform), whose expression is highly upregulated in Ewing tumors. Moreover, we noticed decreased anti-viral immune response in recipient cells treated with ADAR1 KD EVs, compared to the control EVs. We predict that this decrease in anti-viral immune response may be caused by the decreased levels of SINE-ALUs in EVs. However, further experiments are warranted to confirm this.

Overall, this study unravels a novel role for secreted A-to-I edited repetitive RNA, specifically SINE-ALU RNAs, in manipulating the tumor microenvironment.

Dr. Laurence Blavier-Sarte, Dr. Irina Matei, Dr. David Lyden, Professor Yves DeClerck

Introductory Talk and Oral Session: OT05 Cancer Metastasis, Plenary 1, May 9, 2024, 4:00 PM - 5:35 PM

Introduction: There is abundant evidence that tumor-derived extracellular vesicles (TEV) have an important contributory role in establishing the pre-metastatic niche (PMN). Here, we used two models of endogenous release and tracking of TEVs to examine their function in the formation of the PMN and its progression into a metastatic niche (MN).

Method: Human neuroblastoma (NB) and melanoma (MEL) cells engineered to release green fluorescent protein (GFP)-labeled TEVs were orthotopically implanted into immunodeficient mice and their capture by host cells in the PMN and MN was monitored over time. GFP+ TEV-capturing cells were isolated by flow cytometry and their transcriptome analyzed by TaqMan low density array and single cell RNA-sequencing (scRNA-Seq). TEVs purified by differential ultracentrifugation and size exclusion chromatography were incubated with mouse Kupffer cells (KC) and hepatic stellate cells (HSC) and proteins were examined by ELISA and Western blots.

Results: In the lung PMN (MEL model), scRNA-Seq analysis of TEV-capturing cells identified resident alveolar macrophages as the main population (78%), with small clusters of interstitial and recruited macrophages. In the lung MN, macrophages represented 43 % of TEV-capturing cells with the remainder made of endothelial cells (EC), alveolar epithelial cells, dendritic cells (DC) and myeloid-derived suppressive cells (MDSC). In the liver PMN (NB model), TEV-capture was documented in KC (31%) and capsular macrophages but also in hepatocytes, HSC, and sinusoidal EC. In the MN the proportion of KC decreased (24%) and new populations were identified including DC and MDSC. A gene expression analysis in TEV-capturing macrophages revealed a >10-fold increase in IL-18 expression (mRNA), confirmed by proteomics analysis. Consistently, we demonstrated cleavage of pro-caspase-1 and release of mature IL-1β and IL-18 in KC exposed to NB-TEV which was prevented by inflammasome inhibitors Bay-11-7082 and MCC950.

Conclusion: Our data demonstrate that TEV-capture in the PMN involves heterogeneous subpopulations of macrophages and non-macrophage cells which dynamically change as the PMN evolves to a MN. The data also points to activation of the inflammasome as an early event in the establishment of the PMN. Thus, our work provides new insights into the role of TEVs in the earliest stages of metastatic progression.

Dr Pamali Fonseka, Dr Sanjay Shahi, Prof Mark Febbraio, Prof Suresh Mathivanan

Introductory Talk and Oral Session: OT05 Cancer Metastasis, Plenary 1, May 9, 2024, 4:00 PM - 5:35 PM

Introduction

It has been well established that exercise is effective against tumour growth and spread of cancer. However, many cancer patients are unable to perform strenuous physical activities due to the severity of the disease. Hence, we intend to uncover the benefits of plasma extracellular vesicles (EVs) derived from mice that were subjected to exercise on breast cancer progression.

Methods

Plasma EVs were isolated using size exclusion chromatography coupled with ultracentrifugation from mice subjected to high-intensity interval training (HIIT) exercise and sedentary mice. Characterisation of plasma EVs were performed using nanoparticle tracking analysis, transmission electron microscopy and western blotting analysis. Effects of plasma EVs derived from exercise and sedentary mice on metastatic breast cancer were investigated using quantitative proteomic analysis, proliferation, migration and PI/Annexin apoptosis assays, qPCR, IVIS imaging, orthotopic mouse models and immune cell profiling.

Results

Subjection to physical activity increased the number of EVs that were present in the plasma EVs of exercise mice (excEVs) compared to sedentary mice (sedEVs). Proteomic analysis revealed that there were significant changes to the plasma EV proteomic profile when mice were subjected to exercise compared to sedentary mice. When treated with exeEVs, a panel of breast cancer cells showed a significant reduction in migratory ability compared to the sedEVs and PBS treated cells. In order to investigate the role of plasma EVs in vivo, orthotopic mice bearing metastatic breast cancer tumours were given plasma EVs intravenously. Interestingly, there was a significant attenuation in tumour burden and metastasis in the presence of excEVs compared to sedEVs. This observation was further confirmed by qPCR and IVIS imaging of metastatic sites. Importantly, plasma EVs modulated the immune cell profiling of these mice. Moreover, quantitative proteomic analysis of tumours derived from exeEVs treated mice showed upregulation of several proteins that are known to be involved in tumour growth and metastasis suppression compared to sedEVs treated tumours.

Conclusion

For the first time, we were able to show that plasma EVs derived from physically active mice could attenuate aggressive breast cancer tumour growth and metastasis.

Dr Lu Tian, Miss Jingyi Lu, Dr Karen Man-Fong Sze, Dr Goofy Yu-Man Tsui, Dr Daniel Wai-Hung Ho, Prof Irene Oi-Lin Ng

Introductory Talk and Oral Session: OT05 Cancer Metastasis, Plenary 1, May 9, 2024, 4:00 PM - 5:35 PM

Introduction: Small extracellular vesicles (sEVs) play critical roles in mediating intercellular communication during tumor progression in the microenvironment of the primary tumor as well as distant metastatic organs. Previous research has shown that Ral regulates the biogenesis of sEVs during multivesicular body formation in C. elegans and promotes breast to lung metastasis through the upregulation of CD146. In our previous study, we demonstrated that RalA enhances hepatocellular carcinoma (HCC) progression by promoting aggressive features of tumor cells. However, whether RalA regulates tumorigenesis in an sEV-dependent manner in HCC is unclear. Therefore, we aimed to investigate how RalA regulated sEV biogenesis and function in HCC.

Methods: We characterized sEVs from HCC cells by size, morphology, and markers using various technologies. The functions of recipient HCC cells and HUVEC cells upon sEV treatment were examined. In vivo, the role of sEVs in HCC metastasis were demonstrated using orthotopic liver injection and intrasplenic implantation models combined with sEVs injection. Besides, the protein composition of sEVs from RalA manipulated cells were profiled by mass spectrometry and the functions of targeted proteins were validated. Finally, phosphorylation microarrays were used to dissect the downstream signaling in the recipient HCC and HUVEC cells.

Results: We found that RalA upregulated the protein loading in sEVs. Functionally, upregulation of RalA in sEVs enhanced the migration and invasion in vitro and promoted intrahepatic and pulmonary metastasis in vivo. Furthermore, we identified that the surface-associated fibronectin (FN1) was regulated by RalA; blocking the interaction using specific antibody targeting its receptor HSPG, could abolish the promoting effect mediated by sEVs. Additionally, RalA also modulated the functions of sEVs in facilitating angiogenesis, with both receptors of FN1, HSPG and integrinα5β1, contributing to this enhancement. Further analysis using phosphorylation microarrays showed activation of AKT in recipient PLC/PRF/5 HCC cells upon RalA sEVs treatment.

Conclusions: RalA enhances HCC metastasis by upregulating the surface-associated protein FN1 in sEVs, leading to activation of p-AKT signaling in recipient HCC cells and promoting angiogenesis. Overall, our study provides novel insights into the role of RalA/FN1 axis in sEV-mediated tumor progression, highlighting its potential as a therapeutic target for HCC.

Dr Sai Vara Prasad Chitti¹, Mrs Akbar L Marzan¹, Professor Suresh Mathivanan¹

¹La Trobe Institute For Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia, Australia

Introductory Talk and Oral Session: OT05 Cancer Metastasis, Plenary 1, May 9, 2024, 4:00 PM - 5:35 PM

Introduction: Cachexia, characterized by progressive wasting of muscle and fat, is a major cause of mortality in cancer patients, but clinical options against cachexia remain limited due to the multifactorial nature of the disease. Several seminal studies demonstrated that tumour-cell-released small extracellular vesicles (sEVs) containing key cachexins are necessary and sufficient to induce muscle and fat loss. Furthermore, it is now well known that cancer cells secrete more sEVs compared to non-cancerous cells and interestingly, several proteins that are involved in the sEVs biogenesis and secretion are found to be upregulated in cachexia-causing tumours. Hence, we examined whether decreasing the secretion of sEVs from tumour cells can inhibit cancer-induced cachexia.

Methodology: Cortactin (Cttn) was knocked out (KO) using CRISPR/Cas9 technology in colon cancer cells. sEVs were isolated by differential ultracentrifugation and characterised by western blotting, TEM, and nanoparticle tracking analysis. Co-culture and pre-clinical studies were carried out to study the cachectic phenotype. Fluorescence-based high-throughput screening assay was performed to identify the drugs that decrease sEVs secretion.

Results: Loss of Cttn inhibited the release of sEVs. While C26 wild-type (WT) derived sEVs induced atrophy in myotubes and lipolysis in adipocytes, Cttn-KO sEVs did not induce atrophy or lipolysis. Proteomics analysis of sEVs highlighted the enrichment of cachectic proteins in WT sEVs compared to KO sEVs. Follow-up C26 mice pre-clinical studies highlighted that Cttn-KO tumour-bearing mice exhibited stable body weight, reduced tumour burden, and dramatically extended lifespan compared to mice bearing WT tumour. Remarkably, Cttn-KO prevented tumour-induced loss of muscle, fat, and other major organs. Consistent with this, overexpression of Cttn increased sEVs secretion and drastically decreased the lifespan of C26 mice by accelerating tumour-induced weight loss. To use these findings for therapeutic benefit, we screened the library of FDA-approved drugs and identified several drugs that block the release of sEVs. Administration of sEVs inhibitor to the cachexic mice resulted in the abolishment of cancer-associated cachexia and prolonged survival.

Summary/Conclusion: Overall, these findings indicate that decreasing sEVs release from tumours might be a promising approach to treat cancer cachexia, improve quality of life, and extend the lifespan of cancer patients.

Dr. Mohammad Farouq Sharifpour, Dr. Suchandan Sikder, Dr. Yide Wong, Dr. Na'ama Koifman, Dr. Matthias Floetenmeyer, Dr. Robert Courtney, Prof. Jamie Seymour, Prof. Alex Loukas

Introductory and Oral Session: OT06 Diverse Sources of EV Therapies, Eureka, May 9, 2024, 4:00 PM - 5:35 PM

Introduction

Recognized as a superfood microalga, Spirulina stands out as an exceptional cyanobacterium, famous for its many documented health benefits. Aside from its inherent nutritional and therapeutic advantages, Spirulina is increasingly gaining recognition for its untapped potential in biomanufacturing, including the production of biofuels and pharmaceuticals. Despite its acknowledged virtues, our understanding of extracellular vesicles (EVs) in cyanobacteria, including Spirulina, lags behind that of other bacterial counterparts. In an effort to bridge this gap, we successfully isolated, characterised and visualised EVs from Spirulina for the first time. Also, we explored the potential therapeutic benefits of Spirulina EVs (SPEVs) using a mouse model.

Methods

SPEVs were meticulously isolated through a dual approach involving ultracentrifugation and size exclusion chromatography. The EV size distribution and concentration were measured using nanoparticle tracking analysis and tuneable resistive pulse sensing. Purified SPEVs were visualised by TEM and CryoTEM. High-resolution liquid chromatography-mass spectrometry was performed on SPEVs utilising both gel-based and in-solution techniques for sample processing. Building on previous research indicating the immunomodulatory potential of EVs derived from other cyanobacterium, we conducted several in vivo experiments by intraperitoneally injecting SPEVs into mice to evaluate the short-term and long-term immune responses. Additionally, we explored the potential adjuvanticity properties of SPEVs.

Results

We purified a total of approximately 10^12 particles from a 2.25 L culture with an OD680 of 1.3. TEM and CryoTEM analyses revealed the presence of pleomorphic outer-membrane-vesicles (OMVs) and outer-inner-membrane-vesicles (OIMVs) displaying diverse shapes, sizes, and coronation densities. In vivo immune response studies demonstrated a significant increase in neutrophils and M1 macrophages at the injection site following the intraperitoneal injection of SPEVs into mice, indicating a pro-inflammatory effect induced by SPEVs. This finding prompted an additional experiment to investigate the potential adjuvanticity properties of SPEVs. Intriguingly, we observed that SPEVs significantly enhanced the specific IgG response in mice (by over 100-fold) to a model vaccine antigen, Schistosoma TSP2, compared to the antigen administered with EVs from oranges or without SPEVs. Mass spectrometry identified a total of 54 proteins derived from Spirulina EVs, with the top-scored hit belonging to the bacterial porin protein superfamily.

Dr. Piyushkumar Gondaliya, Mr. Adil Ali Sayyed, Julia Driscoll, Irene K Yan, Dr. Tushar Patel

Introductory and Oral Session: OT06 Diverse Sources of EV Therapies, Eureka, May 9, 2024, 4:00 PM - 5:35 PM

Introduction: Although the immune checkpoint PD-L1 has emerged as a key target for cancer immunotherapy, the use of antibodies targeting PD-L1 for cancers such as cholangiocarcinoma (CCA) are limited by poor tumor penetration and systemic toxicity. We have developed an RNA nano-immunotherapeutic approach that uses milk-derived nanovesicles (MNVs) for tumor-cell targeted delivery of PD-L1 siRNA or Cas9/ribonucleoprotein complexes. This strategy aims to improve treatment of cholangiocarcinoma by enhancing the anti-tumor immune response.

Method: we engineered EpCAM aptamer-decorated MNVs for targeted delivery of PD-L1 siRNA (siRNA-tMNVs) or Cas9/ribonucleoproteins (RNP-tMNVs) to achieve targeted PD-L1 silencing in CCA cells. The immunomodulatory effects of these nanovesicles were evaluated in 2D culture, multicellular 3D spheroids, and syngenic mouse model. In the syngenic mouse model, the use of targeted therapy was also evaluated in combination with gemcitabine therapy.

Results: The siRNA-tMNVs and RNP-tMNVs were efficiently taken up by CCA tumor cells and led to a significant reduction in PD-L1 expression. In vitro studies demonstrated the potential of siRNA-tMNVs to enhance T cell and NK cell degranulation, cytotoxicity, and cytokine production when co-cultured with tumor cells. RNP-tMNVs exhibited proficient PD-L1 gene editing capabilities but required higher and repeated dosing. In multicellular CCA tumor spheroids, siRNA-tMNVs reduced tumor cell cytotoxicity when combined with T cells or NK cells. Moreover, the combination of siRNA-tMNVs with gemcitabine substantially reduced tumor burden in an immunocompetent orthotopic tumor model. The combination therapy further increased the infiltration of CD4+, CD8+ T cells, and NK cells compared to control groups, indicating a potentiated anti-tumor immune response.

Summary/Conclusion: The targeted delivery of siRNA-tMNVs and RNP-tMNVs via nanovesicles represents a promising advance in cancer immunotherapy. Our findings show that this approach can boost anti-tumor immune responses and reduce tumor burden, opening up new avenues for effective tumor treatment strategies. Further development of this nanovesicle based RNA nano-immunotherapeutic approach could have a significant impact as a novel modality for cancer treatment.

Postdoctoral Scholar Tatiana Cuellar-Gaviria, Maria Rincon-Benavides, Hatice Topsakal, Ana Salazar-Puerta, Mia Kordowski, Pranav Rana, Orlando Combita-Heredia, Daniel Wozniak, Daniel Gallego-Perez, Natalia Higuita-Castro

Introductory and Oral Session: OT06 Diverse Sources of EV Therapies, Eureka, May 9, 2024, 4:00 PM - 5:35 PM

Introduction: The rise of antibiotic-resistant bacteria is poised to evolve into a significant worldwide health emergency, underscoring the urgency for innovative treatment approaches. Methicillin-resistant Staphylococcus aureus (MRSA) infections are often acquired after invasive medical procedures and are difficult to treat. In this work, we report a nanotechnology-based approach to generate engineered extracellular vesicles (eEVs) loaded with an antimicrobial peptide that mediates the disruption of MRSA-biofilm formation in a murine wound model.

Methods: The antimicrobial peptide LL-37 was used as model cargo. eEVs loaded with LL-37 were generated by electroporation of mouse primary alveolar epithelial cells (mPAEC) with a plasmid coding for LL-37. eEVs were isolated from the transfected cells by size exclusion chromatography and concentrated using VivaSpin concentrator. eEV were characterized via Western blot, cryo-EM and Nanosight and loading efficiency via qRT-PCR and ELISA. Naïve mPAEC were exposed to 3x103 EVs/cell to evaluate the dynamics of eEV uptake and ability to transfer molecular cargo. In vivo evaluations involved the use of tissue-nanotransfection to transfect a LL-37-plasmid into the wound of a murine wound-model infected with MRSA. The impact on bacterial load was assessed after 4-days of daily treatment.

Results: In vitro evaluation showed successful transfection of the LL-37-plasmid into mPAECs. The isolated eEVs had an average size of 144 nm and showed positive expression of EV markers CD63, TSG101 and β-tubulin, and a reduced expression of calnexin. eEVs were effectively packed with LL-37, as wells as transcripts coding for this antimicrobial peptide when compared to the Sham controls EVs. Also, we found that eEVs were readily taken-up by naïve cells and were able to induce upregulation of the LL-37 gene in the recipient cells. In vivo studies showed a significant decrease in the bacterial load in mice tissue-nanotransfected with the LL-37-plasmid. Current studies include a more comprehensive evaluation to determine the role of the eEVs released by the transfected tissue in mediating a paracrine transport of the peptide.

Conclusion: Antimicrobial peptides and their transcripts can be packed within eEVs and are efficiently delivered into recipient cells. This type of antimicrobial eEVs could potentially be harnessed for future therapeutic applications.

Xin Lu, Qing Han, Professor Weiliang Xia¹

¹Shanghai Jiao Tong University, Shanghai, China

Introductory and Oral Session: OT06 Diverse Sources of EV Therapies, Eureka, May 9, 2024, 4:00 PM - 5:35 PM

Introduction: Plant-derived exosome-like nanovesicles (PELNs) are a class of extracellular vesicles characterized by low cost, easy accessibility, and low immunogenicity. Celery exosome-like nanovesicles (CELNs), as one of the PELNs, exhibit many excellent properties.

Methods: The methods used include CELNs isolation, characterization and chemo drug loading; in vitro cell experiments such as co-culture, Western Blot, and flow cytometry; and in vivo experiments using nude mice tumor-bearing model and C57 tumor-bearing mice subcutaneously injected with LLC cells, observing the survival rate, body weight, tumor size, cell population analysis, immunohistochemistry, and spectral live imaging system.

Results: CELNs exhibited high cellular uptake, and their diacylglycerol (DG) content may be a key factor in promoting cellular uptake; they also showed good performance in terms of stability, safety and drug loading capacity. To study the drug delivery potential of CELNs using lung cancer as a model, it was found that chemotherapeutic drugs encapsulated by CELNs could be more efficiently aggregated at the tumor site compared to free drugs. Experimental results of drug delivery to tumor-bearing nude mice showed that CELNs loaded with doxorubicin (DOX) not only reduced DOX cardiotoxicity, but also inhibited tumor growth more effectively compared with model drug carrier liposomes loaded with DOX. CELNs themselves can show therapeutic potential as nanomedicines. In immunocompetent Lewis lung cancer loaded mice experiments, CELNs inhibited the proliferation of lung cancer cells by inhibiting the expression of PD-L1 in tumor cells, activating tumor-infiltrating CD8+ T cells, and promoting the secretion of GzmB, TNFα, and IFNγ. Finally, treatment using CELNs loaded with the chemotherapeutic drug paclitaxel (PTX) was a more effective therapeutic combination than treatment with either CELNs or PTX alone in the Lewis lung carcinoma-loaded mice described above.

Summary: CELNs, as a plant-derived nanomaterial, exhibit superior properties. CELNs can not only be used as a drug delivery platform to improve the efficacy of drug delivery, but also possess the potential to directly inhibit tumor growth as a nanomedicine. CELNs loaded with chemotherapeutic drugs can achieve a synergistic effect between their immunotherapeutic effect and chemotherapeutic drug delivery through a single administration, providing a more effective regimen for cancer treatment.

Doctor Xinxing Du, Doctor Huan Chen, Doctor Cong Hu, Doctor Yanhao Dong, Doctor Xinrui Wu, Doctor Jinyao Liu, Doctor Liang Dong, Doctor Wei Xue

Introductory and Oral Session: OT06 Diverse Sources of EV Therapies, Eureka, May 9, 2024, 4:00 PM - 5:35 PM

Introduction:

Immunotherapy stands as a significant milestone in modern cancer treatment, demonstrating remarkable effectiveness across various cancer types. Nonetheless, the immunosuppressive tumor microenvironment in prostate cancer, characterized by a deficiency of anti-tumor immune cells, remains a critical problem limiting the efficacy of immunotherapy. The approach of turning the “cold” tumor to “hot”, in other words, recruiting immune cells within the tumor microenvironment and activating anti-tumor immune responses offer potential solutions to enhance the effectiveness of the current immunotherapy.

Methods:

Bacteria-derived outer membrane vesicles (OMVs), known for their high immunogenicity, were subjected to FeS functional modifications to create a novel nanodrug. Through a series of in vivo and in vitro experiments, we verified its capability to activate the tumor immune microenvironment and demonstrated its direct cytotoxic effects on tumor cells.

Results:

We successfully engineered a novel nanodrug using FeS-functionalized OMVs. Our experiments, conducted both in vivo and in vitro, demonstrated that this nanodrug has the capacity to induce a significant infiltration of immune cells, particularly CD8+ T cells. Additionally, this nanodrug can release substantial quantities of ferrous ions and hydrogen sulfide within the acidic tumor microenvironment. The released ferrous ions can trigger iron-dependent cell death in tumor cells, while hydrogen sulfide can indirectly promote iron-dependent cell death in tumor cells by inhibiting hydrogen peroxidase and reducing the tumor cell's antioxidant capabilities. Iron-dependent cell death can further activate anti-tumor immune responses through various mechanisms. In mouse model, the combination of this nanodrug and immune checkpoint inhibitor displayed superior anti-tumor effects without exhibiting significant biotoxicity.

Conclusion:

This study has successfully developed a novel nanodrug capable of reshaping the immune microenvironment of prostate cancer, thereby enhancing the effectiveness of immunotherapy and directly targeting and killing cancer cells. Our findings propose an ideal and safe strategy for the treatment of advanced prostate cancer, providing a robust theoretical and practical foundation for potential clinical applications.

Dr. Yunyue Zhang

Introductory and Oral Session: OT06 Diverse Sources of EV Therapies, Eureka, May 9, 2024, 4:00 PM - 5:35 PM

Introduction: RNA interference, facilitated by small interfering RNA (siRNA), is a powerful therapeutic strategy. However, the oral administration of siRNA is currently not possible due to their instability in gastrointestinal tract and poor permeability across the biological barriers of intestinal mucosa. Milk extracellular vesicles (mEVs) possess desirable properties to serve as potential carriers for oral delivery of siRNA based on their safety, stability, and capability to permeate across the intestinal epithelium. However, a major challenge hindering the clinical development of mEVs for siRNA delivery is the current absence of non-destructive methods for efficient loading of siRNA.

Methods: Hybrid vesicles (also termed ‘hybridosomes’) fused by mEVs with cationic liposomes were fabricated in this study to combine the bioactivities of mEVs with high siRNA loading efficiency of liposomes. Hybridosomes were engineered by polyethylene glycol (PEG)-mediated and freeze-thaw fusion, and their intestinal stability, cytotoxicity and permeability through intestinal epithelium were assessed. Additionally, gene silencing capability of these systems were investigated in various in vitro models.

Results: Hybridosomes, designed as optimal oral delivery systems with a size range of 180-230 nm, demonstrated efficient loading of siRNA cargo. They exhibited significantly lower cytotoxicity and superior stability in a fed-state simulated intestinal fluid compared to cationic liposomes. Furthermore, hybridosomes significantly increased the transport of siRNA across in vitro intestinal epithelial model, and hybridosomes loaded with GAPDH siRNA successfully induced gene silencing in J774A.1 macrophages. Importantly, anti-TNFα siRNA loaded-hybridosomes and mEVs were both able to downregulate TNFα levels and relieve inflammation in an in vitro co-culture model of intestinal inflammation.

Conclusion: This work demonstrates that mEVs-mediated hybridosomes can act as safe and reliable systems for oral delivery of siRNA therapies.

Mingzhen Zhong, PhD Weilun Pan, Professor Lei Zheng

Introductory and Oral Session: OT06 Diverse Sources of EV Therapies, Eureka, May 9, 2024, 4:00 PM - 5:35 PM

Atopic dermatitis (AD) is a chronic inflammatory skin disease that weakens the skin barrier function, rendering it more vulnerable to external irritants and oxidative stress. Plant-derived extracellular vesicles (PDEV) have emerged as promising natural therapeutic agents for the management of various mammalian diseases. In the study, we investigated the relationship between AD treatment outcomes and skin barrier integrity by analyzing tissue samples obtained from AD patients. The results indicated that restoring the impaired skin barrier can effectively improve the prognosis of AD treatment.

Through metabolomic and drug targeting analyses, we found that extracellular vesicles derived from turmeric (TEV) have antioxidant, anti-apoptotic, and antimicrobial properties targeting keratinocytes. Building on this discovery, we developed a tissue-adhesive hydrogel formulation by encapsulating TEV within a polyphenol-based hydrogel cross-linked using Fe3+ ions and hyaluronic acid-graft-dopamine (HD). The resulting hydrogel, Fe-HD@TEV hydrogel, demonstrates a synergistic effect in restoring the skin barrier in AD by maintaining redox balance and enhancing cellular functions.

Our antioxidant assays displayed that Fe-HD@TEV hydrogel effectively scavenges excessive reactive nitrogen species (RNS) and reactive oxygen species (ROS) in the affected area. Transcriptomic and immunofluorescence analyses further elucidated that Fe-HD@TEV hydrogel improves the activities of various antioxidant enzymes, upregulates the expression of barrier proteins and antimicrobial peptides (AMPs), and accelerates keratinocyte proliferation, thereby providing a comprehensive reparative effect on the skin barrier. In experimental AD models, the Fe-HD@TEV hydrogel exhibited promising therapeutic efficacy by improving skin hydration, reducing epidermal thickness, regulating skin immunity, and enhancing cellular functions.

These findings highlight the potential of utilizing plant-derived vesicles and polyphenol hydrogel as a novel therapeutic dressing for repairing the skin barrier in AD.

Miss Nidhi Seegobin¹, Miss Marissa Taub¹, Dr Atheer Awad^1,2, Dr Sudax Murdan¹, Prof Abdul Basit¹

¹University College London, London, United Kingdom, ²University of Hertfordshire, Hatfield, United Kingdom

Introductory and Oral Session: OT06 Diverse Sources of EV Therapies, Eureka, May 9, 2024, 4:00 PM - 5:35 PM

Introduction

Inflammatory bowel disease (IBD) affects over 6.8 million people worldwide and is associated with poor clinical outcomes and considerable side effects. EVs can be delivered locally and also have therapeutic effects, thereby enabling a synergistic therapeutic treatment in colitis. The oral delivery of bovine milk derived EVs has demonstrated therapeutic effects in colitis mouse models, including modulation of the gut microbiota, reduction in T lymphocyte infiltration and reduction in gut inflammation. However, the instability of EVs in the upper gastrointestinal (GI) tract and stomach warrants investigation into an additional EV delivery method via the rectum. Here, we propose to deliver bovine milk derived EVs by rectal enema compared to oral gavage in a colitis mouse model to assess if there are enhanced therapeutics effects related to the improved stability by rectal delivery.

Methods

Bovine milk derived EVs were chelated and isolated by ultracentrifugation (Optima L-90K, Beckman Coulter) and size exclusion chromatography (Izon qEV Gen2, 35 nm pore size). Over seven days, DSS C57BL/6 mice received 1mg/day of bovine milk derived EVs by oral gavage or rectal enema. Animal weight and disease activity scoring was monitored daily. At necropsy, the colon and spleen were sampled for macroscopic analysis and for quantification of inflammatory markers.

Results

No significant change in body weight variation, disease activity index, colon and spleen weight or colon length was observed for both oral and rectal administration between the negative control and EV arms of the study. There were also no significant changes in the expression of TNF-alpha, interleukin-1beta, intrinsic nitric oxide or myeloperoxidase.

Conclusion

Bovine-milk derived EVs do not demonstrate therapeutic properties for the treatment of colitis in DSS induced C57BL/6 mice by oral gavage or rectal enema. These findings do not impact their use as a therapeutic drug carrier.

Dr Vincent Hyenne, Dr Kuang-Jin Huang, Dr Jacky G. Goetz

Introductory and Oral Session: OT07 Mechanisms of Biogenesis, Room 105-106, May 9, 2024, 4:00 PM - 5:35 PM

1) Introduction

Exosome secretion is a multis-step process including endosome maturation into multi-vesicular body (MVB), intra-luminal vesicle (ILV) budding, cargo loading, MVB transport to the cell periphery and fusion with the plasma membrane. We have previously shown that the GTPases from the Ral family control MVB maturation and exosome secretion in C. elegans and in mammalian cells (Hyenne et al. JCB 2015). In addition, we demonstrated that the mammalian paralogs RalA and RalB control the levels as well as the protein and RNA content of extracellular vesicles (EVs) secreted by a mouse mammary cancer cell line and thereby their pro-metastatic function in vivo (Ghoroghi et al. eLife 2021). Here, we are investigating the molecular and cellular mechanisms by which Ral drive exosome secretion.

2) Methods

We characterized the proximal interactome of both RalA and RalB using BioID. Among the 400 proteins interacting with either GTPase, we selected proteins related to endosomes and/or also identified in isolated EVs. Subsequently, we confirmed the interactions between Ral and 10 candidates by co-immunoprecipitation. We finally picked EHD4, a dynamin related ATPase, known for its role in membrane tubulation and endosome trafficking for further study.

3) Results

Using a CRISPR knock-out, we demonstrated that EHD4 is required for exosome secretion in mouse mammary carcinoma cells. This phenotype can be rescued by re-expression of or by its close paralog EHD1. We exploited high-pressure freezing volume electron microscopy (using Focus Ion Bim Scanning Electron Microscopy) to examine endosome maturation and found that EHD4 controls the the size of MVBs, similarly to Ral GTPases. We are currently investigating the interactions between Ral and EHD4 and their consequences on MVB maturation and trafficking using fluorescence in vivo microscopy. In parallel and to address the impact of EHD4 on the function of tumor EVs, we performed experimental mammary tumor growth analysis in a syngenic mouse model and observed that EHD4 drastically potentiates tumor growth.

4) Summary/Conclusion

The membrane remodeler EHD4 modulates exosome secretion by controlling MVB maturation, most likely through interactions with Ral GTPases.

Professor Suresh Mathivanan¹

¹La Trobe University, Melbourne, Australia

Introductory and Oral Session: OT07 Mechanisms of Biogenesis, Room 105-106, May 9, 2024, 4:00 PM - 5:35 PM

Introduction:

CRISPR-Cas9 is a widely utilised genome editing technique that has provided an efficient means to study the function of genes in various cells and tissues. Despite this utility, there is currently a limited understanding of the impact of Cas9 expression in mammalian cells, particularly in the biogenesis and secretion of small extracellular vesicles (sEVs). Here, we investigated the changes in the protein cargo and secretion of sEVs upon expression of SpCas9 in human colorectal carcinoma HCT116 and human embryonic kidney HEK293 cells.

Methods:

A panel of cells with varying P53 status were utilised. sEVs were isolated by ultracentrifugation and characterised by Western blotting, EM, NTA and quantitative proteomics. Human and murine cancer cells releasing EVs were implanted in immune-compromised (nude) and immune-competent (C57BL/6) mice, respectively.

Results:

Expression of Cas9 impeded the proliferation of HCT116 cells and increased the abundance of p53 and its downstream target p21. Cas9 expression also increased the abundance of EV-enriched proteins CD63 and CD9 in HCT116 cells and further accelerated the secretion of sEVs. Subsequent label-free quantitative proteomics analysis of whole cell lysates highlighted small but noticeable alterations in the abundance of the few proteins upon expression of Cas9. In contrast, proteomic analysis of sEVs revealed a cell type-dependent differential abundance of several proteins. Knockout of P53 impacted the phenotype of sEVs.

Conclusion:

Overall, the study highlighted the cell-type dependent alterations in the protein cargo and secretion of sEVs upon expression of Cas9.

Dr. Yusuke Yoshioka, Dr. Akira Yokoi, Prof. Takahiro Ochiya

Introductory and Oral Session: OT07 Mechanisms of Biogenesis, Room 105-106, May 9, 2024, 4:00 PM - 5:35 PM

Introduction: Cancer-related EVs, with their ability to act within and distally within the tumor microenvironment, play a major role in tumor progression and metastasis. For example, we found that EVs derived from highly metastatic ovarian cancer cells promote peritoneal dissemination in vivo (Yokoi A et al., Nat Commun, 2017). Therefore, inhibition of EV secretion from cancer cells can be a novel therapeutic tool to inhibit cancer metastasis. This study focused on screening small-molecule inhibitors for EV secretion in ovarian cancer cells. Methods: We used an original screening system based on ExoScreen assay for monitoring CD9-positive EV secretion (Yoshioka Y et al., Nat Commun, 2014). After screening, we used ExoView to measure EV secretion to validate our screening results, and we measured the number of particles of purified EVs by nanoparticle tracking analysis (NanoSight). This screening system and a chemical compound library containing 1271 small molecules identified inhibitors for EV secretion in the ovarian cancer cell line ES-2. Results: Based on the first screening result, 45 small molecules were selected as putative inhibitors for EV secretion. These small molecules were further validated by ExoScreen. As a result of the validation, eight small molecules were found to inhibit EV secretion in ES-2 cells. To confirm the screening result, four cell lines, including two non-cancer cell lines, were treated with these eight molecules, and EV secretion was measured by ExoScreen. Three molecules inhibited EV secretion in a cancer cell-specific manner. Finally, we selected two small molecules as candidate molecules for inhibitors of EV-secretion by ExoView and nanoparticle tracking analysis. To understand the mechanisms of these molecules to inhibit EV secretion, we added these two small molecules to ES-2 cells and performed global gene-expression analysis using a proteomic approach. From the results of proteome analysis, several genes were selected for detailed analysis, and it became clear that two genes could be involved in EV secretion. Conclusion: Here, we identify inhibitors for EV secretion in ovarian cancer cells. Based on these results, we are now analyzing the effects of these molecules on therapeutic effects using a mouse model.

Phd André Cronemberger Andrade, Sarah Razafindrakoto, Lea Jabbour, Florence Gazeau, Amanda Silva Brun

Introductory and Oral Session: OT07 Mechanisms of Biogenesis, Room 105-106, May 9, 2024, 4:00 PM - 5:35 PM

Introduction: Improvement of protocols and methods for therapeutic extracellular vesicles EVs production in bioreactors isolation is essential to meet GMP-aligned quality and quantity. We obtained EVs from adipose stem cells (ASC) that can support the regeneration of injured tissues via several paracrine factors. To improve EV release for therapeutics purposes ASCs were exposed to mechanical stimulation (turbulence) in bioreactor. In our previous studies, we showed that turbulence increases EV yield. Here we investigated which mechanisms are responsible for increase of EV production under turbulence protocol.

Methods: ASC-EVs were produced in bioreactors under turbulence for 4 hours and isolated by tangential flow filtration (TFF). Several pathways are involved in EV secretion like autophagy and lysosomal maturation (Vacuolin-1/PIKfyve), ER-Golgi protein and membrane trafficking (Exo1 and Brefeldin A/ARF1) and mechanical signal (GsMTx4/Piezo1). The cultures were treated with inhibitors: Vacuolin-1, Brefeldin A, Exo1, GsMTx4 to access which pathway is affected under turbulence. EVs were quantified and characterized by nanoparticle tracking analysis (NTA), Western blot and nanoFCM using EV markers. The EV uptake was investigated by immunofluorescence. EV protein cargo and profile were analyzed by proteomics.

Results: After mechanical stimulation of ASC culture in bioreactor the highest inhibition of EV production was with observed with GsMTx4 (53%) treatment followed by Exo1 (36%), Brefeldin A (22%) and Vacuolin-1 (21%). All inhibitors had no impact on cell viability. Proteomics analysis showed an increase of protein expression of Piezo1 in EV samples after mechanical stimulation compare to no stimulated conditions.

Summary/Conclusion: We conclude mechanical sensitive channels (Piezo1) are associated with EV release in ASC culture under turbulence. Stimulation of Piezo1 can improve EV production from large-scale cultures using bioreactors. This new strategy can be applied to safely boost EV yeld for therapeutic purposes.

Professor Sharad Kumar, Dr Ammara Farooq, Dr Natalie Foot, Dr Yoon Lim

Introductory and Oral Session: OT07 Mechanisms of Biogenesis, Room 105-106, May 9, 2024, 4:00 PM - 5:35 PM

Introduction

Ubiquitination is an essential process required for protein turnover, intracellular trafficking, endocytosis, autophagy, and the egress of some viruses. Ubiquitination of membrane proteins serves as a signal to control their recycling and degradation via the endosomal pathway. As the endo-lysosomal compartment and the plasma membrane are sites of EV biogenesis, we have been studying the role of ubiquitination in EV secretion. Recent studies suggest that some members of the Nedd4 family of ubiquitin ligases (E3s) and their adaptors, including Ndfip1, Arrdc1 and Arrdc4, play a role in EV biogenesis (e.g. CDD 2021,97:253-81; JEV 2021,10: e12113: JEV 2022,11:e12188). For example, Nedd4 E3s and Ndfip1 mediate ubiquitination-dependent regulation of iron transporter (DMT1) both via the endocytic and EV-dependent release. Here we investigate the role of specific signals that regulate ubiquitin-dependent EV biogenesis.

Methods

We use standard molecular, biochemical, MS, proteomics, cellular and gene knockout (KO) or knockdown (KD) approaches to study the role of ubiquitination in EV release. EV isolation and characterisation involved differential centrifugation, immunoblotting, TEM and NTA, essentially following MISEV2023 guidelines.

Results

The KO of Nedd4-2 (an E3), Arrdc1 and Arrdc4 in mice, or ablation of these genes by KO/KD in cultured cells, led to significantly reduced EV biogenesis. The Arrdc4 KO mice showed a sperm maturation defect that was linked to reduced EV release. To understand the mechanism of Arrdc4-dependent EV release we identified ubiquitinated residues by MS and discovered that ubiquitination at K270 residue is critical for Arrdc4 function in EV biogenesis. K270 was found to be ubiquitinated with K29 Ub chains by Nedd4-2, thus uncovering a novel function of K29 ubiquitination in EV biogenesis. To further investigate the novel role of K29 Ub chains we utilised TRABID, a deubiquitinase (DUB) for K29/K33 Ub chains. Interestingly, TRABID overexpression not only inhibited Arrdc4-dependent EV biogenesis, but also affected overall EV release, suggesting that K29/K33 Ub chains play a critical role in regulating EV biogenesis.

Summary/Conclusions

Our results identify a novel role of specific ubiquitin chains in regulating EV biogenesis. We propose that inhibition of specific E3 ligases and DUBs as a new approach to control EV biogenesis.

Dr Andrew Lai, Dr Dominic Guanzon, Dr Carlos Palma, Dr Flavio Carrion, Dr Ryan Cohen, Prof Andreas Obermair, Prof Andreas Moller, Prof Carlos Salomon

Introductory Talk and Oral Session: OT08 Biomarker Technologies, Room 109-110, May 9, 2024, 4:00 PM - 5:35 PM

Introduction: Whilst considerable interest in using Extracellular Vesicles (EV) as biomarkers for a wide range of diseases exists, progress in the field is hindered by a lack of development of compatibles EV isolation methods with routine pathology laboratories Here, we introduced an automated method for isolating EV using high-performance liquid chromatography (HPLC) combined with either CaptoCore700 or size-exclusion columns.

Methods: We employed a modular HPLC system with a fraction collector, utilizing CaptoCore700 or size-exclusion columns. Fetal bovine serum (FBS) was used to optimize the system, and we characterized the EV fractions. To assess system reproducibility, we used triplicates of three biological samples, conducting analyses of concentration, size distribution, total RNA, miRNA sequencing, and quantitative proteomics. Finally, we evaluated the system's clinical utility in a group of 140 individuals with ovarian, colorectal, endometrial, lung, or breast cancers. Machine learning algorithms were developed using EV miRNA and protein profiles to accurately classify patients for each condition. All procedures adhered to Good Laboratory Practice principles following ISO17025 guidelines and were accredited by the National Association of Testing Authorities (NATA), Australia.

Results: Our method demonstrated high reproducibility in isolating EVs from human plasma, with an average inter-CV below 10% across technical replicates (particle mean: 3.75%, mode: 7.68%, protein concentration: 8.45%). Characterization of EVs revealed vesicles enriched with CD63, CD81, and CD9, free from lipoprotein contamination, and displaying circular morphology observed through cryo-electron microscopy. The enrichment of isolated vesicles aligned with current techniques and was suitable for next-generation sequencing (NGS) and proteomics. Principal Component Analysis (PCA) on proteomic and miRNA data facilitated the identification of cancer-specific plasma EVs. The miRNAs and proteins within EVs were effectively employed in a classification model, resulting in distinct multi-cancer signatures with over 90% sensitivity and 95% specificity in detecting multiple cancers.

Conclusions: Automated systems for extracellular vesicle isolation hold significant potential in diagnostics due to their high throughput and reproducibility, enhancing the precision of cancer diagnostics, and being compatible with routine pathology laboratories.

Dr. Jina Ko

Introductory Talk and Oral Session: OT08 Biomarker Technologies, Room 109-110, May 9, 2024, 4:00 PM - 5:35 PM

1) Introduction

Extracellular vesicles (EVs) have emerged as a promising source of biomarkers for disease diagnosis. However, current diagnostic methods for EVs present formidable challenges, given the low expression levels of biomarkers carried by EV samples, as well as their complex physical and biological properties. Herein, a highly sensitive double digital assay is developed that allows for the absolute quantification of individual molecules from a single EV. Because the relative abundance of proteins is low for a single EV, tyramide signal amplification (TSA) is integrated to increase the fluorescent signal readout for evaluation. With the integrative microfluidic technology, the technology's ability to compartmentalize single EVs is successfully demonstrated, proving the technology's digital partitioning capacity.

2) Methods

A single layer of antibody-coated and spacer microbeads is introduced to a microwell array for double compartmentalization. A microwell array is fabricated using photolithography and soft lithography. Individual EVs are compartmentalized into each microwell through a flow channel. Each well is sealed with oil and EVs are lysed inside the wells. Once lysed, single EV protein molecules are capture onto each microbead, enabling digital detection. The signal is amplified using TSA and readout using fluorescent microscopy.

3) Results

Our platform was applied to detect single PD-L1 protein molecules from single EVs derived from a melanoma cell line (624-mel) and it is discovered that there are ≈2.7 molecules expressed per EV, demonstrating the applicability of the system for profiling important prognostic and diagnostic cancer biomarkers for therapy response, metastatic status, and tumor progression.

4) Summary/Conclusion

The ability to accurately quantify protein molecules of rare abundance from individual EVs will shed light on the understanding of EV heterogeneity and discovery of EV subtypes as new biomarkers.

PhD Md Khirul Islam, Professor Urpo Lamminmäki, Adjunct professor Janne Leivo

Introductory Talk and Oral Session: OT08 Biomarker Technologies, Room 109-110, May 9, 2024, 4:00 PM - 5:35 PM

Background: Bladder cancer (BlCa) remains a significant global health concern. Current tools for BlCa detection have their own set of limitations, emphasizing the urgent need for a sensitive, fast, and non-invasive diagnostic tool for early detection. This study presents a novel approach for the rapid and high-sensitive detection of urinary extracellular vesicles (EVs) for early BlCa detection using an upconverting nanoparticle-based lateral flow immunoassay (UCNP-LFIA). However, conventional assay for EVs detection is often limited by sensitivity constraints. In our LFIA, we have used UCNPs reporter to enable the detection of low concentration of EVs, resulting in improved sensitivity.

Methods: Isolated EVs from a cancer cell line was used as assay standard. Similarly, isolated uEVs from the urine of BlCa patients and benign sources were used to confirm CD63-positive EVs on urine body fluids. Eventually, minimally processed urinary EVs from bladder cancer (n = 62), benign prostate hyperplasia (BPH) (n = 50), and healthy (n = 30) samples were captured using anti-CD63-antibody and detected with same CD63 antibody conjugated on UCNPs in the microtitration wells. The mixed sample and reporter solution was then allowed to absorb to the lateral flow strip. After 1.5 hours, the strips were read with Upcon reader device that results upconverted luminescent signals. This study was conducted following the guidelines of Helsinki Declaration.

Results: This UCNP-LFIA can measure cancer source derived-EVs with high sensitivity, exhibiting a limit of detection (LOD) 1.9 x 10⁵ /µL. The CD63-CD63-UCNP assay enabled significant discrimination of BlCa patients from benign (2.3-fold, p = 0.007), and healthy (16-fold, p = 0.00001) controls.

Conclusion: The UCNP in the LF platform improves the sensitivity and enables quantitative analyte detection. This UCNP-LFIA may be used for the early detection of BlCa patients from clinically challenging benign as well as healthy conditions with high sensitivity. The UCNP-LFIA technology has the potential to be utilized in simple, rapid, and cost-efficient point-of-care applications. However, more clinical samples are imperative to confirm the effectiveness of this innovative approach.

Dr. Vasiliy Chernyshev, Mr. Alexey Kuzin, Dr. Vadim Kovalyuk, Dr. Pavel An, Mr. Alexandr Golikov, Mr. Sergey Svyatodukh, Mr. Stanislav Perevoschikov, Dr. Irina Florya, Dr. Alexey Schulga, Dr. Sergey Deyev, Dr. Gregory Goltsman, Dr. Dmitry Gorin

Introductory Talk and Oral Session: OT08 Biomarker Technologies, Room 109-110, May 9, 2024, 4:00 PM - 5:35 PM

1) Introduction

Today, the search for biomarkers of diseases and methods for their detection is one of the most important areas in modern healthcare. EVs are known to be associated with the pathogenesis of various diseases, such as cancer, neurodegenerative and cardiovascular diseases. Specific detection of EVs and potential control of their abundance in body fluids may provide a successful therapeutic strategy that involves reducing circulating EVs to normal levels to prevent disease progression. To provide the basis for such research and development, we have for the first time combined photonic integrated circuits (PICs) in the form of a Mach-Zehnder interferometer with microfluidics to create a lab-on-a-chip device.

2) Methods

EVs were isolated from cell culture media of SKOV3 and MDA-MB-231 cells by using ultrafiltration combined with size-exclusion chromatography. To modify the surface of the sensitive part of the chip, 3-aminopropyltriethoxysilane (APTES), and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysulfosuccinimide (EDC-NHS) cross-linking chemistry was used to allow covalent binding of HER2 membrane receptor-specific DARPin. Surface modification and EV capture was performed during controlled fluid flow through microfluidic channels exposed to PICs.

3) Results

Surface modifications were performed on two different sensors on the same chip, but in one case analysis was carried out on HER2-positive EVs (SKOV3) and in the other on EVs containing significantly fewer HER2 receptors (MDA-MB-231). The concentration of EVs in both cases was 4.2×10¹⁰ particles/ml. The first steps of the modification protocol demonstrated approximately the same relative shift in the spectral response of the device. The difference in the abundance of HER2 receptors in the two EV samples analyzed was clearly reflected in the significant shift of the peaks obtained from the Mach-Zehnder interferometer measurements described.

4) Summary/Conclusion

This study demonstrates that such biosensor can be used to quantify biological markers, such as EVs containing a specific membrane protein. The developed platform provides results in real time using microliter volumes of the test sample. This research can be used as a first step towards creating a lab-on-a-chip for diagnostics/monitoring of treatment effectiveness in medicine.

Ms Abha Kumari, Dr Mark Youngblood, Andrew Gould, Dr Yoon-Tae Kang, Li Chen, Karl Habashy, Thiago Reis, Dr Chris Amidei, Dr Rachel Ward, Cristal Gomez, Guillaume Bouchoux, Michael Canney, Dr Roger Stupp, Prof. Adam Sonabend, Prof. Sunitha Nagrath

Introductory Talk and Oral Session: OT08 Biomarker Technologies, Room 109-110, May 9, 2024, 4:00 PM - 5:35 PM

Introduction:

Glioblastoma (GBM) is an aggressive brain tumor with inaccurate or invasive conventional techniques of longitudinal monitoring, requiring alternatives like liquid biopsy. Exosomes are nano-sized vesicles that contain cargo reflective of their cellular origin and have been used for early detection in cancer. However, in GBM, the blood-brain barrier (BBB) leads to a paucity of diagnostic information in blood. This study investigates the effect of opening BBB on circulating exosomes and their potential for early diagnosis of GBM using a microfluidic platform (GlioExoChip). In 20 patients enrolled in a clinical trial NCT03744026, an intracranial pulsed ultrasound emitter- Sonocloud-9 was implanted to open the BBB. We demonstrate that GlioExoChip has high efficiency capture of GBM-specific exosomes using phosphatidylserine and Annexin-V chemistry. Finally, a longitudinal study was performed to study the correlation between tumor growth rate and exosome concentrations.

Methods:

GlioExoChip is a microfluidic platform based on neutravidin-biotin chemistry. The isolated exosomes are characterized using Nanoparticle tracking analysis (NTA), scanning electron microscopy, Bicinchoninic acid (BCA) assay, and western blot. Statistical analyses were performed using Student's t-tests and Pearson correlation.

Results:

GlioExoChip has a high capture efficiency of GBM cell-derived exosomes, i.e., 93.03±3.31% and 96.77±0.72% for GBM6 and U87, respectively. NTA and BCA showed that 15 times more exosomes were captured from patient plasma when compared with healthy. Further, western blot proved the presence of glial protein and Flotillin-1 in exosomes isolated from patients. Exosome concentrations varied with different time points after sonication and had an average twofold increase from BBB opening. In our longitudinal study with 8 patients, an association between the volumetric enhancement of the tumor and exosome concentrations post-sonication shows that the average Pearson correlation among short progression-free survival (PFS) patients was 0.73 (avg. p = 0.39), while poor correlation was seen among long PFS patients (avg. r value = 0.10; avg. p-value = 0.54).

Conclusions:

A microfluidic approach to investigate the role of circulating exosomes using phosphatidylserine-annexin V chemistry in GBM was demonstrated. BBB opening via sonication leads to an increase in the average GBM-specific exosome concentrations in plasma. Thus, exosomes are promising biomarkers for the early detection and longitudinal monitoring of GBM.

Lee-Ann Clegg, MD, PhD Rolf A. Blauenfeldt, Bioinformatician, PhD Jesper Just, MSc in Engineering Rikke Bæk, Professor Peter Kristensen, Professor, MD Grethe Andersen, MSc, PhD Kim R. Drasbek, MSc, Ph.D Malene M. Jørgensen

Introductory Talk and Oral Session: OT08 Biomarker Technologies, Room 109-110, May 9, 2024, 4:00 PM - 5:35 PM

Introduction

Today, stroke cannot be diagnosed based on a physical examination alone but requires a brain scan at the hospital. This delay in treatment initiation may lead to progression of brain injury and long-term disability. Therefore, to improve the treatment outcome it is of utmost importance that time between stroke onset and treatment is minimized. Several EV-based biomarkers for stroke detection have been investigated, however, none are applied in clinical practice. In this study we investigated stroke specific EV biomarkers potential use as an ultra-early stroke diagnostic with the goal to speed up stroke treatment initiation. That may enable prehospital diagnosis, improved patient triage and perhaps ultra-early prehospital immediate treatment initiation and decrease infarct growth in these patients. Thus, lead to better survival. These biomarkers were identified on EVs derived from 1200 plasma samples from stroke patients. Three samples from each patient were used for investigation of biomarker distribution over time. The samples were drawn at <4 hours from stroke onset in the ambulance, repeated at the hospital and again 24 hours after stroke. The biomarker abundance was used to create a stroke prediction model that may distinguish intracerebral hemorrhage (ICH) from acute ischemic strokes (AIS) and non-vascular diseases.

Methods

Study was approved by Dansih regional research ethics commitees. Written consent was obtained by relative or patient. A high-throughput protein microarray-based technology, EV Array, was used to identify a panel of surface markers on plasma EVs, which should be able to distinguish between no stroke, AIS or ICH. Biomarker abundance was compared between groups with Linear Models for Microarray Data. The biomarker panel constituted 49 markers associated with endothelia, neurons, EVs, inflammation, RBCs, BBB, astrocytes and hypoxia.

Results

It was possible to measure biomarkers on EVs related to ICH and AIS with the EV Array. We observed patterns which may help categorize patients into well-defined treatment groups. Results are provided as allowed according to a non-disclosure agreement.

Summary/Conclusions

We suggest that EV Array may shed light on application of EVs in the blood stream of acute stroke patients as diagnostic biomarkers to allow treatment initiation in a pre-hospital setting.

Phd Yunyun Hu, Haonan Di, PhD Ye Tian, Yiyin Weng, Jujiang Guo, Professor Xiaomei Yan

Introductory Talk and Oral Session: OF09 Disease Biomarkers, Plenary 1, May 10, 2024, 10:40 AM - 12:00 PM

Introduction

Breast cancer (BCa) stands as a critical global health concern, ranking highest in both female cancer incidence and mortality rates. However, prevailing screening methods suffer from low sensitivity and high invasiveness, impeding early diagnosis and personalized treatment. Extracellular vesicles show promise as a liquid biopsy alternative to diagnosing BCa. Yet, pinpointing tumor-derived EVs with accuracy is hindered by the extensive heterogeneity among circulating extracellular vesicle and particle (EVP) phenotypes. Here, we conducted a multiplexed analysis of single EVs, revealing that simultaneous recognition of dual surface proteins enhances the diagnostic sensitivity and specificity of BCa.

Methods

EVs were isolated from normal breast cell line (MCF10A), three BCa cell lines (MCF7, SK-BR-3, and MDA-MB-231), and BCa patient plasma by differential ultracentrifugation. Proximity barcoding assay (PBA) was employed to screen potential protein markers for EVs from different BCa cell lines. A dual-laser equipped nano-flow cytometer (nFCM) (488 nm and 647 nm) enabled simultaneous profiling of two surface proteins on individual EVs with minimal steric hindrance. Upon immunofluorescent staining, the proportions and particle concentrations of EV subtypes expressing specific protein compositions for isolated EVs were characterized. Human blood sample collection was approved by the Ethics Committee of Xiamen Maternity and Child Health Care Hospital.

Results

Dual detection of ITGB1 and CD151 (or EpCAM and HER2) on single cell-derived EVs via nFCM allowed classification of EVs into four distinct subsets based on protein compositions. Using t-distributed stochastic neighbor embedding (t-SNE) reduction of seven fluorescent parameters of three EV subsets, successful differentiation of EVs derived from MCF10A and three BCa cell lines was achieved. Single particle enumeration revealed that concentrations of ITGB1-postive and CD151-negative EVs displayed improved clinical relevance for early diagnosis and subtyping of different BCa subtypes compared to a single protein marker.

Summary

This study emphasizes the significance of multiplex tumor marker analysis on single EVs. The utilization of nFCM platform for dual protein co-recognition aids in understanding BCa heterogeneity and enhances diagnostic specificity. Further research in this direction holds promise for advancing breast cancer diagnostics and treatment strategies.

MD. Ryosuke Uekusa, Dr. Akira Yokoi, Dr. Kosuke Yoshida, Dr. Jyuntaro Matsuzaki, Dr. Yusuke Yamamoto, Dr. Hiroaki Kajiyama

Introductory Talk and Oral Session: OF09 Disease Biomarkers, Plenary 1, May 10, 2024, 10:40 AM - 12:00 PM

1)Introduction

High-grade serous ovarian carcinoma (HGSOC) is the most common subtype of ovarian cancer and copy number variations (CNVs) are the dominant genomic events in HGSOC. Extracellular vesicles (EVs) circulate in body fluids and carry pathological genomic information positively. Here, we evaluated the significance of EV-DNA as novel biomarkers for HGSOC patients.

2)Methods

A total of 124 samples from HGSOC patients and cell lines were analyzed. EVs were isolated using the ultracentrifuge method. Comprehensive CNV statuses were analyzed by whole genome sequencing (WGS). In reference to the Ovarian Cancer Moon Shot database, 30 CNVs in HGSOC were selected as dominant targets for analysis by droplet digital PCR (ddPCR). As for predicting response to PARP inhibitors, HGSOC patients treated with olaparib were collected, and DNA was extracted from FFPE tissue at the time of surgery.

3)Results

NTA assays and cryo-electron microscopic analyses showed that the EVs were successfully isolated from all samples. WGS detected correlating CNVs in cell-DNA and EV-DNA for cell lines, and in tissue-DNA and EV-DNA in ascites for patients. The CNV status was also measured by ddPCR. Copy numbers of RAD51, BRCA1, AKT2, CCNE1, and MSH6 were significantly higher in malignant tissue than in benign tissue (p < 0.0001, p < 0.0001, p = 0.0012, p = 0.002. p = 0.0328, respectively), and were detectable in EV-DNA from ascites. Additionally, the amount of DNA in malignant ascites was significantly higher than in benign ascites (p = 0.016), and increased DNA in ascites itself suggested the presence of malignancy. Furthermore, EV-DNA more accurately reflected the CNV status of tumor DNA than cell-free DNA in ascites. Regarding prediction of PARP inhibitor response, the CNV status in EV-DNA from patients treated with olaparib, a PARP inhibitor, demonstrated a marked difference between responders and non-responders. The equation calculated based on the combination of six genes (RB1, GABRA6, CTNNB1, MYC, RAD51, BARD1) could predict the response to olaparib (AUC: 1.0). The selected genes were validated using EV-DNA from both in vitro and clinical samples.

4)Summary/Conclusion

CNV status of EV-DNA was accurately measured and can be novel diagnostic and prognostic biomarkers for HGSOC.

Ms Chloe James, Ms Johanna Farley, Ms Natasha Borash, Dr Hannah Nazri, Ms Anna Tresso, Dr Shima Bayat, Dr Anup Shah, Dr Joel R Steele, Prof Ralf B Schittenhelm, Dr Shanti Gurung, Prof Caroline E Gargett, Prof Christian M Becker, Prof Beverley Vollenhoven, Dr Thomas Tapmeier

Introductory Talk and Oral Session: OF09 Disease Biomarkers, Plenary 1, May 10, 2024, 10:40 AM - 12:00 PM

Introduction

Endometriosis is characterised by intraperitoneal lesions of endometrial tissue. Although ultrasound detection of severe endometriosis is possible, the diagnosis of peritoneal lesions currently relies on invasive laparoscopy, which leads to a delay of 8-10 years. We have previously shown that peritoneal fluid (PF) of women with endometriosis contains endometriosis specific EVs. Traced in peripheral blood (PB), EVs could serve as biomarkers of endometriosis, particularly for stages I and II, which are difficult to detect by ultrasound.

Methods

Women 18-45 years of age undergoing laparoscopic surgery for endometriosis or unrelated conditions were invited to participate in our study (HREC 20-159A/HREC 20-882A). Following informed consent, we collected PF and PB samples from n = 101 participants (n = 47 controls, n = 54 cases). Exclusion criteria were pregnancy, malignancy, and menopause.

We processed PF and PB samples by differential ultracentrifugation and validated the presence of EVs by nanoparticle tracking analysis (NTA), Western blotting and transmission electron microscopy (TEM). EVs were analysed by untargeted, label-based quantitative proteomics using Tandem Mass Tags (TMT). Data analysis was performed using Proteome Discoverer v2.4 (Thermo Fisher) followed by statistical analysis using R. Candidate biomarker proteins were tested by traditional and automated Western blotting (WES, ProteinSimple).

Results

We ascertained the identity of EVs by TEM and NTA (mode size 121.8 ± 18.0 nm (blood, n = 5) and 155.9 ± 37.2 (PF, n = 6)), and signals for syntenin and ALIX in immunoblots as well as expression of CD9, CD63 and CD81 in capture bead-flow cytometry. Proteomic analysis identified 9145 protein groups across all sample groups, with 602 significantly regulated between comparisons (adjusted P-value <0.05). In PF, 533 proteins changed significantly in abundance (245 up/288 down), while in blood, only four proteins changed in abundance.

We tested the presence/absence of two of these as candidate markers on PB EV protein preparations from n = 32 samples to date; our best marker showed a sensitivity of 67% and a specificity of 90%; upon exclusion of severe endometriosis cases, sensitivity improved to 80%.

Summary/Conclusions

Our findings suggest the feasibility of defining a protein signature of endometriosis based on EV cargoes, which could serve as a noninvasive biomarker of the condition.

Dr Nanthini Jayabalan, Dr Andrew Lai, Dr Dominic Guanzon, Dr Soumyalekshmi Nair, Mrs Katherin Scholz-Romero, Valeska Ormazabal, Professor Aasa Handberg, Dr Flavio Carrion, Professor Harold McIntyre, A/ Professor Martha Lappas, Professor Carlos Salomon

Introductory Talk and Oral Session: OF09 Disease Biomarkers, Plenary 1, May 10, 2024, 10:40 AM - 12:00 PM

Introduction: Gestational diabetes (GDM) is linked to increased placental glucose uptake, which can lead to fetal overgrowth. Our previous research showed that women with GDM produce a higher number of extracellular vesicles (EVs) from adipose tissue (AT), and this positively correlates with fetal birth weight. These findings led us to hypothesize that EVs derived from AT in GDM women may influence placental nutrient uptake, contributing to fetal overgrowth.

Methods: We obtained omentum samples from BMI-matched women with normal glucose tolerance (NGT, n = 25) and GDM (n = 35) at delivery. EVs were isolated from AT explant media and characterized following MISEV guidelines. We constructed a small RNA library from AT EV RNA using the Illumina TruSeq® Small RNA Library Prep Kit. For in vitro studies, we isolated human primary placental trophoblasts (PHT) from fresh placentas (n = 6). We evaluated the effect of EVs on glucose uptake in PHT using 2-NBDG. We also overexpressed selected EV-miR mimics in PHT and assessed their impact on glucose uptake. In vivo effects of AT-EVs were determined in pregnant mice, with placenta and pup weights recorded, and fetal liver and blood collected for further analysis.

Results: We found over 150 significantly different microRNAs in AT EVs from GDM compared to NGT. Notably, hsa-miR-146a-5p, hsa-miR-515-5p, and hsa-miR-516b-5p were identified as candidate microRNAs targeting genes associated with glucose metabolism, insulin signaling, amino acid transport, and inflammation. AT EVs from GDM increased glucose and amino acid uptake in PHT, similar to the effects observed with hsa-miR-515-5p overexpression in PHT. In vivo, AT EVs from GDM raised blood glucose concentration and fetal growth compared to EVs from AT NGT, with no changes in placental weight, placental efficiency, or maternal insulin observed. Lastly, overexpression of hsa-miR-515-5p in AT EVs from NGT mimicked the effect on fetal growth observed with AT GDM EVs.

Conclusion: Our findings suggest that AT-EVs may mediate placental glucose uptake and amino acid transfer, contributing to excessive placental nutrient transfer and resulting in fetal overgrowth.

Dr. Huixian Lin, Dr. Chunchen Liu, Prof. Bo Li, Prof. Lei Zheng

Introductory Talk and Oral Session: OF09 Disease Biomarkers, Plenary 1, May 10, 2024, 10:40 AM - 12:00 PM

Quantification of protein-specific extracellular vesicle (EV) subpopulations at the single-vesicle level is of great significance for cancer diagnosis. Although individual vesicle analysis has been implemented by novel emerging technologies, developing an easy-to-operate and cost-effective single EV analysis approach to promote clinical applications is still challenging. Herein, we constructed a versatile droplet digital immuno-PCR (ddiPCR) assay which integrates the high specificity of immuno-PCR and superior sensitivity of droplet digital PCR to profile the surface proteins of single EVs for multi-subpopulation EVs counting. The clinical application of the ddiPCR assay was validated by simultaneous profiling the EV proteins of CD9/CD63/CD81, HER2, EpCAM in a breast cancer cohort, and CD9/CD63/CD81, GPC-3, EpCAM in a hepatocellular carcinoma cohort (HCC). The results demonstrated that the counting of multi-subpopulation EVs could significantly distinguish patients with breast tumor or HCC from healthy controls. Furthermore, with the assistance of machine learning algorithm and under the best combination of sEV subpopulations, our method exhibited great performance in differentiating breast cancer from healthy individuals. Therefore, this study provides a promising strategy to count multi-subpopulation EVs at the single-vesicle level for cancer diagnosis.

Ms Afsareen Bano, Dr. RASHMI BHARDWAJ

Introductory Talk and Oral Session: OF09 Disease Biomarkers, Plenary 1, May 10, 2024, 10:40 AM - 12:00 PM

Introduction

Escalating oral cancer mortalities throughout the world spawns inevitable demand for early screening of most susceptible groups such as tobacco consumers through a non-invasive source such as salivary exosomes. Thus, exosomal proteomics will improve our understanding of oral cancer prognosis in high-risk groups, even before symptoms manifest.

Methods

Salivary exosomes isolated from selected study groups (HC: Healthy control, TC: Tobacco consumers, and OC: Oral cancer) were characterized by Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), Western blot, and EXOCET assay. Total protein was estimated by Bradford assay, and ELISA detected expression of exosomal cytokine (IL-8). Amino acid analysis was carried out to study the concentration pattern of various amino acids among three study groups. FTIR absorption spectrum was exclusively analyzed for protein peaks among the three study groups. The study was approved by the Human Ethical Committee of the institution (HEC/2021/295).

Results

TEM and NTA show that salivary exosomes lie in the acceptable size range i.e., 30-150 nm along with the expression of CD63. There was a significant difference in the concentration of IL-8 among the Control and Tobacco consumer groups (p-value < 0.0001) as well as among the Healthy and Oral cancer groups (p-value = 0.0002). Also, IL-8 concentration levels were significantly different among Tobacco consumers and the Oral cancer group (p-value < 0.0001). Elevated protein peaks (1315: Amide III, 1525: Amide II, 1645: Amide I) were obtained in FTIR absorption spectra in the Oral cancer and Tobacco consumer group compared to the Control group. Protein (Amide III) peaks in the absorption spectrum at 1315 (due to C-N stretching) were significantly different among control and tobacco consumers (p-value = 0.0415). Amino acid analysis reveals significant differences among the three study groups (p-value = 0.0036).

Conclusion

A combinational approach studying exosomal cytokines, amino acid composition, and protein spectrum signifies its importance in screening tobacco consumers from the control group. Our results provide valuable insights for improved prognosis, risk assessment, and personalized strategies for tobacco-induced oral carcinogenesis.

Dr Simon Swift, Yevetta Xiang, Dr Priscila Dauros-Singorenko, Professor Hsin-Chih Lai

Introductory Talk and Oral Session: OF10 Pathogen Host Response II, Eureka, May 10, 2024, 10:40 AM - 12:00 PM

1) Introduction

Parabacteroides goldsteinii (Pg) is a Gram-negative bacterium and anaerobe. The presence of Pg in the gut microbiome is correlated with positive health outcomes when considering chronic inflammatory conditions. The lipopolysaccharide of the Pg outer membrane has anti-inflammatory properties.

2) Methods

For EV preparation Pg MTS01 was grown in 500 mL Brain Heart Infusion Salt broth (BHIS) at 37°C in an anaerobic atmosphere to stationary phase over 2-3 days. Bacteria were removed by centrifugation at 7000 ×g for 10 minutes and filtration at 0.22 µm. Cell free supernatants were concentrated using a 100 kDa cut-off centrifugal filter (Amicon) and isolated using size exclusion chromatography (SEC; qEV70, Izon). A media only control was subjected to the EV isolation procedure. EV characterisation was by nanoparticle tracking analysis (Nanosight, NS300) for size, protein content estimation and transmission electron microscopy. Inflammatory properties were measured using THP-1 dual monocytes (InVivoGen) activated with Pam3CSK4 and measuring recombinant secreted alkaline phosphatase activity to monitor activation of the NF-κB pathway.

3) Results

Three independent preparations of EVs from Pg cultures grown to an optical density at 600 nm of ≈1.0. EVs were isolated in early SEC fractions identified by a peak in particle counts and protein content. Later SEC fractions contained more protein and fewer particles. EV peaks were absent in the BHIS control, but similar levels of protein were observed in later fractions. NTA identified 1-6 x10¹² particles per ml of culture, and < 0.1 x10¹² particles per ml of BHIS. NTA gave a peak particle size as 75 to 105 nm. TEM of SEC fractions selected for EV peaks identified the presence of cup-like structures ≈80 nm in diameter characteristic of EVs, that were not present in later fractions or matched BHIS control fractions. Pg EVs were tested to show low proinflammatory activity and the ability to suppress Pam3CSK4 of THP-1 dual monocytes.

4) Summary/Conclusions

The carriage of anti-inflammatory LPS in Pg EVs may have a direct effect on target cells, and/or may allow carriage of other factors without provoking inflammation. The Pg EVs may find applications in biotechnology and drug delivery.

Ms Dominika Kozakiewicz, Dr Agnieszka Razim, Dr Sabina Górska

Introductory Talk and Oral Session: OF10 Pathogen Host Response II, Eureka, May 10, 2024, 10:40 AM - 12:00 PM

Introduction

There is a great concern about allergies, particularly airway allergies, whose prevalence is rising dramatically worldwide. It is necessary to create a solution that can treat/prevent allergies easily and safely. Probiotics have been extensively researched and are generally considered safe. However, people with a weakened immune system may be at risk of side effects or even bacteremia. A relatively new research direction is using EVs from probiotic bacteria. Only a few studies have investigated the properties of probiotic-derived vesicles, especially bifidobacterium-derived, in allergy. These studies have been conducted to analyze the physicochemical and immunomodulatory properties of B. adolescentis CCDM 368 and B. animalis CCDM 366 derived EVs.

Methods

The bacterial culture was inoculated 1:8 with MRS for 20 h at 37°C. The culture was centrifuged for 20 min at 4°C and 12 000 x g. The supernatant was filtered through a 0.22 µm filter. EVs were isolated by ultracentrifugation (3h at 4°C and 150 000 xg). The pellet was dissolved in 500 µL of sterile HEPES in 0.9% NaCl. EVs were fractionated using chromatography methods. Thermal, long-term stability and batch-to-batch variability were analyzed using dynamic light scattering. EVs were fixed with 4% PFA and applied to grids. Images were obtained using a JEOL JEM F-200 TEM. Human embryonal kidney (HEK) 293 cells, stably transfected with TLR and NOD receptors, were used to determine the recognition pathways. The investigation of the immunostimulatory properties of EVs was conducted by using BMDC and splenocytes from naïve mice ex vivo.

Results

The basic physicochemical properties of EVs were investigated. Their size and zeta potential were determined using DLS. The lipid content does not change between batches. Additionally, EVs were visualized using TEM. Vesicles are recognized by TLR2, but not by TLR4, TLR5, and NOD2 receptors. A broad range of cytokine levels was determined for EVs' stimulated splenocytes and BMDC.

Conclusions

Studies provided EVs characterization in terms of their physicochemical properties, content, immunoreactivity, thermal and long-term stability, batch-to-batch variability, and cytotoxicity. Studies of the changes in cytokine production by the stimulated cells indicated the potential anti-allergy properties of extracellular vesicles derived from both strains.

Dr Ella Johnston, Dr Lauren Zavan, Associate Professor David Greening, Professor Andrew Hill, Associate Professor Maria Kaparakis-Liaskos

Introductory Talk and Oral Session: OF10 Pathogen Host Response II, Eureka, May 10, 2024, 10:40 AM - 12:00 PM

Introduction: Bacteria produce extracellular membrane vesicles (MVs) via a range of mechanisms and contain DNA, RNA and proteins. Due to their biological cargo, MVs can mediate a range of functions that include promoting pathogenesis and horizontal gene transfer. However, the effects of bacterial growth conditions and the mechanisms of MV biogenesis on MV production, composition and functions are not well understood. In this study, we examined how the mechanisms of MV biogenesis and altered bacterial growth conditions affected the composition and biological functions of MVs.

Methods: Helicobacter pylori MVs produced using acidic or neutral pH conditions were examined using proteomics to determine the effect of pH on their proteome. To examine the effects of the mechanisms of MV biogenesis on their cargo composition, MVs were isolated from three Pseudomonas aeruginosa strains that produced MVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis and analysed using proteomics. The effects of altered growth conditions on the ability of MVs to mediate horizontal gene transfer were examined using P. aeruginosa MVs produced during planktonic or biofilm growth conditions harbouring a plasmid encoding for gentamicin resistance, and examining their ability to confer antibiotic resistance to recipient P. aeruginosa bacteria.

Results: The size and proteome of MVs was significantly altered when produced during different pH growth conditions, as MVs produced during acidic conditions were smaller and enriched in cargo compared to MVs produced during neutral pH conditions. Furthermore, MVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome, suggesting that the mechanisms of MV biogenesis defined their cargo. Finally, we identified that bacterial growth conditions influenced the ability of MVs to mediate horizontal gene transfer, as biofilm-derived P. aeruginosa MVs were more efficient in mediating horizontal gene transfer of antibiotic resistance to P. aeruginosa bacteria compared to planktonic-derived MVs.

Summary: Our findings show that bacterial MVs produced during different growth conditions and via different mechanisms of biogenesis vary in their composition and functions, revealing that bacteria produce bespoke MVs during challenging growth conditions and via multiple mechanisms with enhanced biological functions.

Hitoshi Tsugawa, Student Shogo Tsubaki, Dr Takuma Araki, Dr. Yusuke Yoshioka, Dr. Juntaro Matsuzaki, Dr Hitoshi Tsugawa

Introductory Talk and Oral Session: OF10 Pathogen Host Response II, Eureka, May 10, 2024, 10:40 AM - 12:00 PM

Introduction: Klebsiella pneumoniae, a commensal bacteria in the gastrointestinal tract, can cause pneumonia and liver abscesses by invading the lung and the liver, particularly in older adults. The mechanism whereby K. pneumoniae translocates from the gastrointestinal tract to distant organs is unclear. This study aimed to elucidate the role of extracellular vesicles (EVs) in bacterial translocation to the liver. Method: K. pneumoniae-derived EVs (Kp-EVs) were collected using ultracentrifugation and characterized using transmission electron microscopy. Proteins contained within Kp-EVs were identified using shotgun-nano liquid chromatography-mass spectrometry (LC/MS). The biological functions of Kp-EV were analyzed using mouse bone marrow-derived macrophages (BMDMs). To examine the localization of Kp-EV in vivo, DiR-stained Kp-EVs were administered to mice via intravenous injection and traced using in vivo imaging system analysis. Results: Kp-EVs accumulated in the liver, spleen, and lung, consistent with the organs to which K. pneumoniae translocates. Elongation factor Tu (TufA) was identified as a localization protein within Kp-EVs. We purified green fluorescent protein-labeled Kp-EV (Kp-EV-GFP) by constructing a recombinant fusion protein of TufA and GFP. We then examined the localization of Kp-EV within the BMDMs using Kp-EV-GFP. Kp-EV-GFP distributed within BMDM cytoplasm and did not co-localize with organelles such as Golgi, endoplasmic reticulum, and early endosomes. Treatment with Kp-EV increased the population of CD206, M2-phenotype marker-positive BMDMs and strongly induced mRNA expression of M2-phenotype markers, including Arg1, transforming growth factor-β (TGF-β), and interleukin 10 (IL-10). Kp-EVs also enhanced phagocytosis activity of BMDMs. The intracellular K. pneumoniae count within Kp-EV-treated BMDMs was significantly higher than that of non-Kp-EV-treated BMDMs. Conclusion: Kp-EVs facilitate bacterial translocation from the gastrointestinal tract to the liver and lung by enhancing survival of intracellular bacteria by inducing macrophage polarization to the M2 phenotype.

Mr Lewis Timms, Dr Daniel Radford Smith, Prof Daniel C. Anthony, Associate Prof Naveed Akbar, Prof Robin P. Choudhury

Introductory Talk and Oral Session: OF11 EVs in Tissue Function II, Room 105-106, May 10, 2024, 10:40 AM - 12:00 PM

Introduction

Acute infection or disease, such as acute myocardial infarction (AMI), mobilises neutrophils and monocytes from bone marrow and spleen that need to be replenished. Recurrent AMI and infection are associated with long-term bone marrow reprogramming and lower bone marrow haematopoietic stem & progenitor cells (HSPC). Here, we hypothesised that endothelial cell-derived extracellular vesicles (EC-EV) enriched with vascular cell adhesion molecule-1 (VCAM-1) are responsible for preparing the bone marrow niche for myelopoiesis after acute inflammatory challenges as part of the acute phase response.

Methods

Basal-state [mouse s.END1] EC-EV and ‘inflammatory’ EC-EV (TNF-α-stimulated, 10ng/mL, n = 8) were isolated using ultracentrifugation (120,000g, 2 hours with washing) and characterised by Nanoparticle Tracking Analysis, cryo-transmission electron microscopy, and Western blotting. Wild-type mice (n = 6) received intraperitoneal injections with lipopolysaccharides (LPS, 1mg/kg) or intravenous injections with sEND.1 EC-EV (109 particles) from control or inflammatory conditions. Bone marrow was harvested 72 hours post-injection. Bone marrow HSPC were assessed by flow cytometry. In colony forming unit experiments, wild-type bone marrow cells (n = 6) were harvested, exposed to LPS (100ng/mL), or sEND.1 EC-EV from control or inflammatory conditions (109 particles), and granulocyte-monocyte colonies were quantified after 10 days. To test long-term bone marrow reprogramming, wild-type mice (n = 6) received a tail-vein injection of LPS, wild-type sEND.1 EC-EV or VCAM1-knockout sEND.1 EC-EV for 24 hours, followed by an intraperitoneal LPS injection for 72 hours.

Results

Isolated EC-EV had a peak diameter of 110nm, were ALIX+, TSG101+ & CD9+, and ATP5A-. ‘Inflammatory’ EC-EV particles increased 2-fold vs. control (P<0.0001) and were VCAM-1+. ‘Inflammatory’ EC-EV elevated haematopoietic stem cells (P<0.01), granulocyte-monocyte progenitors (GMP, P<0.001), monocytes (P<0.05) and neutrophils (P<0.05) in bone marrow. Similarly, ‘Inflammatory’ EC-EV increased granulocyte-monocyte colony formation in vitro (P<0.05). Mice receiving ‘inflammatory’ EC-EV followed by secondary LPS contained fewer GMP (P<0.05) and neutrophils (P<0.05), indicating diminished myelopoiesis. These effects were VCAM-1-dependent, being abrogated by knockout sEND.1 EC-EV (P = 0.99).

Conclusion

Inflammatory EC-EV induce bone marrow myelopoiesis in vivo and in vitro. This is reversed following subsequent LPS stimulation and appears to be EC-EV-VCAM-1-dependent. This suggests EC-EV prime the bone marrow and HSPC niche, which may alter immunological responses in cardiovascular and metabolic disease.

Yuxia Zhang, Associate Researcher Taotao Tang, Professor Rining Tang, Professor Bicheng Liu

Introductory Talk and Oral Session: OF11 EVs in Tissue Function II, Room 105-106, May 10, 2024, 10:40 AM - 12:00 PM

Introduction:

Vascular calcification (VC) is a serious complication in chronic kidney disease (CKD), recognized in KDIGO guidelines as a leading risk factor for cardiovascular diseases. Despite acknowledgment, the mechanisms of VC in CKD remain unclear. The kidneys intricately regulate blood vessel pathophysiology. Clinical studies suggest a correlation between elevated blood complement levels in CKD and arterial calcification, hinting at complement involvement in CKD-associated VC. Renal-origin complement, a significant source of circulating complement, activates in CKD. Extracellular vesicles (EVs), mediating intercellular communication, transport complement-related proteins in various physiological and pathological processes. This study explores how renal-origin EVs carrying complement mediate CKD-associated VC.

Methods:

A CKD-associated VC model was induced by feeding C57BL/6j mice an adenine and high-phosphate diet for 16 weeks. Techniques, including WB, PCR, and immunofluorescence, detected complement activation and localization in renal tissues. EVs were isolated from renal tissues and plasma. WB examined C3 and tubular markers of EVs. Immunofluorescence investigated tubular-derived EVs uptake in calcified arteries. Tubular-derived EVs, C3a, C3aR receptor inhibitors, and autophagy inhibitors intervened in high-phosphate-induced vascular smooth muscle cells (VSMCs). PCR and WB assessed calcification indicators, complement receptors, and autophagy-related indicators. Alizarin Red staining observed calcification nodules. Transcriptome sequencing was performed on control, high-phosphate, and renal tubular-derived EV intervention groups of VSMCs. An in vivo model infused renal tubular-derived EVs and injected C3aR inhibitors and autophagy inhibitors, measuring complement activation and VC indicators.

Results:

The CKD-associated VC model revealed complement C3 activation in renal tubules, secreted into the blood through EVs. Immunofluorescence indicated tubular-derived EV uptake by the calcified vascular wall. In vitro, tubular-derived EVs enhanced VSMC osteogenic differentiation and upregulated C3aR, mimicking C3a intervention effects. C3aR receptor inhibitors produced opposing effects. Transcriptome sequencing suggested renal tubular-derived EVs downregulated VSMC autophagy-related pathways; in vitro autophagy inhibition reversed osteogenic differentiation. In vivo, reinfusing renal tubular-derived EVs exacerbated complement activation and VC, while injecting C3aR inhibitors and autophagy inhibitors alleviated CKD-associated VC.

Conclusions:

This study confirms that tubular-derived EVs carrying C3 downregulate autophagy in VSMCs, mediating CKD-associated VC. These findings provide insights into understanding CKD-associated VC initiation and progression, revealing potential therapeutic targets.

Ms Xiangju Wang, A/Prof Chang Seong Kim, Mr Benjamin Adams, Dr Monica Ng, A/Prof Helen Healy, Dr Andrew Kassianos

Introductory Talk and Oral Session: OF11 EVs in Tissue Function II, Room 105-106, May 10, 2024, 10:40 AM - 12:00 PM

Introduction: Acute kidney injury (AKI) is a devastating public health issue. Tellingly, 20-50% of AKI survivors progress to chronic kidney disease (CKD) (kidney damage persisting for ≥3 months), increasing their risk for premature death. AKI-to-CKD transition is characterised by the irreversible loss/death of kidney tubular epithelial cells (TEC). Here, we investigated the role of small extracellular vesicles (sEV) in propagating the sustained tubular loss of AKI-to-CKD transition.

Methods: Kidney tissue and urine were obtained with ethics approvals and informed consent from AKI patients at the time of diagnostic kidney biopsy. AKI patients were stratified into two groups based on follow-up kidney function (estimated glomerular filtration rate; eGFR) ≥3 months post-AKI episode: AKI recovery/non-transition to CKD (AKI−CKD; eGFR≥60ml/min/1.73m²) and AKI transition to CKD (AKI+CKD; eGFR<60 ml/min/1.73m²). Control kidney tissue was obtained from the healthy portion of tumour nephrectomies and control urine from age/sex-matched healthy donors. Kidney tissue was frozen for immunofluorescent (IF) microscopy, while urine was collected for isolation of urine-derived sEV (usEV) by size exclusion chromatography (qEV columns). The purity of usEV preparations was confirmed using size/concentration (50-150nm; qNano) and morphology (electron microscopy), with human primary proximal tubular epithelial cells (PTEC) co-cultured with usEV in cell death profiling studies (lipid peroxidation assay/microscopy).

Results: IF staining showed widespread ferroptotic cell death (ACSL4 expression) in AKI+CKD biopsies compared with AKI−CKD biopsies and control tissue. Quantitative analysis revealed significantly increased ACSL4 expression intensity, % ACSL4+ PTEC and absolute numbers of ACSL4+ PTEC in AKI+CKD biopsies. usEV were examined as a clinical representation of sEV released into the tubular lumen/urinary space. Numbers of usEV (particles/µmol urine creatinine) were significantly increased in AKI+CKD urine compared with control urine. There were no significant differences in usEV mean diameter between groups, with characteristic cup-shaped morphology identified in all populations. In functional co-culture studies, only AKI+CKD usEV induced significantly increased PTEC ferroptosis (↑lipid peroxidation) compared with control usEV.

Conclusions: Our data identify a novel sEV-mediated mechanism of ferroptosis propagation in AKI-to-CKD transition. This concept of how tubular pathology is propagated from the initiating insult into a ‘wave of death’ provides novel therapeutic and diagnostic targets for AKI-to-CKD transition.

Phd Lélia Borowski, Cécile Devue, Paul Alayrac, Jean Sébastien Silvestre, Chantal M. Boulanger, Xavier Loyer, Stéphane Camus

Introductory Talk and Oral Session: OF11 EVs in Tissue Function II, Room 105-106, May 10, 2024, 10:40 AM - 12:00 PM

Cardiovascular diseases remain the leading cause of death worldwide. Following myocardial infarction (MI), inflammatory cells are mobilized to the injured myocardium from distant compartments to coordinate tissue remodeling. Although soluble mediators are well known to influence local inflammation, their short half-life conditions their restricted range of action. Extracellular vesicles (EVs) encapsulate biologically active substances that are eventually delivered to target cells, making them ideally fitted for long-range inter-organ communication. In this work, we aimed at determining how cardiomyocyte-EVs (CM-EVs) released during MI shape the inflammatory response and cardiac remodeling.

We have developed two genetic mouse models expressing CM-specific Cre recombinase alone (Myh6-Cre) or in combination with ubiquitously expressed mT/mG fluorescent reporters (Myh6-Cre/mTmG) allowing the determination of CM-EV composition and tropism on one hand, and the characterization of information transfer from CM-EV to target cells on the other.

Intracardiac and circulating EVs were isolated from MI or sham-operated control mice by differential ultra-centrifugations. We determined the protein composition of purified CM-EVs by proteomic analysis. We assessed CM-EV biodistribution in-vivo, we monitored GFP fluorescence in various remote organs after MI by flow cytometry and 2-photon microscopy and defined the identity of CM-EVs target cells by flow cytometry. To investigate the interaction between CM-EV and their target cells, we incubated CM-EVs with primary peritoneal macrophages and visualized interaction using imaging flow cytometry. We then monitored GFP expression by flow cytometry as a proxy for functional transfer of biological material from CM-EVs to macrophages.

First, we showed that our EV comply with proteins MISEV recommendations. Proteomic analysis shows that EV MI were enriched with mitochondrial proteins. Then, we demonstrate CM-EVs are preferentially targeting spleen and lung macrophages after infarction but not the liver. We validated an interaction between EV and resident macrophages and confirmed CRE transfer from CM-EVs to macrophages as shown by GFP expression in recipient macrophages.

Our data show that CM-EVs generated upon MI establish long-range, inter-organ communication routes and target innate macrophages from the spleen and lungs.

Mr Adam Wells, Dr Pauline Marie, Dr Claudia C. Mendes, Dr Shih-Jung Fan, Dr Mark Wainwright, Dr. Preman Singh, Dr. Bhavna Verma, Professor Clive Wilson, Dr Deborah Goberdhan

Introductory Talk and Oral Session: OF12 Modelling EV Biogenesis, Room 109-110, May 10, 2024, 10:40 AM - 12:00 PM

Introduction

Rab11-exosomes are a population of ESCRT-dependent exosomes formed by Rab11-positive endosomes. This exosome subtype appears to share conserved regulatory mechanisms from flies to human cancer cells such as HCT116 cells. Uniquely, Rab11-exosomes can also be imaged at high-resolution whilst still within the endosomes of living Drosophila secondary cells. This provides unique opportunities to study the mechanisms of exosome biogenesis in vivo. Therefore, using this Drosophila model we outline novel mechanisms of subtype-specific biogenesis for Rab11-exosomes.

Methods

Giant multivesicular-endosomes (MVEs), ILVs and key regulatory factors were visualised in Drosophila secondary cells through ex vivo imaging using fluorescently-tagged gene-traps and transgenes. Imaging was conducted using widefield or confocal microscopy and putative regulatory molecules were tested by analysing changes in exosome biogenesis following SC-specific RNAi/transgene expression, driven by the GAL4-UAS-GAL80 system.

Results

We find that Rab11-exosome biogenesis occurs at distinct microdomains on MVE surfaces which are defined by Rab11, Rab19 and E-cadherin localisation. These microdomains additionally recruit ESCRT-0 proteins and colocalise with budding ILVs in live cells. Knockdown of any of these microdomain-associated proteins significantly reduces Rab11-exosome biogenesis, thereby validating the functionality of microdomains in biogenesis.

Rab11-exosome biogenesis is also controlled by significant cancer-associated signalling pathways, including the PI3K/Akt/mTOR pathway and stress-associated SUMOylation. Cell-type specific inhibition of either PI3K/Akt/mTOR signalling or SUMOylation significantly reduced Rab11-exosome biogenesis ex vivo. Inhibiting these pathways also lead to reduced microdomain formation. By contrast, overexpressing upstream elements of the PI3K/Akt/mTOR pathway actively stimulated exosome biogenesis and microdomain formation.

Finally, by examining conserved targets of SUMOylation, we find that SUMOylation controls a novel method of mRNA loading in Rab11-exosomes.

Conclusions

We show that Rab11-exosomes form at novel MVE microdomains which function directly in exosome biogenesis. We also show that the formation of these microdomains and of Rab11-exosomes is controlled by important regulators of cell-growth and cell-stress signalling, namely the PI3K/Akt/mTOR and SUMOylation pathways. Additionally, SUMOylation controls a novel mRNA loading pathway in Rab11-exosomes. Overall, these results provide new insights into exosome biogenesis regulation and act as a strong basis for further study of Rab11-exosome control in human cancer cells.

Ioannis Isaioglou, Lama AlAbdi, Yossef Lopez de los Santos, Muhammad Tehseen, Mansour Aldehaiman, Gloria Lopez-Madrigal, Norah Altuwaijri, Maya Ayach, Ashraf Al-Amoudi, Rachid Sougrat, Vlad-Stefan Raducanu, Amani Al-Amodi, Hessa Alsaif, Firdous Abdulwahab, Amal Jaafar, Tarfa Alshidi, Adriana Montaño, Kara Klemp, Ellen Totten, Wesam Kurdi, Samir Hamdan, Stephen Braddock, Fowzan Alkuraya, Jasmeen Merzaban

Introductory Talk and Oral Session: OF12 Modelling EV Biogenesis, Room 109-110, May 10, 2024, 10:40 AM - 12:00 PM

Exosomes are small extracellular vesicles that mediate communication and transport of biomolecules among cells. In recent years, an increasing number of studies have revealed the vital role of exosomes and their cargo in neurodevelopment. Here, we demonstrate for the first time that abnormalities in exosome processing underlie a neurodevelopmental disorder in humans. Our findings are based on two separate case studies – one from the United States and one from Saudi Arabia where babies presented with a novel form of encephalopathy causing death. Exome sequencing revealed that the infants presented with different mutations in the VPS25 gene, a crucial component of the ESCRT-II complex.

Through various studies: bioinformatic analysis, size exclusion chromatography experiments, and co-immunoprecipitation (Co-IP) followed by mass spectrometry, we discovered that the mutations did not interfere with the formation and composition of the ESCRT-II complex. Rather, there is evidence that it changed how the complex interacted with other proteins. The Co-IP mass spectrometry data showed that the ESCRT-II mutation reduced its interactions with components of other ESCRT family members. These alterations increased the quantity of lysosomes and multivesicular bodies in the cytoplasm of the mutated cells, as revealed by transmission electronic microscopy.

Based on these findings, we examined the exosome biogenesis, and identified a dramatic decrease in the exosome release rate by the cells carrying the VPS25 mutation. Furthermore, an analysis of the protein cargo of the produced exosomes showed that about 33% of the exosomal proteomic profile changed in exosomes derived from cells carrying the VPS25 mutation. These data, in combination with recent reports that exosome cargo is vital for the development and maintenance of neurons, offer insight into the clinical profile of the patients explored in our study. Interestingly, we also identified an increased number of double and multi-layer exosomes from the affected patient cells, as revealed by Cryo-EM analysis, a key discovery in the context of growing interest in extracellular vesicle morphology.

In conclusion, by exposing the role played by VPS25 in a human disease, we believe our study demonstrates the ways in which abnormal exosome processing can be implicated in neurodevelopmental disorders.

Mr. Joseph Trani, Dr. Aashiq H. Kachroo, Dr. Christopher L. Brett

Introductory Talk and Oral Session: OF12 Modelling EV Biogenesis, Room 109-110, May 10, 2024, 10:40 AM - 12:00 PM

Introduction:

We embark on a high-throughput exploration, combining synthetic and systems biology by leveraging Saccharomyces cerevisiae (baker's yeast) as a model to enhance our understanding of evolutionarily conserved proteins (orthology) in extracellular vesicle (EV) biology. This research aims to develop a novel high-throughput research pipeline to express and identify all human proteins that localize to yeast EVs.

Methods:

We developed a high-throughput method for expressing over 15,000 human proteins C–terminally tagged to GFP in yeast. This includes segregating cDNAs, covering the near–complete human genome, into 18 pools (∼900 genes each) and employing a Gateway technology-based en-masse cloning method to engineer humanized yeast strains. Oxford nanopore sequencing validates our cloning and transformation efficiency, while fluorescence microscopy and Western blotting (WB), confirm human protein expression. and mass spectrometry (LC MS/MS) is used to identify human proteins in immunoprecipitated samples from yeast cells and EVs. We use Nano flow cytometry (NanoFCM) to measures GFP content in EVs, while NTA and TEM assess vesicle size and morphology. Human proteins found in yeast EVs are individually cloned and characterized using this pipeline to validate our findings.

Results:

We first conducted a proof-of-concept study that revealed cloning and transformation efficiency rates of ∼99%. Both LC MS/MS and WB analysis confirm that a range of GFP-tagged human proteins are expressed in yeast cells. We visualized these cells using fluorescent microscopy and found a wide range of subcellular localization patterns that include punctate structures resembling MVBs, sites of EV biogenesis. We collected EV samples and used NanoFCM analysis to detect a GFP+ population within small EVs (50-200 nm diameter), which we validated using NTA. By conducting proteomic analysis on EV samples, we identified a subset of human proteins that we validated in isolation.

Summary/Conclusion:

Here, we established a novel synthetic biology pipeline to engineer yeast EVs to include human proteins. Humanized yeast strains provide useful models to study orthology including the potential roles of evolutionarily conserved EV proteins that likely play fundamental roles in biogenesis, cell recognition, uptake, back-fusion, and cargo delivery.

Ms Stephanie Rutter, Ms Amy Hodge, Ms Dilara Ozkocak, Dr Julian Ratcliffe, Dr Taeyoung Kang, Dr Niall Geoghegan, Dr Pradeep Rajasekhar, Dr Georgia Atkin-Smith, Dr Ivan Poon

Introductory Talk and Oral Session: OF12 Modelling EV Biogenesis, Room 109-110, May 10, 2024, 10:40 AM - 12:00 PM

1) Introduction:

Apoptosis and the clearance of apoptotic cells are essential to maintain physiological homeostasis. Notably, uncleared apoptotic debris can release pro-inflammatory material and trigger unwanted inflammation. Previous work from our group identified a series of protein regulators that controls the efficient dismantling of apoptotic cells into large EVs known apoptotic bodies, and its role in facilitating cell clearance. Unexpectedly, recent microscopy analysis revealed a previously uncharacterized morphological step, whereby adherent apoptotic cells can retract and leave behind actin-rich membrane remnants, which round into adherent EVs, herein known as the “FOotprint Of Death” (FOOD).

2) Methods:

We established a variety of 3D imaging approaches to visualise FOOD formation including confocal microscopy, SEM, and lattice light-sheet microscopy (LLSM). Moreover, we developed a novel purification approach to isolate FOOD to performed proteomic analysis, and visualise intravesicle contents using TEM. In addition, we used a model of infleunza A viral infection to induce apoptosis.

3) Results:

Confocal microscopy and SEM analysis revealed that a variety of cell types including fibroblasts, endothelial, and epithelial cells generate FOOD during intrinsic apoptosis. High resolution LLSM revealed distinct morphological stages of FOOD formation including (i) the retraction of the cell where membranous material and F-actin fibres are deposited, (ii) the gradual exposure of phosphatidylserine on membranes, and (iii) the rounding of membranes into vesicle-like structures. Mechanistically, we identified ROCK1, a protein kinase and positive regulator of apoptotic membrane blebbing, as a potential regulator in FOOD formation. Proteomic analysis identified a series of proteins, including adhesion-associated proteins, enriched in FOOD. Finally, we identified the capacity for cells to generate FOOD during influenza A infection, and traffic viral components into FOOD vesicles.

4) Summary/Conclusion:

Collectively, this data has identified a new morphological step that occurs during apoptosis, coined FOOD, that also represents a novel mechanism for the generation of adherent extracellular vesicles. Overall, this work contributes to our understanding of the complex and highly coordinated changes to dying cell morphology.

Dr. Kazuki Takahashi^{Department of Molecular Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shinjuku-ku, Japan}, Dr. Yusuke Yoshioka^{Department of Molecular Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shinjuku-ku, Japan}, Dr. Naoya Kuriyama^{Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japan}, Dr. Shinsuke Kikuchi^{Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japan}, Professor Nobuyoshi Azuma^{Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japan}, Professor Takahiro Ochiya^{Department of Molecular Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shinjuku-ku, Japan}

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Aortic aneurysm (AA) is a chronic disease characterized by localized expansion of the aorta. Although AA are generally asymptomatic, they can threaten human health by sudden death due to rupture, which leads to 150,000 ∼ 200,000 deaths per year worldwide. Among AAs, abdominal aortic aneurysm (AAA) has a reported prevalence of 2.8% of the USA's population in the latest research. In addition, ruptured AAA is associated with approximately 90% mortality risk. AAA diagnosis is made by ultrasound or computed tomography (CT), and many cases are detected incidentally. In addition, not all centers can diagnose AAA, so a more convenient screening test is essential. The biomarkers of AAA have been investigated, but various factors cause AAA and do not have a high diagnostic potential biomarker. Although some reports have used extracellular vesicles (EVs) and microRNAs within EVs, a biomarker with high diagnostic potential has yet to be recognized. In this study, we investigate whether EV-associated microRNA could be diagnostic biomarkers for patients with AAA.

Method: We compared microRNA in small EVs (sEVs) from human serum between AAA and non-AAA patients (controls). Diabetes mellitus (DM) was a known suppressor of AAA, so the study was divided patients according to the presence of DM. We collected serum samples from DM+AAA (N = 20), DM+non-AAA (N = 10), non-DM+AAA (N = 12), and non-DM+non-AAA (N = 12). The sEVs were isolated according to MISEV 2018 guidelines. The sEVs were characterized by nanoparticle tracking analysis, and CD9 and CD63 were confirmed by Western blotting.

Result: The significantly different 34 and 39 microRNAs were detected from DM and non-DM AAA patients compared to their respective controls (Log2 fold change > |0.5|, p < 0.05). The Receiver operatorating characteristic (ROC) curve of microRNA in sEVs alone shows an area under the curve (AUC) of 0.85 in DM and 0.87 in non-DM. KEGG pathway analysis of significantly different microRNA showed enrichment of “extracellular matrix (ECM)-receptor interaction” and “Axon guidance” pathways.

Summary/Conclusion: The sEVs-associated microRNA in the serum helps diagnose AAA. The pathway of “ECM-receptor interaction” and “Axon guidance” are associated with ECM degradation and angiogenesis.

Mr. Kittinun Leetanaporn, Mr. Jitti Hanprasetpong, Miss Wararat Chiangjoing, Mr. Sitthiruk Roytrakul, Miss Piyatida Molika, Mrs. Raphatphorn Navakanitworakul

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Background:

Cervical cancer is one of the most lethal cancers worldwide, especially in developing countries, where patients are presented at the locally advanced stage (LACC). The mainstay treatment of LACC is concurrent chemoradiation (CCRT), in which the response rate is about 60-70%. Therefore, exploring the biomarker that could enhance the predictability of response status remains necessary.

Methods:

A cohort of 63 patients diagnosed with LACC undergoing CCRT was divided into discovery (23 response, 10 non-response) and validation (20 response, 10 non-response) cohorts. Plasma samples were collected at three pivotal time points: initial diagnosis, 1-month post-treatment initiation, and 3-month post-treatment initiation, at which the patient was determined response status clinically. For EV isolation, the discovery cohort's plasma underwent a dual-phase method employing ultracentrifugation (UC) and size exclusion chromatography (SEC), while the validation cohort's samples underwent UC alone. Subsequently, the isolated extracellular vesicles were subjected to a quantitative proteomics analysis.

Results:

A total of 149 unique EV proteins were identified in the discovery cohort. The functional analysis of protein changes across each time point unveiled significant associations with the complement cascade, chylomicron remodeling, and proteins linked to insulin-like growth factor (IGF) and mitogen-activated protein kinase (MAPK) pathways. Notably, 73 of these proteins were also observed in the validation cohort (out of 204). Subsequent analysis of these shared proteins, utilizing the elastic-net model, revealed a subset of 23 proteins that played a pivotal role in determining response status after treatment, including SERPINA3, HBA1, FN1, and FGG, concurrently serving as cervical cancer prognostic gene markers in the TCGA public tumor dataset. Consequently, the top 11 out of 23 influential proteins from the discovery cohort significantly improved the area under the curve for CCRT response prediction from 0.694 (clinical only) to 0.871 (protein with clinical; p = 0.026) in the validation cohort.

Conclusion:

Analysis of plasma EV revealed a set of proteins that improved the predictability of CCRT response in cervical cancer patients. This finding suggested the potential utility of EV proteins for refining treatment strategies and improving outcomes in this challenging clinical context in the future.

Miss Chun Liu, Dr Chaminda Jayampath Seneviratne, Prof Sašo Ivanovski, Dr Pingping Han

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Extracellular vesicles (EVs) are nanoscaled lipid bilayered particles derived from most cells of different species, including host and bacterial derived EVs (BEVs) [1]. Oral bacterial derived BEVs contain a variety of microbial molecules, including enzymes, toxins, and microbial-associated molecular patterns (MAMP) [1], that can be transported to recipient host cells locally and systematically to cause periodontitis or other systemic diseases [1, 2]. In terms of host response, host derived EVs with encapsulated pro-inflammatory cytokines may contribute to the modulation of immune and inflammatory processes in oral disease pathogenesis [3]. Limited studies explored both dental plaque derived BEVs and saliva host EVs cytokine profiles. This study aims to a) understand the BEV component by comparing 16S sequencing profiles from 3D-mimicking saliva biofilm and b) assess the potential of immunoaffinity-enriched host EVs from saliva as diagnostic markers for periodontitis.

Methods

For BEV profiling, oral biofilms were cultured on 3D polycaprolactone (PCL) scaffolds and 2D plates. BEVs were isolated using size exclusion chromatography (SEC) and characterized using TEM, NTA, AFM, BCA, FTIR, DiO tracking and LPS assay, followed by genomic DNA qPCR and 16S sequencing. Simultaneously, host derived EVs were enriched from 12 non-periodontitis and 20 periodontitis patients’ saliva using SEC and bead-based immunoaffinity capture. And characterized by TEM, BCA, NTA, CD9 ELISA, FTIR, FACs cytometry and bacterial pathogens qPCR. Inflammatory cytokines (IL-6, IL-1β, IL-8 and IL-10) in host EVs were compared between 12 non-periodontitis and 20 periodontitis.

Results and Discussion

16s sequencing results suggest that BEVs exhibit strong enrichment ability and sensitivity with genera Capnocytophaga, porphyromonas and veillonella, phylum Firmicutes and Bacteroidota, and species Alloprevotella_tannerae, Capnocytophaga_sputigena, Veillonella_atypica and Prevotella_melaninogenica. Moreover, immunoaffinity-enriched salivary EVs from periodontitis patients displayed elevated pro-inflammatory cytokines (IL-6, IL-8) and reduced anti-inflammatory IL-10 compared to non-periodontitis individuals.

Conclusion

Investigating BEVs from oral biofilm and cytokine signatures in salivary host EVs could enhance our understanding of periodontitis pathogenesis, leading to more accurate diagnosis and targeted therapeutic interventions.

Dr Juliana Müller Bark, Dr Lucas Trevisan França de Lima, Dr Xi Zhang, Dr Daniel Broszczak, Dr Paul J. Leo, Dr Rosalind L. Jeffree, Dr Benjamin Chua, Dr Bryan W. Day, Professor Chamindie Punyadeera

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Glioblastoma (GBM) is a highly aggressive cancer with poor prognosis that needs better treatment modalities. Identification of novel prognostic biomarkers would lead to better stratification of risk and may prevent disease relapse. Cancer-derived small extracellular vesicles (sEVs) are emerging as biomarkers and contain unique macromolecules (DNA/RNA/proteins. sEVs are released from cells into the surrounding environment. Brain-derived sEVs can cross the blood-brain barrier and are easily isolated from biofluids. Studies have demonstrated the potential clinical utility of plasma-derived sEVs in glioma patients. However, little is known about the clinical utility of saliva-derived sEVs in GBM.

Methods

sEVs were isolated from whole mouth saliva of GBM patients at two time points, pre and posttreatment. sEVs were isolated using differential centrifugation/ultracentrifugation. sEVs were characterized by concentration, size, morphology, and EVs cell surface protein markers. Protein cargo in sEVs was discerned using mass spectrometry.

Results

We found no difference in pre and post-operative salivary sEVs in terms of size and concentration. We found four highly abundant proteins (ALDOA, 1433E, ECH1 and TM11B) in preoperative saliva samples from GBM patients with poor outcomes. The protein abundance of ALDOA was confirmed by western blotting. In addition, functional enrichment analysis of pre and postoperative saliva samples showed significant enrichment in several pathways, including those related to the immune system, cell cycle and programmed cell death.

Conclusion

sEVs isolated from saliva samples of GBM patients revealed GBM-related proteins. Our data encourage further studies on salivary small EVs as a source for prognostic biomarkers to evaluate GBM non-invasively.

Professor Sung Shin, Dr. Hye Eun Kwon, Dr. Mi Joung Kim, Professor Heungman Jun, Professor Sang Jun Park, Professor Jun Gyo Gwon

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

The severity of kidney allograft fibrosis is one of the most important factors affecting long-term graft survival after deceased-donor kidney transplantation. In this study, we tried to identify and validate urinary exosomal miRNA biomarkers which may reflect the grade of interstitial fibrosis and tubular atrophy (IFTA).

2) Methods

We collected urine samples from 109 deceased donors at the time of solid organs recovery from May 2019 to June 2021, and a zero-day biopsy was performed before transplantation at four medical centers in Korea. Among 109 specimens, 34 showed no IFTA on zero-day biopsy (No IFTA group) and the other 75 allografts showed IFTA score 1 or more than 1 on zero-day biopsy (IFTA group). Urinary exosomes were isolated by ultracentrifugation and the levels of miRNAs were quantified by qRT-PCR.

3) Results

After reviewing previous reports and electronic databases, a total of six miRNAs (miR-19, miR-21, miR-29c, miR-150, miR-200b, and miR-205) were chosen as potential biomarker candidates for IFTA. miR-16-5p was used as an endogenous control. Among the six candidates, relative expression levels of miR-21, miR-29c, miR-150, and miR-205 were significantly higher in the IFTA group whereas miR-19 expression level was significantly lower in the IFTA group compared with the No IFTA group. ROC analysis of miR-21 (AUC, 0.762; 95% CI, 0.658–0.846; p<0.001) and miR-29c (AUC, 0.825; 95% CI, 0.727–0.898; p<0.001) showed good diagnostic accuracy for predicting IFTA.

Although there were no differences in patient survival, graft survival, and rejection between two groups, the eGFR level of No IFTA group at 1week post-transplant was higher than IFTA group (41.34 vs. 28.65, p-value = 0.012) and the improvement patterns of eGFR over time showed significant difference (Time*Group p-value = 0.031).

4) Conclusion

In conclusion, urinary exosomal miRNAs are potent biomarker candidates to determine the IFTA severity of kidney allograft before recovery.

Ms. Chitra Bhardwaj, Dr. Priyanka Srivastava, Dr. Minakshi Rohilla, Dr. Seema Chopra, Dr. Anupriya Kaur, Dr. Inusha Panigrahi

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Recurrent pregnancy loss (RPL) is one of the common and adverse pregnancy complication, affecting 2-5% couples around the globe. Endocrine factors, uterine anomalies, chromosomal abnormalities and infections are some of the common causes, however, in 50% of cases the cause is still unknown. During pregnancy miRNAs enclosed in extracellular vehicles (EVs) are circulated in maternal blood and plays an important role in maternal-fetal communication. Therefore, profiling of these EVs will provide insights into the molecular pathways involved in trophoblast cell invasion, migration and placentation during embryo growth and thus provide better understanding of RPL pathogenesis.

Methods: EVs were isolated from the maternal plasma samples of RPL patients collected at <22 weeks of pregnancy (n = 10) and gestational aged-matched healthy pregnant females as controls (n = 5), using Total Exosome Isolation Reagent. Transmission electron microscopy, Nanoparticle tracking analysis and Flow cytometry were performed to determine morphology, size, concentration and EV markers. Total RNA was extracted and processed for miRNA sequencing using Illumina NovaSeq 6000 platform. Partek Flow software was used to process the raw files and DESeq2 to identify differentially expressed miRNAs (DEM). Target prediction and enrichment was done using Target Scan and miRTarBase. Gene ontology and KEGG pathway analysis was done using CytoScape.

Results: A total of 2043 DEMs were identified, out of which 559 were known miRNAs, while 1484 were novel miRNAs. Further, 66 miRNAs were significantly differentially expressed between patients and controls (log2 FC ≥ 1, P < 0.05), among which 29 were upregulated and 37 were downregulated. Chromosome 19 miRNA cluster (C19MC) and Chromosome 14 miRNA cluster (C14MC) are placenta specific and identified in maternal circulation only during pregnancy. We found 6 miRNAs from C19 cluster (5 upregulated, 1 downregulated) and 2 from C14 cluster (1 upregulated, 1 downregulated) in our patients. KEGG pathway analysis showed molecular links for dysregulated immune pathways, cell cycle pathways and perinatal lethality.

Conclusion: Exosomal miRNAs from C19C and C14C could be used as predictive biomarkers for idiopathic RPL patients as they play a crucial role in placentation, embryonic development and regulation of immune response at feto-maternal interface.

Ms. Hannah O'Toole, Ms. Neona Lowe, Ms. Visha Arun, Ms. Anna Kolesov, Prof. Tina Palmieri, Prof. Nam Tran, Prof. Randy Carney

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Severe wound infection (sepsis) following burn trauma is a global complication with high mortality. Diagnosing sepsis is often complicated by confounding clinical manifestations. Current investigations into sepsis biomarkers using cytokine/chemokine markers lack the sensitivity and specificity required for prompt treatment. Circulating extracellular vesicles (EVs) are released by sepsis-associated pathogens and play a role in the downstream inflammatory response, thus they have promise to be potent sepsis biomarkers. This study applies label-free Raman spectroscopic analysis of EVs isolated from patient plasma as for rapid, sensitive, and specific detection of sepsis.

Methods: Plasma-derived EVs from septic (n = 8) and non-septic burn patients (n = 6) were isolated via size-exclusion chromatography (SEC) and characterized via resistive pulse sensing (RPS), nanoparticle tracking analysis (NTA), hybrid immunofluorescence/interferometric imaging, cryo-electron microscopy (cryoEM), and Raman spectroscopy. For Raman spectral acquisition, 2µL of SEC-isolated EVs were dried on quartz substrates and analyzed using a custom-built inverted confocal Raman system. Five random spectra (90s each) were sampled from each patient sample (25mW laser power at sample, 785nm incident wavelength). Spectra were background corrected, smoothed, and normalized for downstream analysis by principal component analysis (PCA).

Results: PCA and quadratic discriminant analysis (QDA) effectively differentiated EV Raman profiles of septic from non-septic burn patients. The first five principal components achieved 97.5% sensitivity, 90.0% specificity, and 94.3% accuracy. Of the 70 patient spectra collected, just 1 was falsely predicted as non-septic and 3 were falsely predicted as septic, yielding a 2.5% false negative rate and a 10% false positive rate. The global average Raman spectrum of the difference between septic and non-septic burn patient EVs was compared with spectra of glycoconjugate biomarkers of four top bacterial strains associated with sepsis morbidity in burn patients and showed fitting with lipopolysaccharides specific to K. pneumoniae and E. coli.

Summary/Conclusion: These proof-of-concept results suggest that Raman spectroscopy of circulating EVs can provide sensitive, specific, and label-free detection of sepsis for clinical use. Increasing patient samples analyzed and exploring additional bacterial EV biomarkers and underlying patient microbiome differences are follow-up work interests. Nevertheless, this work demonstrates the promising application of EVs as biomarkers in clinical trauma care.

Md.PhD Yoshito Takeda, MD Hanako Yoshimura, MD.PhD Yuya Shirai, MD Takahito Enomoto, PhD Jun Adachi, MD.PhD Atsushi Kumanogoh

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

BA is a complex respiratory disease characterized by airway inflammation, and variable airflow limitation and is complicated by chronic rhinosinusitis with nasal polyps (CRSwNP). Novel biomarkers (BMs) are urgently needed for bronchial asthma (BA) with various phenotypes and endotypes. To identify novel BMs reflecting tissue pathology from serum extracellular vesicles (EVs)

Methods

In a next-generation proteomics-based approach, we performed data-independent acquisition (DIA) of serum EVs from four healthy controls, four eosinophilic BA patients (EA), and four atopic asthma (AA) patients to identify novel BMs of BA. We confirmed EA-specific BMs via DIA validation in 61 BA patients and 23 controls. To further validate these findings, we performed DIA in six chronic rhinosinusitis without nasal polyps and seven chronic rhinosinusitis with nasal polyps patients. EVs were isolated using the phosphatidylserine (PS) affinity method according to MISEV 2018.

Results

We identified as much as 3032 proteins, of which 23 and 38 were significantly changed in EA and AA, significantly. By ingenuity pathway analysis, protein signatures from each phenotype reflected disease characteristics. Validation revealed five EA-specific BMs, including galectin-10 (Gal10), eosinophil peroxidase (EPO), bone marrow proteoglycan, eosinophil-derived neurotoxin, and arachidonate 15-lipoxygenase. Notably, the potential of Gal10 in EVs was superior to that of eosinophils in terms of diagnosis, the detection of airway obstruction and bronchial wall thickening. In rhinosinusitis patients, 1752 and 8413 proteins were identified from EVs and tissues, respectively. Among 11 BMs identified from both EVs and tissues among patient with nasal poyps, five proteins (including Gal10 and EPO) showed significant expression correlation between EVs and tissue. Moreover, Gal10 in EVs release implicated in eosinophil extracellular trapped cell death in vitro and in vivo.

Conclusions

We identified novel biomarkers for bronchial asthma via state-of-the-art proteomics of serum EVs and tissues. Novel BMs such as Gal10 from serum EVs reflect disease pathophysiology in BA and may represent a new target of liquid biopsy.

Ms Yu Jin Lee, Dr Jie Ni, Dr Valerie Wasinger, Mr Qi Wang, Dr Joanna Biazik, A/Prof Peter Graham, Prof Yong Li

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Breast cancer (BC) is the leading cause of cancer-related death among women worldwide. Currently, common methods for detecting BC, mammograms, are inaccurate and not reliable for detecting small lesions or dense breast tissue. Surgical biopsies cannot provide accurate and real-time information if the target cannot be imaged or is partially sampled. Small extracellular vesicles (sEVs) are secreted by all cell types, containing various biological cargoes that reflect their cellular origin. sEVs are an important intercellular communicator, participating in all stages of cancer metastasis, immunity, and therapeutic resistance. Studying sEVs in liquid biopsy for BC diagnosis is a new developing research area. Therefore, disease specific proteins contained in sEVs are considered as a superior choice for non-invasive liquid biopsy biomarker source in BC.

Aims: The objective of our study was to identify potential sEV protein biomarkers from BC cell lines and human BC plasma samples for BC diagnosis by LC-MS/MS proteomics.

Methods: sEVs derived from three BC cell lines (MDA-MB-231, MCF-7, and SK-BR-3), one normal breast cell line (MCF-10A), BC patients' plasma (n = 3), and non-cancer controls (n = 3) were isolated using ultracentrifugation (UC). The plasma samples were obtained from Health Precincts Biobank. Human ethics approval was obtained from the UNSW Australia Human Research Ethics Advisory Panels. The isolated sEVs were characterised by nanoparticle tracking analysis, transmission electron microscopy, flow cytometry, and western blotting. The total proteome was profiled by LC-MS/MS from BC cell lines and plasma samples to identify potential sEV protein biomarkers.

Results: We identified sEV protein CEBPZ, CHMP1A, CLTA, IFITM1, SEC13, VTA1, BDH2, INS, LAMA3, and TPX2 as potential biomarkers in BC cell lines, while we identified 6 potential sEV protein biomarkers including TUBB1, HPR, ITIH1, IGHA1, LPA, and APOA4 in human BC plasma samples.

Conclusion: This study identified a group of potential sEV protein biomarkers using BC cell lines and human BC plasma samples. These biomarkers identified need to be validtated in a large set of clinical samples for further assessing their clinical significanece in BC diagnosis of liquid biopsy.

Postgraduate Zehong Peng, Postgraduate Yuning Wang, Postgraduate Xinrui Wu, Postgraduate Xingxing Du, Postgraduate Cong Hu, Postgraduate Yanhao Dong, Postgraduate Qi Chen, Postgraduate Yang Ge, Professor Kun Qian, Associate Research Fellow Liang Dong, Professor Wei Xue

Poster Pitches (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:45 PM - 1:00 PM

Introduction

The Prostate Imaging Reporting and Data System (PI-RADS) scoring system is used for multi-parametric magnetic resonance imaging to evaluate prostate cancer lesions, which are divided into levels 1-5. Prostate biopsy is clinically recommended for patients with PI-RADS score≥4, but the positive biopsy rate is not satisfactory. This study utilized key metabolites in serum extracellular vesicle (EV) metabolomics for non-invasive early diagnosis of prostate cancer (PCa) with PI-RADS score≥4. We also compared the diagnostic value of serum EV metabolomics with EV-free serum metabolomics.

Methods

We separated EV and EV-free serum components using size exclusion chromatography (SEC) and obtained their metabolic fingerprints using the ferric nanoparticle-assisted laser desorption/ionization mass spectrometry (FeNPALDI-MS) detection platform. We compared the diagnostic value of EV and EV-free serum metabolic fingerprints and screening key metabolites using machine learning and differential analysis respectively. Simultaneously, we combined key metabolites in EV with prostate-specific antigen (PSA) to further improve diagnostic efficiency. Finally, we discovered multiple metabolic pathways associated with the progression of PCa.

Results

We successfully separated EV and EV-free serum and constructed their metabolic fingerprints, with area under the curve (AUC) of 0.72 and 0.55 respectively. Using the same criteria for differential analysis and four types of machine learning, we identified 8 key metabolites from EV and 6 differential metabolites from EV-free serum. We found that the diagnostic ability for PI-RADS≥4 PCa of key metabolites in EV is better than that of EV-free serum, with mean AUC values of 0.75 and 0.63. Combining 8 key metabolites in EV with PSA, we obtained AUC values of 0.89 for both the training and testing groups, indicating their great clinical predictive ability for PI-RADS≥4 PCa, and identified ten metabolic pathways related to PCa progression.

Conclusions

In this study, we successfully developed a non-invasive and accurate method for diagnosing PI-RADS≥4 PCa using key metabolites in EV combined with PSA. The diagnostic efficacy of EV was superior to that of EV-free serum, suggesting the potential value of serum EV in subsequent prostate cancer diagnosis.

Mr Abolfazl Jangholi, Dr Sarju Vasani, Prof Liz Kenny, Prof Sudha Rao, Prof Riccardo Dolcetti, Prof Chamindie Punyadeera

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Head and neck cancers (HNC) are highly immunosuppressive and clinically challenging, which in turn necessitates multidisciplinary management approaches. Despite aggressive treatment modalities, 20-50% of HNC patients experience relapses. Small extracellular vesicles (sEV) which are widely found in various body fluids, play a crucial role in communication between cells. Saliva and plasma-derived sEV carry molecular cargo indicative of tumour development, metastasis, immune suppression, and therapy resistance. Thus, plasma and saliva sEVs could serve as minimally invasive biomarkers of tumour progression. We aim to develop a sEV signature that can be used as prognostic biomarkers for HNC.

Methods: We collected plasma and saliva samples from HNC patients (n = 36) at the time of diagnosis and followed for up to 2 years. The plasma and saliva sEVs from HNC patients with and without recurrence were isolated using size exclusion chromatography and ultracentrifugation methods, respectively. We assessed the morphology, size, concentration, and markers of salivary and plasma sEVs. We unravelled the protein cargo of plasma and salivary sEVs using SWATH mass spectrometry (SWATH-MS).

Results: No significant differences were observed in the size and concentration of plasma and saliva sEVs derived from HNC patients with and without recurrence. Using quantitative SWATH-MS technology, we identified 38 and 33 (p<0.05) differentially abundant proteins in plasma and salivary sEVs, respectively. We found proteins regulating HNC metastasis, migration and invasion. In addition, we have also observed immune-associated proteins involved in immune tolerance and therapy resistance to be significantly different between recurrent and non-recurrent HNC.

Conclusion: The protein profiles of plasma and salivary sEVs are promising for the development of non-invasive and valuable biomarkers for accurate prediction of prognosis and appropriate selection of therapy. Also, a combination of plasma and saliva sEVs-derived biomarkers proves highly beneficial in finding the strategies to early detect and prevent recurrence.

Doctor Qiu-Yun Fu, Professor Gang Chen

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Small extracellular vesicles (sEVs) residing at tumor tissues are valuable specimens for biopsy. Tumor heterogeneity is common across all cancer types, but the heterogeneity of tumor tissue-derived sEVs (Ti-sEVs) is undefined. This study aims to discover the spatial distributions of Ti-sEVs in oral squamous cell carcinoma (OSCC) tissues and explore how these vesicle distributions affect the patients’ prognosis.

Methods

Four spatial distinct sites of the OSCC were sampled: tumor (Tu, n = 39), tumor margin (TM, n = 36), and para-tumor (PT, n = 30), as well as a distant control mucosa (CM, n = 41). Ti-sEVs of OSCC were isolated by collagenase dissociation, followed by ultracentrifugation. Ti-sEVs paired plasma sEVs were isolated by ultracentrifugation. Sirius red staining was used to determine status of collagen fibers, and mass spectrometry-based proteomics was employed to determine the degradation effect of Ti-sEVs on the tumor extracellular matrix. Ti-sEVs (n = 33) of patients before and after neoadjuvant anti-PD-1 immunotherapy (NCT04649476) were harvested for assessing their temporal variations.

Results

Multi-regional sampling enabled us to uncover that Ti-sEVs’ accumulation at peritumoral sites correlates with a higher disease-free survival rate, and conversely, sparse peritumoral Ti-sEVs tend to forecast a higher risk of relapse. Of those relapsed patients, Ti-sEVs strongly bind to extracellular matrix and subsequently degrade it for allowing themselves enter the bloodstream rather than staying in situ. In advanced OSCC patients, the quantity and spatial distribution of Ti-sEVs prior to anti-PD-1 treatment, as well as the temporal variance of Ti-sEVs before and after immunotherapy, strongly map the clinical response and can help to distinguish the patients with shrinking tumors from those with growing tumors.

Summary

Our work elucidates the correlation of spatiotemporal features of Ti-sEVs with patients’ therapeutic outcomes and exhibit the potential for using Ti-sEVs as a predictor to forecast prognosis and screen the responders to anti-PD-1 therapy.

Rejection after kidney transplantation

Professor Sung Shin, Dr. Mi Joung Kim

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

This study aimed to identify urine-derived exosomal miRNAs as diagnosis biomarkers associated with antibody-mediated rejection (ABMR) after kidney transplantation.

2) Methods

Exosomal miRNAs using differential ultracentrifugation were isolated in urine from 24 donors and kidney transplant recipients with 12 ABMR, 8 T cell-mediated rejection (TCMR), 5 BK virus nephropathy (BKVN), and 11 no major abnormality (NOMOA). We characterized exosomes using transmission electron microscopy, nanoparticle tracking analysis, Western blotting and analyzed miRNAs using RNA sequencing to identify exosomal miRNA candidates in the discovery cohort. We also quantified the expression of miRNA candidates using RT-qPCR in a validation cohort consisted of 20 ABMR, 5 TCMR, 8 NOMOA, and 8 donor. The diagnostic potential of the exosomal miRNAs was evaluated by receiver operating characteristic (ROC) curves.

3) Results

Four miRNAs consisting of miR-221-3p, miR-455-3p, miR-126-3p, and miR-215-5p were significantly higher in the ABMR group than in NOMOA group. Additionally, miR-221-3 and miR-215-5p were highly expressed in the tissue of patients with ABMR. Compared to each miRNA biomarker, the four combined exosomal miRNAs (miR-221-3p, miR-455-3p, miR-126-3p, miR-215-5p) improved the prediction performance determined by ROC curves in the validation set.

4) Conclusion

Urinary exosomal miRNAs can be potent biomarkers for ABMR after kidney transplantation.

Dr. Ayako Muraoka, Dr. Akira Yokoi, Dr. Kosuke Yoshida, Mrs. Masami Kitagawa, Dr. Hiroaki Kajiyama

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction: The use of assisted reproductive technology (ART) for fertility treatment is increasing substantially. Evaluation methods of oocyte quality that can be used to predict successful pregnancy outcome are desired. Follicular fluid (FF) contains extracellular vesicles (EVs) that have small non cording RNAs (ncRNAs). These can be used to evaluate oocyte quality. Here, we investigated specific small RNAs in FF-EVs that could be used to predict successful pregnancy outcome in ART.

2) Methods: From 20 ovarian follicles of 15 patients with infertility who were undergoing ART, FF was collected from each patient's oocyte. EVs were isolated from each FF sample, and small RNA-sequencing was performed. Potential candidate small RNAs were identified by comparing patients who became pregnant (n = 8) with those who were not (n = 12).

3) Results: The EVs in FF were successfully isolated and characterized by nanoparticle tracking assays and cryo-electron microscopic analyses. Small RNA-sequencing identified 430 small RNAs that could be analyzed for as candidate biomarkers for determining the success rate of ART. We focused on micro RNAs (miRNAs) and P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) among small ncRNAs. In pregnant women, two miRNAs were significantly increased in the FF-EVs, while five miRNAs and seven piRNAs were increased in non-pregnant women. Furthermore, we found that a specific combination of 3 miRNAs could predict successful pregnancy precisely using receiver-operating characteristics (ROC) curves analysis (area under the curve (AUC): 0.96). Functional prediction using these specific miRNAs revealed that these miRNAs are involved in follicular development.

4) Conclusion: Our results demonstrate that small RNAs in FF-EVs could be used as novel non-invasive biomarkers to predict pregnancy outcome in ART. Further studies are required to examine the functional importance of these small RNAs in FF-EVs to elucidate their pathological mechanism and process of follicle development.

drg. Dhanni Gustiana¹, Dr. Cynthia Retna Sartika², Mrs. Rima Haifa³, Ms Marsya Nilam Kirana³, Mrs Nisa Zulfani³, Ms Karina Kalasuba³, Mrs Ditta Kalyani Devi³, Mrs Vinessa Dwi Pertiwi³

¹RSUD Tangerang Selatan, Tangerang, Indonesia, ²Faculty of Pharmacy Universitas Padjadjaran, Jatinangor, Indonesia, ³Prodia StemCell Indonesia, Central Jakarta, Indonesia

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Temporomandibular disorders affect the jaw muscles and/or temporomandibular joint (TMJ), with a particular focus on bone and joint issues. Physical therapy has long been used to address TMJ disorders, but evidence supporting its efficacy is limited. Physical therapy typically includes exercise instruction, manual techniques, patient education (covering posture, relaxation, and parafunctional habits), and tissue health modalities. However, the effects of physical therapy are often temporary. Recently, regenerative medicine treatments, such as intra-articular administration of Umbilical Cord-derived Mesenchymal Stem Cell (UC-MSC) secretome, have shown promise in reducing pain and improving jaw mobility.

Methods: The study involved three subjects diagnosed with acute TMJ dysfunction, consisting two males (Patient 1 and Patient 2) and one female (Patient 3). Patients received intra-articular injections of UC-MSC secretome. Patients 1 and 2 were administered 5 cc in the right joint, while Patient 3 received 4 cc in the right joint and 1 cc in other joints. Pain severity and quality of life were assessed before and one-month after injection of UC-MSC secretome using the Visual Analog Scale (VAS).

Results: Following the administration of the UC-MSC secretome, the patient demonstrated significant improvements in their medical condition. In all subjects, the pain during therapy was significantly reduced. The VAS pain at rest demonstrated notable changes: from 8 to 4 for Patient 1, from 9 to 2 for Patient 2, and from 7 to 5 for Patient 3. The positive results highlight substantial advancements in the patient's comprehensive well-being and functional capability of TMJ patients, including improvements in masticatory function and the absence of joint sounds in the TMJ.

Conclusion: The UC-MSCs secretome has demonstrated exceptional efficacy in pain relief, with no adverse events reported. The UC-MSC secretome is of utmost importance due to its anti-inflammatory properties, which provide substantial alleviation of the discomfort commonly associated with TMJ dysfunction. Moreover, it has the ability to enhance joint function, thereby benefiting overall TMJ health. Continued monitoring and further research are essential to elucidate the long-term benefits and broader applications of this innovative therapeutic modality.

Dr Lisa Hasibuan¹, Dr Cynthia Retna Sartika^2,3, Mrs. Rima Haifa², Miss Atikah Anwar Hasibuan², Mrs. Ditta Kalyani Devi², Mrs. Adina Novia Permata Putri²

¹Immanuel Hospital, Bandung, Indonesia, ²Prodia StemCell Indonesia, Central Jakarta, Indonesia, ³Faculty of Pharmacy, Universitas Padjajaran, Jatinangor, Indonesia

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Chronic ulcers, characterized by their prolonged healing process exceeding three months, present a complex challenge, encompassing various wound types such as those associated with diabetes, arterial insufficiency, and pressure. Conventional treatments often prove insufficient in effectively addressing these persistent wounds. However, emerging therapies utilizing stem cells offer a promising avenue in wound care. Stem cells possess an extraordinary capacity for differentiation into diverse cell types, providing a regenerative potential that surpasses conventional approaches. Furthermore, stem cell therapy has shown efficacy across a range of medical conditions, with minimal reported adverse effects. Recent research indicates that mesenchymal stem cells extend their therapeutic capabilities beyond mere cell-to-cell interactions, involving a diverse array of bioactive substances known as the secretome or conditioned medium (CM). This study delves into the evolving landscape of MSC-based therapies and their application in treating chronic ulcers

Methods:

A 57-year-old female patient diagnosed with a chronic ulcer, who had undergone skin graft but no improvement, underwent therapy involving the administration of Umbilical Cord-derived Mesenchymal Stem Cell (UC-MSC) and UC-MSC secretome. The methodology employed in this study entailed intralesional injections, with a total of four injections administered at weekly intervals. Initially, UC-MSC injections were delivered at a dosage of 2x10⁶ cells. Subsequently, for the following three sessions, patients received 1 cc of UC-MSC secretome. The parameters observed in this study are ulcer depth and ulcer pigmentation

Results:

After therapy, significant improvements were observed in the ulcer. Notably, there was a substantial reduction in ulcer depth and pigmentation. Ulcer depth decreased from 1 cm at the baseline to 0.3 cm post-therapy and the pigmentation shifted from slough to epithelial tissue. These enhancements are attributed to the multifaceted mechanisms of action exerted by UC-MSCs and UC-MSC secretome, which include promoting inflammation suppression, augmenting collagen deposition, and modulating the immune response

Conclusion:

The results showed that UC-MSC and UC-MSC secretome have therapeutic potential in facilitating tissue regeneration within chronic ulcer management. Further trials involving larger and more diverse populations are necessary to establish the positive benefits of UC-MSC and UC-MSC secretome in chronic ulcer management

Senior Expert Ikuo Kawauchi¹

¹Fujifilm Holdings, Tokyo, Japan

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

The International Conference on Harmonization of Pharmaceutical Regulations (ICH) has issued guidelines for each topic in the areas of quality, efficacy, and safety of pharmaceutical products, and these guidelines are widely used, especially for small molecule drugs and biologics. On the other hand, since cells are outside the scope of the various ICH guidelines, stakeholders are using consensus standards mainly developed by the International Organization for Standardization (ISO) for regenerative medicine voluntarily, and in some countries, regulatory authorities recommend the use of these standards.

International standards in the field of regenerative medicine are mainly developed in ISO/TC 276 (technical committee for “biotechnology”), and the standards issued or under consideration can be categorized as “analytical methods,” “peripheral industries,” and “manufacturing. A representative standard for analytical methods is ISO 23033” (General requirements and considerations for the testing and characterization of cell therapy products), which was jointly developed by Japan and the U.S. Since cells are not in the scope of the ICH Q6 series, it is positioned as its cell version. Standards related to products and services of peripheral industries cover ancillary materials such as reagents and culture media, equipment systems including consumables, and transportation, and specify the roles of both suppliers and users. The standard related to manufacturing is “cell manufacturing process management system” that prescribes the PDCA (Plan-Do-Check-Act) cycle, in which risk assessments regarding unstable factors are conducted, management plans are formulated, trial manufacturing is performed, and reviewed, and risk assessments are conducted again, when necessary, to achieve consistency in quality of cell manufacturing throughout the product lifecycle based on ISO 9001 (quality management system). The system is being developed as a Japanese Industrial Standard (JIS).

For cell culture, which is a part of production process of extracellular vesicles, it is possible to utilize the existing standards mentioned above. On the other hand, it is also necessary to consider the development of new standards specifically for extracellular vesicles. I would like to introduce“ fit for purpose selection of purification methods,” which is now under study at ISO/TC 276, and discuss standardization issues that should be considered.

Doctoral Researcher Ehsanollah Moradi, Ph.D. Päivi Järvinen, Ph.D. Markus Haapala, Associate Professor Tiina Sikanen

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

Tissue-selective isolation and characterization of extracellular vesicles (EV) is a challenging task, requiring many purification and enrichment steps. In recent years, both magnetic bead-based assays, as well as microfabricated plasmonic and electrochemical sensors, have been introduced for tissue-selective EV isolation via immunocapturing. However, there is still a dire need for platforms enabling in situ activity-based characterization of the exosomal enzymes, such as the cytochromes P450, that catalyze the majority of drug metabolic reactions but manifest large inter-individual variations.

2) Methods

Here, we present the development of a microfluidic chip enabling tissue-selective EV isolation via immunocapturing as well as on-chip determination of exosomal P450 activities. The platform also incorporates integrated gold sensors facilitating quantitation of the isolated EVs through Electrochemical Impedance Spectroscopy (EIS). The chip performance is demonstrated with EVs isolated from 3D cultures of human hepatoma cells and primary human hepatocytes (PHH), and human plasma. Prior to application, samples are purified and enriched (5-fold) by ExoEasy kit.

3) Results

The tissue-selectivity of the microchip was demonstrated using hepatocyte, cardiomyocyte, and erythrocyte-selective antibodies and the feasibility for EV quantitation (based on EIS) cross-validated against nanoparticle tracking analysis revealed a good correlation between the two methods. The P450 activities of the PHH-secreted EVs were characterized using an in-house enzyme activity assay setup and were shown to qualitatively correlate with the P450 activity of the PHH spheroids. For example, the exosomal CYP3A4 activity declines after initiation of the culture but gradually increases as the spheroids mature from Day 6 onward, following the same trend as cellular CYP3A4 activity. The feasibility of the chip for measuring P450 activities of liver EVs isolated from human plasma was additionally demonstrated with respect to CYP1A2, CYP2C9, CYP2D6, and CYP3A4 with ten randomized blood samples from volunteer donors.

4) Conclusion

This microfluidic platform is a promising tool for rapid and tissue-selective EV isolation, enzyme activity characterization, and on-chip quantitation for normalizing exosomal enzyme activities to tissue-specific EV concentration. With a view to P450 phenotyping, this chip could provide a viable alternative for probe drug administration.

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, France

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

EV-based vaccines offer numerous advantages, including their ability to mimic natural antigen presentation pathways, stimulate both innate and adaptive immune responses, and confer long-lasting immunity. Furthermore, EVs can be easily modified to enhance their immunogenicity and can be derived from various cell types, such as pathogen-infected or cancer cells, facilitating the development of personalized vaccines. As research in this field advances, the potential of harnessing EVs as vaccines holds immense promise for the prevention and treatment of a wide range of infectious diseases, cancers, and other immune-related disorders. However, to fully realize the potential of EV-based vaccines, standardization plays a crucial role in advancing EVs research.

Methods

A standardized protocols was established for the isolation, characterization, and functional analysis of EVs. We used automation methods for these processes and compared it with the manual processing. Comparison of experienced versus naïve users was also performed. We also measured immune responses by flow cytometry in the context of infectious disease. We compared the performance of manual, liquid antibody processing versus the use of dry-reagents.

Results

Automated density-based separation EVs (from plasma and uring) reduces variability their recovery when using orthogonal methods. Reproducibility, recovery, and specificity is the highest with automation. Analysis by mass-spectrometry revealed the higher variability in the content of EV linked to manual processing. Not only automation increases reproducibility but reduces errors. Immune monitoring to follow treatment efficacy is limited by the variability of multi-center studies. Standardizing the process with dry reagents enable to include immune profiling in the One Study. The data demonstrates the robustness of the method and the significantly reduced variability of flow cytometry-based immune monitoring.

Conclusion

By establishing standardized EV preparation, counting, characterization, and production methods, as well as standardized approaches to assess their biological effects, we can significantly increase the reproducibility of EV-based vaccines. Integrating these processes from the early phases of R&D to the immune profiling in research clinical trials would improve their generalizability and enhance the overall advancement of EV-based vaccine research.

Parvez MD SORWER ALAM, Takahiro Kochi, Prof Shin-ichi Kano, Prof Atsuo Sasaki, Prof Kazuhiko Tabata, Dr. Eisuke Dohi

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

Extracellular vesicles have an advantage as biomarkers, because simultaneous measurement of cell-specific markers and contents is possible in principle. However, the profiles of miRNAs in human serum/plasma in published papers vary widely from study to study, and the profiles of miRNAs in blood-derived EVs for the same disease are inconsistent. Several reasons have been postulated, including the effects of platelet contamination and diurnal variation. In addition to these, the limitations of Bulk analysis should also be considered. In order to utilize extracellular vesicles as biomarkers, analysis at the single-particle level is required. In this study, we attempted to stain tetraspanins on the surface of extracellular vesicles with antibodies and detect internal miRNAs with gold particle molecular beacons using NanoFCM at the single-particle level.

2) Methods

Human plasma (pool, citric acid) 1ml were aliquot stored -80C and EVs were isolated with iZon qEV (Gen2 qEV1 35nm). Fraction 7-11 were concentrated with Amicon Ultra 4 (10k). Then, isolated EVs were incubated with several detergent (Saponin, SLO or ReagentX) to load the Gold-particle molecular beacon targeted to has-miR-223-3p (Zhao J et al., J Am Chem Soc 2020). And stained EVs were analyzed with NanoFCM.

3) Results

With previously reported methods such as MgCl2 and other detergents, only about 1% of EVs could be detected by the gold particle beacon. However, when Reagent X was used, the EVs detected by the gold particle beacon could be improved to 10%, and the EVs that were positive could be double-stained with CD9.

4) Summary/Conclusion

The EV used in this study was recovered by SEC, but most of the previously reported literature recovered by ultracentrifugation. It has been reported that the ultracentrifugation can damage the integrity of EV membranes, which may have affected the efficiency of loading.

It is desirable to develop a system to detect miRNAs in EVs with higher sensitivity and multiplex manner.

Ms Harleen Kaur, Professor Nathan Bartlett, Doctor Renee V Goreham

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Infectious diseases are a primary cause of severe health problems that burden the health system and our economy. A current and obvious example is the recent coronavirus pandemic, an airborne respiratory pathogen that has affected the global population for over two years. The recent pandemic has brought the danger to the forefront and there is an urgent need for surveillance testing for viruses to monitor and track infections within the community. Currently, the main method of detection follows an intrusive sample collection from high up in the nasal cavity. Therefore, the development of a cheaper, and easier detection platform is required. This project has isolated and characterized extracellular vesicles derived from primary human bronchial epithelial cells for the first time. Transmission electron microscopy analysis of extracellular vesicles showed the diameter size range of 50 nm to 200 nm for isolated extracellular vesicles. Nanoparticle tracking analysis determined a concentration of 1.24 x 10-9 particles/mL, confirming the presence of extracellular vesicles. Extracellular vesicles are heterogeneous in nature, secreted by all cell types and are modulators in pathological processes. A gold-plated electrode will be modified with a thiol-based aptamer to target CD63, a generic marker for extracellular vesicles, as a detection platform. Changes in current and resistance at the gold surface after each modification step will be probed using electrochemical techniques, mainly voltammetry and electrical impedance spectroscopy. The next step will isolate and compare extracellular vesicles derived from primary human bronchial epithelial cells that have been exposed to a virus (OC43). From here, biomarkers specific to cells exposed to the virus will be used to develop novel aptamers. These aptamers will be used to engineer a highly sensitive device that can be used for early detection and monitoring of viral detection.

Kimberly Luke, Casey Scott-Weathers

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction.

Prostate cancer (PCa) is the most frequently diagnosed non-cutaneous urological cancer in men. The Prostate Specific Antigen (PSA) test is the current standard. However, false positive results are common because PSA is an organ-specific, but not cancer-specific, biomarker. Current PSA screening tests do not discriminate between malignant (PCa) and benign (benign prostatic hyperplasia, BPH) conditions, resulting in a high rate of false-positive results. Extracellular vesicles isolated from both PCa cell lines and from PCa patients contain PSA (EV-PSA). We propose that EV-PSA can better distinguish between PCa, benign prostatic hyperplasia (BPH), and healthy controls when compared to plasma PSA.

Methods.

EVs were enriched from cell lines (PSA expressing or control) or IRB-approved plasma from healthy controls, BPH samples, and PCa samples using silicon carbide resin matrix and characterized by nanoparticle tracking analysis and automated western analysis. Quantification of PSA from EVs was performed by ELISA and automated western analysis. A prototype EV immunocapture lateral flow strip was developed using antibodies against tetraspanins to enrich plasma EVs and EV-PSA detected on the test line using an anti-PSA conjugate.

Results.

EVs demonstrated high levels of TSG101 confirming enrichment from cell lines and plasma samples. The relative abundance of CD9, CD63, and CD81 was compared between control, BPH, and PCa derived EVs. There was at least a 10-fold increase in PSA from EVs purified from LNCaP cells when compared to EVs from healthy (no PCa) plasma. PSA levels in EVs from healthy controls and BPH samples were significantly lower than those from PCa EVs. Using antibodies for tetraspanins to immunocapture EVs from plasma, levels of EV-PSA was detected with anti-PSA conjugates in PCa subjects and not in BPH or healthy controls.

Summary/Conclusions.

PSA levels in EVs differs from that PSA in plasma, and better distinguishes between malignant, benign or healthy subjects. Characterization of tetraspanins in EVs containing PSA was used to develop an immunocapture strategy in a two-phase lateral flow assay. Immunocapture of EVs and detection of EV-associated PSA by lateral flow may provide a new differential diagnostic for a familiar biomarker with improved accuracy.

Dr Renee Goreham

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Extracellular vesicles are membrane-enclosed vesicles released by living cells but of interest is how they resemble the cell they came from. These vesicles hold great potential as alternative targets for bacteria detection and as markers for disease. The nano-size characteristic, compared to parent cells, facilitates easy conjugation with targeting ligands named aptamers, enhancing detection strategies. The presence of numerous extracellular vesicles in the extracellular environment is particularly significant, offering abundant target sites for detection. Leveraging these advantages, our research group uses extracellular vesicles and custom designed nanoparticles as promising markers for pathogen and disease detection, employing strategies developed within our group.

Aptamers are small strands of DNA that specifically target and bind to an entity of interest, for example extracellular vesicles. Our group combines customised nanomaterials with aptamers to engineer different types of aptasensors. For example, the indirect detection of Escherichia coli bacteria by binding outer membrane vesicles to indium phosphide quantum dots with a zinc sulphide shell conjugated to aptamers. The quantum dots photoluminesce and therefore are used as a assay via fluorescent emission or through förster resonance energy transfer. Furthermore, we employ a similar approach using a gold substrate and electrochemical impedance spectroscopy to capture breath-derived extracellular vesicles for lung cancer detection. Upon successful capture of disease-specific extracellular vesicles, an electrical response confirms the detection, enabling the development of a disease breathalyser.

By harnessing nature's own nanoparticles with nanotechnology, our research group aims to pioneer novel strategies for the detection of pathogens and diseases.

Mr. Casey Scott-Weathers, Ms. Kaitlyn King, Kimberly Luke

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

HIV is a major source of morbidity and mortality. Diagnosis can be difficult during early infection and monitoring is complicated by anti-retroviral therapies (ART). Current antigen/antibody (Ag/Ab) tests rely on detection of anti-HIV antibodies, which can be obfuscated by Vaccine Induced Sero-Positivity (VISP) in vaccination study patients. VISP occurs when the experimental vaccine elicits an immune response indistinguishable from infection by Ag/Ab tests. HIV proteins are present within plasma derived EVs of viremic and non-viremic patients. Enhanced detection of HIV proteins via EV enrichment from plasma and detection by lateral flow could lead to reliable early diagnosis, compatibility with ART, and clarification of VISP.

Methods

EVs from HIV+ or HIV- cell lines were isolated using Exoquick. EVs from HIV+, vaccinated, and HIV- subjects were isolated using silicon carbide resin. Isolated EVs were characterized by NTA and BCA. Following characterization, the EV marker TSG101 and HIV proteins p24, Nef, gp120, Tat, and Vpu were examined by ELISA or Chemiluminescence Western Protein Analyzer. Detection of HIV proteins from EVs was demonstrated on a prototype immunocapture lateral flow device in which EVs were immunocaptured, lysed to liberate EV cargo, and HIV proteins detected on the test line.

Results

EVs from HIV+ cell lines showed p24 levels 17 times greater than EVs from HIV- cell lines by ELISA. EVs purified from HIV+ sources showed higher levels of HIV proteins by ELISA or Chemiluminescent Western protein analysis when compared to HIV- or vaccinated subjects. P24, Tat, Nef and gp120 were detected in EVs, with differential detection between HIV+ and HIV- EVs found in several proteins. HIV+ samples showed greater detection of p24 protein via lateral flow strips.

Summary/Conclusion

Chemiluminescent Western protein analysis results demonstrate that purified EVs from HIV+ plasma samples are enriched for HIV proteins and can distinguish between vaccinated or HIV- individuals. Additionally, immunocapture of EVs by lateral flow results in enrichment of EV-associated HIV proteins to detectable levels. Antibody pair development for lateral flow is underway for additional HIV antigens. This EV-HIV LF test could improve point-of-care testing accuracy, promote participation in vaccine trials, and enable regular testing of high-risk individuals.

Mr. Rufus Vinod, Ms. Priyadharshini Parimelazhagan Santhi, Mrs. Erica Routila, Ms. Marina Alexeeva, Dr. Kjetil Søreide, Dr. Kim Pettersson, Dr. Janne Leivo

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Gastrointestinal (GI) cancer constitutes around 20% of all cancer diagnoses globally and contributes to 22.5% of cancer-related deaths. Among GI cancers, Colorectal cancer (CRC) is the third most common cancer, and Pancreatic cancer (PCa) is the sneakiest one with a 5-year survival rate of 12% only. Early detection of these cancers can significantly improve the chances of a successful treatment. Our technical approach is based on the use of fluorescent nanoparticles to detect specific glycoconjugates (sugar-based molecules) associated with extracellular vesicles directly from patient serum or cell culture spend medium (CCSM). These glycoconjugates are thought to be associated with the development and progression of GI cancers, and their presence in the cancer-derived EVs may be an early indication of the presence of the disease.

Methods: EVs isolated with SEC (qEV, Izon) from CCSM were immobilized on the Maxisorp plates, having hydrophilic interactions with the glycoproteins present on the EVs surface. Five different CRCs and four PCa were utilized, in the glycoprofiling whereas, HEK293 and MCF10A, cell lines were used as a negative control. To check the cross-reactivity OVCAR3, A549, Endo p1-19, and Liver cirrhosis ascites were also used. An immunometric assay was conducted to rule out glycoprotein expression levels in the EVs with lectins and antibodies coated onto Europium-doped nanoparticles.

Results: A cluster map analysis was conducted among the tested panel of lectins and antibodies, it was observed that the majority of CRC and PCa cell lines exhibited expression of N-linked high mannose oligosaccharides and fucosylated oligosaccharides. Specifically, DC-SIGN binding with mannose oligosaccharides and UEA-I binding with fucosylated oligosaccharides were found to be significantly elevated in both CRC and PCa cell lines.

Conclusion: Glycoprofiled expression analysis of CRC and PCa-associated EVs in CCSM shows promise as a tool for early detection and diagnosis of these diseases in serum samples.

Master Letícia Pedrini, Master Paula Meneghetti, Doctor Vera Lúcia Chiocolla, Doctor Ana Claudia Torrecilhas, Doctor Blima Fux

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Toxoplasma gondii is a protozoan parasite causative of toxoplasmosis. The parasite can cause abortions and neonatal losses in goats, generating a great economic impact in agribusiness. The extracellular vesicles (EVs) can contribute to the pathogenesis of the infection, playing a role in modulating the host response and facilitating the dissemination of the parasite. The spontaneous release of EVs in culture medium can contribute to improving the diagnosis. The goal of this study was to develop a standardized protocol for the chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) technique for the diagnosis of toxoplasmosis in goats, using EVs derived from T. gondii as the antigen.

Methods: A total of 146 animals were examined. CL-ELISA made use of EVs and total parasite antigens to sensitize the ELISA plates. The Nanoparticle Tracking Analysis (NTA) method was used to characterize EVs released by T. gondii. CL-ELISA was used to compare the efficacy of EVs to that of conventional ELISA using conventional antigen.

Results: T. gondii EVs were used to standardize the CL-ELISA assay. The EVs from the parasite were added to the plate at an amount of 103 particles/well (as determined by NTA). Meanwhile, the antigen concentration was 3 µg/well. Conventional ELISA results revealed that 78 (53.4%) of the 146 tested sera were negative. However, when the CL-ELISA method was used with extracellular vesicles (EVs) from these 146 sera, 24 (16.4%) of the results were negative, but 9 (6.1%) of the results were negative when CL-ELISA was used with parasite antigen. When compared to conventional ELISA results, these data show an increase in positive sera of 83.6% (EVs) and 93.8% (Antigen).

Summary/Conclusion: This study highlights the diagnostic efficacy and sensitivity of EVs in detecting toxoplasmosis in goats using a CL-ELISA. The comparative analysis sought to identify potential benefits or enhancements provided by EVs over the conventional approach in the detection and examination of T. gondii infection.

Financial Support: FAPESP, CAPES, CNPq, FAPES.

Samuel Walker^{Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA}, PhD James McGrath^{Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA}, MD, MBA Jonathan Flax^{Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA; Department of Urology, University of Rochester Medical Center, Rochester, NY, USA}

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: The extracellular vesicle (EV) field has grown astronomically in recent years paralleled by the development of numerous analytical techniques. Bulk assays lack the sensitivity to evaluate rare biomarkers relevant to disease and single-EV technologies are often undermined by complicated detection schemes, expensive instrumentation, and incomplete analyses. Here we introduce and carefully validate the ‘catch and display for liquid biopsy’ [CAD-LB] platform, a simple and accurate single-EV assay for quantification and protein biomarker assessment. Critically, conditioned media and plasma samples require no upstream purification steps prior to CAD-LB processing.

Methods: EVs and target surface proteins are labeled with carboxyfluorescein succinimidyl ester (CFSE) and fluorescent antibodies, respectively, while in conditioned media or plasma solutions. These solutions are pipette injected into a simple microfluidic device resulting in EV capture on ultrathin nanoporous membranes and visualization using confocal microscopy. Fluorescent nanoparticles and EVs with well-defined biomarker profiles were used to rigorously characterize and validate CAD-LB.

Results: CAD-LB's dynamic range was defined for nanoparticle/purified EV solutions: 10²-10⁶ (total particles) and nonpurified plasma-derived EVs: 10²-10⁴. EV quantification was validated using purified EVs and two general EV labels: CFSE & MemGlow; these labels target distinct EV components (lumen and membrane, respectively) yet both resulted in a linear counting response and returned counts in sound agreement. Nanoparticle mixtures served as colocalization controls which elucidated false positive/negative rates inherent to CAD-LB; following this, empirical colocalization guides were created for subsequent EV experiments. Biomarker detection was validated using two biomarkers (CD9 and ICAM-1) interrogated in distinct experimental models. Reference assays (Western blot and immunocytochemistry, respectively) confirmed that CAD-LB faithfully recapitulated biomarker expression relationships among observed samples. Finally, we show that CAD-LB consistently detected rare and clinically relevant (PD-1/PD-L1) EV biomarkers in bladder cancer patient plasma at levels higher than isotype controls.

Conclusion: This work thoroughly evaluates and establishes CAD-LB as a novel EV analysis tool. The rapid methodology requires only a simple pipette injection of labeled solutions, uses low-cost materials, and offers high data density. CAD-LB provides a dynamic range spanning 4 orders of magnitude and remarkable sensitivity which yields quantitative data unavailable to bulk assays.

Ph.d Zihao Ou

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Bacterial extracellular vesicles (BEVs) are nano-size particles secreted by bacteria that carry various bioactive components. These vesicles are thought to provide a new window into the mechanisms by which bacteria affect their hosts, but their fundamental proprieties within human remain poorly understood. Here, we developed a single-vesicle analytical platform that enabled BEV detection in complex biological samples of host. Using this platform, we found the presence of BEVs in the host circulation and they were mainly derived from gut microbes. We showed that the levels of circulating BEVs in humans significantly increased with aging due to an age-related increase in intestinal permeability. Significantly different levels of BEVs in blood were also found in patients with colorectal cancer and colitis. Together, our study provides new insights into circulating BEV biology and reveals their potential as a new class of biomarkers.

Ms. Ekaterina Moiseeva, Dr. Vasiliy Chernyshev

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

A tumor-derived extracellular vesicles (T-EVs) test is a promising early-stage cancer diagnostic tool. However, conventional methods for isolation and detection are labor-intensive and require highly qualified professionals and sophisticated instruments. Additionally. the majority of these methods cannot selectively isolate tumor associated protein enriched EVs among other non-specific EVs present in the sample. Paper-based microfluidic fluorescence assays are simple, inexpensive, and sensitive methods for sensing biomolecules, which can be further improved through covalent bioconjugation of the molecular recognition elements onto the cellulose matrix. Here, in this work, we first applied functionalization of cellulose acetate membrane with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) as surface activation for aptamer immobilization of specific EV detection by fluorescent assay on a paper-based microfluidic system.

2) Methods

The chemical efficiency of cellulose acetate functionalization is explored through scanning electron microscopy-energy dispersion X-ray spectrometry (SEM-EDX) and attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. The application of this novel immobilization technique is tested using fluorescein isothiocyanate (FITC) labeled aptamers in a sandwich immunoassay for human EpCAM-positive EVs. The results are evaluated by fluorescence tomography.

3) Results

ATR-FTIR analysis and EDX confirm the surface functionalization of cellulose membrane with free amino groups. Moreover, silanization did not significantly change the membrane's pores and channels, according to SEM results. The APTES-GA-functionalized cellulose spontaneously and irreversibly capture the FITC-labeled aptamer and exhibits strong fluorescence. Moreover, reaction conditions and washing procedures are optimized to prevent non-specific binding of EVs and oligonucleotides. The successful selective EpCAM-EVs detection is demonstrated in a sandwich immunoassay. It showed a fluorescence signal an order magnitude higher than the background signal and half of order magnitude higher compared to non-specific EVs.

4) Summary/Conclusion

The two stage bioconjugation protocol of cellulose acetate based on APTES silanization and GA crosslinking with NH₂-aptamer has been successfully used in the microfluidic device fabricated for EV-based liquid biopsy. The functionalized cellulose surface efficiently binds exosome specific aptamer, providing sensitive and T-EVs selective low-cost paper-based fluorescence assays.

Associate professor Eun-jae Lee, Professor Yong Shin

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Despite the progressive nature of Parkinson's disease (PD) with varying rates of progression, objective blood biomarkers for early diagnosis and precise disease monitoring are currently unavailable. Brain-derived exosomes circulating in the blood hold potential as indicators of brain disease, providing an opportunity for early and accurate PD diagnosis.

Methods

We developed a rapid (<35 min) and cost-effective method using transferrin-conjugated magnetic nanoparticles (TMNs) to isolate brain-derived exosomes from the plasma of patients with neurological disorders. This technique necessitates no expensive ingredients or intricate equipment for extracellular vesicle (EV) extraction. The methodology was applied to patients with PD, multiple sclerosis (MS), and dementia, with ethical approval from the Institutional Review Board.

Results

Successful isolation of EVs was achieved from 33 human plasma samples, including those from patients with PD, MS, and dementia. Quantitative polymerase chain reaction analysis evaluated eight exosomal miRNA profiles as potential biomarker candidates. Six exosomal miRNA biomarkers (miR-195-5p, miR-495-3p, miR-23b-3P, miR-30c-2-3p, miR-323a-3p, and miR-27a-3p) consistently correlated specifically with all stages of PD.

Conclusion

These findings suggest that the TMNs method provides a practical, cost-efficient means to isolate EVs from biological samples, facilitating non-invasive PD diagnoses. Furthermore, the identified miRNA biomarkers in these exosomes may serve as innovative tools for the precise diagnosis and monitoring of neurological disorders, including PD.

Mr. Gaoge Sun¹, M.D. Tian Li³, Ying Zhang¹, Hang Yin^1,2

¹School of Pharmaceutical Sciences, Tsinghua University, Beijing, China, ²Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China, ³Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, China.

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

Diffuse intrinsic pontine glioma (DIPG) is a deadly malignant childhood brain tumor, often misdiagnosed due to reliance on imaging and clinical symptoms. The presence of the H3K27M mutation in over 80% of DIPGs complicates diagnosis, with brainstem biopsies posing risks to patients. In light of these challenges, exploring extracellular vesicles (EVs) as potential liquid biopsy targets shows promise. EVs reflect physiological and pathological states of progenitor cells, are capable of crossing the blood-brain barrier and circulating in the bloodstream. Our study aims to analyze DIPG-EVs using multi omics approaches, such as proteomic, transcriptomic, and lipidomic analyses, to unveil their composition and identify potential biomarkers for improving clinical diagnosis of DIPG.

2) Methods

DIPG and primary pontine progenitor cell cells (PPCs) were cultured in DMEM medium with NSC growth factors while MO3.13 cells were cultured in DMEM with 10% FBS. EVs were isolated and characterized following MISEV2018 guidelines utilizing differential ultracentrifugation. Characterization techniques included TEM, Western blotting, and NTA. The presence of ganglioside GD2 in EVs was validated through dot blotting, quantification via mass spectrometry, and nanoscale flow cytometry (nFCM).

3) Results

Our research involved analyzing the proteomics, lipidomics, and transcriptomics of DIPG cell lines and their associated EVs, comparing findings with the PPC and differentiated MO3.13 cells lacking the H3.3 K27M mutation. Mass spectrometry analysis confirmed the presence of ganglioside GD2, notably acetylated GD2, in DIPG-derived EVs. Gangliosides, enriched in the brain's nervous system, play crucial roles in nerve regeneration and signal transmission. The clinical utility of detecting GD2 in plasma EVs was demonstrated through Western blotting and single particle nanoflow experiments. Furthermore, we successfully isolated and identified H3K27M mutations in EVs from patient plasma, indicating the potential for molecular analysis in DIPG diagnosis using EVs.

4) Summary

The diagnostic challenge posed by DIPG spurred our exploration of EV analysis for DIPG cell lines and patient plasma samples, revealing the detectability of mutated H3K27M and highly expressed acetylated GD2 in plasma EVs. Our goal is to further investigate EVs originating from the brain to enhance the sensitivity, specificity, and accuracy of DIPG diagnosis.

Dr. Jae-a Han¹, M.D. Haekyung Lee², Hee-Sung Ahn³, Dr. Soon Hyo Kwon³, Dr. Kyunggon Kim³, Dr. Seongho Ryu¹

¹Soonchunhyang Institute of Med-Bio Science (SIMS), Soonchunhyang University, Cheonan-si, South Korea, ²Division of Nephrology, Department of Internal Medicine, Seoul, South Korea, ³Asan Institute for Life Sciences, Asan Medical Center, Seoul, South Korea

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Studies suggest that obesity and diabetes is linked to cardiometabolic diseases. However, study in this field faces hardships as there are substantial prognostic heterogeneity involved. To profile the molecular mechanisms that contribute to the different responses to bariatric surgery dependent on the presence of diabetes, we sought to identify extracellular vesicular proteomic signatures of obesity with or without diabetes before and after bariatric surgery.

Methods: Extracellular vesicle proteins in 30 morbidly obese patients with (n = 12) or without (n = 18) diabetes were sampled before or 6 months after bariatric surgery and were compared to those in 37 healthy controls, analyzed by using liquid chromatography-tandem mass spectrometry.

Results: A total of 46 proteins differentially expressed in healthy controls, non-diabetic obese patients, and diabetic obese patients. Bariatric surgery led to comparable weight loss in both non-diabetic and diabetic obese patients. In non-diabetic and diabetic obese patients, respectively, 13 and 14 proteins altered significantly after bariatric surgery; mostly involved in gas transport and reactive oxygen species metabolic process in non-diabetic obese patients and immune system in diabetic obese patients. In both non-diabetic and diabetic obese patients after bariatric surgery, extracellular vesicle proteins were involved in complement activation and endopeptidase inhibitor action showing similar expression patterns. However, proteins associated with lipid clearance and vasoconstriction responded differentially to bariatric surgery when the patient also had diabetes.

Conclusions: Non-diabetic and diabetic obese patients exhibited both common and distinctive extracellular vesicle protein signatures before and after bariatric surgery. These protein expression profiles may shed light on the underlying molecular mechanisms of obesity and diabetes as well as serve to provide potential therapeutic targets.

Valentin Vautrot¹, Isen Naiken¹, Carmen Garrido¹, Pr Côme Lepage², Dr Jessica Gobbo³

¹INSERM 1231, Label “Ligue National contre le Cancer” “and Label d'Excellence LipSTIC, DIJON, France, ²Federation Francophone de Cancérologie Digestive (FFCD), EPICAD INSERM 1231, DIJON, France, ³INSERM 1231, Label” “Ligue National contre le Cancer” “and Label d”Excellence LipSTIC, Department of Medical Oncology, Early phase unit INCa CLIP²; Center Georges-François Leclerc, DIJON, FRANCE

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Duodeno-pancreatic neuroendocrine tumors (DPNETs) are increasing in incidence and prevalence. DPNETs are slow-growing tumors, implying a need for regular follow-up. There is an unmet need for a biomarker to monitor DPNETs progression in order to avoid delays in treatment. Chromogranin A and other biomarkers (urinary 5-hydroxy-indolacetic acid and pancreatic polypeptides) used in current practice lack specificity, making computed tomography scan and Magnetic Resonance Imaging the primary means of monitoring disease in these patients. Many new potentials circulating and histological biomarkers are currently under investigation, such as cell-free DNA, delta-like protein 3, and immunosuppressive molecules (PD-L1/HSP70). In this context, circulating biomarkers such as small extracellular vesicles (sEV) have garnered increasing interest over the last decade. We measured circulating sEV in the plasma of patients with DPNETs. We also investigated the dynamics of sEV-PD-L1 and sEV-HSP70 from 35 patients enrolled in the PRODIGE 31 – REMINET clinical trial (NCT02288377).

Methods

Plasma samples were drawn before treatment; after one month of treatment and disease progression. sEV were isolated by precipitation kit. Their size and concentration were analysed by nanoparticle tracking analysis (NS300, Malvern) and transmission electron microscopy (TEM). Isolated sEV were tested for the expression of sEV markers (TSG101, CD9, CD63, CD81, Alix) by Western blot. In addition, HSP70 and PD-L1 concentration in sEV and soluble forms were measured using ELISA.

Results

HSP70 and PD-L1 in soluble or vesicular forms were detected in all patients. The percentage change in soluble PD-L1 and soluble HSP70 between baseline and 1 month post-treatment was not significantly different between progressive and stable patients (p = 0.5611 and 0.3462). However, we demonstrated an association between increasing sEV-PD-L1 and disease progression (p = 0.0462). On the contrary, level of sEV-HSP70 was not associated with disease progression.

Conclusion

We demonstrate for the first time the usefulness of EV analysis in the monitoring of disease progression in patients with DPNETs. These preliminary results merit confirmation in a larger cohort, and could enable cost-effective detection of disease progression with a simple liquid biopsy-based technique in DPNETs surveillance.

Visiting Researcher Yuta Shimizu^1,2, Researcher Fumi Asai², Researcher Keidai Miyakawa², Assistant Professor Kenji Takahashi³, Director Tatsutoshi Inuzuka²

¹Baylor Genetics, Houston, United States, ²H.U. Group Research Institute, Akiruno, Japan, ³Asahikawa Medical University, Asahikawa, Japan

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer-related deaths worldwide. Most PDAC is diagnosed at advanced stages or metastasis, as there are no typical early symptoms. In addition, the low sensitivity and specificity of CA19-9, a biomarker currently available for PDAC, contribute to the late diagnosis of this deadly disease. There is a strong need for useful biomarkers for PDAC diagnosis. We recently developed a unique immunoprecipitation method called EViSTEP™ that utilizes a chelating-based reagent and CD9/CD63 antibodies that isolate extracellular vesicles (EVs) from body fluids with high purity and high yield. Furthermore, the EViSTEP-optimized RNA-seq pipeline was developed for RNA-seq data processing and bioinformatics analysis. In this study, we sought to identify candidate RNAs for diagnosis of PDAC using the EViSTEP-optimized RNA-seq pipeline. METHODS: We prepared EVs from 1000 µL of healthy control (HC) (n = 29), Intraductal papillary mucinous neoplasm (IPMN) (n = 28) and PDAC (n = 25) serum, using EViSTEP. The resulting EVs were RNA-extracted and reverse-transcripted with GenNext RamDA-seq Single Cell Kit, libraries were synthesized with Nextera XT DNA library prep kit, and RNA-seq data were obtained using Nextseq 550 system. RESULTS: The total number of RNAs detected by RNA-seq was 53983, the average number of mapping reads was 7.7 M reads/sample, and about 70% of transcripts in serum EVs were mapped by non-coding RNA and messenger RNA. There were no major changes in RNA distribution by disease. 12 RNAs such as G0S2, PROS1, and ORM1 were significantly upregulated in PDAC compared to the non-PDAC group (HC and IPMN). In addition, RNAs such as SOCS3 were found to be highly expressed in stage I of PDAC. The Area Under Curves for PDAC diagnosis ranged from 0.606 to 0.750 for each RNA. The result of GO analysis showed that RNAs upregulated in PDAC were enriched in platelet alpha-granule-related molecules. CONCLUSIONS: Several PDAC-specific candidate RNAs identified by the EViSTEP-optimized RNA-seq pipeline have the potential to be valuable for the early diagnosis of PDAC.

Ms Emily Paterson¹, Dr Simon Scheck^1,2, Dr Simon McDowell², Dr Nick Bedford², Associate Professor Jane Girling³, Dr Claire Henry¹

¹University of Otago, Wellington, New Zealand, ²Te Whatu Ora - Capital and Coast, Wellington, New Zealand, ³University of Otago, Dunedin, New Zealand

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Endometriosis is a common gynaecological condition that affects one in ten women, also affecting gender diverse people. Currently, laparoscopic surgery is required to confirm an endometriosis diagnosis, contributing to an eight-year diagnostic delay in Aotearoa New Zealand. Non-invasive biomarkers are critically required to streamline diagnosis and identify the patients that will benefit most from surgery. Plasma EVs as biomarkers of endometriosis have yet to be investigated via flow cytometry.

Methods

Plasma was prepared from whole blood collected in EDTA vacutainer tubes from consenting patients aged 16-45 undergoing surgery for suspected endometriosis at Wellington and Kenepuru Hospitals.

EVs were isolated from 500µL of plasma using qEVoriginal 70nm columns (Izon Science), pooled and stained for endometrial stromal cell markers with anti-CD10-BB515 (BD Biosciences) and anti-CD90-BV711 (BioLegend), or anti-CD140b-PE (BioLegend) overnight at 4°C. EVs and reagent controls were then incubated with 2.5µM Aco-430 (Acoerela) for 60 minutes at room temperature.

Assays were performed on a three laser (violet-blue-red) Aurora full spectrum flow cytometer (Cytek Biosciences) equipped with the enhanced small particle detector. Samples diluted 1:200 were acquired on the lowest flow rate for 240 seconds, in technical duplicate. 8-Peak Rainbow beads (Spherotech) and polystyrene beads sized 70-400nm (Thermo Fisher) were used to calibrate scatter and fluorescence data. Compensation beads and Aco-430 liposomes were positive controls for unmixing (SpectroFlo). The total EV population was gated based on Aco-430 fluorescence, then CD10, CD90 or CD140b gates were applied. Data was expressed as CD10+, CD90+, CD140b+ or CD10+CD90+ EVs as a proportion of the total EV population.

Results

The lower limit of detection of EVs was 100nm and the EV gate set the upper limit at 1000nm. CD10 and CD90 were suitable for multiplexing, however CD140b+ EVs were a low proportion of the total EV population and performed poorly when multiplexed. Preliminary results suggest there is no difference in proportions of CD10+, CD90+, CD10+CD90+ or CD140b+ EVs between surgically confirmed endometriosis cases and controls.

Conclusions

Spectral cytometry can be utilised to measure CD10+, CD90+, CD10+CD90+ and CD140b+ EVs in plasma samples, and the proportions of these EVs do not differ between endometriosis cases and controls.

Professor Tengku Ain Fathlun Kamalden¹, 2 Nur Musfirah Mahmud¹, 3 Ying Jie Liows¹, 4 Sujaya Singh¹, 5 Samarjit Das²

¹UM Eye Research Centre, Department of Ophthalmoogy, Universiti Malaya, Kuala Lumpur, Malaysia, ²Department of Anaesthesiology and Department of Pathology, Johns Hopkins School of 37 Medicine, Baltimore, United States of America

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Diabetic retinopathy (DR) is a major complication of diabetes mellitus, affecting a significant percentage of the diabetic population. Early diagnosis and intervention are critical for preventing irreversible vision loss. Currently, diagnosis is made when retinal damage has already occurred clinically as observed during examination. There are no available laboratory methods available to detect changes before the onset of clinical damage to the eye in diabetic retinopathy.

Tear fluids are easily accessible and have been shown to contain a concentrated amount of extracellular vesicles. This study aimed to investigate the tear fluid-derived miR-15a in extracellular vesicles in various stages of diabetic retinopathy. Tears samples were collected from 135 diabetic patients (36 with DR and 50 without DR) and 49 healthy controls. EVs were isolated, and the expression of exosomal miR-15a was quantified using droplet digital PCR (ddPCR). The expression of EV miR-15a was found to be modulated by diabetes and DR patients compared to healthy controls. This finding suggests that tear fluid-derived miR-15a may be involved in the mechanistic pathways leading to diabetes and its complications and is a promising early disease biomarker in diabetic retinopathy, offering a simple non-invasive detection method with high sensitivity and specificity.

Zhenxun Wang¹, Ph.D Bodeng Wu², Qiaoting Wu¹, Jiawei Li¹, Jiaming Chen¹, Quan Zhong¹, Phd Xin Zhang², Prof. Lei Zheng², Prof. Yu Wang¹

¹Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China, ²Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, China

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Hepatocellular carcinoma (HCC) is the most common type of liver cancer and characterized by poor prognosis and difficult early diagnosis. Extracellular vesicles (EVs) are cell-secreted phospholipid bilayer-bound structures that present in body fluids that can be used as biomarker of liquid biopsy.

Methods: Benign and tumour tissue with paired plasma samples of HCC patients were collected. EVs were isolated by using iodixanol density cushion (IDC) and size-exclusion chromatography (SEC) combined with ultracentrifugation and characterised with Nanoparticle tracking analysis, Western blot (CD9, CD63, TSG101, Calnexin) and Transmission electron microscopy. Differential proteins in HCC tissue EVs was selected by LC-MS/MS analysis. Western blot, ELISA, nano-flow cytometry (nanoFCM) were used to detected specific protein.

Results: Our study described a serious of differential proteins enriched in tumor derived EVs, like FAP, CD34, TOM1 etc. by using proteomics. Besides, we highlight specific protein cargo were also enriched in plasma-enriched EVs by western blot and Elisa rather than the plasma. Then we find that in HCC serum EVs (n = 45), specific proteins was highly expressed compared with normal serum(n = 13). We also find that after treatment, specific proteins expression significantly increased in HCC patient EVs by using western blot and nano-flow cytometry.

Summary/Conclusion: Our study provides a valuable EVs cargo reference for the liquid biopsy, and has great potential to improve early diagnosis of HCC.

Dr Ben Johnson¹, Mr Winston Lay¹, Dr Tamkin Ahmadzada², Mr Richard Zelei¹, Dr Anthony Linton¹, Dr Elham Hosseini-Beheshti¹

¹Asbestos And Dust Diseases Research Institute, Concord, Sydney, Australia, ²The University of Sydney, Camperdown, Sydney, Australia

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Pleural mesothelioma (PM) is an aggressive asbestos-related thoracic cancer associated with poor prognosis. A lack of reliable diagnostic biomarkers, long disease latency period and differing pathological subtypes make it a difficult disease to diagnose. Therefore, improved minimally-invasive diagnostic biomarkers are urgently needed. Extracellular vesicles (EVs) constitute a promising liquid biopsy diagnostic biomarker given their abundance and stability in blood circulation, and enrichment of disease-specific cargo. We have previously identified eight circular RNA (circRNA) biomarker candidates that are over-expressed in mesothelioma cells. Here, we investigated the expression of four circRNA candidates in different mesothelioma-derived EV subpopulations to assess their potential utility as diagnostic biomarkers of mesothelioma.

Methods:

Different EV subpopulations (10K, 18K and 100K pellet) were isolated from the conditioned media of cultured mesothelioma and non-malignant mesothelial cells using our previously published protocol. The EV subpopulations were characterized by transmission electron microscopy, western blot and nanoparticle tracking analysis. Total RNA was extracted from the EV samples using TRIzol reagent and the resulting RNA was subjected to a reverse transcription reaction to generate complementary DNA (cDNA). The cDNA was combined with circRNA-specific primers and probes in a 96-well plate and the EV-derived circRNAs were detected and quantified via droplet digital PCR (ddPCR).

Results:

Our results demonstrate an enrichment of all circRNA biomarker candidates in the 100K EV subpopulation derived from mesothelioma cell lines. The 100K EV subpopulation exhibited more than a two-fold higher circRNA expression level compared to the tumour cell lysate control. Additionally, the four circRNA biomarker candidates were found to be overexpressed in the mesothelioma cell-derived 100K EV subpopulation compared to their non-malignant counterparts by up to three-fold, whilst a modest overexpression was detected for one of the circRNA candidates in the mesothelioma cell-derived 10K and 18K EV subpopulations compared to their non-malignant cell control counterparts.

Summary/Conclusion:

CircRNA-associated EVs constitute a promising biomarker candidate for detection of PM with greater sensitivity than their tumour cell lysate counterparts. This finding provides the rationale for prospective studies aiming to validate the specificity and sensitivity of small-EV (100K EV)-enriched circRNAs in plasma samples isolated from PM patients and asbestos-exposed individuals.

Dr. Eisuke Dohi¹

¹National Center Of Neurology And Psychiatry, Kodaira city, Japan

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

Among blood circulation factors, ncRNAs have been searched as biomarkers in various diseases. However, even in animal experiments, where experimental conditions are easily standardized, inconsistency among studies has been observed even in control groups. This has been attributed to animal handling, feeding, diurnal variation, intestinal flora, reagents for analysis, and other factors at each facility. Recently, the contamination of human plasma samples with platelets and platelet-derived extracellular vesicles (EVs) has been reported as an important new factor. Therefore, the authors investigated conditions to reduce contamination of platelet-derived extracellular vesicles and attempted to detect circulating miRNAs in blood that fluctuate within a day.

2) Methods

Male C57L/B6 mice aged 12-16 weeks were deeply anesthetized and blood was collected from the inferior vena cava every 4 hours, three at each time point, using an ACD as anticoagulant. Plasma was obtained by serial centrifugation at 22°C within 10 minutes of blood collection. Visible hemolyzed samples were excluded. The hemolysis in used samples was examined with the expression level of miR-451a and miR-23a-3p. The platelet contamination was examined with western blotting. RNA was extracted from 200µL of plasma, and miRNAseq was performed. The diurnal variation of miRNA was analyzed with MetaCycle.

3) Results

Western blotting showed no platelet contamination in the plasma sample. The quality of sequence data were assessed with FASQC. 250 miRNAs were detected and 97.6% of them were reported in previous studies that examined miRNAseq on the mice serum EVs extracted with ExoQuick. The analysis with MetaCycle showed diurnal variation of 15 miRNAs in the plasma, 12 of which showed similar trend. Referring to the previous reports, 6 of which had Exomotif and 6 of which had tissue-specific scores of 0.95 or higher.

4) Summary/Conclusion

Sample condition strongly influences the search for biomarkers circulating in trace amounts of blood. Examination of conditions that may affect the sample, such as platelets, platelet-derived extracellular vesicles, and diurnal variation, is considered important in the search for precise biomarkers. The physiological significance of diurnally fluctuating blood miRNAs may also be important, and this is a research question to be solved in the future.

CEO, R&D Se-Hwan Paek¹, Associate Research Engineer Taekmin Kim¹, Research Engineer Dayeon Choi¹, Research Director Seung-Cheol Choi¹

¹SOL Bio Corporation, Seoul, South Korea

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

ISEV Poster Abstract

1) Introduction

More than hundreds of exosome biomarkers have been reported to discriminate samples of patients with certain diseases, such as cancers, from those of healthy people. However, the markers reported may not consistently achieve the intended diagnostic goals. This could result from the dynamic nature of bulk exosomal heterogeneity, surpassing the concentration change of the target marker across sample groups (e.g., cancerous vs. healthy).

2) Methods

To overcome the problem in discovering biomarkers for, for instance, prostate cancer, we investigated a novel approach of quantifying a change of the marker composition within a certain exosome subclass. We introduced an index, defined as the component ratio of the third marker contained in a double pan-exosome tetraspanins-positive subclass, which was determined for each exosome subclass consisting of any two tetraspanins in rotational manners.

3) Results

We found that the index (R1) for CD81 as the third marker within the CD9-CD63 subclass showed significantly decreasing responses to the samples from the cancer cell lines (PC3 and LNCaP) as compared to those from the normal (HEK293). On the other hand, the index (R2) for CD9 as the third marker within the CD63-CD81 subclass showed relatively increasing responses to the cancer samples. Overall, R1 and R2 could distinguish the cell origins regarding cancerous property.

4) Summary/Conclusion

We have investigated a novel concept for exosome marker-based cancer tracking by monitoring an index (R) indicating the component ratio of the third marker within a specific double tetraspanins-positive subclass in samples. This held promise for further clinical sample testing for early cancer detection.

Funds:

TIPS Program (Project No.: S3012455) and Post-TIPS Program (Project No.: 20178922) funded by the Ministry of SMEs and Startups, Korea, and the National Research Foundation (Project No.: NRF-2020R1A2C1007131) of Korea (NRF)

Min Eon Park¹, You Yeon Choi¹, Ki Moon Seong¹

¹Korea Institute of Radiological & Medical Sciences (KIRAMS), Seoul, KOREA

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Atherosclerosis is a chronic inflammatory disease caused by hypercholesterolemia, which can increase in cancer patients after radiation therapy and in patients exposed to radiation after a nuclear accident. microRNAs are a group of multifunctional non-coding RNAs that play important roles in various physiological processes, including atherosclerosis. Recently, our previous study showed that miR-126-5p within endothelial cell EVs mediates inflammatory signaling to activate monocytes in radiation-induced vascular injury. Additionally, circulating endothelial EV content confirmed its potential as a diagnostic and prognostic biomarker for atherosclerosis after radiation exposure. In this study, we identified target genes of miR-126-5p in EVs and revealed a novel regulatory mechanism of cholesterol efflux, an important aspect of lipid metabolism. Using the LDLR(-/-) mouse model exposed to ionizing radiation as a model system, we observed a significant increase in miR-126-5p within EVs. Through screening, ATP-binding cassette subfamily G member 5 (ABCG5) was identified as a target gene of miR-126-5p, which manages cholesterol efflux. Overall, our findings reveal a novel regulatory mechanism of cholesterol efflux by targeting ABCG5 by miR-126-5p in EVs upon radiation exposure. [This study was supported by the grant (No.50091-2024) From the Nuclear Safety and Security Commission, Republic of Korea]

Min Eon Park¹, You Yeon Choi¹, Ki Moon Seong¹

¹Korea Institute of Radiological & Medical Sciences (KIRAMS), seoul, KOREA

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Extracellular vesicles (EVs) play pivotal roles in intercellular communication and have emerged as potential biomarkers for various diseases, including leukemia. Radiation exposure is a well-known risk factor for leukemia development, and understanding the miRNA profiles within EVs post-exposure could elucidate molecular mechanisms underlying radiation-induced leukemogenesis. We performed hematological and histological analyses presenting that high-dose radiation over 1 Gy induced leukemia in the AKR/J mice. All animal experimental procedures followed the Korea Institute of Radiology Institutional Animal Care and Use Committee (IACUC) approved protocols. We also carried out a comprehensive microRNA (miRNA) screen of EVs isolated from the plasma of irradiated AKR/J mice using small RNA sequencing. EV size and range of isolated EVs were assessed by nanoparticle tracking analysis (NTA), which showed a characteristic size distribution consistent with EV populations typically ranging from 30 to 150 nm in diameter. The expression profile of miRNAs can explain the molecular mechanism of radiation response in leukemogenesis. Through further validation, some candidate miRNAs significantly expressed in EVs may facilitate the development of noninvasive diagnostic tools and therapeutic strategies for radiation-related leukemia. [This study was supported by the grant (No.50091-2024) From the Nuclear Safety and Security Commission, Republic of Korea]

Diane Nelson¹, Mahir Mohiuddin, Investigator Jennifer Jones, Jeffrey Fagan, Jerilyn Izac, Sumeet Poudel, Bryant Nelson, Lili Wang

¹Nist, United States

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: This investigation extends our previous comparative analysis of extracellular vesicle (EV) isolation protocols—ultracentrifugation (UC), tangential flow filtration (TFF), size-exclusion chromatography (SEC), and precipitation (P)—by incorporating state-of-the-art findings using the Oni nanoimager. This study aims to elucidate the impact of isolation techniques on EV phenotype and diameter, offering a new dimension to EV research.

Methods: EVs were isolated from Jurkat cell culture supernatants using ultracentrifugation (UC) and tangential flow filtration (TFF). Total protein content in EV isolates was measured with a Bradford assay. The concentration and size distribution were evaluated through microfluidic resistive pulse sensing (MRPS). Additionally, the Oni nanoimager was used to phenotypic and morphological characterization as well as diameter measurement.

Results: Preliminary results found that UC resulted in higher yields of > 1×1010 EV/mL whereas TFF resulted in yields of > 1×109 EV/mL. UC had higher total protein contaminants than TFF. For each method, more than 90% of the EV population had an average size distribution of 50-200 nm and median size of 120 nm based on MRPS and Oni. The ONI imager confirmed the presence of EV protein markers (CD63, CD81, CD9) in each EV isolate; ∼7% of EVs were triple positive across both isolation techniques. The UC method showed superior efficiency in EV capture.

Summary/Conclusion: Incorporating Oni nanoimager insights into our comparative study of EV isolation protocols has not only validated our initial findings but also broadened our understanding of EV phenotypes, morphology, and size distributions, highlighting the nuanced effects of isolation techniques on EV purification and characterization. This approach allowed for a detailed examination of EV populations, leveraging the nanoimager's high-resolution imaging capabilities. This innovative approach enhances the reliability of EV research, offering a refined methodology for future investigations into EV biology and their potential clinical applications. Our results advocate for the integration of advanced imaging techniques, like the Oni nanoimager, in the standardization and optimization of EV isolation and purification protocols.

Dr. Marie Pauwels¹, Dr. Nele Plehiers¹, Dr. Charysse Vandendriessche¹, Prof. Matthew JA Wood², Dr Lien Van Hoecke¹, Prof Roosmarijn E Vandenbroucke¹

¹VIB-UGent, Gent (Zwijnaarde), Belgium, ²University of Oxford, Oxford, UK

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Central nervous system (CNS) disorders present formidable challenges in treatment due to restrictive barriers hindering therapeutic agent access to the brain. Overcoming these barriers is pivotal for effective therapy. Nanoparticle-mediated delivery, particularly via extracellular vesicles (EVs), shows promise in circumventing these obstacles. EVs, naturally secreted by diverse cell types, possess unique capabilities to ferry biological cargo across key CNS barriers, including the blood-cerebrospinal fluid (CSF) barrier which consists of a single layer of choroid plexus epithelial cells that separate blood from CSF.

Here, we investigated the brain-targeting characteristics and functional cargo delivery of choroid plexus epithelial cell-derived EVs. Interestingly, we observed that these EVs exhibit the capacity to home to the brain after peripheral administration. Moreover, these vesicles were able to functionally deliver cargo into the brain, showing the therapeutic potential of choroid plexus-derived EVs as a brain drug delivery vehicle via targeting of the blood-CSF interface. Additionally, engineered EVs derived from HEK293T cells, incorporating a brain-targeting element via a solute carrier transporter domain and a blood-CSF barrier-targeting anti-folate receptor 1 (FOLR1) single domain antibody (VHH), were evaluated. In vivo evidence demonstrated effective brain targeting and successful therapeutic cargo delivery using these VHH-engineered EVs.

Overall, our findings highlight the therapeutic promise of brain-targeting EVs as delivery vehicles via the blood-CSF barrier, signifying a substantial advancement in the field of CNS drug delivery.

Ms. Geetika Raizada, Dr. Benjamin Brunel, Mr. Joan Guillouzouic, Dr. Eric Le Ferrec, Dr. Eric Lesniewska, Dr. Wilfrid Boireau, Dr. Céline Elie-Caille

¹FEMTO-ST Institute, CNRS, University of Franche-Comté, Besançon, France

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Extracellular vesicles (EVs) have shown great potential as biomarkers since they are involved in numerous biological processes. In the context of cytotoxicity, it has been found that exposing cells to toxicants led to changes in protein expression and cargo of the EVs they produce. In this study, we used Raman spectroscopy on lEVs to detect changes in their molecular signature, induced by cell exposure to Benzo[a]pyrene (B(a)P).

Methods:

lEVs were isolated from Human microvascular endothelial cells (HMEC-1) (two cell culture conditions: control & treated) by ultracentrifugation at 10000 x g for 30 mins. We used Surface Plasmon Resonance imaging (SPRi) to adsorb lEVs and used Atomic Force Microscopy (AFM) to qualify and determine the surface coverage of the objects on the biochips. Raman spectra were acquired with a Raman microscope, measuring about 5 regions of interest of 20x20 pixels (spatial resolution of 3 µm), for each sample.

Results:

With the help of SPRi, we controlled the density of the objects on the chip surface. The particle density was calculated by using AFM analysis; it was found to be around 5.7 x 106 /mm2 ± 6.9 x 105 standard error for the control condition and 4.2 x 106 /mm2 ± 3 x 105 for the treated condition. Comparing control and the treating condition, significant differences were found in the high frequencies region of Raman spectra (2800 to 3000 cm-1), corresponding to lipid modifications. Two types of spectra were detected in the control sample, one of them resembling the spectra of mitochondria. A Support Vector Machine (SVM) model was trained on the pre-processed spectral data to differentiate between EVs from cells exposed or not to B(a)P at the spectrum level; this model could achieve the sensitivity of 88% and 99.5% specificity. Whilst 100 % accuracy was achieved at the sample level.

Summary/Conclusion:

By using Raman spectroscopy along with SVM, we were able to clearly discriminate the spectra coming from the two conditions. This method can reveal the effect of B(a)P on the molecular signature of the EVs.

Dr Rana Rahmani¹, Dr. Sanduru Thamarai Krishnan^1,2, Dr. David Rudd^1,2, Ehud Hauben^4,5, Prof. Nicolas H. Voelcker^1,2,3

¹Monash Institute of Pharmaceutical Sciences, Monash University, Australia, ²Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, Clayton 3168, Australia, ³Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia, ⁴The Basil Hetzel Institute for Translational Health Research, Australia, ⁵Discipline of Surgery, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Australia

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Although treatments for advanced colorectal cancer (CRC) have improved, patients without spreading cancer still have the best survival rates. We need tools to find blood markers that can help identify CRC types, track cancer progression, and check how well treatments are working.

Methods: We studied the exosomes, small extracellular vesicles (EVs), from blood samples to find signatures of CRC. Exosomes were isolated by plasma precipitation kits and characterized using NTA, Western blot, and SEM. A special method called nanostructured porous silicon (pSi) SALDI-HR-MS was used for precise detection of substances in these Evs. This method is quick and effective for analyzing small amounts of patient samples.

Results: In our study, we found specific proteins and lipids including PG (O-34:0), PC (36:4), PE (O-38:5), SM (36:1), and Cer (34:2), and proteins such as PP2A and ATF3. Analyzing lipid and protein data provided potential biomarkers for CRC. This method can be expanded to study other diseases once we validate these markers.

Conclusion: In conclusion, our study highlights the potential of nanostructured porous silicon SALDI-HR-MS as a rapid and effective tool for identifying specific biomarkers in small blood samples, offering insights into colorectal cancer progression and classification.

Dr Richard Lobb¹, Dr Alain Wuethrich¹, Associate Professor David Fielding², Professor Andreas Möller³, Professor Matt Trau¹

¹University of Queensland, Brisbane, Australia, ²Department of Thoracic Medicine, Royal Brisbane and Women's Hospital, Brisbane, Australia, ³5JC STEM Lab, Li Ka Shing Institute of Health Sciences, Department of Otorhinolaryngology, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, China

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Despite screening and therapeutic advancements, the global cancer burden is steadily rising, with 1 in 5 men and 1 in 6 women developing cancer in their lifetime. Furthermore, 1 in 8 men and 1 in 10 women will die from untreatable progression of cancer, making cancer one of the leading causes of death worldwide. There is a significant unmet clinical need to identify patients at an early-stage and develop novel therapies to reduce cancer mortality. Early detection of cancer is key as most early stage, localized cancers have the best chance of long-term survival. We hypothesized that circulating extracellular vesicles (EVs) derived from patient plasma carry specific molecular profiles to classify early-stage cancer.

Methods

EVs were purified from 500 µL of plasma using qEV Legacy columns, followed by elution with PBS. High EV fractions were collected and characterized for size and tetraspanin (CD9, CD63, and CD81) profiles using nanoFCM, and transmission electron microscopy. Purified EVs were captured using a nanotechnology platform developed for detection of surface protein and glycosylation markers enriched on cancer-derived EVs. This platform integrates single-particle active surface-enhanced Raman scattering to profile protein and glycosylation phenotypes at the resolution of single EVs.

Results

By profiling the proteins CD63, THSB2, VCAN, and TNC, and aberrant glycan expression, we successfully acquired unique EV molecular profiles to differentiate patients with early-stage cancer at a detection sensitivity of ≈12 EVs µL−1. This was clinically confirmed using positron emission tomography and tissue biopsy.

Summary/Conclusion

Given trace amounts of cancer derived EVs available in blood samples, coupled to the biological sample complexity and the vast heterogeneity of EV populations, this combined approach profiling EV surface biomarkers, coupled to the added specificity afforded by our SERS multiplexed protein mapping approach significantly improved cancer detection in clinical samples.

Associate Prof. Shona Pedersen¹

¹College of Medicine, Qatar University, Doha, Qatar

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Alzheimer's disease (AD) is a complex multifactorial disease without readily available clinical biomarkers. Blood-derived proteins are routinely used for diagnostics; however, in-depth plasma profiling is challenging due to the dynamic range of protein concentrations in plasma. Extracellular vesicles (EVs) carry cell-specific biological material and have been shown to cross the blood-brain barrier, and thus may provide a source for AD biomarkers. Methods: Plasma-derived EVs were investigated for AD-related protein biomarkers from 10 AD patients, 10 Mild Cognitive Impairment (MCI) patients, and 9 healthy controls (Con) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). EVs were enriched using 100,000 × g, 1h, 4°C as wash, and characterized according to the MISEV2018 guidelines. Results: Some AD patients presented with highly elevated FXIIIA1 and FXIIIB. A panel of proteins was identified discriminating Controls from AD patients. Three proteins were particular for AD; ORM2, RBP4, and HYDIN. Conclusion: EVs provide an easily accessible matrix for possible AD biomarkers. Seven of the MCI patients with protein profiles similar to the AD group progressed to AD within a 2-year timespan.

Dr Soumyalekshmi Nair, Anas Emerizal, Dominic Guanzon, Andrew Lai, Flavio Carrion, Yaowu He, Aase Handberg, Lewis Perrin, Gregory Rice, John Hooper, Carlos Salomon

Poster Pitches (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:45 PM - 1:00 PM

Introduction: Ovarian cancer (OVCA) ranks among the deadliest cancers, with 80% of cases diagnosed at advanced stages (Stage III and IV), resulting in 5-year survival rates of 42% and 26%, respectively. The current standard of care for OVCA involves tumor cytoreductive surgery, followed mainly by platinum-based chemotherapeutic regimens. In this study, we identified a set of circulating miRNAs within extracellular vesicles associated with overall OVCA survival that regulate responses to chemotherapy drugs in vitro.

Methods: This study included a cohort of n =  60 OVCA patients with up to 5 years of post-diagnosis follow-up information available. Extracellular vesicles (EV) were isolated and characterized using nanoparticle tracking analysis, protein abundance assessment (CD63, CD9, Alix, TSG101, and CD81), and morphology analysis using NanoSight, Western blot, and electron microscopy, respectively. The EV miRNA profile was analyzed using next-generation sequencing. We identified the association of EV miRNA expression with patient survival through Kaplan-Meier analysis. Cell migration and apoptosis were determined in SKOV3 cells, both in the absence and presence of miRNA mimics, alkylating agents, PARP inhibitors, or platinum-based chemotherapy drugs using a real-time imaging system.

Results: We identified dysregulation of 17 miRNAs in circulating EVs significantly associated with ovarian cancer patient survival. Moreover, miRNAs including miR-451a, let-7a-5p, miR-26a-5p, miR-486-5p, miR-21-5p, and let-7i-5p significantly decreased the migratory ability of SKOV-3 cells. Additionally, EV miRNAs altered the chemotherapeutic response of SKOV-3 cells to carboplatin, olaparib, and melphalan. MiRNAs such as miR-451a, miR-26a-5p, miR-486-5p, miR-21-5p, miR-16-5p, and miR-182-5p significantly increased apoptosis in response to carboplatin. Conversely, cells transfected with miR-26a-5p, miR-486-5p, miR-21-5p, miR-16-5p, let-7i-5p, and miR-182-5p showed a significant increase in apoptosis in response to olaparib, while let-7a-5p and miR-26a-5p overexpression resulted in significantly decreased apoptosis in response to melphalan. Overall, we identified that EV miRNAs associated with patient survival significantly regulate cell migration and chemoresistance in SKOV-3 cells.

Conclusion: This study suggests that sEV-associated miRNAs may play a role in OVCA patient survival by regulating tumor progression, metastasis, and responses to chemotherapy. This can enhance our understanding of the mechanisms behind cancer progression and chemoresistance, leading to the development of new therapies for OVCA patients.

Ms. Akane Sato, Mr. Kyo Okita, Dr. Etsuro Ito

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction. Gastric cancer is a common disease with high morbidity and mortality in East Asia, and 5-FU is routinely used in clinical practice as a first-line treatment for advanced gastric cancer. However, many patients develop drug tolerance during treatment. Drug tolerance becomes a barrier to improving treatment efficacy. Tumor-derived EVs may be involved in the development of 5-FU tolerance, for example in breast cancer. The expression of GRP78 (glucose-regulated protein 78 kDa) is increased in tumor tissues of gastric cancer patients, and GRP78 is involved in promoting cancer progression through EVs-mediated secretion. Based on these findings, we hypothesized that GRP78 secreted via EVs induces tumor cells that accept EVs to acquire stem cell-like characteristics, resulting in the appearance of 5-FU tolerance.

Methods. Ultrafiltration and polymer precipitation methods were used to isolate EVs from the conditioned medium of 5-FU-treated human gastric cancer cells. Confirmation of EVs was determined by particle size measurement and Western blotting. The acquisition of cancer stem cell-like properties in EV-treated cells was evaluated by cell viability assay and wound healing assay. Trace amounts of GRP78 in EVs were quantified by our proprietary thio-NAD cycling ELISA.

Results. Using the thio-NAD cycling ELISA, the limit of detection for GRP78 protein was 2.3 pg/mL. The amount of conditioned medium required for sample collection was approximately one-hundredth of the volume used for conventional analysis. We found that the concentration of GRP78 protein in exosomes increased in accordance with the concentration of 5-FU (0-50 µM) administered to the cells. A higher concentration of GRP78 protein in exosomes led to the acquisition of stem cell-like properties in tumor cells that accepted the exosomes. The cell viability and wound healing assays showed that tumor cells accepting exosomes derived from 5-FU-treated gastric cancer cells acquired stem cell-like characteristics, such as increased migration ability and increased cell viability.

Summary/Conclusion. These results indicate that 5-FU administration to gastric cancer cells secretes GRP78 protein-rich exosomes and that these exosomes promote the acquisition of cancer stem cell-like properties in the recipient tumor cells. Although the detailed mechanisms remain to be investigated, they may involve the PI3K/Akt signaling pathway.

Miss Piyatida Molika, Assoc. Prof. Dr. Raphatphorn Navakanitworakul

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Mesenchymal stem cells (MSCs) are a type of cells that have shown great potential in the field of regenerative medicine. MSCs have been discovered to possess tumor tropism, they can specifically target and home to tumor tissues. This ability allows MSCs to directly interact with tumor cells and exert anti-tumor properties, making them a promising tool for targeted cancer therapy. MSC-derived exosomes have emerged in recent years for the treatment of what were otherwise considered incurable diseases. Cervical cancer (CC) is the fourth most common cancer in female worldwide, especially in developing country including Thailand. In this study, we aimed to determine the effect of bone marrow MSC-derived exosome on cervical cancer spheroid model. Recently, 3D tumor models have become the gold standard in pre-clinical cancer research due to their capacity to better mimic the architecture and microenvironment of tumor tissue. MSC cells were grown in FBS-free DMEM medium for 72 hr. The medium was collected, and MSC-exosome were isolated and purified through 10k amicon column and size exclusion chromatography. The number and size distribution of MSC-exosome was measured using nanoparticle tracking analysis. The MSC-exosome was characterized using western blotting and transmission electron microscopy (TEM). Then, the MSC-exosome were treated to evaluate the effect of MSC-exosome on cervical cancer, HeLa and SiHa spheroids. The major population of MSC-exosome showed in size range 101-150 nm. Moreover, CD63, CD9 are showed expression on surface marker of MSC-exosome with negative of cytochrome C marker. To visualize the morphology under TEM, MSC-exosome expression regular red-cell like shape with double membrane layer. In addition, pkh67 labeled MSC-exosome showed ability to penetrate through 2D cervical cancer cell lines and attached on nuclear surface within 3-6 hr. Moreover, the internalized cells showed increase the proliferation rate than the control. These finding demonstrate that MSC-exosome and their cargo can be transferred and transmitted biomolecule between cells. This supports our initial hypothesis that MSC-exosome may altered the activity of cervical cancer recipient cells.

Professor Wen-wei Chang, Miss Hui-Yu Jiang, Dr. Wan-Hua Tsai, Professor Hsueh-Te Lee

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Breast cancer is the most common cancer in women worldwide. Among its subtypes, triple-negative breast cancer (TNBC) is notable for its aggressive nature, high rates of recurrence and metastasis, and reduced sensitivity to radiation therapy. Probiotics, beneficial microorganisms that promote human health when consumed in sufficient doses, have shown immunomodulatory effects and the potential to inhibit tumor cell growth. Extracellular vesicles (EVs), secreted by cells, facilitate the transfer of biomolecules between cells. While EVs derived from cancer cells have been linked to cancer progression, the anti-cancer potential of probiotic-derived EVs remains under-researched. This study investigates the anti-cancer potential of EVs from LP-0601, a Lacticaseibacillus paracasei strain, in TNBC cells. LP-0601-EVs were successfully isolated from culture media using an ultrafiltration method and validated through nanoparticle tracking analysis and transmission electron microscopy. The uptake of LP-0601-EVs by TNBC cells was observed using a lipophilic fluorescent dye labeling method. We noted a growth inhibition effect of LP-0601-EVs on TNBC cells, particularly to the radioresistant subline, as evidenced by MTT and colony formation assays. Mechanistically, LP-0601-EVs induced apoptosis and altered the cell cycle distribution in TNBC cells, as indicated by the activation of caspase-8 and caspase-3 and the downregulation of cyclin A2, cyclin B1, and cyclin-dependent kinases (CDK1, CDK4, and CDK6). Furthermore, treatment with LP-0601-EVs reduced the ability of TNBC cells to form tumorspheres and downregulated the expression of cancer stemness-related proteins, including BMI1, EZH2, and Nanog. We also used mass spectrometry analysis to identify the potential peptide targets in anti-caner activity of LP-0601-EVs. Our data suggest that LP-0601-EVs could potentially be developed as an adjuvant agent in TNBC therapy due to their ability to suppress both cancer cell proliferation and cancer stem cell activity.

Keywords: Lacticaseibacillus paracasei; extracellular vesicles; triple negative breast cancer; cell cycle; cancer stem cells

PhD Student Silvia López-Sarrió, PhD student Clara Garcia-Vallicrosa, PhD Student Guillermo Bordanaba-Florit, PhD Maria Azparren-Angulo, Postdoctoral researcher Félix Royo, Principal investigator Juan Manuel Falcón-Pérez

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

In a quest to advance and improve current liver cancer treatments, efforts are being concentrated on the development of new culture models for studying this tissue physiology and therapy approaches. In general, three-dimensional (3D) culture display better physiologic aspects of liver tissue compared with two-dimensional (2D) culture systems. In this work, we present the characterization of extracellular vesicles (EVs) released by hepatocyte spheroids as quality control. In addition, we also explore the potential of EVs treatment to target tumoral cells in mixed spheroids.

2) Methods

For the spheroids formation we used primary hepatocytes obtained by perfusion from mice that express a green fluorescent membrane tagged protein in their hepatocytes. Spheroids were formed by seeding 5000 cells/well in ultra low affinity 96 well plates. Vesicles in the conditioned culture media were characterized by different techniques and tracked by flow cytometry. Mixed spheroids formed by primary hepatocytes and tumoral cells were also cultured and characterized, and the specific capture of tumoral EVs by cells were assayed by confocal microscopy and flow cytometry.

3) Results

Spheroid cultures of primary hepatocytes retain better hepatic functionality and maintain viable cells on the surface, while massive death cell occurs in the inner core. They release EVs to the extracellular medium, although the release decrease along the time of culture. In addition, mixed spheroids self organise in a characteristic way depending on the tumour cell lines employed, and they also show differences in the capture of extracellular vesicles of tumoral origin.

4) Summary/Conclusions

In this study, we created a 3D cell culture model of primary hepatocytes, which can be useful for studying the relationship between normal and tumour cells. EVs can be tracked in the extracellular medium as an indicator of cellular status. In addition, EVs from tumour cells show target specificity towards spheroid populations, a phenomenon that can be explored for potential liver cancer treatments.

Keywords: Extracellular vesicles (EVs), spheroid culture, primary hepatocytes.

Dr Sai Vara Prasad Chitti, Akbar Marzan, Prof Suresh Mathivanan

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Cancer cachexia is a complex metabolic disorder that includes progressive muscle wasting and loss of fat stores. Though cachexia accounts for 30-40% of cancer-related deaths, currently there are no established treatment for cancer-induced wasting. In addition to pro-inflammatory and pro-cachectic factors, tumour-derived small extracellular vesicles (sEVs) are also proposed to induce muscle wasting and lipolysis. Furthermore, it is now well known that cancer cells tend to secrete more sEVs compared to non-cancerous cells and interestingly, several proteins that are involved in the sEVs biogenesis and secretion are found to be upregulated in cachexia causing tumours. Hence, we examined whether blocking the secretion of sEVs from tumour cells can inhibit cancer-induced cachexia.

Methodology: Cortactin (CTTN) was knocked-out (KO) using CRISPR/Cas9 technology in colon and pancreatic cancer cells. sEVs were isolated by differential ultracentrifugation and characterised by western blotting and nanoparticle tracking analysis. Co-culture and pre-clinical studies were carried out to study the cachectic phenotype. Fluorescence-based high-throughput screening assay was performed to identify the drugs that decreases sEVs secretion.

Results: Loss of CTTN inhibited the release of sEVs. While C26 wild type (WT)-derived sEVs induced atrophy in myotubes and lipolysis in adipocytes, CTTN-KO sEVs did not induce atrophy or lipolysis. Proteomics analysis of sEVs highlighted the enrichment of cachectic proteins in WT sEVs compared to KO sEVs. Follow-up C26 mice pre-clinical studies highlighted that CTTN-KO tumour-bearing mice exhibited stable body weight, reduced tumour burden, and dramatically extended lifespan compared to mice bearing WT tumour. Remarkably, CTTN-KO prevented tumour-induced loss of muscle, fat, and other major organs. Consistent with this, overexpression of CTTN increased sEVs secretion and drastically decreased the lifespan of C26 mice by accelerating tumour-induced weight loss. To use these findings for therapeutic benefit, we screened the library of FDA-approved drugs and identified several drugs that blocks the release of sEVs. Administration of sEVs inhibitor to the cachexic mice resulted in the abolishment of cancer-induced cachexia and prolonged survival.

Summary/Conclusion: Overall, these findings indicate that blocking sEVs release from tumour might be a promising approach to treat cancer-cachexia, improve quality of life, and extend the lifespan of cancer patients.

Xue Liu

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular matrix (ECM) stiffening is an important feature of tumor stroma and is related to tumor invasion, metastasis, drug resistance and prognosis. Recently, small extracellular vesicles (sEVs) play an important role in mediating cell communication. However, the interaction between EVs and ECM is rarely reported. It attracts researchers’ attention whether EVs produced by carcinoma-associated fibroblast (CAF) can mediate cell-ECM communication. In this study, we investigated the role of the OSCC-derived CAF sEVs mediating collagen crosslinking and promote epithelial-mesenchymal transition.

Methods: Four primary CAFs and normal fibroblast (NF) were isolated from human oral squamous cell carcinoma (OSCC) and normal gingival tissue for culture and purification. sEVs were isolated from the conditioned medium of four CAFs and NF by differential ultracentrifugation. Transmission electron microscopy (TEM) to characterize the morphology of sEVs. Western blot (WB) was used to identify sEVs protein expression. Nanoparticle tracking analysis (NTA) detects the range of particle size and concentration. The concentration of CAF sEV-LOX was determined by ELISA. Twelve hours after CAF sEVs was added to NF, ELISA determined the contents of Pyridinoline, dihydroxylysinonorleucine (DHLNL) and hydroxylysinonorleucine (HLNL) to evaluate collagen crosslinking in vitro. Immunofluorescence staining (IF) and WB examined the expression of the EMT markers in OSCC spheroids treated with CAF sEVs.

Results: CD63 and LOX proteins were present on the surface of CAF sEVs, whereas αLOX was not present on the surface of NF sEVs. The concentration of sEV-associated LOX (sEV-LOX) was determined by ELISA using intact CAF sEVs. Levels of PYD, DHLNL, and HLNL levels were significantly higher in the glucose and CAF sEV groups than in the PBS-treated group. E-cadherin, N-cadherin, vimentin in UM-SCC6 spheroids showed that CAF sEVs groups significantly decreased E-cadherin expression and significantly increased N-cadherin, vimentin compared with the PBS group. BAPN rescued E-cadherin expression and downregulated N-cadherin and vimentin in UM-SCC6 spheroids.

Conclusions: αLOX located on the surface of CAF sEVs, and directly mediated the collagen crosslinking and promoted the EMT of OSCC. These findings elucidate a critical mechanism underlying tumor ECM alteration and reveal a novel role of sEVs in ECM remodeling and its effect on cancer cells.

Dr. Yi Xu

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Hepatocellular carcinoma (HCC) is a major threat to human health worldwide, especially for East and Southeast Asian people. Small extracellular vesicles (sEVs) play a key role in exchanging cargoes between cells in tumor microenvironment. This study aimed to elucidate the functions and mechanisms of HCC derived sEV-clathrin light chain A (CLTA) in remodeling microvascular niche.

Methods:

CLTA level in the circulating sEVs of HCC patients was analyzed by enzyme-linked immunosorbent assay (ELISA). The functions of sEV-CLTA in affecting HCC cancerous properties were examined by multiple functional assays. Mass spectrometry was used to identify downstream effectors of sEV-CLTA in human umbilical vein endothelial cells (HUVECs). Tube formation, sprouting, trans-endothelial invasion and vascular leakiness assays were performed to determine the functions of sEV-CLTA and its effector, basigin (BSG) in HUVECs. BSG inhibitor, SP-8356, was tested in a mouse model of patient-derived xenografts (PDXs).

Results:

Circulating sEVs of HCC patients had markedly enhanced CLTA levels than control individuals and were reduced in patients after surgery. HCC derived sEV-CLTA enhanced HCC cancerous properties, disrupted endothelial integrity and induced angiogenesis. Mechanistically, sEV-CLTA interacts with BSG, thereby alleviating the interaction between BSG and FBXO22 and preventing BSG from polyubiquitination and degradation induced by FBXO22 in HUVECs. Blocking BSG with antagonists either alone or in combination with sorafenib could suppress the development of HCC PDXs.

Conclusions:

The study demonstrated the role of CLTA in remodeling premetastatic microvascular niche by stabilizing BSG via its transfer to endothelial cells by sEVs. The findings point to the clinical relevance of the potential application of circulating sEV-CLTA in liquid biopsy for early detection of HCC. This study also provides insights into a new therapeutic strategy by inhibiting BSG and blocking its mediated effect on neoangiogenesis.

Research Fellow Adnan Shafiq, Shinya Sato, Alissa Weaver

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Abstract:

Cancer metastasis is intricately influenced by both tumor cell attributes and the microenvironment. Extracellular vesicles (EVs) play a key role in tumor metastasis by both promoting cancer cell migration and invasion and influencing cells in the tumor microenvironment. To identify key cargoes driving metastasis of head and neck squamous cell carcinoma, we performed a quantitative proteomics analysis of EVs purified from matched mouse oral cancer (MOC) cell lines with different metastatic abilities.

Methods:

Small EVs were purified by the cushion density gradient method from MOC1 and MOC2 oral cancer cell lines. MOC1 is a nonmetastatic cell line that is “immune-hot”, recruiting T-cells into tumors. By contrast, MOC2 is a metastatic cell line that is “immune-cold”. EVs were analyzed by isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry. The data were analyzed by STRING and Gene Set Enrichment Analysis (GSEA) methods to identify enriched functional and interacting groups of proteins.

Results:

iTRAQ proteomics analysis of MOC1-and MOC2 cell-derived exosomes revealed that MOC1 cell-derived small EVs contained many RNA binding proteins, including those involved in ribosome biogenesis, RNA processing, and nonsense-mediated decay (NMD) as well as apoptosis. By contrast, MOC2 cell-derived small EVs were enriched with factors related to cell migration, angiogenesis, ECM organization, and wound healing.

Conclusion:

We performed a comparative proteomics analysis of matched oral carcinoma cell lines that have distinct indolent (MOC1) versus metastatic (MOC2) phenotypes. We identified increased expression of RNA binding proteins in MOC1 cell-derived small EVs, suggesting increased RNA content. By contrast, MOC2 cells-derived small EVs were significantly enriched for proteins associated with cell migration, invasion, and tumor angiogenesis. Future work will investigate the RNA content of MOC1 small EVs and its role in modulating the immune microenvironment, along with the role of MOC2 small EV cargoes in promoting tumor cell metastasis and angiogenesis.

Mr Tae-Kyu Jang, Dr Eunyi Moon

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

The primary cilia (PC) are microtubule-based organelles that help detect and transmit environmental signals. Ciliogenesis is influenced by Ciliary extracellular vesicle (EV) formation. Chemokine receptor CXCR4 is required for ciliogenesis in Kupffer's vesicle. CXCR4 is specific for stromal-derived-factor-1 (SDF-1 also called CXCL12), which usually increased in various cancer cells. Cells survived from anticancer drug treatment is one of the most serious obstacle in cancer chemotherapy. Here, we investigated whether CXCR4 could affect cancer cell death via PC formation enhanced by hedgehog signaling using HeLa human cervical cancer cells and anticancer drug, vinblastine (VBL). Cancer cell death rate by VBL was reduced under serum-deficiency (SD) condition which increase the frequency of ciliated cells. VBL enhanced CXCR4 expression, which was more increased under SD condition to activate hedgehog signaling compare to the condition with FBS. CXCR4 expression was increased by the treatment with smoothened agonist (SAG). Cell death by VBL was decreased by the overexpression of pEGFP-SMO or SAG treatment increasing PC formation. In addition, SDF-1, CXCR4 ligand, increased PC formation, which contribute to the decrease in cell death by VBL. Cells survived from repeat treatment with VBL show the increased CXCR4 expression compared to wildtype cells. PC formation and cell death by VBL were attenuated by the inhibition of CXCR4 expression with siRNA. Taken together, data demonstrate that VBL-mediated cancer cell death could be regulated by CXCR4 expression-mediated PC formation. It suggests that CXCR4 could be a novel chemokine to crosslink PC formation and cancer cell death.

“This research was supported by the Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT(grant number 2021R1A4A5033289 and RS-2023-00244570), Republic of Korea.”

Dr Tung Him Ng, Ms Aijun Liang, Prof Judy Wai Ping Yam

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Small extracellular vesicle (sEV) can be utilized as drug delivery vehicle attributed by its ability to target and accumulate in different tissues. The selectivity is mainly determined by integrins located on sEV surface. Evidence showed that lung targeting sEV had elevated level of integrin β4. Here, we aim to clarify the role of integrin β4 in directing sEV to lung, and try to devise a drug-loaded integrin β4-enriched sEV to target pulmonary metastasis of hepatocellular carcinoma (HCC).

Methods: Small EVs were isolated from cells, fluorescently labelled and injected into mice. Tissue distribution of the labelled-sEV was examined. Pulmonary HCC metastasis model was established by intravenously injecting p53-/- mouse hepatoblast into nude mice. The mice were treated with drug-loaded sEV. Treatment efficacy was evaluated.

Results: Small EVs from metastatic HCC cells had higher level of integrin β4 compared to those from normal liver and non-metastatic HCC cells. The level correlated with the tendency to localize in lung. When integrin β4 in sEV was reduced upon knockdown, the lung targeting ability was largely compromised. In contrast, overexpression of sEV-derived integrin β4 remarkedly enhanced its lung targeting ability. Then, sEVs from 293FT cells were overexpressed with integrin β4 and loaded with doxorubicin (Dox) by electroporation. The morphology, composition and lung targeting ability of the sEVs and cytotoxicity of Dox were not affected. Treatment using the Dox-loaded sEV significantly reduced tumor cell colonization in lung comparing to treatment using saline or equal amount of free Dox in the murine metastasis model. The treatment efficacy was as effective as using ten-fold amount of free Dox.

Summary: Our results demonstrated the ability of integrin β4 to direct sEV to lung. The lung targeting sEV could be loaded with chemotherapeutics and used in the treatment of pulmonary metastasis of HCC.

Dr Darren Toh, Ms Hui Sun Leong, Ms Fui Teen Chong, Ms Mengjie Ren, Dr Gopalakrishna Iyer

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Cancers of the head and neck (HNSCC) are primarily driven by EGFR overexpression. Previously, we reported the expression of an EGFR splice-variant isoform D (IsoD) in extracellular vesicles (EV) isolated from HNSCC primary cell line and demonstrated the co-application of IsoD-containing EVs and tyrosine kinase inhibitor (TKI) increases the sensitization of cancer cells previously resistant to TKI treatment. We extended the study to understand the sensitization mechanisms conferred by IsoD-containing EV by examining the mechanisms of IsoD EV uptake in target cells. We find that EV binding and endocytosis are required to exert an IsoD EV-sensitizing effect.

Methods

We over-express IsoD in HEK293T cells and culture them in defined serum-free media; EVs are purified and concentrated by an Amicon Ultra 15, 50kD MWCO filter. The isolated EVs are characterized using NTA and labeled with CellTrace Violet dye. Cancer cells are grown in EV-free RPMI and co-treated with labeled IsoD EVs and inhibitors of clathrin-mediated endocytosis, PitStop2, for the indicated time. The cells are then examined by immunofluorescence. In addition, siRNA against RAB5A was also applied to target cells to reduce early endosome trafficking. Immunofluorescence studies were conducted on these cells after they were treated with EVs containing IsoD. Viability assay (CellTiter-Glo) was used to determine target cell viability during IsoD EV and TKI co-treatment in the presence or absence of the inhibitors.

Results

We observed increased EV accumulation on the cell surface in cells treated with inhibitors (PitStop2 and siRNA5A) compared to no inhibitor controls. This suggests that PitStop2 and siRNAB5A inhibit the endocytosis of EVs in target cells. Importantly, the inhibition of endocytosis reduces the sensitizing effect of IsoD EV in TKI co-treatment of target cells, suggesting that endocytosis of IsoD-containing EV is required to confer its sensitizing effect.

Summary/Conclusions

Clathrin-mediated endocytosis and endosomal trafficking are required for the sensitizing effect conferred by IsoD-containing EVs during TKI co-treatment of HNSCC cancer cells.

Dr. Xiaoxin Zhang

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Intorduction

Emerging evidence have proved that the interplay between tumor and its microenvironment facilitate tumor metastasis, including the hepatocellular carcinoma (HCC). Surgical resection is not available for HCC patients with distant metastasis, and therapeutic options are quite limited to multi-kinase inhibitors, which tremendously influence prognosis of late-stage HCC patients. Therefore, identifying novel target for HCC metastasis and is essential to improve the survival.

In recent years, EV mediated metabolic regulation has been a growing interest in cancer metabolism. For instance, the ectosomal pyruvate kinase M2 isoform (PKM2) was found to facilitate glycolysis reprogramming in monocyte-to-macrophage differentiation and tumor microenvironment remodeling. Besides, the hepatic stellate cells secreted hexokinase (HK1) through exosome was hijacked by tumor cells to accelerate glycolysis in HCC progression. Compared to directly receiving metabolite or substance from the EV, we proposed that the tumor cells are more likely to obtain proteomics alteration to exert long-term metabolic reprogramming.

Methods

EVs were extracted from the metastatic HCC cells, and the non-metastatic HCC cells. The DEPs will be identified by LC-MS/MS and the differentially metabolic pathways will be identified by GO and KEGG analysis. Meanwhile, the metabolomic screening will also be conducted to clarify the differences of metabolite between metastatic and non-metastatic HCC EVs.

The function of G6PD regulating pancreatic cancer cells: siRNA knocks down the expression of G6PD.Combing with CCK8, plate cloning, Brdu, Transwell and so on, we verify the effect of G6PD on the proliferation, apoptosis, invasion, and metastasis of pancreatic cancer cells. In the meantime, parallel validation of G6PD inhibitors was conducted.

Results

Our study found that key enzyme involved in pentose phosphate pathway (PPP), rather than glycolysis, was extremely enriched in metastatic HCC derived small extracellular vesicles (sEVs). Inhibition the enzymic activity of sEVs-derived G6PD significantly reduce the invasion and metastasis of HCC tumor cells. Besides, the bioinformatics analysis also indicated that G6PD was associated with activation of cancer-associated fibroblast.

Conclusion

The study found the dysregulated metabolicin HCC cell and pre-metastasis niche.

Mr. Chun-Fan Lung, Ph. D Student Chun Fan Lung

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Lung adenocarcinoma, a prevalent form of non-small cell lung cancer, frequently progresses to an advanced stage accompanied by resistance to EGFR tyrosine kinase inhibitors like gefitinib, contributing to poor patient outcomes. Exosomes, crucial mediators of intercellular communication, have surfaced as potential influencers in drug resistance mechanisms. This study delved into understanding the involvement of exosomes in gefitinib-resistant lung adenocarcinoma.

Exosomes were meticulously isolated from cell lines, and an extensive screening ensued to discern miRNAs, RNAs, and proteins showcasing significant differences in expression between exosomes and the cell line's intrinsic RNA and gene products (fold change > 2 or < -2). Within this comparative analysis, 18 miRNAs within exosomes exhibited noteworthy expressions, among which 16 displayed downregulation in gene interaction, including ERK1/2, mTOR, DDX5, AMPK, and AKT.

Our findings emphasize the potential roles of ERK1/2 and mTOR encapsulated within exosomes in fostering gefitinib resistance in lung adenocarcinoma. Moreover, targeting exosomal miRNAs emerges as a promising strategy to counter resistance. Further investigations are imperative to authenticate these observations, paving the way for novel therapeutic interventions against lung adenocarcinoma.

Dr Takahiko Sakaue, Kalpana Deepa Priya Dorayappan, Dr Wafa Khadraoui, Dr Muralidharan Anbalagan, Dr Adrian Suarez, Dr Casey Cosgrove, Dr Larry J. Maxwell, Dr Hironori Koga, Dr David O'Malley, Dr David Cohn, Dr Selvendiran Karuppaiyah

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Endometrial cancer (EC) is the leading gynecologic malignancy in the U.S., with obesity implicated in 57% of cases. This study explores the molecular workings of oncogenic protein expression (TMEM205, STAT5, and FAS) and their role in regulating exosome secretion. Understanding these mechanisms is crucial for unraveling pathways involved in obesity-related EC, informing the development of innovative strategies for prevention and treatment.

Methods:

The isolated exosomes were quantified using nanoparticle tracking analyzer (NTA) and their size measured by Transmission electron microscopy (TEM). TMEM205, STAT5, FAS and PIAS3 expression was confirmed by IHC, ELISA and RT-PCR in patient and high fat diet treated mouse tissue. Endometrial hyperplasia was developed in immunocompetent mice using high fat diet (HFD; 45 kcal% fat diet) for 16 weeks.

Results:

Our study investigates exosome secretion and the regulation of oncogenic proteins in adipose and uterine tissues from obese EC patients compared to non-obese EC samples. Analyzing the effects of a 45% kcal high-fat diet (HFD) on mice over 24 weeks, we observed higher body weight and increased adipose tissue in the HFD group, along with enlarged uterine horns and heightened inflammation. This correlated with elevated exosome secretion and increased expression of TMEM205, FAS and STAT5, while the tumor suppressor gene PIAS3 was downregulated. The identified small molecule inhibitor, DAP-5, selectively targeting TMEM205 and exosome secretion, demonstrated a significant reduction in body weight and adipose tissue accumulation when administered to HFD mice. Additionally, DAP-5 treatment restored normal uterine morphology and reduced the expression of exosome-related proteins.

Conclusion:

This study offers central insights into the mechanisms underlying obesity-mediated TMEM205 expression and exosome secretion, shedding light on their role in the pathogenesis of EC. Additionally, it provides pre-clinical evidence supporting the initiation of the first in-human studies for exosome-targeted therapies aimed at preventing obesity-mediated EC.

Phd Xin Zhang, Quan Zhong, Jiaming Chen, Zhenxun Wang, Bin Xu, Boyan Boyan Huang, Jinsheng Zheng, Tianyu Wu, Yu Wang, Lei Zheng

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Background: Intrahepatic and lung metastasis is a hallmark of Hepatocellular carcinoma (HCC). Tumor shedding EVs to naturalize pre-metastatic niche formation caused wide attention, while mechanism of EVs activating metastasis are remains unclear.

Methods: HCC based cell dependent xenograft (CDX) model were established and Early growth response-1 (EGR1)-rich Extracellular Vesicles (EVs) tail vein injection (i.v.) were performed to study the relationships between EGR1-rich EVs and HCC intrahepatic and lung metastasis.

Results: EGR1-EVs injection induced several CAFs-associate factors and pro-inflammatory cytokines, such as IL-6, CXCL12, IL-8, IL-1β, α-SMA and TGF-β etc. in the tumor tissues. Besides, we found that EVs-EGR1 inhibited TGF-β induced-LX2 invasion and activation of resident fibroblasts to be cancer associated fibroblast (CAFs) in vitro. Consistent with this, in clinical samples of metastatic HCC and non-metastatic HCC, EGR1 positive EVs (EGR1+EVs) significantly decreased in metastatic HCC plasma.

Conclusions: These findings uncovered extracellular vesicle-dependent inhibition effects of EGR1 on HCC metastasis. Monitoring EGR1+EVs level in plasma provides an attractive approach for distinguishing metastatic HCC, making it a potential diagnostic and therapeutic biomarker.

PhD Darshak Bhatt, Msc Annemarie Boema, PhD Silvina Bustos, PhD Andreia Otake, PhD Alexis Carrasco, Professor Patricia Reis, Professor Roger Chammas, Professor Toos Daemen, PhD Luciana Andrade

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Antineoplastics induce modifications in tumor cells that are also reflected in its secretome, including EVs, which may lead to therapy resistance. We previously demonstrated that EVs secreted by melanoma during chemotherapy induce a nuclear reprogramming in the tumor microenvironment leading to tumor recurrence. Then, we hypothesized that EVs secreted during the therapeutic period constitutes a potential predictive factor of response. Since oncolytic viral (OV) therapy has been explored as an adjuvant for advanced/unresectable melanoma, we aimed to determine whether EVs released under virotherapy impact in tumor response. To that, we used an

OV expressing a transgene for hmgb1, a known DAMP (damage-associated molecular pattern), with the goal of generating more potent OVs to eliminate tumor burden. Methods: B16F10 murine metastatic melanoma cells were infected or not with Semliki Forest Virus (SFV)-based replicon, containing hmgb1 as a transgene or GFP as control (SFV-HMGB1 and SFV-GFP, respectively). 24h after infection, conditioned media was collected to EVs isolation by differential ultracentrifugation. Size distribution and concentration quantification were determined by nanoparticle tracking analysis. The presence of CD9, CD63, and calnexin were determined by western blot. For in vivo experiments, naive B16F10 cells were educated in vitro with EVs from infected and non-infected cells for 3 days and treated or not with SFV-HMGB1 or SFV-GFP particles. Cells were injected s.c in C57BL/6 mice (Ethics of Animal Experiments approval #1808/2022) and tumor growth was measured using a caliper. For EVs miRNA analysis, EVs were treated with Proteinase K and RNAse A before RNA extraction. miRNA content was conducted through hybridization. Results: EVs from SFV-infected cells impaired melanoma growth in comparison to the control group. Surprisingly, the presence of the transgene hmgb1 in SFV particles was able to abolish the inhibitory effect and, indeed, boosted tumor growth. Moreover, at the molecular level, EVs secreted by cells infected with SFV-HMGB1 particles were enriched with miRNAs related to tumor growth and cancer. Conclusion: The presence of hmgb1 in oncolytic SFV genome counteracts the efficacy of virotherapy that seems to be associated with a specific loading of a pro-tumoral miRNA profile. Funding: FAPESP (2020/09176-8).

Extracellular vesicles and breast cancer Mina Mobin Rahni, Immunology Marzieh Ebrahimi, Extracellular Vesicles Faezeh Shekari

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Breast cancer is a prevalent malignancy affecting women globally, characterized by changes in estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. These alterations are also evident in extracellular vesicles (EVs) isolated from peripheral blood. Notably, elevated HER2 expression, particularly the extracellular domain (HER2-ECD), is a significant marker. Recent studies on EVs reveal their ability to transport surface-bound biological molecules, creating a corona and enabling the diffusion of these molecules throughout the body. This newfound understanding offers valuable insights into the breast cancer diagnostic and therapeutic approaches.

Methods

First, conditioned media from two cell lines, HER2-negative (MCF7) and HER2-positive (SKBR3), was collected over 52 days. To ensure optimal conditions, fresh complete medium was added at least 24 hours prior to reaching confluency, followed by standard incubation. Conditioned media, obtained from cells with confluency exceeding 80%, was promptly stored at -80°C. Extracellular vesicles (EVs) were isolated from the conditioned media through high-speed centrifugation (20,000 g), and their characteristics were assessed following the MISEV 2018 guidelines. The presence of HER2-ECD in both cell secretions and EVs was determined using ELISA method. Subsequently, HER2-negative EVs were incubated in HER2-positive cell secretions, followed by EVs isolation and identification of HER2 in EVs-corona and functional assessments on MCF7 cell line.

Results

The results of our investigation reveal that three essential EV markers were appropriately expressed in EVs derived from both HER2-negative and HER2-positive sources. The isolated EVs exhibited a medium size. The distinct presence of HER2 in EVs originating from the HER2-negative and HER2-positive cell lines, along with the impact of HER2-enriched EVs on the MCF7 cell line, underscores the significance of HER2 localization within EVs.

Conclusion

Our discovery of the HER2-ECD in EVs corona marks the beginning of a promising journey with potential applications in both the biological significance of circulating HER2-ECD and diagnostic use.

Dr. Jen-Lung Chen, Ms. Hsin-Yi Tsai, Assistant Professor Ming-Wei Lin

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Cancer cells possessing the ability to self-renew and maintain stemness may cause cancer recurrence and contribute to chemoresistance. Glucose-regulated protein 78 (GRP78) is thought to regulate the tumor microenvironment, leading to cancer chemoresistance and metastasis. Our previous study revealed that GRP78-containing extracellular vesicles (EVs) induce endothelial migration and angiogenesis. However, whether GRP78-rich EVs contribute to gastric cancer stemness and metastasis are unclear. Here, this study aims to explore the molecular mechanism of GRP78-rich EVs in gastric cancer cancer stemness and metastasis.

Methods: GRP78 was overexpressed or knocked down in human gastric cancer cells. The EVs-derived from human gastric cancer cells were isolated and purified by using qEV size exclusion chromatography. The concentration and size of EVs were measured by scatter-based nanoparticle tracking analysis. The biomarkers of EVs, including CD63, CD9 and CD81, were identified by flow cytometry. The GRP78 concentrations in EVs were measured using an ultrasensitive ELISA. Cancer stemness abilities were evaluated by sphere formation analysis and soft agar colony formation analysis. The signal transduction molecules and cancer stemness-related markers were evaluated by Western blot or flow cytometry. The cell viability was evaluated by Cell Counting Kit-8, and cell migration was measured by wound healing assay.

Results: The concentrations of GRP78 in EVs-derived from GRP78-overexpressed cells was significantly higher than GRP78-KO cells. GRP78-rich EVs promoted the malignant behaviors of gastric cancer and expression of stemness marker proteins, including CD44, CD24, Sox2 and Nanog. Moreover, Co-incubation with GRP78-rich EVs promoted gastric cancer cell chemoresistance to 5- Fluorouracil.

Conclusion: Taken together, the present study points to the importance of GRP78-rich EVs in cancer stemness and metastatic potential in gastric cancer, which may be potential targets for anticancer therapy.

Dr. SANJAY SHAHI, Prof. Suresh Mathivanan

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Breast cancer is the second most common cancer in women worldwide. Most of the breast cancer related death is attributed to metastasis. While complete mechanisms of metastasis are yet elusive, small extracellular vesicles (sEVs) are known as key drivers of cancer metastasis and organotropism. In this project we targeted cortactin, a protein involved in sEVs secretion, to block EVs and inhibit breast cancer metastasis.

Methods: In attempt to block sEVs release via molecular approach, we knocked out cortactin from mouse (4T1.2) and human (MDA-MB-231) triple negative breast cancer cells via CRISPR/Cas9 gene editing technology. The wild-type and cortactin knockout cells, as well as EVs secreted from them, were characterized in vitro. The effect of cortactin knockout in the breast cancer progression and metastasis was evaluated in respective preclinical mice models.

Results: Knockout of cortactin compromised the sEVs secretion from breast cancer cells. Depletion of cortactin lead to significant reduction in proliferation, colony formation and migration of human and mouse breast cancer cells. Proteomic analysis of the cells and sEVs released from wild-type and cortactin knockout human breast cancer cells revealed significant alteration in biological pathways of cells, as well as cargo of EVs. Furthermore, animal model of murine and human breast cancer cells unravelled significant inhibition of breast cancer growth and metastasis upon loss of cortactin.

Conclusion: Overall, these observations signify that sEVs mediated regulation of breast cancer progression may be controlled by cortactin. Moreover, targeted sEVs secretion significantly diminished the aggressiveness of breast cancer, establishing sEVs as potential target for combating breast cancer progression and metastasis.

Dr Andrew Lai, Dr Priyakshi Kalita-de Croft, Dr Dominic Guanzon, Dr Soumyalekshmi Nair, Mr Nihar Godbole, Dr Flavio Carrion, Dr Shayna Sharma, A/Prof Margaret Cummings, Prof Lewis Perrin, Prof John Hooper, Prof Ken O'Byrne, Prof Sunil Lakhani, A/Prof Fernando Guimaraes, Dr Arutha Kulasinghe, Prof Andreas Moller, Prof Carlos Salomon

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Patients diagnosed with epithelial ovarian cancer (EOC) often present with advanced-stage disease, where treatment options are limited. In this study, we investigate the miRNA content of circulating extracellular vesicles (EV) in EOC patients and integrate this data with spatial information from the tumour microenvironment (TME).

Methods: We enrolled a cohort of 48 patients with varying clinical outcomes: recurrence of the disease (n = 9), deceased (n = 17), and disease-free (n = 21). We isolated and characterized EVs from plasma using size-exclusion chromatography, assessing their size, protein abundance, and morphology through nanoparticle-tracking analysis, western blot, and electron microscopy, respectively. We prepared a small-RNA library and validated the expression of specific miRNAs using RT-qPCR. To gain insights into the TME, we employed the NanoString-GeoMX Digital Spatial Profiler (DSP) platform on a subset of formalin-fixed tumor samples. This platform enabled profiling of EOC tissue for various protein markers related to immune cell profiling, immuno-oncology (IO) drug targets, immune activation status, immune cell typing, and pan-tumor protein modules.

Results: A total of 1218 sEV-associated miRNAs were identified across the samples. Among these, 49 miRNAs exhibited significant differences between disease-free and recurrent cases. Notably, hsa-miR-3202-1, hsa-miR-3202-2, hsa-miR-4516, hsa-miR-139-5p, and hsa-miR-6865-5p were over 20-fold higher in patients with cancer relapse. Additionally, hsa-miR-4740-3p was 17-fold higher in deceased patients compared to disease-free individuals, while hsa-miR-106b-3p and hsa-miR-144-3p were five-fold higher in deceased patients compared to those with recurrent disease. Utilizing NanoString DSP, we identified 54 differentially expressed proteins between the tumor and its microenvironment in EOC. Within the EOC group, 32 proteins were dysregulated in deceased patients compared to disease-free ones. Furthermore, the recurrent disease group exhibited 26 highly expressed proteins compared to the deceased group. Notably, IO drug target molecules such as B7-H3, Beta-2-microglobulin, CD14, CD34, CD44, and CD45RO were among the most dysregulated in the TME. An integrative analysis pairing miRNAs with mRNAs (NanoString) via Ingenuity Pathway Analysis revealed a regulatory network of 10 miRNAs potentially influencing the expression of proteins associated with ovarian cancer progression.

Conclusions: Our findings suggest that circulating EV in EOC patients may transfer oncogenic miRNAs to cells within the TME, thereby promoting cancer progression.

Ms Diana Huber, Mrs Tsima Abou Kors, PhD Linda Hofmann, Prof Monika Pietrowska, PhD Marta Gawin, Prof Ramin Lotfi, Prof Thomas K Hoffmann, Prof Cornelia Brunner, Prof Marie-Nicole Theodoraki

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

As one of the most immunosuppressive cancers, head and neck squamous cell carcinomas (HNSCC) show an increased NF-κB activation with downstream production of immunosuppressive and tumor-promoting factors. Plasma-derived small EVs (sEVs) from HNSCC patients contain molecules, which can contribute to the immunosuppressive tumor microenvironment (TME). Here, we investigate the influence of plasma-derived sEVs from patients with HPV-positive and HPV-negative HNSCC on macrophage function.

Methods

sEVs were isolated from plasma of HNSCC patients and healthy donors by size-exclusion chromatography (EV-Track: EV200068). Monocytes from buffy coats were used to generate primary macrophage cultures, which were incubated with plasma-derived sEVs to investigate their effects on the proteome, analyzed by mass spectrometry. To examine the difference between non-HPV- and HPV-induced HNSCC, RNA sequencing of macrophages, incubated with sEVs from HPV-positive and -negative HNSCC patients, was performed.

Results

Incubation with sEVs changed the proteome of macrophages in a time-dependent manner, supposedly by modulating processes rather than by transferring proteins from sEVs. Activation of p65-dependent NF-κB signaling was transiently increased by HNSCC-sEVs, which was confirmed by p65 translocation assays. HPV-tumor status of sEV donors affected the RNA profile of treated macrophages and several NF-κB-related genes were differentially expressed after incubation with HPV-positive or HPV-negative sEVs, respectively, indicating an HPV-dependent modulation of NF-κB pathway by HNSCC sEVs. Furthermore, uptake of EVs showed different dynamics depending on the HPV status of EV donors.

Summary/Conclusion

Plasma-derived sEVs from HNSCC patients alter immunosuppressive properties of macrophages. Importantly, the HPV-status of the patients has to be considered. The sEV dependent NF-κB activation may be useful for future therapeutic strategies on modulation of tumor-associated macrophages through targeting sEVs in the TME or suitable molecules within this pathway.

Ms Daniela Likonen

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Background

Malignant melanoma (MM) originates in the epidermis; during progression, cells invade into the dermis and become metastatic through the lymphatic system. Immunotherapy has become a clinically validated treatment for Malignant MM. However, despite its remarkable success, the majority of patients will experience only partial response followed by relapse of resistant tumors. One of the mechanisms for tumor escape is immunotolerance induced by tumor microenvironment (TME) cells; however, whether the immunomodulatory features of TME cells are regulated by MM extra-cellular vehicles (EVs) is currently unknown.

Aims

We aimed to evaluate the immunoregulatory effect of MM-secreted melanosomes on TME cells, with a focus on human dermal lymphatic endothelial cells (HDLEC).

Material and methods

Melanosomes were isolated from patients-derived MM cells and molecularly characterized. Tumor induced lymphocytes (TILs) were incubated with HDLEC with or without prior incubation with melanosomes. The Immunogenic activity of the TIL's was analyzed using apoptosis assays and ELISA for LDH and IFNγ. RT-PCR of candidate genes and RNA-seq were used to evaluate the effect of EVs on HDLEC and the TILs, respectively.

Results

Melanosomes secreted by MM cells are taken by HDLEC and induce tumor infiltrating lymphocytes (TILs) inactivation by ∼25%. Transcriptionally, T-cell exhaustion and CTLA-4 were among the most regulated pathways in TILs. Mechanistically, melanosomes express CEACAM1, a known checkpoint inhibitor and lead to CEACAM1 overexpression in HDLEC. The immune inactivation induced by melanosomes is partially reversed upon inhibition of CEACAM1 by blocking antibody.

Conclusions: MM secreted melanosomes induce immune tolerance in lymphatic endothelial cells, at least in part through CEACAM-1. Overall, this work highlights a novel mechanism by which MM induces immune tolerance in the TME cells.

Professor Yoon-Jin Lee, Ms. Shinwon Chae, Ms. Haekang Yang, Mr. Chul Won Seo, Mr. Chang Yeol Lee, Professor Sang-Han Lee, Dongsic Choi

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Most cancer cells adopt a less efficient metabolic process as an aerobic glycolysis with high level of glucose uptake followed by lactic acid production, known as the Warburg effect. This phenotypic transition enables the cancer cells to get the increased cellular survival and proliferation in the harsh low-oxygen tumor microenvironment. Also, resulting acidic microenvironment brings the inactivation of immune system such as T-cell impairment favoring the escape by immune surveillance. Although tumor-derived EVs deliver the parental oncogenic materials to adjacent cells contributing the oncogenic reprogramming, metabolic effects of EVs are not well addressed.

Methods: In this study, we established the prostate cancer cell line PC3-AcT, resistant from the cellular death in the acidic culture media driven by lactic acid. Quantitative proteomics between EVs derived from PC3 and PC3-AcT cells identified the 1139 confident EV proteins with abundant canonical EV proteins such as ALIX, syntenin-1, CD9, and CD81 but depletion of calnexin and cytochrome c. Also, metabolic phenotypes were addressed by glycolytic enzyme expression, glucose uptake, ATP generation, and cell proliferation.

Results: We revealed that the increased cellular growth of PC3-AcT cells is mediated by the activation of glucose energy metabolism such as hexokinases, PFKP, and PDH. From proteomic analyses, we found PC3-AcT EVs are equipped with the glycolysis-related proteins abundantly and particularly enriched with heparin-binding proteins and ApoB100 for the facilitated EV uptake. PC3-AcT EVs were readily taken up by PC3 cells and PC3-AcT EVs encourages the parental PC3 cells to acquire the increased cell proliferation and survival in acidic culture media. Also, PC3 cells primed by PC3-AcT EVs showed the increased expression of HK-1 and HK-2, PFKP, and PDH as the similar level of PC3-AcT cells and increased glucose comsuption with ATP generation representing the acquired metabolic reprogramming by EVs. These Warburg phenotypic transition was inhibited by EV uptake inhibitors such as EIPA and Dynosore validating the EV uptake is key regulating step in these metabolic reprograming.

Conclusion/Conclusion: Our study first revealed that EVs derived from prostate cancer cells could contribute the energy metabolic reprograming and their acquired metabolic phenotypic transition favors the cellular survival in tumor microenvironment.

Ms Nathalia Leal Santos, Roger Chammas, Luciana Andrade

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Melanoma is a highly aggressive and resistant form of skin cancer. To date, advanced-stage patients often relapse to standard-of-care approaches, prompting the search for innovative therapies. Recently, modulation of tumor-derived Extracellular Vesicles (EVs) cargo, with enrichment of antitumoral molecules, has been showing good perspectives as adjuvants. We recently demonstrated that melanoma-derived EVs enriched with the tumor suppressor miRNA, miR-195-5p, could inhibit tumor growth and sensitize cells to targeted therapy, through miR-195-5p/BCL2-L1 axis. As target prediction analysis showed that this miRNA may also regulate DNA damage response (DDR)-related genes, we aimed to analyze the effect of miR-195-5p-loaded EVs in 3D tumor growth and response to radiotherapy (RT), which is often used in unresectable cases and as palliative care in metastatic melanoma patients. Methods: EVs were isolated from the conditioned media of primary (A375 and WM-1366) and metastatic (SKMel-28, SKMel-147, and UACC-62) melanoma cells using differential ultracentrifugation followed by size exclusion chromatography. Nanoparticle tracking analysis, cryo-electron microscopy and western blot (CD63 and CD9) were used for EVs characterization, according to MISEV2018 guidelines. MiR-195-5p was loaded into isolated vesicles by electroporation, which was confirmed by RT-qPCR post RNAse A treatment. 3D tumor spheroids were observed 72 hours after plating 1.000 cells/well in 96 multiwell plates coated with 1% agarose layer. Three days-old spheroids were submitted to fractionated RT for 5 days (5 x 3Gy) and EVs (10^7/spheroid) were added 1h prior to the last three doses. Results: EVs electroporation resulted in a 100x increase in intravesicular miR-195-5p levels and RT-qPCR confirmed its up-regulation in recipient cells post miR-195-loaded EVs treatment. MiR-195 EVs alone restrained spheroids growth from 15 to 50%. Combined RT/miR-195 EVs treatment suppressed the up-regulation of RT resistance-related genes, including ALDH1, ATM, OCT4, SOX2 and PDL-1, as well as the lncRNAs LINC00473 and LINC00511, and reduced cells proliferation and clonogenic capacity, resulting in 5 to 60% smaller spheroids diameter upon re-growth challenge, compared to RT/Control EVs treatment. Conclusion: mir-195-EVs may enhance the efficacy of RT when used as an adjuvant in melanoma treatment. Funding: FAPESP (2021/13681-2).

Nao Nishida-Aoki

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

In tumor tissues, cancer extracellular vesicles (EVs) are secreted to interstitial spaces of varieties of non-cancerous cells and extracellular matrix. The tissue structure will introduce target cellular preferences and a physical barrier that will affect the dynamics of EVs. As EVs transmit intercellular signals via reaching other cells, identifying the targeted cell types, the amount, and the distance of reach are crucial to understand their roles in establishing a tumor-favorable environment. However, current functional studies based on cell culture or animal studies using isolated EVs cannot address the questions. This study aims to investigate cancer EV transfer within physiological tissue context of primary and metastatic tumors.

Methods:

To observe detailed cancer EV transfer within tissue architecture, I established ex vivo tissue platform with highly-sensitive EV labels. Cancer EVs were labeled with Halo-tag fused to CD63, stably expressed in human and mouse breast cancer cell lines. For tissue model, precisely-cut tissue slice culture (TSC) method was used (Nishida-Aoki, et al., JoVE, 2020). Mouse breast tumors and organs were sliced into 250 µm thickness and cultured at air-liquid interphase. To validate EV uptake, TSCs were supplemented with PKH67-labeled breast cancer EVs collected by ultracentrifugation. To establish the EV transfer platform, the breast cancer cells with EV labels were sparsely seeded onto tumor or organ TSC and cultured till the cells were settled.

Results:

Cancer EVs collected from breast cancer cells expressing CD63-Halo-tag were successfully labeled with approximately 80% efficiency of the total EVs. Cancer EVs supplemented onto tumor and tissue TSC were incorporated into live cells TSC with uneven distribution. The EVs secreted from seeded cancer cells on TSC, localized mostly at the surface of TSC but some invaded inside, were incorporated unevenly into proximal cells, and distributed sparsely.

Summary:

By integrating EV labeling and TSC techniques, I established ex vivo physiological platforms for observing EV transfer at primary and metastatic tumors. Currently, I am analyzing the EV uptaking cells within the tissue and will present the results at the conference. This research will provide spatial investigation on physiological EV-mediated cell-cell communications occurring locally at primary and metastatic tumors.

Dr. Baldev Singh, Dr. Pankaj Gaur, Dr. Jeyalakshmi Kandhavelu, Mr. Yanjun Zhang, Mr. Zihao Zhang, Dr. Shivani Bansal, Mr. Meth Jayatilake, Mr. Yaoxiang Li, Dr. Pritha Bose, Dr. Seema Gupta, Dr. Partha Banerjee, Dr. Vivek Verma, Dr. Baldev Singh

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Pancreatic cancer (PaCa) is an aggressive GI malignancy that is deemed to become the second leading cause of cancer-related deaths by 2030. Extracellular vesicles (EVs) are Nano-sized membranous structures secreted by cells that serve as conduits for cellular cross-talk. Recent work by us and others has shown that cancer-derived extracellular vesicles (cEVs) facilitate modulation of surrounding cells and the tumor microenvironment (TME). Macrophages are an essential component of the TME, and their interaction with tumor EVs plays a major role in the tumor development and progression. The current study aims to ascertain how PaCa-derived EVs affect the phenotype, function, and behavior of the macrophages.

Materials and methods: EVs were isolated using size exclusion chromatography from the conditioned medium of a non-tumorigenic established pancreatic epithelial cell line (hTERT-HPNE), one established pancreatic cancer cell (PANC-1) and one PDX cell line (PPCL68). The EVs were characterized as per ISEV guidelines using cryo-electron microscopy, nanoparticle tracking (NTA), and immunoblot analysis. Macrophages were generated from the bone marrow cells of C57/BL6 mice using macrophage colony stimulating factor (M-CSF) and co-cultured with isolated normal or PaCa cells derived EVs. Flow cytometry and multi-omic analyses were performed to determine the effect of EVs on macrophage phenotype, function and behavior. Small RNA sequencing was performed to characterize the miRNA profile of PaCa EVs.

Results: PaCa derived EVs treatment of mouse bone marrow derived macrophages (BMDMs) resulted in increased expression of CD206 and higher secretion of immunosuppressive cytokines including TGF-beta and IL-10. Functionally, PaCa EV educated macrophages were also able to suppress the T cells proliferation under in-vitro and in-vivo conditions. RNAseq and metabolomic analysis revealed a pro-tumor metabolic phenotype of PaCa EVs treated macrophages. Furthermore, miRNA profiling of PaCa EVs showed that the EV cargo was enriched with various pro-tumor and/or immune-modulatory miRNAs.

Conclusions: Our findings suggest that PaCa-derived EVs are enriched with immuno-modulatory miRNAs that have potential to influence macrophage function and phenotype that could ultimately facilitate tumor growth and metastasis. Taken together these results, demonstrate a novel mechanism by which pancreatic cancer cells alter immune cells to both induce an immunosuppressive environment and facilitate metastasis.

Mr. Yanjun Zhang, Dr. Baldev Singh, Dr. Pritha Bose, Dr. Jeyalakshmi Kandhavelu, Mr. Zihao Zhang, Dr. Shivani Bansal, Dr. Sunil Bansal, Mr. Meth Jayatilake, Mr. Yaoxiang Li, Dr. Shu Wang, Dr. Baldev Singh

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Tumor-associated macrophages (TAMs) are the most abundant immune cell population within the tumor microenvironment (TME) and major contributors to its immunosuppressive nature. Interestingly, the main source of TAMs is the circulating monocytes instead of organ-specific resident macrophages. Extracellular vesicles (EVs) are membrane-bound nanoparticles containing bioactive molecules released by all cell types. Recently, the role of extracellular vesicles (EVs) in immune modulation has gained increasing credence. Our present study aims to examine the role of pancreatic cancer cell-derived EVs in monocyte recruitment to TME and their differentiation into the TAM phenotype.

Material and methods: EVs were isolated from one non-tumorigenic pancreatic epithelial cell line hTERT-HPNE, one pancreatic cancer cell line PANC-1, and one pancreatic PDX cell line PPCL-68 by size exclusion chromatography (SEC). The EVs were characterized per ISEV guidelines using cryo-electron microscopy, nanoparticle tracking (NTA), and immunoblot analysis. The effect of EVs on the monocyte behavior, differentiation, and proliferation was investigated using flow cytometry, western blot, and cytokine profiling in the co-culture experiment with different EV types and the human monocyte cell line THP1. RNASeq analysis and metabolomics analyses were performed to gain insights into functional alterations.

Results: We observed that THP1 monocytes, which typically grow as suspension cultures, became attached to the cell culture plates and adopted a macrophage-like morphology after being co-cultured with pancreatic cancer cell-derived EVs. Flow cytometry and cytokine profile analysis confirmed expression of macrophage marker CD68 and showed that monocytes treated with pancreatic cancer cells-derived EVs secreted an elevated level of anti-inflammatory cytokines such as IL10 and IL4 compared to untreated control and hTET-HPNE EVs treated monocytes.

Conclusion: Our results suggest a novel finding that pancreatic cancer-derived EVs carry specific cargo that, upon uptake, facilitate the differentiation of monocytes into anti-inflammatory TAMs, suggesting EVs may be important enablers for monocyte recruitment to TME and their subsequential differentiation to TAMs. Further studies examining the mechanisms behind the effect of pancreatic cancer-derived EVs on monocytes are ongoing and will be presented at the conference.

Kesara Nittayaboon, Kittinun Leetanaporn, Prof. Surasak Sangkhathat, Prof. Sittiruk Roytrakul, Assoc. Prof. Raphatphorn Navakanitworakul

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Butyrate resistant cells in colorectal cancer (CRC) patients was addressed to play an important role in the acquisition of chemo-resistant in colorectal cancer. Our previous study showed that butyrate-resistant colorectal cancer cells (BR cells) showed a cross-resistant to chemotherapy including 5-Fluorouracil (5-FU) and Oxaliplatin (Oxa) in both monolayer and spheroid culture. The mechanism of drug resistant has been revealed. However, the link between parental (PT cells) and BR cells still missing. Extracellular vesicles or EVs are known as a key cell-cell communication which transport various molecules including DNA, RNA, especially proteins between donor and target cells. EVs also involving in mediated drugs resistant in cancer such as melanoma, and lung cancer. In this study, we determined proteomic data from EVs-derived from PT and BR cells to investigate the mechanism behind butyrate- and chemo-resistant features. The EVs were isolated from PT and BR culture medium using ultracentrifugation method, then characterized them by western blotting and transmission electron microscopy. The proteomic data were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). We found that the unique protein expressed in BR cells are related to chemo-resistant phenotype. The functional enrichment analysis illustrates that BR cells showed a higher expression in catalytic activity, binding protein, and transcription activity. The results from STITCH database showed the correlation between protein and drugs interaction in BR cells more than PT cells. Moreover, the up regulated proteins in volcano plot are related to carcinogenesis. Our finding supports the hypothesis that EVs not only promote tumor progression, and metastasis but also effect the tumor microenvironment.

Mrs Akbar Marzan, Dr. Sai Chitti, Prof Suresh Mathivanan

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Cancer cachexia is a wasting syndrome that results in dramatic loss of whole-body weight, predominantly due to loss of skeletal muscle mass. While it's well-established that cancer cells themselves can trigger cachexia through the release of pro-cachectic and pro-inflammatory factors, the regulatory mechanisms and key cachexins involved remain unclear. Therefore, a comprehensive analysis of the protein content in small extracellular vesicles (sEVs) and secretome, coupled with identifying those pathways that are dysregulated in atrophied muscle is crucial for developing new therapies.

Methods: Initially, C26 and EL4 cells were established as cachexic and non-cachexic representative models by treating adipocytes and myotubes with C26-derived conditioned media (CM). Subsequently, the secretome and small extracellular vesicles (sEVs) released by both C26 and EL4 cancer cells were analysed using label-free quantitative proteomics. Finally, the impact of CM, which contains secreted factors and EVs from cachexia-inducing C26 cells on C2C12 myotubes was examined using mass spectrometry-based proteomics approach.

Results: The proteomic profile shed light on tumor-derived factors that contribute to weight loss by influencing protein and lipid loss in various tissues. Functional enrichment analysis revealed enrichment of proteins implicated in biological processes such as muscle atrophy, lipolysis, and inflammation in both the secretome and sEVs derived from C26 cancer cells. Furthermore, the impact of cachexia inducing CM on C2C12 myotubes was studied. Substantial changes in the protein profile of C2C12 cells were identified upon exposure to C26-derived CM. Functional enrichment analysis revealed an abundance of proteins associated with inflammation, mitochondrial dysfunction, muscle catabolism, ROS production, and ER stress in CM-treated myotubes. Furthermore, pronounced downregulation in muscle structural integrity, development, and/or regenerative pathways was observed.

Conclusion: The proteins found in abundance in C26-derived sEVs and secretome have the potential to serve as therapeutic targets and biomarkers of cancer cachexia. Additionally, the proteins significantly prevalent in atrophied muscle may function as markers for muscle wasting, and the dysregulated biological processes could be employed for therapeutic benefits in the context of cancer-induced muscle wasting.

Prof Muriel Meiring, Ms Noluthando Gasa

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Cells actively release extracellular vesicles (EVs) upon activation and apoptosis. EVs mediate intercellular communication in normal physiology and pathology. Cancer cells, like normal cells, secrete cancer-specific EVs into the bloodstream. EVs can be used to monitor disease progression and response to treatment. This research investigated circulating EVs in the plasma of patients with cervical cancer before, during, and after treatment.

Aim: To quantify and characterize extracellular vesicles in the systemic circulation before, during, and after the treatment of cervical cancer patients.

Objectives: To count and characterize circulating EVs in cervical cancer patients before, during, and after treatment.

Design and methods: EVs were isolated from plasma by size exclusion chromatography. Flow cytometric analysis was used to characterise the presence of EVs (CD63). To characterize cancer-derived EVs, CD133 was used; furthermore, to characterize platelet-derived EVs, CD41 was used.

Results: CD63+ events had a significant decrease in week six compared to baseline. There was a significant decrease in CD133+ events in week six compared to baseline. CD41+ events also indicated a significant decrease in week six compared to baseline.

Conclusion: This study demonstrated that there is a significant decrease in circulating EVs after treatment compared to baseline. These findings suggest that EVs can possibly be used to monitor patient's response to treatment.

Mr Harrison Rudd, Dr Geeta Upadhyay

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Pancreatic cancer remains challenging to treat with high mortality rates and late detection. Small extracellular vesicles (sEVs) are nano-sized, membrane bound secreted organelles containing nucleic acids and proteins and have been shown to play key roles in tumor progression and metastasis. In this study, we report the novel discovery of matrix metalloproteinase-3 (MMP-3), neutrophil gelatin-associated lipocalin (NGAL)-MMP-9 and interleukin 6 (IL-6) glycoforms in pancreatic cancer-derived (CD) sEVs. We also define the implication of these pancreatic CD sEVs on migratory and invasive capacity, metastatic potential, cancer stem cell marker expression/function, and tumorigenicity.

Our sEV samples were isolated using a standardized workflow consisting of size exclusion chromatography (SEC) followed by a three-pronged characterization approach using western blot, transmission electron microscopy (TEM)/immunoTEM, and nanoparticle tracking analysis (NTA). Using a comprehensive range of assays, we demonstrated the presence and activity of MMPs in pancreatic CD sEVs. Our findings indicate that sEV-derived MMP-3 contributes significantly to the aggressive metastatic and transformative capacity of pancreatic cancer cells, underscoring its potential as a critical target for therapeutic interventions and as a promising biomarker for diagnostics.

The implications of this research are multifaceted. The identification of specific CD sEV-derived cargos, such as MMP-3 and IL-6 glycoforms, presents promising opportunities for improved diagnostic accuracy and sensitivity. These sEV-derived biomarkers hold the potential to serve as reliable indicators of disease progression and treatment response, facilitating early detection and monitoring. Moreover, the discovery of these cargos in CD sEVs opens new avenues for targeted, personalized drug delivery approaches.

In conclusion, the identification and functional characterization of MMP-3 in pancreatic CD sEVs represents a significant advancement in cancer research. Future studies include investigating patient-derived organoid (PDO)-derived sEVs and comparing the landscape of sEV cargo in 2D and 3D in vitro models. Additionally, we plan to study the effect of these CD sEVs on immune function of PD T-cells. Embracing these findings may pave the way for a shift toward more personalized approaches in combating pancreatic cancer and other aggressive malignancies.

Doctor Lin-Zhou Zhang, Professor Gang Chen

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Immune checkpoint molecule programmed cell death 1 (PD-1) expressed on cell surface impairs antigen-driven activation of T cells, thus plays a critical role in tumorigenesis, progression, and poor prognosis of oral squamous cell carcinoma (OSCC). Additionally, increasing evidence indicates that PD-1 carried on small extracellular vesicles (sEVs) also mediates tumor immunity, though their contributions to OSCC are yet unclear. Here, we investigated the biological functions of sEV PD-1 in OSCC patients.

Methods

The cell cycle, proliferation, apoptosis, migration, and invasion of oral cancer CAL27 cell lines treated with or without sEV PD-1 were examined in vitro. We further performed mass spectrometry to investigate the underlying biological process, combined with immunohistochemical study of oral cancer xenograft mice model and OSCC patient samples.

Results

In the present study, OSCC cell line CAL27 cells were treated with sEV PD-1 in vitro to investigate the roles of sEV PD-1. We revealed that sEV PD-1 resulted in suppressed proliferation and arrested cell cycle with promoted resistance to apoptosis in CAL27 cells. Additionally, we also found out that sEV PD-1 significantly enhanced migration and invasion of tumor cells. To explore the potential mechanism, we performed mass spectrometry analysis and it showed that sEV PD-1 treatment initiated significant mediation of tumor cell senescence and EMT, which was further confirmed with in vitro assays. The onset of EMT was proved subsequent to senescence. The abovementioned effects were reversed when sEV PD-1 was pre-blocked with anti-PD-1 antibody or surface PD-L1 was disrupted in CAL27 cells, indicating that the effects resulted from the ligation of sEV PD-1 to tumor cell PD-L1. Moreover, with mass spectrometry and small molecule inhibitor, we identified that the activation of p38 MAPK was attributed to sEV PD-1-induced senescence-initiated EMT. Last, the close correlations among circulating sEV PD-1, senescence and EMT of tumor, and lymph node metastasis were verified in both oral cancer xenograft mice model and OSCC patient samples.

Conclusion

The results demonstrate that circulating sEV PD-1 trigger senescence-initiated EMT in a PD-L1-p38 MAPK-dependent manner, contributing to tumor metastasis. It suggested inhibition of sEV PD-1 as a promising therapeutic target for treatment of OSCC.

Mr Abdulwahab Teflischi Gharavi, Ms Raheleh Tahmasvand, Dr Amirabbas Rahimi, Dr Saeed Irian, Prof Mona Salimi

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1)Introduction: Tumor immune microenvironment (TIME) is featured by tumor-promoting macrophages, anti-inflammatory cytokines and immunosuppressive cells such as T-regs and Th2 as well as tumor suppressive cells such as Th1, making a circumstance in favor or against tumor progression. Small extracellular vesicles (sEVs) loaded with biologically active molecules, such as miRNAs are involved in TIME reprogramming and pose a hurdle for efficient response to cancer immunotherapy. Here we aimed to evaluate the immuno-modulatory effect of breast cancer cell-derived EVs on PBMCs isolated from non-metastatic breast cancer patients.

2)Methods: sEVs were isolated from MCF-7 cell line by ultracentrifugation. To characterize sEVs, DLS, TEM, Western blotting and flowcytometry techniques were employed. PBMCs were isolated from blood samples of breast cancer (BC) patients and healthy individuals. To track the internalization of sEVs into PBMC cells, the extracellular vesicles were labeled using PKH26 red fluorescent labeling kit and tracked using confocal microscopy. Different amounts of the collected sEVs ie., 0.25, 0.5, 1, 2, 5, 15 and 45 µg were then incubated with PBMCs for 24 or 48 hours. The amount of the cytokines IFN-γ and IL-4 was evaluated by ELISA. CD4, CD25 and FOXP3 were subjected to flowcytometry. The quantity of miR-155-5p and miR-181a-5p was measured using stem loop-based qPCR.

3)Results: The characterized sEVs were well-uptaken by PBMCs after 20 h. Our findings revealed that 15 and 45 µg of sEVs slightly increased IFN-γ levels. However, the initial stimulation of PBMCs using PHA followed by exposure to 1, 2 and 5 µg of sEVs for 24 h caused a reduction in IFN-γ levels secreted from PBMCs in a concentration-dependent manner. Notably, we could not detect IL-4 upon incubation with sEVs. By changing the time from 24 to 48 hours and using 1, 0.5 and 0.25 µg of sEVs, we obtained the same results. Moreover, sEVs had no noticeable effect on the population of T-reg cells in PBMCs. Finally, an alteration in miR-155-5p and miR-181a-5p expression levels were detected in both sEVs and PBMCs.

4)Summary/Conclusion: Overall, we demonstrated an immunomodulatory effect for the BC-derived sEVs, which might be attributed to the role of miR-155-5p and miR-181a-5p.

Lin Wang

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Cervical cancer is a leading cause of death in developing countries. Although the placenta is a tumor-like organ, the placental development including invasive function is well controlled. One mechanism is that extracellular vesicles (EVs) released from the placenta contribute to this regulation. Placental EVs carry functional proteins and regulatory RNAs. Our previous study reported that placental EVs inhibited ovarian cancer growth in vitro and in vivo. Whether the inhibitory effect induced by placental EVs also applies to cervical cancer. In this study, we investigated the functions of placental EVs on human cervical tumor tissues in vitro.

Methods: Human cervical tumor tissues (n = 7) were dissected and co-cultured with the first-trimester placentae collected from elective abortion for 24 hours by a 2D cultural model. The tumor tissues were then fixed with 4% PFA for histology or proteins were extracted by RIPA buffer. The levels of senescence by measuring γH2AX, p21, and p16, or proliferation by measuring PCNA were examined with western blots and IHC. Placental-specific miRNAs which are associated with cancer cell death were measured in the cervical tissues after treatment.

Results: Histological analysis showed signs of necrosis in cervical tumor tissues that had been treated with placental conditioned media, compared to controls. Additionally, the expressions of γH2AX, p21, and p16 were significantly increased, while the levels of PCNA were significantly reduced in cervical tumor tissues that had been treated with placental conditioned media. The levels of placental-specific miRNA-143-3p, miRNA-519a-5p, and miRNA-199a-3p were significantly increased in cervical tissues after the treatment.

Conclusions: Placental EVs are involved in the regulation of placental development. The delivery of cargo in EVs significantly impacts the functions of target cells. Here we showed increased senescence and reduced proliferation in cervical tumor tissues that had been treated with placental explants. The delivery of placental-specific and death-associated miRNAs contributed to this effect.

Li-Ying Wu, Dr. Luize Lima, Dr. Sunyoung Ham, Student Mina Lim, Dr. Edna Chai, Prof. Yong-Soo Choi, Prof. Andreas Möller

Poster Pitches (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:45 PM - 1:00 PM

Introduction

The high immunogenicity of melanoma predestined it for immunotherapies as effective treatment for advanced melanoma1. Thus, understanding the immune regulation occurring in the melanoma tumour microenvironment is critical for developing new therapies for patients resistant to current treatments. High levels of CD155 are found in melanoma, and it has been implied in the resistance to immunotherapy4. Initial data suggests that CD155 can be transferred from melanoma cells to immune cells in vivo. However, how CD155 is transferred from melanoma cells to immune cells, and implications of immune cells acquiring CD155, are unclear. Extracellular vesicles (EVs) are secreted by all cells, including melanoma, and taken up by all type of cells, including immune cells5. Thus, we hypothesize that CD155 is loaded into EVs, and transferred from melanoma to immune cells, impacting on their immune response.

Methods

Mouse melanoma B16F10 cells and human melanoma A375 cells are used in this study to investigate the role of cancer-derived CD155-EVs in the tumour microenvironment. EVs isolated from B16F10 or A375 were used to treat NK-92MI cells to investigate the function of cancer-derived CD155-EVs on NK cells. Further, EVs isolated from B16F10 were used to treat B16F10-CD155KO subcutaneous tumour in CD155 KO mice to determine the role of cancer-derived CD155-EVs in vivo.

Results

Our data demonstrates that CD155 is transferred from melanoma cells specifically to NK cells in vivo. Moreover, CD155 is enriched in melanoma-derived EVs, including microvesicles (MVs) and small extracellular vesicles (sEVs). Functionally, it appears that there is a difference if CD155 is contained in MVs or sEVs, as the CD155 abundance on NK cells specifically increased upon melanoma-derived MV treatment, but not when CD155 sEVs are used.

Summary

Our data shows that CD155 is contained in EVs and can be specifically taken up by NK cell. It also appears that only MV-CD155 is displayed by NK cells, but not sEV-CD155. Further work will evaluate the role of CD155-containing EVs in immunoregulation we expect that this data will demonstrate an important, novel role of immune regulation by CD155-containing EVs in melanoma immune responses.

Maulee Sheth, Dr Manju Sharma, Maulee Sheth

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Extracellular biophysical cues such as matrix stiffness affect tumor progression in vivo. However, it remains unclear how cells in a 3D tumor microenvironment (TME) perceive and translate matrix stimuli into intracellular signals driving cancer. Small extracellular vesicles (sEVs)-mediated intercellular communication facilitates interplay and rewiring between different components of the TME. Here, we engineered a 3D spheroid culture system with varying mechanobiological properties to study the role of sEVs in matrix stiffness triggered oral squamous cell carcinoma (OSCC) invasiveness by means of piezo1 activity.

Methods

Cal27 spheroids were generated using liquid overlay in sEV-depleted media. Matrix stiffness was defined as soft and stiff for 3 and 12 mg/mL Matrigel, respectively, and characterized by atomic force microscopy (AFM) and shear rheology. Day 14 spheroids were dissociated and stained with Piezo1 and AF594-secondary or CD44-APC. Flow data were acquired using Cytek Aurora and analyzed using FlowJo. sEVs were extracted from culture media using a Lab-on-a-Chip device(1). Nanoparticle tracking analysis (NTA) was performed using Nanosight NS300. sEVs were single stained with Piezo1-FITC, CD44-APC, and CD63-FITC. Imaging flow cytometry (iFCM) was performed using ImageStreamX Mark II and analyzed using IDEAS. Single sEVs immobilized on EV profiler chips and stained with a mix of CD63-Cy38 and Piezo1-FITC or CD44-APC were imaged using dSTORM with a Nanoimager S Mark II and analyzed using CODI.

Results

Mechanical characterization indicated a 3-fold increase in stiffness at the intermolecular level and 8-fold increase at the bulk level using AFM and rheology, respectively. Cellular flow cytometry demonstrated significant increase in piezo1 and CD44 expressions with increasing stiffness. NTA indicated a 5-fold increase in particle concentration between soft and stiff cultures. iFCM showed a 3.5- and 2-fold increase sEVs with Piezo1 and CD44, respectively, from soft to stiff conditions. dSTORM indicated presence of Piezo1 and CD44 in sEVs with increasing positivity with stiffness.

Conclusion

3D extracellular matrix stiffness alters sEVs production and composition, and thereby drives cancer progression by employing biomechanical Piezo1 and CD44 signatures from parental spheroids. This provides premise for exploring sEV-Piezo1 as a potential therapeutic target or prognostic marker against OSCC.

(1) Sharma et al. Sci. Rep. 2023

M.D. Takaaki Tamura, Ph.D. Tomofumi Yamamoto, Ph.D. Akiko Kogure, Ph.D. Yusuke Yoshioka, M.D., Ph.D. Shinichi Sakamoto, M.D., Ph.D. Tomohiko Ichikawa, M.D. Takahiro Ochiya

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Prostate to bone cancer metastases induce mixed lesions containing areas of bone destruction and formation that are directed by osteoclasts (OCs) and osteoblasts (OBs), respectively. OCs play an essential role in the tumor invasion areas even in osteogenic bone metastases of prostate cancer (PCa). OC-derived extracellular vesicles (EVs) are reported to regulate OB activity in normal bone homeostasis; however, there is no study investigating the role of EVs from OC educated by PCa cells in the tumor bone microenvironment.

Methods: We prepared four types of OCs and EVs from these cells; OCs differentiated from RAW264.7 cells (OC cells), OC cells co-cultured with normal prostate epithelial cells (OCN cells), osteolytic PCa cells (OCP cells), and osteoblastic PCa cells (OCC cells). OC differentiation was induced in the presence of RANKL. EVs were purified from culture supernatant using ultracentrifugation. To observe the changes in PCa-educated OCs, we investigated the response to denosumab (anti-RANKL antibody) and signaling pathways in OCP and OCC cells. Further, to reveal the function of EVs from PCa-educated OCs, we added EVs to normal OCs and mineralizing OBs (MC3T3-E1). The expression levels of OC marker genes such as TRAP, CTSK, and NFATC1 and OB marker genes such as ALP and BGLAP were compared by qPCR. TRAP and ALP staining was also conducted. Next-generation sequencing (NGS) was performed to identify EV-delivered miRNAs and target genes regulating OC and OB activity.

Results: OCP and OCC cells showed denosumab resistance. Some signaling pathways, which are reported to promote OC differentiation independent of the RANKL pathway, were increased. Moreover, OCP and OCC cell-derived EVs significantly promote OC activity and inhibited OB activity; these EVs up-regulated OC marker genes and down-regulated OB marker genes. NGS identified some candidate miRNAs as EV components regulating OC and OB activity.

Summary/Conclusion: We report the role of EVs derived from OCs educated by cancer cells for the first time. OCs educated in the tumor invasion areas may release EVs, promoting OC activity and inhibiting OB activity and leading to further bone destruction. Some animal studies are ongoing.

Mr Quan Zhou, Prof. Jing Wang, Dr. Zhen Zhang, Dr. Alain Wuethrich, Dr. Richard Lobb, Prof. Matt Trau

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Melanoma continues to be a leading cause of mortality among skin cancers. Despite advancements in targeted therapy, patients frequently develop resistance, leading to disease progression within a year. This resistance may result from the epithelial-to-mesenchymal transition (EMT)-like phenotype switching of melanoma cells. Tracking EMT-related phenotypic changes on extracellular vesicles (EVs) has potential to inform early about response to targeted therapy and melanoma progression. However, the knowledge on protein biomarkers carried by melanoma EVs involved in the EMT-like process remains unexplored.

Methods

We develop a microfluidic biosensor for simultaneous detection of EMT-associated protein biomarkers (E-cadherin, N-cadherin, THBS1, and ABCB5) on melanoma EV surfaces during targeted therapy. This biosensor integrates surface-enhanced Raman scattering and alternating current electrohydrodynamics-induced nanomixing enhancement for improved specificity and sensitivity. This biosensor enables the analysis of 28 samples per run without the need for EV pre-isolation.

Results

This biosensor successfully tracks the EMT-like phenotype switching on EVs derived from melanoma cell lines treated with mitogen-activated protein kinase inhibitor (MAPKi). Longitudinal monitoring of patients who receive MAPKi therapy and develop resistance, this biosensor shows its ability to identify the EMT-like phenotype switching on circulating EVs.

Conclusion

This biosensor provides a highly sensitive and specific tool for tracking the EMT-like phenotype switching during targeted therapy in melanoma by monitoring EV phenotypic evolution, which potentially can be used to predict the development of resistance to targeted therapy, enabling timely intervention and personalized treatment.

Mr. Zhixian Chen, Prof. Judy Yam

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Small extracellular vesicles (sEVs), also called exosomes, have been established with cardinal significance in the intercellular communication within tumor microenvironment.

Methods: ROS detection kit and different methods evaluating DNA damage were used to examine effects on recipient normal liver cells after treatment with HCC cell-derived sEVs

Results: In our study, we found an increasing trend of reactive oxygen species (ROS) level in normal liver cell line treated with sEVs derived from hepatocellular carcinoma (HCC) cells with an increasing metastatic proclivity. Similar trends were also found in mitochondrial stress and indicators reflecting oxidative DNA damage including 8OHDG and γH2AX. ROS-induced DNA damage has been recognized as an important mechanism contributing to tumorigenesis. We also identified a key protein target from sEV proteomic analysis data, which potentially mediates the above-mentioned, EV-induced effects by enhancing lipid oxidation in recipient cells.

Conclusion: These results suggest that HCC-derived sEs activate ROS-induced DNA damage in surrounding normal liver cells which might provoke their cellular transformation potential. Our research may provide new evidence for sEV-driven mechanism in mediating development of HCC.

Mr. Joaquín Jurado-Maqueda, Alessandra De Feo, Prof. Katia Scotlandi

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

MicroRNAs (miRNAs) and proteins within extracellular vesicles (EVs) stand as promising biomarkers for the early detection of cancer and prediction of treatment response. These EV-shuttled biomolecules may directly contribute to the mechanisms promoting chemotherapy resistance. Poor response to neoadjuvant MAP (methotrexate, doxorubicin and cisplatin) chemotherapy is a common issue affecting the survival of patients with metastatic osteosarcoma, the most common malignant bone tumor among children and adolescents. In this study, we performed next-generation RNA sequencing and MS-based proteomics to explore the miRNA and protein cargo of EVs derived from drug-resistant osteosarcoma cells.

Methods

EVs were isolated from cell supernatants of drug-sensitive osteosarcoma U2OS cells and their resistant versions to doxorubicin (DX), cisplatin (CDDP) and methotrexate (MTX) using ExoQuick-TC and purified using Sephadex G-25 columns. EVs were quantified and characterized by Bradford assay, Nanoparticle Tracking Analysis, and immunoblotting for EV markers. EV-miRNAs were extracted by ExoQuick RNA Purification kit. cDNA libraries were constructed using QIAseq miRNA kit and sequenced on NextSeq 500. For proteomics, RIPA buffer was added to EV samples followed by sonication and digestion, and proteins were profiled by UHPLC-MS/MS. Data analysis was performed using in-house scripts and Bioconductor packages in R. Selected miRNA and proteins were validated by RT-qPCR and immunoblotting.

Results

EV size was ∼90-130nm and they contained CD81, ALIX, HSP90 and no calnexin. A total of 8 miRNAs were differentially expressed in DX-EVs, 375 in CDDP-EVs and 22 in MTX-EVs compared to the drug-sensitive cell-derived EVs. Interestingly, we found several EV-miRNAs, such as miR-184, miR-21-5p and miR-9-3p which could promote a chemoresistant phenotype in recipient cells and thus be associated with the mechanisms of resistance to DX, CDDP and MTX in osteosarcoma. Furthermore, we could find common overrepresented EV-proteins associated with negative regulation of cell adhesion and chemoresistance such as ANXA1, CTSB, PSAP and VGF.

Summary/Conclusion

We identified chemoresistance miRNA and protein signatures in EVs that could be proposed as a minimally invasive approach for predicting and monitoring patient response to chemotherapy. Further validation in plasma-EVs of clinically annotated patients pre-/post-chemotherapy with informed consent is currently ongoing and should be ready for the congress date.

Mr Vivek Dharwal, Dr Vivek Dharwal, Mr Jiawei Chang, Dr Zaklina Kovacevic, Dr Elham Hosseini-Beheshti

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Extracellular vesicles (EVs) are membrane bound heterogenous vesicles that are released by cells to facilitate cell-to-cell and cell-to-matrix interactions. Emerging evidence suggest that EVs derived from cancer cells, when compared to healthy cells, have distinct cargo that drives tumour microenvironment modulation, immunomodulation, cancer metastasis and angiogenesis. Pleural mesothelioma (PM) is a rare cancer that has poor prognosis and low survival due to its long latency period. The pathology of PM is poorly understood and the role of EVs in its pathogenesis is yet to be explored. In this project, we investigated the effects of mesothelioma-derived EVs on lung fibroblast function and tumour microenvironment.

Methods

We isolated three subpopulations of EVs from three cell lines, VMC23 (epithelioid), MSTO-211H (non-epithelioid) and Met5A (non-malignant) as a control, via centrifugation followed by ultracentrifugation. A full characterisation of EV subpopulations (2.8K, 10K and 100K) was completed using transmission electron microscopy, nanoparticle tracking analysis, western blotting and proteomics. MRC5 lung fibroblasts were then treated with each EV subpopulation isolated (10ug/ml) for 72 h. Uptake of EV was analysed by fluorescent microscopy. Cancer associated fibroblast (CAF) expression after EV treatment was analysed via western blots. The functional effect of EV treated fibroblast was assessed using proteomics and RNA sequencing.

Results

Our results confirm three distinct EV subpopulations derived from the three cell lines in vitro. All EV subpopulation exerted a strong effect on the conversion of fibroblasts to CAF. PKH26 staining indicated that the EVs derived from PM cells were internalised by fibroblasts. We demonstrate that the unique cargo of different EV subpopulations modulate fibroblast through different signalling pathways involving tumour microenvironment modulation.

Summary/conclusion

Our findings identify the diverse function of EVs in the tumour microenvironment, which provides insight into PM pathogenesis and thereby paving way for diagnostic and therapeutic studies.

Mireia Gomez

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

According to the World Health Organization (WHO), breast cancer is the most frequently diagnosed malignancy and represents the leading cause of cancer mortality in women worldwide. Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a poor prognosis due to the absence of targetable receptors. In these tumors, high levels of the CD133 protein, a cancer stem cell marker, and a bad prognostic marker, correlate with adverse outcomes and resistance to chemotherapy. Its role in cancer invasion and metastasis remains however elusive. Lack of sensitive diagnostic tools and knowledge in mechanisms of emergence and progression are major.

Extracellular vesicles (EVs) released by cancer cells are acknowledged to mediate intercellular communication and contribute to tumor growth and metastasis, but the underlying mechanisms and cellular components are far from understood. Recently, we have reported that CD133 promotes the biogenesis and secretion of extracellular vesicles (EVs) from membrane protrusions with functional roles. The major goal of this project is to unravel the nature and function of CD133-EVs and associated components in cancer invasion and metastasis.

EVs from two triple-negative breast cancer cell lines and one hormone-dependent cell line were isolated and characterized The three cell lines secrete a heterogeneous population of EVs, that carry EV-specific markers while also bearing CD133. Moreover, our data indicate that TNBC-derived EVs, bearing CD133, promote branching and sprouting in endothelial cells, a key feature in cancer invasion. We are currently evaluating in vitro the function of EVs on migration and epithelial-mesenchymal transition potentially related to the metastatic capacity of cancer cells and in vivo the ability of EVs derived from TNBC cells to disseminate throughout an organism and promote metastasis at specific distant organs. Furthermore, we want to correlate the possible association of CD133 overexpression in primary tumors and metastases with clinical and biological parameters relevant to patient care. For this purpose, we have focused on the study of the content of EVs from healthy and TNBC patients at different stages of chemotherapy treatment.

Dr Venkatesh Kumar Chetty, Dr Jamal Ghanam, Prof. Dr Dirk Reinhardt, Dr Basant Kumar Thakur

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

In the bone marrow microenvironment (BMM), it is known that the reciprocal interaction between acute myeloid leukemia (AML) cells and bone marrow-derived mesenchymal stem cells (BM-MSCs) is crucial for AML progression and contributes to treatment failure or success. In recent years, small extracellular vesicles (sEVs) released by AML cells have been reported to influence the trilineage differentiation of BM-MSCs. However, it remains elusive which biological cargo from AML-sEVs is responsible for this effect.

Methods

sEVs were isolated from cell-conditioned media of leukemia cell lines (MV4-11 and K562) using a combination of tangential flow filtration (TFF), size exclusion chromatography (SEC), and ultrafiltration (UF). In parallel, sEVs were also isolated from the blood plasma of 15 pediatric healthy donors and AML patients using SEC and UF. sEVs were characterized according to MISEV2018 guidelines using NTA, micro-BCA, TEM, and western blot.

Results

We found that AML-sEVs increased the viability and proliferation of BM-MSCs. Conversely, key proteins (CxCl2, Scf, Col1A1, and Angpt1) that are important for normal hematopoiesis were downregulated in BM-MSCs. In addition, we revealed that AML-sEVs significantly reduced the differentiation of BM-MSCs to osteoblasts without influencing adipogenic or chondrogenic differentiation. Next, LC-MS/MS proteomics elucidated that various proteins, including Y-box-binding protein 1 (YBX1), were upregulated in both AML-sEVs and BM-MSCs treated with AML-sEVs.

Clinically relevant, we uncovered that YBX1 is considerably upregulated in most pediatric AML patient-derived sEVs compared to healthy controls, and their expression level is independent of the AML subtype. Furthermore, YBX1 expression was significantly increased on BM-MSCs treated with AML-sEVs. Next, overexpression of YBX1 in BM-MSCs notably decreased their osteogenic differentiation, highlighting the involvement of YBX1 in BM-MSCs’ osteogenic differentiation. Interestingly, treatment of BM-MSCs with sEVs isolated from AML cells with the downregulation of YBX1 remarkably rescued the osteogenic differentiation of BM-MSCs.

Summary/ Conclusion

Altogether, our data demonstrate for the first time that YBX1 containing AML-sEVs is one of the key players that disrupt the normal function of BMM by reducing the osteogenic differentiation of BM-MSCs. Further research focusing on the functional interaction of AML-sEVs with other BMM components is vital to developing a new therapeutic strategy for AML.

Dr Sarah Tassinari¹, Dr Federica Collino², Benedetta Bussolati¹

¹Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy, ²8 Department of Clinical Sciences and Community Health, University of Milano, Milano, Italy

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Extracellular vesicles (EV) are important in tumor progression and diffusion. The analysis of the profile, the origin, as well as of the functional characterization of different EV types within the tumor microenvironment and matrix may provide insight into their role in tumor progression. In this study, we aimed to compare EVs from decellularized and fresh colorectal cancer (CRC) biopsies, to gain information on their compartmentalization and possible function.

Methods

EVs were extracted from 110 fresh tumor biopsies and 46 CRC decellularized tissues. For each CRC sample, EVs also were extracted from 10-cm far normal mucosa used as control in order to specifically identify tumor-associated EVs. EVs were isolated after enzymatic digestion and ultracentrifugation and analyzed by NTA, TEM, super-resolution microscopy, and bead-based cytofluorimetric analysis. Functional assays on tumor cells (angiogenesis, infiltration, motility) were performed.

Results

NTA and TEM confirmed the presence of intact EVs in our preparations. Tetraspanin expression was confirmed by super-resolution microscopy and FACS. EVs of platelet and lymphocyte origin were the most abundant type of EVs within the matrix and were increased in normal healthy mucosa in metastatic patients with respect to low-stage tumors.

In addition, large EVs (oncosomes) expressing colon cell markers were specifically present in fresh biopsies but not in decellularized tumor tissue. TEM identified large EVs in the intercellular space. In vitro, large EVs from tumor biopsies increased cancer cell invasion and movement. Overall, CD25 (T-reg marker) was the exclusive marker overexpressed in matrix-derived and fresh biopsy-derived EVs. Analysis of lymphocyte infiltration within tumors confirmed the T lymphocyte regulatory phenotype.

Conclusion

In conclusion, EVs from decellularized tissues, entrapped in the extracellular matrix, may embody the microenvironment alterations more prominent in the tumor surrounding tissue. Tumor-released EVs, at variance, may be involved in tumor functional properties. Finally, T-reg released EVs expressing CD25 appear as an interesting tumor-associated marker.

Graduate Student Yuhan Qiu¹, Undergraduate Student Rebecca Yu¹, Graduate Student Andrew Chen¹, Postdoc Matt Lawton¹, Graduate Student Pablo Llevenes¹, Lab Manager Manohar Kolla¹, Postdoc Naser Jafari¹, Kiana Mahdaviani², Naomi Ko², PI Stefano Monti¹, PI Gerald Denis¹

¹Boston University School Of Medicine, Boston, United States, ²Boston Medical Center, Boston, United States

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Type 2 Diabetes (T2D) is a chronic disease characterized by insulin-resistant adipose tissue. Patients with triple negative breast cancer (TNBC) and comorbid T2D have higher risk of metastasis and shorter survival. However, adipocyte metabolism is often ignored in medical oncology world and mechanisms that couple T2D to TNBC outcomes are unknown. Here we hypothesize that exosomes, small vesicles secreted by tumor microenvironment (TME) breast adipocytes, drive epithelial-to-mesenchymal transition (EMT) and metastasis in TNBC via miRNAs.

Methods: Exosomes were purified from conditioned media of 3T3-L1 mature adipocytes, either insulin-sensitive (IS) or insulin-resistant (IR), then characterized and quantified by NanoSight. Murine 4T1 cells, a TNBC model, were treated with exosomes in vitro (3d). In in vivo model, IHC detected TME differences (angiogenesis, EMT and proliferation). Metastases in distant organs were quantified by clonogenic assay, and profiled by RNA-seq. Exosomal RNAs were extracted and then profiled to identify potential candidates responsible for driving metastasis.

Results: In primary tumors, vimentin (EMT), ki67(proliferation) and CD31 (angiogenesis) were elevated in IR exosome group vs. control and IS exosomes groups. Clonogenic assay of brain metastases showed more mesenchymal morphology and RNA-seq analysis revealed EMT pathway enrichment in IR exosome group. Several highly differentially expressed miRNAs between IS and IR potentially regulate metastasis.

Summary/Conclusion: IR adipocyte exosomes modify TME, increase EMT and promote metastasis to distant organs, likely through miRNA pathways. We suggest metabolic diseases (e.g., T2D) reshape TME, promoting metastasis and decreasing survival. Therefore, TNBC patients with T2D should be closely monitored for metastasis, with metabolic medications considered.

Parvez MD SORWER ALAM, Dr. Eisuke Dohi

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

miRNAs are present in extracellular vesicles and are thought to be responsible for homeostasis in vivo through intercellular communication. Some studies have used miRNAs of EVs in the blood circulation as biomarkers, but in human disease studies, there are cases in which results do not agree among studies, and it is necessary to consider the effects of handling of biological materials and EV extraction methods. This is very important in the study of EV homeostasis in vivo. In this study, we conducted a systematic review of studies using mice, for which experimental conditions are easy to set up, and analyzed blood EVmiRNA profiles to examine the consistency of these blood EVmiRNA profiles.

2) Methods

A systematic review was conducted by searching PubMed, WOS, SCOPUS, and GEO for articles examining EV miRNAs in rodent blood. Of the 1392 articles obtained from the search results, 33 were identified based on the presence or absence of raw data and English language content. From these, 6 references were selected based on the inclusion criteria; such as male adult C57/B6 mice, serum, and EV recovery by ExoQuick, and a total of 14 serum EVmiRNA control data were analyzed. Consistency of miRNA profiles within and between studies was examined.

3) Results

Regarding mouse serum EV miRNA profiles, the correlation between mice within studies was relatively well maintained at 0.4-0.95, but the correlation between studies was low with a range of -0.5 to 0.5.

4) Summary/Conclusion

Even in studies of mice, where experimental conditions were relatively easy to match, the profiles of blood EVmiRNAs differed between studies. This may be due to the influence of confounding factors that have not been considered previously. For example, the effects of contaminating platelets and platelet-derived extracellular vesicles, diurnal variation, and contamination with extracellular vesicles derived from bacterial flora in vivo. How to control for the effects of confounding factors not previously considered and careful reporting of experimental conditions will be important for future biological studies, in addition to making the most effective use of the experimental data that will be shared.

Ms Inge Varik, Ms Katariina Johanna Saretok, Dr Ileana Quintero, Dr Maija Puhka, Dr Aleksander Trošin, Ms Kristine Roos, Dr Paolo Guazzi, Dr Agne Velthut-Meikas

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Numerous studies have identified the presence of extracellular vesicles (EVs) in follicular fluid (FF). While most investigations have focused on the collective pool of EVs in FF, EV subtypes may serve distinct functions within the ovary. In this study, we isolated large (LEVs) and small EVs (SEVs) from human FF, and characterized their molecular composition and biophysical characteristics. Our study explored the impact of EV subpopulations on KGN granulosa cell proliferation, viability, and steroid hormone production, presenting novel insights into FF EV composition and functionality.

Ethical approval was obtained from the Research Ethics Committee of the University of Tartu. FF samples, with participant consent, were collected at Nova Vita Clinic and East Tallinn Central Hospital located in Tallinn, Estonia. EV subpopulations were purified from human FF pools by size exclusion chromatography followed by tangential flow filtration (pore size 200 nm). The characterization of EVs involved nanoparticle tracking analysis, transmission electron microscopy, immunoblotting, and imaging with the ExoView R100 platform. In functional assays, KGN cell line was treated with 10⁶-10⁸ LEVs or with 10⁷-10⁹ SEVs for 24 hours, after which cell viability and proliferation were determined with a bioluminescent assay and steroid hormones were measured from cell media via ELISA.

Human FF contains approximately ten times more SEVs than LEVs, with mean diameters of 93 nm and 299 nm, respectively. Immunoblotting revealed the presence of CD9, CD81 and HSP70 in both EV subtypes, along with the depletion of albumin and ApoA1 when compared to the FF profile. ExoView analysis showed a lower proportion of CD9+CD63+CD81+ particles in LEVs than SEVs. Functional studies concluded that EV subtypes had no impact on KGN cell viability or proliferation. Steroid hormone measurements indicated elevated testosterone production when treated with SEVs.

This study highlights differences in tetraspanin marker composition among human FF EV subtypes. While FF EV subtypes do not influence KGN cell proliferation or viability, they seem to impact testosterone production. These findings enhance our understanding of the characteristics and the functional roles of EV subtypes in follicular fluid.

Ph.D. Asako Shimoda, Professor Emeritus Kazunari Akiyoshi

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) are known to copy biological information from their origins and convey it distant cells. EVs are involved in various kinds of cellular events including cancer progression, immune responses, viral pathogenesis and treatment responses. Consequently, they have been gathering attention in diagnostic and therapeutic applications. However, EVs are heterogeneous mixtures of different biogenesis, size, and the cargos, it is still hard to distinguish EV populations. In order to solve the problem, we are focusing on EV surface glycans to evaluate EV heterogeneity. In this study, we used an evanescent-field fluorescence-assisted lectin microarray system and imaging flow cytometry (IFCM) for both comprehensive and specific analysis of EV surface glycan-lectin interactions.

Methods: EVs were isolated from human two cancer cell lines, adipose-derived mesenchymal stem cells (MSCs), and human plasma by ultracentrifugation. Lectin microarray was used for high-throughput analysis of EV surface glycan patterns. Characterization of lectin-glycan interactions and tetraspanin expression on EVs in a single particle level was performed by IFCM. Furthermore, we captured EVs by various kinds of lectin-coupled beads and compared glycan profiles between them.

Results: A lectin microarray method was useful for exhaustive and simultaneous analysis of EV surface glycans. Surface glycan patterns on EVs were different depending on the cell type and EV size, indicating that EV glycans can be used as one index for clarifying EV subpopulations. Interactions between EVs stained with lipid and fluorescent lectins can be successfully detected by IFCM, and the percentages of EVs positive to lectins were depending on cell types. In addition, sialic acids-recognizing lectin-coupled magnetic beads can be captured specific EV populations.

Conclusion: Our results indicate that lectin-based glycan analysis will help assess EV heterogeneity and this technique can be used as potential tool for the discovery of biomarkers.

Lauren Litchfield, Dr Rebecca Wellburn, Dr Sarah Bajan, A/Prof Yoke Lin Fung, A/Prof John-Paul Tung

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Platelet concentrates (PCs) are used to treat patients who are bleeding or at risk of bleeding with thrombocytopenia or who have non-functioning platelets. In Australia, PCs are stored at room temperature (20-24°C) for up to 7 days. During this storage, platelet activation and degradation occurs resulting in the release of bioactive proteins, lipids and extracellular vesicles (EVs) that can affect the recipient. This storage lesion is highly dependent upon the specific processes used to collect and manufacture PCs and, therefore needs to be investigated in the Australian context.

Methods:

This study was approved by Lifeblood's Human Research Ethics Committee. PCs (n = 9) were prepared by pooling buffy coats from four ABO compatible whole blood donations. The resultant pools then underwent leucofiltration, centrifugation, resuspension in platelet additive solution, and irradiation. Samples were collected at days (D) 2, 5, and 7 of storage and underwent centrifugation. The resulting supernatant was aliquoted and frozen at -80°C. Thawed aliquots underwent nanoparticle tracking analysis (NTA) on the Nanosight NS300. Normality was assessed and confirmed using Shapiro-wilk test. Statistical analysis was then completed using repeated measure one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Significance was determined at P<0.05.

Results:

D2 is the first day when PCs are available for issue and transfusion. D2-PCs contained 3.52 ± 0.34 x 10¹⁰ extracellular particles / mL with a mean size of 120.8 ± 12.6nm. There was no increase in extracellular particle size or concentration over the remaining 5 days of PC storage: D5-PCs contained 3.24 ± 0.98 x 10¹⁰ extracellular particles / mL with a mean size of 120.0 ± 12.7nm while D7-PCs contained 3.47 ± 0.65 x 10¹⁰ extracellular particles / mL with a mean size of 120.2 ± 13.3nm.

Summary/Conclusions:

Extracellular particles were observed in PC. The length of storage did not affect their concentration or size. In line with MISEV2018 guidelines, further investigations are needed to identify if the extracellular particles present in PCs are EVs, and if so, to assess their phenotype or cargo and if these change throughout storage.

Miss Irumi Amarasinghe, Dr Ebony Monson, Dr Eduard Wilms, Mr William Phillips, Dr Shuai Nie, Miss Abbey Milligan, Dr Donna Whelan, Prof Andrew Hill, Prof Karla Helbig

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Cell-to-cell communication is essential for the effective coordination of biological processes, often being facilitated by the release and uptake of small particles. Small lipid enclosed particles such as extracellular vesicles (EVs) mediate intercellular communication through transfer of their cargo and are actively explored for their role in various diseases and their potential therapeutic and diagnostic applications. Though EVs are the dominantly studied extracellular communicators, lipid droplets (LDs), which are also lipid-enclosed small particles, remain unexplored in this space. Our laboratory has recently demonstrated that LDs transfer between cells and can directly influence the antiviral response of neighbouring cells. Mechanisms underpinning this LD movement remain undescribed as does their relationship to EVs. Therefore, this study aimed to explore this connection further.

Methods

To understand if LDs are hijacking EV secretion pathways, proteomic and lipidomic comparative analysis using a primary immortalised astrocyte model was performed. Western blot and super resolution microscopy were used to confirm protein targets.

Results

Mining of published databases revealed a significant overlap of up to 96% in the proteomes of EVs and LDs, with both groups sharing proteins involved in biogenesis, cargo recruitment and transport. Using a primary astrocyte model, we confirmed that there was approximately an 80% proteome overlap; with 48 of the 50 common EV protein markers present in the astrocyte EVs being also observed in the LD proteome. The annexin, heat shock proteins and ALIX were highly abundant on LDs and EV markers ALIX and TSG101 were confirmed to be present on LDs via western blot and super resolution microscopy. EV associated proteins SDC4 and ARRDC1 were not identified on astrocyte LDs. Lipidomic analysis of astrocytic EVs revealed a distinct lipidome to isolated LDs, with the EV lipidome being similar to that of whole cell lysate.

Conclusion

To date, there has been little research investigating the similarities of protein cargo between LDs and EVs and their potential interactions. Here, we have demonstrated for the first time that LDs carry proteins involved in EV biogenesis and release which may allude to their transport between cells.

Ji Hui Hwang, A/Prof John-Paul Tung, Prof Damien Harkin, Prof Robert Flower, Dr Natalie Pecheniuk

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Fresh frozen plasma (FFP) and cryoprecipitate are used to treat bleeding patients. FFP can be slowly thawed (1–6°C), and the cold-insoluble precipitate is removed and refrozen as cryoprecipitate. The extracellular vesicle (EV) content of FFP and cryoprecipitate is dependent on their specific manufacturing processes, so needs to be investigated in the Australian setting.

Methods:

Units of FFP and cryoprecipitate (n = 5 each) were thawed, pooled, aliquoted, and stored at -80°C. For EV isolation, thawed aliquots underwent centrifugation (2000g; 10,000g) and size-exclusion chromatography (SEC; qEVoriginal 70 nm columns on Izon automated fraction collectors). Collected fractions were concentrated (Amicon Ultra-15 filters,100 kDa cutoff). Pooled FFP and pooled cryoprecipitate, as well as their fractions (FFP-EV and cryo-EV) underwent nanoparticle tracking analysis (NTA; Nanosight NS300), protein quantification (bicinchoninic acid protein assay), cryogenic transmission electron microscopy (cryo-TEM; Jeol Cryo ARM 200), and western blot (anti-CD9, anti-CD81, anti-flotillin 1, and anti-fibronectin).

Results:

FFP had higher NTA particle concentrations than cryoprecipitate (1.20 X 10¹¹ vs. 6.75 X 10¹⁰ particles/mL; P = 0.0014). A reduction was observed following EV isolation (FFP-EV: 3.0 X 10¹⁰ particles/mL, p < 0.0001; cryo-EV: 1.12 X 10¹⁰ particles/mL, P = 0.0009). Average mode particle sizes were similar between FFP and cryoprecipitate (163nm vs 171nm, P>0.05). Mode particle size increased in cryoprecipitate following EV isolation (216 nm, P = 0.0273).

Protein concentrations in FPP and cryoprecipitate (79,672.7 µg/mL and 84,292.5 µg/mL, respectively) were reduced following EV isolation (FFP-EV: 67.6 µg/mL; and cryo-EV: 390.6 µg/mL) suggesting successful enrichment (FFP-EV: 4.4 x 10⁸ particles/µg vs FFP: 1.5 x 10⁵ particles/µg protein; and cryo-EV: 2.9 x 10⁷ particles/µg vs 8.0 x 10⁵ particles/µg protein).

Morphological assessment of FFP-EV and cryo-EV using cryo-TEM revealed circular particles ranging approximately from 50-100 nm and containing a membrane bilayer, indicative of EVs.

Western blot analysis of FFP-EV and cryo-EV confirmed the presence of EV-associated proteins (CD9, CD81, and flotillin), while fibronectin was used to assess major components of co-isolated non-EV protein.

Summary/Conclusions:

These results allowed us to classify the SEC-isolated particles as EVs, confirming the presence of EVs in FFP and cryoprecipitate. Successful enrichment of these EVs will allow further characterisation of their phenotypes and cargo.

Ms. Geetika Raizada, Mr. Joan Guillouzouic, Dr. Eric Le Ferrec, Dr. Eric Lesniewska, Dr. Wilfrid Boireau, Dr. Céline Elie-Caille

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Researchers have been exploring the role of extracellular vesicles in the context of environmental toxicology. It has been found that they play a role in triggering oxidative stress when they originate from cells exposed to the toxicants like polycyclic aromatic hydrocarbons (PAHs) (N. van Meteren, et al, 2020). Under the toxic conditions, the EVs undergo molecular changes and it has been noted that large EVs (lEVs) might also contain mitochondria which contributes in triggering the cell death. In this study, we are interested in developing a morpho-nanomechanical approach to study the biomechanical properties of EV, especially lEVs subsets that are derived from Benzo[a]Pyrene (B[a]P) treated endothelial cells.

Methods:

lEVs were isolated by ultracentrifugation (10 000 g for 30 min) from the Human microvascular endothelial cell line (HMEC-1) which was treated either with B[a]P (treated condition) or DMSO (control condition).

We combined fluorescent optical microscopy with Atomic Force Microscope (AFM) to perform nanomechanical mapping of fluorescent and non fluorescent tagged lEVs, adsorbed on positively charged mica substrate. This enabled us to characterize respectively the lEVs that contained mitochondria or not.

Results:

We observed a noticeable difference in lEVs morphology, size and elasticity between control and BaP treated conditions. The lEV subpopulations present in both samples ranged from 200 nm to more than 700 nm in diameter, with more bigger ones in the case of treated cells. We measured the Young's modulus of 100 lEVs from the treated and 96 lEVs in the control conditions; and the lEVs populations coming from treated conditions were stiffer EVs than control. We also observed the intrinsic heterogeneity in the elasticity across a single vesicle that ranged from 0.1 to 30 MPa and from 0.1 to 10 MPa for treated and control conditions respectively.

Conclusion:

With this study, we were able to establish the size profiles and elasticity maps of the EVs subsets, containing or not mitochondria, in both control and BaP treated conditions. Those results reveal the potential of using AFM to detect a specific lEVs morpho mechanical phenotype in a context of cytotoxicity.

Phd Student Mariane Shouky, Graca Raposo

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

Skin wound healing is a complex event that involves interaction and communication between skin cells. It consists of four phases: hemostasis, inflammation, re-epithelialization, and tissue remodeling. Orchestrated cross-talk between the epidermal and dermal skin cells, keratinocytes and fibroblasts respectively, plays a dominant role in the re-epithelialization phase: migration and proliferation.

Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures that are released by cells into the extracellular environment to act as signaling organelles. They play a crucial role in intercellular communication, allowing cells to exchange proteins, lipids and genetic material.

In addition to the aforementioned types of EVs, MBsomes (MBs), could be classified as a new class of large ectosomes that are released by cells, at the final stage of cytokinesis, to act as postmitotic signaling organelles. Whether MBs serve as a mean of intercellular communication in skin wound healing, to our knowledge, is not investigated yet.

The aim of this project is to decipher the biogenesis, release and signaling function of MBs, the new EV to be, in parallel with other EVs, in skin wound healing. Our hypothesis is that, MBs, released from hyperproliferating epidermal skin keratinocytes, along with other EVs, are a mean of epidermal/dermal cross-talk in skin wound healing.

2) Methods

To address our hypothesis, several approaches were used, including purification of EVs by differential ultracentrifugation followed by SEC, and purification of MBs by differential ultracentrifugation followed by sucrose cushion, from epidermal keratinocytes. Characterization of EVs and MBs size and concentration by NTA, protein markers by WB, morphological validation by TEM, surface markers expression by IEM, identification of the cargoes of MBs and EVs by transcriptomics and proteomics. In addition, their function in skin wound healing processes: migration and proliferation, was also assessed by scratch-wound assay and KI67 nuclear staining, respectively.

3) Results

Our results show that epidermal keratinocytes release EVs and MBs. Interestingly, epidermal keratinocytes EVs and MBs promote migration and proliferation of dermal fibroblasts via ERK activation.

4) Summary/Conclusion

Primary epidermal keratinocytes release EVs and MBs which play a role in the epidermal/dermal crosstalk in skin wound healing via ERK activation.

Xiaomei Yan, Yunyun Hu, Haonan Di, Dr. Ye Tian

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

Advancements in isolation methods have uncovered the diverse landscape of extracellular particles (EPs). Non-vesicular extracellular particles (NVEPs), distinct from extracellular vesicles (EVs), lack a lipid bilayer membrane. They constitute a unique type of EPs prevalent in extracellular spaces and body fluids, with potential roles in maintaining homeostasis and facilitating cellular communication. However, prevalent EP separation methods often co-isolate NVEPs and EVs, while many characterization techniques lack the specificity to detect EVs or smaller NVEPs, impeding the identification of extracellular cargo and secretion mechanisms of specific biomolecules. Establishing a rapid and efficient method to differentiate EVs and NVEPs in complex samples is crucial for evaluating EP isolation methods and pinpointing carriers of particular biomolecules.

2) Methods

EPs from colorectal cancer cell lines were isolated via differential ultracentrifugation (UC). High-resolution iodixanol density gradient centrifugation was employed to separate EVs from NVEPs. A laboratory-built nano-flow cytometer (nFCM) with two spatially arranged lasers was applied to characterize the size, purity, particle concentration, DNA, lipophilic membrane, and specific proteins of both EP populations at the single-particle level. This analysis utilized side scattering and fluorescence detection using SYTO 16, di-8-ANEPPS, and immunofluorescent staining to identify distinguishing features between EVs and NVEPs. EP samples obtained via UC were further purified through UC, ultrafiltration (UF), and size exclusion chromatography (SEC) to evaluate their effectiveness in eliminating NVEPs.

3) Results

Differences in morphology (via TEM), biophysical properties, and molecular compositions were evident between EVs and NVEPs. SYTO16 DNA staining proved to be a distinct biochemical feature distinguishing between the two. Increasing centrifugation force and duration in UC enhanced NVEP yield. SEC and UF showed limited efficacy in NVEP elimination. SEC was utilized to remove residual antibodies upon immunofluorescent staining, followed by DNA labeling and nFCM analysis. Multiplexed analysis of DNA and protein facilitated the identification of carriers for specific biomolecules within EPs.

4) Summary

This study showcases nFCM as a potent platform for differentiating EVs from NVEPs via DNA analysis on single EPs. Multiparameter analysis of DNA and proteins could unveil intra-subtype heterogeneity of NVEPs and EVs, supporting fundamental and translational research on specific EP types.

Ms Jiyoung Goo, Ms Somi Park, Ms Hyeyeong Ku, Ms Jeongmin Lee, Mrs Jeong Hee Kim, Mr In-San Kim, Mr Cherlhyun Jeong

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1. Introduction

Cells secrete exosomes through various biogenesis pathways, resulting in distinct molecular compositions even when originating from the same cells. Current characterization assays can only provide ensemble averaging. Therefore, developing a single exosome characterization assay is a solution to identify exosome heterogeneity. In this study, we focus on quantifying exosomal membrane proteins using a unique methodology based on single-molecule fluorescence binding imaging.

2. Methods

Liposomes were generated using DOPC lipids and extruded through a 100 nm diameter filter. Cholesterol-Cy3-conjugated single-strand DNA and Cy5-conjugated single-strand DNA were purchased. Exosomes were derived from HEK293T cells, and following the MISEV2018 guidelines, purification was performed through stepwise centrifugation-tangential filtration with diafiltration by PBS until at least an 8-10 fold volume exchange. Fluorescence signals were imaged in a prism-type total internal reflection fluorescence microscopy.

3. Results

Our TIRF based single-molecule fluorescence imaging proved to be suitable for the quantification assay using liposome experiments as exosome mimic models. DNA hybridization mimicked the binding of membrane proteins and aptamers or antibodies. We acquired information from these individual traces of fluorescent signals over time. The number of CD63 proteins in HEK293T cell-derived exosomes was identified using CD63 aptamers and antibodies. We further confirmed the presence of other membrane proteins using specific antibodies.

4. Summary/Conclusion

Consequently, we developed a membrane protein quantification assay for individual exosomes utilizing single-molecule and particle fluorescence imaging techniques. Our results demonstrated the effectiveness of this technique in exosome mimic models, quantifying various membrane proteins, including CD63. Through this technology, we aim to elucidate the distribution of membrane proteins in exosomes derived from various cells, enabling their potential use in future biopsies or quantification studies.

Mr Liam Hourigan, Mr William Phillips, Mr Chaomei Chen, Mr Amirmohammad Nasiri Kenari, Mr Krishna Chaitanya Pavani, Mrs Lesley Cheng, An Hendrix, Mr Andrew Hill

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

The extracellular vesicle (EV) field has grown at an exponential rate in recent years, but what of the direction and consequences of this growth? It's challenging for researchers to maintain an awareness of the various subdomains in the field, their relation to one another and future directions. Traditional reviews are unable to encapsulate this breadth of information, the more quantitative methodology termed scientometrics is apt for this task.

Methods

A dataset of 52,286 articles published between 1987-2023 and relating to EVs was downloaded from Web of Science, then enriched with additional data from Dimensions and the EV-Track knowledgebase. This was analysed using co-citation networking in the program CiteSpace which delineated clusters of interest. These clusters were further analysed with comparison being made to a global map of science derived from 20 million PubMed articles. These findings were synthesized using the language of intellectual structures and Kuhnian scientific paradigms.

Results

17 clusters of interest were identified in a local map of the EV field using CiteSpace. Trends in the translational research, cancer data, and nomenclature usage were examined between these clusters and across the field as a whole. To provide further validation for the intellectual structures identified in the local map, these were compared against a global map of science. An overarching paradigm dubbed the ‘MISEV paradigm’ was identified along with two exemplary nested paradigms. How this paradigm relates to the field, its growth and the field's growth is explored.

Conclusions

The EV field cannot continue to grow at an exponential rate, at some point the growth will plateau due to physical constraints. The MISEV paradigm exerts an enormous influence on the field, yet there are a variety of other paradigms that operate within and around its bounds, and with the field's growth trajectory these too will grow and change. Thus far, scientometric methods have seen limited application in the EV field, but these findings demonstrate their uniquely quantitative perspective can yield insights outside the scope of any traditional review or expert opinion.

Dr Kosuke Yoshida, Dr Akira Yokoi, Dr Kazuhiro Suzuki, Dr Yukari Nagao, Dr Ryosuke Uekusa, Ms Masami Kitagawa, Dr Eri Inami, Dr Takao Yasui, Dr Hiroaki Kajiyama

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

High-grade serous ovarian carcinoma (HGSOC) is one of the leading causes of death. HGSOC frequently spreads to the peritoneal cavity, and thus, it is important to elucidate the molecular mechanisms underlying the peritoneal dissemination. Here, we developed a novel method using cellulose nanofiber sheets, that can capture extracellular vesicles (EVs) from microfluid. This technique allowed us to evaluate the EV profiles on the surface of each organ in the peritoneal cavity. In this study, we investigated a new concept, which spatially diverse EVs are involved in the HGSOC development.

2) Methods

First, we retrospectively reviewed the surgical video of 24 cases with stage III HGSOC who underwent diagnostic laparoscopy. Second, we collected EVs on the liver surface and peritoneum from 36 spots from 12 patients using the cellulose nanofiber sheets. Then, we performed microRNA sequencing to evaluate the diversity of the EV profile. Finally, we performed in vitro analyses. HepG2 and HOSE1 cells-derived EVs were collected through ultra-centrifuge, and we investigated the function of HepG2-derived EVs on ovarian cancer cells (ES-2 and SKOV3 cells).

3) Results

Diagnostic laparoscopy revealed that only one patient (6%) experienced metastasis on the liver surface, whereas 18 patients (82%) had metastasis on the diaphragm. Then, we confirmed that the cellulose nanofiber sheets surely captured EVs. Subsequently, microRNA sequencing for microfluid on the peritoneum showed that the EV microRNA profile on the liver surface was different from that on the abdominal wall, suggesting the spatial diversity of intraperitoneal EVs. Moreover, we confirmed that HepG2-derived EVs suppressed the migration and invasion abilities of the ovarian cancer cells (p < 0.01). Furthermore, the expression of CDH1 in the ovarian cancer cells was increased when co-cultured with HepG2 cells.

4) Summary/Conclusion

We clinically showed that HGSOC rarely spread to the liver surface, which had a unique EV miRNA profile. Moreover, hepatic cell-derived EVs acted as tumor suppressors in ovarian cancer cells. Therefore, spatially diverse EVs might contribute to the formation of peritoneal dissemination.

Dr Chris Pridgeon, Ms Julia Monola, Ms Kerttu Airavaara, Dr Daniel Palmer, Prof. Marjo Yliperttula, Dr Riina Harjumäki

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Hypoxia, or physiological normoxia, alters cellular phenotype and that of the EVs that arise from them. The effects of hypoxia, upon the contents of EVs may alter their behaviour in vivo, which is important in cancer mechanistic studies. In addition, there is disagreement in the literature whether hypoxic culture increases the rate of EV production. The majority of studies combining EVs and hypoxia use only transient periods of hypoxia, typically less than 3 days. We examined the relationship between duration of either acute (2 days) or chronic (1 month) 5% O2 hypoxia, and the effects on EV production and phenotype in two cancer cell lines, HepG2 and PC3.

Methods: Hypoxic HepG2 and PC3 lines were established that could be cultured indefinitely in 5% O2 and cryopreserved without exposure to oxygen. Cells were cultured under atmospheric oxygen or exposed either to acute hypoxia (2 days) or chronic hypoxia (1 month). Cell number and medium amount were kept constant between different conditions. EVs were purified by differential centrifugation and their number and size distribution was measured using nanoparticle tracking analysis. EVs and their parent cells were then subjected to proteomic analysis.

Results: Relative to normoxic (atmospheric oxygen), the number of EVs produced by PC3 cells increased in chronic hypoxia but was not significantly different in acute hypoxia. The number of EVs produced in HepG2 cells was not significantly changed by hypoxic culture of any duration although the protein content was increased in hypoxic conditions. A biphasic hypoxic response was discernible in the cells and also the EVs produced by both PC3 and HepG2 cells in acute and chronic hypoxia.

Summary: These findings demonstrate that hypoxia only produces an increase in EV production in some cell lines and that these changes can take substantial time to occur. Regardless of cell type, the phenotype of cells and EVs is distinctly altered by hypoxic culture in a manner that changes over time. The phenotype of cells and EVs continues to adapt after the most commonly used period of study for hypoxia, which should be considered in future studies.

Marley Dewey, Assistant Professor George Hussey, Professor Stephen Badylak

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Matrix-bound nanovesicles (MBV) are extracellular vesicles (EV) isolated from the extracellular matrix (ECM). MBV are similar to exosomes in size and shape, but these have distinctly different lipid profiles and miRNA cargo. MBV have demonstrated immunomodulatory and wound healing capabilities in applications such as rheumatoid arthritis and optic nerve repair. MBV have the potential to be used as a therapeutic and diagnostic tool; however, an understanding of MBV biogenesis is lacking and hence their full potential depends on developing an understanding of mechanisms of action. Our goal is to determine MBV biogenesis by answering how MBV bind to the ECM and their relationship with the ECM.

Methods: ECM-related surface markers (integrins) on both MBV and exosomes from the same cell source (human mesenchymal stem cells, fibroblasts) were assessed by western blotting. Additionally, these cells were cultured on glass coverslips and SEM identified MBV location in regard to collagen production. We assessed whether increases in collagen production related to increases in MBV production by applying various concentrations of ascorbic acid to cell cultures. Exosomes were isolated from cell culture media via ultracentrifugation and size exclusion chromatography. ECM deposited by cells was enzymatically digested to release MBV, which were isolated via ultracentrifugation. EV populations were verified by particle tracking analysis, TEM, and surface markers.

Results: We report a process for isolating MBV and exosomes from the same cell source. MBV were enriched in integrins α5, αV, β1, β5, and the presence of ALIX on MBV suggests a potential biogenesis mechanism related to the ESCRT pathway. SEM imaging revealed vesicles bound to collagen fibers produced by cells, even after subsequent washing and preparation steps. Increasing ascorbic acid (1000 uM) concentration increased MBV production while this had no impact on exosome production.

Conclusion: The present work expands on our knowledge of MBV. Tethering of MBV to the ECM may be the result of additional integrins on the surface of MBV, and these could be used to identify MBV from exosomes. Furthermore, there is a relationship between collagen formation and MBV production, which will be examined in the future to further explore MBV biogenesis.

Dr Agne Velthut-Meikas¹, Inge Varik¹, Katariina Johanna Saretok¹, Kristine Rosenberg^1,2, Aleksander Trošin³, Maija Puhka⁴, Ileana Quintero⁴, Paolo Guazzi⁵

¹Tallinn University Of Technology, Tallinn, Estonia, ²Nova Vita Clinic, Tallinn, Estonia, ³East Tallinn Central Hospital, Tallinn, Estonia, ⁴University of Helsinki, Helsinki, Finland, ⁵HansaBioMed Life Sciences Ltd, Tallinn, Estonia

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Ovarian follicle is the environment of oocyte maturation, where the oocyte is nurtured by the granulosa cells. The dominant follicle forms a cavity filled with follicular fluid (FF). Ovarian cells exchange metabolites, steroids, and proteins via FF –a molecular signaling mechanism that is necessary for oocyte maturation and ovulation. The importance of extracellular vesicles (EVs) in follicular intercellular communication has not been extensively investigated. While previous studies have focused on the pool of FF EVs, EV subtypes may serve distinct functions within the ovary. This study aimed to investigate the differential impact of small and large EVs (SEVs and LEVs, respectively) on the transcriptome of the human granulosa-like tumor cell line KGN: a stable and widely used model for the ovarian granulosa cells.

Methods

Ethical approval was obtained from the Research Ethics Committee of the University of Tartu, Estonia, and all patients provided a written informed consent. FF was collected from preovulatory follicles (>18 mm diameter) of IVF patients. SEVs and LEVs were purified using size exclusion chromatography followed by tangential flow filtration (cut-off 200 nm). EV characterization involved nanoparticle tracking analysis, transmission electron microscopy and Western blotting. For gene expression studies, KGN cells were incubated with 109/ml SEVs or 108/ml LEVs for 24h. Total RNA was extracted using the Qiagen miRNeasy kit, and sequencing libraries were prepared with the Revvity NextFlex Rapid Directional RNA-seq kit. Single-end sequencing was performed with the Illumina NextSeq2000 instrument. Identification of differentially expressed genes (DEGs) was conducted with the R package DESeq2 v1.42.0, reporting DEGs with FDR ≤ 0.1.

Results

SEVs changed the expression levels of 1006 genes in KGN. Enrichment analysis categorized 642 downregulated genes into pathways related to cholesterol biosynthesis, cell cycle and extracellular matrix organization, while 364 upregulated genes were enriched into translation-related pathways. Treatment with LEVs resulted in 86 DEGs, with 63 downregulated genes enriched into extracellular matrix and adhesion pathways, and 23 upregulated genes which similarly to SEVs were enriched into translation-related pathways.

Summary

These findings enhance our understanding of the intercellular communication within the ovarian follicle and indicate the potential involvement of SEVs in regulating ovarian steroidogenesis.

Dr. Igor V Kurochkin¹, Lausonia Ramaswamy

¹Central Research Laboratory, Sysmex Co., Kobe, Japan

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: We explored here if the tetraspanin-containing plasma particles are differentially sensitive to various detergents and their combinations, the feature that could be utilized for a simple and fast enrichment step of the particle subpopulations or their subdomains for biomarker analysis.

Methods: Human plasma samples were fractionated by size-exclusion chromatography using various resins. Fraction material was treated with 0.5% Triton X-100, DRM reagent disrupting lipid rafts but preserving protein native structure and a combination of both. CD9 was quantified using sandwich ELISA with capturing and detecting antibody recognizing the same epitope thus detecting structures containing at least two CD9 molecules.

Results: Fractionation of human plasma on either Sepharose CL-2B or CL-4B resulted in a major peak of CD9 in the void column volume consistent with the size of extracellular vesicles and a shoulder eluted in the fractions suggestive of very small vesicles or large proteins. This shoulder was not likely the result of a non-specific binding of plasma particles to Sepharose that could slow their elution, because nearly identical elution profiles were observed on columns packed with the resin having a different polymer structure, Toyopearl HW-65 and HW-75. Treatment with Triton X-100 resulted in a significant loss of the CD9 signal in early (70%) and late (50%) fractions. DRM reagent had no effect on the CD9 signal in early fractions after their pretreatment with Triton X-100 but significantly decreased the signal in Triton X-100 pretreated late fractions. Approximately 30% of the CD9 signal in both fractions was resistant to a combinatorial treatment with Triton X-100 and DRM.

Summary/Conclusion: This study reveals that CD9 molecules are incorporated into different extracellular structures with a broad size distribution consistent with the presence of very small and large vesicles. These structures could be classified as Triton X-100 sensitive and Triton X-100 resistant. Triton X-100 material, in turn, consists of lipid rafts and distinct from lipid raft domains. Future studies will explore whether disease biomarkers are present predominantly in distinct CD9-positive structures. Selective detergent extraction would provide then a fast and simple strategy for the enrichment of these structures for diagnostic purposes.

Director, Computational Oncology Unit Ahmed Fadiel², Process Development Lead Shuaizhen Yuan¹, Associate Scientist Eileah Loda¹, Ceo Adam Koster¹, Chair, Medical Scientific Advisory Board Frederick Naftolin¹, Director, Medical Affairs Matthew Peterson

¹Interactome Biotherapeutics, Grand Rapids, United States, ²University of Chicago, Chicago, USA

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Extracellular Vesicles (EVs), particularly exosomes, serve as critical messengers in intercellular communication, orchestrating a myriad of physiological and pathological processes. This study characterized the EV's of commonly utilized stem cell EV's to characterize their physical properties and surface/interior markers using advanced techniques with the aim of uncovering the multifaceted heterogeneity inherent within EV subpopulations.

Mesenchymal stem cells sourced from umbilical cord (MSC-UC) and platelet lysate (MSC-PL) provided the basis for harvested EV's from the conditioned media, which underwent meticulous EV extraction protocols. Through ultracentrifugation, distinct exosome subpopulations were delineated. Their biophysical properties were characterized by using tunable resistive pulse sensing. Additionally, the molecular contents of these subpopulations were probed through Western blot and mass spectrometry analyses. All studies were repeated at least three times and showed sufficiently constant results for commercial development.

Our findings unveil a plethora of protein markers and functional signatures within the isolated exosome fractions, providing insights into their intricate roles in cellular communication and disease pathogenesis. Examples are presented in the figures.

This investigation substantiates the utility of the methods utilized to expose exosome heterogeneity and functional diversity, not only for advancing our understanding of fundamental biological processes but also for harnessing their therapeutic potential in clinical settings. In conclusion, our research contributes to the burgeoning field of exosome biology, emphasizing the critical importance of unraveling the intricate roles of exosomes in health and disease. These insights underscore the necessity for further investigation into the diverse functions and therapeutic potentials of exosomes extracted using different extraction and processing methods, shaping the future of personalized medicine and disease management.

Dr Wei Heng Chng¹, Mr Ram Pravin Kumar Muthuramalingam¹, Dr Yub Raj Neupane¹, Dr Chenyuan Huang¹, Dr Wei Jiang Goh¹, Dr Choon Keong Lee¹, Bertrand Czarny², Assistant Professor Jiong-Wei Wang¹, Associate Professor Giorgia Pastorin¹

¹National University of Singapore, Singapore, ²Nanyang Technological University, Singapore

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

The use of extracellular vesicles (EVs) for therapeutic purposes has been well-documented. In particular, EVs isolated from mesenchymal stem cells (MSC-EVs) have demonstrated beneficial effects in the treatment of several human diseases including wound healing and tissue repair. Our lab has developed a cost-effective and time-efficient method to produce EV mimetics from cells, known as cell-derived nanovesicles (CDNs). Our production method reduces the duration required by half and increases the yield of the generated EV-mimetics. The therapeutic potential of these MSC-CDNs were evaluated and compared with MSC-EVs in the treatment of myocardial infarction and wound healing.

Methods

Mesenchymal stem cells (MSCs) were sheared using membrane-fitted spin cups and purified by size exclusion chromatography to produce MSC-CDNs. MSC-CDNs were administered intravenously prior to reperfusion in the mouse ischemic/reperfusion model. The cardiac infarct areas and cardiac functions were evaluated. To investigate the wound healing activity, MSC-CDNs were topically applied onto excision wounds on mice and the wound recovery was evaluated by monitoring the wound size.

Results

Our CDNs exhibited a high level of similarity as compared to their natural EVs counterparts with respect to their physical, biochemical, and pharmacological activities. CDNs exhibited minimal immunological responses were administered intravenously to mice. Our MSC-CDNs, similar to MSC-EVs, reduced the infarct area of the heart within 24 hours of treatment in a mouse ischemia/reperfusion model. The treatment with MSC-CDNs significantly improved the cardiac function within 7 days. Further investigations of the mouse hearts revealed that the MSC-CDNs reduced the level of apoptosis, modulate inflammation, and reduced the oxidative stress in the infarct area. The miRNA composition of MSC-CDNs was analysed to identify potential miRNAs responsible for the cardioprotective activities. In another separate study, MSC-CDNs promoted cell proliferation of human dermal fibroblasts and angiogenesis of human dermal microvascular endothelial cells. The topically applied MSC-CDNs accelerated excision wound closure at a recovery rate similar to treatment with MSCs or MSC-EVs.

Conclusion

The shearing of cells to produce CDNs provides an alternative scalable method to obtain comparable EV mimetics for clinical applications. Similar to EVs, CDNs have intrinsic therapeutic activities including tissue repair and wound healing.

Dr. Farrukh Aqil, Raghuram Kandimalla, Disha Moholkar, Margaret Wallen, Chuanlin Ding, Ramesh Gupta

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Immunotherapy has gained increased attention in cancer treatment. Considering the versatile mechanisms of tumor evasion from the host immune system, RNAi therapy can be utilized to enhance the immune response against tumors. Bovine milk/colostrum serve as biocompatible and abundant sources for exosomes for delivery of biologics such as siRNA. Here, we demonstrate exosome mediated delivery of siPD-L1 for lung cancer immunotherapy.

Methods: Exosomes were isolated from colostrum powder by rehydration and ultracentrifugation and analyzed for size, pdi and charge by Zetasizer, and characteristic surface markers by Western blot. Firstly, the exosomes were functionalized with tumor-targeting ligand, folic acid (FA), then complexed with polyethyleneimine (FA-EPM) and siPD-L1. siRNA entrapment efficiency of the EPM was assessed using 5’-32P-labeled siRNA as a tracer. siPD-L1 candidates were screened for efficacy in human and murine lung cancer cells (A549 and LLC-1). Exosomal formulations of the lead siPD-L1 candidate are being tested in vivo against syngeneic orthotopic lung tumor xenografts produced by luciferase-expressing LL/2-Luc2 cells in B6 mice.

Results: The average particle size of exosomes was 66±2.5 nm, with pdi of 0.27±0.01 and a zeta potential of -9.2±0.70 mV. Western blot analysis revealed the presence of several exosomal surface markers, including CD81, TSG101, and Alix. The entrapment efficiency of the EPM for siPD-L1 (1 – 20 µg) was found to be >90%. Biodistribution studies of exosomes and EPM, with or without the FA, revealed similar tissue distribution; however, FA-EPM exhibited enhanced tumor targeting compared to EPM, due to the overexpression of folate receptors on tumor cells. Four different siPD-L1 sequences were screened in vitro. Following 48 h of EPM-siPD-L1 treatment, analysis of protein lysates by western blot showed a significant downregulation of PD-L1 expression, ranging from 55% to 80% in LLC-1 cells and over 80% in A549 cells. An in vivo mouse study with syngeneic orthotopic Lewis lung carcinoma (LL/2-Luc2) tumors is underway to validate the effective knocking-down of PD-L1 and its effect on tumor inhibition and immune markers.

Conclusion: In summary, our tumor targeted EPM technology loaded with an immunomodulatory target gene represents a novel nanoplatform for delivery of siRNA therapeutics.

PhD Chih-Ming Pan, MS Yu-Ting Liao, PhD Shao-Chih Chiu

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

The chemotherapeutic drug has been commonly prescribed for patients with Glioblastoma (GBM), but drug resistance is one of the main reasons for failure in treating GBM. Previously, MSI1 overexpression has been observed in GBM tissues and is considered a well-established marker for tumor metastasis and recurrence, and needs to be removed. Here, we try to use engineered exosomes (EXO) as the drug delivery system and treat tumor cells with the combination of chemotherapeutic drugs and siRNA of the MSI1 gene to suppress the tumor progression. We designed the anti-HLA-G-EXO which be exploited to simultaneously deliver an anticancer drug and MSI1 inhibitor oligonucleotide (siMSI1) to HLA-G-expressing cancer cells. We establish the method of isolation and characterization of exosomes and develop the manufacturing procedure of exosome formulations of siMSI1 and chemo-therapeutics. Using imaging analysis and flow cytometry, we found that fluorescent-labeled anti-EXO was significantly more uptake by cancer cells than without anti-HLA-G expression, and caused a stronger cytotoxic effect. We evaluated the anti-tumor therapeutic efficacy of drug-loaded anti-HLA-G EXO in a xenograft GBM mouse model and confirmed the presence of anti-HLA-G-EXO in the GBM tumor of mice in a biodistribution study. The strategy for co-delivering the functional siRNA and anticancer drug by exosomes foreshadows a potential approach to reverse the drug resistance in GBM and thus enhance the efficacy of the cancer treatment.

PhD student Trinh Tran, Doctor Dai Phung, Brendon Yeo, Rebecca Prajogo, Migara Jayasinghe, Yuan Ju, Eric Yeo, Doctor Boon Cher Goh, Doctor Wai Leong Tam, Doctor Minh Le

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Antisense oligonucleotides (ASOs) can be designed to match individuals' genetic alterations and increase the likelihood of therapeutic success while minimizing dose-related side effects and unnecessary treatments. However, it is challenging to deliver ASOs to the right tissues for specific therapeutic purposes. This challenge can be significantly mitigated through the utilization of extracellular vesicles.

Methods

In this study, we employed red blood cell extracellular vesicles to deliver ASOs, strategically designed to target activating mutations inherent to non-small cell lung cancer (NSCLC) in a specific and individualized manner.

Results

Our data demonstrate the efficiency of RBCEVs as carriers for delivering ASOs to cancer cells, where they effectively inhibit the expression of their targets. Specifically guided by the mutational profile identified in tumor cells obtained from NSCLC patients, we have successfully developed ASOs that selectively inhibit EGFR point mutations, including L858R and T790M, while sparing the wild-type EGFR gene. Through empirical comparison, cancer cells harboring the T790M gatekeeper mutation, which displayed reduced responses to standard TKIs, were effectively restrained in their growth by the mutation-specific ASOs. Furthermore, we have demonstrated the remarkable ability of ASO-loaded EVs to suppress tumor cell growth in mouse models of cell line xenografts and patient-derived xenografts.

Conclusion

Overall, our findings present an effective and adaptable therapeutic platform in which RBCEVs serve as a potential carrier for RNA drugs in the treatment of NSCLC. By harnessing the principles of personalized and precision medicine, this approach holds the potential to advance NSCLC treatment strategies.

Ms Sua Kim, Dr. Dokyung Jung, Professor Moon-Chang Baek

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Immune checkpoint blockade (ICB) therapy has shown promising effects in cancer immunotherapy by blocking immune checkpoints such as programmed cell death-1 (PD-1) / programmed cell death ligand-1 (PD-L1). However, patients with various types of cancer are resistant to ICB therapy due to several factors, such as the immunosuppressive tumor microenvironment (TME). Tumor-derived extracellular vesicles (EVs) contribute to TME immunosuppression and EV PD-L1 promote immunosuppression by regulating the response to anti-PD-1/PD-L1 therapy. Although various strategies to inhibit EV PD-L1 have been studied, we investigated a new strategy for predictive medicine. The number of breast cancer patients expressing an immune checkpoint called B7-H4 is approximately four times the number of patients expressing PD-L1, and the expression of PD-L1 and B7-H4 in breast cancer patients is negatively correlated with each other. B7-H4 is part of the B7 family of ligands and inhibits T cell activity by binding to the B7-H4 receptor present on the surface of T cells. Additionally, EV B7-H4 promotes tumor growth by suppressing anti-cancer immunity. Here, we discovered that an FDA-approved endothelin receptor A (ETA) antagonist inhibits not only EV B7-H4 but also cellular B7-H4. Moreover, we confirmed that ETA antagonist regulates B7-H4 independently of ETA-mediated signaling. Finally, we found that ETA antagonist represses B7-H4 expression by regulating post-translational modification (PTM). Collectively, we provided a new aspect of the anti-cancer effect of ETA antagonist in addition to its function to inhibit EV PD-L1. Therefore, we expect that this drug will have great potential in anti-cancer treatment for breast cancer patients widely.

Liang Dong, QI Chen

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introdution&objectives

Due to the low immune cell infiltration and the suppressive tumor immune microenvironment (TIME), the efficacy of immunotherapy for prostate cancer is currently quite limited.

This study aims to design a cancer vaccine using supramolecular peptide hydrogel encapsulated with ex vivo tumor tissue-derived exosomes (TEXs) and adjuvants to inhibit the growth and recurrence of prostate cancer, as well as improve the effect of immune checkpoint inhibitors.

Materials&methods

Here, we design an amphiphilic self-assembling peptide, KKFE8, capable of forming hydrogels in-situ after subcutaneous injection. TEXs are extracted through the lysis and ultracentrifugation of tumor tissue collected from prostate cancer patients. The positively charged self-assembler KKFE8 can adsorb negatively charged TEXs, CDA (a STING agonist), and GM-CSF through electrostatic interactions to create the Gel-Vaccine system.We then assess the therapeutic effects and related molecular mechanisms of gel vaccines in different mouse models of prostate cancer.

Results

Significant tumor regression was observed in mice vaccinated with the Gel-Vaccine across multiple mouse models of prostate cancer. This effect was even more pronounced in mice treated with the combination of aPD-1. We detected a higher number of DC aggregates at the site of gel vaccination, with an increased presence of mature and migratory DCs. Furthermore, we observed an elevated count of mature DCs, along with an increased presence of CD4+ and CD8+ T lymphocytes in the lymph nodes. Additionally, a greater number of CD8+ T cells with cytotoxic capabilities and fewer regulatory T cells infiltrates were detected within the tumor tissue.

Conclusion

TEXs are enriched with tumor antigens, making them potential stimulators of anti-tumor immune responses. After subcutaneous inoculation, the exosome-gel scaffold can be triggered in situ to form an artificial secondary lymphoid organ, continuously recruiting antigen-presenting cells through cytokines and achieving highly efficient antigen processing induced by adjuvants. This orchestrated process induces immune infiltration in the TIME. When combined with aPD-1 immunotherapy, it triggers a potent and long-lasting tumor-specific immune response, effectively inhibiting prostate cancer growth and recurrence.

This innovative approach presents a novel strategy for the future treatment of prostate cancer: tailoring gel vaccines based on individual tumor-derived exosomes, thereby creating individualized therapies.

Dr. Shi-Wei Huang, Dr. Mei-chih Chen, Dr. Yu-Chuan Lin, Dr. Chih-Ming Pan, Dr. Chung-Chun Wu, Miss Chen-Yu Lin, Miss Pei-Ying Lin, Miss Yu-Ting Chiang, Miss Yu-Han Huang, Miss Wan-Yu Mao, Miss Steffany Rusli, Professor Shao-Chih Chiu, Professor Der-Yang Cho

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

The successful benefits of chimeric antigen receptor-T (CAR-T) cells therapies have been achieved in patients with blood cancers. However, there are restrictions on utilizing ex vivo CAR-T in the treatment of solid tumors. The in vivo generation of CAR-T has been suggested as a potential strategy to overcome the barriers of ex vivo CAR-T therapeutics. Accordingly, the purpose of this study is to develop a CAR transgene delivery system using T cell-directed exosomes to generate in vivo CAR-T for the treatment of solid tumors.

2) Methods

HEK-293 cells were pre-engineered to produce CD3ε Nb harboring exosomes (Exo). The CD3ε Nb-Exo produced from HEK-293 cells were purified through VHH-affinity resin, and subsequently loaded with a nanobody-based CAR with secreting BiTE (Nb-CAR.BiTE) DNA transgene via electroporation. The transfection rate of the Nb-CAR.BiTE transgene loaded in Exo to T cells was determined using human PBMCs and whole blood in vitro, and PBMC-humanized mice in vivo. The effectiveness of anti-tumor activity of Nb-CAR.BiTE transgene-loaded exosomes was assessed through a co-culture system containing transfected PBMCs and various types of solid tumor cell lines. The efficacy of employing exosomes to generate in vivo Nb-CAR.BiTE T cells for the treatment of solid tumors were evaluated, by performing PBMC-humanized mice with xenografted human colon or pancreatic tumors.

3) Results

The CAR transgene-loaded CD3ε Nb-Exosomes selectively transfect CAR into CD3+ cells, resulting in effective outcomes against solid tumors both in vitro and in vivo. Mechanistically, CD4+ cells played a vital role, while CD8+ cells and CD3- cells contributed to a minor effect in the enhanced anti-tumor activity facilitated by the engineered exosomes. The CD3-targeted strategy enhanced the capacity for memory CAR-T formation, correlating with prolonged immune surveillance to eliminate the same tumor cells upon rechallenge.

4) Summary

Our findings suggest that CAR-T cells can be generated and sustained by CD3-targeted exosomes, representing a potential off-the-shelf immunotherapy for treating malignancies.

Graduate student Jihoon Han, Graduate student Yeongha Hwang

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) introduction

Cancer vaccines offer a promising avenue in cancer immunotherapy by inducing systemic, tumor-specific immune responses. Tumor extracellular vesicles (TEVs) are nanoparticles naturally laden with tumor antigens, making them appealing for vaccine development. However, their inherent malignant properties from original tumor cells limit their direct therapeutic use. This study introduces a novel approach to repurpose TEVs as potent personalized cancer vaccines. We show that inhibition of both YAP and autophagy not only diminishes the malignancy-associated traits of TEVs but also enhances their immunogenic attributes by enriching their load of tumor antigens and adjuvants.

2) Method

TEV preparation and isolation

When the cancer cells reached 80–90% confluence, they were incubated in serum-free media containing verteporfin. Cell supernatants underwent a series of centrifugation steps to eliminate microvesicles and cellular debris. Subsequently, the resultant supernatants were filtered through a 0.22-µm pore filter dialyzed with PBS via tangential flow filtration using a filter, and then centrifuged at 150,000 × g for 2 h. The pellets were reconstituted in PBS and preserved at 4°C.

TEV characterization and In vitro function test

The size and number of TEVs were measured using ZetaView. TEVs were visualized through the use of cryogenic transmission electron microscopy. In vitro TEV functions were verified by western blot, dsDNA quantification assay, calreticulin analysis, proteomics, transwell assay, and dendritic cell maturation test.

In Vivo Tumor Models

Therapeutic vaccination model, prophylactic vaccination model, tumor recurrence model, and personalized cancer vaccine model were established by subcutaneously inoculating E.G7-OVA tumor cells into the left flank of C57BL/6 mice. Five days after inoculation, TEVs were subcutaneously injected adjacent to the tumor inoculation site or on the opposite site.

3) Results

These revamped TEVs, termed attenuated yet immunogenically potentiated TEVs (AI-TEVs), showcase potential in inhibiting tumor growth, both as a preventive measure and a possible treatment for recurrent cancers. They prompt a tumor-specific and enduring immune memory.

4) Summary/Conclusion

In summary, we have developed an improved form of TEVs as a cancer vaccine that can elicit robust tumor-specific responses and effectively suppress cancer growth in prophylactic as well as post-surgical settings.

Yang Lu, Songbo Qiu, Professor Zhen Fan

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

The use of vaccines to eliminate infectious diseases is one of the greatest achievements in human history; however, the dream of using vaccines to prevent or treat human cancer remains largely unfulfilled. We hypothesized that pre-existing anti-viral immunity acquired through natural viral infection or through vaccination can be redirected to cancer cells if infectious virus-related antigens can be specifically delivered to targeted tumors and if the viral antigens can be subsequently presented on cancer cell surface in complex with major histocompatibility complex class I (MHC-I). To test this hypothesis, we developed tumor-targeting extracellular vesicles (EVs) for delivery of MHC-I-compatible peptides to tumors. We engineered a murine cell model (in C57BL/6 background) to express cell membrane-anchored single chain variable fragment (scFv) antibody against human epidermal growth factor receptor-2 (HER2). EVs released by the engineered cells can specifically target cancer cells expressing HER2. In our proof-of-concept experiments, an H2-Kb-compatible ovalbumin (OVA) peptide (257-264), SIINFEKL, was loaded onto the EVs via direct pulsing in an H2-Kb-restricted manner. Cell treatment with SIINFEKL-loaded EVs led to detection of SIINFEKL presentation in several murine tumor cell lines of C57BL/6 background (MC38 murine colon tumor cells and E0771 mammary tumor cells) but not in murine tumor cells of Balb/c background (4T1 murine mammary tumor cells). The SIINFEKL-loaded EVs activated T cells from OT-1 mice that express a transgenic T cell receptor recognizing the 8-mer SIINFEKL peptide. By contrast, the SIINFEKL-loaded EVs did not activate naïve T cells from C57BL/6 wildtype mice. Control EVs without loading of SIINFEKL peptide did not activate OT-1 T cells. Co-culture of OT-1 T cells with MC38/HER2 cells induced apoptosis (detected by annexin-v apoptosis flow cytometry) only after treatment with SIINFEKL-loaded EVs. In summary, tumor-targeting EVs pulsed with MHC-I-compatible peptides can be used to redirect pre-existing noncancer T cell-mediated immunity to cancer cells through delivery of MHC-I-compatible peptides to the targeted cancer cells. We are currently expanding our studies using the EVs to deliver influenza-related H2-Kb peptides to murine tumors expressing human HER2 in a human HER2-immunotolerant transgenic mouse model following vaccination with influenza virus as a novel type of cancer immunotherapy.

Dr. Sung-Min Kang, Dr. Dokyung Jung, Ms Soojeong Noh, Ms Sanghee Shin, Ms Minju Kim, Professor Byungheon Lee, Professor Kyungmoo Yea, Professor Moon-Chang Baek

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Natural killer (NK) cell-derived small extracellular vesicles (sEVs) exhibit inherent anti-tumor activity and offer the advantages of cell-free therapy. Here, our aim was to genetically engineer NK-sEVs to express the anti-tumor cytokine interleukin 15 (IL15) and the monoclonal antibody cetuximab on their surface, creating a potent anti-tumor immunotherapy with tumor-targeting capabilities. We generated IL15 and cetuximab-tethered NK-sEVs (eEVs) through lentivirus-based modification. The anti-tumor effect of eEVs was evaluated by analyzing their direct modulation of cancer cell viability and measuring their indirect enhancement of NK cell-mediated cytotoxicity. The targeting ability of eEVs was assessed through explorations of tumor-specific targeting in established tumor models. Additionally, we conducted a comprehensive evaluation of the anti-tumor efficacy of eEVs in a mouse lung tumor model, which also included investigating the potential synergistic effects of combining eEVs with anti-PD-1 antibody (αPD-1 Ab). We observed that eEVs, obtained through the engineering of NK cells, demonstrated significantly enhanced cytotoxicity against lung cancer cells compared to control NK-sEVs. The stimulation of NK cells by eEVs resulted in an increased immune cell-mediated anti-tumor efficacy. The eEVs exhibited specific targeting abilities against cancer cells in the mouse model. Furthermore, treatment of eEVs alone or in combination with the immune checkpoint inhibitor drug αPD-1 Ab effectively reduced tumor growth and size in the lung tumor mouse model. Overall, our study highlights NK-sEVs engineered through genetic modification exhibit potent anti-tumor efficacy and tumor-targeting capabilities, demonstrating potential as a novel immunotherapeutic strategy.

Ms Seong A Kim, Ms Yeji Lee, Dr. In-San Kim

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: The therapeutic landscape of colorectal cancer (CRC) is influenced by microsatellite instability (MSI) and its implications in treatment response. MSI, resulting from impaired DNA mismatch repair (MMR), is characterized by the accumulation of insertions or deletions in microsatellites of DNA. High levels of MSI (MSI-H) in CRC correlate with an enhanced response to cancer therapy, whereas microsatellite stable (MSS) tumors typically show a limited response, even to oxaliplatin, a first-line drug for CRC. Here, we suggested a therapeutic strategy for therapy-resistant MSS-CRC to make it MSI-H for a better treatment response. Synthetic immunogenicity refers to the induction of immunogenicity, where a combination of two events results in immunogenic cell death of cancer cell; Induction of cell toxicity and disruption of the cell repair mechanism.

Method: Considering the resistance developed after oxaliplatin treatment is derived from the efficient repair system of CRC, we disrupted MLH-1, an MMR system component of CRC, using siRNA, following the oxaliplatin treatment to induce synthetic immunogenicity. We further hypothesized that synthetic immunogenicity induced unrepairable DNA damage and this damaged DNA can be delivered to extracellular vesicles (EVs) of CRC, which can increase the stemness of T cells in tumor microenvironment. To test this hypothesis, we prepared EV from CRC, receiving synthetic immunogenicity, following methods in accordance with the MISEV2018 guidelines.

Results: We first observed that MLH-1 was highly expressed in MSS-CRC compared to MSI-H-CRC cell lines, indicating their DNA repair efficiency that could develop chemotherapy resistance. This was successfully inhibited by siMLH-1 treatment. By inducing synthetic immunogenicity, we observed an accumulation of cytosolic DNA. Furthermore, we found that inducing synthetic immunogenicity in CRC increased the total DNA amounts in EVs secreted from CRCs in vitro. We also observed that this DNA-accumulated EVs increased the stemness and ameliorate the exhaustion of CD8 T cells. In vivo studies revealed that these EVs elicited a potent antitumor effect in murine CRC model.

Conclusion: We demonstrates that the induction of synthetic immunogenicity in MSS-CRC leads to the delivery of cancer DNA via EVs to CD8 T cells, thereby enhancing T cell stemness and enhancing the antitumor immune response.

Dr. Jacob Orme, Henan Zhang, Prashanth Lingamaneni, Yohan Kim, Roxane Lavoie, Jacob Hirdler, Elizabeth Bering, Joanina Gicobi, Heather Dale, Lisa A Kotschade, Matthew S. Block, Svetomir N. Markovic, Haidong Dong, Fabrice Lucien, Annie T. Packard, Jeffrey L. Winters, Sean S. Park

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Immune checkpoint inhibitors (ICI) are the most important systemic therapy for melanoma. However, two thirds of melanomas respond develop resistance to ICI. Tumor-derived extracellular vesicles (EVs) expressing PD-L1 (evPD-L1) drive checkpoint inhibitor resistance and correlate with inferior survival (PMID 30089911). We previously showed that therapeutic plasma exchange (TPE) removes immunosuppressive evPD-L1 (PMID 32817395). Thus, we hypothesized that TPE-mediated removal of evPD-L1 could overcome immunotherapy resistance in refractory melanoma.

Methods: In this clinical trial (NCT04581382), we prospectively enrolled 17 patients with metastatic melanoma with progression despite anti-PD-1/PD-L1 immunotherapy (PMID 35551087). Patients were selected based on elevated plasma PD-L1 levels by ELISA (≥1.7ng/mL). Each patient received radiotherapy to at least one—but not all—metastatic lesions followed by three consecutive days of TPE and continued checkpoint inhibitor. The primary endpoint of the study was to evaluate safety. Secondary endpoints included RECIST-based response and PD-L1 kinetics.

Results: Mean age of patients in the study was 62 (SD 13). 11 (65%) were male. Mean baseline sPD-L1 was 25.46ng/mL. Mean evPD-L1 was 7.99x106 per mL (total 3.2x1010 particles). The treatment was well-tolerated with one (6%) grade 3 adverse event from a line infection after TPE. Levels of PD-L1 were significantly reduced by TPE (mean 78% reduction, p = 0.0002). Two patients (12%) experienced complete response (CR), one (6%) partial response, two (12%) stable disease, and 12 (70%) progressive disease. In one case, immunotherapy was discontinued after two years due to no detectable lesions.

Summary/Conclusion: TPE appears to be a reliable method for reducing PD-L1 from circulation. Consistent TPE may re-sensitize cancers to immunotherapy, but future clinical trials of serial TPE are needed.

MS course Na-Eun Kim, Dr. Dokyung Jung, Professor Moon-Chang Baek

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Cancer cells exhibit increased expression of immune checkpoint proteins as a strategy for immune evasion, with CD47 emerging as a prominent anti-phagocytic signal that attenuates innate immunity within the tumor microenvironment. Ongoing research aims to target CD47, either through blocking or downregulating its expression. However, anti-CD47 antibody therapy has not yet been applied to patients due to reported hematopoietic side effects, such as severe anemia. Recognizing the need to find alternative CD47 inhibitors without adverse effects, we happened to identify vitamin B as a potent CD47 inhibitor. Vitamin B is one of the essential nutrients, but its relationship with CD47 is not fully understood. Our findings demonstrate that vitamin B not only suppresses CD47 expression in extracellular vesicles (EVs) but also significantly enhances the phagocytosis of M1 macrophages following the pre-treatment of cancer cells with vitamin B. As a result, vitamin B emerges as a promising therapeutic agent capable of enhancing innate immune responses by inhibiting CD47 expression in both cellular and EVs. Especially, we propose the potential for a synergistic effect when combining vitamin B with other immune checkpoint blockade therapies.

Associate Professor Han Liu

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Osteoporosis (OP) is a systematic bone degenerative disease characterized by low bone mass and fragile bone microarchitecture. Traditional treatment for OP such as systematic drug administration has limited efficacy and long-term toxicity. Advances in synthetic biology make bacterial extracellular vesicle (BEVs)-based therapeutic strategies a promising alternative for the treatment of OP. Here, we constructed a recombinant probiotics Escherichia coli Nissle 1917-pET28a-ClyA-BMP-2-CXCR4 (ECN-pClyA-BMP-2-CXCR4), in which BMP-2 and CXCR4 were overexpressed in fusion with BEVs surface protein ClyA. Subsequently, we extracted engineered BEVs-BMP-2-CXCR4 (BEVs-BC) for highly effective treatment of OP. The engineered BEVs-BC exhibit good bone targeting in vivo. In addition, BEVs-BC has good biocompatibility and remarkable ability to promote osteogenic differentiation of BMSCs. Finally, the synthetic biology-based BEVs-BC significantly prevented the OP in an ovariectomized (OVX) mouse model. In a word, we constructed BEVs-BC with both bone-targeting and bone-forming in one-step using synthetic biology, which provides an effective strategy for OP and has great potential for industrialization.

Dr. Rostyslav Horbay, Daniel Panting, Michaela van der Meerwe, Maria Dimancheva, Dr Eric LaCasse, Dylan Burger, Dr Shawn Beug

¹Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and University of Ottawa, 401 Smyth Rd, Ottawa, Ontario, K1H 8L1, Canada, Ottawa, Canada, ²Kidney Research Centre, The Ottawa Hospital Research Institute and University of Ottawa, 401 Smyth Rd, Ottawa, ON, K1H 8L1, Canada,

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction. We have shown that SMAC mimetics (SMCs), a class of drugs that antagonize members of the Inhibitor of Apoptosis protein family, synergize with immune ligands and immunostimulatory agents to eradicate tumors. We have discovered that this synergy can be attributed to SMC-mediated release of 30-150 nm small extracellular vesicles (sEV) that carry inflammatory cytokines from immune and cancer cells.

Methods. sEVs were isolated by differential ultracentrifugation and were validated per the latest ISEV recommendations via Zetaview NTA, ONI Nanoimager, and Western blotting. We used cell culture approaches to decipher the impact of SMC-induced sEVs on cancer and immune cells, including viability assays, direct and transwell co-culture conditions, ELISA, and flow cytometry.

Results. The use of SMCs increases the rate of sEV release in immune cells by ∼5X (t-test, P<0.05, n = 4), but does not significantly alter the secretion rate in cancer cells). SMC treatment on immune cells leads to packaging of proinflammatory cytokine into sEVs, particularly TNFα. The packaging of TNFα into immune sEVs is an ESCRT-dependent process as reflected by the presence of Syntenin and Alix via Wester blotting, but tumor cell-derived sEVs were missing these key ESCRT proteins. Both immune and cancer cell-derived sEVs were positive for the exosomal markers Flotillin-1 and HSC70. The resultant TNFα-bearing immune-cell derived sEVs induce the death of cancer cells in the presence of a SMC (ANOVA, n = 4, P<0.0001). Interestingly, Syntenin-targeting drugs increased the effects of SMCs to induce death of cancer cells (67% SMC only compared to 14% of the combination, n = 2), but not the death of immune cells. Furthermore, the SMC-stimulated exosome release led to packaging of other inflammatory mediators within immune cell-derived sEVs, which can polarize macrophages into the pro-inflammatory M1 subset (from a baseline level of ∼4% to ∼15%, n = 2). Lastly, we observed packaging of RIG-I within tumor-derived sEVs from SMC-treated cancer cells. This finding implicates that SMCs can stimulate innate immune cells to be in a pro-inflammatory state.

Conclusion. Our data demonstrates that SMC treatment increases cytokine-carrying sEV release from immune cells, which then eradicates cancer cells in a therapeutic favorable manner.

Lucas Walther³, Jacky Goetz¹, Karola Rittner², Dr Vincent Hyenne¹

¹INSERM U1109, Strasbourg, France, ²Transgene SA, Illkirch-Graffenstaden, France, ³INSERM U1109 and Transgene SA, Strasbourg, France

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Virus-based therapies bear promising potential in various pathologies, in particular in cancer where they can be used as oncolytic or immunotherapeutic vectors. We hypothesize that immune cells infected with therapeutic virus release modified EVs with anti-tumor immunomodulatory potential. To test this hypothesis, we studied how armed poxvirus affect EVs secretion from human PBMCs and whether those EVs could improve or mediate anti-tumor potential of such viruses in vivo.

Methods: Because poxviruses are structurally and functionally extremely close to extracellular vesicles (EVs), we first compared six EVs isolation methods and found that only 0.1um filtration (combined with SEC or ultracentrifugation) reliably allowed to segregate poxvirus from small EVs (characterized by NTA, cryo EM, cytometry and mass spectrometry) secreted by immune cells (PBMCs) infected by therapeutic virus. We used this method to distinguish them from EEVs or viral-like particles and to study the content and function of EVs.

Results: We observed that viral infection with 3 different strains of poxvirus significantly stimulates the secretion of small EV secretion in addition to modifying their content, characterized by M/S proteomics. In particular, enrichment of actin-related proteins in EVs secreted from infected cells suggests that the actin cytoskeleton could contribute to virus-induced EV secretion. We further showed that EVs isolated from infected cells contain viral proteins and viral encoded therapeutic payloads, suggesting that they could contribute to viral therapies. We thus tested the function of virus-induced EVs in an EG7-OVA mouse lymphoma model in vivo and found that EVs isolated from infected cells similarly reduce tumor growth when compared to mice treated with therapeutic virus only. Current work aims at validating such effect in additional mouse tumor model while characterizing the immunomodulatory role of those EVs.

Summary/conclusion: Our results show that virus-induced EVs contribute to anti-tumoral therapy response. We are currently manipulating EV secretion levels in order to improve therapy efficiency.

Dr Kartini Asari¹, Siena Barton¹, Sadman Bhuiyan¹, Kol Thida Mom¹, Amirah Fitri¹, Dr Mozhgan Shojaee¹, Dr Carlos Palma¹, Dr Sara Nikseresht¹, Dr Ramin Khanabdali¹, Professor Gregory Rice^1,2

¹INOVIQ Ltd, Notting Hill, Australia, ²UQ Centre for Clinical Research, Herston, Brisbane City, Australia

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Chimeric antigen receptor (CAR)-T cell immunotherapies offer exciting opportunities to improve clinical outcomes in the treatment of cancers resistant to frontline therapies, due to their exquisite specificity. While FDA-approved for the treatment of an array of intractable hematological malignancies, there remains considerable challenges including solid tumor infiltration and risk of cytokine release syndrome (CRS) toxicity. These safety considerations suggest that refinement of therapeutic approaches is warranted. To date, there is a paucity of data on the application of CAR-T cell-derived EVs on cancer cells. This study aimed to test the hypothesis that CAR-T derived EVs are a less immunogenic and safer treatment approach as an alternative to cell-based therapy.

Methods: CAR-T engineered cells were cultured in xeno-free conditions and conditioned medium (CM) was collected via stepwise centrifugation to remove cells and debris. Engineered EVs (EEVs) were isolated and enriched from CM via ion-exchange chromatography to ensure maximum EV recovery. A terminal 0.22 µm sterile-filtration step was performed to ensure sample sterility, and nanoparticle tracking analysis (NTA) applied to determine particle size and concentration. MCF-7 breast adenocarcinoma and K-562 chronic myeloid leukemia cells were treated with increasing concentrations of EEVs in growth and starvation conditions for up to 3 days, prior to determination of efficacy via MTS assay.

Results: While both breast and blood cancer cell lines exhibited consistency in treatment response when treated in either growth conditions or starvation conditions, their response to culture conditions differed. Exposure of breast cancer cells to EEVs resulted in a significant decrease in cell viability under growth conditions at Day 3 (p ≤ 0.05), whereas, in blood cancer cells, a significant decrease was observed when cells were treated under starvation conditions at Day 2 (p ≤ 0.01).

Summary/Conclusion: Our preliminary findings have shown that EEVs enriched from CAR-T cells reduce cell growth in breast and blood cancer cells in vitro. These findings highlight that use of EEVs is a viable refinement of CAR-T therapy, and further upscaling of EEVs from such sources will enable development of a safer method of eradicating cancer cells using this promising technology.

Phd Qianbei Li, Professor Lei Zheng

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Inflammatory bowel disease (IBD) is a chronic and multifaceted group of intestinal disorders characterized by intestinal barrier dysfunction. Restoration of intestinal barrier function is crucial in the treatment for IBD. While probiotic-based therapies have shown promise in repairing the intestinal barrier, concerns about potential infections and limited understanding of their underlying mechanisms necessitate the exploration of alternative therapies. In this study, we demonstrated the role of Lactobacillus plantarum (L. plantarum) and the effect of watermelon diet on intestinal barrier function in IBD. We observed a significantly lower abundance of L. plantarum in the intestines of patients with IBD. Notably, L. plantarum exhibited protective effects on intestinal epithelial cells by releasing bacterial extracellular vesicles (L. plant-EVs), thereby inhibiting DSS-induced apoptosis of intestinal epithelial cells through modulation of the mPTP-CytC-Caspase-9-Caspase-3 axis. Meanwhile, we confirmed that watermelon diet could ameliorate intestinal dysfunction by effectively up-regulating the abundance of L. plantarum in human intestine. In addition, watermelon could ameliorate intestinal dysfunction relying on intestinal flora in mice and increase the levels of L. plant-EVs in bacterial culture supernatants. Overall, our findings provide novel insights into the mechanism of intestinal probiotics and present a new therapeutic strategy based on fruits diet for maintaining intestinal barrier function.

Deborah Brandt Almeida, Ms Jenicer Kazumi Umada Yokoyama Yasunaka, Doctor Verônica Feijoli Santiago, Doctor Simon Ngao Mule, Ms Paula Menegheti, Doctor Giuseppe Palmisano, Doctor Ana Claudia Torrecilhas, Doctor Mauro Cortez

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

INTRODUCTION: Leishmania amazonensis manipulate the host immune response to survive, and cause leishmaniasis. Amastigotes release extracellular vesicles (Evs), which participate in inducing CD200 from infected macrophages, ligand that inhibits cellular activation, and the inducible nitric oxide synthase/nitric oxide (iNOS/NO) mechanism. OBJECTIVE: We explored the content of these structures and whether the release of amastigote-Evs is affected by different pH conditions or Ca2+ inhibitors. MATERIALS AND METHODS: Leishmania amastigotes were incubated in axenic amastigote media (AA) for vesiculation by 1h and the supernatants were filtered through a 0.45-mm sterile cell strainer, concentrated and used for different assays: Proteomics assays (nano LC-MS/MS). Amastigotes were also incubated in PBS, RPMI or AA, prepared at different pH. BMM stimulation assays for CD200 induction following Immuno-precipitation/ western blot (IP/WB) or then analyzed by SDS-PAGE silver staining and WB assays using a polyclonal antibody against Leishmania-Evs. Nanoparticle tracking analysis (NTA) was also included to verify the purity and quantity of Evs. RESULTS: Proteomic analysis shows 25 proteins shared in the replicates, obtaining a total of 56 on the replicate 1 and 63 from the replicate 2. Most of the shared proteins were considered to have catalytic activity (molecular function) and localized in the nucleus (cellular component analysis). When amastigotes were incubated in different culture media, the induction of CD200 is affected when pH is higher than 5.2. Amastigotes incubated in different pH (4.5, 5.2, 7.0, and 8.0) showed an impairment of Evs release at lower pH (4.5), with a more dramatic effect at higher pH (7 or 8). Regarding the role of Ca2+, Evs´s quantity and profile size were affected when the inhibitors EDTA, EGTA, and BAPTA-AM were included. On the three conditions, the quantities of Evs analyzed by NTA were lower, with a more dramatic effect visualized with EDTA. Moreover, the Evs profile in the control, showing more concentrated EVs ranging from 100 to 200 nm, changed with the different treatments, suggesting an essential role of Ca2+ during amastigote -Evs release. CONCLUSION: pH and Ca2+ are crucial for amastigote-Evs release and highlight the unique factors that must be considered in studying parasite Evs.

Assistant Professor Yumi Kumagai^1,2, Special Appointed Professor Isao Nagaoka^1,3, Professor Etsuo Susaki¹

¹Dept. of Biochemistry and Systems Biomedicine, Graduate School of Medicine, Juntendo University, Bunkyo-ku, Japan, ²Biomedicine Research Core Facility, Graduate School of Medicine, Juntendo University, Bunkyo-ku, Japan, ³Faculty of Medical Science, Urayasu, Japan

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

Extracellular vesicles (EVs), secreted in response to microbial infection, play a crucial role in modulating immune and infectious responses. Sepsis, a life-threatening multiple organ dysfunction caused by a systemic dysregulated inflammatory response to infection, poses significant challenges in therapeutic interventions. Despite extensive trials, achieving satisfactory therapeutic outcomes remains elusive. Our prior research unveiled the potential of LL-37, a human cathelicidin host-defense peptide, in improving the survival rates of septic mice subjected to cecal ligation and puncture (CLP). In this study, we explore the capacity of LL-37 to modulate neutrophils, the primary defender against pathogens, focusing on its ability to enhance the secretion of anti-inflammatory EVs and promote cell motility.

2) Methods

EVs isolated from the peritoneal exudates of CLP mice and the supernatant of LL-37-stimulated mouse bone marrow neutrophils were subjected to analyses through flow cytometry, interferometric microscopy, and Western blotting. The antibacterial efficacy of the EVs was evaluated by incubating them with Escherichia coli isolated from mouse cecum. Furthermore, the administration of neutrophil-derived EVs to CLP mice was followed by histological assessment of tissue samples using hematoxylin and eosin staining. Neutrophils were transfected with an EGFP-encoded plasmid via electroporation, and their motility was monitored through 3D imaging with a time-lapse function using a Lattice light sheet microscope.

3) Results

Administration of LL-37 to CLP mice resulted in enhanced neutrophil infiltration and EV release at the site of inflammation, within the peritoneal cavity. In vitro stimulation of neutrophils with LL-37 led to elevated secretion of EVs (LL-37-PMN-EV) containing antimicrobial molecules such as CRAMP and lactoferrin, consistent with their observed antibacterial activity. Injection of LL-37-PMN-EV into CLP mice significantly improved survival rates and reduced bacterial burdens. Notably, lung tissues from LL-37-PMN-EV-injected mice exhibited reduced inflammation. 3D imaging with a time-lapse function revealed heightened neutrophil motility following LL-37 treatment.

4) Summary/Conclusion

LL-37 orchestrates the chemotaxis of neutrophils to inflammatory sites and promotes the secretion of anti-bacterial/anti-inflammatory EVs in septic mice, thereby attenuating bacterial loads and conferring protection against lethal septic conditions.

Dr Sin-Yee Fung¹, Kam-Leung Siu¹, Man Lung Yeung², Prof Judy Wai Ping Yam³, Prof Dong-Yan Jin¹

¹School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong, ²Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong, ³Department of Pathology, The University of Hong Kong, Pokfulam, Hong Kong

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

SARS-CoV-2 is the causative agent of COVID-19. Severe COVID-19 is characterized by exacerbated release of proinflammatory cytokines interleukin (IL)-1β and IL-6. SARS-CoV-2 accessory protein ORF3a is capable of inducing the secretion of IL-1β. Macrophages are thought to be major producers of proinflammatory cytokines. However, it remained debated if macrophages are susceptible to SARS-CoV-2 infection. The relevance of proinflammatory cytokine release in macrophages to the life cycle of SARS-CoV-2 remains to be established. We hypothesized that a certain secretory viral factor might be responsible for proinflammatory response in macrophages.

Methods

Extracellular vesicles (EVs) from ORF3a-overexpressing HEK293T cells were obtained by differential centrifugation. Purified EVs were added to THP-1-derived macrophages to establish the impact of secretory ORF3a on proinflammatory cytokine release.

Results

Existence of ORF3a in EVs were detected by Western blotting and immuno-electron microscopy. Its transfer to neighboring cells was confirmed. Feeding phorbol-12-myristate-13-acetate-differentiated THP-1 cells with ORF3a EVs triggers proinflammatory cytokine release.

Conclusion

Taken together, our demonstration of exosomal ORF3a-mediated proinflammatory cytokine release from macrophages suggests that SARS-CoV-2 can modulate cellular machinery of EV biogenesis to facilitate viral pathogenesis, and also provides a novel strategy for targeted therapy to control cytokine storm in severe COVID-19.

Dr. Xiao Deng¹

¹National Institute For Materials Science, Tsukuba, Japan

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Nanosized outer-membrane vesicles (OMVs) secreted by Gram-negative bacteria are known to play crucial roles in cell physiology and their interactions with the environment. Here, we discovered that OMVs secreted by sedimentary sulfate-reducing bacteria under energy-starving conditions significantly promoted the bacterial capability of utilizing solid electron donors via extracellular electron uptake (EEU). Proteomic analysis of the OMVs secreted under conditions of both organic abundance and scarcity revealed that OMVs from organics-starving conditions contained abundant redox-active cytochromes, previously identified on bacterial surfaces for mediating direct EEU from solid electron donors. Using electrochemical analysis, we compared bacterial EEU rates on electrodes with and without OMV supplementation and found that these cytochrome-containing OMVs highly enhanced bacterial EEU from electrodes acting as the sole electron donor. Importantly, confocal and electron microscopy analyses revealed that the cytochrome-containing OMVs boosted the formation of a thick and electrically conductive biofilm on the electrode surface. In contrast, OMVs secreted under conditions with sufficient organics neither possessed surface cytochrome nor enhanced EEU or biofilm formation on electrodes. These results reveal the essential role of redox-active OMVs in supporting the survival of sedimentary bacteria under energy-limited conditions. Other environmental and industrial implications of our findings will be presented at the conference.

Master Seoah Park¹, Jongsoo Mok², Junghoon Choi¹, Hye-Min Yu³, Hye-Jin An³, Ga-Hyun Choi³, Yeon-Seon Lee³, Ki-Jin Kwon³, Sung-Jun Choi³, Soo-Jin Kim³, Joonghoon Park^1,2

¹Graduate School of International Agricultural Technology, Seoul National University, Korea, ²Institute of Green Bio Science & Technology, Seoul National University, Korea, ³Schofield Biome Research Lab, HK inno.N, Korea

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Bacterial extracellular vesicles (bEVs) hold promise for targeted interventions in human health. In this study, we conducted a comparative characterization of bEVs from multiple bacterial strains to predict and evaluate their potential therapeutic applications.

Methods: We isolated bEVs from 8 bacterial strains (L. rhamnosus, L. plantarum, L. fermentum, L. paracasei, Lc. lactics, L. acidophilus, S. thermophilus, L. salivarius), then characterized them for yield, size, protein and lipid content. We applied the bEV-induced differentially expressed genes (DEGs) to in silico analyses to predict bEV indications. The collagen produced by bEV was measured in NIH3T3 cells, and the protein levels of pSmad3, Hsp47, and Mmp1 were quantified. mRNA levels of collagen producing/degrading genes by bEVs were also measured in HS68 cells.

Results: The bEV size (45 to 82 nm) and yield (0.75 to 3.15 x 109 particles/mL) varied by strains, and the size corresponded to increased cell uptake (p < 0.05). The protein (0.006 to 0.014 pg/particle) and lipid content (1.6 to 16.3 ug/particle) in bEV varied by strains, and the lipid content was associated with the cytotoxicity (p = 0.0371). bEV-induced DEGs were ranged from 1,255 (L. fermentum) to 1,954 genes (L. plantarum), and Connectivity Map and KEGG analyses suggested that bEVs from L. rhamnosus, L. fermentum, and L. acidophilus promoted skin tightening and collagen synthesis via PI3K-Akt activation (p < 4.6 x 10-4) and/or TGFb inhibition (p = 5.4 x 10-4). As predicted, the three bEVs significantly promoted the collagen synthesis in NIH3T3 cells by more than 29% (p < 0.001). In addition, pSmad3 and Hsp47 were increased by more than 1.6-fold (p < 0.05) and Mmp1 decreased by more than 1.6-fold (p < 0.05). In HS68 cells, the three bEVs dose-dependently increased the mRNA levels of COL1A1, COL1A2, or COL1A3 by up to 23% (p < 0.05), and decreased MMP1 by up to 78.6% (p < 0.05).

Conclusion: In this study, we performed comparative analyses of bEVs and presented a systematic methodology for the discovery of putative indications. It holds significant promise for advancing the understanding and clinical applications of bEVs.

Mx Madeleine Rogers¹, Dr Athena Andreosso¹, Dr Jagan Billakanti², Dr Sandip Kamath³, Prof Donald McManus¹, Prof Malcolm Jones¹, Dr Catherine Gordon¹, A/Prof Severine Navarro¹, A/Prof Severine Navarro⁴

¹QIMR Berghofer Medical Research Institute, Herston, Australia, ²Cytiva, Brisbane, Australia, ³Medical University of Vienna, Vienna, Austria, ⁴Centre for Childhood Nutrition Research, Brisbane, Australia

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Food allergy is considered the “second wave” of the allergy epidemic after asthma and allergic rhinitis. The absence of early childhood pathogen stimulation embodied by the Hygiene Hypothesis is one explanation, and the eradication of parasitic helminths could be at play. Parasitic infections with Schistosoma spp. have been found to have a negative correlation with allergic diseases. Schistosomes achieve host immunomodulation through the release of excretory/secretory products such as extracellular vesicles (EVs). Schistosome EV-induced tolerogenic processes are modulated by internal and membrane-bound cargos (protein, microRNA, metabolites, lipids). Research on S. mansoni egg-derived EVs is minimal, while studies on S. japonicum focus the modulatory mechanisms of egg EVs in the liver. Unfortunately, these studies do not follow the MISEV2018 guidelines, making it difficult to confirm the nanoparticles identified are in fact EVs. Finally, the “native” structure of S. mansoni egg EVs has not been characterised.

Methods

We isolated EVs from cultured eggs of S. mansoni using a novel scalable liquid chromatography method. Purified EVs were characterised using cryogenic transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Lipid-proteomic, metabolomic, and microRNA cargo were analysed using mass spectrometry and small RNA sequencing.

Results

We report the discovery of a novel population of globular lipoprotein-like nanoparticles that are distinct from other helminth-derived EVs in their structure, size, and cargo. These nanoparticles (10 - 30 nm) exist in low abundance, are not enclosed by typical membrane structures or have a typical circular shape, and appear to aggregate significantly. Trans-well/culture of S. mansoni eggs and co-culture of egg-derived nanoparticles with gut-relevant pro-inflammatory or pro-tolerogenic DCs resulted in distinct pro-tolerogenic skewing of DC RNA expression.

Conclusions

Current knowledge of S. mansoni egg-derived EVs is limited. This study provides novel insights into the extracellular nanoparticles produced by S. mansoni eggs in culture and suggest they may not produce typical exosome-like nanoparticles like that of its other life stages. By elucidating the molecular mechanisms by which S. mansoni egg EVs may promote tolerance in the gut microenvironment, this work provides an exciting avenue for the identification of novel therapeutic moieties that promote tolerance and treat food allergy.

Professor Flavio Carrion, Dr Soumyalekshmi Nair, Katherin Scholz-Romero, Dr Carlos Palma, Dr Andrew Lai, Dr Dominic Guanzon, Professor Bernardo Morales, Associate Professor Martha Lappas, Professor Carlos Salomon

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (sEVs) play a pivotal role in intercellular communication in both normal physiology and various diseases, including gestational diabetes mellitus (GDM). When EV are isolated from plasma, a heterogeneous population is obtained with diverse origins. While the administration of human EVs to experimental animals has generally been well-tolerated, the effects of circulating EVs derived from human samples and murine immune cells have not been extensively studied. This study aims to assess the impact of circulating EVs from women with normal glucose tolerance (NGT) and GDM on the activation and proliferation of mouse splenocytes.

Methods: Immune cells were isolated from each spleen and labeled with the fluorescent probe CellTraceTM Violet (CTV) at a concentration of 5 µmol/L. CTV-labeled T-cells were cultured with or without Concanavalin A (ConA) at a concentration of 10 µg/ml, along with total circulating EVs (100-µg/ml) isolated from plasma samples obtained from non-pregnant women, NGT, or GDM patients in complete RPMI medium. After 4 days of culture, cells were stained with CD3-FITC, CD25-PE, and LIVE/DEAD™ Fixable Near-IR, and data were acquired using the Navios EX flow cytometer (Beckman Coulter). Data analysis was performed using the Beckman Coulter Kaluza Analysis Software.

Results: EVs were characterized according to the recommendations of ISEV based on size, the abundance of proteins associated with EVs, and morphology, as assessed through nanoparticle tracking analysis, western blots, and electron microscopy (MISEV2018). EVs were positive for CD9, TSG101, and PLAP, confirming the presence of vesicles of placental origin in our preparations. The heterogeneity of placenta-derived EVs was assessed by detecting EVs using fluorescence-tagged tetraspanin and PLAP antibodies. The CD63+/PLAP+, CD63+/CD81+, CD81+/PLAP+, CD9/CD81+, CD9+/PLAP+, and CD81+/CD9+ populations were significantly higher in GDM compared to NGT. ConA increased the activation of CD3+/CD25+ cells, with no significant differences observed in the absence or presence of EVs from non-pregnant, NGT, or GDM sources. Similarly, no differences in cell proliferation were observed in the absence or presence of EVs.

Conclusions: Our findings demonstrate that the administration of human sEVs to mice does not elicit toxic or immunogenic effects, suggesting that sEVs may be well-tolerated when used for therapeutic purposes.

Miss Jordan Fyfe, Dr Danielle Dye, Dr Pat Metharom, Professor Marco Falasca

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Pancreatic cancer (PC) is a lethal malignancy fuelled by aberrant immune interactions. Cells within the PC tumour microenvironment (TME) engage in a complex molecular exchange to form a pro-tumorigenic, immunosuppressive landscape that favours tumour growth. Small extracellular vesicles (sEVs) have been highlighted for their role in this intercellular communication, serving as proxies of the cell in which they are produced. Accordingly, tumour-derived sEVs transfer oncogenic cargoes to surrounding cells, fostering tumorigenic transformation, pre-metastatic niche formation, and immune evasion to promote disease progression. However, the precise mechanisms by which this occurs are yet to be fully understood. Sphingosine-1-phosphate (S1P) is an endogenous bioactive lipid involved in various physiological processes, including cell proliferation, migration, survival, and immune behaviour. In the present study, we investigated whether S1P is involved in sEV-induced immune suppression.

Comprehensive targeted lipidomic analyses were performed using HPLC-ESI-MS/MS on sEVs isolated from non-malignant/normal PC cell lines, patient-derived xenograft (PDX) cell lines, and the plasma of pancreatic cancer transgenic mice (KPC). Immune cells were obtained from the peripheral blood of healthy human volunteers and treated with PC cell-derived sEVs and/or antagonists specific for S1P receptors (S1PR)1-3. Flow cytometry was used to analyse the expression of pro- and anti-inflammatory markers in the treated cell populations.

We have identified that sEVs from malignant – but not healthy – pancreatic cells are enriched in S1P. Our results revealed that PC-derived sEVs induce pro-tumorigenic immune phenotypes by upregulating both pro- and anti-inflammatory markers. Pre-treatment of cells with either S1PR1 or S1PR3 inhibitors did not modify sEV activity. Interestingly, dual inhibition of S1PR1/3 with VPC23019 reduced the expression of anti-inflammatory markers compared to the sEV-only treated cells. Additionally, VPC23019 significantly increased pro-inflammatory marker expression, where selective S1PR1 or S1PR3 inhibition showed no change compared to sEV-treated groups.

Our studies have revealed that S1P is selectively enriched in PC-derived sEVs. We have demonstrated that S1P actively participates in the modulation of immune activity induced by PC sEVs, and that this can be augmented by inhibiting S1PR1/3 signalling. Together, this has provided novel insight into the molecular crosstalk facilitating PC progression and immune dysregulation.

Dr Caitlin Boyne, Mr Jordan Marsh, Dr Sally Shirran, Dr Alan Stewart, Dr Simon Powis

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1. The immunopeptidomes of EV in plasma (the peptides presented on HLA-I molecules), remain relatively unknown. Alterations in the ‘signatures’ of peptides could potentially be used to indicate the onset or presence of disease. However, a signifiant proportion of plasma EV are derived from platelets. We therefore undertook a study to start to map the immunopeptidome of platelet derived EV to use as a reference in future studies.

2. Purified platelets from a healthy donor with known HLA-I typing were activated with thrombin, the EV isolated by size exclusion chromatography, and HLA-I molecules isolated by immunoprecipitation with pan-HLA-I monoclonal antibody W6/32. Peptides released from HLA-I by acidification were analysed and sequenced by mass spectrometry on a Orbitrap instrument.

3. Over 3000 peptides were detected as predicted binders of HLA-I alleles HLA-A*02:01, -A*11:01, -B*07:02 and -B*18:01. Pathway analysis indicated peptides were derived from proteins associated with platelet function and megakaryocytic differentiation.

4. This is the first characterisation of the HLA-I immunopeptidome of a major subset of EV present in plasma. As such it will provide a database of peptides that can be utilised when both global and specific subsets of plasma EV are studied for their immunopeptidomes in both health and disease.

Miss YEJI LEE, Miss Jiyoung Goo, Mr In-San Kim

Poster Pitches (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:45 PM - 1:00 PM

1. Introduction

Due to its unique anatomical location, the liver serves as a crucial defense organ against sepsis through immunoregulation. This process triggers an inflammatory response and aids in the effective removal of microbes. Both Kupffer cells and infiltrating MoMFs are characterized not only as inflammatory cells but also as crucial players in tissue repair. HIF1α has emerged as a pivotal mediator of monocyte reprogramming. Indeed, HIF1α exhibits an increase in expression within MoMFs localized at the lesion site, and Hif1α leads to resolution of necrotic lesions. Activating HIF1α in MoMFs can play an immune regenerative role in sepsis-induced liver injury.

Loading HIF1α into EVs under normoxia is challenging. We have developed a platform capable of efficiently loading HIF1α into EVs ensuring effective functionality as a transcription factor. This strategy has the potential to open avenues for interventions in sepsis-induced liver injury.

2. Methods

EVs were harvested from HEK293T cells and isolated using sequential centrifugation, tangential filtration, and ultracentrifugation methods in accordance with the MISEV2018 guidelines. We induced a sepsis in C57BL/6 mice by LPS(5mg/kg). Subsequently, a 30µg of scHIF1α-EVs was administered, and immune profiling was assessed using FACS, scRNA-seq, and Visium.

3. Results

We engineered HIF1α to remain stable under normoxic conditions by deleting its ODD domain. To develop EVs encapsulating the HIF1α, we fused HIF1α to the C-terminus of the ΔI-CM and attached the CD81 tetraspanin to the N-terminus of the ΔI-CM. The functionality of scHIF1α encapsulated within EVs(scHIF1α-EVs) was validated using a reporter system and qRT-PCR.

We assessed the therapeutic efficacy of scHIF1α-EVs in sepsis model. It was observed that only the scHIF1α-EV group showed an increased survival rate and decreased liver toxicity.

MoMFs differentiation induced by scHIF1α-EVs was confirmed through FACS and scRNA-seq analysis. Increased expression of Nr1h3 and C/EBPb, both associated with differentiation, was observed. We confirmed that scHIF1α-EVs induced resolutive macrophages zonated near the portal vein through Visium.

4. Summary/conclusion

We have demonstrated that scHIF1α-EVs plays a vital role in promoting the differentiation of resolutive MoMFs, which are critical for tissue recovery during the liver damage. This facilitates the recovery from Sepsis-induced Liver Injury.

Ms Michaela Klaczynski, Ms Birgit Hirschmugl, Ms Barbara Darnhofer, Ms Katharina Eberhard, Mr Harald Köfeler, Mr Karl Kashofer, Mr Christian Wadsack

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Preeclampsia (PE) is a pregnancy-related disease affecting 3-8% of pregnant women worldwide and posing a significant risk to maternal and neonatal health. Fetal complications associated with PE are thrombocytopenia and neutropenia, suggesting an immature innate immune system during pregnancy. Placental small extracellular vesicles (sEVs) are known to contribute to the maternal immune dysregulation in PE. Here, we aim to elucidate the contribution of placental sEVs in the development of the fetal immune system.

Methods

Primary endothelial cells (ECs) were isolated from fetal vessels of placental chorionic plate from term (T, n = 6), preterm (PT, n = 6) and early onset PE (n = 5) pregnancies. Conditioned media from cultured ECs were used to isolate sEVs by differential ultracentrifugation. The characteristics of EC-sEVs were determined by size and concentration using Nanoparticle Tracking Analysis (NTA) and validated by EV-specific markers (Western Blot). In depth characterization of EC-sEVs, miRNA and proteomic cargo were determined by miRNAseq and nanoLC-MS/MS. A lectin microarray was applied to profile the glycosylation pattern of EC-sEVs and respective parent ECs cell membrane.

Results

The pathological origin of the ECs did not alter the size of secreted sEVs. However, we observed a 2.5-fold higher number of released sEVs into the conditioned media from PE- and PT-ECs. miRNAseq revealed six up- and one downregulated miRNAs (fold change cut-off ≥ 1.5, p-value ≤ 0.05) in the PE EC-sEVs compared to both T and PT. Pathway enrichment analysis of overrepresented miRNAs in PE EC-sEVs revealed 620 target genes, indicating their potential involvement in regulating the IFN-γ pathway. Proteomic analysis of EC-sEVs revealed 1272 proteins; however, no significant changes were observed between the groups. The glycosylation pattern of both T and PE EC-sEVs corona showed an enrichment of sialic acid compared to the cell membrane of parent ECs.

Conclusion

We propose a distinct immune regulative miRNA signature on endothelial derived placental sEVs in PE. The altered sialic acid pattern on both T and PE EC-sEVs corona indicates the role of glycans in the intercellular communication in the fetus. This to be verified working hypothesis is underlined by the fact that fetal derived hematopoietic cells express sialic acid receptors.

Dr Mee Chee Chong, Dr Anup D. Shah, Associate Professor Ralf B. Schittenhelm, Dr Anabel Silva, Dr Patrick F. James, Professor Jason Howitt

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Habitual physical exercise induces beneficial health adaptations, which are thought to be partly mediated by signaling molecules contained within extracellular vesicles (EVs). Recent studies have reported EV proteome changes during high intensity or exhaustive aerobic exercise. However, it remains unclear whether EV protein cargo is modulated by moderate intensity exercise and influenced by aerobic fitness of an individual. Here, we examined the changes of small EV proteome after moderate intensity exercise in aerobically unfit and fit individuals.

Methods: Following ethical approval and written informed consent, we collected blood plasma from unfit and fit healthy males before and after an acute bout of 20-min cycling exercise at 70% VO2max. Small EVs were isolated using differential ultracentrifugation and characterized using western blot and NTA. Small EV protein cargo was examined using quantitative proteomic analysis. Gene ontology functional enrichment and Reactome pathway analyses were performed on significantly altered EV proteins in response to exercise.

Results: We observed that small EV proteome is significantly altered during moderate intensity exercise. We further identified exercise-derived EV protein cargo is distinctly modulated by aerobic fitness, with a greater number of proteins in small EVs significantly altered in fit individuals as compared to unfit individuals. Functional enrichment and pathway analysis revealed that the majority of the significantly altered small EV proteins are associated with the innate immune system. This includes the damage-associated molecular patterns (DAMPs) that are identified as stress response proteins, suggesting the priming of immune function following exercise-evoked stress.

Summary/Conclusion: Our findings suggest that moderate intensity exercise can be an adjuvant to elicit acute positive challenge to the innate immune system through the secretion of immune proteins and DAMPs via small EVs. This provides a mechanism to enhance immune surveillance in the body through regular exercise, as indicated by a greater response in aerobically fit individuals.

Phd Qianbei Li, Professor Lei Zheng

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Obesity, linked to gut microbiota imbalances, is marked by excessive white fat accumulation and lipid metabolism disorders. Akkermansia muciniphila (Akk), a common gut commensal, has been shown to reduce fat storage and convert white adipocytes to a brown-like phenotype. This study highlights the role of Akk in ameliorating glucose intolerance, hyperlipidemia, endotoxemia, liver steatosis and obesity-associated mental disorders in a high-fat diet (HFD) murine model. A particular focus was on Amuc_1100, a membrane protein of A. muciniphila, identified in the secreted extracellular vesicles. Amuc_1100 promoted lipolysis and the browning of white adipocytes via the AC3/PKA/HSL pathway in 3T3-L1 preadipocytes. These findings offer new insights into the therapeutic potential of Akk and its components in treating obesity and associated metabolic disorders.

Msc Johanna Matilainen, Viivi Berg, Maija Vaittinen, Janne Tampio, Ville Männistö, Jussi Pihlajamäki, Tanja Turunen, Marjo Malinen, Pirjo Käkelä, Dorota Kaminska, Veera Luukkonen, Anne-Mari Mustonen, Uma Thanigai-Arasu, Kristiina Huttunen, Reijo Käkelä, Sanna Sihvo, Petteri Nieminen, Kirsi Rilla

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Obesity is associated with chronic low-grade inflammation and dysregulated production of inflammatory cytokines in adipose tissue (AT). Recently, AT has been demonstrated to also secrete extracellular vesicles (EVs), which act as rapid responders to inflammatory stimuli, and increased AT EV secretion is also realized in human circulation in obesity. The inhibition of calpains has been shown to exert anti-inflammatory and fibrotic effects in AT in mice in vivo, but also to reduce EV secretion in certain cell types in vitro. However, effects of calpain inhibition on human AT function, or EV secretion from adipocytes, have not been studied. In our experiments, we aim to investigate calpeptin's effects on EV-mediated communication and function of human adipocytes, potentially revealing therapeutic potential against metabolic diseases.

Methods:

Human SGBS adipocytes were differentiated, and AT from subjects undergoing bariatric surgery were cultured ex vivo prior to calpeptin treatments. EVs were isolated by standard ultracentrifugation, and studied by nanoparticle tracking analysis, electron microscopy and mass spectrometry. Various analyses, including RNA-sequencing, mass spectrometry (MS) and confocal microscopy were utilized to study the effects of calpeptin on SGBS cells.

Results:

In our study, calpeptin exerted anti-inflammatory effects on mature SGBS cells, while reducing EV secretion. Furthermore, our preliminary results suggest that calpeptin lowers EV secretion also from patient AT cultured ex vivo. Surprisingly, despite the anti-inflammatory effects, adiponectin expression and secretion were downregulated in SGBS cells, suggesting suppressed insulin sensitivity. Further analyses revealed that calpeptin altered insulin signalling via downregulated PI3K/Akt pathway, leading to disrupted trafficking of Glut4 glucose transporter. MS analysis of EVs and conditioned medium treatments of hepatocytes will reveal the effects of calpeptin on EV proteome and hepatocyte-mediated responses.

Summary/Conclusions:

To our best knowledge, this is the first study investigating thoroughly the effects of calpeptin on human adipocyte function and metabolism, with special emphasis on EV secretion and EV-mediated communication. Our results suggest that calpeptin reduces EV secretion and suppresses inflammatory responses in human adipocytes, but does not improve glucose tolerance, opposing to previous in vivo studies with mice. Thus, our findings introduce new aspects to the effects of calpeptin in AT in humans.

Dr Soumyalekshmi Nair, Lilian Kessling, Dominic Guanzon, Andrew Lai, Flavio Carrion, David Simmons, Mireille Van Poppel, Harold David McIntyre, The Dali Core Investigator Group, Gernot Desoye, Carlos Salomon

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Normal pregnancy relies on the development of maternal insulin resistance and enhanced insulin secretion to allocate nutrients for fetal growth, whereas gestational diabetes (GDM) exacerbates maternal insulin resistance and inadequate beta cell compensation, leading to hyperglycaemia. The aim of the present study is to determine the association between circulating extracellular vesicles (EV) and insulin sensitivity across gestation in normal glucose tolerant (NGT) and GDM pregnancies.

Method: DALI (vitamin D and lifestyle intervention for GDM prevention) lifestyle study is a multicenter randomized controlled trial in which plasma samples were obtained from NGT (n = 67) and GDM (n = 63) women longitudinally at three time points during pregnancy. The insulin sensitivity of the patients at these time points were determined using the Homeostatic model assessment (HOMA-IR) index. EVs were isolated from the plasma samples using size exclusion chromatography and characterized by size, protein abundance and morphology using nanoparticle tracking analysis, western blot and electron microscopy, respectively (MINSEV2018). The miRNA and protein profile in the sEVs were analysed using next generation sequencing and mass spectrometric analysis.

Results: The concentration of EV in maternal circulation increased significantly across gestation and correlated significantly with the BMI, HOMA-IR and weight gain across gestation for NGT and GDM women. 50 proteins that are differentially expressed between NGT and GDM. This includes proteins serum amyloid P-component (SAMP) and Pappalysin-1 (PAPP-A). Using linear mixed modelling analysis, 390 EV-associated proteins have significant correlation to changes in HOMA-IR during gestation in NGT and GDM patients. This included proteins such as afamin, annexin and SAMP. Moreover, 6 miRNAs (miR-30c-5p, miR-574-5p) were significantly different between NGT and GDM pregnancies. Additionally, 24 miRNAs (miR-503-5p, miR-32-5p, miR-199-5p) were significantly correlated with HOMA-IR across gestation in NGT and GDM. Using bioinformatic analysis, we identified that the differentially expressed proteins and miRNAs in sEVs target insulin signalling and glucose homeostasis pathways.

Conclusion: This study suggests that molecular content of circulating EV is associated with the changes in insulin sensitivity across gestation. The molecular content of circulating EV might be involved in metabolic regulation during pregnancy and be utilized as biomarkers for early screening or therapeutic targets for GDM.

MD, PhD Melissa Razo-Azamar, PhD Rafael Nambo-Venegas, PhD Iván Rafael Quevedo, PhD Gregorio Juárez-Luna, PhD Carlos Salomon, MD, PhD Martha Guevara-Cruz, PhD Berenice Palacios-González

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Pancreatic β-cell function impairment is a key mechanism for developing gestational diabetes mellitus (GDM). Maternal and placental exosomes regulate maternal and placental responses during hyperglycemia. Studies have associated exosomal micro RNAs (miRNAs) with GDM development. To date, no studies have been reported that evaluate the profile of miRNAs present in maternal and placental exosomes in the early stages of gestation from pregnancies that develop GDM.

We assessed whether early pregnancy serum maternal and placenta-derived exosomes miRNA profiles vary according to pancreatic β-cell function in women who will develop GDM.

Methods

A prospective nested case-control study was used to identify exosomal miRNAs that vary in early pregnancy stages (<18 weeks of gestation) from women with normoglycemia and those that developed GDM based on their pancreatic β-cell function using HOMA-%β index. Early pregnancy serum maternal and placenta-derived exosomes were isolated to obtain miRNA profiles. Potential target and pathway analysis were performed to identify molecular and metabolic pathways associated with the exosomal miRNAs identified.

Results

In early pregnancy stages, serum maternal exosome size and concentration are modified in GDM group and fluctuate according to HOMA-%β index. Serum maternal exosomal hsa-miR-149-3p and hsa-miR-455-3p in GDM are related to insulin secretion and signaling, lipolysis, and adipocytokine signaling. Early-pregnancy serum placenta-derived exosomes hsa-miR-3665 and hsa-miR-6727-5p in GDM are related to regulate genes involved in response to pregnancy's immunological tolerance and pathways associated with placental dysfunction.

Conclusions

Early serum exosomal miRNAs differ depending on their origin (maternal or placental) and pancreatic β cell function. This research provides insights into the interactions between maternal and placental exosomal miRNAs and may have implications for identifying potential biomarkers or therapeutic targets for GDM.

Pin Hsuan Chu, Dr. Han-Yi E. Chou, Dr. Tien-Jyun Chang, Dr. Shiau-Mei Chen

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Liver-pancreatic crosstalk is pivotal for fine tuning glucose homeostasis, however, the alteration of hepatic EVs and their effect on pancreatic islets during progressive diabetes pathology remain largely obscure. Here we establish animal and primary cell models of nonalcoholic fatty liver disease (NAFLD) and type II diabetes (T2DM) to analyze their EV profile and functional implications on isolated pancreatic islets.

Methods: NAFLD and T2DM were induced using the CL57BL/6N mice under high-fat, high-sugar chow with/without intraperitoneal streptozotocin administration (HF/HS, HF/HS+STZ compared to normal chow Ctrl groups). Their respective primary murine hepatocyte (PmH) cultures were established and EVs were purified by size exclusion. All EVs were characterized by NTA and transmission electron microscopy, and functional analysis on intact islets including EV uptake, survival and glucose stimulated insulin release were performed. These studies were compared to in vitro primary human hepatocytes (PhH) models and analyzed for their miRNA and protein profiles.

Results: PmH from both HF/HS and HF/HS+STZ mice exhibited significantly higher cytoplasmic lipid accumulation and lower survival rate relative to the Ctrl group. EVs isolated from both HF/HS and HF/HS+STZ groups show a dramatically increase in amount and population heterogeneity, accompanied by lower lipid fluidity and reduced uptake by intact islet than the Ctrl EVs. Albeit short term glucose stimulated insulin secretion shows no significant variation among the three groups, miRNA and protein profiling suggest factors to modulate pancreatic adaptation under insulin resistance.

Summary: Our study shows that hepatic EV production is altered in NAFLD and T2DM models, this change in crosstalk between liver and pancreas may exacerbate NAFLD and promote progression of T2DM. Our data also suggest that ceramide inhibitors may help reverse these effects, shedding light to future directions for pharmacological intervention.

Principal Researcher Eun Hee Han, Ms. Hye Min Kim, Dr. Jin Young Min, Mr. Min Sung Park1

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction

Extracellular vesicles (EVs) play a key role in cell-to-cell communication, especially in transporting microRNAs (miRNAs) that influence gene expression. In the kidney, miRNAs contribute to various diseases, including kidney diseases and diabetic nephropathy. This study explores the impact of methylglyoxal-lysine dimer (MOLD), a toxic advanced glycation end product, on renal disease, focusing on miRNAs within EVs from mesangial cells.

2) Methods

EVs were isolated from kidney mesangial cells, with and without MOLD exposure, and underwent miRNA sequencing to identify differentially expressed miRNAs. miR-130a-3p was selected for further study due to its significant upregulation post-MOLD treatment. We used the TargetScan database for miRNA target prediction and PPRE reporter assays to investigate the suppression of PPAR-γ expression by miR-130a-3p. RNAscope analysis in mouse tissues with diabetic nephropathy was also performed.

3) Results

24 miRNAs showed over 2-fold expression change after MOLD treatment, notably miR-130a-3p. TargetScan analysis identified PPAR-γ as a target of miR-130a-3p. PPRE reporter assays confirmed miR-130a-3p's role in suppressing PPAR-γ expression. RNAscope analysis in diabetic nephropathy-induced mouse tissues showed that increased miR-130a-3p levels corresponded with decreased PPAR-γ mRNA and increased TGFbeta mRNA.

4) Summary/Conclusion

This study elucidates the role of EV-mediated miR-130a-3p in MOLD-induced kidney disease. The upregulation of miR-130a-3p by MOLD and its subsequent suppression of PPAR-γ suggests a novel mechanism for renal injury. These findings underscore the significance of miRNA pathways in renal pathologies, offering new perspectives for targeted therapeutic strategies against kidney diseases associated with toxic advanced glycation end products like MOLD.

Ph.D. Jimin Kim, M.S. Seul Ki Lee, M.S. Haedeun You, M.S. Sang-Deok Han, Ph.D. Tae Min Kim, Ph.D. Soo Kim

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

1) Introduction: Nonalcoholic steatohepatitis (NASH) is a chronic liver disease associated with metabolic syndrome. Extracellular vesicles (EVs) are essential signaling mediators containing functional biomolecules. EVs are secreted from various cell types, and recent studies have shown that mesenchymal stem cell-derived EVs have therapeutic potential against immune and metabolic diseases. In this study, we tested whether EVs derived from induced mesenchymal stem cells (iMSC-EVs) can block NASH progression.

2) Methods: EVs were generated from induced mesenchymal stem cells. The proteome signature of iMSC-EVs was analyzed. iMSC-EVs were intravenously administered into two different mouse models of NASH (methionine/choline-deficient diet-induced and ob/ob mice). The repression of NASH by iMSC-EVs was assessed by analyzing the expression of molecular markers in NASH mice and relevant in vitro models.

3) Results: Proteome analysis revealed that iMSC-EVs carry cargo proteins with the potential to regulate lipid metabolism. iMSC-EVs inhibited free fatty acid release from adipose tissues by downregulating the lipolytic genes in NASH. In addition, iMSC-EVs reduced hepatic steatosis by modulating AMPK signaling, which plays crucial role in improvement of metabolic homeostasis in NASH. Moreover, iMSC-EVs reduced CD36 expression, contributing to the blockade of free fatty acid transport to the liver of NASH mice. Finally, iMSC-EVs reduced inflammation, endoplasmic reticulum stress, and apoptosis while promoting hepatic regeneration of the NASH liver.

4) Summary/Conclusion: In conclusion, iMSC-EVs can potentially serve as cell-free therapeutics for NASH owing to their multifaceted modality.

PhD student Karen Lahme, PhD Wiebke Sachs, PhD Desiree Loreth, Stephanie Zielinski, Johannes Brand, PhD Kristin Surmann, Professor Uwe Völker, Thorsten Wiech, Professor Tobias N. Meyer, Lars Fester, Professor Catherine Meyer-Schwesinger

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Membranous nephropathy (MN) is an autoimmune glomerulonephritis of kidney podocytes induced by circulating autoantibodies directed against podocyte foot process proteins. The morphologic hallmark of MN is the glomerular antigen-autoantibody deposition and is characterized by the accumulation of aggregated proteins in injured podocytes. The upregulation of the ubiquitin-proteasome (UPS) occurs during disease progression and correlates with podocyte injury in rodent models. Here we analyzed the podocyte-derived urinary extracellular vesicle (EV) formation in human nephrotic patients and in experimental murine MN.

Methods: EVs were isolated from mouse and human urine by differential ultracentrifugation, followed by ultrafiltration (human urine) or by dialysis (mouse urine). EVs were quantified by nanoparticle tracking analysis, characterized by electron microscopy, ImageStream, and immunoblotting. The proteostatic content of human urinary EVs was further investigated by mass spectrometry. Immunohistology from human nephrotic patient biopsies was performed.

Results: Both human and murine podocyte-derived urinary EVs contain UPS components. The amount of podocyte-derived urinary EVs increases in human MN and in murine models of MN. The proteostatic content changes in a disease-dependent manner, mirroring the disease-associated proteostatic situation of podocytes. Human EV abundance and content differ depending on the underlying nephrotic syndrome. Protein aggregate removal depends on proteasome functionality in injured podocytes, as well as on the possibility of EV formation and release.

Conclusion: Analyses of the genesis and release of podocyte-derived urinary EVs have the potential to give insight into the proteostatic status of podocytes, possibly reflecting origin (and prognosis) of the underlying injury.

Neslihan Erdem, Nathaniel Hansen, Min Talley, Heather Zook, Kevin Jou, Jose Ortiz, Nagesha Guthalu Kondegowda, David Arribas-Layton, Fouad Kandeel, Enrique Montero, Helena Reijonen, Rupangi Vasavada, Patrick Pirrotte, Tijana Jovanovic-Talisman, Hsun Teresa Ku

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Type 1 Diabetes (T1D) is an autoimmune disease characterized by the destruction of the endocrine beta cells in the pancreas. Despite extensive research, there is still no definitive cure for T1D. Recent evidence demonstrates that the exocrine compartment, which includes acinar and ductal cells, is impaired in T1D patients. T1D ductal cells display increased MHC Class II molecule, suggesting potential interactions between ductal and immune cells. However, very little is known about the responses of normal human ductal cells to T1D proinflammatory cytokines and whether primary ductal cells can secrete extracellular vesicles (EVs) for cell-cell communication. We therefore aimed to determine the effects of T1D proinflammatory cytokines on primary human ductal cells and examine their EVs.

Methods: Human ductal cells were isolated from cadaveric donors without apparent diseases (City of Hope, IRB #01046) and cultured in a spheroid suspension culture. The effects of graded doses of proinflammatory cytokines on ductal cells were evaluated by flow cytometry and qRT-PCR. Ductal EVs were isolated from conditioned media by size exclusion chromatography (SEC) according to MISEV 2018 guidelines and the protein cargo of EVs was determined by mass spectrometry.

Results: A combination of IL-1β (25 IU/mL), TNF-α (250 IU/mL), and IFN-γ (250 IU/mL) was sufficient to increase cellular stress and MHC Class II expression but not apoptosis in normal primary human ductal cells. SEC fractions 1-3 were highly enriched with EVs, as evidenced by the presence of EV markers and the absence of non-EV markers. Ductal EVs were confirmed by transmission electron microscopy and nanoparticle tracking analysis. Proteomic analysis demonstrated differences in protein cargo in EVs obtained from ductal cells treated with or without proinflammatory cytokines.

Summary/Conclusion: Normal primary human pancreatic ductal cells are responsive to T1D-mimicking proinflammatory cytokines by increasing cellular stress, MHC class II expression, and secrete different protein cargo in EVs. Our results suggest that pancreatic ductal cells are not naïve bystanders in T1D. Further studies are underway to understand the role of ductal cells on immune and beta cell function. Our results may have implication in the utility of ductal EVs as biomarkers and therapeutic targets for T1D.

Henry Louie, Ilva D. Rupenthal, Odunayo O. Mugisho, Lawrence W. Chamley

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: The nucleotide-binding oligomerisation domain-like receptor protein 3 (NLRP3) inflammasome is a multiprotein complex that forms part of the innate immune system. Recent studies have shown that the inflammasome pathway is upregulated in diabetic retinopathy (DR) resulting in the breakdown of the retinal pigment epithelium (RPE) barrier. It has been hypothesised that small extracellular vesicles (sEVs) may transport inflammasome-related cargos between cells which in turn contributes to DR pathogenesis. This study aimed to characterise sEVs secreted by retinal pigment epithelial cells (ARPE-19) grown under normal and DR-like conditions and assess their effects on inflammasome activation.

Methods: sEVs were isolated from ARPE-19 cell culture medium (basal sEVs) by ultracentrifugation at 100,000 g for 70 min at 4oC. To mimic DR-like conditions, ARPE-19 cells were exposed to high glucose and pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) for 72 h (HG+Cyt sEVs). sEVs were characterized for size, concentration and morphology using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Western blotting was used to determine the sEV cargo (NF-κB, IL-1β, NLRP3, cleaved caspase-1). Immunocytochemistry was performed to assess inflammasome markers (NLRP3, cleaved caspase-1) in response to sEV addition onto ARPE-19 cells grown under normal and DR-like conditions.

Results: HG+Cyt treatment increased sEV concentration relative to basal conditions. While sEV markers (CD9 and TSG101) were equally expressed in both groups, inflammasome markers (NLRP3 and cleaved caspase-1) were increased in HG+Cyt sEVs relative to basal sEVs. When added to normal ARPE-19 cells, HG+Cyt sEVs induced inflammasome activation, while basal sEVs added to injured ARPE-19 cells reduced inflammasome activation.

Conclusion: These findings support the hypothesis that sEVs can mediate inflammasome activation through their immunomodulatory cargo depending on the disease state and culture conditions. Further studies are necessary to determine whether other sEV cargo, such as miRNA, may also play a role in mediating the inflammasome.

Pregnant dams during early pregnancy sufficient to cause obesity in offspring?

Phd Student Taylor Hollingsworth¹, Pharm.D., Ph.D. Thea Golden¹, M.D. Rebecca Simmons¹

¹University Of Pennsylvania, Philadelphia, United States

Poster Pitches (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:45 PM - 1:00 PM

Introduction:

Studies in humans and animal models demonstrate that maternal obesity increases the risk of offspring developing obesity. Our previous embryo transfer studies reveal that preimplantation embryonic exposure to obesity is sufficient to induce obesity and glucose intolerance in adulthood, suggesting this process starts very early in development. The molecular mechanisms remain unknown. EVs are a potential mechanism by which molecular information from the mother is transferred to the embryo. Emerging data show that EVs from obese mice are sufficient to induce an abnormal metabolic phenotype, including obesity and insulin resistance. Consequently, we hypothesize EVs from obese pregnant dams are sufficient to lead to the development of obesity in adulthood.

Methods:

Female mice were fed standard chow (lean) or a diet with 60% of calories derived from fat (obese), from weaning to 14 weeks of age, then mated with males. On embryonic day (E) 4.5, dams were sacrificed, blood collected, and spun at 2,000xg for 10 minutes at 4°C for plasma separation. 1mL plasma was spun at 2,000xg for 10 minutes at 4°C. The supernatant was then spun at 20,000xg for 30 minutes at 4°C. The pellet (LEVs) was washed in 1mL filtered PBS and spun again at 20,000xg for 30 minutes at 4°C. LEV pellet was resuspended in 100uL filtered PBS. EV isolation was confirmed by cryo-electron microscopy, nanoparticle tracking, and protein measurement. 1x10⁹/100uL of LEVs from the lean (LLEVs) or obese (OLEVs) groups were administered via intraperitoneal injection to dams from E0.5 to E4.5. PBS was used as a control. Dams delivered pups spontaneously and offspring were weaned between 21 and 24 days. Weekly weight measurements were recorded.

Results:

Offspring from the OLEV group weighed significantly more than the LLEV group and controls at 14 weeks of age in both males and females. No significant difference was observed between LLEV offspring and controls in both sexes.

Summary:

Exposure to EVs from an obese dam in early pregnancy is sufficient to cause obesity in offspring. Ongoing experiments include assessing glucose tolerance and energy expenditure.

Ms. Kobe Abney¹, Pharm.D., Ph.D. Thea Golden¹, Dr. Yu-Chin Lien¹, Dr. Nadav Schwartz¹, Dr. Rebecca Simmons¹

¹University Of Pennsylvania, United States

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

In the U.S., Black, cis-women disproportionately experience adverse pregnancy outcomes driven by placental dysfunction. Our lab's preliminary data demonstrated significant metabolic and transcriptional variations in second-trimester placentas from healthy Black women compared to white. Whether these differences contribute to adverse pregnancy outcomes remains unclear. Extracellular vesicles (EVs) are actively involved in mediating cellular communication, shuttling cargo between maternal and placental cells. While studies have identified distinct EV profiles in pregnancies with adverse outcomes, the potential racial dependence of these distinctions remains unexplored.

This study aims to investigate whether circulating plasma-derived EVs from Black women can serve as early indicators of adverse outcomes in pregnancy. In a nested case-control cohort, 23 women (83% Black, 17% white) with pre-eclampsia, gestational hypertension, gestational diabetes, and preterm birth were matched with controls based on race, age, BMI, and health insurance. Cases exhibited a significantly lower gestational age at delivery (37.5 weeks) and higher BMI in the first trimester (30.5) compared to controls (39.4 weeks, 28.4).

Methods

To characterize the EV signature, first-trimester EVs were isolated from 1 mL of plasma using differential- and ultra-centrifugation, as well as size-exclusion chromatography, yielding small particles (60-200 nm) and large particles (200-500 nm). Cryo-electron microscopy showed the morphological characteristics of EVs, and western blot analysis confirmed CD9+ particles within the specified size ranges. Nanoparticle Tracking Analysis (NTA) was used to assess EV size distribution and concentration from each sample. To elucidate EV cargo, mitochondrial DNA was isolated from each sample and quantified using qPCR.

Results

NTA revealed no significant differences in size and concentration of EVs from Black women with adverse outcomes versus controls. The number of mtDNA copies per small EV from Black women with an adverse outcome was significantly higher compared to controls. Additionally, large EVs from those with an adverse outcome carried more mtDNA copies than controls however, this was not statistically significant.

Conclusion

These findings collectively contribute to establishing a profile of EVs derived from Black women experiencing adverse pregnancy outcomes. This signature holds promise as a supplementary tool, alongside clinical methods, to predict the likelihood of an unfavorable pregnancy outcome.

Miss Cottrell Tamessar^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Miss Chishan Burch^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Miss Piper Miller^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Miss Jane Durbidge^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Miss Tegan Bryde^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Miss Shanu Parameswaran^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Associate Professor Geoffry De Iuliis^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Doctor Judith Weidenhofer^{3 Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 4 School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, The University of Newcastle, Ourimbah, NSW 2258, Australia.}, Dr Hui-ming Zhang^{5 Central Analytical Facility, Research and Innovation Division, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Professor Sarah Robertson^{7 Robinson Research Institute and School of Biomedicine, The University of Adelaide, SA 5005, Australia.}, Doctor Elizabeth Bromfield^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia. 6 Bio21 Institute, School of BioSciences, The University of Melbourne, VIC 3010, Australia.}, Doctor David Sharkey^{7 Robinson Research Institute and School of Biomedicine, The University of Adelaide, SA 5005, Australia.}, Professor Brett Nixon^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Doctor John Schjenken^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}

¹Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, Newcastle, Australia, ²School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, Callaghan, Newcastle, Australia, ³Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, Newcastle, Australia, ⁴School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, The University of Newcastle, Ourimbah, Central Coast, Australia, ⁵Central Analytical Facility, Research and Innovation Division, The University of Newcastle, Callaghan, Newcastle, Australia, ⁶Bio21 Institute, School of BioSciences, The University of Melbourne, Parkville, Melbourne, Australia, ⁷Robinson Research Institute and School of Biomedicine, The University of Adelaide, North Adelaide, Adelaide, Australia

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: As well as delivering sperm to fertilize oocytes, seminal fluid induces molecular changes in the female reproductive tract to promote reproductive success. Transforming growth factor-β (TGFβ) and other factors in seminal fluid have been identified as signaling agents but do not fully explain the female response. Seminal fluid extracellular vesicles (SFEVs) likely have signaling potential given their suggested roles in immune regulation. However, their signaling capacity remains elusive. To investigate the impact of SFEVs on female reproductive tract signaling, we used a well-established human ectocervical epithelial (Ect1) cell culture model.

Methods: All procedures described in this abstract had human ethics approval and semen samples were obtained with informed written consent. Human SFEVs were isolated from the semen of normozoospermic donors (in accordance with World Health Organization criteria) using sequential density-gradient ultracentrifugation and characterized via immunoblotting, nanoparticle tracking analysis, and transmission electron microscopy. Ect1:SFEV interactions were evaluated by immunofluorescence with biotin-labelled SFEVs (n = 3). Gene expression in Ect1 cells 8h after incubation with SFEVs was analyzed by transcriptomics (n = 4) and qPCR (n = 8) using untreated Ect1 cells as a control. Transcriptomic data were analyzed using Ingenuity Pathway Analysis.

Results: SFEVs rapidly interacted with Ect1 cells with biotinylated protein cargo from SFEV's detected on Ect1 cells within 5-minutes post-incubation. Following 8h of co-incubation, SFEVs substantially altered gene expression profiles in Ect1 cells (>1.5FC, 216 genes induced, 211 suppressed, FDR<0.05). Amongst the top 10 most differentially up-regulated genes, common functions ascribed were immune response, inflammatory response, and angiogenesis with several cytokines, including CXCL1 (p≤0.01, 7.9FC), IL1A (p≤0.01, 2.6FC) and IL6 (p≤0.01, 2.8FC), confirmed by qPCR as being induced by SFEVs. Bioinformatic analysis revealed that 55% of activated (Z-score>2) pathways were immune signalling pathways. Upstream regulator prediction of signaling agents carried by SFEVs identified both TGFβ1 (Z-score = 4.8) and Nuclear factor-κB (NF-κB, Z-score = 5.8) as predicted SFEV cargo that may influence these gene expression changes. The presence of TGFβ and NF-κB2 (p52), but not NF-κB1 (p50) subunits in SFEVs was confirmed via immunoblotting.

Conclusion: This study highlights SFEVs may enact an important role in shaping the immune environment of the female reproductive tract to promote reproductive success.

Miss Nidhi Seegobin¹, Miss Victoria Chris^1,2, Miss Marissa Taub¹, Dr Sudaxshina Murdan¹, Prof Abdul Basit¹

¹University College London, London, United Kingdom, ²University of Oxford, Oxford, United Kingdom

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Inflammatory bowel disease (IBS) impacts over 6.8 million people globally, often leading to poor outcomes due to the side effects of current treatments. To address this, localized drug delivery of negatively charged nano-sized carriers such as bovine-milk derived extracellular vesicles (EVs) to positively charged inflamed gut tissue is proposed. This study evaluates EV drug carrier stability in the gastrointestinal (GI) tract for oral targeted drug delivery to treat IBS.

Methods

Bovine milk derived EVs were obtained by ultracentrifugation, and HEK293 cell derived EVs were obtained by ultrafiltration of the cell culture media. Both EVs were then purified by size exclusion chromatography (SEC). EVs and liposomes (positive control) were loaded with the polar dye laurdan and were purified by SEC. The laurdan loaded EVs were then incubated with simulated GI fluids, and fluorescence spectra analysis was conducted to assess changes in generalised polarity (GP) within the lipid bilayer. Bovine milk derived EVs were loaded with riboflavin (hydrophilic) and acridine orange (lipophilic) by sonication and passive diffusion, respectively. The riboflavin loaded EVs were purified by SEC and incubated in simulated GI fluids prior to SEC and fluorescence analysis.

Results

EVs and liposomes exhibited an increase in GP values (P<0.001) compared to the control in gastric pH and fluid indicating lipid bilayer damage. Milk EVs and liposomes also showed instability (P<0.001) in the simulated intestinal fluid. A significant reduction in fluorescence (P<0.001) was observed for the lipophilic drug due to EV release in saliva, gastric and intestinal fluids. No significant release was reported for simulated colonic fluids. There was no significant release of hydrophilic drug from EVs in all fluids, indicating cargo stability.

Conclusion

Animal milk and Human stem cell derived EVs behave similarly in simulated GI fluids. EVs are unstable in acidic and enzymatic conditions, characteristic of the stomach and small intestine. EVs are stable in the colonic environment. Hydrophilic cargo located in the aqueous core of the EV is stable throughout the GI tract, but hydrophobic cargo is only stable in colonic fluids. This suggests a need for colonic-targeted delivery to maintain EV integrity and ensure effective oral drug delivery.

Dr. Allen Wei-Lun Huang¹, Ms. Tzu-Ching Kao², Dr. Sin-Tian Wang², Ms. Yi-Wen Chiu², Dr./Prof. Pin-Nan Cheng³, Dr./Prof. Chi-Yi Chen⁴, Prof. Kung-Chia Young²

¹Center of Applied Nanomedicine, National Cheng Kung University, Tainan, Taiwan, ²Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ³Department of Internal Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ⁴Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chia-Yi Christian hospital, Chiay, Taiwan

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Extracellular vesicles (EVs) have been shown to play important roles in both physiological and pathological conditions via transmitting cargos of DNA, RNA, proteins, and lipids. The lipid-metabolism dysfunction of hepatocytes might affect production, releasing, size distribution and cargo contents of EVs, however, it remains unclear the characteristics of hepatocyte-secreting EVs in the progression of metabolic-associated fatty liver disease (MAFLD).

Methods:

First, lipid-metabolism dysfunction was induced with two mechanisms in cultivated primary human hepatocytes (PHH), including fatty acid overloads by oleic acid (OA) and impairment of cholesterol-to-cholesteryl ester (CE) transition by TMP-153, an inhibitor of sterol O-acyltransferase. Next, the hepatic steatosis, cellular senescence and EVs secretion were investigated, by lipid profiling, filipin and SAβG staining, senescent biomarkers, and nanoparticle tracking analysis. In this study, we isolated EVs using the ultra-centrifugation method. Then, the contents of isolated EVs were examined using qPCR, Western blotting and ELISA. In addition, the EVs of serum sample of MAFLD patients were also analyzed.

Results:

Hepatosteatosis with prominent triglyceride accumulation was induced in PHH under both conditions. Moreover, additional CE deposit was founded in OA induction group but not in TMP-153 induction group. The OA-treated PHH tended to secret smaller size EVs (≤200 nm) when the TMP-153-treated cells exhibited significantly senescent phenotypes and secreting EVs in a broad range (50∼1000 nm). Furthermore, the cargos in the isolated EVs induced by TMP-153 contained substantially elevation of pro-inflammatory factors at both protein and mRNA levels. Finally, the EVs were validated in the serum samples from MAFLD patients stratified by age groups.

Summary/Conclusions:

Lipid metabolism dysfunction might trigger hepatocytic cellular senescence that elevates the release of pro-inflammatory EVs in the MAFLD progression by aging.

Postgraduate student Keren Esther Kristina Mantik^1,2, Researcher Sohee Moon¹, Sujin Kim¹, Researcher, MS Bon-Sang Gu¹, Postgraduate student Jubi Lee^1,2, Postgraduate student Chan-Young So^1,2, CEO Shingyu Bae⁴, Professor, MD, PhD Ju-Hee Kang^1,2,3

¹Department of Pharmacology and Research Center for Controlling Intercellular Communication, College of Medicine, Inha University, 22212, South Korea, ²Program in Biomedical Science and Engineering, Inha University, 22212, South Korea, ³Institute of Sports & Arts Convergence (ISAC), Inha University, 22212, South Korea, ⁴BioDrone Research Institute, MDimune Inc, South Korea

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Cachexia, linked to various underlying diseases such as cancer, chronic kidney disease, and chronic heart failure, exacerbates their progression. This metabolic syndrome involves weight loss, skeletal muscle wasting, and decreased muscle strength, contributing to physical disability and reduced quality of life. This study aims to investigate the efficacy of stem cell-derived artificial nano vesicles (CDV) in preventing or treating muscle atrophy linked to cachexia. Using a unique extrusion technique, CDV were produced and applied to cachexia models to assess their potential in inhibiting muscle atrophy, enhancing muscle cell mitochondrial activity, and promoting muscle regeneration.

Methods: CDV were created with a unique extrusion method and tested in in-vitro cachexia models. The proliferative and differentiating effects of these CDV on myogenic cells were assessed. In inflammatory models induced by TNF-α and IFN-γ, the expression levels of myogenetic transcription factors and muscle atrophic transcriptional factors were quantified. Mitochondrial function in muscle cells following CDV treatment was evaluated through measurements of mitochondrial oxygen consumption rate. Additionally, CDV's impact on cancer cachexia models was studied by co-culturing myotubes with colon cancer cells (CT26) and analyzing results with immunofluorescence.

Results: CDV demonstrated a concentration-dependent promotion of C2C12 myogenic cell proliferation. Pre-treatment with CDV increased the expression levels of MyoD, myogenin, and MHC proteins associated with muscle growth, while suppressing the expression of MAFbx, a muscle atrophy-inducing protein, in inflammatory muscle atrophy models. Furthermore, CDV enhanced mitochondrial function in muscle cells, mitigating inflammation-induced impairment of oxygen consumption rate. In cancer cachexia models, CDV restored reduced MHC levels, resulting in increased muscle length, number, and thickness.

Summary/Conclusion: This study introduces a novel approach that utilizes CDV from mesenchymal stem cells for the treatment and/or prevention of cachexia. CDV treatment demonstrated promising effects in promoting myogenic cell proliferation, facilitating muscle growth, and improving mitochondrial function in in-vitro cachexia models. These findings suggest the therapeutic potential of CDV against cachexia. Further research is needed to elucidate the mechanisms underlying the inhibitory effects of CDV on muscle atrophy and to explore their translational potential through animal studies.

Dr. Andrea Galisova, Dominik Havlicek, Ayca Tunca, Ondrej Sedlacek, Daniel Jirak

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Targeted cancer therapy is essential for efficient elimination of tumor mass without prominent side effects on healthy tissues. Extracellular vesicles (EVs) are being considered as promising drug delivery vehicles due to their tailorable targeting properties while being fully biocompatible. We developed cancer targeted EV-like mimetics (MIMS) created by extrusion of genetically engineered cells. MIMS were labeled fluorescently and with fluorinated polymers for highly-specific 1H/19F magnetic resonance imaging (MRI), what can enable monitoring of drug delivery.

Methods

HEK293 cells were genetically engineered to display a targeting peptide (RGD). MIMS were prepared by sonication and extrusion of the cells (1um, 400nm, 200nm; 7x each). To prepare labeled MIMS, a synthetized fluorinated polymer FluoroPoly (70 mg/mL) was added during extrusion. Vesicles were incubated with a fluorescent dye DiR (5 µM, 15min) before isolation by Iodixanol density gradient ultracentrifugation (100,000g, 2h; wash, 2h). The resulted MIMS were characterized according to the MISEV2018 guidelines by Western blot (targeting), NTA (size), optical imaging and 4.7T MRI. 19F-MR-labeled and unlabeled MIMS were subcutaneously injected into C57BL/9 mice and visualized by fluorescence and 1H/19F-MR imaging.

Results:

Vesicles extruded from the engineered cells contain targeting peptides on the surface as confirmed by Western blot. The presence of myc-tag (downstream) and ALFA-tag (upstream) confirmed expression of the entire peptide, which is essential for proper targeting. Fluorescently and fluorine labeled MIMS in solution gave a strong fluorescent and 19F-MR signals confirming their excellent imaging properties. The size of 148±11nm is suitable for the EPR effect (passive accumulation in tumors). In mice, DiR-labeled MIMS were localized on both injection sites by fluorescence imaging. Importantly, only the 19F-MR-labeled MIMS were specifically visualized by 19F-MR imaging. Next, targeted therapy of 19F-MR-labeled MIMS loaded with chemotherapeutic agents will be examined on a mouse model of breast cancer.

Conclusion

We show the development of biocompatible EV-based delivery vehicles with targeting properties towards cancer tissue. Loading vesicles with highly fluorinated polymers equipped them with excellent imaging properties and allowed them to be tracked with specific heteronuclear MR imaging both in vitro and in vivo. This versatile platform can contribute to improvement of image-guided therapy of cancer.

Dr Chunlian Qin, Dr Danyang Li, Dr Ning Hu, Dr Lizhou Xu

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction: Cardiac oxidative stress is a significant phenotype of myocardial infarction disease, a leading cause of global health threat. There is an urgent need to develop innovative therapies. Nanosized extracellular vesicles (nEVs)-based therapy shows promise, yet real-time monitoring of cardiomyocyte responses to nEVs remains a challenge.

2) Methods: In this study, a dynamic and label-free cardiomyocyte biosensing system using microelectrode arrays (MEA) was constructed. Cardiomyocytes were cultured on MEA devices for electrophysiological signal detection. Various types of nEVs were isolated from different sources such as E. coli, gardenia, HEK293 cells and mesenchymal stem cells (MSC). The nEVs were characterized according to the MISEV2018 guideline. For studying their interactions dynamically, the cells were treated with E. coli-nEVs, Gardenia-nEVs, HEK293-nEVs, and MSC-nEVs, respectively, and the electric signals of the MEA bio-chip were recorded in a real-time fashion.

3) Results: Size and morphological characterization of these particles confirmed their nanosized vesicular properties, despite the difference in their origins. Those nEVs showed no significant effect on cell viability. The MSC-nEVs treatment could significantly improve recovery from oxidative stress infarction (n>30) without increasing complications such as arrhythmias as illustrated by an in vitro cellular oxidative stress injury model. However, E. coli-nEVs and gardenia-nEVs induced severe paroxysmal fibrillation, revealing distinct biochemical communication compared to MSC-nEVs. Principal component analysis identified a lower level of dissimilarity between MSC-nEVs and PBS (mean interclass distance 1.080) than the other two types of nEVs.

4) Summary/Conclusion: This study establishes a fundamental platform for assessing biochemical communication between nEVs and such as cardiomyocytes, offering new avenues for understanding nEVs' functions in cardiovascular system. It can also serve as a useful tool for assessing nEVs uptake in cells, real-time nEVs perception, and quantitative evaluation of the effects of nEVs-based therapy, which is of significantly importance for discovering and assessing EV-based potential drugs.

Miss Shiana Malhotra, Dr Renee Goreham, Dr Thomas Nann

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Contamination of water by pathogens particularly E. coli presents a major challenge to human health today. These outbreaks burden healthcare systems and result in major financial damage to water-treatment industries. The economic costs can be avoided by timely detection of bacteria but most conventional techniques are time consuming and require high pathogen concentrations for effective detection. In this work, we capture and quantify outer membrane vesicles (OMVs) released by E. coli.

Methods: E.coli BW25113 OMVs were isolated using IZON qEV size exclusion columns. Single stranded DNA aptamers Ec2 and Ec3 were used as detection probes to target outer membrane proteins of OMVs. These aptamers were pre-conjugated to InP/ZnS quantum dots (QDs) synthesised using hot injection method serving as fluorescent labels. The QD-aptamer conjugate was then incubated with isolated OMVs for 30 minutes. Detection was quantified by change in optical properties of QDs at each step of the experiment by using photoluminescence spectroscopy and lifetime measurements.

Results: Isolated OMVs were well within the expected size range of 50-200 nm. The synthesised InP/ZnS QDs were orange emitting, size ranging from 2- 6 nm with emission maximum of 546 nm. Upon conjugation to aptamers Ec2 and Ec3, emission peaks of the QDs showed redshifts to 552 nm and 554 nm respectively. Further changes in spectral properties upon incubation with OMVs suggested changes in surface chemistries of QDs that altered their lifetimes. To probe these, lifetime measurements were done at each step of the strategy. The results indicated reduction in lifetimes i.e., faster relaxation of QD excited state electrons to the ground state from 121.72 ns for only QDs to 75.54 ns for QD-aptamer to 37.70 ns post conjugation to much larger OMVs. This provides promising prospects in the application of fluorescent QDs in bacterial detection via their OMVs.

Summary and future work: Successful binding of E.coli OMVs to InP/ZnS QD-aptamer probe has been confirmed as a sensitive detection strategy and ongoing work includes optimization and incorporation of this strategy to design an ELISA-based bacteria sensing platform.

Dr Linda Hofmann, Dr Annika Betzler, Prof Thomas Hoffmann, Prof Cornelia Brunner, Prof Marie-Nicole Theodoraki

Poster Pitches (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:45 PM - 1:00 PM

Introduction: Small extracellular vesicles (sEVs) have emerged as promising liquid biomarkers for a variety of solid tumors, including head and neck cancer. Current methods for analyzing sEVs, such as bead-based flow cytometry, analyze the bulk sample instead of single vesicles. As sEV populations in plasma are very heterogenous and represent a mixture released from different cell types, single vesicle analysis is highly desired for more specific diagnostic approaches. Single vesicle flow cytometry is challenging due to the small size of sEVs (50-150 nm) and requires machines with high sensitivity. This project aimed to establish single vesicle surface staining for the quantitative and standardized analysis of sEVs from plasma of head and neck cancer patients.

Methods: Plasma from patients with head and neck cancer was prepared from citrate blood by centrifugation at 1000xg and 2500xg for 10 min each. Saliva was collected using Salivettes. Informed consent was obtained as per ethics approval by Ulm University. sEVs were isolated by ultrafiltration and size exclusion chromatography (plasma) or ultracentrifugation (saliva) and thoroughly characterized according to the MISEV2018 guidelines (EV-Track IDs EV200068, EV210344). Staining protocol establishments and measurements were performed using spectral flow cytometer Aurora (Cytek).

Results: The flow cytometer settings (side scatter and fluorescence detector gains, trigger threshold) for optimal sEV measurement were determined using Nanosphere polystyrene size standards. Flow rate, light scatter and fluorescence were calibrated using reference material (Apogee, Rosetta, MESF beads) to be able to quantitatively determine sEV concentration, size and fluorescence intensity of the biological samples. Measurement of serially diluted sEVs were performed to determine the optimal sample dilution without swarm detection. Based on our previous work, panels for measurement of total sEVs as well as tumor-derived sEVs and the immune checkpoint PD-L1 were established. Respective antibodies were titrated and appropriate controls (unstained, isotype, detergens) were included.

Summary/Conclusion: Our established panels will be used for our future sEV-based biomarker studies on plasma- and saliva-sEVs for the identification and analysis of tumor-specific changes. They can further be applied by other researchers and are comparable interlaboratory due to the standardized and calibrated procedure.

Student Zehan Zeng, PhD Weilun Pan, Professor Jinxiang Chen

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

i. Introduction

Extracellular vesicle (EV) membrane proteins have shown significant clinical potential for the early diagnosis of tumors. However, the rapid isolation and detection of tumor-specific EV subpopulations from plasma still remains challenges. In order to address this, we have designed an integrated chip that incorporates self-sacrificial metal-organic frameworks (MOFs) made of ZIF-8 and aptamers (ZIF-8@Apt) for the specific capture of EVs. Additionally, we have employed a “fuel”-driven DNAzyme amplification approach for highly sensitive detection of these EVs.

ii. Methods.

Specifically, after capturing EVs with ZIF-8@Apt, numerous DNA nano-fuel structures are in situ assembled on the EV surface. Subsequently, the ZIF-8@Apt@EV@fuels composites are lysed by adjusting the pH to release Zn2+ and the EV's nucleic acids as cofactors and additional fuels for subsequent detection. Finally, a DNAzyme detection system is introduced, where the nano-fuel structures on the EV membrane and the leaked nucleic acids (miRNA-21 in this study) initiate the DNAzyme with the assistance of Zn2+.

iii. Results.

Due to the extraordinary surface area and small space hindrance of ZIF-8@Apt, the chip can effectively enrich the target EVs with an efficiency of 92.4%. With the fuel-based multi-step signal amplification strategy, the chip can capture and detect plasma EV membrane proteins within 60 minutes, achieving a significantly enhanced detection sensitivity of 500 particles/µL. Moreover, the chip's signal can be read using either fluorescence spectroscopy or imaging, offering a rapid, highly sensitive, and high-throughput analysis of plasma EV subpopulations for various applications.

In clinical cohort analysis for glioblastoma, this chip effectively differentiates between cancer patients and healthy individuals, achieving an area under curve (AUC) of 0.92. By changing the aptamers and fueling structures, the chip could be applied to analyze diverse EV protein profiles, offering a universal EV analysis platform.

iv. Summary.

Overall, this study presents an integrated method for the rapid and accurate analysis of plasma EVs, facilitating early diagnosis and disease monitoring.

Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Yulin Cao, Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Yuxuan Jiang, Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Qiubai Li, Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Yong Deng

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Fluorescent labeling detection of extracellular vesicles (EVs) is crucial for deciphering their biological functions within organisms. Insufficient sensitivity of the wide-field optical imaging used in current fluorescent molecular imaging (FMI) technology in detecting EVs necessitates the urgent development of reliable visualization techniques. Herein, we have enhanced the scanning method and image processing of fluorescence molecular tomography to develop a fluorescence molecular projection (FMP) imaging system and investigated the efficacy of FMP in detection of in vivo EVs.

Methods

EVs derived from mesenchymal stem cells were prepared and characterized following the MISEV2018 guidelines. C57BL/6J mice were injected with DIR-labeled EVs (30ug dissolved in 150ul PBS) through the tail vein, and then detected using our developed FMP and an existing FMI system, respectively. For developing FMP imaging, a biaxial galvanometer was employed to control the focused near-infrared beam (749 nm) for intensive scanning of the mice's surface. Concurrently, fluorescence projections were acquired using an EMCCD equipped with a 780 nm center wavelength filter. A second scan was performed using a 750 nm filter to obtain the excitation projections, and a bright field image of the mice was captured without any filter. The fluorescence projections were normalized against their corresponding excitation projections to generate normalized Born images. The final image was derived by normalizing all Born images pixel-wise and merging it with the mouse's bright field image.

Results

Our proposed FMP method, unlike the established FMI method, employed a focused near-infrared beam rather than wide-field light. The focused laser beam has the capability to penetrate deeper and excite lesser doses of the fluorescent target. Our results demonstrated that the FMP's specific scanning and image processing methods effectively imaged and analyzed the distribution of EVs in the bone marrow of the lower limbs, spine, and kidneys. Conversely, the FMI barely identified the same dose of EVs distribution in mice. Moreover, the FMP precisely and quantitatively identified the isolated femur and tibia, while the FMI still struggled to achieve similar results.

Summary/Conclusion

Our developed FMP enhances EVs detection sensitivity over the FMI system, potentially advancing measurement precision and fluorescence imaging technology development of EVs.

Associate Professor Ryo Ishihara, Hinako Yokohari, Ren Ogata, Kotomi Katori, Kentaro Doi, Kurumi Omiya, Tadaaki Nakajima, Eri Shimura, Takeshi Baba

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Extracellular vesicles (EVs) are promising biomarkers in liquid biopsy (Y. Yoshioka et al., Jpn. J. Clin. Oncol., 2018). Various EV analysis methods have been reported including single EV detection (C. Liu et al., Nano. Lett., 2018) and several EV analysis kits are commercially available. However, the EV analysis methods are generally time- and cost-consuming. For the facile EV analysis toward cancer point-of-care testing (POCT), surface-functionalized power-free poly(dimethylsiloxane) microchips (SF-PF microchips) have been developed (R. Ishihara et al., Membranes., 2022). In this study, we introduce the strategy of EV detection sensitivity improvement on the SF-PF microchips.

Methods:

The SF-PF microchip with partial low-height regions was prepared as follows. First, to immobilize the antibody that specifically captures EV the inner surface of the microchannel was modified with poly(2-aminoethyl methacrylate) (PAEMA) by UV grafting. Then, the antibody was immobilized on the PAEMA-grafted surface by dehydration condensation reaction and the microchip was degassed. The EV was detected with laminar flow-assisted dendritic amplification (K. Hosokawa et al., Anal Chem., 2007) on the SF-PF microchip and the limit detection was qualitatively evaluated.

Results and Summary

The EV signal increased with decreasing channel height, reaching a maximum at 8 µm. This can be attributed to the increase in the contact efficiency between the EV and antibody immobilized on the inner surface of the microchannel as the channel height was partially lowered. To increase the amount of EVs captured at the detection region, the detection protocol was also optimized. These two improvements reduced the LOD by 29-fold and the LOD was 0.63 × 10^11 particles/mL. Since the concentration of EVs in the blood is 1.2 × 10^11–6.0 × 10^11 particles/mL, the proposed method is expected to be able to detect EVs in blood. The detection time in the improved protocol was 19 min and the sample volume used was 2.0 µL. Although some of the reported EV detection methods using microchips have femtomolar or attomolar LOD levels, the proposed method is advantageous with respect to device portability. The SF-PF microchip might contribute to the establishment of EV-based cancer POCT.

Research Scientist Prashant Kumar, Brian Dobosh, Rabindra Tirouvanziam

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Conventional flow cytometry (FCM) is a valuable technique for precise measurement of cells and particles in the multi-micron range, providing accurate size, concentration, and phenotype data with high reproducibility. However, its utility is limited when dealing with nanoscale particles such as extracellular vesicles (EVs). To address this limitation, we undertook the optimization of various parameters for nanoflow cytometry (nFCM), including EV concentration, fluorescent membrane dye labeling concentration, antibody concentration, and incubation timing. The goal of this study is to describe a generic experimental design toward optimization of custom nFCM experiments.

Methods: EVs from various sources (HEK293T transfected with plasmids encoding the antigen of interest as positive controls or primary human neutrophils, monocytes or H441 lung epithelial cells) were isolated through differential centrifugation at 800 g, 3000 g, 20,0000 g followed by ultrafiltration (Amicon filters; 300 kDa cut off) or ultracentrifugation at 150,000 xg for 2 hours in a 70 Ti rotor, following MISEV recommendations. Nanoparticle tracking analysis (Malvern NS300) was used to estimate EV size distribution and concentration, and validated by transmission electron microscopy. EV samples were diluted and analyzed by nFCM (Cytek Aurora). A total of 5x10^6 EVs were labeled with 2 mM di-8-ANEPPS in the presence of Pluronic F127. Antibodies were titrated to a different concentration range, utilizing a fixed number of EVs. Calibration beads were used to ensure run-to-run reproducibility. Data were analyzed using FlowJo V10 software.

Results: We successfully titrated the concentrations of antibodies, dye and EVs, yielding reproducible single-particle resolution, fluorescent intensity, and populations of CD66b+ (neutrophil-derived), CD115+ (monocyte-derived), EpCAM (epithelial-derived) EVs. Based on these proof-of-concept study, we created a template for researchers to determine the optimal concentration for all dyes and antibodies for use in custom nFCM analysis of their EV population of interest.

Summary/Conclusion: This study offers innovative perspectives on nFCM, introducing two innovations: 1) using empirical methods, we optimized multiple experimental parameters to enhance data reproducibility; and 2) we addressed the steric hindrance resulting from the binding competence of three distinct monoclonal antibodies to their respective target antigens when these reagents are employed concurrently.

Dr. Tamás Visnovitz, Ms Kinga Dóra Kovács, Dr. Tamás Gerecsei, Dr. Beatrix Péter, Dr. Sándor Kurunczi, Ms Anna Koncz, Dr. Krisztina Németh, Ms Dorina Lenzinger, Dr. Krisztina V Vukman, Ms Anna Balogh, Ms Imola Rajmon, Dr. Péter Lőrincz, Dr. Inna Székács, Prof. Edit I Buzás, Dr. Róbert Horváth

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

Extracellular vesicles (EVs) attract substantial attention in biomedicine because of their ubiquitous potential role in cellular responses. Over the past decade, EVs have emerged as mediators of intercellular communication and important players of cellular homeostasis. Our study introduces an innovative approach: the direct nanoinjection of EVs into the cytoplasm of different cells and cell lines.

Methods

Utilizing robotic fluidic force microscopy (robotic FluidFM), we performed single cell nanoinjections in our experimental setup. This advanced technique enabled the direct delivery of GFP-positive EVs and EV-like particles into live cells, including HeLa, H9c2, MDA-MB-231, and LCLC-103H cell lines. The nanoinjected cells (both fixed and live) were monitored by confocal microscopy, providing insights into cellular responses. In fixed cells, our recently developed lactadherin-based membrane labelling approach was applied.

Results

Our nanoinjection platform demonstrated remarkable cell selectivity, emerging as a potent tool for studying the intracellular fate of the nanoinjected EVs. We also successfully nanoinjected mRNAs, plasmids, sEVs from bone marrow derived mast cells (BMMCs), HEK 293T-PalmGFP cell-derived small EV like particles (sEVLPs), and plasmid-containing EVLPs into live HeLa cells. Furthermore, sEVs from BMMCs were injected into MDA-MB-231, LCLC-103H, and H9c2 cells. Confocal microscopy revealed the deposition of EVs and EVLP clusters in the cytoplasm without immediate spreading. Following a 1-hour incubation, sEVs were transported within membrane-enclosed compartments towards the cell periphery, persisting in the cytoplasm even 24 hours after-nanoinjection. Injected EVs differed from endocytosed EVs as they avoided lysosomal co-localization and degradation.

Conclusion

Our proof-of-principle data show the efficacy of the robotic FluidFM platform for precisely targeting individual living cells with isolated EVs. This novel approach not only expands the technological repertoire for studying EVs but also holds promise for further study of intracellular EV cargo delivery at the single-cell level. Potential applications of this methodology may lead to better understanding of cellular responses and to advancing therapeutic interventions.

Dr. Maria Gracia Garcia Mendoza, Dr. John Nolan, Erika Duggan, Patrick Nolan, Kate Pilkington, Dr. Haley Pugsley

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

1) Introduction: Extracellular vesicles (EVs) are cell-derived, membrane-bound small particles less than a micron in size. EVs are potentially valuable biomarkers and therapeutic agents in many disease environments, including cancer, autoimmunity, and neurodegenerative disorders. Due to their small size and heterogeneity, EVs can be difficult to characterize. The single vesicle flow cytometry (vFC™) assay is a method developed with rigor and reproducibility capable of single EV measurements.

2) Methods: Cytek® Aurora™ instruments were outfitted with the Enhanced Small Particle (ESP™) Detection option to increase the side scatter sensitivity. Aurora systems were qualified using a set of hard-dyed beads (vCal™ nanoRainbow beads, Cellarcus Biosciences, Inc.) and calibrated with antibody capture beads (vCal™ nanoCal beads, Cellarcus Biosciences, Inc.) and a synthetic vesicle size standard (Lipo100™, Cellarcus Biosciences, Inc.) enabling spectral unmixing, followed by the conversion of fluorescence measurements to calibrated units for reporting. The multicolor vFC™ assay (Cellarcus Biosciences, Inc.), new to the Aurora system, was performed according to the manufacturer's workflows and protocols, using reference EVs, HEK 293T-derived recombinant EVs (Sigma), platelet-derived EVs (Cellarcus Biosciences, Inc.) and relevant positive and negative controls, as recommended by the MIFlowCyt-EV guidelines.

3) Results: Our results demonstrate that the sensitivity and resolution of the Aurora full spectrum flow cytometer, as evaluated using calibration beads and unmixing, were superior to the sensitivity and resolution observed when the data were analyzed in the conventional manner with compensation to address spillover. Using single vesicle flow cytometry and an Aurora spectral flow cytometer, we were able to detect vesicles as small as ∼90 nm with as few as ∼10 MESF of PE immunofluorescence. Implementing ESP™ light scatter detection on the Aurora system also improved the ability to resolve EVs with different light scatter efficiencies.

4) Conclusions: Here, we present a comprehensive workflow to analyze EVs, which includes instrument qualification, fluorescent channel calibration, sizing, and surface cargo measurements. These data help to address the requirements for improved standardization in reporting and reproducibility of EV flow cytometry data produced by spectral flow cytometry.

Lecturer Yutaka Naito, Professor Kazufumi Honda

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Intercellular communication plays an essential role in cancer initiation and progression through direct physiological contact and indirect interactions, such as growth factors. Recent studies have revealed that extracellular vesicles (EVs) are also regulatory players in such communication. However, few experimental systems can detect the spatiotemporal changes in EVs secreted by each cellular component under the presence of various cell types. Here, we analyze whether EV components change over time under direct and indirect coculture conditions in vitro. We focused on CD63, a conventional surface marker of EVs, and generated cancer cell lines expressing CD63 extracellular Achilles GFP (CD63exAch), which fused Achilles GFP into the extracellular loop domain of CD63. The EVs derived from cancer cells expressing CD63exAch are detectable by immunoprecipitation methods and the ExoCounter system, which can digitally count the number of EV particles using anti-GFP antibodies. We believe this system contributes to understanding the mechanisms of cancer progression and exploring new biomarkers reflecting the status of tumour stroma. We will also show whether the coculture of CD63exAch cancer cells and fibroblasts affects EV secretion and its molecular components.

Ms. Willemijn de Voogt, Dr. Richard Wubbolts, Dr. Pieter Vader

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles are endogenous, cell-derived nanocarriers that are naturally able to functionally deliver RNA cargo to recipient cells. This makes them promising candidate therapeutic RNA delivery systems. However, how EV-RNA cargo is trafficked post-uptake and subsequently released remains inadequately understood. Visualizing trafficking of EVs and their RNA cargo separately may help to illuminate these mechanisms. Currently, methods to fluorescently label endogenously loaded EV-RNA in a specific and sensitive manner are lacking. Here, we improved a click-chemistry-based metabolic labelling approach to label, visualize and track total RNA cargo in EV donor cells, isolated EVs and EV recipient cells.

Methods: We generated MDA-MB-231 donor cells stably overexpressing Uridine-Cytidine Kinase 2 (UCK2), which facilitates phosphorylation and, consequently, incorporation of nucleosides into RNA. Cells were treated with 5-Ethynyl Uridine (5-EU). Subsequently, EVs were isolated using size exclusion chromatography. 5-EU containing RNA was fluorescently labelled using click-chemistry. The number of RNA+ EVs was quantified by dSTORM imaging using a Nanoimager-S (ONI). Uptake of EVs and their 5-EU-RNA cargo was quantified by flow cytometry, while intracellular trafficking of EVs and 5-EU-RNA over time was assessed using fluorescence microscopy.

Results: MDA-MB-231 UCK2+ donor cells exhibited a 17-fold increase in 5-EU fluorescent signal after 2h compared to MDA-MB-231 wildtype cells. Quantitative cluster analysis of dSTORM images from isolated EVs revealed that using the metabolic labelling approach, ∼10% of the EVs stained positive for RNA cargo. MDA-MB-231 UCK2+ donor cell-derived RNA was detected in recipient cells by flow cytometry after coculturing or addition of isolated EVs. Finally, we show that our approach allows for assessing intracellular trafficking of EVs and RNA cargo separately, as well as their coincidence.

Summary/Conclusion: 5-EU treatment of MDA-MB-231 UCK2+ donor cells followed by click-chemistry enables efficient RNA labelling in donor cells. Moreover, by using this approach, we can specifically label endogenously loaded EV-RNA in isolated EVs and recipient cells. We utilized this system to localize the EVs and EV-RNA cargo post-uptake over time. This may help to elucidate the mechanisms that underly functional RNA delivery by EVs

Mr Jakub Tomaszewski¹, Bsc Wiktoria Klimek², PhD Hanna Kozłowska³, PhD Małgorzata Czystowska-Kuźmicz¹

¹Department of Biochemistry, Medical University of Warsaw, Warsaw, Poland, ²Faculty of Biology and Biotechnology, Warsaw University of Life Sciences, Warsaw, Poland, ³Laboratory of Advanced Microscopy Techniques, Mossakowski Medical Research Institute Polish Academy of Sciences, Warsaw, Poland

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular Vesicles (EVs) are small membranous entities that are released from living cells either as a mean of intercellular communication, stress response, and in other situations, many of which are yet to be described. The study of their release, distribution and uptake necessitates labeling Evs for the purpose of imaging and optical tracking. In this three most commonly used approaches to EVs labeling: lipophilic dyes, fluorescent protein expression and surface immunofluorescence were assessed and compared.

Methods: THP1 cell line (ATTC: 88081201) was used as the source of EVs for the study. Evs smaller than 200nm (small EVs, sEVs) were then isolated from the conditioned culture media by Size Exclusion Chromatography (SEC), followed by an appropriate staining protocol. Both cell lines: THP-1 CD63 conjugated with eGFP and tdTomato were used to obtain Evs dyed with fluorescent proteins. Size and concentration of the obtained vesicles were characterized before and after staining using Nanoparticle Tracking Analysis (NTA, ZetaView, Particle Metrix). Stained Evs were then added to a THP-1-derived macrophage culture, and incubated for 12 hours. The efficiency of Evs uptake was assessed with the ELYRA PS.1 (Zeiss imaging system PALM and SR-SIM). The three staining methods were compared based on their price, efficiency, influence on Evs size and influence on the cellular uptake rate.

Results: Constitutive expression of membrane-bound fluorescent proteins proved to be the cheapest and most efficient of all methods. Due to the nature of this technique, genetic labelling didn't affect the vesicle size. The uptake rate was influenced mostly by immunofluorescent labeling. Lipophilic stains increased the size of Evs the most.

Summary/Conclusion: Three most common Evs labeling techniques were successfully compared using super-resolution imaging. Presented comparison provides a viable guideline for laboratories intending to implement EVs imaging.

Mr Satendra Jaysawal^1,2, Dr. Rocky Chowdhury^1,2, Mr. Rajindra Napit^1,2, Ms. Jasmine Catague^1,2, Mr. Haben Melke^1,2, Dr Cuong Pham³, Dr. Wei Duan^1,2

¹School of Medicine, Deakin University, Geelong, Australia, ²Institute of Mental and Physical Health and Clinical Translation (IMPACT), Deakin University, Geelong, Australia, ³Molecular Imaging and Theranostics Laboratory, Baker Heart and Diabetes Institute, Melbourne, Australia

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction:

Fluorescence polarization (FP) is a powerful technique employed in molecular biology and analytical chemistry for monitoring molecular interactions. This abstract highlight the integration of aptamers, single-stranded DNA or RNA molecules with high affinity and specificity for a target, into fluorescence polarization assays as a novel biosensing strategy for the detection of the CD9 protein expressed on the surface of sEVs.

Method:

In this study, a capture antibody, Biotin-labelled anti-human CD63 antibody, was coated on a neutravidin-coated black well plate, which was then incubated with 2.0 X 108 sEVs (from serum or MDA-MB-231 CD9-KO #14 cell culture media) for 16 hours. For selectivity and specificity tests, a control sample was prepared using 1% Triton X-100 to treat and lyse sEVs immobilized on the well. Subsequently, all wells, containing both sEVs and lysed sEVs, were washed, and 5nM of folded FAM-conjugated aptamer (A17-CD aptamer specific to CD9 protein and random 43 as a negative aptamer) was added. The fluorescence intensity using a FITC filter was used to measure the fluorescence polarization, where millipolarization (mP) units were used to express the ΔFP values.

Result:

The average change in fluorescence polarization values for all control samples were as follows: FAM-Random 43 aptamer (not binding to CD9) control: 0.37 mP, Triton X-100-treated serum sEVs (lysed sEVs) control: -0.10 mP, and MDA-MB-231 CD9-KO #14 sEVs (lacking CD9 expression) control: -0.97 mP. The experimental sample FAM-CD9-A17-CD aptamer exhibited ΔFP values of 6.93 mP.

Conclusion:

All control values were significantly lower compared to those in the wells containing immobilized sEVs from human serum with FAM-CD9-A17-CD aptamer demonstrating the capability to selectively detect sEVs from serum expressing CD9 and CD63 proteins on its surface with A17-CD aptamer using FP technique.

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, Lyon, France

Sponsor Exhibition, Poster Session and Lunch (Friday), Exhibit hall-Doors 14-15, May 10, 2024, 12:00 PM - 2:00 PM

Introduction

The world of extracellular vesicles (EVs) is highly diverse, with variations in size, internal composition, and membrane-bound proteins. Understanding this diversity and its significance is crucial for unraveling the physiological role of EVs. In this study, we explore the use of flow cytometry to characterize EVs and enhance our comprehension of their functions.

Methods

To assess the potential of flow cytometry in detecting, characterizing, and isolating EVs, we employed nano flow cytometry for analysis and sorting. Engineered EV (GFP), EVs isolated from biological samples and beads of various sizes were used in this study. EVs isolation was performed using ultracentrifugation or SEC. EVs were analyzed for their size and for the fluorescent reporter to test the sensitivity of the flow cytometer. FCM PASS software was used for light scatter standardization.

Results

By utilizing flow cytometry, we compared the quality of EV samples obtained through ultracentrifugation. Through side scatter analysis in the violet channel (V-SSC), we successfully identified the isolated EVs. Furthermore, we were able to resolve particles as small as 40 nm, highlighting its effectiveness in characterizing small EVs. To confirm the heterogeneity of the EV preparation, EV preparations were sorted. Post-sort data revealed distinct profiles based on scatter and fluorescence characteristics. Finally, we highlight the superior sensitivity of the last generation of flow cytometer dedicated for nanoparticles. EV-related data was further analysed using the FCM PASS method. Overall, these findings strongly support the use of flow cytometry for counting, characterizing, and sorting EVs.

Conclusion

The field of EVs is rapidly advancing, necessitating a deeper understanding of their heterogeneity. This knowledge is crucial for comprehending their physiological roles and involvement in diseases. In this study, we successfully applied flow cytometry to analyze EVs, employing various flow cytometry tools. Further research will enable the development of tailored flow cytometry protocols for EV characterization based on specific requirements.

Ph.D candidate Youngju Seo¹, Ibio hyejin Kang, Ibio, Mechanical Engineering Jaesung Park

¹Postech, South Korea

Introductory Talk and Oral Session: OF13 Stem Cell EV Therapy, Plenary 1, May 10, 2024, 4:00 PM - 5:35 PM

Introduction

Mesenchymal stem cell-Extracellular vesicles have drawn attention as promising therapeutic agents; however, their practical utility is limited by low EV yields. Cell culture under fluidic flow has been proposed to address this challenge to enhance EV secretion. However, the precise mechanism of increased EV production in response to flow conditions has yet to be thoroughly studied. Here, we investigated the mechanism of higher release of EVs from mesenchymal stem cells under flow conditions, focusing on the correlation between intracellular calcium ions and EV production.

Methods

First, we prepared the shaking cell culture system using the orbital shaker, optimizing to exert 0.05 dyne/cm2 of shear stress to cells. Then, there are changes in the calcium ion levels of cells in various culture conditions. The intracellular calcium ion concentration under shaking conditions and static conditions were confirmed. The source of increased calcium ions was investigated by altering calcium ion concentration in culture media and the presence of a calcium ion channel blocker, which is assumed to influence intracellular calcium ion levels. Further, we collected EVs from the cells in various culture conditions by ultracentrifuged twice and characterized them. The molecular marker based on single vesicle analysis and therapeutic effect on acute kidney injury model in vitro was analyzed.

Results

The results confirmed that when MSCs are exposed to shear stress, rather than being released from ER-stored calcium ions, extracellular calcium ions are transported into cells through plasma membrane-located ion channels. When evaluated by single-vesicle analysis of MSC-marker expression, the numbers of the EVs produced and released under shear stress were about 10 times higher than those under static conditions. In addition, EVs produced through the high-production method maintain their function as a therapeutic effect.

Summary/conclusion

The observation suggested that extracellular calcium ions are transported through plasma membranes, increasing the intracellular calcium level and producing 10 times higher EV production under the flow conditions. Although the condition used for this study is partially optimized, the results may contribute to the production of therapeutic agents of MSC-derived EVs by overcoming the current problem of EV shortage.

Miss Cynthia Garcia Guerrero, Patricia Luz-Crawford, Ana Maria Vega-Letter, Carolina Pradenas, Alexander Ortoff, Jose Barraza, Fernando Figueroa, Maroun Khoury, Aliosha Figueroa, Yeimi Herrera

Introductory Talk and Oral Session: OF13 Stem Cell EV Therapy, Plenary 1, May 10, 2024, 4:00 PM - 5:35 PM

Introduction

Osteoarthritis (OA) is a progressive degenerative condition linked to cartilage loss, mineralization, and joint inflammation due to disrupted mitochondrial homeostasis in chondrocytes, leading to increased oxidative stress. Mesenchymal Stem Cells (MSCs) have been studied for their regenerative potential in OA mediated by their paracrine mediators. Between them, small extracellular vesicles (sEVs) and mitochondria (MITO) show promising therapeutic potential as recent findings support that sEVs protect against oxidative damage, while mitochondrial transfer rescue cells with dysfunctional mitochondria. Our proposal suggests that sEVs and MSC-derived mitochondria remain localized in the damaged area without migrating outside the joint, effectively reducing the clinical development and progression of OA without triggering any immune response.

Methods

sEVs and mitochondria were obtained from Human Umbilical Cord (UC-MSCs) through ultracentrifugation processes. sEVs were characterized and quantified using Nanoparticle Tracking Analysis and flow cytometry. To asses biodistribution, sEVs and MITO were labeled with DIR or Mitoview720 respectively and intra-articularly injected into mice. The migration was assessed using near-infrared fluorescence analysis.

For in vivo assessment, mice received intra-articular collagenase VII injections to induce OA (CIOA). The CIOA model was then treated with UC-MSCs, sEVs or MITO by intra-articular injection. Knee samples obtained post-euthanasia were analyzed via X-ray-microtomography for bone mineralization and histopathology for OA clinical progression. To assess immunogenicity, popliteal lymph nodes were extracted post-sEVs or mitochondria administration to assess T lymphocyte pro- or anti-inflammatory populations by flow cytometry.

Results

Our results demonstrate that sEVs from UC-MSCs exhibited a size of approximately ∼150nm and the presence of CD63, CD9, and CD81 markers. The vesicles and mitochondria remained concentrated in the joint cavity without redistributing to other tissues even a week after intra-articular administration. The sEVs and mitochondria derived from UC-MSC did not trigger any immunogenic response following their administration. In the CIOA model, sEVs and mitochondria significantly reduced joint mineralization and decrease the clinical damage score obtained from joint histology.

Summary

sEVs and mitochondria derived from MSCs exhibited non-migratory and non-immunogenic behavior post-administration and significantly attenuated joint cartilage damage and mineralization. This suggests their capacity to impede OA progression and facilitate joint repair, suggesting the possibility of joint regeneration.

Scientist Jieun Lee

Introductory Talk and Oral Session: OF13 Stem Cell EV Therapy, Plenary 1, May 10, 2024, 4:00 PM - 5:35 PM

Introduction: There are 800,000 stroke victims per year each year who have significant neurological deficits indicating a critical unmet medical need for new therapies to treat ischemic stroke. Exosomes-derived from adult stem cells such as MSCs have shown promising therapeutic potential in a variety of animal models of stroke and other ischemic diseases. However, manufacturing adult stem cell exosomes at scale presents challenges of purity, identity, and stability. We therefore explored the use of hESC-derived-clonal vascular progenitor cell lines (eEPC) as an exosomes source because of the increased scalability and regenerative capacity of embryonic cells. We selected eEPC-exosomes with the highest activity in both scratch wound and tube forming angiogenic assays for preclinical studies. Our results indicate that eEPC-exosomes can effectively mediate angiogenesis and tissue regeneration in rodent model of stroke.

Methods: We developed large-grade protocols for exosome production and purification which combine methods of TFF and SEC. We characterized exosome cargo by screening RNA-seq, and proteomic mass spectrometry analysis. The angiogenic activity of a panel of eEPC-exosomes was assessed in vitro using live-cell imaging scratch wound and vascular tube forming assays. We further assessed the most active exosomes in a rat middle cerebral artery occlusion model of stroke. We assessed neurological recovery following injury in ischemic animals and assigned a neurological severity score.

Results: The human eEPC-exosomes were directly taken up by HUVECs and promoted the migration, proliferation, and tube formation of endothelial cells. We found that eEPC-exosomes showed higher angiogenic potency than primary MSC-derived exosomes. The eEPC-exosomes were enriched with angiogenic miRNAs(miR-126), anti-inflammatory miRNA(miR-146), and anti-apoptotic miRNAs(miR-21). In vivo, mNSS scores showed that sensorimotor function in ischemic MCAO rats was significantly increased by intravenous administration of eEPC-exosomes and exceeded recovery by treatment with umbilical cord stem cells. We investigated the potential mechanism of eEPC-exosomes in alleviating ischemic stroke injury and inflammation by assessing the expression of endothelial, neuronal, and inflammatory markers.

Conclusion: These data demonstrate the potential for using eEPC lines as a highly scalable source of therapeutic exosomes. We anticipate that eEPC-exosomes will be a valuable resource for developing EV therapies for stroke and other ischemic diseases.

Mr Naveen Kumar¹, Dr Ishmael Inocencio, Dr Tamara Yawno, Dr Dandan Zhu, Associate Professor Rebecca Lim

¹Hudson Institute Of Medical Research, Clayton, Australia

Introductory Talk and Oral Session: OF13 Stem Cell EV Therapy, Plenary 1, May 10, 2024, 4:00 PM - 5:35 PM

Introduction: Infection of the fetal membranes and amniotic fluid, termed chorioamnionitis, causes inflammatory brain injury and is a major risk factor for preterm birth. The combination of impaired and incomplete brain development greatly increases the risk of lifelong neurological impact. Currently, there is no cure for perinatal brain injury, and neuroprotective therapies that address inflammation are in dire need. Human amniotic epithelial cells (hAEC) and their extracellular vesicles (EVs) both demonstrate anti-inflammatory effects and mitigate brain injury. However, hAEC lack proliferative ability, thus making hAEC-EV manufacturing difficult to scale. We addressed this limitation by developing immortalised hAEC (ihAEC) lines to serve as a continual EV source and reduce donor-to-donor variation. We evaluated the therapeutic efficacy of ihAEC-derived EVs compared to umbilical cord mesenchymal stem cell EVs (hUC-MSCs) in a murine model of antenatal inflammation and postnatal hyperoxia.

Methods: ihAEC-EVs and UC-MSC-EVs were isolated via tangential flow filtration and size exclusion chromatography. Perinatal brain injury was induced in C57BL6 mice via intra-amniotic lipopolysaccharide (LPS) at E16 and hyperoxia exposure (65% O2) at birth (E21). Injured mice were randomly assigned to receive intravenous (IV-ihAECEVs (n = 9), IV-hUC-MSCEVs (n = 10)) or intranasal (IN-ihAECEVs (n = 14), IN-hUC-MSCEVs (n = 11)) delivery of treatments. Treatments were administered on postnatal day 1 and compared to vehicle (IV-vehicle (n = 11), IN-vehicle (n = 14)) and healthy controls (n = 12). All mice were euthanised on postnatal day 14 for brain histological analysis.

Results: LPS/hyperoxia injury reduced IBA-1 positive cells in the cerebral cortex for both intravenous (p = 0.0155) and intranasal (p = 0.0307) cohorts. TUNEL-positive cells increased in dentate gyrus for the intravenous cohort (p = 0.0155). IV-hUC-MSCEVs treatment significantly reduced TUNEL-positive cells compared to LPS/hyperoxia injury (p = 0.0155). TUNEL-positive cells in the cerebral cortex decreased following IV and IN delivery of ihAECEVs and hUC-MSCEVs, but this did not reach statistical significance.

Conclusion: Region-specific brain injury was observed following antenatal inflammation and postnatal hyperoxia. IV and IN ihAEC- and UC-MCS EVs administration did not induce or exacerbate brain injury, indicative that EV exposure is safe in 1-day-old mice. Further research on dosage and therapeutic modality will be undertaken.

Miss Eliana Lara Barba, Miss Yesenia Flores Elías, Mr Felipe Bustamente Barrientos, Miss María Jesús Araya, Miss Yeimi Herrera Luna, Miss Noymar Luque Campos, Ms Ana María Vega Letter, Ms Patricia Luz Crawford

Introductory Talk and Oral Session: OF13 Stem Cell EV Therapy, Plenary 1, May 10, 2024, 4:00 PM - 5:35 PM

Introduction: MSCs are multipotent, fibroblast-like cells that exert various biological functions, including immunosuppressive activity, making them attractive for autoimmune disease treatment. Their immunomodulatory activity is mainly mediated by paracrine factors. However, the release of small extracellular vesicles (sEVs) by these cells has been demonstrated as the principal mechanism by which MSCs perform their biological effects. Our studies on human umbilical cord MSCs showed that metabolic reprogramming to glycolysis significantly improves their immunoregulatory capacity over proinflammatory T cells (Th1, Th17) through the induction of T regulatory cells (Treg). Therefore, in the present study, we evaluated the role of sEVs from glycolytic and non-glycolytic UC-MSCs in their immunosuppressive properties in vitro and in vivo.

Material and Methods: First, we obtained different fractions of glycolytic or non-glycolytic MSC secretomes, including their sEVs, characterized by NTA and FACS for specific sEV markers, and evaluated their immunosuppressive activity over PBMCs. Proinflammatory T cells and the induction of T regulatory cells were also assessed. Furthermore, sEV internalization in T cells was evaluated by FACS and confocal microscopy techniques. Moreover, we assessed the sEV effect on memory T-CD4 cells cultured in the presence or absence of sEVs. The phenotype of proinflammatory and anti-inflammatory cells in memory T-CD4 cells was evaluated by FACS, and IL-10 production was assessed by ELISA. Finally, we evaluated sEVs immunosuppressive activity in vivo in a mouse model of Delayed-type hypersensitivity.

Results: We found that the MSC glycolytic conditioned medium and their sEVs significantly decreased the proliferation of CD4+T cells, reduced Th1 cells, and induced Treg cells in vitro. Our internalization experiments showed that sEVs are incorporated into memory T-CD4+ cells and decrease the percentage of Th1 and Th17 cells, while having no effects on the percentage of Treg cells, with an increase in IL-10 production. In vivo assays show that glycolytic sEVs-MSC significantly decrease the inflammatory response, reducing proinflammatory T cells, specifically Th1 cells.

Conclusion: Glycolytic MSC-sEVs can be internalized and specifically modulate activated T cells, regulating the frequency of T cell proliferation and phenotype, exhibiting in vitro and in vivo immunomodulatory effects on T cells, demonstrating the potential of this immunosuppressive tool.

Dimitrios Kapogiannis, Maja Mustapic, Carlos Nogueras-Ortiz, Apostolos Manolopoulos, Francheska Delgado-Peraza, Pamela Yao, Krishna Pucha, Mark A Espeland, Luigi Ferrucci, Stephen R. Rapp, Susan M. Resnick

Introductory Talk and Oral Session: OF14 Biomarkers of Brain Disease, Eureka, May 10, 2024, 4:00 PM - 5:35 PM

Introduction: The APOE ε4 allele is the most robust genetic risk factor for late-onset Alzheimer's disease (AD). However, many ε4 carriers survive to old age without cognitive impairment. We used neuronal-enriched Extracellular Vesicle (NEV) biomarkers to determine biological factors that predict cognitively healthy status after age 80 in the presence of e4 (APOE ε4 “escapees”) or in ε3/ε3 individuals with average AD risk.

Methods: We isolated NEVs from 676 women with ε4 or ε3/ε3, participants in the Women's Health Initiative (WHI)/ Long Life Study (LLS), who provided blood samples at baseline and 13-17 years later and eventually became cognitively impaired (Mild Cognitive Impairment or dementia) before age 80, after age 80 or remained unimpaired. In 1352 EDTA plasma samples, we performed immunoaffinity capture for neuronal marker L1CAM, and quantified protein biomarkers for core AD pathologies (total tau, p181-Tau, Aβ42), neuronal response to neuroinflammation (pSer536-NFκB, TNFR1), insulin resistance (pSer312-IRS1) and mitochondrial energy generation (Complex V). Neuronal cargo enrichment was confirmed by Flow Cytometry Analysis for neuronal markers (VAMP2, Tuj1, L1CAM). Analysis used repeated measures mixed models. The geometric mean of CD81, CD9, and CD63 measured on intact EVs by electrochemiluminescence was used to normalize for EV yield; age and years of education were additional covariates.

Results: At baseline, ε4 “escapees” did not have different NEV-associated levels of core AD biomarkers or pSer312-IRS1 compared to women with ε4 who eventually developed MCI/dementia. However, ε4 “escapees” had higher NEV-associated pSer536-NFκB, TNFR1 and Complex V compared to women with ε4 who became impaired after or before age 80 (p = 0.015 and 0.025; 0.017 and 0.024; and 0.004 and 0.09 (trend), respectively). Compared to ε3/ε3 women who remained unimpaired, ε4 “escapees” had higher NEV-associated pSer536-NFκB and TNFR1 at baseline (p = 0.003 and 0.005, respectively) and during LLS visit (p = 0.037 and 0.033, respectively).

Summary/Conclusion: This large NEV biomarker study provides insights into the mechanisms underlying cognitive resilience in the presence of APOE ε4. Augmented TNFR1/NFκB pathway response to neuroinflammation and mitochondrial energy production at baseline characterize ε4 “escapees”. These findings motivate novel hypotheses and open avenues for AD prevention and treatment.

Dr. Hao Tian, Dr. Yanling Cai, Mrs. Fang Wang, Professor Chuanzhi Duan, Dr. Haitao Sun

Introductory Talk and Oral Session: OF14 Biomarkers of Brain Disease, Eureka, May 10, 2024, 4:00 PM - 5:35 PM

Introduction: Early diagnostic methods for intracranial aneurysms are scarce, and most patients are asymptomatic before the rupture of aneurysms. The aneurysmal subarachnoid hemorrhage caused by the rupture has a high mortality and disability rate. Exploring the heterogeneity of plasma single extracellular vesicles (EVs) between patients with unruptured intracranial aneurysms (IAs) and healthy individuals using proximity barcoding assay (PBA), to identify potential biomarkers related to the progression of IAs which would be used for the early diagnosis and further exploration of potential molecular mechanisms of unruptured IAs.

Methods: This study included 35 unruptured IA patients diagnosed with digital subtraction angiography and 20 matched healthy individuals without IAs. PBA was used to bind antibody-rolling circle DNA conjugates to the surface proteins of single EVs, thereby converting the surface proteins of EVs into DNA sequences that can be sequenced, which can be used to achieve the separation and labeling of single EVs. Consequently, surface proteome data of single EVs were detected using high-throughput sequencing which was further analyzed by bioinformatics.

Results: The levels of PECAM1, ITGB1, and CD151 on the surface of plasma EVs were significantly upregulated (FDR<0.05, |log2FC|>0.585) after the occurrence of IA. In addition, 34 double-protein combinations with early diagnostic potential for IA were identified on individual EVs (P<0.01, AUC>0.7). After further dimension reduction and clustering of the surface protein characteristics of single EVs, a group of EVs with significant differences between the two groups was identified (p = 0.02), with characteristic surface proteins including PECAM1 and ITGB1.

Conclusion: Surface proteins of plasma EVs in patients with IAs showed heterogeneity compared with healthy controls, and plasma EV surface PECAM1 and ITGB1 are potential biomarkers for unruptured IAs. The surface protein of plasma EVs is expected to be used for the early diagnosis of IAs.

Hülya Torun^{Stanford and Koç University}, PhD Ugur Parlatan, Chris Nguyen, BS Tim Valencony, MS Furkan Kaysin, PhD Ozgur Albayrak, MD Ibrahim Kulac, MD, PhD Candidate Oguz Baran, MD, PhD Candidate Goktug Akyoldas, MD Ihsan Solaroglu, PhD Utkan Demirci, PhD, DVM Demir Akin, PhD Mehmet Ozgun Ozen

Introductory Talk and Oral Session: OF14 Biomarkers of Brain Disease, Eureka, May 10, 2024, 4:00 PM - 5:35 PM

Introduction:

Glioblastoma, the most aggressive adult brain tumor, requires sophisticated laboratory settings and prolonged and costly molecular testing for accurate diagnosis. The current average survival of 14 months underscores the urgency for rapid, accurate, and cost-effective diagnostic strategies. Our study directly addresses this urgency through the introduction of a transformative diagnostic paradigm - a liquid biopsy approach merging Surface-Enhanced Raman Spectroscopy (SERS) analysis of exosomes with artificial intelligence (AI).

Methods:

We conducted a prospective study to evaluate the efficacy of a SERS and AI-based liquid biopsy analysis of plasma exosomes for glioblastoma detection. The methodology involved the application of SERS for the identification of glioblastoma plasma exosome signatures from clinical samples. 17 glioblastoma, 20 meningioma, and 30 healthy plasma samples were collected within informed written and oral consent under the Koc University IRB approval. Exosomes were isolated using Exosome Total Isolation Chip (ExoTIC) and characterized according to MISEV 2018 guidelines utilizing TEM, cryo-EM, flow cytometry, NTA, and western blotting techniques.

SERS was employed to analyze the isolated plasma exosomes, generating spectral data on molecular signatures indicative of each condition. Deep learning algorithms were integrated into the analysis pipeline to facilitate rapid and accurate differentiation between glioblastoma, meningioma, and healthy plasma exosome SERS signatures.

Results:

Our liquid biopsy approach utilizing SERS and AI demonstrated a remarkable 87% prediction accuracy in distinguishing glioblastoma exosomal signatures from those of meningioma and healthy individuals. This result signifies a substantial advancement in the precision and speed of glioblastoma diagnostics compared to traditional methods.

Summary/Conclusion:

This pioneering liquid biopsy technique emerges as a front-line solution for glioblastoma detection, differentiation, and monitoring. The ongoing integration of an expansive library of tumor signatures signifies a significant leap forward in analytical technologies tailored for neuro-oncology. The study demonstrates our approach's technological superiority as well as its real potential to revolutionize the glioblastoma detection landscape and, ultimately, to improve patient care. The presentation aims to engage the audience in understanding the study's design and results, highlighting the possible transformative impact of this novel methodology.

***Information disclosure: A patent application is pending, therefore limiting information disclosure

Dr. Thamara Dayarathna, Dr. Austyn Roseborough, Dr. Janice Gomes, Dr. Reza Khazaee, Dr. Shaun Whitehead, Dr. Hon Sing Leong, Professor Stephen Pasternak

Introductory Talk and Oral Session: OF14 Biomarkers of Brain Disease, Eureka, May 10, 2024, 4:00 PM - 5:35 PM

Introduction: Currently there are no widely accepted diagnostic tests for identifying the earliest stages of Alzheimer's disease (AD) which could transform clinical trials and clinical treatment and monitoring guidelines. Although there is much excitement in the field of biomarker development, most proposed tests for AD are expensive, require highly specialized sample handling, invasive approaches or radiation exposure. The goal of this project was to develop non-invasive AD biomarker assessment using nanoflow cytometry to examine proteins on circulating brain-derived extracellular vesicles in plasma.

Methods: Plasma samples were collected from a population of individuals clinically diagnosed with MCI and mild, moderate or severe Alzheimer's Disease and cognitively intact control subjects. We evaluated more than 40 potential biomarkers which have previously been investigated in AD. Plasma was incubated with fluorescently-conjugated antibodies against candidate biomarkers including oligomeric amyloid, fibrillar amyloid, amyloidβ-42, pTau-T181, pTau-T217, pTau-S235, Ubiquitin, Neurofilament, α-synuclein and synaptophysin. After incubation, labelled events were quantified using the Apogee Micro Plus nanoscale flow cytometer for single event analysis. Using Receiver-Operator Curve (ROC) analysis, individual markers and various combinations to distinguish diagnostic groups was determined.

Results: Nanoscale flow cytometry quantification of pTau-T181, pTau-T217, pTau-S235, pTau-T231, Aβ-42 (among others) successfully distinguished MCI and AD plasma from healthy controls. Using ROC analysis, our most sensitive antibodies (pTau- T231) separate controls from AD patients with an AUC of 0.96 (Sens: 0.95/Spec: 1.0). Other prominent antibodies include pTau 181 with an AUC of 0.93 (Sens: 0.92/Spec: 0.92). The most promising combination of antibodies combines beta-amyloid42 and pTau-181 to separate AD patients from controls with an AUC of 0.960 (Sens: 0.93, Spec: 0.92). Isolation of EVs from plasma and validation of marker labeling was performed with Western Blot, ELISA and transmission electron microscopy.

Conclusion: Here we demonstrate the use of nanoscale flow cytometry to directly quantitate proteins on circulating extracellular vesicles in plasma from MCI and AD patients. The assays are rapid, inexpensive, directly quantitative, and require minimal sample volume and manipulation. Future studies are required involving a larger population of patients who have had ‘gold standard’ biomarkers (Amyloid and tau CSF measurements), longitudinal follow-up and/or autopsy confirmed diagnosis of AD.

Ms. Sanskriti Rai, Mr. Rishabh Singh, Dr. Prahalad Singh Bharti, Dr. Roopa Rajan, Dr. Saroj Kumar

Introductory Talk and Oral Session: OF14 Biomarkers of Brain Disease, Eureka, May 10, 2024, 4:00 PM - 5:35 PM

Introduction: MicroRNAs (miRNAs) are a class of small non-coding RNAs known to circulate freely or encapsulate within small extracellular vesicles (sEVs). miRNAs from plasma-derived small extracellular vesicles (PsEVs) and whole plasma are minimally invasive potential biomarkers for early detection, prognosis, and disease monitoring; however, there is heterogeneity in miRNA expression in both. This study systematically compares the diagnostic potential of miRNAs in PsEVs and plasma to address the need for precise molecular diagnostics of Parkinson's disease.

Method: 60 subjects [20 each from PD, age-matched (AMC), and young controls (YC)] were recruited after obtaining written informed consent (Ethical Ref. No.: IECPG-766/30.11.2022). PsEVs were isolated using chemical precipitation method and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western-blot of the sEVs marker (CD-9 and Flotillin), Calnexin and neuronal marker (CD171). Small RNA sequencing was done using Illumina single-end sequencing, and miEAA 2.0 software was used for miRNA enrichment analysis and annotation. Quantitative PCR validation was done in Plasma and PsEVs.

Results: 336 miRNAs specific to PsEVs and 432 in plasma were identified. In both groups, 245 miRNAs were common with 18% upregulated and 16% downregulated miRNAs (log2FC ±1.2). miEAA analysis revealed that PsEVs had CSF-specific miRNAs but were not observed in plasma. Both groups contained nerve-specific miRNAs. Out of 245, miR-339-3p and miR-23b-3p were selected which regulate genes involved in alpha-synuclein metabolism, neuroinflammation, mitochondrial function, and synapse assembly based on differential read counts and downstream analysis. The expression of miR-339-3p was significantly upregulated in PsEVs (Log2FC = 1.6829, p = 0.0005) and downregulated in plasma (Log2FC = -1.2304). miR-23b-3p also showed opposite regulation, with higher diagnostic accuracy in plasma (AUC = 0.8086; Sensitivity = 93.75%, Specificity = 68.75%; p = 0.0029) and in PsEVs (AUC = 0.7278; Sensitivity = 84.62%, Specificity = 69.23%; p = 0.0483) respectively.

Conclusion: This is the first direct assessment of the miRNA profile in whole plasma and PsEVs for biomarker targets in Parkinson's disease. Our results indicate that the overall PsEVs-derived miRNA profile is comparable with the entire plasma. However, some miRNA expressions were the opposite. These findings emphasize the importance of exploring plasma and PsEVs for comprehensive Parkinson's disease diagnostics.

Funding: DHR (GIA/2020/000595), CSIR [09/006(0519)/2019-EMR-I]

Keywords: sEVs, Plasma, Parkinson's disease, Biomarker, miRNAs

Ph.d Yaoyao Lu, research assistant Nathalie Majeau, Ph.D Gabriel Lamothe, Research assistant Joel Rousseau, Professor Jacques-P Tremblay

Introduction Talk and Oral Session: OF15 Engineering EVs, Room 105-106, May 10, 2024, 4:00 PM - 5:20 PM

Introduction:

Therapeutic genome editing has the potential to cure diseases by directly correcting genetic mutations in target pathological tissues and cells. Recent progress in the CRISPR/Cas9 system provided a breakthrough in gene editing tools due to its high versatility and efficiency. However, its safe and effective distribution to the target organs in patients is a major hurdle. Our study aimed to encapsulate the CRISPR/Cas9 ribonucleoprotein into extracellular vesicles (EVs) by using protein palmitoylation modification, which is the reversible addition of fatty acids to the cysteine residues of the protein. It is an important post-translational modification that regulates protein function, including the localization to membranes, protein stability, intracellular trafficking, protein interactions, and protein conformation. In this study, we demonstrate that the palmitoylation modification on SpCas9 mediated the components CRISPR/Cas9 to be encapsulated into EVs, which permits gene editing in the recipient cells.

Methods:

We obtained the different peptide sequences from the N-terminus of the palmitoylation substrate proteins, which is the ideal substrate for palmitoyltransferase, the enzyme that catalyzes palmitoylation. We first fused oligonucleotides coding for those peptides to the 5’end of both the eGFP and SpCas9 genes. This promoted the palmitoylation and encapsulation of the resulting proteins into EVs. The encapsulation efficiency of palmitoylated eGFP and SpCas9 was verified by flow cytometry and western blotting. Meanwhile, the packaging efficiency of the components of CRISPR/Cas9 was verified in reporter cells.

Results:

The palmitoylation modification permitted 87.2% of eGFP entry into EVs. The N-terminus palmitoylation modification did not negatively affect the activity of SpCas9 and was successfully encapsulated into EVs. EVs coated with VSV-G, which encapsulated the components of CRISPR/Cas9 restored 10.5% gene editing in double-cutting sites of Ai9 reported cells and 98.6 % gene restoration in surrogate reporter cells with a single-cut with modification.

Conclusion:

Our study provides a novel approach to encapsulating the CRISPR/Cas9 system into EVs. This may open an effective avenue for using EVs as vehicles to deliver CRISPR/Cas9 for genome editing.

Hui Yang, PhD Rui Hao, PhD Candidate Zitong Yu, PhD Candidate Shi Hu, PhD Yanhang Hong, Professor Yi Zhang, Sihui Chen

Introduction Talk and Oral Session: OF15 Engineering EVs, Room 105-106, May 10, 2024, 4:00 PM - 5:20 PM

Introduction

Small extracellular vesicles (sEVs) are lipid bilayer-enclosed nanovesicles that serve as a critical intercellular mediator in both physiological and pathological activities. Due to their significant potential in drug delivery, sEVs have been widely used to load exogenous cargoes by electroporation for biomedical research. However, commercially bulky systems often suffer from limitations of restricted electric fields, inconsistent stimulation, metal contamination, damage to sEVs, etc. Alternatively, nanofluidics offer the capability to precisely manipulate fluids and objects at subcellular scales, enabling more homogeneous physical treatment while minimizing interference with sEVs. To address the demand for effective sEV loading with various cargoes, a nanoengineering platform, which is referred to as ExoNP, is proposed for nondestructive mechanoporation of sEVs, thus providing a versatile technology for sEV-based cargo loading.

Methods

ExoNP is an ultrahigh-pressure nanofluidic device integrating 62,400 nanochannels, developed to mechanically stimulate sEVs at a flow rate of 150 µL/min, with a total volume ranging from tens of microliters to milliliters. As sEVs (1012 sEV/mL) rapidly pass through height-confined nanochannels, they experience mechanical compression and fluid shear, inducing transient membrane mechanoporation, thereby facilitating convective influx of cargo molecules into the sEVs from surrounding medium.

Results

To date, we've successfully loaded doxorubicin and dextran into sEVs. Additionally, short RNAs, specifically miR-29a and miR-494, were loaded into sEVs without affecting their integrity or functionality. The loaded microRNAs in ExoNP-treated sEVs are significantly enriched over 10 times compared to the control group. Furthermore, we've also successfully loaded biomacromolecules, such as mRNAs and proteins, into sEVs using this platform. This demonstrates the technology's straightforwardness as a promising solution for loading large biomolecules into sEVs to rapidly promote clinical validation and drug production. Detailed protocols and results will be presented at the conference.

Summary/Conclusion

In conclusion, we've introduced an ultrahigh-throughput nanofluidic platform that effectively loads various cargoes into sEVs through mechanoporation. This technology paves the way for the development of diverse sEV-based nanomedicines. Its implementation provides new opportunities to streamline the manufacturing practice of sEV-based therapeutic drugs by efficiently loading them directly, greatly simplifying the current development process and ensuring compliance with current clinical application regulatory requirements.

Phd Tamas Beke-Somfai, Tasvilla Sonallya, PhD Imola Cs. Szigyarto, PhD Tunde Juhasz, Kinga Ilyes, Priyanka Singh, Delaram Khamari, DSc Edit Buzas, PhD Zoltan Varga

Introduction Talk and Oral Session: OF15 Engineering EVs, Room 105-106, May 10, 2024, 4:00 PM - 5:20 PM

Introduction

Host defense peptides (HDPs) exhibit significant potential as biomaterials with applications in both antimicrobial and anticancer fields. Their biological functions involve disrupting or lysing cell membranes. These peptides engage in various membrane interaction mechanisms, such as the carpet, toroidal pore, and barrel stave models. Additionally, cell-penetrating peptides play a role in loading cargo and facilitating the uptake of small molecules and nanoparticles. While much study has been done on the mechanics of these peptides' interactions with model membranes, our understanding of their interactions with extracellular vesicles (EVs) remains restricted. There are various aspects where the interplay between EVs and HDPs could be relevant, spanning from their cooperative presence at infection sites to potential functions in EV cargo loading.

Methods

A series of HDPs were selected: Indolicidin, Aurein 1.2., Dermcidin (DCD-1), DHVAR 4, Bactenecin, Protegrin-1, Transportan, Buforin IIb, KLA, Temporin-La, LL37, FK16, Mellitin, Polybia MPI, Histatin 5, PNC-28, CM15, Buforin II, Gramicidin, Arg-1, Macropin I, Lasioglossin LL-III, R8, Penetratin. Polarised light spectroscopy (Linear dichroism), flow cytometry, nanoparticle tracking analysis, zeta potential and freeze-fracture TEM were used to investigate these interactions.

Results

Biophysical investigations have unveiled distinct mechanisms employed by various host defense peptides (HDPs). These mechanisms encompass vesicle penetration, lytic actions, and the removal of protein corona. Biophysical studies categorize host defense peptides (HDPs) into eight groups based on their mechanisms. LL37 and Lassioglossin removes surface proteins effectively. Melittin disrupts membranes strongly, and Octaarginine and Penetratin use a mechanism with lower disruptive affinity on original vesicle composition.

Summary

These insights provide an overview of the surface interactions of Host defense peptides with EVs, allowing us to gain a wide perspective on the molecular level interactions, which may be useful in tailoring the surface of EVs with short HDPs and manipulating for bioengineering.

Professor Yoon-Kyoung Cho, Chaeeun Lee, Sumit Kumar

Introduction Talk and Oral Session: OF15 Engineering EVs, Room 105-106, May 10, 2024, 4:00 PM - 5:20 PM

Introduction:

Extracellular vesicles (EVs) hold immense potential for drug delivery, yet challenges persist in optimizing their membrane permeability without compromising functionality. This study introduces the tonicity control (TC) method, utilizing hypotonic solutions to transiently permeabilize EV membranes for efficient cargo loading. The TC method was evaluated for loading doxorubicin (Dox), dextran of varying sizes, single-stranded DNA (ssDNA), and miRNA-497 as cargo models to assess loading efficiency. Additionally, the ability of miRNA-497 and Dox-loaded EVs to penetrate tissue spheroids and impact their size was demonstrated, highlighting the potential of this method for targeted drug delivery.

Methods:

EVs from HEK 293T and A549 cells were isolated using the Exodisc, enabling efficient separation and loading. Hypotonic solutions were employed for transient permeabilization, facilitating cargo influx (Dox, dextran, ssDNA, miRNA-497). Comparative analyses against traditional loading methods (e.g., sonication, extrusion) evaluated efficiency, membrane integrity, and cargo retention.

Results:

The TC method demonstrated superior efficacy, achieving 4.3-fold and 7.2-fold higher loading compared to sonication and extrusion, respectively. Dynamic light scattering confirmed EV swelling and increased permeability post-hypotonic exposure, indicating successful cargo uptake. TC-treated EVs maintained particle count, protein concentration, and surface markers, preserving EV characteristics. Notably, miRNA-497-loaded EVs exhibited enhanced therapeutic efficacy in inhibiting lung cancer cell proliferation compared to conventional methods.

Conclusion:

The TC approach offers a breakthrough in EV cargo loading, enabling gentle and efficient loading of diverse molecules while circumventing structural damage. Surpassing conventional methods in particle retention and cargo delivery, this method holds promise for innovative EV-based therapeutics, potentially revolutionizing targeted and effective drug delivery systems in biomedical applications.

Hanguo Jiang, Professor Zhijie Chang

Introductory Talk and Oral Session: OF16 Cancer Biology, Room 109-110, May 10, 2024, 4:00 PM - 5:35 PM

Introduction

Cancer cells secrete amounts of small extracellular vesicles (EVs) to immerse neighbor cells in the cancer-dominant microenvironment. Cancer-derived EVs (CDEVs) induce the pre-malignant of normal epithelial cells adjacent to cancer, called the process of cancerization. However, it remains unknown how the CDEVs twist the cell fate of recipient cells, in particular the normal epithelial cells around cancer.

Methods

EVs of cancer cells and normal epithelial cells were isolated through ultracentrifugation and identified by NTA, WB, TEM, and ExoView. The normal cells were treated with CDEVs and EVs derived from normal cells (NDEVs) respectively. Whole genome bisulfite sequencing (WGBS) and RNA-seq were utilized to demonstrate the CDEVs’ induction of DNA methylation reprogramming and transcriptional regulation of EVs-recipient cells. In addition, the cargo transported by EVs was determined by LC-MS and total RNA-seq. IHC of mouse models and clinical samples, RT-PCR, and WB were used to investigate the proteins that function in CDEVs-induced DNA methylation reprogramming. Cancerization was assayed by H&E staining in the hypoplasia tissues of mice treated with CDEVs.

Results

In this study, we revealed that the DNA methylation pattern of normal epithelial cells, in both CpG island and non-CG sites, was reprogrammed by the isolated CDEV, leading to cancerization. The hypermethylation of tumor suppressor genes and hypomethylation of oncogenes occurred concomitantly during the gradual cancerization. GO and KEGG analyses showed that multiple cancer-promoting pathways, including PI3K-AKT, MAPK, Hippo, Rap1, and cGMP-PKG were activated during DNA methylation reprogramming. Interestingly, we observed that DNMT1, a methyltransferase, and CREPT, an oncoprotein in multiple cancers, were upregulated by CDEVs. Deletion of CREPT impaired the expression of DNMT1 induced by CDEVs and partially impeded the CDEVs-induced DNA methylation alterations at the same sites. Moreover, deletion of CREPT rebuilt the DNA methylation pattern and reduced cancerization caused by CDEVs.

Conclusion

Our results reveal a new epigenetic mechanism for cancerization and local recurrence. CDEVs increase the tendency to cancerization of normal epithelial cells by inducing DNA methylation reprogramming.

Mr Samuel Wan Ki Wong, Miss Claudia Wing Lam Tam, Mr Nicolas Cheuk Hang Lau, Dr Xiaowen Mao, Prof Judy Wai Ping Yam

Introductory Talk and Oral Session: OF16 Cancer Biology, Room 109-110, May 10, 2024, 4:00 PM - 5:35 PM

1) Introduction

Numerous studies have explored the functions, mechanisms, and clinical implications of sEVs in hepatocellular carcinoma (HCC). However, the underlying mechanism of sEV biogenesis remains elusive. Our observations indicate that metastatic HCC cells secrete more sEVs compared to non-metastatic cells and normal liver cells. In this study, we aim to identify key mediators involved in the biogenesis of HCC-derived sEVs and investigate their clinical significance in HCC.

2) Methods

To identify a kinase that regulates sEV secretion by metastatic HCC cells, we conducted a kinase inhibitor library screening through bead-based Amplified Luminescent Proximity Homogenous Assay (ALPHA). Candidate inhibitors that suppressed sEV secretion were identified. The targeted kinase was knocked down in HCC cells to examine its role in sEV secretion. The sEV collected from control and knockdown cells were functionally characterized by in vitro assays and animal models and subjected to proteomic profiling.

3) Results

We identified that MRT68921, an inhibitor of Unc-51-like kinase 1 (ULK1), effectively reduced sEV secretion. The involvement of MRT68921 was corroborated by the significant reduction in sEV secretion by stable ULK1 knockdown cells compared to control group. Notably, a decreased level of synaptosomal-associated protein 23 (SNAP23), a known regulator of transport vesicle docking and fusion, was detected in ULK1 knockdown cells. Functional studies and phosphoproteomics predicted that ULK1 phosphorylates and stabilizes SNAP23 through Ser-6, leading to increased sEV biogenesis. Clinically, positive correlation of ULK1 and SNAP23 was detected in HCC clinical samples. Additionally, mass spectrometry revealed a drastic alteration in the protein composition of sEV collected from ULK1 knockdown cells. sEVs released from ULK1-knockdown metastatic cells exhibited reduced levels of anion exchange protein 2 (AE2) and compromised oncogenic properties. AE2 is involved in intracellular pH regulation, but its function within sEVs has never been reported. Strikingly, our results demonstrated that suppression of sEV-AE2 diminished the oncogenic effects of metastatic cell-derived sEV in proliferation and metastasis induction.

4) Conclusions

This study unveils the non-autophagic role of ULK1, mediated by SNAP23, in promoting sEV secretion and altering sEV proteome. ULK1 upregulation facilitates the release of oncogenic sEVs that contribute to sEV-induced tumorigenesis and dissemination in HCC.

Mrs. Aditi Patel^{Ahmedabad University, Ahmedabad, Gujarat, India}, Dr. Shanaya Patel^{Ahmedabad University, Ahmedabad, Gujarat, India}, Ms. Vaishnavi Patel^{Ahmedabad University, Ahmedabad, Gujarat, India}, Dr. Vivek Tanavde^{Ahmedabad University, Ahmedabad, Gujarat, India}

Introductory Talk and Oral Session: OF16 Cancer Biology, Room 109-110, May 10, 2024, 4:00 PM - 5:35 PM

Introduction: One of the major confounders of Oral Squamous Cell Carcinoma (OSCC) in the Indian subcontinent is chemo-resistance. There is an urgent need to identify non-invasive and effective biomarkers to identify patients at risk of chemoresistance. In this study, we have identified the clinical relevance of a novel Extracellular vesicle (EV) encapsulated miRNA and unraveled its underlying mechanism responsible for the development of chemoresistance in OSCC.

Methods: Salivary EVs were derived from OSCC chemo-resistant and chemo-responding patients (n = 100) and healthy volunteers (n = 50) using a modified method with a starting volume of 200-500ul. EV size, concentration, and purity were assessed using transmission electron microscopy (TEM), flow cytometry (CD63, CD81, CD9, and CD47), and nanocyte tracking analysis (NTA). Small RNA sequencing was performed in the training cohorts from RNA isolated using a TRIzol-based in-house modified method. These findings were substantiated in the validation cohort using real-time PCR. Further, EVs derived from the supernatant of anti-miR-1307-5p lentiviral transduced cancer stem cell (CSC) subpopulation were assessed for their functional role in inducing proliferation, migration, invasion, tumor re-formation, angiogenesis, and epithelial-mesenchymal transition using in-vitro, ex-vivo and in-vivo models.

Results: Exclusive upregulation of miR-1307-5p was observed in salivary EVs of chemo-resistant OSCC patients (FC: 4.82 ± 2.38, p-value: 0.01) and patients with multiple relapses (FC:3.15± 1.2, p-value:0.05). Longitudinal monitoring of EV miRNA levels pre- and post-treatment demonstrated decreased miRNA levels in chemo-responders, while non-responders and relapse patients experienced an increase. EVs derived from the supernatant of anti-miR-1307-5p transduced CD44+ cells hindered the proliferation, migration, and invasion of CSCs. Furthermore, EVs hindered tube formation in human umbilical vein endothelial cells, and the chick chorioallantoic membrane (CAM) assay further validated their ability to inhibit micro-vascular formation. Combinatorial treatment of supernatant-derived EVs and cisplatin on CD44+ cells resulted in a 69.23% reduction in proliferation. In vivo, the study demonstrated that when injected into the buccal cavity of SCID mice, supernatant-derived EVs showed a reduction in tumor growth and metastatic potential.

Conclusion: Thus, we propose EV-derived miR-1307 as a candidate biomarker for predicting and monitoring the chemotherapeutic response and as a potential target to reverse the drug resistance.

Phd Candidate Nai Yang Yeat, Li-Heng Liu, Yu-Hsuan Chang, PhD Kui-Thong Tan, PhD Ruey-Hwa Chen

Introductory Talk and Oral Session: OF16 Cancer Biology, Room 109-110, May 10, 2024, 4:00 PM - 5:35 PM

Introduction: Exosomes are originated from a diversion of endocytic pathway and elicit pleiotropic pro-tumor functions. In late endocytic trafficking, multivesicular bodies (MVBs) are directed to lysosome for degradation or secreted as exosomes. However, the mechanism controlling the choice between these two fates remains elusive. In this study, we pointed out the critical proteins in directing the fate of MVBs, validated the interacting proteins participate in the selection process, uncovered the tumor-promoting exosome cargoes and successfully mapped the signaling pathway that contributes to the cancer metastasis and reshape of immunosuppressive tumor microenvironment.

Methods: A ubiquitylome analysis was performed to identify WDR4, a substrate adaptor of Cul4 ubiquitin ligase, promotes exosome biogenesis through the degradation of its substrate PTPN23, a Bro1 family protein associated with ESCRT complex. PTPN23 as an ESCRT-associated protein, playing opposite role with ALIX, which inhibits exosome secretion. We further perform TurboID-based biotinylation and LC-MS/MS to uncover the specific interacting proteins of ALIX and PTPN23. Moreover, we utilized LC-MS/MS to discover the proteome of WDR4-derived exosomes and distinguish the factors that promote tumor metastasis.

Results: Unlike other ESCRT components, PTPN23 inhibits, rather than promotes, exosome biogenesis. PTPN23 competes with another Bro1 family protein ALIX for binding syntenin, thereby reducing the exosome-promoting ALIX-syntenin complex. Furthermore, PTPN23 and ALIX oppositely control the fate of MVBs towards lysosome degradation and exosome secretion, respectively. ALIX, but not PTPN23, recruits actin and a set of actin-binding proteins. ALIX-deficiency leads to the formation of aberrant F-actin structure around MVBs, thereby impeding MVB trafficking to cell periphery and promoting a degradation fate. Functionally, the WDR4- and ALIX-dependent exosomes preferentially load a set of pro-tumor proteins including MCAM and NRP1, which enhance cancer cell migration, invasion, metastasis and reshape an immunosuppressive tumor microenvironment.

Summary/Conclusion: Our study highlights a novel mechanism in determining the fate of MVBs and uncovers the tumor-promoting factors that are favorably sorted in WDR4- and ALIX-dependent exosomes in the context of cancer metastasis and the reshape of immunosuppressive tumor microenvironment. These results provided a new insight in tuning the exosome production and a potential therapeutic target for cancer treatment.

Mr Keyang Xu

Introductory Talk and Oral Session: OF16 Cancer Biology, Room 109-110, May 10, 2024, 4:00 PM - 5:35 PM

Introduction

Small extracellular vesicles (sEVs) play crucial roles in facilitating intracellular communication by transporting cargo content to target cells. sEVs from breast cancer (BC) have been implicated in promoting pre-metastatic niches formation and metastatic tumor cells proliferation. However, whether sEVs contribute to the enhanced BC growth and metastasis under obesity conditions has not been explored.

Methods

In this study, we employed proteomics analysis to analyze the protein profiles of circulating sEVs in obese human subjects and high-fat diet-induced obesity (DIO) mice. The diameters of purified sEVs were assessed using transmission electron microscopy and nanoparticle tracking analysis. Western blotting was performed to examine the expressions of sEV markers, differentially expressed proteins in sEVs, and downstream targets in BC cells after sEV treatments. In vitro migration and invasion assays were conducted to evaluate the impact of sEVs on BC metastasis. Additionally, we utilized both wild type and Rab27a knockout mice to investigate the effects of sEVs on BC growth and metastasis.

Results

Here, we found that extracellular matrix protein 1 (ECM1) protein levels were significantly increased in the circulating sEVs under obesity conditions. Loading of ECM1 protein into sEVs was associated with integrin-β2 levels in donor cells. Knockdown or overexpression of integrin-β2 in the cells did not affect the cellular ECM1 protein levels but significantly reduced or increased ECM1 protein in the sEVs released by these cells. Interestingly, circulating sEVs purified from high-fat diet-induced obesity mice (D-sEVs) delivered more ECM1 protein to BC cells compared to sEVs from control diet mice. In turn, the BC cells secreted more ECM1 protein, which promoted cancer cell invasion and migration. Furthermore, overexpression of ECM1 in BC cells increased matrix metalloproteinase 3 (MMP3) and S100A/B protein expressions. Treatment with D-sEVs significantly enhanced ECM1-mediated BC metastasis and growth in various mouse models, including Rab27a knockout mice, as evidenced by elevated levels of tumor MMP3 and S100A/B proteins.

Conclusion

The identification of ECM1 protein in circulating sEVs as a promoter of BC development under obesity conditions not only unveils a novel mechanism but also indicates the potential for utilizing sEV-based strategies in the treatment of obesity-associated breast cancer.

Highly sensitive & specific, multi-omics compatible, pan-tumor liquid biopsy platform

Dr. Yanan Zhang, Dr. Alexander Koepp, Adela Brzobohata, Dr. Emanuele Puca, Dr. Roberto De Luca, Dr. Cesare Di Nitto, Dr. Teresa Hemmerle, Dr. Yingchao Meng, Dr. Stavros Stavrakis, Dr. Alexander Ring, Prof. Dr. Andreas Wicki, Prof. Dr. Julia Furtner, Dr. Caroline Hertler, Dr. Marcel Buehler, Prof. Dr. Michael Weller, Dr. Emilie Le Rhun, Prof. Dr. Dario Neri, Dr. Tobias Weiss

Introductory Talk and Oral Session: OS17 Cancer Biomarkers, Plenary 1, May 11, 2024, 10:40 AM - 12:00 PM

Introduction

Blood-based detection of cancer has the potential to improve cancer care by providing minimally invasive diagnosis and cost-efficient longitudinal disease monitoring. Extracellular vesicles (EVs) have emerged as a promising liquid biopsy resource of cancer, potentially in early diagnosis, pathological condition monitoring, and tailored treatment development based on cancer biology and the predicted treatment response of individual cases. In terms of “precision medicine”, tumor-derived EVs afford a possibility to measure temporal tumor burden levels and early evidence of recurrence or therapy resistance. However, there are currently no approved biomarkers for specific detection and isolation of tumor-derived EVs in the circulation for most cancer types.

Methods

Plasma/serum-EVs are purified by ultracentrifugation or size exclusion chromatography from 140 primary brain tumors (including glioblastoma, IDH-mutant glioma, meningioma), 100 brain metastasis, 80 extracranial cancers and 80 healthy donors, then characterized in accordance with ISEV-guidelines, by nanoparticles tracking analysis, electron microscopy and western blot. TNC+ circulating EVs are recognized by super-resolution microscopy and nanoscale/imaging flow cytometry. TNC-EVs transcripts and TERT/BRAF mutation are evaluated using digital droplet PCR. Diagnostic, disease monitoring and prognostic performance of TNC+ EVs are validated in 4 independent clinical trials with newly diagnosed glioblastoma and metastatic NSCLC.

Results

Here, we present the first pan-cancer potential EV-associated RNA- and protein-level biomarkers based on tumor-specific tenascin-C (TNC). TNC+ EVs enabled single marker- and blood-based detection of a variety of extracranial and intracranial neoplasms, including gliomas, with sensitivities and specificities exceeding 90%, outperforming previous liquid biopsy strategies. Furthermore, we show that for glioblastoma, one of the most challenging tumors to detect in blood, plasma TNC+ EVs can inform about tumor burden, are prognostically relevant, and offer molecular insights into tumor-associated DNA and proteins.

Summary/Conclusion

Our results provide a simple, versatile, and affordable platform that is applicable across cancers and compatible with widespread PCR or flow cytometry detection methods. Importantly, as an identifiable cancer hallmark, it could fill the information gap of current liquid biopsy platforms.

Dr Fanqin Bu, Dr Guangyu Ding, Dr Yunzi Wu, Dr Chenjie Xu, Dr Liyi Bai, Professor Xintao Qiu, Professor Pengfei Yu, Professor Yibin Xie, Professor Li Min

Introductory Talk and Oral Session: OS17 Cancer Biomarkers, Plenary 1, May 11, 2024, 10:40 AM - 12:00 PM

Introduction: Glycosylation is a crucial biochemical modification for extracellular vesicles (EVs), which participates in the pathogenesis and progression of gastric cancer (GC). The characterization of EV glycosylation profiles presents a promising frontier for liquid biopsy techniques, yet its application in clinical diagnostics and prognostics for GC patients requires further exploration.

Methods: We developed a lectin microarray to depict the lectin-affinity glycosylation patterns (LAGPs) of plasma EVs. A total of 84 plasma samples were analyzed, representing a spectrum of GC stages, benign gastric diseases (BD), and non-disease controls (NC). The efficacy of the microarray was rigorously evaluated for its potential in cancer diagnosis, prognosis, and the prediction of responses to immunotherapy.

Results: Distinct LAGPs were identified within the different patient groups, enabling the establishment of group-specific glycosylation signatures. Machine learning algorithms, including linear discriminant analysis (LDA), were applied to these signatures, achieving an exemplary 100% accuracy in classifying advanced and early-stage GC, BD, and NC samples. Furthermore, the development of a LAGP-based nomogram demonstrated considerable predictive capability for patient survival outcomes at 200, 300, and 500-day intervals, with corresponding AUCs of 0.793, 0.914, and 0.988. The predictive validity of LAGPs was also confirmed for immunotherapeutic responsiveness, with AUC values ranging from 0.866 to 1.000 across different supervised machine-learning algorithms.

Conclusion: This investigation delineates the variable LAGPs associated with the oncogenesis and progression of GC and establishes a comprehensive clinical tool for the screening of GC patients, monitoring survival outcomes, and prediction of responses to immunotherapy.

Dr Byeong Hyeon Choi, MD Jun Hee Lee, Dr Ok Hwa Jeon, Mr Chang Gun Kim, Professor Yeonho Choi, Professor Yong Park, Professor Ji-Ho Park, Professor Sunghoi Hong, Professor Hyun Koo Kim

Introductory Talk and Oral Session: OS17 Cancer Biomarkers, Plenary 1, May 11, 2024, 10:40 AM - 12:00 PM

Introduction

Lung cancer is the leading cause of cancer-related deaths worldwide. Liquid biopsies are a promising non-invasive method for cancer diagnosis and prognosis, as they provide a comprehensive analysis of tumor-derived biomarkers. Previously, we identified that the lung tumor-draining pulmonary vein blood (TDPV) has higher levels of small extracellular vesicles (sEVs) than peripheral blood. Moreover, we identified GRIP and Coiled-Coil Domain Containing-2 enriched sEVs (sEV-GCC2) as diagnostic biomarkers for lung adenocarcinoma. However, prognostic research using sEV-GCC2 in lung cancer surgery-received patients remains unclear. In this study, we investigated the prognostic values of sEV-GCC2 in TDPV for lung cancer surgery-received patients.

Methods

This study analyzed eight rabbits (four healthy controls and four VX2 rabbit lung cancer models) and 120 human participants, including 50 controls (mean age 63.2 ± 7.2 years, male: female = 24:26) and 70 primary lung adenocarcinoma patients (mean age 65.8 ± 9.0 years, male: female = 32:38) who underwent surgery. The mean follow-up duration was 33.65 ± 22.97 months. Ten milliliters of peripheral blood were collected in lung cancer patients under general anesthesia before surgery. To acquire TDPV, a 23-gauge needle was used to puncture the pulmonary vein immediately before division of this vein. Carcinoembryonic antigen (CEA), CD63, circulating-GCC2, and sEV-GCC2 levels were measured and statistically evaluated by immunochemistry and enzyme-linked immunosorbent assay. Kaplan–Meier and Cox regression analyses were used to assess survival analysis.

Results

The diagnostic accuracy of sEV-GCC2 in TDPV was significantly higher than that of CEA, sEV-CD63, and circulating-GCC2 (AUC of sEV-GCC2: 0.899, sEV-CD63: 0.765, circulating GCC2: 0.735, and CEA: 0.505 in TDPV; all p < 0.001). sEV-GCC2 in TDPV was a more significant predictor of pathological up migration (AUC of 0.836 versus 0.768, p < 0.0001) and pathological tumor size than in peripheral blood. Higher concentrations of sEV-GCC2 in TDPV were correlated with poor overall survival (91.24 vs 70.09 months; p < 0.05) and disease-free survival (80.13 vs. 42.73 months; p < 0.05).

Conclusion

The analysis of sEV-GCC2 in TDPV might be clinically significant for the better pathological correlation and prognosis of lung surgery-received patients.

Student Tong Liao, PhD Weilun Pan, Professor Lei Zheng, Professor Bo Li

Introductory Talk and Oral Session: OS17 Cancer Biomarkers, Plenary 1, May 11, 2024, 10:40 AM - 12:00 PM

1)Introduction

Programmed cell death ligand-1 (PD-L1) positive extracellular vesicles (EVs) in the bloodstream have emerged as a promising candidate for predicting outcomes in cancer immunotherapy. However, due to the heterogeneous specialization and varying abundance of PD-L1 markers on EVs, simultaneous analysis of protein and RNA expressions on PD-L1 EV populations at the single-vesicle level in plasma is necessary and challenging towards clinical application. In this study, we present an integrated technology called sEV-PREDICT, which enables the in-situ and concurrent detection of PD-L1 protein and mRNA expression in plasma EVs without requiring an isolation process.

2)Methods

sEV-PREDICT is an amphiphilic nanosensor that utilizes cationic liposomes as highly efficient membrane fusion carriers. It comprised a hydrophobic lipid membrane anchored with PD-L1 aptamers, encasing CRISPR-Cas13a units within a hydrophilic inner core. Thus, sEV-PREDICT could recognize PD-L1 proteins present on EVs by surface aptamers, while simultaneously delivering CRISPR-Cas13a units into the interior of the EVs via membrane fusion, facilitating the detection of PD-L1 mRNA. To achieve accurate quantification, the sEV-PREDICT incorporated nano-flow cytometry, allowing for precise determination of the proportion of plasma PD-L1-positive EVs at the single EV level.

3)Results

On nano-flow cytometry, sEV-PREDICT was verified to effectively classify the PD-L1 positive EVs into three subpopulations based on the expressions of PD-L1 protein and mRNA. This system exhibited excellent detection performance, allowing for centrifuge-free analysis of PD-L1 postive EV in plasma with a low sample volume requirement (5 µL) in 2 h. In a clinical cohort (n = 164) with twenty-one cancer types, sEV-PREDICT demonstrated outstanding clinical diagnostic efficacy by accurately distinguishing advanced cancer patients from healthy individuals, with an area under curve (AUC) of 0.970. Moreover, with the assistance of machine learning algorithms, sEV-PREDICT provided a precise index for predicting the outcomes of immunotherapy according to diverse cancer phenotypes, clinical stage identification, and therapeutic efficacy monitoring.

4)Conclusion

Overall, sEV-PREDICT offers a simple, isolation-free, and high-throughput approach for analyzing PD-L1 positive EVs at the single-vesicle level, thereby expanding the analysis strategy for EV subpopulations and providing more accurate disease information for clinical decision-making.

Seiko Ikezu, Post doctoral fellow Victor Santos, Postdoctoral fellow Mohammad Abdullah, Technician Justice Ellison, Research associate Zhi Ruan, Professor Tsuneya Ikezu

Introductory Talk and Oral Session: OS18 Neurobiology, Eureka, May 11, 2024, 10:40 AM - 12:00 PM

Introduction: We previously identified the novel mechanism of pathological tau transfer via extracellular vesicles (EVs) in Alzheimer's disease (AD). Targeting EV secretion to mitigate tau transfer is therefore a promising therapeutic approach for AD. P2RX7 regulates microvesicle shedding or secretion of multivesicular body-derived exosomes through purinergic signaling. In this study, we aim to investigate the effect of abrogating P2rx7 on EV secretion and tau transfer from microglia or astrocytes in vitro and cognitive function and tau pathology development in PS19 tauopathy mouse in vivo.

Methods: Microglia and astrocytes were cultured from C57BL/6 (WT) and P2rx7–/– mice and incubated with human tau (hTau) before evaluating the secretion of EV and EV associated tau by nanoparticle tracking and ELISA, respectively. PS19:P2rx7–/– transgenic mice at 9 months of age were evaluated for the fear conditioning test and their brain tissues were assessed for the hippocampal/cortex volume and tau pathology using immunofluorescence against aggregated tau (Alz50) or phosphorylated tau (AT8) and ELISA (total tau and pTau Ser396).

Results: Deletion of P2rx7 significantly suppressed secretion of EVs from microglia and astrocytes and the hTau level in EVs secreted from hTau-phagocytosed microglia and astrocytes compared to WT group. PS19:P2rx7–/– mice showed significant improvement in contextual and cued memory. Those results were aligned with significant reduction in tau pathology showing preserved cortical and hippocampal volume and reduction in ALZ50 and AT8 in the hippocampal regions and pTau Ser396 in Sarkosyl-insoluble fractions compared to PS19 mice.

Conclusions: Our study demonstrated that P2RX7 regulates EV mediated tau transfer from microglia or astrocytes, and abrogation of P2rx7 ameliorates cognitive dysfunction and tau pathology development in PS19 mice, suggesting P2RX7 as a potential therapeutic target for AD.

Dr Yang Yu, Dr Wenjun Xiao, Associate Professor Zhigang Li

Introductory Talk and Oral Session: OS18 Neurobiology, Eureka, May 11, 2024, 10:40 AM - 12:00 PM

Introduction: Alzheimer's disease (AD) is a neurodegenerative disorder responsible for 70% of global dementia cases, posing a significant threat to the health of the elderly. Previous studies has highlighted the deposition and aggregation of amyloid-beta as a major pathological alteration in the AD brain. The activation-mediated amyloidogenesis processing by beta-secretase 1 (BACE1) is a pivotal step in this process. However, the mechanisms underlying the continuous accumulation of amyloid-beta in the brain of AD remain unclear. Circulating extracellular vesicles (EVs) serve as communication mediators between organs and may play a pivotal role in the progression of AD. However, whether circulating EVs in AD can promote the generation and aggregation of amyloid-beta in the brain has not been elucidated.

Methods: We employed size-exclusion chromatography and subsequent ultracentrifugation to isolate plasma EVs from wild-type and APP/PS1 (AD model) mice (WTEVs and APPEVs). EV purity was confirmed using nanoflow cytometry, electron microscopy, and Western Blot. Differential protein analysis through mass spectrometry proteomics and bioinformatics identified unique proteins in EVs. JAK2-STAT1-BACE1 signaling pathway activation was assessed via Western Blot and Confocal microscopy. The distribution of DiR-labeled EVs after intravenous injection was visualized in various organs. Amyloid-beta plaques was analyzed through brain tissue sections and fluorescence staining.

Results: Compared to WTEVs, the APPEVs exhibited elevated concentration. These APPEVs activated the JAK2-STAT1 signaling pathway in neurons, leading to an upregulation of BACE1 expression and activity. This cascade promoted amyloid precursor protein (APP) beta-cleavage in lipid rafts, inducing substantial amyloid-beta generation. Proteomic analysis revealed complement C1q in APPEVs as a key protein activating the JAK2-STAT1-BACE1 pathway. Furthermore, in vivo experiments demonstrated that intravenously injected APPEVs crossed the blood-brain barrier, activating JAK2-STAT1-BACE1 signaling in neurons, and enhancing amyloid-beta production and aggregation in brain. Inhibition of C1q mitigated these effects in both in vitro and in vivo experiments.

Conclusion: During the progression of AD, circulating EVs containing complement C1q are delivered to neurons, activating their JAK2-STAT1 signaling pathway. This activation upregulates the expression of BACE1, subsequently enhancing the beta-cleavage of APP in lipid rafts. These events lead to a substantial increase in amyloid-beta production, exacerbating the pathological progression of AD.

Dr. Zhengrong Zhang, Dr. Kaiwen Yu, Dr. Hanmei Bao, Dr. Michael DeTure, Ms. Clara Scholes, Dr. Yang You, Dr. Seiko Ikezu, Dr. Dennis Dickson, Dr. Xianlin Han, Dr. Junmin Peng, Dr. Tsuneya Ikezu

Introductory Talk and Oral Session: OS18 Neurobiology, Eureka, May 11, 2024, 10:40 AM - 12:00 PM

Introduction: Extracellular vesicles (EV) play crucial roles in pathological protein transfer between cell-to-cell in Alzheimer's disease (AD). While apolipoprotein E (APOE) ε4 allele is a genetic risk factor strongly associated with late-onset AD, its impact on the cargo composition of EV in AD remains largely unknown. The purpose of this study is to perform comprehensive lipidomic and proteomic profiling of brain-derived extracellular vesicles (BDEV), stratified by APOE genotype (APOE3/3 vs. APOE4/4).

Methods: Frontal cortical tissue samles of AD patients with APOE3/3 (n = 20) or APOE4/4 (n = 20) were obtained from Mayo Clinic Brain Bank. EV were isolated from brain tissue with discontinuous sucrose gradient ultracentrifugation, examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The samples were tested for multidimensional mass spectrometry for shotgun lipidomic analysis and data-independent acquisition (DIA) liquid chromatrography-mass spectrometry (LC-MS)-based proteomics.

Results: There were no significant differences in size distribution and morphology between APOE4/4 and APOE3/3 BDEV by TEM and NTA. We identified 17 lipid classes and 173 distinct lipid molecules in the BDEV lipidome. APOE4/4 EV exhibited increased levels of Linoleic acid (18:2) and dihomo-γ-linolenic acid (20:3), precursors of proinflammatory mediator arachidonic acid (20:4), which are significantly associated with Braak stages over APOE3/3 EV. DIA LC-MS identified more than 4,000 unique proteins. BDEV of APOE4/4 females showed enrichment in abnormal fatty acid or lipoprotein transport and cell type specificity in microglia and astrocytes compared to those of APOE3/3 females, while BDEV of APOE4/4 males were enriched for neurotransmitter regulation and astrocytes and neuron compared to those of APOE3/3 males. Multi-omic analysis shows significant correlation of specific lipid group with unique protein networks in while BDEV of APOE4/4 over APOE3/3 background.

Conclusion: Both proteomic and lipidomic analyses of BDEV identified metabolic dysfunction influenced by APOE genotype. These molecular alterations may indicate enhanced potency of tau seeding activity or transfer in APOE4/4 EVs over APOE3/3 EVs, which may be regulated by both lipids and proteins. The study also provides the significance for lipidomic and proteomic profiling of BDEV for understanding the EV-mediated disease progression in Alzheimer's disease.

Natayme Rocha Tartaglia, Francesca Farina, Morgane Fontaine, Johanna Cormenier, Damarys Loew, Florent Dingli, Heike Rohweder, Chantal Bazenet, Emmanuel Brouillet, Lorena Martin-Jaular, Frédéric Saudou, Clotilde Théry, Christian Neri

Introductory Talk and Oral Session: OS18 Neurobiology, Eureka, May 11, 2024, 10:40 AM - 12:00 PM

Extracellular vesicles (EVs) mediate cell-to-cell communication via several mechanisms such as extracellular ligand/receptor-mediated signaling and fusion with the target cell membrane to deliver bioactive cargos to recipient cells. EVs may either gang up against cells or contribute to cell protection, which may involve a variety of EV subtypes. However, several questions remain unanswered about the role of EV subtypes in nervous systems in normal or disease conditions, e.g. in neurodegenerative diseases. To address these questions, we used Huntington's disease (HD) as a well-characterized neurodegenerative and repeat expansion disease.

To characterize EV remodeling in self-renewal/neurogenesis, which is altered in HD, we analyzed the features of small EVs (SEVs) secreted by mouse striatal cells derived from HD knock-in mice and by human HD iPS cell-derived neural stem cells (NSCs). We purified SEVs using differential ultracentrifugation or size exclusion chromatography. SEVs were characterized using electron microscopy, quantification of soluble mutant huntingtin (mHTT), nanoparticle tracking analysis, total protein dosage, western blot analysis using a panel of EV markers (e.g. ALIX, syntenin-1, and tetraspanins), and functional assays. Changes in protein composition of human SEVs were analyzed using label-free quantitative proteomics.

To characterize EV remodeling in neuronal circuits that are known to be affected by HD, we analyzed EV trafficking in cortico-striatal connections isolated from HD knock-in mice and reconstituted in microfluidic devices.

Our data suggest that NSCs and adult neurons may re-organize the secretion, function, and transport of SEVs in response to mHTT. We will discuss the implications of our findings for understanding the role of EV remodeling in neural stem cell niches and neuronal circuits that are exposed to chronic neurodegenerative insults.

Dr. Anthony Yan-Tang Wu, Ms. Wendy Wan-Ting Wong, Ms. Shannon Yu-Hsuan Yeh, Ms. Angela Yun-Fei Zhang, Dr. Charles Pin-Kuang Lai

Introductory Talk and Oral Session: OS19 EV Tracking, Room 105-106, May 11, 2024, 10:40 AM - 12:00 PM

Introduction

Extracellular vesicles (EVs) are released by cells to mediate intercellular communication, and play a role in various (patho)physiological processes. EVs can be categorized by size into small (sEV; < 200nm) and large (big EV; bEV; > 200nm) to transport bioactive cargoes such as nucleic acids and proteins. While a majority of EV studies focuses on a specific population of EVs (sEV or bEV), simultaneous tracking of multiple EV subpopulations, especially in vivo, has not been achieved due to a lack of available methods.

Methods

We introduce PalmSORBET system, a pair of spectrally distinct, multiplexed EV reporters based on bioluminescence resonance energy transfer (BRET). PalmSORBET system consists of a novel Soret band-mediated near-infrared BRET reporter PalmSORET, and our established PalmGRET. PalmSORET and PalmGRET label inner membrane of cells and EVs through a palmitoylation signal of growth-associated protein 43. Both reporters enable multi-resolution imaging of EVs via bioluminescence (BLI), BRET-mediated fluorescence (BRET-FL), and fluorescence (FL). PalmSORET contains a palmitoylated iRFP713 fused to Rluc8.6-535SG (modified from Nishihara et al.), whereas PalmGRET comprises a palmitoylated EGFP fused to nanoluciferase. PalmSORET catalyzes BBlue2.3 to emit peak BLI (415nm) and BRET-FL signals (713nm), while PalmGRET reacts with its substrate fluorofurimazine to emit peak BLI (460nm) and BRET-FL signals (507nm) for in vivo EV imaging. Furthermore, PalmSORET and PalmGRET can be respectively excited at 690nm and 488nm for super-resolution EV imaging.

Results

PalmSORET and PalmGRET label bEVs and sEVs without affecting their mean size and size composition. PalmSORBET simultaneously detects bEVs and sEVs in a mixture, and has a limit of detection of 100 ng in vitro. PalmSORBET further enables concurrent visualization of intravenously co-injected bEVs and sEVs in vivo within 30 min, followed by ascertaining their respective ex vivo organ distribution. Moreover, PalmSORBET allows detailed biodistribution analysis of intravenously co-administered bEVs and sEVs at 10 µg for each.

Conclusion

We present PalmSORBET as a sensitive and versatile method for multiplexed tracking of bEV and sEV. The multimodal imaging strategy of PalmSORBET enables multi-resolution imaging of bEV and sEV from whole animal to super-resolutions while providing orthogonal analyses to elucidate important dynamics of EV subpopulations.

Dr Basant Kumar Thakur, Prof. Dr. Cremer Christoph, Dr Jamal Ghanam, Prof. Dr. Dirk Reinhardt, Dr. Xiaomin Liu, Xingfu Zhu, Dr. Venkatesh Kumar Chetty

Introductory Talk and Oral Session: OS19 EV Tracking, Room 105-106, May 11, 2024, 10:40 AM - 12:00 PM

Introduction

Small extracellular vesicles (sEVs) are crucial players in intercellular communication in various diseases, including cancer, where they transport essential cargo molecules between cells. However, the limitations due to the lack of high-resolution imaging techniques hindered a comprehensive understanding of sEVs, including their biogenesis and functional attributes. We addressed this knowledge gap by applying single-molecule localization microscopy (SMLM) that has a spatial resolution of around 20nm for sEV imaging.

Methods

sEVs were isolated using a combination of tangential flow filtration (TFF), size exclusion chromatography (SEC), and ultrafiltration (UF), collectively known as TSU. According to MISEV2018 guidelines, sEVs were characterized using NTA, TEM, and western blot. 5-ethynyl-2′-deoxyuridine (EdU) was employed to label DNA cargo associated with sEVs (EV-DNA) that are released by metabolically active donor cells. Following the sEV treatment in the recipient cells, boron dipyrromethene- azide (BODIPY-azide) was utilized to label EV-DNA-EdU via click chemistry, and Alexa647-conjugated nanobody against GFP (Alexa647-Nb) to label CD63-eGFP+-sEVs.

Results

Following EV-DNA labeling using BODIPY-azide, we showed BODIPY's buffer-independent blinking feature by performing EV-DNA imaging in the recipient cells using SMLM. Then, the spatial distribution of CD63-eGFP+-sEVs in the recipient cells using Alexa647-Nb was visualized at a nanometer scale. Next, through simultaneous labeling of EV-DNA (BODIPY-azide) and CD63+-sEVs (Alexa647-Nb), we demonstrated the association of EV-DNA and CD63-eGFP+-sEVs in the recipient cells at the nanoscale resolution using dual-color SMLM. Functionally important, we illustrated the potential of dual-color SMLM imaging for the first time to elucidate the intricate interactions between EV-DNA and a cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS) within the recipient cells.

Summary/ Conclusion

Altogether, our data illustrated that dual-color SMLM imaging has a promising scope to study how sEVs are being uptaken along with its EV-DNA cargo in the recipient cells and their molecular interaction with different cellular compartments, which would enable us to understand the biological function of sEVs in various diseases.

Dr. Koushik Debnath^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Sadiq Umar^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Kasey Leung^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Chun-Chieh Huang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Miya Kang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Yu Lu^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Prof. Praveen Kumar Gajendrareddy^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Prof. Sriram Ravindran^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}

Introductory Talk and Oral Session: OS19 EV Tracking, Room 105-106, May 11, 2024, 10:40 AM - 12:00 PM

Introduction:

Cells intricately package molecules and biological information into nano-sized lipid bilayer vesicles called Extracellular Vesicles (EVs). These EVs serve as crucial nanoscale mediators for intercellular signaling. EVs derived from stem cells possess inherent anti-inflammatory and protective properties that make them excellent candidates as alternatives to stem cell therapy. Despite their significance, understanding the in vivo biodistribution and pharmacokinetic profiles of EVs upon administration into living subjects remains a significant challenge. Current EV labeling technologies have a poor life span for detection or compromise the integrity and functionality of the EVs, limiting their full potential. To address this challenge, we introduce a pioneering concept termed “Endovesiclosis.” This approach enables efficient loading of nanoparticles into EVs and facilitates non-invasive visualization using fluorescence imaging.

Methods:

In this study, as a proof-of-principle approach, we generated small (<10 nm) Quantum Dots (QDs) for fluorescence imaging with guanidium-rich surfaces and with positive, negative, and zwitterionic surface charges. The ability of these nanoparticles to enter mesenchymal stem cell-derived EVs (MSC-EVs) in a dose-dependent and saturable manner without compromising EV integrity was evaluated quantitatively and qualitatively using fluorescent imaging, quantitation, and electron microscopy. The mechanism of entry (active versus passive), the charge dependence, and temperature dependence were evaluated. The functionality of the labeled EVs (anti-inflammatory activity of MSC-EVs) was evaluated in vitro on primary macrophages and in vivo in a rat calvarial defect model.

Results:

Our findings demonstrate that only positively charged particles are endovsiclosed by the EVs in a dose-dependent and saturable manner. The process is inhibited in surfaces that are not guanidium-rich. The formulated nanoparticles effectively reside within EVs until the vesicles are degraded by harsh environmental conditions (such as treatment with detergent) or taken up (endocytosed) by cells. The nanoparticles do not compromise MSC-EV functionality in vitro and in vivo.

Conclusion:

Through our research, we have established a method to label EVs with nanoparticles, enabling effective in vitro and in vivo EV tracking without compromising EV surface integrity and bio-functions. We envision that this approach can extend towards using other nanoparticles and holds promise for advancing therapeutic applications and understanding EV biology.

Ms. Willemijn De Voogt, Dr. Sander Kooijmans, Mr. Kevin Harrijvan, Ms. Soultana Karakyriakou, Dr. Richard Wubbolts, Dr. Pieter Vader

Introductory Talk and Oral Session: OS19 EV Tracking, Room 105-106, May 11, 2024, 10:40 AM - 12:00 PM

Introduction: Extracellular vesicles (EVs) represent a heterogeneous population of endogenous intercellular delivery vehicles for biomolecules including proteins, nucleic acids and lipids. EVs intrinsically possess properties such as low immunogenicity and high cargo delivery efficiency that make them attractive carrier systems for delivery of biological therapeutics. An important challenge in employing EVs for drug delivery, however, is a lack of understanding of the intracellular trafficking and processing mechanisms of different EV subpopulations within recipient cells. Here, we define distinct subpopulations of EVs based on unique surface protein expression, which we individually trace in recipient cells using HaloTag fusion proteins compatible with bright, photostable fluorophores.

Methods: dSTORM imaging of CD63, LAMP2, ITGA2 and CD147 on MDA-MB-231 EVs was performed using a Nanoimager-S (ONI) to quantify coincidence of these proteins on single EVs. Additionally, EVs immunoprecipitated using CD63, LAMP2, ITGA2 and CD147 antibody-coated protein G Dynabeads were characterized by western blot. HaloTag fusion proteins were expressed in EV donor cells to evaluate the uptake kinetics of EV subpopulations using flow cytometry, along with their subcellular localization using fixed and live cell imaging.

Results: Immunofluorescence images of EV donor cells showed a clear endosomal staining pattern for CD63 and LAMP2. In contrast, ITGA2 and CD147 predominantly localized at the plasma membrane. Cluster analysis of dSTORM images showed low coincidence of CD63/LAMP2 and ITGA2/CD147 on individual EVs, whereas CD63 and LAMP2 coincidence, as well as ITGA2 and CD147 coincidence, was high. This observation was supported by EV immunoprecipitation followed by western blotting, revealing that the majority of ITGA2 and CD147 protein was not pulled down with CD63-positive and LAMP2-positive EVs and vice versa. HaloTag fusion constructs for these proteins allowed successful assessment of uptake kinetics of individual EV subpopulations. Interestingly, we observed variations in coincidence between the different EV subpopulations and recipient cell early, late and recycling endosomes.

Summary/Conclusion: We show that CD63- and LAMP2-positive EVs are distinct from ITGA2- and CD147-positive EVs. Moreover, these EV subpopulations show different intracellular trafficking patterns post-uptake. Next, using cargo delivery reporter systems, we will evaluate the functional implications of the observed differences in trafficking between the EV subpopulations.

Phd Student Amelie Chane, PhD Student Meline Macher, PhD Student Sarada Muduli, Doctor (Dr.) Ilia Platzman, Professor (Prof.) Joachim Spatz

Introductory Talk and Oral Session: OS20 Hybrid & Artificial EVs, Room 109-110, May 11, 2024, 10:40 AM - 12:00 PM

Introduction

Atopic dermatitis (AD) is a common chronic inflammatory skin disease characterized mainly by skin barrier dysfunction and a T helper 2 (Th2) cell-directed immune response. Current treatments remain limited and are often associated with potentially harmful long-term side effects. Extracellular vesicles (EVs) from human adipose-derived stem cells (hASC) appear to be of great interest in the treatment of AD due to their pro-regenerative and immunomodulatory effects and have been initially tested in mouse models. However, their biomedical implementation remains hampered due to their heterogeneity and difficulties associated with their scale-up production. To overcome these limitations, we apply bottom-up synthetic biology to produce bioinspired minimalistic fully synthetic EVs (synEVs). Particularly, we are focusing on their immunomodulatory potential for the treatment of AD.

Methods

SynEVs are assembled by lipid film swelling and subsequent extrusion and their biophysical and biochemical properties were designed to mimic the natural hASC EVs. The synEVs are functionalized by adding surface peptides and encapsulating miRNAs. Using ELISA, ELISPOT, flow cytometry and qPCR measurements the effects of synEVs on in vitro stimulated T cells are investigated.

Results

We demonstrated the successful assembly of synEVs with respect to size, surface charge, lipid and protein compositions. Currently, we are evaluating their immunomodulatory effects on T cell activation, proliferation, secretion and differentiation. Initial results indicate a slight decrease in T cell proliferation when treated with synEVs, opening up promising prospects regarding their immunomodulatory potential. Further analyses will be carried out up to the conference to assess the hypothesis that synEVs can, in particular, limit the differentiation of T cells into Th2 cells and significantly reduce the secretion of interleukins.

Summary/Conclusions

Using the principles of bottom-up synthetic biology we have designed and developed bioinspired synEVs for potential AD treatment. In this research, we particularly focus on the immunomodulatory properties of synEVs to regulate the Th2 immune response in AD.

Mr. Tanner Henson, Alessandra Arizzi, Hyehyun Kim, David Wang, Neona Lowe, Conary Meyer, Keerthana Ananda, Dr. Erkin Seker, Dr. Randy Carney, Dr. Aijun Wang, Dr. Cheemeng Tan

Introductory Talk and Oral Session: OS20 Hybrid & Artificial EVs, Room 109-110, May 11, 2024, 10:40 AM - 12:00 PM

Introduction: Extracellular vesicles (EV) are decorated with membrane proteins from the parent cells, allowing them to target specific cells in therapeutic applications. Native EVs, however, have high protein heterogeneity, making dosing difficult and quality control and assurance challenging for clinical translation. To solve this issue, we created a novel platform to synthesize artificial nanovesicles (ANV) that mimic native EVs’ protein display. The platform combines cell-free synthetic biology, modular transmembrane domains, and single EV measurements. The system will reduce heterogeneity by including only the proteins of interest, creating a new frontier of engineering artificial extracellular vesicles for treating various diseases.

Methods: Sonication was used for generating ∼75 nm liposomes. Soluble/extracellular domains of various membrane proteins were synthesized using cell-free protein synthesis. When the liposomes, cell-free systems, and plasmids were incubated overnight, the proteins were synthesized and inserted into the liposome membrane through a universal membrane tag. Protein insertion was validated using flow cytometry, western blotting, ELISA, and super-resolution microscopy. Artificial EV's function was characterized by neuroprotection and cell uptake studies.

Results and Discussion: We have tested and compared three molecular scaffolds to attach extracellular domain proteins to liposomes, transferrin, aquaporin-Z and Strep-tag/streptavidin. To quantify the effectiveness and robustness of protein attachment to liposomes, the second extracellular domain of CD9 was attached and analyzed through flow cytometry, western blot analysis, ELISA, and super-resolution microscopy. We find that the AquaporinZ-molecular scaffold showed robust signals of all tetraspanins in western blots. Furthermore, it generates a high percentage of liposomes with protein attached, as shown in single-liposome flow cytometry. The most robust molecular scaffold is then used to attach a broad class of proteins (>40, found on native MSC EV) to liposomes. High throughput cell-screening assays show distinct uptake of the artificial extracellular vesicles that were decorated with certain proteins.

Summary/Conclusion: My work demonstrates that cell-free protein synthesis can generate artificial EVs in a robust manner. As a result, it significantly accelerates the workflow of prototyping artificial EVs. The artificial EVs will have a broad impact on the translation of EVs as therapeutics.

Assistant Professor Akhil Srivastava, Anjugam Paramanantham, Yariswamy Manjunath, Rahmat Asfiya, Siddharth Das, Grace McCully, Assistant Professor Jussuf Kaifi

Introductory Talk and Oral Session: OS20 Hybrid & Artificial EVs, Room 109-110, May 11, 2024, 10:40 AM - 12:00 PM

Introduction: Engineering Extracellular Vesicles (EV) is a widely explored strategy for developing efficient anti-cancer therapeutic systems. In one such attempt, our laboratory has developed a novel EV-iron oxide nanoparticle (IONP) hybrid system- ‘ExoFeR,’ that can induce ferroptosis and reverse chemoresistance. Ferroptosis is a newly discovered form of non-apoptotic regulated cell death (RCD) that depends on iron (Fe) ion (Fe3+/ Fe2+) enrichment in the cells. Known is the fact that physiological dysregulation of apoptosis in cancer often leads to chemotherapeutic resistance, leading to poor prognosis. Alternative RCD approaches like ferroptosis can circumvent dependence on apoptosis and change the treatment outcome by overcoming therapeutic resistance.

Materials and methods: To formulate ExoFeR, EVs from normal lung fibroblast were loaded with IONPs by passive diffusion. Induction ferroptosis by ExoFeR was confirmed in Non-Small Cell Lung Cancer (NSCLC) A549 cells and NSCLC Patient Derived Tumoroids (PDTs) by Prussian blue, FerroOrange, and Mito-FerroGreen dyes. Transmission electron microscopy (TEM) and expression levels of ferroptosis markers like GPX4 and SCL7A11/xCT were evaluated to assess ferroptosis induction. Finally, after ferroptosis induction, A549 cells and resistant NSCLC PDTs were tested for sensitivity to chemo drugs.

Results: Physicochemical characterization by TEM, zeta sizer, and inductively coupled plasma mass spectrometry indicated the successful formulation of ExoFeR. Reduction in the expression level of SLC7A11/xCT- and GPX4 proteins in ExoFeR-treated NSCLC cells and PDTs suggested intrinsic induction of ferroptosis. Further, sensitization of chemo drug (cisplatin) was also observed in ferroptotic A549 cells and PDTs, which was noted by a reduction in cell viability as compared to non-ferroptotic equivalents.

Summary/Conclusion: We report here the synthesis of ExoFeR. This novel EV-IONP-based system can intrinsically induce ferroptosis and overcome chemoresistance by circumventing the conventional apoptosis-based resistance. Our finding is expected to develop a potent cancer treatment strategy for efficiently treating resistant NSCLC tumors.

Professor And Executive Director Eduardo Marban, Dr Ahmed Ibrahim, Dr Russell Rogers, Dr Alessandra Ciullo, Dr Ke Liao

Introductory Talk and Oral Session: OS20 Hybrid & Artificial EVs, Room 109-110, May 11, 2024, 10:40 AM - 12:00 PM

INTRODUCTION: FDA- or EMA-approved noncoding RNA (ncRNA) drugs (n = ∼20) target disease-causing pathways by small interference or other known mechanisms; all are 18-35 nucleotides in length, and chemically-modified. We sought to create new FDA-compliant ncRNA drugs based on bioactive EV cargo. EVs derived from cardiac stromal/progenitor cells contain numerous ncRNA species of unknown significance. Most are uncharacterized fragments annotated by homology to a class of ncRNA (e.g., YRNA, transfer RNA [tRNA], or long noncoding RNA [lncRNA]). We identified bioactive natural EV ncRNAs and used them as bioinspiration for synthetic RNA drugs that conform to the structural conventions of the FDA-approved ncRNA armamentarium.

METHODS: Unbiased RNA sequencing of EV cargo, focused on species 20-200 nucleotides in length, was used to identify exceptionally plentiful ncRNAs of unknown function, which we screened individually for bioactivity. Selected species were synthesized by solid phase chemistry, coated in DharmaFECT® transfection reagent, and exposed in vitro to macrophages and/or lymphocytes in primary culture. Using recipient cell transcriptomics as the readout, species that induced salutary changes in gene expression (e.g., activation of anti-inflammatory and/or anti-fibrotic gene pathways) were further characterized in vivo in preclinical disease models. The most promising such ncRNAs served as templates for structure-activity optimization of new chemical entities (NCE), which were further tested in vitro and in vivo.

RESULTS: Using the approaches described above, we have discovered and characterized six NCE lead compounds (chemically-modified mutant ncRNAs, each 24-35 nucleotides in length), which are in various stages of translation:

1) TY1, derived from yREX1, a 5´ fragment of the human YRNA4 gene. Disease-modifying bioactivity (DMbioactivity) in animal models of scleroderma and systemic sclerosis

2) TY2, derived from yREX3, a 3´ fragment of the human YRNA4 gene. DMbioactivity in animal models of myocardial infarction

3) TT1, derived from tREX1, a bioactive fragment of a human tRNA codon-matching glutamate. DMbioactivity in mouse model of Duchenne muscular dystrophy

4) TL1, TL2, TL3, derived from lncRNA BCYRN1. DMbioactivity in animal models of myocardial infarction

TY1 has already entered the FDA approval process.

SUMMARY: Our drug discovery paradigm—mining therapeutic EVs for bioactive ncRNAs as templates for regulatory-compliant NCEs—is potent and generalizable.

Martina Trentini, Dr. Luca Lovatti, Prof. Dr. Kathrin Becker, Dr. Giulia Brunello, Prof. Dr. Barbara Zavan

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

The term plant-derived nanovesicles (PDNVs) is commonly used to identify all vesicular fractions obtained from plant tissues. A previous study has shown that Apple-derived Nanovesicles (ADNVs) favour tissue regeneration, reducing inflammation by acting on the NFkB/IL-1b signalling pathway in dermal fibroblasts. Bone healing is determined by the differentiation of osteoprogenitor Mesenchymal Stem Cells (MSCs), which is in turn influenced by macrophage-MSC cross-talk. The aim of this in vitro study was to understand the role of ADNVs in bone healing, by observing their effect on macrophages and MSCs respectively.

Methods

ADNVs were obtained by ultracentrifugation of freshly produced apple juice, and subsequent purification by ultracentrifugation gradient. The ADNV-enriched fraction was characterised by Nanoparticle Tracking Analysis (NTA) and Transmission Electron Microscopy (TEM) analysis. The polarization of THP-1-derived macrophages towards M1 or M2 phenotype was monitored, before and after treatment with ADNVs for 24 hours. Osteogenic differentiation and new bone formation were assessed on adipose-derived Mesenchymal Stem Cells (AdMSCs) cultured on hydroxyapatite scaffolds, common bone grafting material, by RT-qPCR and cytofluorimetry of differentiation markers, Scanning Electron Microscopy analysis and Alkaline Phosphatase assay after 3, 7 and 21 days of culture and repeated ADNVs inoculations.

Results

ADNVs were found to be biocompatible and to drive macrophage polarization towards the pro-regenerative tissue resident M2 phenotype. Data highlighted an increase in CD163 and ARG-1 expression and morphological changes towards star-like and amoeboid shapes. The differentiation of ADNVs towards osteoblasts, already induced by the release of Ca2+ from hydroxyapatite, was further improved by ADNVs treatment, as confirmed by SEM imaging and ALP assay. Furthermore, treatment with ADNVs enhanced angiogenesis, a crucial part of bone regeneration, and induced extracellular matrix turnover through the production of collagen fibres (COL1, COL2) and inhibition of metallopeptidase activity (MMP1, MMP8, MMP9).

Conclusions

ADNVs positively correlate to M2 polarization of THP-1-derived macrophages. M2 macrophages have a crucial role in bone regeneration, abating inflammation and aiding the healing process through the secretion of osteoinductive factors. ADNVs themselves have osteoinductive properties, as they improve AdMSC differentiation towards osteoblasts.

Professor Kewei Zhao, Xuejun Tan, Bowen Gao, Yukun Xu, Yue Cao, Qing Zhao, Tianxin Qiu, Mingzhen Zhang

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Ulcerative colitis(UC) is a recurrent inflammatory bowel disease with deficiencies in both Chinese and Western medical treatments. Atractylodes macrocephala(AM) is a traditional Chinese medicine commonly used clinically in the treatment of UC, with anti-inflammatory and immunomodulatory effects. Atractylodes macrocephala derived EV-like particles(AM-EVLPs)are bioactive components extracted from AM with the properties of AM Chinese medicine.

Methods and Results: In the present study, we isolated and characterized AM-EVLPs, and found that AM-EVLPs were internalized and absorbed by macrophages and inhibited the expression of inflammatory factors; they were enriched in the colon, and they had significant therapeutic and prophylactic effects in the UC disease model. Combined analysis by fecal flora analysis, metabolomics, transcriptomics and bioinformatic analysis showed that AM-EVLPs could treat UC by regulating the intestinal flora and were closely related to the TH17 signaling pathway. This result was further validated by immunohistochemistry and antibiotic-induced depletion of the intestinal flora. Subjects with ulcerative colitis have also been recruited clinically (with ethical approval) with significant results.

Conclusion: In conclusion, our study not only successfully isolated high-quality AM-EVLPs, but also verified that AM-EVLPs can be used to treat UC by regulating the intestinal flora and TH17 signaling pathway, and the initial clinical translation was accomplished. AM-EVLPs have the potential to be a novel natural treatment for UC.

Bertrand Czarny

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Exosomes are extracellular membrane vesicles (EVs) produced from the plasma-membrane of most eukaryotic cells, and are currently intensively studied for practical use in medicine. Most studies on bacterial EVs to date have focused on outer membrane vesicles (OMVs) that can be retrieved from Gram-negative bacteria. Such OMVs contain primarily components of the outer membrane including endotoxin (lipopolysaccharide) and a cargo mainly comprising of constituents of the periplasmic space. OMVs have attracted much interest in vaccine industry as they may induce protective immune responses. and being used as antigens to prevent invasive meningococcal infections. EVs secreted from Gram-positive bacteria are derived from the plasma membrane and the mechanism initiating this so-called “blebbing” remains not clear. Bacterial EVs (BEVs) have been reported to be produced by a number of pathogenic Gram-positive bacteria for which new vaccines are urgently needed, including group A streptococci, Staphylococcus aureus and Streptococcus pneumoniae (Pn), and one advantage of using exosomes of Gram-positive origin is the lack of endotoxin, in contrast to OMVs from Gram-negative bacteria.

BEVs are difficult to isolate and characterize, rendering their study and therapeutic use challenging, especially for future clinical trials. To achieve the production of BEVs in large scale needed for vaccine production, we proposed to produce them directly from bacteria by a physical process to obtain bacterial extracellular vesicles mimetics (BEVMs) and we demonstrated the capacity to preserve the key components responsible for the function and characteristics of the natural vesicles. This method also allow us to mix bacteria from different capsular serotypes with different virulence properties. Hence, unlike conventional methods, the inclusion of a large spectra of BEVs that can protect against all pneumococci circulating in the population –and causing disease– should be possible.

In vivo studies was conducted and confirmed the protection by BEVMs as a potential vaccine using animal models. The cross protection of different strain was also evaluated in this study and show encouraging results.

In conclusion the BEVs or their mimetics carries a huge potential for vaccine development and can potentially provide a better protection against Streptococcus pneumoniae.

Dr Dinesh Kesavan

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Streptococcus pneumoniae, a common cause of both invasive and non-invasive human infectious diseases, has a high occurrence rate globally and is linked to significant sickness and death, particularly in children and older adults. While S. pneumoniae can persist in the respiratory system, specific individuals exhibit more susceptibility to developing pneumococcal disease than others. Pneumococcal disease, including pneumonia, meningitis, and bacteremia, can cause invasive and noninvasive conditions like acute otitis media or sinusitis. Vaccinations against pneumococcal disease may successfully prevent the transmission of invasive infections caused by S. pneumoniae. S. pneumoniae comprises over 90 serotypes, each exhibiting unique antigenic properties and inflammatory responses. Globally, there are variations in the prevalence of disease-causing pneumococcal serotypes from an epidemiological perspective. Currently, two pneumococcal vaccines are available: capsular polysaccharide (PPSV23) and conjugate (PCV13). These vaccines protect against only 23 out of the 100 known varieties of capsules. However, they have several problems, including a higher occurrence of non-vaccine serotypes due to the rapid switching and recombination of pneumococci capsules. Therefore, it is imperative that we immediately develop novel vaccines capable of protecting against a wide range of serotypes. Pneumococcal vesicles exhibit significant potential as novel vaccine candidates for pneumococcal infections. These vesicles include several antigenic components derived from the cell membrane and exhibit decreased pathogenicity compared to living organisms. Recent studies have shown that pneumococcal vesicle-based vaccinations can elicit a potent immune response without adverse cytotoxic effects. In this study, we isolated bacterial EVs (BEVs) and bacterial extracellular vesicle mimetics (BEVMs) from four serotypes of S. pneumoniae and studied their immunological responses to various immune cells. The study also involved combining several serotypes of BEVs and BEVMs to determine their impact on immune cell responses. The relationship between protein levels and gene expression varied among cytokines and chemokines. These findings demonstrated that the BEVMs stimulated the release of proinflammatory cytokines, with a more significant effect shown upon combining the serotypes. In the future, BEVMs could become one vaccine that protects several S. pneumonia serotypes.

Ms. Megha Sharma^{All India Institute of Medical Sciences (AIIMS), New Delhi, India}, Dr. Rupesh K. Srivastava^{All India Institute of Medical Sciences (AIIMS), New Delhi, India}

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Glucocorticoids are anti-inflammatory steroid medications widely used for the treatment of various inflammatory conditions like RA, SLE, Colitis, Cancer, Covid-19 etc. Prolonged glucocorticoids usage is known to increase the risk of osteoporosis. Glucocorticoid induced osteoporosis (GIOP) is the most common cause of secondary osteoporosis & iatrogenic osteoporosis. The role of probiotics in modulating bone health has already been established by our group. But to date, the role of probiotics in ameliorating GIOP is relatively unexplored. One of the mechanisms by which probiotics regulate bone health is via the release of extracellular vesicles. Thus, in the present study we investigated the potential of probiotic Lactobacillus rhamnosus UBLR-58 derived extracellular vesicles (Lr-EVs) in enhancing osteoblastogenesis in Dexamethasone (DEX) treated MC3T3-E1 cell line & murine bone marrow mesenchymal stem cells (BMSCs).

Methods: Extracellular vesicles were isolated from Lactobacillus rhamnosus UBLR-58 culture medium (MRS Broth) by differential ultracentrifugation & characterized by Nanoparticle tracking analysis (NTA) & Transmission electron microscopy (TEM). Effect of DEX & Lr-EVs on the cell viability of MC3T3-E1 & murine BMSCs was evaluated via MTT assay. For osteoblastogenesis, MC3T3-E1 cells & murine BMSCs were cultured in osteoblastogenic medium & treated with DEX; DEX + Lr-EVs. ALP, ARS staining & qRT-PCR were performed to evaluate osteoblastogenesis.

Results: DEX treatment significantly decreased the cell viability of MC3T3-E1 & BMSCs in a dose dependent manner while no significant change in cell viability was observed in Lr-EVs treated cells. Moreover, Osteoblastogenesis was significantly decreased in DEX treated MC3T3-E1 cells & BMSCs. Interestingly, in the presence of Lr-EVs, osteoblastogenesis was significantly increased in DEX treated MC3T3-E1 & BMSCs as indicated by ALP & ARS staining of osteoblast cells & qRT-PCR of osteoblastogenic genes: Runx2, ALP & bglap.

Summary/Conclusion: Taken together, our results for the first time establish the osteoprotective potential of probiotic Lr-EVs in preventing GIOP via modulating the effects of Dexamethasone on bone forming (osteoblasts) cells ex vivo. Our results thereby propose probiotic EVs as a viable therapy for the management and treatment of GIOP.

Ms Melissa Tan, Dr Brenda Wan Shing Lam, Dr Waqas Muhammad Usman, Dr Thach Tuan Pham, Dr Chang Gao, Dr Harwin Sidik, Ms Rachel Tan, Dr Minh TN Le

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Gene therapy offers a promising solution for treating neurological disorders, but its application is restricted by the need for a safe and effective delivery method to the central nervous system. Red blood-cell derived extracellular vesicles (RBCEVs) have the potential to overcome hurdles in drug delivery as they are non-immunogenic, non-cytotoxic, redosable, and amenable for nucleic acid loading. Therefore, our study explores the potential of RBCEVs as a novel delivery vehicle for gene therapies to the CNS.

Methods: Fluorescent-labeled RBCEVs were administered by intrathecal and intracisternal injection to determine biodistribution in C57BL/6 mice. RBCEVs were loaded with nanoplasmids with the CMV early enhancer/chicken beta actin (CAG) promoter for gene expression or antisense oligonucleotides (ASOs) for gene knockdown in the nervous system. Uptake of RBCEVs by specific CNS cell types were identified by immunostaining. Toxicity of nanoplasmid-loaded RBCEVs was evaluated in rhesus macaque (Macaca mulatta) following intrathecal delivery.

Results: We demonstrate that RBCEVs can be distributed within the CNS and taken up by major CNS cell types including neurons, astrocytes, and microglia. Furthermore, RBCEVs can deliver nanoplasmids with the CAG promoter to healthy mice by intrathecal and intracisternal injection, enabling expression of both cytosolic and secretory proteins in the CNS. Green fluorescent proteins and luciferase signal can be observed in the brain and spinal cord. Secreted nanoluciferase can be detected in cerebrospinal fluid, as well as brain and spinal cord tissues. RBCEVs can also deliver smaller payloads to the CNS, such as Gapdh ASO for gene knockdown. Notably, delivery of nanoplasmid-loaded RBCEVs to the CNS of rhesus macaque did not result in observable toxicity.

Conclusion: Our study presents the first evidence that RBCEVs can be internalized by major cell types in the CNS and deliver DNA cargos safely and functionally to the CNS. These findings can contribute to the advancement of targeted gene delivery for the treatment of neurological disorders.

PhD Marina Trouillas, Mrs Marine De Taddeo, Mr Pierre Maincourt, Mrs Muriel Nivet, MD Guillaume Valade, Mrs Claire Langle, Mrs Marion Grosbot, Mrs Sylvie Goulinet, PhD Philippe Mauduit, MD, PhD Sébastien Banzet, PhD Juliette Peltzer

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Despite advances in the field of regenerative medicine, the management of severe burns remains difficult and can lead to delayed healing with the formation of pathological scars. Mesenchymal stromal cells (MSCs) secretome has emerged as a promising new therapeutic option for tissue repair thanks to its immunomodulatory and remodeling properties similar to those of MSCs themselves. The secretome is composed of bioactive soluble factors and Extracellular Vesicles (EVs) containing lipids, proteins, and small RNAs. MSC can modify their secretome based on various environmental stimuli. We have already shown that IL-1β priming can direct MSC secretome towards a wound healing phenotype. However, studies are needed to better understand which compounds or entities in the MSC's secretome are responsible for their biological effects. The objective of the study was therefore to evaluate different entities of the IL-1b-primed MSC secretome on their capacity to promote angiogenesis, cell migration and proliferation in vitro.

Methods: MSCs were primed with IL-1β, and the secretome, named conditioned medium (CM) was collected after 72 hours. Tangential flow filtration was used to isolate and concentrate EVs or EVs with soluble factors from the CM, using filters with respective cut-off sizes of 500 or 10 kDa. EV characterization was assessed by NTA, cryo-electron microscopy and phenotypic analysis. Efficiency of different doses of products related to number of secretory MSC was evaluated in migration, proliferation and angiogenesis tests under serum-depleted conditions, using keratinocytes, dermal fibroblasts and endothelial progenitor cells.

Results: Functional tests indicated an enhance migration and proliferation properties of keratinocytes and fibroblasts under stressed conditions with product containing both EVs and soluble factors. Similar results were obtained for in vitro angiogenesis potential.

Summary/Conclusion: Our study emphasizes the importance of both soluble factors and extracellular vesicles to provide functional activities in wound healing. Proteome and miRNome characterization will help us in the future to better identify active compound in our product. This study will help us optimize scalable production methods for a product dedicated to enhancing severe burn healing.

Dr Yuanyuan Jiang, Dr Shipin Zhang, Dr Sai Kiang Lim, Dr Wei Seong Toh

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: This study aims to compare the efficacy of mesenchymal stem cells (MSC) exosomes with hyaluronic acid (HA) against HA alone in the treatment of temporomandibular joint osteoarthritis (TMJ-OA) in a rabbit model.

Methods: Nine age-matched New Zealand rabbits were randomly assigned to Sham, OA-HA, and OA-HA+Exo groups. TMJ OA was induced using intra-articular injections of mono-iodoacetate (MIA) (0.5mg MIA in 200µL PBS) in bilateral TMJs for OA-HA and OA-HA+Exo groups. After 2 weeks of OA induction, weekly interventions were administrated for the next 3 weeks: 200µL PBS with 50µL HA for OA-HA group, and 0.2mg exosomes in 200µL PBS with 50µL HA for OA-HA-Exo group. Rabbits in Sham group received only needle pricks. TMJ OA pain was assessed weekly using an algometer. The magnetic resonance imaging (MRI) was performed at 2, 4, 6, and 10 weeks to evaluate the effect of MSC exosomes for joint inflammation. At the 10th week, functional connectivity (FC) analysis of functional MRI assessed nociceptive changes in the rabbit brain. After all rabbits were euthanized, dissected TMJs were evaluated grossly and histologically with micro-computed tomography (micro-CT), immunohistochemistry, and transcriptomics.

Results: Compared to HA-treated osteoarthritic TMJs, those treated with MSC exosomes showed improved head withdrawal threshold, MRI signal intensities and significantly better macroscopic score (P < 0.05), OARSI histologic score (P < 0.01) and synovial membrane inflammatory score (P < 0.01). Transcriptomic analysis revealed preferential gene expression of PCNA, TGFβ1, and IGF1 over TAC1, TNF-α, and MMP13 with exosome treatment (P <0.05). Micro-CT indicated a higher ratio of bone volume over total volume (BV/TV) in the OA-HA+Exo group as compared to OA-HA group (P < 0.01). The whole-brain voxel-by-voxel analysis showed that several regions displayed altered resting RVM (rostral ventromedial medulla) FC in OA-HA group, while OA-HA+Exo rabbits exhibited FC similar to the sham group (P > 0.05).

Conclusion: Addition of MSC exosomes with intra-articular HA injections proved superior to HA alone in the rabbit TMJ OA model in terms of pain suppression, condylar cartilage & subchondral bone degeneration and synovial inflammation.

Mr. Aliosha I. Figueroa-Valdés¹, Mr. Nicolás Georges², Ms. Catalina Adasme-Vidal¹, Ms. Yeimi Herrera-Luna³, Ms. Patricia Luz-Crawford^{1, 3}, Mr. Maroun Khoury^1,2,4,5,6, Ms. Francisca Alcayaga-Miranda^1,2,4,5,6

¹IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Santiago, Chile, ²Universidad de los Andes, Centro de Investigación e Innovación Biomédica (CiiB), Laboratory of Nano-Regenerative Medicine, Santiago, Chile, ³Universidad de los Andes, Centro de Investigación e Innovación Biomédica (CiiB), Laboratory of Molecular and Cellular Immunology, Santiago, Chile, ⁴Consorcio Regenero, Chilean Consortium for Regenerative Medicine, Santiago, Chile, ⁵Universidad de los Andes, Faculty of Medicine, School of Medicine, Santiago, Chile, ⁶Cells for Cells, Santiago, Chile,

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Knee osteoarthritis (OA) imposes a significant global burden, profoundly affecting life quality. Our previous research employing small extracellular vesicles (sEVs) derived from umbilical cord mesenchymal stromal cells (UC-MSCs) demonstrated promising in vivo OA mitigation. Acknowledging the need for a regulatory-compliant sEV-based therapy, we have conducted an extensive characterization and in vitro studies, delving deeper in the functional capacities of UC-MSC-derived sEVs, seeking to establish therapy-release criteria and potency testing.

Methods

sEVs were isolated via differential centrifugation and characterized according to MISEV 2018 guidelines using nanoparticle tracking analysis, flow cytometry, western blot, and transmission electron microscopy. Profiling of sEV-miRNAs was performed utilizing an HTG/EdgeSeq array, followed by bioinformatic analyses to identify OA-related targets. To assess macrophage polarization, specific markers including anti-inflammatory markers CD206 and CD163, as well as pro-inflammatory markers CD86 and HLA-DR, were evaluated via flow cytometry. An ELISA analysis aims to determine the production of cytokines such as IL1-β, TNF-α, IL-6, TGF-β, IL-10, VEGF, and IL-12 in sEVs-treated macrophages, aimed at improving their phenotype classification and as evaluation of experiment reproducibility. Furthermore, the chondroprotective effects of sEVs were evaluated on chondrocytes exposed to an apoptotic agent.

Results

sEVs displayed an average size of approximately 150nm and exhibited markers CD63, CD81, CD9, Flotillin-1, Syntenin-1, CD90, and CD44, with a limited presence of HLA-A/B/C MHC-class I antigens, lacking Calnexin, TOMM20, and HLA-DR/DP/DQ MHC-class II antigens. The characteristic cup-shaped morphology was observed in the sEVs. Analysis of sEV-miRNA profiles demonstrated consistency among samples from three UC-MSC donors, with three predominant miRNAs constituting nearly 70% of total miRNA reads, potentially targeting genes associated with immune system regulation, angiogenesis, and extracellular matrix modulation. Functionally, sEVs induced a notable shift in macrophage polarization towards an anti-inflammatory (M2) phenotype. Additionally, chondrocytes treated with sEVs exhibited enhanced chondroprotection when exposed to an apoptosis inducer compared to untreated cells.

Summary/Conclusions

Therapy using umbilical cord mesenchymal stromal cell-derived small extracellular vesicles demonstrates potential for knee osteoarthritis. sEVs consistently induced anti-inflammatory macrophage polarization and enhanced chondroprotection, reinforcing a promising off-the-shell OA treatment.

Mr. Satyajit Ghosh, Dr. Surajit Ghosh

Poster Pitches (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:45 PM - 1:00 PM

1) Introduction:

Aging and neurodegenerative diseases lead to reduced adult neurogenesis and increase in quiescent Neural Stem Cell (NSC), impacting the brain's regenerative abilities. Current treatments involve stem cell transplants or neurodegeneration prevention, yet efficacy remains limited. Our solution involves harnessing the brain's NSC through Exo-pep-11, an engineered extracellular vesicle (EV). This novel approach targets NSCs via Epha4 receptors, offering promising therapeutic benefits by stimulating NSC proliferation and differentiation, potentially mitigating disease severity.

2) Methods:

In this study, a novel peptide was designed using computational techniques and synthesized through solid-phase peptide synthesis. The peptide was characterized and purified using mass spectroscopy and HPLC, respectively. Rat adipose tissue stem cell-derived extracellular vesicles (EVs) were isolated using ultracentrifugation, and the peptide was functionalized on the exosome surface using EDC/sulfo-NHS acid amine coupling. The engineered EVs were comprehensively characterized via TEM, DLS, FTIR, and Western blotting. Exo-pep-11 cellular uptake assays were conducted in NSC cultures isolated from the rat Subventricular Zone (SVZ). These findings were further validated through multiple assays, including co-immunoprecipitation and in-vivo uptake analyses. BrdU assays were employed to assess cellular proliferation. Expression analysis of Nestin, ID1, Tuj1, and TH was conducted using immunocytochemistry, immunohistochemistry, and Western blotting.

3) Results:

The engineered extracellular vesicles (EVs) were successfully functionalized, as evidenced by an increase in mean diameter (177.3 ± 14.89 nm to 195.76 ± 8.37 nm) observed via DLS, TEM and FTIR peak analysis. 5-FAM tagged Exo-pep-11 was internalized by Neural Stem Cells (NSCs), colocalizing with Nestin and Epha4, specific NSC markers. Exo-pep-11-treated NSCs exhibited elevated Nestin and reduced quiescent marker ID1 expression compared to controls. The specificity of uptake was demonstrated through decreased internalization upon Epha4 antibody treatment, confirmed via Co-immunoprecipitation. Furthermore, treated cells displayed enhanced proliferation, as evidenced by BrdU cell proliferation assay. Western blot analysis of proteins from Exo-pep-11-treated rat Subventricular Zone (SVZ) revealed increased Nestin and decreased ID1 expression. Neurogenesis was affirmed in the olfactory bulb, displaying elevated levels of both Tuj1 and TH compared to controls.

4) Conclusion:

Exo-pep-11 enhances NSC proliferation, reduces quiescence, and boosts neuronal markers, indicating neurotherapeutic potential.

Anders Boysen, Doctor Bradley Whitehead, Doctor Anne Louise S. Revenfeld, Doctor Anna Karina Juhl, Doctor Reza Yarani, Doctor Yonglun Luo, Doctor Thor Petersen, Doctor Peter Nejsum

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Mesenchymal stem cells (MSCs) have been shown to hold great potential in ameliorating multiple conditions. The beneficial effects of MSCs are mediated, in part, by extracellular vesicles (EVs). Autologous sampling for MSCs is normally invasive and produces discomfort for the individual. Further, primary cells can be harder and more tedious to culture. In contrast, urine derived stem cells (USCs) are a facile, non-invasive, and non-exhaustive source of stem cells. Their potential as an EV source is less investigated than their cousin adult stem cell subtypes.

Methods: We isolated USCs from fresh urine from male and female donors. At passage 0 USCs were characterized by flow cytometry. Cell conditioned media (without growth factors) was collected at passage 3-5. Viability was determined at harvest. EVs were enriched using size exclusion chromatography (SEC). Enrichment was validated by nanoparticle tracking analysis (NTA) and the presence/absence of CD63. Immunomodulatory capabilities of EVs were evaluated on PBMCs from non-treated newly diagnosed multiple sclerosis patients. Recombinant protein expression potential was evaluated by transfection and mRNA/LNP delivery.

Results: Isolated cells were positive for canonical MSC markers and void of negative markers by flow cytometry. USCs showed similar viability after 72 hours, cultured in a media void of any EV contaminating growth factors, as the seeding viability. EV enrichment fractions had the most particles and were highly enriched for CD63. Cytokine profile for EV treated patient PBMCs is being analysed. USCs were easily transfected and expressed protein after mRNA/LNP delivery.

Summary/Conclusion: We see USCs as a unique, non-invasive/-exhaustive, robust, and easily genetically manipulated primary cell source. Further, the advantages that USCs show make them ideal as an autologous cell source to further optimize biocompatibility that can be expanded extensively.

Ethics.

All donors and patients gave written consent and the study was approved by the danish national research ethics commitee.

Dr Shanti Gurung^1,2, Ms Diem-Mai Pham Diem-Mai Pham¹, Ms Molly McLaughlin^1,2, Dr Jill C. Danne⁴, Dr Joel R. Steele³, Professor Ralf B. Schittenhelm³, Professor Jerome A. Werkmeister^1,2, Professor Caroline E. Gargett^1,2

¹The Ritchie Centre/Hudson Institute Of Medical Research, Clayton, Australia, ²Obstetrics and Gynaecology, Monash University, Clayton, Australia, ³Proteomics and Metabolomics Platform, Monash University, Clayton, Australia, ⁴Monash Ramaciotti Centre for Cryo-Electron Microscopy, Clayton, Australia

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Small extracellular vesicles (sEVs) contain bioactive molecules from the donor cells and are attractive as an off-the-shelf therapeutic alternative to mesenchymal stem cells (MSCs) with similar benefits without biosafety concerns. This study explores the protein composition in human endometrial MSC (eMSCs)-derived sEVs for their acellular applicability.

Method: Human SUSD2+eMSCs were isolated from endometrial biopsies from healthy women and expanded with and without TGF-βR inhibitor (A8301). Conditioned media were collected and sEVs were isolated using differential ultracentrifugation. sEVs were characterised for size, morphology, concentration and protein composition, and subjected to label-based quantitative proteomics. sEVs were also explored for anti-inflammatory molecules following priming the eMSC/A8301 with cytokines TNFα/IFNγ or LPS.

Results: eMSC-sEVs were spherical with a diameter of ∼121 nm, expressing TSG101, Alix and Syntenin-1 proteins. Proteomic profiling further validated their identity (CD63, CD9, CD81, flotillin and RAB proteins) and identified 1892 proteins involved in endocytosis, regulation of actin cytoskeleton, cell adhesion (CASP3, MFGE8), migration (integrins, YES1), wound healing (CD151), angiogenesis (EGFR, BSG), collagen synthesis and fibril organisation, chemotaxis and immunoregulation (TGFβ1, Galectins, CD36, PD-L1, CD39, HLA A-C), and anti-coagulation (CD46, CD55, CD59, TFPI, PTX3 and CFI). They also comprise various antimicrobial (B2M, TMSB4X, DEFA3) and antiviral (IFIT1, IFIT3, IFIT5, IFITM3) proteins. On average, eMSCs and A8301/eMSCs produced 0.6+13E+12 and 1.6E+12 sEVs/µg protein, respectively. There were 24 differentially expressed proteins between sEV derived from eMSC treated with and without A8301. CD36, GGT5 (Immune regulators), CEMIP (cell migration), SUSD2 (eMSC marker), TNS2 (proliferation) and TNXB (collagen fibril organisation) were significantly upregulated and WNT5A (fibroblast, fibrosis) and MRC2 (proliferation and anti-apoptosis) proteins were downregulated. TNFα/IFNγ-primed eMSC/A8301-sEVs had additional potent anti-inflammatory cytokines PGE2 and IDO while LPS-primed eMSC/A8301-sEVs contain potent antimicrobial CXCL4.

Conclusion: eMSC/A8301 secrete sEVs and their protein composition can be enhanced for targeted therapy by priming the donor cells with a focused disease environment, similar to the donor cells. This study suggests that eMSC/A8301-derived sEVs are haemocompatible and have potential application for various diseases such as pelvic organ prolapse, chronic wounds, scars and infection. Overall,

eMSC/A8301-sEVs have acellular therapeutic potential, mitigating the biosafety concern of the parent cells.

Miss Mihiri Goonetilleke^1,2, Ms Jeanne Correia¹, Dr Yuan Chen¹, Ms Hannah McDonald¹, Dr Siow Teng Chan¹, Mr Ian Simpson⁵, Dr Ishmael Inocencio¹, Prof. William Sievert^3,4, A/Prof Rebecca Lim¹

¹Hudson Institute Of Medical Research, Clayton, Australia, ²Obstetrics and Gynaecology, Monash University, Clayton, Australia, ³Gastroenterology and Hepatology Unit, Monash Health, Clayton, Australia, ⁴Centre for Inflammatory Disease, Monash University, Clayton, Australia, ⁵Monash Health, Clayton, Australia

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Metabolic dysfunction-associated steatohepatitis (MASH) is a liver disease driven by a chronic inflammatory response following prolonged fat accumulation. MASH can result in hepatocellular carcinoma (HCC). Currently, there is no cure for MASH or HCC. Human amniotic epithelial cells (hAECs) and their extracellular vesicles (hAEC-EVs) have shown therapeutic potential in the treatment of chronic liver injury. Primary hAECs can be isolated from placentas in high numbers but lack proliferative capacity. To overcome this challenge, hAECs were isolated from healthy donors and immortalized (ihAECs). In this study we examined the effects of ihAEC-EVs in an experimental model of MASH and HCC.

Methods: MASH and HCC were induced in 6- to 8-week-old male mice using an experimental ‘western diet’ and a weekly low-dose of intraperitoneal carbon tetrachloride (CCl4) administration for 12 weeks or 24 weeks respectively. Mice were randomized to receive 10µg of ihAEC EVs via oral gavage either twice or thrice a week from week 6 until week 12 for the MASH model or from week 12 to 24 for the HCC model. Obeticholic acid (10mg/kg) administered via oral gavage acted as a comparison. Control mice were fed normal diets. Mice were euthanized at weeks 12, 18 or 24. Serum was collected for metabolic parameter analysis (ALT, AST, insulin and cholesterol) and livers were collected for histological and molecular analysis.

Results: Injury mice demonstrated hepatic fibrosis, inflammation and steatosis typical of NASH and HCC. At 12, 18 and 24 weeks, thrice weekly ihAEC-EV treatment resulted in a significant reduction in liver fibrosis (∼2-fold reduction, p<0.05) and macrophage numbers (1.6-, 3- and 3.6-fold reduction respectively, p<0.05). A reduction in steatosis, serum alanine transaminase (ALT) and aspartate aminotransferase (AST) was also observed following ihAEC-EVs treatment.

Conclusion: Orally administered ihAEC-EVs reduced hepatic fibrosis and steatosis in an experimental model of chronic inflammatory liver injury. The reduction of inflammation and fibrosis suggest potential to prevent the progression of MASH and eventually, HCC. Efficacy following thrice weekly administration suggests that repeated administration is required for circulating EVs to prevent the progression of disease. Overall, our findings support the clinical translation of ihAEC-EVs for inflammatory liver injury.

Duvvada Srinivas

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

One of the revolutionary events in the history of mankind is the discovery of a vaccine that saved millions of lives. Vaccines can induce both humoral and cell-mediated immune response and memory to the particular antigen of viruses/bacteria/fungi which are pathogenic. It is very difficult to build a universal delivery system for different vaccines. The occurrence of frequent infections caused by IBDV,IBV, and NDV among avians. The prevalence of Dengue. New viruses like COVID-19 are creating havoc. It is therefore imperative to look for novel ways and formulations to design vaccines to overcome these limitations. Bacterial (probiotic Nissle Ecoli) outer membrane vesicles (OMV) as a vaccine delivery vehicle using ClyA as a carrier, to improve immunogenicity and enhance the persistent release of immune-dominant vaccine candidates.

Methods

We used different immunoinformatic tools to predict antigenic fragments of IBDV, and NDV viruses and selected only those predicted regions. The antigen is tagged with ClyA which directs antigen to the outer membrane of bacteria. Antigen-specific B-cell response will be evaluated by the total IgG ELISA method in which anti-serum from immunized mice will be used to evaluate the specific antibodies against the given peptides. Cytokine secretion assay in lymph node cells and splenocytes to assess the levels of cytokines. Dynamic Light Scattering (DLS), Scanning Electron Microscopy (FESEM), TEM, Ultracentrifugation, and Ultrafiltration were employed for Biophysical characterization and purification of OMVs

Results

Those antigenic sequences predicted by in-silico analysis for VP2, and HN genes expressed in hyper vesiculating Nissle Ecoli 1719 purified them, and the recombinant OMVs (rOMVs) expressing these antigens were immunized to mice both subcutaneously and orally then examined their potential to induce persistent antigen-specific B and T-cell responses and produce virus-neutralizing antibodies by immunized mice. Mice immunized with rOMVs vaccine formulation showed an antigen-specific immune response, both serum IgG, IgA, and S-IgA.

Summary

Engineered probiotic bacterial OMVs can be used to deliver a wide variety of antigens, ligands, and drugs for vaccine and targeted(drug/cancer) therapy. Naturally, secreting nano carriers optimized for activating robust immune response makes OMVs a potential platform for efficient vaccine delivery.

4th grade in Ph.D Yusuke Nishiguchi¹, Ph.D Mamoru Ueda², Ph.D Hirohito Kubo², Ph.D Junichiro Jo³, Ph.D Yoshiya Hashimoto³, Ph.D Toshihiko Takenobu²

¹Graduate school of dentistry department of oral and maxillofacial surgery, Osaka Dental University, Osaka/Chuoku Otemachi, Japan, ²Second Department of Oral and Maxillofacial Surgery, Osaka Dental University, Osaka/Chuoku Otemachi, Japan, ³Department of Biomaterials Osaka Dental University, Hirakatashi/Kuzuhahanazonocho, Japan

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction

Extracellular vehicles (EV) have recently been attracting attention for their usefulness in various medical fields. These structures include various functional molecules (e.g., proteins, mRNA) and are less than 1000 nm in diameter. EVs isolated from stem cell have been shown to be valuable for bone regeneration. In addition, it is well known that the characteristics of EV change depending on the culture medium. This study is to investigate the effect of extracellular vesicles isolated from human dedifferentiated fat cells (DFAT) from the buccal fat pad on osteoblast differentiation using osteoinductive medium.

2)Methods

Adipose tissue was collected from the buccal fat pad exposed during jaw deformity surgery performed in our department, and DFAT was established using the ceiling culture method. After reaching 90% confluence, DFAT was changed to common medium with 2% exosome depleted FBS (DFAT-ctrl-CM) or osteoinductive medium (DFAT-OI-CM) and cultured for 3 days. The collected culture supernatants were concentrated, and Ev was purified by size chromatography. The morphology of the extracted Ev was observed using transmission electron microscope (TEM). Nanoflow cytometry was performed to detect the specific markers of Ev (CD9, CD63). Osteoblast induction was performed with DFAT using each EV and osteogenic differentiation induction medium. ALP activity was measured on the 7th and 14th day, and gene expression was quantified by RT-PCR. miRNA was extracted from each EV and small RNA-seq analysis (Illumina NovaSeq 6000) was performed.

3) Results

DFAT-Ev was CD9 and CD63 positive by nanoflow cytometry. Particles with a diameter of approximately 100 nm, which appear to be lipid bilayer were confirmed by TEM. The expression of ALP activity, RUNX2, and CollagenI in the DFAT-OI-Ev-added osteoblast differentiation medium group was significantly higher than that in the DFAT-ctrl-Ev-added osteoblast differentiation medium group. Small RNA-seq analysis revealed the expression of many osteogenic differentiation-related miRNA, and it became clear that there were differences between the two groups.

4) Conclusion

Small RNA-seq analysis suggested that the properties of Ev may differ depending on the culture medium. It was suggested that addition of DFAT-OI-Ev to the osteoinductive medium promoted osteogenic differentiation of DFAT.

Dr. Angliana Chouw^1,2, Dr. Cynthia Retna Sartika^1,2, Miss Geofanny Facicilia¹, Miss Annisa Nur Arofah¹, Miss Riska Agustina¹, Miss Zulfa Maulidah¹

¹Prodia Stemcell Indonesia, Jakarta, Indonesia, ²Universitas Padjajaran, Sumedang, Indonesia

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Background:

Umbilical cord mesenchymal stem cell (UC-MSC) Secretome holds great promise for therapeutic applications due to its immunomodulatory, tissue-regenerative, and anti-inflammatory properties. However, before clinical translation, a comprehensive understanding of its safety and efficacy profile are crucial, including its acute toxicity following different administration routes. Determining the acute toxicity of UC-MSC Secretome is essential to establish safe dosage ranges and guide further preclinical and clinical investigations. This study investigates the acute toxicity of UC-MSC Secretome in rat following different administration routes.

Method:

40 female Sprague-Dawley rats were randomly divided into four different groups based on route of administration: intravenous, intranasal, intraperitoneal, and intramuscular. The test substance was administered in accordance with the prescribed protocol.

A total of 1-mL UC-MSC Secretome was administered to each group. Test animals were observed individually for the first 30 minutes after administering the UC-MSC Secretome, followed by observation every 4 hours for 24 hours and daily up to 14 days. The animals were monitored for weight, toxic, and clinical signs for 14 days. Organ analysis was conducted at the end of observation by isolating the tissue from heart, liver, kidney, spleen, and lung.

Result and Discussion:

Single injection of UC-MSC Secretome through intravenous, intraperitoneal, intramuscular, and intranasal did not cause acute toxicity for 14 days of observations. In addition, there were no significant signs of clinical toxicity, or changes in laboratory and histopathology data in all concentrations of UC-MSC Secretome.

Conclusion:

This study concluded that there were no acute toxicity effects of UC-MSC Secretome delivered via intravenous, intraperitoneal, intramuscular and intranasal methods.

Mrs Maimonah Al-Masawa¹, Associate Professor Dr Angela Min Hwei Ng¹, Dr Jhi Biau Foo², Dr Chee Wun How³, Dr. Jia Xian Law¹

¹Centre For Tissue Engineering and Regenerative Medicine, Faculty of Medicine, National University Malaysia, Kuala Lumpur, Malaysia, ²School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, Malaysia, ³School of Pharmacy, Monash University, Malaysia

Poster Pitches (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:45 PM - 1:00 PM

Introduction: Atopic dermatitis (AD) presents a growing global concern. It is characterized by inflammatory skin manifestations, compromised skin barrier function, and dysregulated immune response. Existing treatments predominantly address symptoms without curative options, highlighting the urgent need for innovative therapeutic approaches. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have gained attention for their potential in immunomodulation and tissue repair akin to their parent cells. MSC preconditioning is a common strategy to enhance MSC functionality. This study investigates the therapeutic potential of naive umbilical cord MSC-EVs (NEVs) and interferon gamma preconditioned MSC-EVs (IFNEVs) for AD. Methods: MSCs isolated from umbilical cords were culture expanded and characterized according to ISCT guidelines. For the preconditioned group, MSCs were preconditioned with interferon gamma (IFN-γ), followed by two washes with PBS and addition of serum free media. EVs were separated from the conditioned media by a combination of differential centrifugation and tangential flow filtration. Characterization of EVs was conducted according to the MISEV2018 guidelines, employing nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot (WB). The study examined the effects of different concentrations of the two EV preparations (NEVs and IFNEVs) on the proliferation and viability of skin keratinocytes in vitro. Additionally, the influence of these preparations on the gene expression of skin keratinocytes exhibiting (AD)-like features induced by cytokines was assessed. Results: Both EV preparations exhibited positive expression for CD63, CD73, HSP70, and TSG101, and demonstrated a cup-shaped morphology. The mode diameter was approximately 60.8±1.8 nm for NEV and 61.4±2.9 nm for IFNEV. Our results indicated that neither NEV nor IFNEV altered keratinocyte viability. Low concentrations of NEV preparations, but not IFNEV, enhanced the proliferation of keratinocytes. Furthermore, NEV preparations, but not IFNEV, restored the expression of key epidermal structural proteins like filaggrin and loricrin and decreased the expression of inflammatory mediators such as CCL17. Conclusion: These findings highlight the modulatory effects of NEV preparations on atopic dermatitis-like features in vitro, showing promise for AD treatment. The limited impact of IFN-γ-preconditioning suggests the need for further investigation.

Dr Sik Loo Tan¹, Dr Zahrah Shamim¹, Omar Maged¹, Nik Aizah¹, Dr Qi Hao Daniel Looi², Dr JhiBiau Foo³, Professor Tunku Kamarul^1,4

¹Department of Orthopaedic Surgery, National Orthopaedic Center for Excellence in Research and Learning (NOCERAL), Faculty of Medicine, Universiti Malaya, 50603, Kuala Lumpur, Malaysia., Malaysia, ²My CytoHealth Sdn. Bhd., Lab 6, DMC Level 2, Hive 5, Taman Teknologi MRANTI, Bukit Jalil, Kuala Lumpur 57000, Malaysia, Bukit Jalil, Malaysia, ³School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, Subang Jaya 47500, Selangor, Malaysia, Subang Jaya, Malaysia, ⁴Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, Bertam 13200 Kepala Batas, Pulau Pinang, Malaysia., Bertam, Kepala Batas, Malaysia

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

This study aims to elucidate, in a high-fat diet(HFD) and streptozotocin(STZ)-induced Type II Diabetes(T2D) rat model, the effects of T2D on the tendon, articular cartilage, and subchondral bone, and subsequently assess the effects of intravenous mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) administration on tendon and cartilage degeneration in T2D rats.

Methods

Fifteen Sprague Dawley rats were utilized; three rats were fed with a normal diet(G1, as control rats), while the remaining 12 rats were induced to develop T2D through an HFD regimen followed by STZ(60 mg/kg) and nicotinamide(120 mg/kg) injection. OGTT was conducted to confirm the T2D status of all rats. At week 8(W8), intravenous administration of three different doses(n = 3 for each dose) of MSC-EVs (in mg/kg bw; G3:0.25mg, G4:0.50mg, and G5:1.00mg) was initiated at three-day intervals until W12. Three T2D rats served as a negative control(G2) without MSC-EVs administration. At W12, all rats were euthanized, and all knee, Achilles tendon(AT) and supraspinatus(Ssp) tendons were harvested for analysis.

Results

T2D led to a significant increase in the total cell count in AT and Ssp tendons, along with evident collagen fiber crimping observed in G2. Furthermore, a significant increase in total collagen content(in Ssp tendons) was evident in G2 compared to G1. However, no significant changes were observed in the biomechanical properties of AT in G2 compared to G1. X-ray microscope imaging analysis revealed a significant difference in bone volume, exclusively in the non-load-bearing region of the distal femur, with no significant differences observed in other bone morphometric parameters. A significantly higher modified Mankin scores were observed in G2 compared to G1. Treatment with MSC-EVs significantly attenuated the number of rounded cells and total cell count in both AT and Ssp tendons, while restoring their histological appearance to a state closer to that of G1. Interestingly, a significant increase in cartilage thickness was observed in MSC-EVs treated G5 compared to G1, G3, and G4.

Conclusion

In conclusion, T2D disrupts histological and cellular characteristics in tendons and cartilage. Intravenous administration of MSC-EVs in T2D rats ameliorated histological features closer to the normal tendon, reduced tendon cellularity, and improved cartilage thickness.

Dr. Tomohiro Umezu, Mamoru Yanagimachi, Ph.D. Masakatsu Takanashi, MD, Ph.D. Yoshiki Murakami, MD, Ph.D. Masahiko Kuroda

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction

In recent years, there has been active development in drug delivery systems (DDS) based on the properties of extracellular vesicles (EV). However, several challenges persist in supplying EVs for clinical applications, such as cost, quality, and storage. Consequently, we identified that exosome-like vesicles can be efficiently extracted from acerola juice at a low cost.

The objective of this study is to orally administer ApoB siRNA and ApoA1 mRNA to the target tissue (liver) using acerola-derived nanovesicles as DDS carriers.

2) Methods

Acerola juices were obtained from Nichirei Foods Inc. Exosome-like nanovesicles (AELNs) derived from acerola juices were extracted using the exoEasy Maxi kit (Qiagen).

ALENs (1.09 × 10^9 particles/ml) and synthetic ApoB siRNA (100 pmol) or ApoA1 mRNA (100 pmol) were mixed in 50 µL of PBS. After incubation on ice for 30 min, oral administration was conducted with total volume 200 µL sample, including 10mg/kg of ApoB siRNA-AELN complex, using a sonde in C57BL/6 female mice (8 weeks old, n = 5 per cohort).

3) Results

The acerola juice yielded 1 × 10^12 exosome-like nanovesicles in 8 mL. In vivo kinetics of acerola vesicle-nucleic acid drug complexes were analyzed using a mouse model. PKH26-labeled acerola vesicles were orally administered, and fluorescence signals were observed in the stomach, intestinal tract, liver, kidney, and notably in brain tissue, including the cerebrum, indicating central nervous system penetration.

To explore clinical applications, oral administration of siRNA-AELN complexes targeting the ApoB gene was conducted. Knockdown efficiency was assessed by qPCR over time. 24 hours post-administration, ApoB gene expression showed a decrease in the liver and small intestine, confirming siRNA-induced knockdown. Additionally, an elevation in ApoA1 protein levels was noted in the liver and serum following oral administration of the ApoA1 mRNA-AELN complex.

4) Summary/Conclusion

This research highlights three key aspects: 1) emphasis on plant-derived exosomes, offering cost-effectiveness and stable supply; 2) the creation of a highly safe drug delivery system using plant-derived vesicles, typically consumable by humans; and 3) the pioneering development of the world's first orally administrable nucleic acid medicine.

Ph.d Student Diksha Choudhary

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

INTRODUCTION: The obesity epidemic is a complex, multifactorial, and largely preventable disease, affecting over a third of the world's population today. Recently, growing evidences has implicated Plant-Derived Extracellular Vesicles (PDEVs) as an important therapeutic agent because PDEVs display very similar features to mammalian exosomes and PDEVs might enhance plant miRNA delivery to in vitro and in vivo models. In this study, we report that treatment with Ginger-derived Extracellular Vesicles (GEV)s reduced lipid accumulation in 3T3-L1 cell line by affecting the major genes participating in adipocyte differentiation and PPAR signaling pathway.

Methods: We extracted EVs from ginger juice following standard ultracentrifugation method. After isolation GEVs were characterized with the help of DLS and TEM. GEVs uptake ability by host cells was studied under Confocal Microscopy. To check the effect of GEVs on lipid accumulation the NIH-3T3-L1 cells were treated with GEVs for specific period of time. After that genes associated with adipogenesis were further quantified using RT-PCR. Further, the miRNA profile of GEVs were analyzed using high-throughput sequencing followed by bioinformatic analysis. miRNA gene targets (genes involved in adipocyte differentiation) were identified in mouse and human with the help of miRanda and enrichment analysis was done with DAVID.

Results: The in-vitro study showed that GEVs treated 3T3-L1 cells demonstrated a dose-dependent decrease in lipid accumulation. In bioinformatic analysis with the help of intaRNA, specific miRNAs were identified that targe genes and pathways associated with adipogenesis in human and mouse. Furthermore, studies were conducted to decipher the molecular mechanism which highlight the role of GEVs in decreasing the mRNA expression of transcription factors (PPARϒ, C/EBPδ) and terminal differentiation genes (LPL, SCD1) which play key role in adipocyte differentiation.

Conclusion: Our findings support GEVs is composed of multiple factors like lipids, protein and nucleic acid (miRNAs) and have potential to correct multiple dysregulated pathways during the development of obesity. Further, our studies demonstrated that GEVs plays crucial role in the downregulation of genes which participating in PPAR pathway. These positive effects could be related to the miRNAs enriched in the GEVs. In summary, our results provide natural therapeutic agent against obesity.

Graduate Student Zhu Zhao¹, Dr. Jerome Lacombe¹, Dr. Frederic Zenhausern¹

¹Univ Of Arizona, Phoenix, United States

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Extracellular vesicles (EV) have shown great promise as drug delivery system (DDS). However, their complex and costly production limit their development for clinical use. Interestingly, plant kingdom can also produce EV-like nanoparticles that can be easily isolated and purified from a large quantity of raw material at high yield. Here, we deeply characterized olive-derived nanoparticles (ODNPs) physical and biological features to demonstrate their promising potential to be used as a DDS.

Methods

ODNPs were isolated from raw fruits after blending, serial centrifugations and sucrose gradient ultracentrifugation. Size, surface charge and morphology were characterized by nano-tracking analysis (NTA), dynamic light scatters (DLS) and CryoEM. ELISA and lipidomics analysis were performed to assess surface biomarkers and membrane composition, respectively. Effect of physical stress and storage conditions on ODNPs size and yield, were also investigated by NTA. Cell viability tests and immunofluorescence imaging were performed on both 2D and 3D cell culture to explore the cytotoxicity and internalization of ODNPs. Finally, anticancer agent doxorubicin (Dox) was loaded into ODNPs and treatment efficiency assessed by viability assay.

Results

NTA indicated an average size of 100 nm and yield of 1012 ODNPs/ml. CryoEM and lipidomics showed the presence of a lipid bilayer predominantly made of glycerolipids, sterol lipids and fatty acyls. ODNPs are enriched in PEN1 and TET8 compared to raw fruits. ODNPs size and yield stay unchanged after exposure to 70°C for 1 h, pH 5-10, 0-10 mM salt and 50-100 nm extrusion. They are also stable in water at 4°C for a month and the addition of trehalose is required for long-term freezing storage. ODNPs are internalized by both 2D and 3D cell culture without triggering cytotoxicity. Dox-loaded ODNPs decreased cell viability by 90%, compared to only 70% for free Dox at the same dox concentration. Interestingly, this high cytotoxicity biological effect of Dox-loaded ODNPs is stable after stored for 2 weeks in the fridge.

Conclusion

ODNPs are promising DDS candidates as demonstrated by their biocompatibility, high resistance to stress, good stability in minimal environment, and improvement of anticancer drug efficacy.

Su Jin Kang, Ph.D Won Jong Rhee

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Extracellular vesicles (EVs) are small, membrane-bound structures released by cells into their surrounding environment. Recent research has witnessed a significant surge in the utilization of plant-derived EVs. Plant-derived EVs offer several advantages, including high productivity, low production cost, diverse biological functions, and low cytotoxicity. In this study, our aim was to expand the potential therapeutic applications of red cabbage-derived extracellular vesicles (Rabex). Furthermore, we embarked on engineering Rabex to achieve precise targeting and modulation of inflammation.

In the inflammatory bowel disease (IBD) model, specifically the well-established acute colitis model induced by dextran sulfate sodium (DSS), we provided compelling evidence that engineered Rabex possesses substantial anti-inflammatory effects in vivo. To enhance the therapeutic effects of Rabex, we engineered its surface with hyaluronic acid (HA) to target CD44 receptors on immune cells and intestinal epithelial cells.

Engineered Rabex with HA (Rabex_HA) showed increased delivery efficiency to both cell types and exhibited no cytotoxicity in THP-1 cells. In the IBD model, Rabex_HA demonstrated enhanced therapeutic efficacy compared to Rabex alone, validating the targeting effects and potential of engineered Rabex_HA for IBD treatment.

Our findings not only underscore the therapeutic promise of Rabex but also illuminate the diverse applications of plant-derived EVs. In conclusion, engineered Rabex holds promise as a targeted nanomedicine for the effective and safe treatment of IBD.

Mr. Yasir Mohamed Riza, Dr. Faisal Alzahrani, Dr. Rami Mosaoa

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Background: Extracellular vesicles, especially those ranging from 20 to 200 nm in diameter, have shown promise in therapeutic and diagnostic applications due to their safety, cost-effectiveness, and scalability. This pilot study investigates the chemo-resistive effects of seaberry and garlic-derived extracellular vesicles in human liver (HEPG2) and breast (MCF-7) cancer cell lines and their synergistic potential with the chemotherapeutic agent cisplatin. Additionally, we compared these effects with those on normal breast cells and analyzed the metabolite cargo of both extracellular vesicles.

Methods: We evaluated the combined impact of seaberry and garlic extracellular vesicles with cisplatin on HEPG2 and MCF-7 cell lines. The cytotoxic effects were quantified and compared against the effects of cisplatin alone and on normal breast cells. The concentration-dependent response of the cell lines to the treatment was assessed. Furthermore, the metabolite cargo of both types of extracellular vesicles was analyzed using Ultra-High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (UHPLC-MS/MS).

Results: The study observed a significant increase in the efficacy of cancer cell inhibition with the combined treatment, especially notable in the MCF-7 cell line, which showed the lowest IC50 value of 4.481, indicating enhanced inhibition compared to cisplatin alone. This synergistic effect, which was directly proportional to the concentration of the treatment, was contrasted with the effects on normal breast cells. The UHPLC-MS/MS analysis of the extracellular vesicles' metabolite cargo provided further insights into the mechanistic aspects of their chemo-resistive properties.

Conclusion: This pilot study suggests that extracellular vesicles from seaberry and garlic, combined with cisplatin, can synergistically enhance the inhibition of liver and breast cancer cells while exhibiting a different impact on normal breast cells. The UHPLC-MS/MS analysis of the vesicles' metabolite cargo sheds light on their potential roles in this enhanced efficacy. These findings open new avenues for research into the clinical applications of natural extracellular vesicles as adjuncts in cancer treatment and warrant further exploration to fully understand and utilize this novel therapeutic approach.

Dr. Ramesh Gupta, Dr. Raghuram Kandimalla, Ms. Disha Moholkar, Dr. Margaret Wallen, Mr. Jeyaprakash Jeyabalan, Dr. Wendy Spencer, Dr. Neetu Tyagi, Dr. Farrukh Aqil

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Exosomes are emerging as key nano biocompatible materials for delivery of both small molecules and biologics. Based on their ability to cross the blood brain barrier, exosomes offer an efficient means for delivery of drugs to the brain. Bovine milk and colostrum serve as biocompatible, abundant, and cost-effective sources of natural exosomes for delivery of drugs as reported in our series of papers. In this study, we used colostrum exosomes for delivery of curcuminoid mixture, curcumin (CUR) and bisdemethoxycurcumin (BDMC), to target the brain of AD mice.

Methods: Exosomes were isolated from standardized bovine colostrum powder by rehydration and differential centrifugation and characterized for the exosome characteristics such as size, PDI, zeta potential, and presence of hallmark exosome markers. One-year old 3xTg-AD mice were randomized and treated with ExoCUR/BDMC and free CUR/BDMC at a combined dose of 20 mg/kg. Age-matched AD and WT mice treated with PBS served as controls. Test regimens were given by oral gavage daily, animals were euthanized after 3 weeks, and brain tissues were analyzed for modulation of key markers of AD by Western blot. In a separate study, WT mice were treated with ExoCUR/BDMC or free CUR/BDMC for analysis of brain curcuminoid levels by UPLC.

Results: The average particle size of the exosomes was <70 nm as measured by Zetasizer and confirmed by atomic force microscopy. Whole brain lysates of 3xTg-AD mice treated with ExoCUR/BDMC showed decreased levels of pTau and amyloid precursor protein (APP) compared to untreated AD mice, while free CUR/BDMC was ineffective. Furthermore, the levels of brain-derived neurotrophic factors (BDNF) in AD mice treated with ExoCUR/BDMC, not free CUR/BDMC, returned to the basal levels of age-matched untreated WT mice. Brain tissue analysis by EMSA (electrophoretic mobility-shift assay) showed significant reduction in the hallmark anti-inflammatory molecule, NFκB accumulation only with ExoCUR/BDMC. The favorable modulation of the key AD biomarkers with exosomal formulation of CUR/BDMC, not with free CUR/BDMC corroborate the higher brain levels of the curcuminoids.

Conclusions: In summary, oral exosomal delivery of small drug molecules like CUR/BDMC can effectively target the mouse brain for neurological diseases, including AD.

Li Chan-gu, Professor Fu Qing-Ling

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Background:

Extracellular vesicles (EVs) are reported to show great potential as drug carriers. However, the lack of a large-scale and cost-effective EVs purification method are limited for clinical application. Bovine milk contains abundant EVs (mEVs), which shows low immunogenicity, biosafety and cost-effectiveness. Thus, mEVs show promising potential as as carriers for drug delivery.

Previous studies have demonstrated that miR146a-5p is able to alleviate allergic airway inflammation (AAR) by inhibiting the function of group 2 innate lymphoid cells (ILC2s), a subset of ILCs crucial in the initiation and maintenance of AAR. However, the utilization of miR146a-5p, similar with other oligonucleotides, faces challenges such as degradation susceptibility and difficulty in crossing cytomembrane. In this study, we aim to examine the effects of mEVs delivering miR146a-5p to ILC2s (miR146a-5p-mEVs) in ILC2-AAR model.

Methods:

mEVs were purified from bovine milk using a combination of ultracentrifugation and tangential flow filtration method. Extrusion was used to load miR-146a-5p into mEVs. After assessing the efficiency of loading and the stability of miR146a-5p in mEVs, we evaluated the therapeutic effects of miR146a-5p-mEVs on ILC2s function using human peripheral blood mononuclear cells (PBMCs) derived from pateints with allergic rhinitis, and ILC2s-dominate AAR mouse model. The levels of type 2 cytokines and the frequency of ILC2s were determined. Inflammatory cell infiltration and mucus production of lung tissue were evaluated by pathological examination.

Results:

We successfully purified high-yield EVs from bovine milk, which shows biocompatibility and high efficiency in delivering miRNA into cells, and miR146a-5p in mEVs shows great stability. miR146a-5p-mEV were found to significantly inhibit the function of ILC2s both in human and mouse model. The level of T helper 2 cytokines and ILC2s were decreased both in human PBMCs and mice. Moreover, inflammatory cell infiltration and mucus production in the mice lung were alleviated after the treatment of miR146a-5p-mEV.

Conclusion:

We successfully loaded miR146a-5p into mEVs via extrusion. miR146a-5p-mEVs were able to prevent ILC2-dominant allergic airway inflammation, suggesting that mEVs have potential to be nano-carriers to deliver miRNA.

PhD Student Abhinay Kumar Singh, Dr. Win-Ping Deng

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction

Despite the advancement in chemotherapeutic drugs for colon cancer treatment, it is still a life-threatening disease worldwide due to drug resistance. Therefore, an urgently needed to develop novel drugs for colon cancer therapies. Extracellular vesicles (EVs) from vegetable and germinated brown rice (VGBR) contains a lot of biomolecules like proteins, miRNAs, and mRNA that may be exerting anti-oxidative, anti-inflammatory, anti-microbial and anti-tumor activities in oral cancer. In this study, we investigate EVs-derived from VGBR (EVs-VGBR) anti-proliferative, anti-metastatic and apoptotic activity to explore its anti-cancer activity against colon cancer cells and its underlying mechanism.

2) Method

Here, in-vitro studies were performed to determine the antiproliferative activity of EVs-VGBR through MTT and colony formation assays. Wound healing and transwell migration assay were used to evaluate the metastasis. Flow cytometry and protein expression were used to investigate the involved molecular mechanism by evaluating the cell cycle and apoptosis. We also characterized the EVs-VGBR through NTA and TEM. The in-vivo anti-cancerous activity of EVs-VGBR was assessed by xenograft mice model of colon cancer cells.

3) Results

We found that EVs-VGBR significantly inhibited the proliferative capacity and metastasis of colon cancer cells in-vitro. In addition, EVs-VGBR induced cell cycle arrest through upregulating/downregulating p21 in colon cancer cells. Annexin-v assay indicated that colon cancer cells had entered early or late apoptosis after treatment with EVs-VGBR. Furthermore, a mechanistic protein expressions study revealed that EVs-VGBR regulated p53-pathway to maintain the apoptosis and increasing the expression of Bax and caspase-9 to inhibit the colon cancer cells. In vivo study, we found that EVs-VGBR significantly reduced the xenograft tumor growth in NOD/SCID mice with no adverse effect on the kidney and liver.

4) Conclusion

Collectively, EVs-VGBR has the potential to inhibit colon cancer through inhibiting proliferation, migration, and cell cycle kinase by upregulating p21 protein expression and promoting the apoptotic protein through p53-pathway.

Dandan Su, Manchun Li, Professor Hongbo Chen, Fang Cheng

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: The gut microbiota are crucial factors influencing host health and disease by modulating both innate and adaptive immune responses. Parabacteroides goldsteinii MTS01, a newly discovered gut commensal bacteria strain, demonstrates immunomodulatory effects in inflammatory diseases. However, numerous uncontrollable risks constrain the clinical use of live bacteria therapy. Outer membrane vesicles (OMVs) derived from bacteria may play a crucial intermediary role in immune-related diseases due to their biological characteristics.

Methods: The OMVs derived from Parabacteroides goldsteinii MTS01 (Pg OMVs) were purified under a set of procedures. The composition of Pg OMVs was resolved through multi-omics sequencing. Their immunoregulation in vitro were analyzed by flow cytometry analysis, ELISA, western blot, and so on. In addition, imiquimod induced psoriasis-like mouse were treated with the oral administration of Pg OMVs or the subcutaneous injection of PF-127 hydrogel loaded with Pg OMVs to verify whether Pg OMVs could modulate the inflammation in the skin lesions.

Results: Our study showed that Pg OMVs can effectively inhibit M1 macrophages, and reduce the maturation of dendritic cells and the release of inflammatory factors, thereby inhibiting the differentiation of Th17 cells and decreasing the proliferation of keratinocytes. Mechanistically, we found that Pg OMVs are rich in pentadecanoic acid, an odd-chain fatty acid, which serves as a key substance mediating the immunomodulatory effects of Pg OMVs. In imiquimod-induced psoriasis mice, we observed that both oral administration and subcutaneous injection of Pg OMVs effectively downregulated inflammation at the skin lesions and systematically, leading to a significant improvement in the symptoms of psoriasis.

Summary: Our research comprehensively investigated the multifaceted immunomodulatory functions of Pg OMVs secreted by gut bacteria, which modulate skin inflammation not only through the gut-skin axis but also via local administration. This study indicates the potential for Pg OMVs as a promising therapeutic alternative to live bacteria therapy for the treatment of psoriasis and other inflammatory skin diseases.

Dr Ishmael Inocencio, Mr Naveen Kumar, A/Prof Rebecca Lim, Dr Tamara Yawno

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Infection of the placental membranes, termed chorioamnionitis, commonly causes inflammatory fetal brain and lung injury. This greatly increases the risk of long-term adverse outcomes including cerebral palsy and bronchopulmonary dysplasia. Human amniotic epithelial cells (hAECs) possess anti-inflammatory and proangiogenic properties and can protect against inflammatory organ injury in the perinatal population. However, complexities surrounding storage and transport has hindered clinical uptake. hAEC-derived extracellular vesicles (hAEC-EVs) demonstrate comparable therapeutic efficacy but are easier and cheaper to store and transport, thereby offering a more readily available and affordable therapeutic option for chorioamnionitis-associated injury. Most investigation into the therapeutic potential and safety of EVs has been performed in small animal models. Physiological parameters (oxygenation, heart rate and blood pressure) are important indices of safety, especially for translation into first-in-human trials. However, this cannot be easily measured in rodents. We aimed to investigate the physiological response and therapeutic potential of intravenous hAEC-EVs in a large animal model of fetal inflammation.

Methods: hAECs were cultured in chemically defined media. Conditioned media containing hAEC-EVs was concentrated using tangential flow filtration and hAEC-EVs isolated using size exclusion chromatography. Fetal sheep at 99 days of gestational age (dGA) were surgically exteriorised. Arterial and venous catheters were implanted for continuous in-utero physiological recordings and intravenous (IV) treatment administration. Fetuses were returned to the uterus and randomly allocated to: 1) control, 2) injury or 3) injury + treatment. Fetal inflammation was induced in groups 2 and 3 via IV lipopolysaccharide (LPS). Group 3 received IV hAEC-EVs and group 1 received saline. Physiological recordings were collected until 113 dGA. Fetuses were then euthanised and brains and lungs collected for histological analysis.

Results: Fetal physiology was not different between all groups. Alveolar thickness and epithelial sloughing (indices of lung injury) and microglial activation increased (marker of neuroinflammation and injury) following LPS challenge but decreased following hAEC-EV administration.

Conclusion: Stability of physiological parameters suggests acute systemic exposure to hAEC-EVs is safe. Further, decreased indices of cerebral and pulmonary injury suggest therapeutic potential for fetal brain and lung injury. Future studies will focus on optimisation of dosage and timing of hAEC-EV administration.

Haobo Wang, Dr Wen Chen, Dr Yan Wang, Dr Danyang Li, Dr Lizhou Xu

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction: As a medicinal and edible resource, gardenia contains a variety of important bioactive components such as iridoid terpene glycosides dominated by gardeniflorin and carotenoids dominated by crocin glycosides. Gardenia-derived extracellular vesicles show great potential in containing bioactive compounds which could be developed as future drugs for disease treatment.

2) Methods: Density gradient ultracentrifugation on sucrose was used to extract gardenia-derived extracellular vesicles (gEVs). Their size and concentration were characterized by NTA and the morphologies were observed by TEM. HPLC and GC-MS were used to detect key chemical components in gEVs. Cellular uptake studies were conducted by treating the cells with the isolated gEVs. An in vitro Parkinson's disease injury model was constructed using MPP+ induced PC12 cells for exploring the biological activity of gEVs.

3) Results: The collected gEVs were purified into two specific populations, gEVs1 (8-30%) and gEVs2 (30-45%), based on their densities. The particle size of gEVs1 was smaller than that of gEVs2 as illustrated by NTA and TEM observation despite that both groups had similar vesicle structures and morphologies. The concentrations of Geniposide, crocin1 and crocin2 in gEVs1 were higher than those in gEVs2 as shown by HPLC data, showing the isolation difference affects the final amount of active ingredients of in gEVs. Cellular uptakes of gEVs by Caco-2, Hepg2 and PC12 cells were confirmed by confocal microscopy images. The gEVs were found to be biologically safe to the recipient cells. The gEVs showed therapeutic effect to the MPP+ induced Parkinson's disease model, with a dose-dependent protective effect for PC12 cells. gEVs1 showed 20% more protective effect than the other group in terms of viability.

4) Summary/Conclusion: Gardenia-derived gEVs are novel, natural phytosome-like nanovesicles containing a variety of bioactive components with potential health-promoting activity and may become a new drug against Parkinson's disease.

Professor Kewei Zhao, Doctor Yue Cao, Master Xuejun Tan

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Morinda Officinalis (MO) contains various components with anti-osteoporotic activities. Morinda Officinalis-derived extracellular vesicle-like particles (MOEVLPs) are active components isolated from MO, but no relevant studies have investigated their anti-osteoporosis effect and mechanism.

Methods: Differential centrifugation and ultracentrifugation were used to isolate MOEVLPs from MO. Transmission electron microscopy (TEM), flow nano analyzer, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), agarose gel electrophoresis and thin-layer chromatography were employed to characterize MOEVLPs. PMOP mouse models were utilized to examine the anti-PMOP effect of MOEVLPs. H&E and immunohistochemical staining were used for drug safety and osteogenic effect assessment. Mouse embryo osteoblast precursor cells (MC3T3-E1) were used in vitro experiments. CCK-8 kit, alizarin red staining, proteomic, bioinformatic analyses and western blot were used to explore the mechanism of MOEVLPs.

Results: In this study, MOEVLPs from MO were successfully isolated and characterized. Animal experiments demonstrated that MOEVLPs exhibited specific femur targeting, were non-toxic, and possessed anti-osteoporosis properties and the ability to strengthen bone formation. In vitro experiments, results revealed that MOEVLPs did not significantly enhance osteogenic differentiation in MC3T3-E1 cells. Instead, MOEVLPs promoted the proliferation of MC3T3-E1 cells. Proteomic and bioinformatic analyses suggested that the proliferative effect of MOEVLPs was closely associated with the mitogen-activated protein kinase (MAPK) signaling pathway, particularly the altered expression of cAMP response element-binding (CREB) protein and 90 kDa ribosomal S6 kinase 1 (RSK1). Western blot results further confirmed these findings.

Conclusion: Our studies successfully isolated high-quality MOEVLPs and demonstrated that MOEVLPs can alleviate PMOP by promoting osteoblast proliferation through the MAPK pathway. MOEVLPs have the potential to become a novel, natural anti-PMOP drug.

Mr. Dulla Naveen Kumar, Ms. Aiswarya Chaudhuri, Ms Deepa Dehari, Dr. Dinesh Kumar, Dr. Ashish Kumar Agrawal

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Cancer is a leading cause of death worldwide, particularly skin cancer, where cases have risen quickly in the past decade. Melanoma is a form of skin cancer with the most aggressive and poor prognosis; due to its nature, and it is difficult to treat. Fewer therapeutic options are available for melanoma treatment, particularly chemotherapeutics that include dacarbazine (DTIC) as the cornerstone. However, DTIC produces several dose-related toxicities, like liver toxicity, which compelled scientists to search for or develop a new drug to treat melanoma effectively. Dihydroartemisinin (DHA) is a semisynthetic active metabolite of all artemisinin compounds which is well-known for its anti-malarial activity. Still, after some studies, it was found that apart from its anti-malarial activity, it also shows anti-cancer activity. Despite its effective anti-cancer property, DHA has some pharmaceutical issues like low solubility and toxicity that hinder the therapeutic activity of DHA. To address these issues, we developed a sEVs formulation of DHA that will improve its anti-cancer efficacy and decrease metastasis in vitro and in vivo.

Methods: sEVs were isolated, and DHA was loaded via the sonication method. The DHA-loaded sEVs were characterized in terms of size, PDI, and zeta, via particle size analyser and morphology via SEM/AFM and crystallography via XRD. The developed formulations were tested for in vitro and in vivo efficacy study via cytotoxicity, uptake assay, apoptosis assay, MMP assay, migration assay, Colony formation assay, Oral bioavailability and in B16F10 induced melanoma model

Results: The particle size, PDI and Zeta potential were found to be 90-103 nm, 0.119-0.123, and -23 to -28 mV, respectively. The entrapment efficiency and DL% were found to be 17% and 76% respectively. Both in vitro and in vivo studies confirmed that the DHA delivery via sEVs improved oral bioavailability and efficacy, and reduced toxicity The in vitro efficacy was confirmed via cytotoxicity and apoptosis assay. Interestingly, sEVs-DHA significantly improved the animals' life span and decreased melanoma cells' metastasis.

Conclusion: Despite its good in vitro and in vivo results, western blotting and xenograft models are required to elucidate the mechanism of action, mimicking human cancer.

Ms Wensi Zhu, Ms Linxiao Han, Ms Ludan He, Dr Chih-Jung Chang, Jian Zhou

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Acute lung injury (ALI) is a disease with the highest mortality rate among respiratory diseases. However, there is still a lack of effective treatment for ALI. Parabacteroides goldsteinii is a newly discovered probiotic that exerts anti-inflammatory effects by altering the gut microbiota. However, the function and role of Parabacteroides goldsteinii-derived exosomes (Pg-Exos) in ALI is still unknown.

Methods: After the ALI mouse model was established by instilling bleomycin (BLM) into the airway, Pg-Exos were administered intragastrically. The morphology of lung tissue was observed by HE staining, and the secretion of inflammatory factors in bronchial alveolar lavage fluid was detected by ELISA. RNA sequencing was further performed on the lung tissue to detect the differential gene expression. The impact of Pg-Exo on the gut microbiota was analyzed through Metagenomic Next-Generation sequencing. The role of Pg-Exo was verified through fecal microbiota transplantation (FMT) experiment.

Results: Compared with mice only instilled with BLM (BLM group), the mice treated with Pg-Exos (BLM_Exo group) had less lung damage, with less inflammatory cell infiltration and lower inflammatory factors secretion (IL-1β, IL-6). Moreover, Pg-Exos could significantly inhibit the activation of the NLRP3 inflammasome signaling pathways and inflammasomes. Gut microbiota analysis revealed that after instillation of BLM, gut microbial dysbiosis was observed. After treatment with Pg-Exos, there was a significant difference in the species abundance of the gut microbiota in the BLM group and the BLM_Exo group, and the proportion of Akkermansia muciniphila increased significantly in the BLM_Exo group. Furthermore, compared with the germ-free mice giving feces from the BLM group, the weight loss of the germ-free mice giving feces from the BLM_Exo group was significantly reduced, whereas the survival rate was significantfly improved.

Conclusion: Parabacteroides goldsteinii-derived exosomes alleviate the inflammatory infiltration and lung damage of ALI by changing the composition of the gut microbiota (increasing the proportion of Akkermansia muciniphila) and inhibiting the production of inflammasomes.

Dr Ningcen Li, Prof Bo Li, Prof Lei Zheng

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Rheumatoid arthritis (RA) is a common, chronic, inflammatory and debilitating disease that mainly affects joints, manifested as pain and swollen joints, seriously damaging physical function and the quality of life. Reducing inflammatory response is the main treatment goal for RA, but due to the non-specific distribution of anti-inflammatory drugs and increased metabolic burden on the body, many adverse reactions usually occur. Therefore, there is an urgent need to explore new drug delivery systems and other complementary strategies with practical targeting capabilities.

Methods: Extracellular vesicles from medicinal plants have been widely used in drug delivery systems and clinical studies as a new natural nanoscale drug carrier. In this experiment, we combined Polygonum cuspidatum (a traditional Chinese herb) derived nanoparticles (PDNPs) with acupuncture to treat complete Freund's adjuvant (CFA) induced RA mice. Preparation of PDNPs with anti-inflammatory effects using ultrahigh-speed centrifugation method. PDNPs were injected through the ankle joint and acupuncture is performed at the Zusanli (ST36) acupoint to promote targeted and effective delivery of PDNPs to specific damaged areas, thereby improving treatment effectiveness.

Results: PDNPs were extracted and purified by ultrahigh-speed centrifugation. PDNPs were spherical with a particle size of 229.93 nm and a zeta potential of -14.1 mV. PDNPs combined with acupuncture can alleviate the foot swelling of mice, reduce cartilage damage and inhibit the development of arthritis, playing a synergistic role. After administration, the fluorescence in the ankle and plantar areas gradually increased, indicating that PDNPs may target inflamed joints. Acupuncture can better promote PDNPs to reach arthritic joints. Further research shows that PDNPs combined with acupuncture can restore the balance of macrophages and regulate the production of inflammatory cytokines, which may be an important link in the treatment of RA.

Conclusion: In summary, we successfully developed a combined therapy of PDNPs and acupuncture for targeted RA. This combined treatment with PDNPs and acupuncture provides a perspective therapeutic strategy for RA treatment from the perspective of immunomodulation.

Qi Xiu, Prof. Bo Li, Prof. Lei Zheng

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Burns are one of the most common forms of skin injuries. Deep-second degree burn, easy to cause tissue necrosis, poses a major clinical problem. Polygonum cuspidatum is a widely used herb for curing burn injury for hundreds of years in Asian. However, the bioactive components of Polygonum cuspidatum (PC) are easily degraded by a variety of enzymes in the external environment of human body, which brings difficulties to the delivery of therapeutic components of PC. Herein, we used a deep-second burn mouse model to show that nanoparticles isolated from PC juice using centrifugation procedure resulted in accelerating wound healing. Then we loaded PDNs into thermos-responsive hydrogel PF-127 to create a PDN-gel composite as an in situ forming delivery system. Our results showed PDNs could accelerate wound healing by promoting the migration of vascular endothelial cells and keratinized epithelial cells, resisting oxidative stress and inducing M0 macrophages to polarize into anti-inflammatory M2 macrophages. And with the help of PF-127, the PDNs will be firmly attached to the wound and gradually released. Furthermore, we identified PDNs mediated activation of nuclear factor erythroid 2-related factor 2 (Nrf2) leads to the expression of a group of antioxidant genes and inhibits the production of reactive oxygen species, thus protecting the skin away from exacerbates tissue damage and inflammatory reactions. In summary, this study designed the thermo-responsive hydrogel PF-127 loaded with PDNs to treat deep second-degree burns on the skin. By optimizing the administration conditions, the therapeutic mechanism was elucidated and the therapeutic effect in mice was verified, which provided an efficient, economical and simple new strategy for the treatment of deep second-degree burns.

PhD Student Amelie Legare, Miss Michele Iskandar, Andre Marette, Stan Kubow

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction: In Autism Spectrum Disorder (ASD), multifaceted behavioral, social, and cognitive deficits are often accompanied with gastrointestinal issues. While the gut-brain interplay mechanisms in ASD remains poorly understood, clinical trials hint at pre- and probiotics' potential in regulating the gut microbiota and alleviating ASD-related symptoms. Notably, polyphenol-rich cocoa prebiotic has been linked to amelioration of behavioral deficits in children with ASD. However, limited bioavailability of crucial prebiotic-derived metabolites might hinder their effectiveness. Utilizing bacterial membrane vesicles (MVs) seems promising, as they may facilitate improved their delivery to target organs. Here, we produced a novel symbiotic bacterial MV, by co-incubating Escherichia coli Nissle 1917 with a polyphenol-rich cocoa powder.

2) Methods: Probiotic strain E. coli Nissle 1917 was grown in Luria-Bertani (LB) broth with either polyphenol-rich powder or vehicle. In addition, two negative controls, containing LB with or without cacao, were produced. For all groups, including controls, MVs isolation protocol inspired by Watson, D.C. et al (2021) (EV-TRACK ID: EV210211) was performed. Briefly, tangential flow filtration combined with size-exclusion chromatography was used. Particle size and concentration were quantified using tunable resistive pulse sensing and purity was assessed by transmission electron microscopy. Untargeted metabolomics was performed by UPLC-MS/MS and metabolites were mapped against the KEGG database.

3) Results: Our preliminary results suggest that bacterial MVs derived from cocoa-treated E. coli, are enriched in numerous metabolites involved in tryptophan metabolism. In fact, 5-hydroxyindoleacetaldehyde and kynurenate, antagonist of amino acid receptors NMDA, seems to be found in higher proportion in the cocoa-treated MV compared to the vehicle-treated group. Interestingly, the serotonin precursor, L-tryptophan, is uniquely present in the cocoa-treated MV.

4) Summary/Conclusion: In summary, the enrichment of pre- and probiotic-derived neuroactive metabolites within bacterial vesicles presents a compelling prospect, suggesting an innovative avenue to potentially enhance bioavailability and unlock more robust therapeutic benefits in the management of ASD. Further investigations are warranted to elucidate their proteomic and transcriptomic profiles, to shed light on their molecular mechanisms. Moreover, exploring their potential implications in in vitro and animal models of ASD will offer valuable insights into the development of improved therapeutic interventions.

HoChung Jang, Dr. Yoosoo Yang

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Extracellular vesicles (EVs), playing a crucial role in macromolecule transmission and intercellular communication, are increasingly recognized as robust nanovesicles for targeted drug delivery. While accumulating evidence demonstrates the unique modes of action of EVs that enable them to reach various targets, few studies have investigated the mechanisms underlying their potential properties for promoting tumor-targeted drug delivery following oral administration. In this study, we present the development of a promising approach to facilitate gut-to-tumor oral drug delivery using milk-derived EVs (mEVs) that exhibits exceptional stability within various digestive enzymes in the gastrointestinal (GI) tract. The tumor-activated prodrugs (FDX), comprising a cathepsin B-specific cleavable FRRG peptide and doxorubicin, are complexed with mEVs, resulting in FDX@mEVs. In colon tumor-bearing mice, FDX@mEVs orally administered demonstrate substantial absorption in the intestines through neonatal FC receptor (FcRn)-mediated transcytosis. Subsequently, mEVs significantly enhance the pharmacokinetic (PK) properties of prodrugs along with an increase in half-lives and area under the curves (AUC) owing to their outstanding stability in the bloodstream, leading to remarkable tumor accumulation via the enhanced permeability and retention effect. Ultimately, the oral administration of FDX@mEVs leads to significant antitumor efficacy in colon tumor models while reducing the potential risk of side effects due to the minimal cytotoxicity of FDX towards off-target tissues expressing relatively low levels of cathepsin B. Collectively, this study provides valuable insights into a promising strategy for gut-to-tumor oral drug delivery with mEVs as more effective, safe and convenient approach to cancer therapy.

Professor Hanne Winther-Larsen

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Secretion of extracellular vesicles (EVs) from the bacterial body is associated with a range of phenotypes including cell-cell communication, host-pathogen interactions and antimicrobial resistance development. EV may also elicit a humoral and antibody mediated immune response after immunization of a host making them an interesting vaccine candidate against an infectious agent. We have investigated the use of EV as a potential vaccine against several intracellular bacterial pathogens creating problems in farmed animals including fish. Immunization with EV revealed protection against some diseases, but not in others where we hypothesized that the EV was part of the microbial pathogenesis of the disease. To circumvent possible negative side effects of natural BEV with the host, we have explored the immunogenic properties of recombinant BEV (rEV) isolated from E. coli expressing antigens from the infectious bacterial species.

Methods: We have expressed antigens from the fish bacterial species Yersinia ruckeri in E. coli. The rEV were isolated from E. coli using tangential flow and ultracentrifugation and compared to BEV isolated from the native E. coli strain. The rEVs were analyzed by transmission electron microscopy (TEM) and Nano-particle tracking analysis (NTA), and the antigen expression were confirmed by SDS-PAGE and mass spectrometry. The rEVs were exposed on fish cell cultures and the immune modulating properties were evaluated by qPCR.

Results: Transmission electron microscopy revealed the presence of rEVs in the E. coli with similar shape to native E. coli EVs. Y. ruckeri antigens were successfully expressed in the rEVs as verified by SDS-PAGE and proteomic analysis using mass-spectrometry. Increased rEV concentrations were detected E. coli when expressing Y. ruckeri antigens compared to empty vector controls as verified by nanoparticle tracking analysis.

Summary/conclusion: These results show that E. coli has the potential to be utilized as a vector for production of EVs expressing outer membrane antigens from Y. ruckeri and probably antigens from other bacterial species. These rEVs will hopefully circumvent the negative side effects of native EVs isolated from the pathogenic bacteria when used as vaccines and may enhance the repertoire of EVs based vaccines within animal health.

Ph.d Qing Zhao, Ph.D Junjie Feng, Ph.D Lei Zheng, Ph.D Kewei Zhao

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction Although various anti-osteoporosis drugs are available, the limitations of these therapies, including drug resistances and collateral responses, require the development of novel anti-osteoporosis agents. Rhizoma Drynariae displays a promising anti-osteoporosis effect, while the effective component and mechanism remain unclear.

Methods Here, we revealed therapeutic potential of Rhizoma Drynariae-derived nanovesicles (RDNVs) for postmenopausal osteoporosis and demonstrated that RDNVs potentiated osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) by targeting estrogen receptor-alpha (ERα).

Results RDNVs, a natural product isolated from fresh Rhizoma Drynariae root juice by differential ultracentrifugation, exhibited potent bone tissue-targeting activity and anti-osteoporosis efficacy in an ovariectomized mouse model. RDNVs, effectively internalized by hBMSCs, enhanced proliferation and ERα expression levels of hBMSC, and promoted osteogenic differentiation and bone formation. Mechanistically, via the ERα signaling pathway, RDNVs facilitated mRNA and protein expression of bone morphogenetic protein 2 and runt-related transcription factor 2 in hBMSCs, which are involved in regulating osteogenic differentiation. Further analysis revealed that naringin, existing in RDNVs, was the active component targeting ERα in the osteogenic effect.

Conclusion Taken together, our study identified that naringin in RDNVs displays exciting bone tissue-targeting activity to reverse osteoporosis by promoting hBMSCs proliferation and osteogenic differentiation through estrogen-like effects.

Ph.D Bodeng Wu, Ph.D Weilun Pan, Ph.D Shihua Luo, MD Mingzhen Zhong, Professor Bo Li, Professor Lei Zheng

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction

Disrupted cellular networks and unfavorable microenvironments hinder diabetic wound healing. Conventional clinical interventions inadequately restore imbalanced reparative biofunctions. Current diabetic wound dressings employ cells, cytokines, extracellular vesicles and particles (EVPs), and nanomaterials for microenvironment enhancement. Challenges, such as high production costs, potential immunological risks, and storage limitations, impede clinical translation. In contrast, natural plant-derived nanoparticles (PDNPs) offer a cost-effective, eco-friendly, low immunogenic, and scalable alternative. The cross-kingdom regulatory potential of PDNPs in diabetic wound healing remains unexplored.

2) Methods

Clinical tissue samples were collected and analyzed to gain insight into the pathological characteristics of the wound microenvironment. Turmeric-derived nanoparticles(TDNPs) were isolated, characterized, and subjected to metabolite analysis and target genes prediction. In vitro experiments were conducted to analyze the biological phenomena and mechanisms of TDNPs intervention in fibroblasts and macrophages. TDNPs@aerogel (TAG) was developed and characterized, including elemental analysis, transmission electron microscopy characterization, biocompatibility verification, and release rate assay. A model of diabetic mouse cutaneous ulcer healing was applied to evaluate the overall effect of TAG.

3) Results

Metabolomics indicated turmeric-derived nanoparticles (TDNPs) for versatile pharmacological loading. Drug target analysis demonstrated TDNPs' modulation of multicellular signaling networks in diabetic wound healing. TDNPs promoted fibroblast proliferation and migration, enhanced antioxidant capacity, and reduced apoptosis through Nrf2/HO-1 activation. Uptake by macrophages led to decreased pro-inflammatory cytokines via TLR4/MyD88 inhibition. Additionally, TDNPs restored intracellular communication in re-educated macrophages and fibroblasts, facilitating extracellular matrix formation and tissue remodeling. For diabetic wound management, TDNPs were loaded into an ultralight, high-swelling, breathable aerogel (TAG) composed of cellulose nanofibers (CNF) and sodium alginate (SA). TAG featured customizable wound accessibility, water-adaptable adhesiveness, and sustained TDNPs release, exhibiting outstanding in vivo performance in diabetic wound healing.

4) Summary/Conclusion

This work highlights the potential of TDNPs in regenerative medicine and the significance of their application form to provide a promising solution for clinical situations.

Dr Weilun Pan, Master Mingzhen Zhong, Prof Lei Zheng

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Methicillin-resistant Staphylococcus aureus (MRSA) infections in subcutaneous tissues pose a global health challenge due to their antibiotic resistance and tendency to recur, making them difficult to eliminate. Accurate detection, effective treatment, and proper repair of infected subcutaneous tissues remain critical in clinical practice.

Plant-derived extracellular vesicles (PDEV) have emerged as promising therapeutic agents as they offer a wide range of sources and multifunctionality. Through metabolomics and drug target analysis, we discovered that turmeric-derived extracellular vesicles (TEV) contain small molecules with excellent antibacterial, antioxidant, and skin tissue repair effects. In this study, we in situ assembled TEV with two-photon responsive ultra-small AuAg nanoclusters (TEV@AuAg, TA), and loaded them into hyaluronidase-responsive dissolving microneedles, forming the TEV@AuAg@Microneedles (TAM) system. As MRSA releases large amounts of hyaluronidase, TAM can selectively and responsively trigger the release of TA at the infection site to detect active/non-active bacterial infections by two-photon imaging. Additionally, the two-photon irradiation on AuAg nanoclusters induces massive reactive oxygen species (ROS) and exert synergistic antibacterial effects with TEV.

Transcriptomics and PCR results showed that TAM exerted multiple effects on MRSA. It inhibited protein synthesis in MRSA cell walls, induced lipid peroxidation, suppressed the tricarboxylic acid cycle, and hindered bacterial information amplification, communication, and biofilm formation. Transcriptomics in keratinocytes revealed that TAM have the capability to enhance endogenous antioxidant systems, secretion of antimicrobial peptides, and expression of growth factors, offering favorable capabilities in the repair of damaged skin tissues. In the subcutaneous infection model, TAM has demonstrated the ability to detect bacterial lesions within 10 minutes and provide effective treatment to the infected area. Immunofluorescence, Giemsa staining, histochemistry, and PCR assays confirmed that TAM can rapidly clear bacterial infections, improve the oxidative microenvironment, reprogram inflammatory macrophages, and promote the regeneration of skin tissue.

This study represents a multidisciplinary integration project that utilizes natural PDEV as therapeutic agents. By employing in-situ membrane engineering technology combined with minimally invasive enzyme-responsive microneedles, these PDEV are empowered to achieve two-photon monitoring and synergistic treatment of subcutaneous infections. This research aims to provide novel insights and strategies for the engineering application and clinical translation of PDEV.

Jitendra Kumar¹

¹ICAR-National Dairy Research Institute, Karnal-132001, India

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Jitendra Kumar Ph.D*, Priya Sharma, Suneel Kumar Onteru Ph.D & Dheer Singh Ph.D

Jitendra Kumar Ph.D*

Molecular Endocrinology, Functional Genomics & System Biology Laboratory, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal-132001(Haryana), India

Email:[email protected]

https://orcid.org/0000-0001-6301-9989

Abstract

Antibiotic therapy stands as the most significant and common method for managing bacterial-infected bovine mastitis. However, in many cases, infections cannot be cured due to the failure of antibiotic treatment, which leads to chronic and recurrent infections because of the low bioavailability of antibiotics at the infection site. Additionally, overdoses of antibiotics may lead to the development of resistance against multiple pathogens.

To address these challenges, our team conducted research to develop a novel therapeutic approach for treating bacterial infections. Bovine milk extracellular vesicles were explored for their unique potential as both drug delivery vehicles and treatment modulators against the contagious pathogen. Well-characterized milk extracellular vesicles were utilized to deliver antibiotics, and this formulated antibiotic was tested against bacterial infections in mastitis-afflicted cows, characterized by high bacterial and somatic cell counts in milk.

Our findings suggested that milk extracellular vesicles loaded with antibiotics exhibited significantly higher therapeutic efficacy compared to antibiotics administered without extracellular vesicles, even at the same dosage and frequency of treatment. Furthermore, milk extracellular vesicles loaded with antibiotics reduced the number of somatic cell counts and bacterial load compared to antibiotics without extracellular vesicles. Therefore, the utilization of milk extracellular vesicles as unique nanomaterials for delivering drugs against bacterial infections in dairy animals could be promising tools in the modern healthcare system.

Professor Sujata Mohanty, Ms Meenakshi Mendiratta, Ms Mohini Mendiratta, Dr Suchi Gupta

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Mesenchymal Stem cells (MSCs) are recognized widely for their regenerative potential & immunomodulation, partly via paracrine secretions. Recent studies have shown that apoptotic induction can be a strategy to enhance their regenerative capabilities. This study investigates apoptosis induction to enhance regenerative abilities in MSCs-derived Small Extracellular Vesicles (MSCs-sEVs) and aims to identify the MSCs tissue source more responsive to this induction.

Methods:

Tissue-specific MSCs (Bone Marrow & Wharton's Jelly) were isolated after obtaining donor consent (IC-SCR/140/23 (O)) and cultured in serum-free media. Apoptosis was induced in tissue-specific MSCs using 0.5µM staurosporine (STS) for 12h. Uninduced MSCs were termed as Viable, and sEVs were isolated from both groups i.e., Viable MSCs (V-sEVs) and Apoptotic MSCs (Apo-sEVs) after 48 hours of culture via ultracentrifugation. They were further characterized as per MISEV 2018 guidelines. In vitro immune response assessment was performed via T cell proliferation, T regulatory cell induction & macrophage polarization assay. Further, mitochondrial bioenergetics was studied using MitoSOX red–based staining & Seahorse assay in H2O2 treated HuH7 cells. These findings were validated in vivo in the CCL4-induced Chronic Liver Disease (CLD) model (422/IAEC-1/2023) in C57BL/6 mice via H&E staining, biochemical parameters such as ALT, AST, bilirubin and fibrotic, pro-inflammatory and anti-inflammatory markers.

Results:

The concentration of sEVs released by Apo-MSCs was significantly higher. As per immune response assessment, Apo-sEVs were able to significantly suppress the proliferation of CD8+ T cells, their differentiation towards T regulatory cells, and polarized macrophages towards M2 phenotype. In terms of tissue specificity, it was observed that WJ-sEVs were faring better. Apo-EVs were more effective in suppressing mitochondrial reactive oxygen species & significantly improved oxidative phosphorylation (ATP production) and glycolysis over V-sEVs. Furthermore, in vitro findings were corroborated in an in vivo model of Chronic Liver Disease (CLD), wherein Apo-sEVs treatment demonstrated improvement in fibrosis and inflammation, demonstrated through histopathological studies, biochemical analysis, and a reduction in fibrotic and pro-inflammatory markers.

Conclusion:

This study underscores the potential efficacy of Apo-sEVs as a minimal manipulation strategic intervention for enhancing the therapeutic efficacy of MSCs. WJ-MSCs-sEVs can be considered a prime candidate for managing inflammation and immune-related disorders.

Dr Emma Symonds, Rachelle Smith, Mr Alexander Brown, Associate Professor Margaret Currie, Dr Kathryn Hally, Dr Kirsty Danielson

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Autologous fat grafting (AFG) is an increasingly popular tool for oncoplastic breast reconstruction because it uses natural tissue with low donor site morbidity and can help ameliorate post-radiation and surgery-associated pain. The major caveat for AFG is the variable retention rate of the graft resulting from a failure of the graft to integrate into the recipient environment. We have previously shown that adipose-derived stem cell extracellular vesicles (ASC-EVs) modulate macrophages to more anti-inflammatory phenotypes, increase tube formation by HUVECs, and increase macrophage-HUVEC co-localisation in 3D multicellular models. Together this suggests that ASC-EVs might represent a novel therapeutic option for improving AFG retention. We now explore the effects of ASC-EVs on fibroblasts and breast cancer cells, and in multicellular systems including these cell types.

Methods: Fat samples were collected from women undergoing AFG in Wellington, New Zealand (HCEC19/CEN/23). ASCs were isolated by enzymatic digestion, cultured with EV-depleted fetal bovine serum, and used between passages 2-4. ASC-EVs were isolated using size exclusion and quantified by tunable resistive pulse sensing. ASC-EVs were added to all cultures at a concentration of 105 EVs/mL. Recipient cells included CCD1128SK (breast fibroblasts), MCF-7 (ER+/PR+), MDA-MB-231 (triple negative), and SKBR3 (HER2+). Output assays included cell proliferation (CCK8), invasion (hanging drop method), migration (scratch-wound assay), gene expression (RT-qPCR), and confocal imaging.

Results: ASC-EVs increased fibroblast proliferation comparable to TNF-α (p<0.01), and partially rescued a TGF-β induced decrease in proliferation (p<0.001; one-way ANOVA with Tukey post-hoc). They had no effect on fibroblast migration or expression of α-SMA, COL1A1, or FAP. ASC-EVs decreased proliferation of breast cancer cells across all 3 cell lines (p<0.05-0.001; one-way ANOVA with Tukey post-hoc) and had no effect on migration or invasion. In 3D cultures, fibroblasts take up fluorescently labelled ASC-EVs and form scaffolds with HUVECs and macrophages to create vessel-like structures.

Conclusions: We have shown that ASC-EVs have differential effects on cellular proliferation in fibroblasts vs breast cancer cell lines. The decrease in breast cancer cell proliferation, with no change in migration or invasion, suggests that ASC-EVs may be safe for treating a former tumour bed.

Xue Wu, Jiuheng Shen, Youxiu Zhong, Xian Zhao, Peifen Gao, Wantong Zhou, Xudong Wang, Professor Wenlin An

¹National Vaccine & Serum Institute (NVSI), China National Biotech Group (CNBG), Sinopharm Group, Beijing, China

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

The role of extracellular vesicles (EVs) in medical cosmetics has attracted increasing attention, but the promotion of cultured cell-derived EVs has been limited due to the high production costs and low yields of EVs. Although the EVs are very abundant in milk and can be isolated and prepared on a large scale at low cost, the preparation process, the efficacy of milk-derived EVs (mEVs), and their use as delivery vehicles are needed to be further investigated.

Methods

In current study, we prepared multiple batches of mEVs using tangential flow filtration combined with weak anion chromatography, and analyzed the nucleic acid, protein, and lipid composition of mEVs using multi-omics, and also analyzed the efficacy and safety of mEVs and engineered mEVs using cellular, zebrafish, and caenorhabditis elegan models.

Results

The results of multiple batches of mEVs preparation and multi-omics analysis suggested that a large quantity of high-purity EVs with controlled quality could be obtained from milk, and the mEVs did not show significant cytotoxicity in fibroblast. However, the mEVs exhibited strong anti-oxidative stress and free radical scavenging functions in cellular and zebrafish models, which suggest that the mEVs have a good anti-aging effect; the mEVs could promote the proliferation of skin keratinocytes, suggesting that the mEVs have the function of enhancing the skin barrier; the mEVs could significantly inhibit the inflammatory response induced by LPS-treated macrophage 264.7 cells, suggesting that the mEVs can be used for the development of soothing efficacy products;the mEVs could also reduce the production of melanin through the inhibition of tyrosine hydroxylase, suggesting that the mEVs have a strong whitening effect. In addition, the engineered mEVs loaded with conotoxin have a significant anti-wrinkle effect, as confirmed in skeletal muscle cells and caenorhabditis elegan experiments.

Summary

In conclusion, the mEVs can be used as a very promising raw material for cosmetic or medical cosmetic products because of their unique characteristics to modulate the function of skin. We believe that with the in-depth research on the skin-improving function of mEVs, their applications in cosmetics and skin care are becoming more and more imaginative.

Tsuyoshi Kawaharada, Daisuke Watanabe, Shuhei Iida, Amaka Watanabe, Akio Mizushima

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) secreted by mesenchymal stem cells (MSC-EVs) play a crucial role in the paracrine effects of mesenchymal stem cells (MSCs). Notably, microRNAs encapsulated within MSC-EVs (EV-miRNAs) regulate specific signaling pathways involved in angiogenesis, neural repair, immunomodulation, anti-inflammatory effects, and wound healing. A comprehensive evaluation of EV-miRNAs is crucial for identifying potential therapeutic applications of MSC-EVs in regenerative medicine, such as bone/cartilage and urogenital repair, as well as in the treatment of intractable cardiac and lung diseases. This study aims to compare the RNA-Seq profiles of EV-miRNAs from MSC-EVs isolated from both monocultured and co-cultured MSCs derived from dental pulp (SHED), adipose tissue (AD), umbilical cord (UC), and bone marrow (BM). Furthermore, potential therapeutic applications of each isolated MSC-EV, considering their individual characteristics, are discussed.

Methods: Each MSC is cultured under identical conditions, either monoculture or indirect co-culture:

Droplets with an equal total cell number were plated in 4-well plates. After confirming cell adhesion, the medium was replenished to a specified volume. The culture supernatant solution was filtered through a 0.2 μm filter, and MSC-EVs were isolated by the Tangential Flow Filtration (TFF) system. The expression levels of EV-miRNA encapsulated in MSC-EVs were quantified using Next-Generation Sequencing (NGS) and evaluated by visualizing RNA-Seq analysis heatmaps.

Results: A total of 15 comparative cultures, including 4 types of MSC monoculture, 6 types of 2MSC co-culture, 4 types of 3MSC co-culture, and 1 type of 4MSC co-culture, were conducted. The results showed that 3MSC and 4MSC increased the production capacity of EVs compared to MSC monocultures. RNA-Seq analysis heatmaps were used to assess the expression profiles of EV-miRNA; each of the 15 types exhibited a unique profile. Detailed sequencing data, including clustering results from the heatmap dendrogram, are currently being analyzed and will be presented at the annual meeting.

Summary/Conclusions: This study suggests that the synergistic effects of co-culturing MSCs from different tissue origins, which mimics the in vitro environment closer to the in vivo one, and changes in EV-miRNA organization may lead to potential therapeutic applications.

Extracellular Vesicles Seoah Park¹

¹Seoul National University, South Korea

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Methods: We isolated bEVs from 8 bacteria strains using TFF, then characterized them for yield and size using NTA, protein profiles using PAGE-Coomassie blue staining, protein and lipid quantity, and morphology using TEM. Based on the bEV-induced transcriptomic responses, we applied CMap, STRING, and KEGG pathway analyses to predict bEV indications. Predictions for bEV indications were made using Microarray, CMap, STRING, and KEGG analyses. Validation was achieved through collagen assays. The collagen produced after bEV application to mouse NIH3T3 cells was analyzed, and the protein levels of pSmad3, Hsp47, and Mmp1 in the collagen synthesis pathway were quantified. mRNA

Dr Chintan Bhavsar¹, Dr Rui Chen¹, Ms Elaina Coleborn¹, Ms Shuying Li¹, Ms Sarah Wilkey¹, Mr Trent Neilson¹, Dr Katharine Irvine², Dr James Cuffe¹, Dr Sherry Wu¹

¹The University Of Queensland, Brisbane, Australia, ²Mater Research, Brisbane, Australia

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Intravenously administered extracellular vesicles (EVs) are sequestered by the liver and subsequently cleared from the body, irrespective of their origin. This significantly affects their distribution to target sites. Despite the rapid clearance, it has been observed that ∼1% of the injected EVs remain in the circulation for an extended period of time. Here, we aim to characterise the EVs that have long circulatory half-life to understand features needed to achieve better tumour targeting.

Methods: Blood was obtained via retro-orbital plexus or cardiac puncture from wild-type C57BL/6J mice or Sprague Dawley rats. EVs were isolated using differential ultracentrifugation followed by qEV 35 nm Gen 2 columns and subsequently incubated with Kupffer cells (KCs) to counter select EVs that can evade KC uptake. Similar experiments were performed using plasma fluid containing EVs instead of isolated EVs. Unselected EVs were obtained simultaneously to obtain control EVs (CEVs). In vitro uptake efficacy for KC-evading EVs (KCEEVs) and control EVs (CEVs) was assessed using nanoFCM. The biodistribution profile of KCEEVs and CEVs was compared using luminescence bioimaging system in ID8ip1-luc tumour-bearing mice. Mass spectrometry was used to compare the characteristics of KCEEVs and CEVs.

Results: In vitro nanoFCM-based validation studies revealed that KC-counter selection of EVs resulted in ∼80% decrease in KC uptake of EVs compared to CEVs in both species examined (mice and rats). In vivo biodistribution and flow cytometry-based characterisation highlighted ∼40% decrease in liver uptake of KCEEVs (specifically, Kupffer cells) compared to CEVs while tumour localisation remained unchanged between the two groups. Further mass spectrometry characterisation revealed significant differences in EV proteins that could explain this difference in biodistribution pattern.

Summary/Conclusion: Although KC-counter selected plasma EVs did not increase EVs’ tumour localisation, they had significantly lowered liver uptake. Further investigation on strategies to improve tumour localisation could significantly improve their use for delivering cancer therapeutics to tumours.

Phd Student Auriane Drack, Doctor Alin AR Rai, Hien A Tran, Associate professor Jelena Rnjak-Kovacina, Associate professor David Greening

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Stem-cell derived extracellular vesicles (EVs) and their bioactive molecular cargo regulate various phases of tissue repair, yet their clinical translation is hindered by off-target interactions, rapid clearance from circulation and production yield. Here, we unveil a pioneering, dual-therapeutic strategy combining highly scalable stem cell nanovesicles (ScNVs) and natural silk biomaterials, to enable local and sustained delivery of therapeutic factors for cardiac repair.

Methods

ScNVs were generated from human-induced pluripotent stem cells (IPSCs; CERA, CL2, RM3.GT-dtTom) via serial extrusion, and characterized biophysically (size (NTA), morphology (cryoEM)) and biochemically (proteome landscape (mass spectrometry)). ScNVs uptake and functionality on human cardiac fibroblasts (anti-fibrotic) and HUVECs (pro-angiogenic) was assessed in vitro. Different silk fibroin hydrogels were synthesized by photo-crosslinking or β-sheet formation to encapsulate NVs, characterized based on their mechanical properties (Young's modulus), structure (cryoEM) and nanovesicle release-rate (fluorescence intensity and particle yield). ScNVs pro-angiogenic potential under hypoxia was performed on a fibroblast/endothelial cell co-culture assay for 7 days. Finally, the reparative and proteome remodeling effects of ScNV-hydrogels were examined using mass spectrometry, assessing both co-culture and isolated cellular environments.

Results

Previously, our research showcased ScNV's notable signaling capacity for cellular repair and rescue, with direct administration aiding cardiomyocyte survival, angiogenesis, fibroblast de-activation (in vitro), and heart repair following myocardial infarction (in vivo). Here, we furthered this research through the development of a novel td-Tomato-labelled ScNV system to enable time-dependent tracking of delivery and cell remodeling. We encapsulated ScNVs within different silk fibroin hydrogels (800µg/mL hydrogel), revealing a controlled, gradual release of functional nanovesicles over 5 days, with a rapid release within the first 24h. Comprehensive mass spectrometry analysis revealed pro-reparative proteins within ScNVs, and that the synergistic effect of ScNV-hydrogel signaling remodeled recipient cells towards neovascularization, ECM regulation, and tissue repair.

Summary/Conclusions

Overall, we demonstrate a synergistic approach combining nanovesicles and biomaterials, and advance our understanding of their therapeutic functionality using cell-specific assays combined with mass spectrometry. This work highlights the potential of our dual-therapeutic strategy as a potent, scalable, and localized therapeutic for cardiac repair, and represents a significant stride in advancing EVs a viable therapeutic option.

Mr. Aliosha I. Figueroa-Valdés¹, Ms. Catalina Adasme-Vidal¹, Dr. Maroun Khoury¹, Dra. Francisca Alcayaga-Miranda¹

¹IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Santiago, Chile

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Bovine milk contains extracellular vesicles; however, the literature shows contradictory data regarding the presence and biological activities of these vesicles in milk produced and processed on an industrial scale. In the present work, we isolated vesicles (mEVs) from six different sources of commercial milk obtained from a production plant, established their characterization, and probed biological activities in-vitro and distribution in-vivo.

Methods: Milk samples ranged from whey powder, skimmed pasteurized/dried milk powder, whole pasteurized/dried milk powder, skimmed ultra-high temperature (UHT)-treated liquid milk, and whole ultra-high temperature (UHT)-treated liquid milk. The isolation was performed mainly by ultracentrifugation; the characterization was carried out for the determination of particles’ size and concentration by nanoparticle tracking analysis, bovine-CD63 presence by bead-based flow cytometry, and integrity/morphology structure by transmission electron microscopy (TEM). Their biological activity was evaluated by a human monocyte-derived macrophage-polarization assay, a human peripheral blood mononuclear cells proliferation assay, and a menadione-induced cytotoxicity assay in human chondrocytes. A preliminary pilot in-vivo distribution assay of mEVs was established in C57BL/6J mice after 24 h of oral gavage.

Results: Milk-derived particles’ size was between 100 to 200 nm, the obtained concentration ranged between 10¹¹-10¹³ particles/mL and CD63 was detected in all samples. TEM images confirmed the presence of cup-shaped vesicles in the isolated samples. mEVs-treated macrophages polarized to an M2-like/anti-inflammatory phenotype; also, mEVs increased the proliferation of CD3+, CD4+ and CD8+ cells by 5 to 20% and the apoptosis/death of menadione-treated cells decreased in 10 to 30% compared to control. Oral gavage distribution showed mEVs accumulation in liver and spleen.

Summary/Conclusions: It is possible to obtain high yield of biologically active mEVs from different industrial-scale milk sources while maintaining their phenotype, biological activity and biodistribution.

Ms Shital Wakale, Dr Antonia Sun, Dr Yang Chan, Dr Jennifer Gunter, Dr Chamikara Liyanage, Prof Ross Crawford, Dr Song Wu, Dr Hai Hu, Dr Indira Prasadam

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

The current interventions for osteoarthritis (OA) do not significantly delay the progressive degeneration of articular cartilage. Bone marrow mesenchymal cells (BMSCs) possess regenerative capabilities. Recent studies have shown that extracellular vesicles (EVs) derived from BMSCs are a great choice for cell-free therapy applications in several degenerative diseases. This study aims to determine if EVs from young and old bone marrow mesenchymal stem cells (BMSCs) have different effects on delaying the progression of OA.

Methods

BMSCs were isolated from young (2 months) and old (27 months) mice (n = 3). EVs were isolated from the conditioned medium of these BMSCs by ultracentrifugation and further characterised for size and morphology. Primary chondrocytes were stimulated for 24 hours with IL-1β and then incubated for 24 hours with EVs derived from young BMSC and old BMSC. SA-β-Gal activity and quantitative PCR were used to evaluate the chondroprotective effects and senescence of EVs in chondrocytes. The Seahorse assay was used to investigate the effect of EV treatment on IL-1β-induced chdonrocyte's mitochondrial activity. For an in vivo study, intra-articular injections of old and young BMSC EVs were administered to a DMM-induced mouse model for 1, 3, 5, 7, and 10 days post-surgery. A histological examination of the synovial joint was carried out after eight weeks of surgery.

Results

The morphological analysis showed no significant differences in the structure and size of young and old BMSC-derived EVs (n = 3). In vitro and in vivo studies indicate that young BMSC EVs alleviated senescence and induced chondroprotective effects; however, old BMSC EVs did not show the same effects.

Conclusion

EVs from young and old BMSCs have different levels of chondroprotective and senescence-alleviating effects in vitro and in vivo. This might be because of the cargo content of the EVs.

Karyna Tarasova, MSc Belen Arteaga, PhD Harini Nivarthi, MSc Johanna Gamauf, MSc Angkana Kidtiwong, PhD Sinan Gültekin, PhD Mathias Hackl, PhD Regina Grillari, Prof. PhD Christopher Gerner, Ass. Prof. Dr. Iris Gerner, Prof. Dr. Florien Jenner

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Mesenchymal stem cells (MSCs) show promising therapeutic effects for osteoarthritis, mainly mediated by paracrine signals, with fetal and perinatal cells demonstrating greater efficacy than adult cells. This study aims to compare the therapeutic efficacy of fetal and perinatal (Wharton's jelly (WJ) and Amnion) MSCs-derived Extracellular vesicles (EVs) on inflamed adult ovine chondrocytes in vitro.

Methods: To obtain the EVs, ovine fetal umbilical cord derived MSCs (n = 4 biological replicates) and immortalized perinatal MSCs cell lines (WJ (WJ-MSC/TERT273) and amniotic membrane (P-MSC/TERT308)) were seeded and maintained in serum-free medium in the extracellular space of hollow fiber bioreactors. EVs were isolated from the supernatant by tangential flow filtration (300kDa cut off), characterized for particle size distribution and number, and marker proteins. Fetal and perinatal MSCs-derived EVs (1x109 particles/mL) effects on inflamed (1ng/mL tumour necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β)) chondrocytes were assessed in 2D and 3D culture and compared to: healthy, inflamed non-treated and inflamed treated with Dexamethasone. Wound healing and proliferation assays, and a multi-omic approach combining RNA-Seq, miRNA-Seq and Mass Spectrometry-based proteomics were employed as readouts.

Results: Fetal MSCs-derived EVs treated inflamed chondrocytes showed a similar proliferation as healthy chondrocytes, in contrast to EVs derived from perinatal sources, which negatively influenced chondrocytes proliferation. Chondrocytes treated with fetal and perinatal MSCs-derived EVs showed a similar wound closure rate. All three EVs-treatments showed superior immunomodulatory effects compared to Dexamethasone by significantly (p = <0.05) downregulating several pro inflammatory mRNAs ((chemokine (C-C motif) ligand 20 (CCL20), interleukin 6 (IL6) and cyclin dependent kinase inhibitor 1A (CDKN1A)) and miRNAs (miR-146a-5p, miR-146b-5p and miR-34). Intriguingly, Amnion-derived EVs significantly increased matrix metalloproteinase-1 (MMP1) protein expression compared to inflamed cells. Fetal MSCs-derived EVs showed a superior pro-regenerative effect compared to perinatal-derived EVs with significant upregulation of several mRNAs (aggrecan (ACAN), collagen type II alpha 1 (COL2A1), collagen type XI alpha 1 chain (COL11A1)); as well as upregulation of miR140-3p.

Conclusion: Our findings support the hypothesis that MSCs derived-EVs from different cell tissue sources show distinct biological activity emphasizing the necessity to optimize the EVs donor cell for the desired treatment effect and clinical application.

Dr. Nithikan Suthumchai, Miss Panjaree Siwaponanan, Miss Payalak Sudcharee, Dr. Siripakorn Sangkitporn, Miss Acharaporn Dambua, Miss Patcharaporn Boonchu, Miss Phatcharaphon Nopprang, Prof.Dr. Kovit Pattanapanyasat, Prof. La-ongsri Atchaneeyasakul

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Oxidative damage contributes to death of rods, cones, and retinal pigmented epithelial (RPE) cells in retinitis pigmentosa. In this study, we evaluated the effect of bone marrow mesenchymal stem cell-derived small extracellular vesicles (BM-MSC-sEVs) against hydrogen peroxide (H₂O₂)-induced oxidative damage in human ARPE-19 cells.

Methods: Culture supernatant of BM-MSC was collected and used for sEV isolation by tangential flow filtration (TFF). The BM-MSC-sEVs were characterized by nanoparticle tracking analysis (NTA), transmission electron microscope (TEM), and western blot. The BM-MSC-sEVs were later co-incubated with ARPE-19 cells to determine the effect of sEVs on H₂O₂-induced oxidative damage. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and flow cytometry were used to demonstrate the cell viability, cell proliferation, and cell apoptosis.

Results: Characterization of BM-MSC-sEV-like particles revealed an average size of about 102.25±7.89 nm that expressed TSG101, CD63, CD9, and Alix suggestive of sEVs. The BM-MSC-sEVs was internalized in ARPE-19 cells which located near the cytoplasm, pericytoplasm, and nuclei. The cell viability of ARPE-19 cells was significantly higher after incubation with 50 µg/ml of BM-MSC-sEVs (112±3.04%) and cell proliferation was significantly increased 31.00±1.43% compared to untreated control. To investigate the effect of BM-MSC-sEVs treatment on ARPE-19 cells from H₂O₂-induced cell damage, ARPE-19 cell pretreated with BM-MSC-sEVs for 24 hours before H₂O₂ exposure (sEVs-H₂O₂) for 24 hours or H₂O₂ challenge followed by BM-MSC-sEVs treatment for 24 hours (H₂O₂-sEVs). Cell viability was significantly decreased to 37.86±0.61% in cultures treated with H₂O₂ alone as compared with untreated cells. Interestingly, in the H₂O₂-sEVs group, the cell viability was 52.68±0.49%. The pre-treatment of BM-MSC-sEVs before exposure to H₂O₂ increased cell viability (54.60±3.59%) compared with H₂O₂ group. Moreover, the total apoptotic cells of ARPE-19 were significantly reduced when sEVs were added to culture condition in both H₂O₂-sEV and sEV-H₂O₂, compared with H₂O₂ group.

Conclusions: BM-MSC-sEVs effectively protects and rescues the ARPE-19 cells from H₂O₂-induced oxidative damage and could be applied in the development of therapeutic modalities in the future.

MD Kuan-Wen Chen, PhD Chao-Yuan Chang, MD,PhD Chun-Jen Huang

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Background: Mesenchymal stem cells (MSCs) are renowned for their anti-inflammatory prowess, predominantly mediated through exosomes. Previous investigations have suggested that hypoxia preconditioning augments the anti-inflammatory potential of MSCs, yet the underlying mechanism remains elusive. This cellular study aims to delve deeper into this issue, with a specific focus on microRNAs.

Methods: Three distinct experiments were devised. Initially, the impact of exosomes from MSCs (designated as Exo) and hypoxia-preconditioned MSC-derived exosomes (designated as HExo) on inhibiting lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) upregulation in RAW264.7 cells was compared to elucidate the influence of hypoxia preconditioning. Subsequently, the microRNA profiles of HExo and Exo were scrutinized using a microRNA array to identify the specific microRNA involved. Lastly, the inhibitor of the identified microRNA was introduced into HExo (designated as Inhib-HExo), and its effects on IL-6 upregulation induced by LPS in RAW264.7 cells were compared with HExo.

Results: Firstly, the IL-6 concentration in the LPS+HExo group (i.e., RAW264.7 cells treated with LPS [100 ng/mL] plus HExo [1x10⁹ particles/mL]) was significantly lower than in the LPS+Exo group (i.e., RAW264.7 cells treated with LPS [100 ng/mL] plus Exo [1x10⁹ particles/mL]) (p < 0.0001), validating the enhancing effect of hypoxia preconditioning on the anti-inflammatory properties of MSC-derived exosomes. Secondly, microRNA array analysis revealed a notable enrichment of microRNAs, particularly microRNA-21-5p (1.84-fold higher), in HExo compared to Exo. Thirdly, the IL-6 concentration in the LPS+Inhib-HExo group (i.e., RAW264.7 cells treated with LPS [100 ng/mL] plus Inhib-HExo [1x10⁹ particles/mL]) was significantly higher than in the LPS+HExo group (p = 0.0119), indicating that inhibiting microRNA-21-5p negates the impact of hypoxia preconditioning on enhancing the anti-inflammatory capacity of MSC-derived exosomes.

Conclusion: This study elucidates the pivotal role of microRNA-21-5p in mediating the enhancement of the anti-inflammatory effects of MSC-derived exosomes induced by hypoxia preconditioning.

Associate Professor Jian-Jr Lee, Dr En-Wei Liu, Dr Yen-Hong Lin, Ms Min-Hua Yu, Associate Professor Ming-You Shie

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Chronic wounds have become the main axis of health care today, including diabetic foot, peripheral arterial ulcers, autoimmune diseases, varicose veins, bedsores, burns, radiation injuries, and hemiplegia. These chronic diseases will eventually form chronic wounds. It often results in high complications and high mortality, and high mortality is also related to a high amputation rate, thus causing many social medical costs and consumption of nursing manpower. In recent years, with the advancement of biotechnology, the application of exosomes to chronic wounds has begun to be extensively studied on regulating adipose-derived stem cells-derived exosomes are important trends in the treatment of chronic wounds in the future.

Methods: This study demonstrates a biomaterial-based approach to preparing high amounts of extracellular vesicles with high bioactivity from adipose-derived stem cells (ADSC) by stimulation with extracts derived from bioactive silicate ceramics. We further show that collagen matrix-containing engineered ADSC-derived extracellular vesicles are highly effective in the treatment of chronic wound healing by significantly enhancing angiogenesis.

Results: After CS-engineering ADSC-derived extracellular vesicles were placed into the collagen matrix, they were released in large quantities within two days and then became stable for up to 14 days, with the total release amount being approximately 44%. The analysis shows that HDF can phagocytose these released exosomes and enhance the proliferation ability of cells. In addition, it can be observed that exosomes with fluorescent signals are engulfed by cells, and the exosomes can still maintain an intact cellular structure after entering the cells. The matrix loaded with CS-engineering ADSC-derived extracellular vesicles further demonstrates the repair effect in animal models of diabetic wounds, indicating that exosomes play a key role in the wound healing process, such as cell migration, proliferation, and angiogenesis. The cytokines contained in exosomes can accelerate the activation of tissue regeneration and repair and may be used in the clinical treatment of “difficult-to-heal chronic wounds” in the future.

Conclusion: This therapeutic effect is attributed to significantly promoted revascularization by the high content of miRNA and angiogenic-related factors such as VEGF, EGF, and eNOS in CS-engineered ADSC-derived extracellular vesicles, which activate proliferation and enhance wound healing processing.

PhD Student Ana Meliciano, Pedro Vicente, Ana Filipa Louro, Cláudia Diniz, João Jacinto, Paula Marques Alves, Margarida Serra

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: The incidence of cardiovascular-related deaths has been steadily rising, and recent findings have shown the potential of extracellular vesicles (EV) as promising cell-free therapeutic modalities for treating chronic heart failure patients. Our previous research identified human induced pluripotent stem cells (hiPSC) as the best parental cells for generating EV (hiPSC-EV) with superior cardiac regeneration compared to hiPSC-cardiac derivatives. Importantly, we characterized the hiPSC-EV miRNome, pinpointing key miRNAs (miR-302c-3p; miR-200c-3p) and their role in the mechanism of action for cardiac repair. Recognizing the high self-renewal ability of hiPSC, this study introduces a scalable bioprocess for producing clinically relevant doses of hiPSC-EV with improved bioactivity.

Methods: hiPSC were expanded as 3D aggregates in 200 mL stirred-tank bioreactors (STB), operated in perfusion (D = 1.3 day-1) and precise dissolved oxygen control (4% O2). hiPSC-EV were isolated from the conditioned medium using density gradient ultracentrifugation, and characterized for the presence and topology of specific EV-associated markers, and assessed for yield, particle size distribution, and morphology. The bioactivity of EV was evaluated through in vitro cardiac fibrosis and angiogenesis models. Small RNA-Seq was conducted to identify differentially expressed miRNA between hiPSC-EV secreted in the STB and the conventional 2D monolayer static culture. Lastly, the established protocol was successfully transferred to a 2L single-use STB, following engineering principles for scaling-up.

Results: The STB bioprocess yielded a remarkable 6-fold increase in hiPSC-EV isolated per mL of conditioned medium compared to 2D culture systems. The hiPSC-EV produced in STB displayed a cup-shaped morphology, tested positively for EV markers, and were efficiently taken up by recipient cells. Functionally, these hiPSC-EV exhibited enhanced pro-angiogenic and anti-fibrotic activities, as confirmed by a correlation at the transcriptomic level through small RNAseq. Importantly, maintaining a constant power input per volume, the scalability of the bioprocess was successfully validated at a 2L scale (r² = 0.9973), yielding clinically relevant doses of hiPSC-EV (totaling 7.06e+11 hiPSC-EV per bioreactor run).

Summary/Conclusions: In this work, we developed a powerful bioprocess that ensures the efficient and scalable production of clinically relevant numbers of hiPSC-EV in STB, enhancing both EV yields and functionality, thereby potentiating their use in cardiac repair.

Ashok Shetty, Dr Maheedhar Kodali, Dr Leelavathi N Madhu, Dr Shama Rao, Dr. Raghavendra Upadhya, Ms Sahithi Attaluri, Dr Bing Shuai

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: The outcome of traumatic brain injury (TBI) depends on the degree of brain tissue loss and the intensity of secondary neuroinflammatory signaling pathways. This study investigated the proficiency of EVs from human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) for modulating the nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) inflammasome-mediated hyperactivation of p38/MAPK signaling and cyclic GMP-AMP synthase (cGAS)-the stimulator of interferon genes (STING) pathway and interferon-I (IFN-I) signaling.

Methods: Ninety minutes after a unilateral controlled cortical impact injury, adult mice received intranasal (IN) administrations of hiPSC-NSC-EVs (4 billion EVs) or the vehicle. The injured cerebral hemispheres were investigated for neuroinflammatory signaling pathways at 48 and 72 hours post-TBI, and cognitive and mood functions were examined at two months post-TBI.

Results: The concentrations of mediators and end products of NLRP3 inflammasome activation were normalized 48 hours post-TBI in mice receiving hNSC-EVs. Such an effect also prevented the hyperactivation of p38/MAPK signaling, evidenced by normalized concentrations of p38/MAPK, AP1, IL-8, and TNF-a. hiPSC-NSC-EV treatment also inhibited the activation of the cGAS-STING pathway and the downstream JAK-STAT signaling, which was apparent from normalized concentrations of cGAS, pSTING, pIRF3, pJAK1, pSTAT1, and pSTAT-3. Single cell-RNA seq of microglia revealed normalized expression of genes linked to disease-associated microglia and downregulation of genes linked to IFN-I signaling in TBI mice receiving hNSC-EVs. TBI mice receiving hiPSC-NSC-EVs also displayed better cognitive and mood function and hippocampal neurogenesis at two months post-TBI.

Conclusion: Intranasal administrations of hiPSC-NSC-EVs after TBI can restrain the activation of NLRP3-p38/MAPK and cGAS-STING pathways, alleviating chronic neuroinflammation and enduring cognitive and mood impairments.

Dr. Ruijing Chen, Dr. Taojin Feng, Dr. Ming Chen, Dr. Ruijing Chen

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Despite a remarkable regenerative capacity that bone tissue possesses, it remains challenging for bone defect to heal. This study aimed to investigate the osteogenesis capacity of exosomes derived from 3D-cutured human adipose-derived stem cells (hADSCs) in vivo and in vitro.

Methods

Exosomes from 2D- or 3D-cultured hADSCs (2D-Exo and 3D-Exo) were characterized by transmission electron microscopy, nanoparticle tracking analysis and western blotting, then injected to calvarial defect mice through tail veins. The bone defect targeting capacity was evaluated by in vivo fluorescence intensity tracking. The osteogenesis ability was measured by micro-CT analysis. Exosomes were also co-cultured with calvarial osteoblasts and its osteogenesis ability was measured by ARS staining, ALP staining and RT-qPCR. MiRNA sequencing was performed to identify the potential cargo in 3D-Exo that mediated the osteogenesis effects.

Results

Exosomes from 3D-cutured hADSCs showed superior bone defect targeting and bone defect repair capacity comparing to that of exosomes from 2D-cultured hADSCs. Also, intravenous injection of exosomes from 3D-cultured hADSCs could increase bone mass. In vitro study revealed that exosomes from 3D-cultured hADSCs better promoted osteoblast differentiation as compared to exosomes from 2D-cultured hADSCs.

Conclusion

In summary, we have underscored the bone-targeting and pro-osteogenic effects of exosomes derived from hADSCs, and these effects can be further potentiated through the 3D culture of parental cells. The 3D culture of hADSCs leads to an increased expression of miR-3648 in their exosomes, thereby enhancing their osteogenic properties

Phd Pei-Ling Chi

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Vibrio vulnificus, an estuarine bacterium, is a major causative agent of severe wound infections and septicemia, with case-fatality rates surpassing 50% in primary septicemia within 72 hours post-hospitalization. The bacterium's sensitivity to antibiotics, which can exacerbate conditions by inducing cytokine storms with high mortality, necessitates the development of non-antibiotic therapies. This study explores the antibacterial and immunomodulatory efficacy of induced pluripotent stem cell-derived exosomes (iPSC-Exo) against V. vulnificus in vitro and in vivo.

Methods

iPSC-Exo were isolated from iPSC cultures and characterized using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and Nano Flow Cytometry. The impact of iPSC-Exo on Vibrio vulnificus infection was rigorously evaluated using both RAW264.7 cell lines and murine models. To elucidate the protective mechanism of iPSC-Exo against V. vulnificus cytotoxicity, comprehensive assays were employed: phagocytosis was quantified, cellular cytotoxicity was assessed, and cytokine secretion was measured using an ELISA kit. Furthermore, the activity of critical proteins in the pyroptosis signaling pathway—caspase-1, IL-1β, and Gasdermin D (GSDMD)—was analyzed through Western blotting, quantitative RT-PCR, and immunofluorescence techniques. Additionally, the direct bactericidal effect of iPSC-Exo was determined by evaluating the bacterial growth rate, survival metrics (optical density at 600 nm, colony-forming units), biofilm formation capacity, and cellular viability (MTT assay).

Results

iPSC-Exo treatment significantly enhanced macrophage phagocytosis of V. vulnificus and reduced its cytotoxicity. V. vulnificus infection upregulated the P2X7-dependent NLRP3/caspase-1 inflammasome signaling in macrophages, leading to GSDMD transmembrane pore formation and secretion of IL-1β, IL-18, and MIP-2. iPSC-Exo reversed these effects, decreased V. vulnificus colony-forming units in the spleen, and reduced immune cell infiltration and cytokine levels in infected tissues. Additionally, iPSC-Exo combined with ceftriaxone synergistically inhibited V. vulnificus growth and survival in vitro.

Summary

iPSC-Exo effectively inhibits the pyroptotic pathway in V. vulnificus, enhancing macrophage phagocytosis. The combination of iPSC-Exo and ceftriaxone exhibits robust antimicrobial activity against V. vulnificus, providing a promising basis for future development of non-antibiotic or reduced antibiotic treatments for infectious diseases.

Denise Zujur, MSc William Theoputra, PhD Makoto Ikeya

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

While the regenerative capabilities of mesenchymal stem/stromal cell (MSC)-derived extracellular vesicles (EVs) are well-established, the potential of EVs isolated from induced pluripotent stem cell-derived mesenchymal stem /stromal-like cells (iMSCs) remains largely unexplored. iMSCs, characterized by their ability for accelerated in vitro expansion, elimination of invasive biopsies, and possibility for genetic modification, offer a compelling cell source for EV isolation. This study investigates EVs' immunomodulatory and tissue regeneration capabilities derived from iMSCs.

Methods:

EVs from iMSCs were characterized using electron scanning microscopy and flow cytometry analysis. Interferometry was used to quantify EV concentration and size profiling. In vitro evaluations included assessing EV bioactivity in various cell types, and the therapeutic and immunomodulatory properties were evaluated in vivo using a murine wound healing model.

Results:

EVs from iMSCs demonstrated significant bioactivity in vitro, increasing cell proliferation, differentiation, and migration across diverse cell types. Notably, the EVs induced a favorable shift in macrophage phenotype from M1 to M2. In vivo, EV administration in the wound periphery effectively mitigated inflammation by enhancing the M2/M1 polarization ratio and increasing anti-inflammatory factor expression.

Summary/Conclusions:

This study unveils the promising therapeutic potential of iMSC-derived EVs in immunomodulation and wound healing. These findings, emphasizing the unique advantages of iMSCs and their EVs, contribute to exploring novel regenerative medicine approaches.

Dr. Prapatsorn Charoenyingpaisal, Dr. Hsien-Hen Lin, Dr. Toru Okubo, Mr. Hayato Kurata, Mr. Tetsuo Koike, Mr. Yoichi Honma

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Numerous reports have highlighted the potency of Mesenchymal Stem Cell-derived Exosomes and Extracellular Vesicles (MSC-EVs) across diverse disease models. Particularly, both enhancing and inhibitory effects of MSC-EVs on tumors have been reported. This study explores the impact of MSC-EVs on tumors using mice carrying A549 lung cancer cells as a model.

A tumor model was created by subcutaneously transplanting A549 human lung cancer cells into BALB/c nude mice, concurrent with MSC-EVs administration. Tumor volume was calculated by measuring tumor diameter over time until 30 days after the start of MSC-EVs administration. After tumor excision, an analysis was conducted to study the alterations in gene expression.

A significant increase in the tumor volume was noticed in the control group over time, whereas the MSC-EVs administered group recorded a significant decrease. Clustering analysis of gene expression in excised tumors resulted in different clusters between the control and MSC-EVs groups. Further, the pathway analysis of the differentially expressed genes confirmed the presence of gene clusters related to inflammation, cancer cell aggression and migration, inhibition of angiogenesis in tumors, and an immune cell's activity.

These results suggest that, under the test conditions of this study, MSC-EVs have a suppressive effect on cancer cell adhesion and proliferation. Furthermore, suggestions arise that MSC-EVs appear to exhibit suppressive influences on cancer cells and activating effects on immune cells.

Ph.D. Seon-Yeong Jeong, Ph.D. Jimin Kim, M.S. Seul Ki Lee, M.S. Haedeun You, Ph.D. Soo Kim

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall-Doors 14-15, May 9, 2024, 12:00 PM - 2:00 PM

Introduction: The blockage of the coronary blood supply to the myocardium causes myocardial infarction (MI), which can result in irreversible damage such as myocardial loss, pathological remodeling, cardiac dysfunction, and heart failure. Mesenchymal stem cells (MSCs) are thought to be the most promising cell source for cell-based cardiac regeneration therapy. In this regard, MSCs derived from induced pluripotent stem cells (iMSCs) have tremendous advantages in tissue regeneration due to their infinite growth and differentiation potentials. Extracellular vesicles (EVs) isolated from iMSCs, which are key players in mediating the iMSC capacity, have therapeutic effects and several potential advantages. Hyaluronic acid (HA) plays crucial role in the tissue regeneration process. EVs derived from HA primed iMSCs (HA-iMSC-EVs) might have emerged as one of the most promising strategies for restoring cardiac function after MI.

Methods: HA-iMSC-EVs were characterized, and proteomic analysis was performed. Whether HA-iMSC-EVs can restore cardiac damage was investigated using the LAD permanent ligation (PL) rat model. The anti-inflammatory, anti-fibrotic, and anti-apoptotic functions of HA-iMSC-EVs in inhibiting MI progression was investigated using heart tissues, THP-1 macrophages, and neonatal rat cardiac fibroblasts (NRCF).

Results: We investigated the possibility of accelerating cardiac repair using HA-iMSC-EVs with increased cardiac regeneration. First, HA-iMSC-EVs were evaluated for their pro-angiogenic, anti-fibrotic, anti-inflammatory, and anti-apoptotic properties on a variety of cells. Second, in order to validate the effects of HA-iMSC-EVs in vivo, they were injected into hearts of PL rat model. It was shown that by exhibiting an increased ejection fraction and fractional shortening, HA-iMSC-EVs can enhance cardiac function. Furthermore, HA-iMSC-EVs resulted in decreased fibrotic area and increased density of capillary and cardiomyocyte, indicating improved remodeling of the heart tissue.

Summary/Conclusion: In summary, HA-iMSC-EVs have the potential to be an effective therapeutic agent for the cardiac repair.

Dr. Jia Xian Law, Ms. Chiew Yong Ng, Assoc. Prof. Min Hwei Ng, Prof. Ying Yang, Assoc. Prof. Jhi Biau Foo, Dr. Chee Wun How, Assoc. Prof. Kien Hui Chua, Assoc. Prof. Kok Yong Chin, Dr. Rizal Abdul Rani, Prof. Nor Hamdan bin Mohamad Yahaya

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) are membrane-bound vesicles released by cells, containing a rich assortment of biological molecules such as nucleic acids, proteins, and lipids known for their capacity to facilitate tissue regeneration, including cartilage tissue. Given the variability in EV content based on their cellular origin, it is crucial to compare the functional properties of EVs from different sources to identify the most effective for cartilage injury treatment. This study aimed to assess and compare the regenerative potential of EVs derived from human umbilical cord mesenchymal stem cells (MSC-EVs) and human cartilage tissue (C-EVs) in the context of cartilage repair. Methods: EVs were obtained from fifth-passage UC-MSCs and partially digested cartilage tissue using ultrafiltration and tangential flow filtration methods. Characterization of the isolated EVs adhered to the MISEV2018 recommendations. Subsequent experiments evaluated the impact of these EVs on chondrocyte viability, proliferation, migration, and the expression of extracellular matrix (ECM) genes. In vivo experiments utilized monosodium iodoacetate-induced osteoarthritis (OA) rats to assess the efficacy of these EVs. The protein profiles of the EV preparations were analyzed through LC-MS/MS. Results: Results indicated that the isolated EV preparations were enriched with vesicles around 70 nm in size. MSC-EVs exhibited general EV markers (CD63 and HSP70), while C-EVs were positive for lactadherin and putative annexin A2-like protein. MSC-EVs enhanced chondrocyte proliferation, while C-EVs increased the gene expression of type II collagen and cartilage oligomeric matrix protein. In vivo, combined treatment with MSC-EVs and C-EVs partially improved gait analysis, with C-EVs alone demonstrating a reduction in cartilage destruction in OA animals. The EV therapies were considered safe, with no observed adverse events. Variations in protein profiles between MSC-EVs and C-EVs highlighted distinct modulatory effects on human chondrocytes, emphasizing the diverse roles played by different cargoes within these EVs in modulating cartilage regeneration. Summary: In summary, MSC-EVs and C-EVs exhibit distinct roles in the modulation of cartilage regeneration, influenced by their specific protein cargoes.

Miss Mona Belaid, Giorgia Pastorin, Driton Vllasaliu

Poster Pitches (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:45 PM - 1:00 PM

Introduction: Inflammatory bowel disease (IBD) is a chronic and incurable inflammatory condition of the gastrointestinal tract characterised by damage to the intestinal mucosa. The loss of barrier integrity allows antigens and bacteria-derived molecules across the intestinal epithelium and results in the upregulation of pro-inflammatory cytokines such as TNF-α and IL-6. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) show great promise for repair and regeneration of injured tissues and their immunomodulatory effect makes them strong potential therapeutics for IBD. Here, we investigated the anti-inflammatory effect of EVs derived from human bone marrow mesenchymal stem cells in a new in vitro co-culture model that mimics intestinal inflammation.

Methods: A novel model of intestinal inflammation was developed based on the co-culture of intestinal epithelial Caco-2 cells and murine J774A.1 macrophages. To mimic the inflamed intestine, Caco-2 cells were primed with an optimised cytokine cocktail (TNF-α, IFN-γ and IL-1β) and J774A.1 cells were pre-exposed to lipopolysaccharide (LPS) and IFN-γ for 24 h before combining the two cell lines into co-culture. The inflamed model exhibited significant disruption of the epithelial barrier and increased levels of pro-inflammatory cytokines (TNF-α and IL-6). MSC-EVs were isolated by ultracentrifugation onto a 25% sucrose cushion and were characterized by total protein content, concentration, size and protein marker expression using bicinchoninic acid assay, nanoparticle tracking analysis and the Exo-Check antibody array kit, respectively. MSC-EVs were then applied to the inflamed co-culture model of IBD, either on the apical side or the basolateral side. The effect of MSC-EVs on Caco-2 barrier integrity was measured by transepithelial electrical resistance (TEER) and the effect on cytokine release from J774A.1 macrophages was quantified using Luminex assay.

Results: MSC-EVs significantly reduced the production of TNF-α and IL-6 in inflamed J774A.1 macrophages and increased TEER values in the Caco-2 barrier. MSC-EVs applied on the apical side transported across the Caco-2 epithelial cells and showed similar anti-inflammatory activity on cytokine release from macrophages as MSC-EVs applied on the basolateral side of the inflamed co-culture model. This suggests that oral delivery of MSC-EVs could be an effective route of administration for the treatment of IBD.

Summary/Conclusion: MSC-EVs effectively reduced inflammation and restored intestinal barrier integrity in a new co-culture model of intestinal inflammation, which could have potential therapeutic applications in IBD.

Mr. Maksim Vigovskii, Ms. Nataliya Basalova, Ms. Olga Grigorieva, Ms. Uliana Dyachkova, Mr. Vladimir Popov, Ms. Anastasia Tolstoluzhinskaya, Ms. Anastasia Efimenko

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Mesenchymal stromal cells (MSC) are well-established regulators of regeneration and repair in damaged tissues. Many studies have shown the ability of the extracellular vesicles fraction of MSC secretome (EV) to prevent the development of fibrosis as an outcome of tissue injury. However, specific microenvironment in damaged area may affect the composition of MSC secretome. Previously, we have shown that profibrotic conditions induce cellular senescence of MSC. In this study we investigated the antifibrotic potential of EV from senescent MSC (senMSC) both in vitro and in vivo.

Methods: Extracellular matrix of fibroblasts and TGFβ were used for the modeling of profibrotic conditions. EV were separated by ultrafiltration from human adipose-derived MSC secretome. For an in vitro model of fibrosis, TGFβ-induced differentiation of myofibroblasts was used; the differentiation effectiveness was assessed by αSMA level using Western blot and ICC. Lung fibrosis in vivo was induced in mice (4-6 and 20-24 weeks old) by intratracheal injection of bleomycin; after 14 days, EV MSC or EV senMSC were injected intratracheally once. Lung fibrosis was evaluated by MRI and lung histology; the percentage of Lin+ cells in bronchoalveolar lavage (BAL) was analyzed by flow cytometry.

Results: In fibrosis model in vitro, we found a 2-2.5-fold decrease in the antifibrotic efficiency of EV senMSC compared to EV of control MSC. In vivo, we showed that in young mice, normal lung tissue restoration occurs both when EV MSC or EV senMSC were injected (although for the EV senMSC there is a trend towards effectiveness decrease). In aged mice, EV MSC therapy promoted restoration of functional tissue, while EV senMSC were significantly less effective. We found a significant increase in the basal level of Lin+ cells in old mice BAL and in the same group revealed a tendency to decrease Lin+ amount after injection of EV MSC, but not EV senMSC.

Conclusions: The profibrotic microenvironment-induced senescence of MSC leads to a decrease in antifibrotic potential of cell secretome mediated by EV. However, in vivo this functional decline is realized only in old, but not young animals with lung fibrosis.

Ms. Sofia Baptista, Ms. Cristina Manfredi, Dr. Marcin Jurga, Mr. Gabrielis Kundrotas, Mr. Dimitri Stevens, Mr. Domenico Mancuso, Ms. Elisabetta Gramegna, Mr. Rudra Kashyap, Ms. Sandrine Mores, Prof Eugenio Baraldi, Prof Maurizio Muraca, Dr. Beatrice De Vos

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular Vesicles (EVs) are nanometre-sized particles secreted by various cells, acting as cell-to-cell messengers. EVs are considered safer than current cellular therapeutic approaches. Bronchopulmonary Dysplasia (BPD) affecting preterm new-borns is a multifactorial lung disease involving both arrested lung development and lung inflammation, with no current approved treatment.

Methods: EVs are categorised as biological medicinal products defined by an active substance with a biological origin, according to the definition of the Directive 2001/83/EC.

The goal of any phase I clinical trial is to study the safety of the new drug and the goal of any phase II trial is to define dosages. However, the use of EVs for BPD treatment in premature neonates brings several challenges such as the route, timing and number of administrations, and the concentration of EVs in a dose.

EVENEW study: a clinical trial application was submitted to the European Medicines Agency (EMA), including a robust, reproducible production process, PD/PK studies with in vitro and in vivo assays of EXOB-001 activity for the prevention of BPD or attenuation of its severity.

Results: In July 2023, the EMA approved the EVENEW study (EU CT number 2022-500293-34-01), an adaptive, seamless Phase 1/2 trial assessing the safety and efficacy of intratracheal administration of EXOB-001 in preventing BPD. The study is composed of six dose-escalating cohorts with a board actively monitoring safety data throughout the study. Besides safety of EXOB-001 treatment, the trial also will study the prevention of BPD.

Summary/Conclusion: The EVENEW study is the first UC-MSC-EVs trial approved by the EMA for the prevention of BPD in preterms overcoming the hurdles of a new regulatory pathway. The study holds promise that EVs can sustain a therapeutic effect against the short and long-term sequelae induced by BPD. The EV production platform is designed to achieve a reproducible and efficient production of EVs in a scalable way.

Dr Yue Zhang, Professor Tao-tao Tang, Professor Lin-li Lv, Professor Bi-cheng Liu

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as a promising regenerative therapy. However, their clinical application is hampered by the presence of xenogeneic components in the medium. In the present study, we aimed to decipher the regenerative properties of MSC-EVs generated under xeno-free conditions to favor the clinical translation of MSC-EVs. Here we compared the influence of MSC-EVs with culture medium including fetal bovine serum (F-EVs) and clinical-grade human platelet lysate (H-EVs) by using of multi-omics and functional assays. Our study demonstrated that HPL promoted MSC-EVs production without compromising EVs characteristics. Multi-omics sequencing revealed the stability of H-EVs from different umbilical cord donors and global functional alterations for MSC-EVs under different culture conditions. In comparison to F-EVs, the regenerative potential of H-EVs was enhanced especially angiogenesis associated with increased cargo expression. The in vitro studies demonstrated that H-EVs induced more tube-like structure formation and migration. Further, it significantly reduced kidney capillary sparsity in mice with renal ischemic-reperfusion model. Our data provided a comprehensive understanding about H-EVs and demonstrated their potential as a safe and effective regenerative therapy, especially targeting angiogenesis-related disorders.

Ms Yashvi Sharma, Dr Sujata Mohanty

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Wharton's Jelly derived Mesenchymal Stem Cells (WJ-MSCs) have been recognised for their paramount immunomodulatory activity. Studies have reported that hypoxia priming of MSCs improves their therapeutic function, especially via enhancing the release & content of their small Extracellular Vesicles (sEVs). miRNA cargo of MSCs-sEVs has received significant attention as it can target various molecular pathways. Recent literature has shown that miR125b-5p is one of the highly enriched miRNAs in WJ-MSCs-sEVs & is involved in wound healing. This study explores the mechanism of hypoxia priming induced elevation in immunomodulatory & regenerative activity of WJ-MSCs sEVs with respect to miR125b-5p, and validates its functional potential in traumatic wounds.

Methods: WJ-MSCs, isolated with donor consents (IC-SCR/120/21(R)), were cultured in serum-free media and exposed to normoxic (5% CO2; 21% O2), or hypoxic (5% CO2; 1% O2) conditions for 24 hours. sEVs were isolated via ultracentrifugation & characterized following MISEV, 2018 guidelines. Functional validation involved miR125b-5p modulation through lipofection, and evaluation of sEVs for immunomodulatory and regenerative effects via macrophage polarization, MTT, and scratch assay. TargetScan analysis identified the miR125b-5p target and it was confirmed via Dual Luciferase Assay. Excisional wound model was created in Wistar rats (226/IAEC-1/2019) and treated with normoxia, hypoxia & miR125b-5p enriched sEVs. Mechanistic validation employed qPCR, Immunofluorescence, H&E staining, and MT staining for insights into wound healing processes.

Result: Hypoxia priming significantly elevated immunomodulatory and regenerative activity in WJ-MSCs-sEVs, and miR125b-5p enriched sEVs showed further functional enhancement. TargetScan analysis identified IL-6R as a target of miR125b-5p, which was confirmed by dual luciferase assay. H&E and MT staining of the rat wound tissues demonstrated improvement in inflammation & regeneration in the wound milieu upon sEVs treatment, following the order: Normoxia > Hypoxia > miR125b-5p Enriched sEVs. This was further validated by decreased IL-6 & IL-6R expression, in the same order, as identified through qPCR and Immunofluorescence staining of wound tissues.

Conclusion: This study hereby concludes that hypoxia primed WJ-MSCs-sEVs demonstrates enhanced functionality predominantly via miR125b-5p/IL-6R axis, and therefore it can be considered as a minimum manipulation strategy to enhance the regenerative capabilities of MSCs-sEVs.

PHD Student Guillaume Valade, MD Clément DEVAUTOUR, Mrs Marion GROSBOT, Mrs Muriel NIVET, Phd Student Marine DE TADDEO, PHD Ahmad HAIDAR, PHD Patrice DECKER, Mrs Sylvie GOULINET, PHD Philippe MAUDUIT, MD, PHD Sébastien BANZET, PHD Marina TROUILLAS, PHD Juliette PELTZER

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Traumatic Hemorrhagic shock (THS) can quickly induce death by bleeding or later by organ failure. The administration of IL-1β-primed human bone marrow mesenchymal stromal cells (MSC-IL) modulates the inflammatory response in this context and reduces organ dysfunction. We explore the therapeutic potential of concentrated fractions of MSC-derived conditioned medium (CM) enriched in extracellular vesicles (EV) combined or not with soluble factors (SF).These acellular products, are easier to store than MSC and immediately available for emergencies or in isolate areas.

Method

CM are obtained after 72h of MSC-IL secretion, from a pool of 5 donors. Two priming conditions were evaluated: IL-1β at 1 and 5 ng/mL. The CM are purified/concentrated by Tangential Flow Filtration (TFF) with two different filters. Two enrichments are evaluated, depending on filtration conditions: i) purified EV fraction, and ii) the association of EVs and SF. The characterization of EVs is carried out in terms of quantity, size, structure and phenotype. Their immunomodulatory properties are evaluated in vitro by a monocyte anti-inflammatory assay, a mixed leucocyte reaction and a NETosis assay.

Results

EVs’ characterization indicate that they meet the criteria defined in the MISEV 2018. A stronger anti-inflammatory activity is obtained with EV+SF fraction. In contrast, EV and EV+SF induce an equivalent immunosuppressive effect on T lymphocyte proliferation. Finally, preliminary results of NETosis production also show similar capacity of the two fractions to inhibit NETosis by activated neutrophils. Furthermore, the two priming doses used do not show any major difference in efficacy in these models.

Summary/Conclusion

This work indicates that the EV+SF fraction has the most complete immunological effect. In vivo experiments are underway to evaluate if early administration of EV+SF fraction issued from rat IL-1β-primed (at 5ng/ml, as in our previous in vivo study) MSC could moderate THS-induced organ failure in a context of immunological storm.

Dr Sadiq Umar^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Koushik Debnath^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Chun-Chieh Huang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Kasey Leung^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Miya Kang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Yu Lu^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Praveen Gajendrareddy^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Sriram Ravindran^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Dental pulp stem cells (DPSCs) are MSCs and exert their paracrine activity via their extracellular vesicles (EVs). The anatomical location and propensity for inflammatory exposure put DPSCs in a prime location to exert anti-inflammatory function. The objective of this study was to characterize the anti-inflammatory properties of normal and inflammatory-preconditioned DPSC derived EVs and identify the EV resident miRNAs responsible for these functions.

Methods:

Dental Pulp Stem Cell (DPSCs) were procured from Lonza. EVs were isolated from DPSCs under normal culture condition and after LPS pre-conditioning and characterized by immunoblotting, TEM and nano particle tracking (NTA) analyses. The micro-RNA composition of the naïve and pre-conditioned EVs was evaluated by miRNA sequencing followed by informatics based analyses. The anti-inflammatory activity of the EVs was evaluated in vitro on primary mouse bone marrow derived macrophages by qRT-PCR, ELISA and immunoblotting techniques. In vivo effects of the EVs were studied in a rat calvarial defect model at days 1, 3 and 5 post wounding using qRT-PCR and immunohistochemistry (IHC). All Data was analyzed for significance by one way ANOVA and post hoc Tukey's analysis (P<0.05).

Results:

EVs from naïve and LPS pre-conditioned DPSCs showed similar EV characteristics. Both naïve and pre-conditioned DPSC EVs elicited anti-inflammatory activity in vitro and in vivo. LPS pre-conditioned DPSC EVs showed enhanced anti-inflammatory activity compared to other groups by significantly reducing the expression of inflammatory markers IL1β, TNF-α, and iNOS and increasing expression of anti-inflammatory markers IL-10, ARG1 and TGFβ both in vitro and in vivo. miR-Seq analyses revealed significant changes to EV miRNA composition that attributes enhanced EV functionality to select miRNA candidates.

Conclusions:

DPSC EVs possess inherent anti-inflammatory properties, and this is enhanced when DPSCs are inflammation pre-conditioned with LPS. This effect is possibly linked to the EV miRNA composition and provides vital clues for future studies on miRNA-based EV engineering.

Prof. Qing-Ling Fu

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Repeated mild traumatic brain injury (rmTBI) had raised more attraction due to its long-term effect on cognitive and behavior in which acetylated tau significantly increase the risk of developing Alzheimer's disease (AD). Nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme for substrate salvage during deacetylation, plays important role in anti-aging.

Methods: Here we overexpressed NAMPT in cultured mesenchymal stem cells (MSCs) and purified small extracellular vesicles (sEVs) using anion exchange chromatography. NAMPT-sEVs were intranasally delivered in the mice of rmTBI. The neurological function and neuroinflammation were evaluated.

Results: NAMPT-MSC-sEV exhibited the typical characterization of sEVs and had high level of NAMPT. Intranasal delivery of NAMPT-MSC-sEV improved the cognitive function determined by mNSS test, rotar rod test, cognitive function, morris water maze task and novel object recognition test in rmTBI mice. The treatment of NAMPT-sEV suppressed the neuroinflammation, maintained aquaporin protein-4 (AQP4) polarity to facilitate the glymphatic system and promoted the repairment of meningeal lymphatic, which contributed to the clearance of acetylated tau (ac-tau) from brain parenchyma. Furthermore, the treatment of NAMPT-sEVs significantly increased the expression of Sirtuin 1 (SIRT1) which is involved in deacetylating tau in rmTBI mice.

Conclusion: Our results indicate that NAMPT-sEVs reduced the neuron loss and improved the cognitive function of rmTBI mice.

Dr. Reza Yarani, Dr. Rosita Primavera, Dr. Shashank Chetty, Dr. Jing Wang, Prof. Flemming Pociot, Dr. Avnesh Thakor

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Mesenchymal stromal cells (MSCs) act as “mobile drug stores” by releasing therapeutic and regenerative factors – this can include soluble cytokines and factors as well as extracellular vesicles (EVs). In our study, we assessed the ability of EVs derived from adipose tissue MSCs (AD-MSC-EVs) to maintain glycemic regulation and prevent the onset of diabetes in rats by protecting β cells.

Methods: EVs were isolated from cultured AD-MSCs, characterized following MISEV2018 guidelines, quantified, and delivered intra-arterially (IA) directly into the pancreas and intravenously (IV) into rats that were made diabetic by streptozotocin (STZ). A positive control group (Diabetic, no EVs) and a negative control (Healthy, no STZ, no EVs) were also included. Non-fasting blood glucose levels and the weight of rats were checked every day for 30 days. Intraperitoneal glucose tolerance tests (IPGTT), immunohistochemical (IHC), and plasma insulin and cytokine measurements were also performed for all groups.

Results: Following STZ treatment, IV and diabetic group animals became hyperglycemic and lost weight. Although hyperglycemia was milder in the IV group compared to the diabetic group, both lost their insulin response capacity. In contrast, animals in the IA group remained euglycemic and responded to IPGTT equally, as did the healthy group. IHC analysis also showed that the IA group has a tissue structure like the healthy group. Insulin levels and cytokines profiles are also identical in the IA and healthy groups compared with the diabetic and IV groups.

Conclusion: Our data showed IA-delivered AD-MSC-EVs can maintain glycemic regulation in diabetic rats by protecting the pancreatic islets and β cells, the mechanisms of which we are currently investigating in vitro.

Dr. Chun-chieh Huang, Dr Miya Kang, Dr Koushik Debnath, Ms Yu Lu, Dr Sriram Ravindran

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

EV therapeutics requires a reasonable shelf life of native and engineered EVs to be translationally relevant. EV membranes are similar to cellular plasma membranes and freezing, thawing and/or lyophilizing can compromise membrane integrity and EV function. The objective of this study was aimed at exploring the possibility and effectiveness of engineered MSC EVs in a lyophilized state.

Methods:

We have previously developed and characterized osteoinductive engineered EVs (BMP2 EVs) generated by constitutively expressing the growth factor BMP2 in parental MSCs. These EVs served as an ideal test subject for this study based on their established functionality in vitro and in vivo. Lyophilized BMP2 EVs were generated in the presence and absence of varying levels of cryoprotectant DMSO. The ability of fresh and lyophilized EVs to be endocytosed by MSCs was evaluated quantitatively in vitro. Further, the functional efficacy of the lyophilized EVs was evaluated quantitatively in vitro using qRT PCR, luciferase reporter assays and osteoinductive assays. In vivo functionality was evaluated in a rat calvarial defect model. Freshly prepared EVs served as controls.

Results:

Results indicated that BMP2 EVs lyophilized with 10% DMSO showed a comparable endocytosis profile to freshy isolated EVs and the endocytic ability diminished with reduction in DMSO concentration indicating that 10% DMSO may be an ideal amount for preserving the endocytic function of lyophilized EVs. In vitro assays indicated that in terms of inducing osteoinductive gene expression, triggering the promoter activity of BMP2 response element, inducing ALP activity under mineralizing conditions, and in inducing the phosphorylation of SMAD 1/5/8, the lyophilized BMP2 EVs containing 10% DMSO performed on par with that of fresh EVs. These EVs also showed similar in vivo functionality with fresh EVs in the calvarial defect model when evaluated using micro CT and histological approaches

Summary:

Overall, the results indicated that lyophilizing HMSC EVs in the presence of 10% DMSO cryoprotectant preserves its functional activity. Future studies can focus on evaluating other cryoprotective agents as well as EV shelf life after lyophilization.

Post-doctor Ruiyun Tian, Professor Furong Li

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Androgenic Alopecia (AGA) is a model progressive scalp hair loss disease determined by genes. AGA can cause the miniaturization of hair follicles, thus making them lose their periodic circulation and regeneration functions. At present, the clinical treatment methods mainly include drug treatment and surgical treatment, but the results are not ideal. Small extracellular vesicle (sEV) derived from stem cells is regarded as a “cell-free biotherapy” that can replace mother cells. In this project, sEV derived from MSCs was used to verify its safety and effectiveness in promoting hair follicle regeneration in mouse alopecia model and androgenic alopecia model, and it was found that miR-155-5p related to cell proliferation and division was highly expressed in MSC-sEV, and AKT/β-catenin/GSK-3β protein in skin stem cells showed the same upward trend, which was further verified by in vitro and in vivo experiments. The results show that MSC-sEV is effective in promoting hair follicle regeneration, and its safety is verified by the analysis of inflammatory cell infiltration, organ lesions and sEV retention. MiR-155-5p in MSC-sEV can activate AKT/β-catenin/GSK-3β signal pathway in skin stem cells to participate in the regulation of hair follicles from resting stage to growing stage, thus achieving therapeutic effect on AGA. This study provides a solid preclinical data support and theoretical basis for developing effective clinical drugs for AGA by expounding the preclinical research and molecular mechanism of MSC-sEV in the treatment of AGA.

Ms. Ching-fen Yang, Ms. Hoei Ser Chong, Dr. Takaaki Matsuoka

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Stem cell derived exosomes have been used on skin regeneration and rejuvenation, however the exact mechanism is not clear yet. In this study, we investigated and compared the protein and RNA markers of umbilical cord derived mensenchymal stem cell exosomes (um-EXOs) and adipose derived mensenchymal stem cell exosomes (ad-EXOs), and their treatment effect by cell assays and clinical data.

Materials and methods

The stem cells are from volunteered patients, and the cells are well preserved and managed in our clinic. The human dermal fibroblasts are commercial cell lines. The exosomes are isolated by centrifuge method and confirmed the expression of CD markers (CD63 and CD81) by western blot. The protein markers are investigated by western blot and ELISA, and the RNA markers are investigated by RT-qPCR. The effect on skin regeneration and rejuvenation is examined by wound healing assay and collagen production assay, and facial injection treatments on volunteered patients. 3 Patients (1 male and 2 females) accepted facial injection on whole face or specific areas, and the results are confirmed by facial skin analyzer CLREO 1 month after treatment.

Results

The wound healing assay shows both um-EXOs and ad-EXOs are able to improve skin regeneration, and the collagen production assay also shows that stem cell exosomes can improve human dermal fibroblasts collagen production. The results of facial injection show porphyrin, pore and wrinkle indicators are improved after treatment on whole face, and wrinkles and spots are improved on specific facial areas such as eye corners and forehead.

Summary

The investigation of protein and RNA markers shows slightly different profile on um-EXOs and ad-EXOs. Both um-EXOs and ad-EXOs are proved to have effect on facial skin regeneration and rejuvenation, in cell assay level and clinical level, but the results do not have much difference from each other. In future, we still need more patients to better understand the treatment effects of different kinds of exosomes.

Shiyue Lu, Zhe Li, Yuqing Xu, Yuxiao Deng

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction Sepsis is a life-threatening condition characterized by organ dysfunction due to a dysregulated immune and inflammatory response to infection in the host, which can impact multiple organs. Recent research indicates that exosomes derived from MSCs (MSCs-Exo) can modulate cytokine expression, reduce inflammatory cell production, and regulate the inflammatory response.

2) Methods 1. Extraction of MSCs-Exo by ultracentrifugation: Identification of MSCs-Exo by transmission electron microscopy (TEM), particle size detection, and western blotting. PKH67 was used to label exosomes, and the uptake of exosomes by HUVECs was observed. 2. Cell model: LPS(20ug/ml) stimulation of HUVECs for 6h was used as a model to explore the intracellular autophagy of endothelial cells during endothelial injury: autophagy staining kit (MDC), antibody: SQSTM1/P62, LC3, Becline 1. 3. Animal models: the cecal ligation perforation model (CLP) model was used as the mouse sepsis model and hematoxylin-eosin staining (HE) was detected.

3) Results MSC-Exos showed a cup-shaped vesicular structure with a particle size ranging from 30-150 nm. They also exhibited positive expression of CD9 and CD63. PKH67 staining indicated the uptake of exosomes by HUVECs. LPS stimulation of the umbilical vein endothelial cell line (HUVECs) mimicked the endothelial injury model of sepsis. After 6 hours of LPS stimulation, cellular autophagy fluorescence was higher in the LPS group compared to the control group, as evidenced by increased expression of LC3 and SQSTM1/P62, and decreased expression of Becline 1 (p<0.05). Histological examination of lung tissue 6 hours after cecal ligation and puncture (CLP) modeling revealed thickening of alveolar walls, inflammatory cell infiltration, alveolar collapse, and intrapulmonary hemolysis. However, in the CLP+MSC-Exo (20ug) group, HE staining showed mild alveolar wall thickening and inflammatory cell infiltration, with a less pronounced inflammatory reaction compared to the CLP modeling group alone.

4) Summary/Conclusion In this study, exosomes derived from mesenchymal stem cells (MSCs-Exo) are explored as a new treatment for sepsis. It was found that MSCs-Exo can activate autophagy and attenuate the inflammatory response in endothelial cells stimulated by LPS in a CLP model of sepsis in mice. These findings provide a new research direction for the subsequent treatment of sepsis.

Seyedmohammad Moosavizadeh, PhD Student Jiemin Wang, Dr. Ellen Donohoe, PhD Student Aoife Canning, Dr. Aideen Ryan, Professor Thomas Ritter

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Mesenchymal stromal cells (MSCs) have been shown to possess reparative and immunomodulatory therapeutic potential. MSC secrete particles including small extracellular vesicles (MSC-sEV), to mediate their therapeutic effects. Pre-activation or licensing of MSC by cytokine cocktails can upregulate their immunomodulatory and tissue repair activities.

The aim of this study was to characterize naïve- and licensed-MSC-sEV and compare their characteristics and pro-repair efficacy in 2D in-vitro models.

Methods:

Human bone marrow MSCs were isolated, cultured in xeno-free media, and characterized for surface marker expression via flow cytometry and multi-lineage differentiative potential. MSCs were exposed to a licensed medium containing 50 ng/mL TGFb and 50 ng/mL IFNg for 72 hr.

Following, MSC-sEV were isolated from both naïve- and licensed-MSC-conditioned medium using size exclusion chromatography (SEC) columns. MSC-sEV were further characterized following MISEV guidelines for size distribution, morphology, and surface biomarkers with nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and flow cytometry, respectively.

Naïve- and licensed-MSC-sEV were co-cultured with human corneal epithelial cells (HCEpi) and human corneal keratocytes (HCK) in 2D in-vitro scratch models to investigate their effects on human corneal cell migration and wound closure properties.

Results:

MSC showed positive surface expression for CD90, CD73, and CD44, and negative for CD45, CD11b, and HLA-DR biomarkers. Besides, MSC could successfully differentiate into adipocyte and osteoblast lineages.

Isolated naïve- and licensed-MSC-sEV were analyzed by NTA and TEM exhibiting spherical bilayer morphology with a size range of approximately 80nm. Both groups showed similar surface biomarker profiles and were positive for CD9, CD63, and CD81, with higher trend expression in the licensed-MSC-sEV group.

In the wound healing assay, while both naïve- and licensed-MSC-sEV had therapeutic effects and promoted cell migration, licensed-MSC-sEV were shown to be more effective in HCEpi and HCK wound closure models in-vitro.

Conclusion:

sEV were successfully isolated from human bone marrow-derived MSC and showed the desired size and morphology, while licensed-MSC-sEV had slightly increased surface marker expression and had enhanced therapeutic effects on HCEpi and HCK wound healing 2D in-vitro models. Furthermore, the therapeutic and immunomodulatory efficacy of these MSC-sEVs will be investigated on different human 2D and 3D in-vitro models.

Dr Yu Chen Huang, Dr. Chao Yuan Chang, Dr. Chun Jen Huang

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction:

Psoriasis, a chronic inflammatory autoimmune disease affecting the skin and joints, is associated with genetic predispositions and inflammatory dysregulation. Mesenchymal stem cell-derived exosomes (MSC-exosomes) exhibit potent anti-oxidative and anti-inflammatory properties, alleviating psoriasis-like inflammation in mice treated with imiquimod (IMQ). Transcriptomic analysis revealed an abundance of microRNA-100-5p in MSC-exosomes, downregulated in IMQ-treated mouse skin, suggesting its potential role in mediating therapeutic effects by targeting the mTOR signaling axis.

2) Methods:

Adult male Balb/c mice (6-9 weeks old) were randomly assigned to IMQ, IMQ plus MSC-exosomes (IMQ+MSC-Exo), and IMQ plus MSC-exosomes treated with microRNA-100-5p inhibitor (IMQ+MSC-Exoi) groups (n = 6 mice/group). Control groups were run simultaneously. IMQ was topically applied for 6 days, followed by daily topical application of MSC-exosomes or inhibitor-treated MSC-exosomes for an additional 7 days. Clinical skin severity scores, body weight, spleen weight, spleen over body weight, and epidermal thickness were assessed on the 14th day.

3) Results:

Clinical skin severity scores, body weight, spleen weight, and spleen over body weight were comparable among IMQ groups. However, the IMQ+MSC-Exo group exhibited significantly reduced epidermal thickness compared to the IMQ group (p = 0.0021). Conversely, the IMQ+MSC-Exoi group showed significantly increased epidermal thickness compared to the IMQ group (p<0.0001). These findings highlight the critical role of microRNA-100-5p, as its inhibition abrogated the therapeutic effects of MSC-exosomes against psoriasis in IMQ-treated mice.

4) Conclusion:

This study provides clear evidence that microRNA-100-5p is implicated in the therapeutic effects of MSC-exosomes against IMQ-induced psoriasis in mice. Understanding this molecular mechanism may pave the way for targeted interventions in psoriasis.

Prof. Qing-Ling Fu, Lifen Wen, Longxin Huang

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

ackground: Allergic asthma is one of the chronic inflammatory diseases and is generally induced by CD4+ T helper 2 cells (Th2) in the context of persistent inhaled stimuli. Dendritic cells (DCs) are essential to mounting the Th2-mediated airway inflammation by presenting inhaled antigens to prime CD4+ T cells. Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSCs) exhibited great interest in intractable diseases. However, whether MSC-sEV play a role on DCs in airway inflammation is still unclear.

Methods: Mice were sensitized with house dust mites (HDM) to induce airway inflammation, and treated with MSC-sEV. The effects of sEV on murine and human DCs were identified.

Results: MSC-sEV mitigated the accumulation of Th2-associated monocyte-derived DCs (moDCs) in mouse lung in response to HDM. MSC-sEV also decreased the activation of moDCs induced in vitro including the expression of co-stimulatory molecules and cytokines secretion. Furthermore, we identified that DCs were able to take MSC-sEV in vitro and in vivo. Mechanistically, using bulk RNA-sequencing, we found that MSC-sEV played roles in the metabolic pathway of murine DCs. Using extracellular flux analysis, we found that MSC-sEV increased the requirement of oxidative phosphorylation on moDCs. Importantly, MSC-sEV displayed similar effects on human moDCs including decreased co-stimulatory molecular and cytokine production.

Conclusion: MSC-sEV are able to alter the metabolic state of DCs, favoring DCs to maintain OXPHOS (oxidative phosphorylation) rather than glycolysis, thereby reducing DCs-initiated inflammatory responses and attenuating Th2 lung inflammation, suggesting MSC-sEV can be a potential clinical therapy for airway inflammation.

Phd Student Kajal Singh, Mr. Abhinay Kumar Singh, Dr. Yen-Hua Huang

Poster Pitches (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:45 PM - 1:00 PM

1) Introduction

Acute respiratory distress syndrome (ARDS)/Acute lung injury (ALI) is a serious clinical illness with a high mortality rate. Currently, mechanical ventilation and fluid management are the main symptomatic therapy for ARDS/ALI. Most ARDS/ALI patients face a poor prognosis, due to a lack of effective treatment. Even recently, the SARS-CoV-2-induced ARDS/ALI pandemic spread worldwide unabated. However, achieving protection from lung damage, and progenitor stem cell death and calming the subsequent cytokine storm remains a major challenge. Here, we hypothesized that an inhaled/intratracheal administration of pcMSC-derived biomaterial like exosome or secretome will reprogram the progenitor stem cells and ameliorate the inflammatory condition to protect the lung injury and has been a promising prospect for the treatment of ARDS/ALI.

2)Method

Herein, exosome/secretome was isolated from pcMSCs cells and exosome was characterized by TEM, NTA, and protein expression of their markers. In in-vitro study, LPS-induced in-vitro (2D) and in-vivo mimic 3D Lung ARDS/ALI models were created, gene expression, immunofluorescence staining, and IHC were used to evaluate activation of AT2 (type 2 alveolar) cells. For the animal studies, LPS was also used to induce the ARDS/ALI mice model, and lung regeneration were investigated through micro-CT, lung histology, and expression of biomarker of stem cells to identify the reprogramming of stem cells to regenerate the lung after exosomal therapy.

3) Result

pcMSC-derived exosomes/secretomes can reduce inflammation, inhibit apoptosis, and promote cell renewal. It may be possible that the inhaled exosomes or secretome significantly reduce ARDS/ALI lung injury effectiveness over the whole course of the respiratory system in vitro and in vivo. Moreover, we will also investigate whether inhaled/intratracheal administration pcMSC-derived exosomes efficiently neutralize proinflammatory cytokines, cause an alternative landscape of lung-infiltrated immune cells, and alleviate hyperinflammation of lymph nodes.

4) Summary

In summary, an ARDS/ALI mice and in-vitro (2D and 3D) model, pcMSC-derived exosome or secretome show significant therapeutic efficacy by regulation of the multisystem inflammatory syndrome and reduce acute mortality, suggesting a powerful synergic strategy for the treatment of patients with severe ARDS/ALI via non-invasive administration.

Assistant Professor Sukonthar Ngampramuan, Doctor Anyapat Atipimonpat, Associate Professor Hathaitip Sritanaudomchai, Assistant Professor Paranee Yatmark

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Ischemic stroke is the most prevalent condition among populations, resulting from insufficient blood flow in particular areas of the brain due to the blockage of an artery. There are limited therapeutic approaches and time windows available. Mesenchymal stem cells (MSCs) have a paracrine effect through the secretion of extracellular vesicles (EVs), which could positively affect cell repair and recovery. Thus, this study aims to investigate the therapeutic effects of dental stem cell-derived exosomes (DSCs-EXO) therapy to enhance neurological recovery and cerebral blood flow in rats after ischemic stroke.

Methods: DSCs-EXO were isolated and characterized under the “MISEV2018” guidelines. Cerebral ischemic in rats was induced by bilateral common carotid arteries occlusion. Male Sprague-Dawley rats were divided into three groups: sham, bilateral common carotid arteries occlusion (BCCAO), and BCCAO with DSCs-EXO (100µg) treatment. DSCs-EXO was provided intravenously (IV) 3h after the BCCAO procedure. Neurological deficits are assessed using modified neurological severity scores (mNSS) of balance performance on days 1,7 and 14 after BCCAO and treatment. Arteries blockage and insufficient blood flow were determined by a brain CT scan on day 14. All animal experimental protocols and procedures were approved by Mahidol University-Institute Animal Care and Use Committee (COA.NO.IMB-ACUC 2022/004).

Results: The exosomes from DSCs-MSCs medium have a size of around 100 nm and cup-shaped morphology using differential centrifugation and TEM images. Nanoparticle tracking analysis (NTA) showed the size of exosomes from 40.5 nm - 749.5 nm (the average is 178.9 +/-2.6 nm). The western blot analysis showed the expression of exosome protein markers, which are transmembrane protein CD81 cytosolic proteins recovered in EVs TSG101 and HSP70. DSCs-EXO treatment significantly reduced the beam balance score of the neurological deficit test and increased Hounsfield units of cerebral blood flow from CTA imaging of three-dimensional rendering after the injection of contrast media.

Summary/Conclusions: Our results suggest that DSCs-EXO has the therapeutic capabilities to enhance neurological recovery and increase cerebral blood flow in rats after ischemic stroke.

Dr. Olivia Cardenas- Trowers, Ralph Perkerson, Tammee Parsons, Nabanita Halder, Nisha Durand, Abba Zubair, Jing Zhao, Takahisa Kanekiyo

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Vaginal prolapse, the abnormal descent of the vagina, is life-altering and affects millions of women. Unfortunately, the rate of prolapse recurrence after surgical correction is ∼30-46%. Extracellular vesicles (EVs) from induced pluripotent stem cells-derived mesenchymal stem cells (iPSC-derived MSCs) hold promise as an adjunct treatment to prolapse surgery, possibly by suppressing inflammation and fibrosis. We describe our method of establishing a scalable and effective supply chain of iPSC-derived MSC EVs as a potential therapy for vaginal prolapse.

Methods:

iPSC-derived MSCs were seeded and expanded in T175 flasks with standard media until ∼70% confluent. Cells were then cultured in FBS depleted media for 48-72 hours and conditioned media was collected and briefly centrifuged to remove cellular debris. Tangential flow filtration (TFF) was used to process the conditioned media through two consecutive filters with pore sizes of 0.65 µm and 500 kDa to concentrate the EV product. A diafiltration step was performed to exchange the cell culture media for a 0.9% NaCl solution with 25mM trehalose. Particle size and concentration were estimated using Nanoparticle Tracking Analysis (NTA). Protein was extracted from samples using 10x RIPA with protease and phosphatase inhibitors and concentration was assessed via Nanodrop. EV surface marker analysis was performed using western blot, multiplex immunoassay, and flow cytometry. Immune modulation was assessed by stimulating a THP-1 cell line that expresses luciferase upon activation of the NF-kβ pathway with 100 ng/ml LPS.

Results:

TFF for EV purification can concentrate ∼250 mL of cell culture media to ∼8-10 mL of diafiltrated product. iPSC-derived MSC EVs runs produce particles ∼150 nm in size with an average concentration of ∼6x10^10 particles/mL. EV markers such as ALIX, CD9, CD63 and CD81 were detected on western blot and multiplex immunoassay. Modulation of the immune response was shown by suppression of the activation of the NF-kβ pathway by 100 ng/mL LPS stimulation in a modified THP-1 cell line.

Summary/Conclusions:

iPSC-derived MSC EVs have the potential be a novel regenerative therapy to treat vaginal prolapse. Our efforts have successfully generated a pipeline to produce functional iPSC-derived MSC EVs using standard MSC culturing techniques and EV isolation/purification methods.

Professor Jisook Moon

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Extracellular vesicles (EVs) modulate diverse biological phenomena, but the EV-mediated modulation of normal aging has yet to be studied in depth. Here, we investigated whether human placenta-derived mesenchymal stem cells (hpMSCs) and the EVs (hpEVs) from hpMSCs could regulate organismal aging and cellular senescence. The hpMSCs were intravenously infused into aged 18–19-month-old mice and two additional injections were conducted at every six weeks along with behavioral and molecular analyses during each interval. Compared to age-matched controls, the treated mice showed significant improvements in cognitive and locomotive activities, enhanced expression of genes related to neuronal activities, and suppression of aging associated ones in the hippocampus. Furthermore, the hpMSCs delayed the cellular senescence of cocultured human cells, including human neural progenitor cells, in a transwell system suggesting that the hpMSC-mediated effects were due to secreted materials such as EVs. Further studies found that the hpEVs suppressed toll-like receptor 4 (TLR4), a suppressor of the pluripotent transcription factors, by TLR4 targeting microRNAs (miRNAs) and induced expression of pluripotent transcription factors such as SOX2 and C-MYC. These results suggest that hpMSCs and hpEVs rejuvenate cells through both suppression of TLR4 signaling and the upregulation of pluripotency factors.

This research was supported by a grant (C390000) from the Korea Basic Science Institute.

Mr Illayaraja Krishnan, Associate Professor Dr Min Hwei Ng, Dr. Jia Xian Law, Ms Shathiya Rajamanickam, Dr Baskar Subramani, Associate Professor Dr Yogeswaran Lokanathan

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Safety evaluation using healthy subjects is an important component at the preclinical stage before the potential biotherapeutics candidate is further evaluated in an efficacy study using an established animal model. Methods: Wharton's Jelly Mesenchymal Stem Cells (WJMSCs) at passage 3 were characterized based on International Society for Cellular Therapy guidelines. Small extracellular vesicles (sEVs) were isolated using the Tangential Flow Filtration (TFF) method and characterized according to Minimal Information for Studies of Extracellular Vesicles 2018 guidelines. Healthy male Sprague Dawley rats were used for the safety evaluation of sEVs. The control group was injected intravenously with normal saline and the treated group with pooled sEVs with a pre-determined concentration in normal saline every 3 weeks. Acute safety evaluation was conducted on day 14 post-injection and sub-chronic safety evaluation on day 90 post-injection. Weekly monitoring was performed for physiological parameters (body weight, food consumption, water intake, body length, abdominal circumference, and body mass index). Full blood count and serum biochemistry (alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, cholesterol, amylase, creatinine, and lactate acid dehydrogenase) were evaluated every 3 weeks. Necropsy evaluation was conducted by the veterinarian and histopathological analysis by the consultant pathologist on major organs such as lungs, liver, spleen, kidney, and lymph nodes. Results: Food consumption and water intake were found to increase in the treated group compared with the control group. No significant differences between both groups were noted in the full blood count and serum biochemistry profiles. In the necropsy evaluation, mottled appearances at the edges of the liver were observed and there were significant percentage relative organ weight differences between both groups. Histopathological evaluation showed no abnormalities in the spleen and lymph nodes for both groups. Moderate inflammation and mild tubular changes were observed in the kidney of the treated group. Meanwhile, both groups showed severe inflammation in the lungs. A state of recovery was observed in the liver for inflammation and vascular congestion in both groups. Summary/Conclusions: Major safety issue was not detected with the sEVs intravenous injection on healthy male Sprague Dawley rats with moderate inflammation and mild tubular changes observed in the kidney.

Ms Mohini Mendiratta, Ms Meenakshi Mendiratta, Dr. Sandeep Rai, Professor Ritu Gupta, Dr. Sabyasachi Bandyopadhyay, Dr. Hariprasad GuruRao, Professor Sujata Mohanty, Dr. Ranjit Sahoo

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

The key to the efficacy of mesenchymal stem cells (MSCs) in patients of acute graft-versus-host-disease (aGvHD) is linked to its rapid elimination from the circulation and highlights the importance of MSCs-secretome. The current study aims to investigate the mechanism underlying the immunomodulation of secretome derived from hypoxia-primed Wharton jelly (WJ)-MSCs, both in their viable-, and apoptotic state in aGvHD.

Methods:

Human MSCs isolated from WJ were pre-conditioned with 1% O2 for 24 hours followed by generation of apoptotic-MSCs with 1.0µM staurosporine for 12 hours. Culture-conditioned media (CCM) was collected after 48 hours from both viable- and apoptotic-MSCs. The effect of CCM on immune cells derived from patients with aGvHD was assessed by evaluating T-cell proliferation, Treg-induction, and macrophage polarization using flow cytometry. Gene expression and protein levels of immunomodulatory factors were analyzed by qRT-PCR and ELISA. Furthermore, mass spectrometry identified soluble factors in the secretome of hypoxia-primed WJ-MSCs (viable/apoptotic) and their co-culture with aGvHD patient-derived aPBMNCs, offering insights into potential pathways for immune modulation.

Results:

Secretome derived from Apoptotic-WJ-MSCs (hypoxia-primed) was found superior to the viable-WJ-MSCs-CCM (hypoxia-primed) to T-cell suppression (65%, 47%, p: ≤0.0001), Tregs induction (19%, 9%, p: ≤0.001), and macrophage polarization towards the M2 phenotype (70%, 53%, p: ≤0.0001). In contrast, apoptotic WJ-MSCs (hypoxia-primed) demonstrated an inability to inhibit T-cell proliferation. However, they did show the capacity to induce the generation of Tregs (5%) and promote the polarization of macrophages toward the M2 phenotype (58%). Furthermore, the mass spectrometry analysis revealed distinct changes in the secretome composition of WJ-MSCs (hypoxia-primed; viable/apoptotic) when cultured alone compared to their co-culture with aPBMNCs. These findings suggested a metabolic shift in MSCs upon interaction with aPBMNCs, contributing to their immune-modulating capabilities.

Conclusion:

Our results demonstrate the superiority of secretome derived from hypoxia-primed-apoptotic-WJ-MSCs which may serve as a non-cellular approach for managing aGvHD.

Dr Yaoying Long, Dr. Bianlei Yang, Prof. Zhichao Chen, Prof. Qiubai Li

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Stem cell senescence and age-related tissue degeneration increase with decreased nicotinamide adenine dinucleotide (NAD+) levels. CD38, the main NAD+-consuming enzyme, is highly expressed in senescent cells as a potential treatment target. We previously demonstrated that extracellular vesicles (EVs) derived from umbilical cord mesenchymal stem cells (MSC-EVs) rejuvenate senescent mesenchymal stem cells (MSCs) and delay age-related degeneration. Here, to enhance the function of MSC-EVs, we constructed CD38 antigen receptor membrane-modified MSC-EVs (CD38-ARM-MSC-EVs) by transfecting MSCs with lentivirus loaded with a CD38 antigen receptor-CD8 transmembrane fragment fusion plasmid to target senescent cells and alleviate aging. Compared to MSC-EVs, our CD38-ARM-MSC-EVs could highly express CD38 antigen receptor and target senescent MSCs or tissues with high expression of CD38 antigen in vitro and in naturally aged mouse models after intraperitoneal administration. Importantly, CD38-ARM-MSC-EVs could more effectively rejuvenate both replicative and physiologically senescent MSCs. In aged mouse models, stronger therapeutic efficiency in both bone and lung age-related degeneration was observed using CD38-ARM-MSC-EVs than MSC-EVs, combined with or without nicotinamide mononucleotide (NMN), a NAD+ precursor. This study supports that our CD38-ARM-MSC-EVs have advantages over unmodified MSC-EVs in targeting and rejuvenating senescent MSCs and slowing age-related degeneration, suggesting a potentially novel treatment for aging-related tissue degeneration and diseases, especially when combined with NMN.

Ms Tomoko Yamaguchi, Dr Juntaro Matsuzaki, Dr Takeshi Katsuda, Ms Noi Tokuda, Mr Yuzhi Tan, Dr Masaki Kimura, Dr Takahiro Ochiya, Dr Yoshimasa Saito

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Liver cirrhosis is the irreversible stage of liver fibrosis caused by hepatitis viruses, alcohol, diet, drugs, and autoimmune diseases. Hepatic stellate cells (HSCs) play key roles in liver fibrogenesis. Activation of HSCs causes them to produce extracellular matrix. We previously established a novel method to generate liver progenitor cells, named chemically induced liver progenitors (CLiPs), from mature hepatocytes in 2D culture using small molecule inhibitors (Cell Stem Cell 20:41-55, 2017, Elife 8:e47313, 2019). Since the anti-fibrotic effects of stem cell-derived small extracellular vesicles (sEVs) were recently reported (NPJ Regen Med. 30;6(1):19, 2021), we examined the therapeutic potency of human CLiP (hCLiP)-derived sEVs (hCLiP-sEVs) in liver fibrosis.

Methods: hCLiPs were intrasplenically administered to mice with CCl4-induced liver fibrosis and fibrosis regression was evaluated. To consistently collect hCLiP-sEVs of the same quality, hCLiPs were immortalized by overexpression of the CDK4(R24C), Cyclin D1, and TERT genes, and sEVs were obtained from the culture supernatant of immortalized hCLiPs by ultracentrifugation (210,000 ×g, 70 min, 4°C). The size and number of sEVs were quantified by nano-tracking analysis. The expression of CD9 and GM130 was confirmed by immunoblotting. HSCs were activated by TGF-β (5 ng/mL) in vitro. Upon co-culture of hCLiPs and HSCs using the transwell culture system, exposure of HSCs to hCLiP-sEVs, and transfection of hCLiP-sEV-derived total RNA into HSCs, mRNA expression of αSMA in HSCs, an indicator of the activation, was evaluated.

Results: Fibrosis was pathologically improved in the hCLiP transplantation groups with reasonable changes in gene expression. In particular, hCLiP transplantation decreased mRNA expression of profibrogenic markers such as αSMA. Indirect co-culture with hCLiPs decreased αSMA expression in TGF-β-activated HSCs, which was also confirmed when co-cultured with immortalized hCLiPs. Upon exposure to hCLiP-sEVs, the expression of αSMA in HSCs significantly decreased. To test if RNA cargo in sEVs is responsible for the effects of hCLiPs, we transfected hCLiP-sEV-derived total RNA to TGF-β-activated HSCs. Strikingly, the transfected RNA inactivated HSCs almost at the same level as hCLiP-sEV exposure.

Summary/Conclusion: Our results indicate that hCLiP-sEVs are applicable as an anti-fibrotic modality for the treatment of liver fibrosis via their RNA cargo.

PhD. Bi-Xin He, Prof. Qing-Ling Fu, PhD. Shu-Bing Fang, PhD. Chan-Gu Li

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction Type 17 helper T cells (Th17)-dominant neutrophilic airway inflammation is critical in the pathogenesis of steroid-resistant airway inflammation such as severe asthma. Small extracellular vesicles (sEV) derived from human mesenchymal stem cells (MSCs) display extensive therapeutic effects and advantages in many diseases. However, the role of MSC-sEV in Th17-dominant neutrophilic airway inflammation and the related mechanisms are still poorly studied.

Methods We successfully developed a neutrophilic airway inflammation mouse model by sensitization with ovalbumin (OVA) and lipopolysaccharide (LPS) and challenged with 5% OVA, and MSC-sEV were administered intravenously before challenge. Levels of T helper cells and inflammatory cells in lung tissues were analyzed by flow cytometry, and level of Interleukin-17A (IL-17A) in bronchoalveolar lavage fluids (BALF) were analyzed by enzyme-linked immunosorbent assay (ELISA). The mCherry-labeled MSC-sEV were used for evaluation of uptake effects of CD4+ T cells on MSC-sEV. Human CD4+ T cells were polarized into Th17 cells with or without stimulation with MSC-sEV to study the effects of MSC-sEV Th17 polarization. RNA sequencing (RNAseq) was used to explore the signaling pathways involved in the MSC-sEV mediated inhibition of Th17 polarization. The mechanism in Th17 by MSC-sEV was studied by Western blot and confirmed by p-STAT3 activator colivelin. RNA-depleted MSC-sEV were used to evaluate the functional components of MSC-sEV and proteomics analysis was conducted.

Result We found that MSC-sEV significantly alleviated the infiltration of inflammatory cells in peribronchial interstitial tissues and reduced levels of inflammatory cells, especially neutrophils, in BALF of mice with neutrophilic airway inflammation. Consistently, MSC-sEV significantly decreased levels of IL-17A in BALF and Th17 in lung tissues. Furthermore, mCherry-labeled MSC-sEV were taken up by human CD4+ T cells most obviously at 12 h after incubation. More importantly, using RNA sequencing and Western blot, JAK2-STAT3 pathway was identified as an important role in the inhibition of Th17 polarization by MSC-sEV. Proteins but not RNAs in MSC-sEV were mainly involved in the therapeutic effects of MSC-sEV.

Conclusion MSC-sEV were able to ameliorate Th17-dominant neutrophilic airway inflammation by the inhibition of JAK2-STAT3 pathway, suggesting that MSC-sEV could be a potential therapeutic method for the treatment of neutrophilic airway inflammation.

Dr Hala Saneh, Heather Wanczyk, Joanne Walker, Dr Christine Finck

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Background: Despite advances in neonatal care, the incidence of Bronchopulmonary Dysplasia (BPD) remains high among preterm infants. Human induced pluripotent stem cells (hiPSCs) have shown promise in repairing injury in animal BPD models. Evidence suggests they exert their effects via paracrine mechanisms. We aim here to assess the effectiveness of extracellular vesicles (EVs) derived from hiPSCs and their alveolar progenies (diPSCs) in attenuating hyperoxic injury in a preterm lung explant model.

Methods: Murine lung lobes were harvested on embryonic day 17.5 and maintained in air-liquid interface. Following exposure to 95% O2 for 24 hours, media was supplemented with 5x106 particles/mL of EVs isolated from hiPSCs or diPSCs by size-exclusion chromatography. On day 3, explants were assessed using mean linear intercept (MLI) measurements, and VEGFa and antioxidant gene expression. Statistical analysis was conducted using one-way ANOVA and Multiple Comparison Test. EV proteomic profiling was performed and annotations focused on anti-inflammatory, antioxidant, and regenerative pathways.

Results: Exposure of fetal lung explants to hyperoxia induced airspace enlargement, increased MLI, upregulation of antioxidants Prdx5 and Nfe2l2 with decreased VEGFa expression. Treatment with hiPSC-EVs improved histologic changes. VEGFa and antioxidant genes were upregulated with diPSC-EVs, suggesting pro-angiogenic and cytoprotective potential. EV proteomic analysis provided new insights in regard to potential pathways influencing lung regeneration.

Conclusion: HiPSC- and diPSC-EVs have a promising role in attenuating lung changes associated with prematurity and oxygen exposure. Our findings pave the way for a novel cell free approach to prevent and/or treat BPD, and ultimately reduce the global burden of the disease.

Doctoral Researcher Julia Monola, Postdoctoral Researcher Chris Pridgeon, Alisa Jokela, Principal investigator Riina Harjumäki

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Two-dimensional (2D) cell culture in 5% CO2 is not representative of physiological conditions and produces less in vivo-like extracellular vesicles (EVs). Stem cell-derived EVs can enhance the regeneration of damaged tissues, but reproducible production of therapeutic in vivo-like EVs remains challenging. This study aims to optimise the conditions to produce in vivo-like EVs for wound healing applications by comparing the EVs produced by 3D culture methods in physiological oxygen and conventional production methods.

Methods:

Human adipose stem cells were cultured in four conditions: atmospheric oxygen or 5% oxygen, each with 5% CO2 and in either 0,2 % nanofibrillated cellulose hydrogel (NFCh) or ultra-low attachment (ULA) conditions. 2D culture in atmospheric oxygen was used as control. Spheroid formation was visually confirmed and conditioned medium was collected after 3 and 7 days of culture. EVs were isolated by differential centrifugation and stored at -80°C in PBS. Metabolic activity of cells was measured by resazurin assay. Western blotting and immunocytochemistry staining were performed for PECAM-1, E-cadherin, vimentin, β-actin. Real time qPCR was performed for hβ-2M, hCD105, hCD90, NANOG, CD34, CD146, SOX2. The EV yield and size distribution were determined by nanoparticle tracking analysis and other EVs characteristics were evaluated by proteomics and RNA sequencing.

Results:

Cells formed spheroids in all conditions and produced EVs for 7 days after which, the experiment was terminated. The metabolic activity trended higher in cells cultured in ULA conditions than in NFCh and was higher in 5% oxygen than in atmospheric oxygen. The EV yield was greater in ULA plates (size range 110-120 nm) than in NFCh (size range 120-140 nm) and in 5% oxygen compared to atmospheric oxygen.

Summary:

Physiological oxygen conditions and 3D culture affect the metabolic activity of cells and EV yield, the best tested condition was ULA in 5% oxygen. Detailed study of the effects is ongoing. EVs with the most promising characteristics will be selected for further in vitro and in vivo wound healing studies to gain more detailed insight of the role of therapeutic EVs in wound treatment applications.

MSc João Jacinto, MSc Ana Meliciano, MSc Lara Inocêncio, MSc Pedro Vicente, PhD Margarida Serra

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Previous studies have been demonstrated that extracellular vesicles (EV) of immature cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMi) exhibit notable bioactivity towards cardiac regeneration. Yet, the limited proliferation capability of these cells and the prevalent reliance on expanding them in a less clinically relevant 2D platform have constrained the translation of CMi-EV to clinical trials. To overcome these challenges, we recently developed and optimized a scalable bioprocess for the expansion of hiPSC-CMi in bioreactors, yielding cell expansion factors of 10 in 11 days of culture. In this study we leverage the established bioprocess for hiPSC-CMi production to excel the yields of CMi-EV and investigate their therapeutic potential in cardiac regeneration.

Methods

A 2 L stirred tank bioreactor was employed for expanding hiPSC-CMi as 3D aggregates using perfusion feeding. Conditioned media were collected at days 10 and 11 and subjected to OptiPrep density gradient ultracentrifugation to isolate the EVs. The obtained particles underwent quantification through Nanoparticle Tracking Analysis (NTA). Additionally, cell-based assays assessing CMi-EV's potential to promote angiogenesis and cell migration in human endothelial cells, to hamper cardiac fibrosis and inflammation, have been performed. To ensure the structural integrity of the extracellular vesicles, a Transmission Electron Microscopy (TEM) analysis was performed. Complementary assessments included an EV uptake assay and a Western blot analysis.

Results

The NTA results revealed similar particle size distribution over the two samples, yielding 6.75E9and 8.10E9 Particles/day for days 10 and 11, respectively. Western blots confirmed positive EV-associated markers (CD63 and CD9) and high purity, marked by the absence of Argonaute 2 and Calnexin. Cell-based assays demonstrated positive results when compared to the negative control. TEM images displayed the characteristic cup-shaped structure of EV. The uptake assay showed an increased EV uptake along the time points.

Summary/Conclusions

This study highlights the promising role of hiPSC-CMi as a valuable cell platform to produce EV targeted for cardiac regeneration therapeutics. We are now developing novel integrated and scalable approaches for EV purification, paving the way for enhanced translatability and clinical applicability in the field of cardiac regenerative medicine.

Aslan (mehdi) Dehghani, Senior Scientist Eric Black, Senior Scientist Zheng Zhao, Senior Scientist Namitha Haridas, Senior Manager of Process Development Sunandan Saha, Senior Manager of Process Development David Splan, Head of Process Development Services Mark Szczypka, Head of Advanced Bioprocessing David Pollard

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Extracellular Vesicles derived from mesenchymal stem cells (MSCs) have been investigated in several clinical trials for therapeutic applications. Optimization of upstream and downstream process for EV production is a critical step to support clinical trials.

Methods

The USP consisted of expansion, growth, and collection phases for the total of 10 days in single use bioreactor using microcarriers. MSCs were seeded at 3000 cells/cm2 with microcarrier density of 10 cm2/mL in MSC NutriStem® media. At day 4, 50% media exchange was done and at day 7, the EV collection phase was started by switching to EV depleted media.

The DSP started with a clarification step using 5 and 0.6 µm filters. Tangential flow filtration was then performed using 750 KDa hollow fibers to achieve 5X concentration and diafiltration. The impurities were further removed in an anion exchange chromatography step. Second TFF step was performed to exchange the buffer and concentrate the EVs and was followed by sterile filtration.

Results

The MSC harvest was collected at day 10 with the final cell density of 1-5[ ×10]^6 cells/mL and particle concentration of 1-5 [ ×10]^9 particles/mL measured by scattering mode of NTA. More than 93% cell viability was measured throughout the upstream processing.

Multiple analytical techniques were used to measure recovery and purity before and after each unit operations in DSP. We optimized each step using orthogonal single particle analysis techniques such as NTA (scattering and fluorescence) and flow cytometry as recommended by MISEV guidelines. We further analyzed each sample by Analytical HPLC, a semi-quantitative technique to characterize EV samples using 3 different detectors: multi angle light scattering, UV and fluorescence. The purity was assessed by monitoring the protein and DNA concentration throughout the process. Western blot showed the presence of tetraspanin markers and cargo protein. The functionality of the purified MSC-EV was then confirmed by measuring the wound recovery in a dose dependent manner.

Summary/Conclusion

We have developed a bioprocess for MSC derived EVs which provides a scalable and end-to-end platform for EV production. We have optimized each step using multiple orthogonal analytical techniques to achieve the highest yield and purity.

Dr Kartini Asari, Kol Thida Mom, Amirah Fitri, Sadman Bhuiyan, Dr Ramin Khanabdali, Professor Gregory Rice

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) are lipid bilayer membrane-bound particles secreted by cells, with recent studies suggesting possibilities for both diagnostic and therapeutic applications. Despite their significant potential, obtainment of functional EVs at a higher yield from cell conditioned medium remains challenging. The aim of this study was to develop a cost-effective serum/xeno-free EV collect medium for maximizing EV yield and purity for HEK293 cells.

Methods: HEK293 naïve and engineered cells were cultured in growth medium (MEM + 1x GlutaMAX + 10% FBS) in a 37°C humidified chamber at 5% CO2. Upon reaching 80% confluency, the monolayer was rinsed with 1x PBS to remove growth media. Cells were then incubated in four types of serum-free EV collect medium: MEG, MEGaN, MEGaS and MEGaNS. The conditioned medium (CM) was collected after 24 h and centrifuged at 3,000 rcf for 10 min to remove dead cells and debris. Cell morphology was analyzed pre-and-post CM collection via microscopy, prior to determination of cell numbers and viability via automated cell counting. EVs were enriched using the EXO-ACE EV-isolation method according to established in-house protocols. Isolated EVs were subsequently characterized using Nanoparticle Tracking Analysis (ZetaView) for particle number and size distribution. The amount of protein from each group was quantified using Nanodrop One.

Results: Twenty-four hours after EV collect media exposure, morphological changes in adherent HEK293 cell monolayer were observed. Although cells remained highly viable across all EV collect media types, cell numbers at EV harvest were lowest in MEGaNS compared to MEG in both HEK293 naïve and engineered cells. Protein analysis indicated MEGaNS ranked highest, notably in naïve cells. As for EV yield, a 1.8-fold and 1.5-fold increase in nanoparticles per cell relative to cells was observed when MEGaNS was compared to MEG, in naïve and engineered cells respectively.

Conclusion: Our findings revealed that cell number does not necessarily correlate with the quantity of EVs produced. As the highest yield of EXO-ACE enriched EVs was achieved when MEGaNS was utilized for EV collection in both naive and engineered HEK293 cells, this offers a cost-effective alternative for routine collection of nanoparticles for future upscaling productions.

Aslan (mehdi) Dehghani

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Despite the unprecedented therapeutic potentials of extracellular vesicles derived from mesenchymal stem cells, it is still early in their development and transition towards clinical applications. Integrating scalable upstream and downstream cGMP-compatible technologies is critical for developing reliable and cost-effective EV manufacturing pipelines that meet commercial and clinical demands. We have developed an EV bioprocess economic model to support the bioprocess optimization while minimizing financial and technological risks in EV manufacturing.

Methods

To develop any economic model, an actual bioprocess run is required as the base case. Therefore, we performed an end to end and scalable 2L bioprocess run for EVs derived from MSCs as the base case. In summary, the upstream included MSCs expansion, growth, and collection phases (10 days total) in a 2L bioreactor. Downstream processing consisted of a combination of clarification/filtration, tangential flow filtration, and anion exchange chromatography steps. From the experimental recovery data, %15 total EV recovery and production of 1000 EVs per cell were used as inputs for the model at different scales (2, 15, 50 and 150L). Since, a wide range of EV doses have been reported in different pre-clinical and clinical data, we performed our cost analysis based on 3 different EV doses, 2×10^8, 10^9, and 10^10 EVs.

Results

Downstream processing is around 10% of the total cost and the upstream processing is the main contributor (90%) with media costing 50% of that.

We also compared the cost of MSC-EV therapy with MSC therapy alone. At the highest dose (10^10 EVs), the cost of MSC-EV therapy is 10 times higher than MSC therapy, but it becomes comparable or cheaper at the lower doses.

We studied the effect of total EV recovery on the cost. Our analysis suggested that increasing the recovery from 15% to 30% drops the cost to 50%. However, the cost reaches almost a plateau beyond 40% total EV recovery.

Summary/Conclusion

In conclusion, this model is a fundamental tool to guide researchers in the EV bioprocess design. Furthermore, this model will allow us to compare EV based therapeutic approaches against viral vector based and lipid nanoparticle-based drug delivery systems.

Associate Professor Ming-You Shie

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) are promising candidates for drug loading and delivery systems in medicine; however, the lack of scalable manufacturing processing for high quantities and clinically suitable number of EV production limits the translation of EV-based therapies into clinical practice. Current EV production relies heavily on 2D cell culture or bioreactors, which are not only less physiologically relevant to cells but also require substantial medium and space. Furthermore, EV-derived ribonucleic acid cargo in 3D and 2D culture environments remain largely unknown. Methods: In this study, we biofabricated 3D auxetic scaffolds encapsulated with human embryonic kidney 293T (HEK293T) cells and significantly enhanced the EV production yield by applying tensile stimulation in bioreactors. Results: The proposed platform increases EV yields by approximately 110-fold compared to conventional 2D culture, while having properties related to inhibiting tumor progression. Further mechanistic examinations revealed that the mechanosensitivity of YAP/TAZ mediated this effect. EVs from tensile-stimulated HEK293T cells on 3D auxetic scaffolds exhibited superior capability for loading doxorubicin compared to their 2D counterparts for cancer therapy. Conclusion: Our results demonstrate the potential of this strategy for enhancing EV production and optimizing functional performance for clinical translation.

Malvika Gupta, Dr. Mandeep Kaur, Dr. Sowmya Shree Gopal, Dr. Jessica Cardenas, Dr. Amit Srivastava

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Platelets help stabilize the vasculature and maintain the integrity of endothelial cell (EC) barriers. However, their therapeutic application faces logistical challenges primarily due to a limited shelf life. Platelet-derived extracellular vesicles (PEVs) offer a promising alternative, capable of mimicking platelet responses and having an extended storage lifespan. Previous research by our group and others has demonstrated the role of PEVs in restoring endothelial integrity, but these studies lacked an exploration of donor-to-donor variability. In this study, we aim to assess how donor variability influences the effectiveness of PEVs in regulating endothelial cell permeability.

Methods: PEVs isolated from platelets of 20 donors (10 males, 10 females, aged 18-50) were categorized into resting PEVs (representing the baseline state) and activated PEVs (simulating storage conditions in blood banks). Resting PEVs were isolated immediately after platelet isolation, while activated PEVs were obtained by subjecting platelet-rich plasma to a 24-hour rocking regimen. PEV characterization followed MISEV2018 criteria. Their therapeutic effects on human pulmonary endothelial cells were evaluated using XCelligence for trans-endothelial electrical resistance (TEER), while tight junctions (TJ) were analyzed by western blot and immunofluorescence analyses. The PEVs were fluorescently labeled with Calcein AM, and their cellular uptake by ECs was analyzed by flow cytometry.

Results: Isolated PEVs displayed homogeneity, with sizes averaging 154.12±49.65 nm for male donors and 164.2±49.65 nm for female donors. In our in vitro model system, the introduction of thrombin significantly increased endothelial cell permeability, a response effectively attenuated by PEVs treatment. The protective impact was consistent at concentrations of 10^8 and 10^9 PEVs, confirming their versatility. Remarkably, therapeutic outcomes were influenced by donor sex, with PEVs isolated from female platelets providing greater protection than those from males. This sex-specific effect was confirmed by analyzing TJ proteins, revealing more robust protection in the female group. A uniform uptake of PEVs by ECs was observed, with insignificant sex-based differences among donors.

Conclusion: This study highlights donor variability's impact on PEVs regulating endothelial cell permeability, emphasizing the need for personalized approaches in PEV-based therapies. Variations based on donor sex underscore the importance of individual characteristics in optimizing therapeutic effectiveness.

Dr Choon Keong Lee^{Esco Aster Pte Ltd}, Ms Claudine Ming Hui Lim^{Esco Aster Pte Ltd}, Ms Winnie Faustinelie^{Esco Aster Pte Ltd}, Dr Desy Silviana^{Esco Aster Pte Ltd}, Mr Xiangliang Lin^{Esco Aster Pte Ltd}

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

An efficient large-scale production of extracellular vesicles (EVs) is critical to unlock their potential for therapeutic applications. Our study demonstrates the application of an adherent packed bioreactor in scaling up the cell line for continuous EV production. By overcoming the challenges, the bioreactor ensures scalability and demonstrates its ability to maintain the quality of the cell line and the EVs.

Method

500 mL tide motion bioreactor, inoculated with HEK293T cells modified to overexpress CD63, in a 100 mL packed bed volume. A chemically defined medium, connected to the bioreactor via a recirculation system was used to sustain the cell culture. By optimizing the cell seeding density relative to the number of macrocarriers, the cells were exposed to alternating aeration and nutrition phases using the tide motion principle, through the bellow compression cycle. Over a nine-day period, the macrocarriers were retrieved to assess the cell growth and viability. The biochemical composition of the medium and the amount of EVs produced were assessed to evaluate the metabolic activity of the cells.

Results

The cells achieved a 90% attachment efficiency onto the macrocarriers, without using an attachment solution. The cell reached a peak density at 3.5E9, with an initial density of 2.4E8. Cells were observed to form a 3-diemensional (3D) matrix on the macrocarriers and this may suggest that the macrocarriers may provide a platform to mimic the 3D microenvironment of the cells. This facilitated a favorable cell culture environment with the cell viability maintained at a range between 75 – 95%, with consistent metabolic activity profile. This translated to a high cell density that enabled a high yield of EVs produced. The cumulative number of EVs produce reached 3E13 particles at the end of the cultivation.

Summary

The tide motion-based bioreactor demonstrated the feasibility of supporting a high density of the cell culture system. The alternating exposure to aeration and nutrition phases provides a consistent environment for the cells to proliferate and maintaining their viability. This enabled a continuous high yield production of EVs and coupled with downstream processes, this may translate into a labour free automated EV production platform.

Francesca Loria, Sabrina Picciotto, Giorgia Adamo, Andrea Zendrini, Samuele Raccosta, Lucia Paolini, Mauro Manno, Paolo Bergese, Giovanna L. Liguori, Paolo Guazzi, Antonella Bongiovanni, Nataša Zarovni

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: The complex nature of extracellular vesicles (EVs) challenges the establishment of standardized procedures to promote their proper manufacturing, handling, analysis and use. This calls for novel methodological strategies to conduct EV Research and Product Development in a responsible and sustainable manner. Hereby, we introduce the benefit of using a Multi-Criteria Decision-Making (MCDM) tool, named EV decision-making grid (EV-DMG), to guide decision-making in the EV field. To showcase its design and application, we illustrate the adoption of our EV-DMG for streamlining EV quality control (QC) in both research-oriented and industrial settings.

Methods: Distinct EV batches from different EV sources were assessed against specific quantity and quality assessment criteria. Each assessment criterion was given: i) an importance weight, based on its relevance in the decision-making process; ii) an analytical assessment score, based on sample conformity to pre-defined QC acceptance criteria; iii) an assessment criterion score, defined by multiplying given importance weight by given analytical assessment score; iv) a final grade, determined by summing up all assessment criterion scores, leading to sample classification as either batch of good quality, batch of medium quality or batch of bad quality.

Results: The use of our EV-DMG to refine EV QC provided a straightforward and unbiased indication of the discrepancies between the multiple batches analyzed. Samples classified as batches of good quality could be subjected to any pre-determined research or commercial use. Conversely, samples reported as batches of medium quality were tailored to a limited spectrum of applications, specifically excluding sales in industry and functional investigations in research contexts. Samples of bad quality were discarded in any setting.

Summary/Conclusion: This works describes the use of an effective, user-friendly and standardization-oriented model of a MCDM tool that may be exploited in any working environment to support responsible decision-making. Through the identification and weighing of key assessment criteria to compare distinct EV-based preparations for research or commercial goals, our EV-DMG allowed to make quantifiable, objective and sustainable decisions, minimizing costs, meanwhile increasing data reliability and reproducibility.

Aslan (mehdi) Dehghani, Paul Keselman, Prabuddha Mukherjee, Meng Chai, michael Olszowy, Jordan Speidel, Thomas Gaborski, Nick Luey

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Robust and well-established orthogonal techniques for characterization of individual EVs are required to utilize them as therapeutic and diagnostic tools. However, to better utilize any analytical technology, we first need to determine the limitations of that technology.

Methods

We have established capabilities to reliably analyze EVs samples using three different orthogonal techniques; nanoparticle tracking analysis, flow cytometry and analytical HPLC. Using these three platforms, EV samples can be analyzed reliably and in agreement with MISEV and MIFlowCyt-EV guidelines.

Results

A) Scattering and fluorescence mode of NTA suffer from masking effect and photobleaching respectively. We have optimized the labeling and measurement parameters to achieve reliable measurements. We will provide guidelines on how to characterize EVs accurately using any single particle analysis technique.

B) We tested EV standards from Sigma (tested at ISEVxTech 2022) and measured identical particle concentration on 3 orthogonal techniques sc-NTA, fl-NTA and flow cytometry.

C) We compared the size distribution using 5 techniques, sc-NTA (static), sc-NTA (flow), fl-NTA (flow), DLS and Cryo-EM. We found out that fl-NTA (flow) and Cryo-EM (as the gold standard) agreed the most with each other.

D) Different nanoparticle families such as liposomes, lipid nanoparticles, mammalian EVs, and bacterial EVs were successfully characterized using this analytical toolbox.

E) Single particle analysis techniques are less reliable when crude EV samples are tested. We used BSA as a model of non-EV associated protein contaminants. We observed that the free protein concentration must be below 50 µg/mL for scattering and 5 µg/mL for fluorescence NTA to characterize EVs accurately. Therefore, the relevant ratio of EVs compared to other non-EV components is a critical parameter in reliably analyzing crude EV samples.

F) MSC conditioned media can be successfully characterized throughout each step of the purification process including clarification, tangential flow filtration, and chromatography.

Summary/Conclusion

The optimization of labeling and measurement parameters is a critical step to characterize EVs reliably. Here, We provide guidelines on performing systematic and fundamental studies using standard samples and models for both EVs and protein contaminations in the background for any analytical technology.

Mr Abdulwahab Teflischi Gharavi, Prof Keykavous Parang, Dr Saeed Irian, Prof Mona Salimi

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1)Introduction: Recently, nanotechnology has emerged as a promising approach for cancer therapy by providing suitable carriers. Nano-carriers have the capability to transport various types of agents to the recipient cells. Nano-carriers can be classified into two main categories: synthetic nanoparticles and biological/bio-inspired nanovectors. Small extracellular vesicles (sEVs) as a subgroup of biological nanovectors can be manipulated using different approaches to enhance their suitability for targeted therapy in breast cancer. This systematic review aims to widely summarize the in vitro and in vivo findings regarding the therapeutic capabilities of sEVs, with a specific focus on breast cancer treatment with more inclusion criteria.

2)Methods: A comprehensive search was conducted on bibliographic datasets including Web of Science, PubMed and Scopus using relevant queries from several related published articles and the Medical Subject Headings (MeSH) Database.

3)Results: A total of 30 studies were identified based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Our systematic review concluded that sEVs, as a drug delivery system, exhibit strong attachment to the cells and are easily uptaken by tumor cells, aligning with the findings of earlier systematic reviews that have examined the binding efficacy of the targeted sEVs to breast cancer cells. According to our analysis, therapeutic sEVs serve as safe drug delivery systems, introducing cell death in breast cancer cells through multiple mechanisms with minimal side effects. Our review also assessed the role of the sEVs loaded with various anticancer agents in cellular migration and organ metastasis. Furthermore, we highlighted the advantages of sEVs as nano-carries for breast cancer treatment compared to the synthetic counterparts such as liposome or PLGA. This systematic review also categorizes the breast tumor-bearing animal models used to assess the therapeutic potential of the sEV-based drug delivery systems.

4)Conclusion: The findings support the notion that sEVs can be utilized for theranostic approaches such as targeted therapy in breast cancer. However, several improvements are still needed to overcome the remaining hurdles, such as the limited long-term stability of sEVs, inefficient loading of the therapeutics, clearance of sEVs from the circulation, and the transition from bench-scale to clinical production.

Julie Chen, Product Line Manager - Particle Characterization Jeffrey Bodycomb, Ph.D.

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction:

Instrument calibration is one of the standardization requirements in EV (biomarker) research. The process involves comparing the measurement values of an instrument with a known standard to determine its accuracy. Latex standards commonly used for instrument calibration have a number particle concentrations derived from a fraction (1%) solids. This value is not reported as carefully as, for example, particle size. This implies a rather large uncertainty in the calibration standard which is propagated into NTA measurements. In this study, size and number concentration standards are analyzed using orthogonal techniques and subsequently incorporated for NTA calibration. The use of orthogonal techniques will significantly reduce concentration measurement uncertainties.

2) Methods:

150 nm NIST traceable polystyrene latex size standard and 30 nm, 50 nm, and 100 nm diameter Ultra UniformTM Gold Nanospheres Number Standards were obtained from ThermoFisher Scientific and nanoComposix respectively. These standards were analyzed in triplicates using the ViewSizer 3000 multi-energy NTA from HORIBA Scientific. Multiple lasers with individually adjusted laser power settings and camera gains were carefully selected for each sample. Gold nanoparticle NTA size results were verified using Transmission Electron Microscopy (TEM); concentration (number standards only) were verified using single particle Inductively Coupled Plasma Mass Spectroscopy (sp-ICP-MS).

3) Results:

NTA size and concentration from four standards were obtained and plotted as a direct comparison to TEM and sp-ICP-MS reporting. Percent Coefficient of Variation (CoV %) was used to assess differences and the results of each technique were compared. NTA results after calibration by latex standards were similar to NTA concentration measurement of gold nanoparticles.

4) Summary/Conclusion:

Orthogonal techniques serve as independent confirmation to evaluate and qualify NTA's ability to accurately size and count number standards as small as 30 nm in diameter, thereby improving accuracy of EV (biomarker) characterization.

Dr Mingming Zhang, Dr Ran Li, Dr Zhongqi Wang

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction The quality and function of senescent skeletal muscles gradually decline, greatly influencing the life quality of older citizens. Previous research showed that the exosomes secreted by skeletal muscles had a significant impact on bone metabolism. However, the regulatory mechanisms of aging skeletal muscle exosomes on bone metabolism have not yet been clarified.

Objective To explore the effects of senescent skeletal muscle-derived exosomes in regulating bone remodeling.

Methods Skeletal muscle tissues of 2-month-old (Young) and 24-month-old (Old) mice were digested into single cell suspension, and the exosomes were extracted from the suspension, which were divided into young skeletal muscle tissue exosomes (Young-Exo) and senescent skeletal muscle tissue exosomes (Old-Exo). Exosomes were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting. Effects of the two groups of exosomes on osteogenic differentiation and osteoclast formation were detected by intervening primary osteoblasts and bone marrow mononuclear macrophages.

Results The exosomes from the Young-Exo group and Old-Exo group were both circular vesicles with a double-layered membrane structure, approximately 110 nm in diameter, and both expressed exosome-related markers, CD9, CD63, CD81 and TSG101. After primary osteoblasts were cocultured with two groups of exosomes, the staining results showed that the amount of calcium deposition and the staining intensity of alkaline phosphatase in the Old-Exo group were reduced compared with the Young-Exo group (P<0.01). Moreover, the relative mRNA expression levels of osteogenic differentiation-related genes, Alp, Opn, Ocn, Col1a1 and Runx2 were also decreased (P<0.01). After bone marrow monocyte macrophages were cocultured with two groups of exosomes, the TRAP staining results showed that the percentage of osteoclast area in Old-Exo group increased compared with the Young-Exo group (P<0.001). Further, the relative mRNA expression levels of osteoclast generation and function-related genes, Ctsk, Dc-stamp, Atp6v0d2, Mmp-9, Acp5 were increased (P<0.01).

Conclusion Compared with young skeletal muscle-derived exosomes, senescent skeletal muscle-derived exosomes inhibited the osteogenic differentiation of primary osteoblasts, as well promoted the formation and function of osteoclasts.

PhD Ana Salazar Puerta, Neil Ott, Sara Kheirkhah, Jon Stranan, Grant Barringer, Samuel Cortes, Roxanne Vermette, Emily Moser, MS Hallie Harris, PhD William Lawrence, PhD Devleena Das, MD PhD Mana Saffari, MD Amy Moore, PhD Daniel Gallego-Perez

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Peripheral nerve injuries (PNI) often result in long-lasting consequences, including motor and sensory deficits and substantial disabilities. Although gene/cell therapies hold promise for PNI treatment, there is a need for technologies to facilitate their clinical translation and mitigate degeneration. Therefore, we used Tissue Nano-Transfection (TNT) to drive controlled vasculogenic reprogramming in damaged/graftable nerve tissue via the release of engineered extracellular vesicles (EVs). This strategy intends to promote healing by establishing new vasculature, thereby supporting axonal growth, and facilitating repair processes.

Methods: Fibroblasts were transfected in vitro with a cocktail of plasmid DNA (ETV2/FLI1/FOXC2: EFF) to promote the conversion into endothelial cells, confirmed after 7 days via RT-qPCR and immunostaining. EVs were collected at 24h, 5 and 7 days post-transfection. TNT delivered EFF or control plasmids into the epineurium of the sciatic nerve (in C57BL/6-mice) and EVs were collected after 24h, isolated via size extrusion chromatography (SEC), and characterized using qRT-PCR, Western Blot, ZetaView, and Cryo-EM. Additionally, 8-10-week-old C57BL/6-mice (n = 10/group) underwent sciatic nerve crush injury, followed by EFF/control TNT to the epineurium. Tissue response was assessed via histology and RT-qPCR at 7-14 days post-intervention.

Results: Our findings demonstrate that transfected cells release EFF-loaded EVs at 24h, 5, and 7 days. Removing EVs negatively impacted in vitro reprogramming, highlighting their vital role. We hypothesize that EVs create a positive feedback loop, amplifying the reprogramming and enhancing efficiency post-transfection possibly via autocrine and paracrine signaling. Moreover, TNT-transfected epineural-fibroblasts produced EFF-loaded EVs, while likely playing a role in driving vasculogenic reprogramming at the core of the nerve via paracrine signaling. Finally, vasculogenic TNT interventions in crushed nerve tissue at 7 and 14 days post-intervention, led to a significant increase in nerve tissue vascularity and improved axonal growth compared to control, as confirmed by immunostaining and qRT-PCR.

Conclusions: Our research highlights the potential of using TNT for nerve tissue reprogramming, amplified by EVs to enhance local vasculogenesis, likely via autocrine and paracrine signaling, supporting healing and axonal growth. Outcomes from this research could be key to elucidating novel approaches leveraging in vivo EV production to drive therapeutic reparative and regenerative processes beyond nerve tissue.

Ms NATALIYA BASALOVA, Ms Olga Grigorieva, Ms Anastasiya Tolstoluzhinskaya, Ms Uliana Dyachkova, Mr Maxim Vigovsky, Ms Maria Kulebyakina, Mr Vladimir Popov, Ms Natalia Kalinina, Ms Zhanna Akopyan, Ms Anastasia Efimenko

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction.

Mesenchymal stromal cells (MSCs) have the ability to regulate fibrosis, including through microRNA transferring within extracellular vesicles (EV-MSCs). Along with myofibroblasts, one of the most important cellular targets for the regulation of fibrosis is a pool of cells called activated stromal cells (or FAPa+ activated fibroblasts). Activated cells, due to their high invasive and specific secretory activity, as well as the ability to quickly differentiate into myofibroblasts, can ensure the rapid progression of fibrosis. EV-MSCs could contain specific components targeting this pool of cells and thus promoting the control of progressive fibrosis.

Methods.

To explore this hypothesis, EVs were isolated from the conditioned medium of human MSCs and characterized according to MISEV-2018 criteria. MicroRNA inhibition was carried out by transfecting EV-MSCs with synthetic oligonucleotides. The effect of EV-MSCs was assessed in a model of bleomycin-induced pulmonary fibrosis in C57Bl/6 mice (approved by local ethics committee) as well as in vitro models of TGFb-induced fibroblast differentiation.

Results.

We have shown that in mice with established fibrosis, administration of EV-MSCs significantly reduced the area of the deposited extracellular matrix in the lungs as well as the severity of signs of fibrosis according to the Ashcroft scale. We showed that in vivo EV-MSCs stimulated not only the dedifferentiation of myofibroblasts, but also reduced the number of activated FAPa+ stromal cells to the level observed in the intact group. We also showed a decreased expression of other markers of activated fibroblasts revealed earlier by single cell RNAseq such as NTM, RDH10, CHD3 (Grigorieva 2023). Using an in vitro inhibitory assay, we showed that microRNA-29c could be plausible agent transferred by EV-MSCs and responsible for reducing the expression of FAPa in activated fibroblasts.

Summary/Conclusion.

It can be concluded that the pool of activated stromal cells during the development of fibrosis can be regulated by EV-MSCs. One of the key molecular mechanisms may be the transfer of microRNAs, including microRNA-29c. The study was supported by the Russian Science Foundation grant No. 23-15-00198, https://rscf.ru/project/23-15-00198/.

A. E. Tolstoluzhinskaya, Ms Uliana Dyachkova, Mr Maksim Vigovskiy, Dr Nataliya Basalova, Ms Anna Gardzhuk, Dr Anastasia Efimenko, Dr Olga Grigorieva

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

An important role in the fibrosis development belongs to the cross-talk between fibroblasts as the main source of myofibroblast pool, macrophages as key producers of factors involved in fibrosis progression and mesenchymal stromal cells (MSCs) as important regulators of fibrotic processes via paracrine factors secretion, including extracellular vesicles (EVs). The cellular communication between macrophages and MSCs is a crucial aspect of fibrosis pathogenesis, however, mechanisms of this interaction remain poorly understood. We studied the mechanism of paracrine interaction mediated via EVs between MSCs and macrophages during fibrosis development in vitro. We isolated monocytes from human peripheral blood and stimulated their differentiation into macrophages by GM-CSF or M-CSF addition. Macrophages were polarized with either LPS+IFNg combination (M1-type), or IL-4 (M2-type). EVs from human adipose tissue derived MSC (EV-MSC) were added to polarized macrophages, then the expression level of pro- and anti-inflammatory factors genes was analyzed. We observed that EV-MSC reduced proinflammatory factor expression (IL-12p35, IL-6, TNFa) in M1-macrophages and induced the anti-inflammatory factor expression (IL-10) in M2-macrophages. To clarify the role of macrophages in fibroblast-to-myofibroblast differentiation, we added conditioned medium (CM) collected from M1- and M2-macrophages, which were cultivated with or without EV-MSC exposure, to human dermal fibroblasts. Then, we evaluated the main myofibroblast markers such as smooth muscle actin alpha (aSMA)-level by immunoblotting and its incorporation into stress-fibers by immunocytochemical analysis. Addition of CM-M1 led to the increase of aSMA expression without its incorporation into stress fibrils in most fibroblasts. CM-M1 collected from M1 macrophages after incubation with EV-MSC tended to decrease the level of aSMA in fibroblasts compared to CM-M1 from control cells. Exposure to CM-M2 did not change the aSMA expression in fibroblasts whether or not EV-MSC addition to M2-macrophages.

Thus, EV-MSC promoted the anti-inflammatory M2-phenotype development of macrophages and suppressed the pro-inflammatory M1-phenotype. EV-MSC exposure to M1-macrophages reduced their ability to induce fibroblast-to-myofibroblast differentiation, which indicated that cross-talk between MSCs and macrophages could regulate fibrosis development.

The study was supported by the RSF grant 19-75-30007(monocyte polarisation), https://rscf.ru/project/19-75-30007/, and the RSF grant 00198 (fibroblast activation and differentiation) https://rscf.ru/project/23-15-00198/.

Dr. Ming Chen, Dr. Taojin Feng, Dr. Mingming Zhang, Dr. Ruijing Chen, Prof. Yi Li, Prof. Licheng Zhang, Prof. Pengbin Yin, Prof. Peifu Tang

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Extracellular vesicles (EVs) originating from bone tissue play a crucial role in systemic health and inter-tissue communication. This study explores the potential of bone-derived EVs in promoting muscle formation, particularly significant for individuals unable to engage in physical activity due to age or clinical conditions. We focus on the release of therapeutic EVs from human bone marrow mesenchymal stem cells (hBMSCs) via histone deacetylase (HDAC) inhibition.

Methods:

Trichostatin A (TSA), a well-known HDAC inhibitor, was applied to hBMSCs to stimulate the release of EVs. These EVs were analyzed for their contents, and their regenerative effects on human skeletal muscle myoblasts (HSMMs). The study aimed to assess the feasibility of using bone-derived EVs to simulate exercise-induced muscle formation.

Results:

Treatment with TSA markedly enhanced the emission of EVs from hBMSCs. When introduced to HSMMs, these EVs targeted specific muscle regeneration pathways and promoted muscle formation. This finding reveals a novel method for inducing muscle formation, utilizing bone-derived EVs as a therapeutic agent.

Summary/Conclusion:

Our research underscores the potential of bone-secreted EVs in regenerative medicine and nanotechnology. Leveraging FDA-approved HDAC inhibitors to facilitate the release of regenerative EVs from bone cells presents a novel strategy for simulating exercise benefits in muscle tissue. This approach holds significant promise in treating conditions impeding physical activity, highlighting the pivotal role of EVs in nano-communication and tissue regeneration.

Dr. Juntaro Matsuzaki, Ms. Mayu Yoshida, Dr. Koji Fujita, Dr. Masamichi Kimura, Ms. Noi Tokuda, Ms. Tomoko Yamaguchi, Dr. Masahiko Kuroda, Dr. Takahiro Ochiya, Dr. Yoshimasa Saito, Dr. Kiminori Kimura

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

There is an unmet need for antifibrotic therapies to prevent the progression of liver cirrhosis. Previously, we conducted an exploratory trial to assess the safety and antifibrotic efficacy of PRI-724, a selective CBP/β-catenin inhibitor, in patients with liver cirrhosis (EBioMedicine 80:104069, 2022). PRI-724 was well tolerated and exerted a potential antifibrotic effect. Here, we investigated whether measuring the profiles of circulating microRNAs packaged in extracellular vesicles (EV-miRNAs) can be useful during the treatment of liver fibrosis.

Methods:

Eighteen patients who received PRI-724 for 12 weeks in a phase 1/2a study were classified as responders (n = 10) or non-responders (n = 8) based on changes in liver stiffness. Plasma samples were obtained before and after PRI-724 administration. EVs were isolated from 1 mL of plasma via the TIM4-affinity method, using the MagCapture Exosome Isolation Kit PS. The quality of the isolated EVs was checked using the nano-tracking analysis (NanoSight NS300). Total RNA was extracted from plasma EVs and a comprehensive miRNA microarray analysis was performed.

Results:

Three miRNAs (miR-6510-5p, miR-6772-5p, and miR-4261) were identified as predictors of response or non-response to PRI-724, and the levels of three other miRNAs (miR-939-3p, miR-887-3p, and miR-7112-5p) correlated with the efficacy of treatment. Among these 6 miRNAs, only miR-887-3p was expressed in liver tissue. In plasma EVs, levels of miR-887-3p were decreased in responders but not in non-responders. In liver tissue, levels of miR-887-3p were decreased following PRI-724 treatment. In situ hybridization analysis of human liver tissues confirmed that miR-887-3p was expressed in hepatocytes. In addition, transfection of a miR-887-3p mimic enhanced the alpha-SMA expression in the activated hepatic stellate cells in vitro. These data suggest that miR-887-3p would be profibrotic miRNA released from hepatocytes, which can be a non-invasive biomarker for liver fibrosis.

Summary/Conclusion:

Through analyzing plasma EV-miRNA levels in patients who received PRI-724 treatment, we identified plasma EV-miRNAs that reflect the condition of liver cirrhosis.

Dr Marta Prieto-Vila, Dr Yusuke Yoshioka, Professor Takahiro Ochiya

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: The 21st century was disrupted by a viral pandemic directed by SARS-CoV-2. Despite the reduction in the mortality rate thanks to the vaccines, emerging studies indicate persistent long-term effects post-infection, including cardiac damage and fibrosis, even in vaccinated individuals. Such conditions involve excessive fibroblast proliferation and extracellular matrix (ECM) accumulation, leading to decreased cardiac function and potential fatality. In previous studies, we demonstrated that cardiomyocyte (CM)-derived extracellular vesicles (EVs) ameliorated cardiac fibrosis in a chronic hypertension mouse model, showcasing improved cardiac function. Thus EV contained multiple anti-fibrotic and anti-inflammatory microRNAs, suggesting a potential in treating inflammation-induced cardiac damage, as seen in COVID-19.

Methods: To generate a non-infectious disease model, we explored the use of the SARS-CoV-2 Spike protein to activate the immune system, both in vitro and in vivo. The bacterial endotoxin LPS, and viral DNA fragment Poly I:C were used as positive controls. CM-EVs were isolated via ultracentrifugation, and characterized based on MISEV2018 guidelines (morphology, particle number, membrane markers, and internalization). Subsequently, we evaluated their impact on macrophages and inflammation-activated fibroblasts.

Results: LPS, Poly I:C and Spike protein treatments increased M1 proinflammatory phenotype markers at mRNA and protein levels, accompanied by increased secretion of cytokines IL-6 and TNF-α. Direct application of EVs on macrophages resulted in reduced M1 phenotype and associated cytokine secretion without inducing pro-fibrotic cytokines like TGFβ. Notably, CM-EVs also exhibited anti-fibrotic effects by significantly reducing fibroblast activation and inducing ECM degradation. In a chronic inflammatory mouse model, caused by the continuous release of LPS and Spike protein, cardiac function declined by approximately 10%. However, EV treatment reduced fibrotic areas and regulated macrophage infiltration. Intracardiac injection of EVs showcased substantial improvement, resembling the non-treated group. Even tail vein injections displayed enhanced function.

Conclusion: This research indicates promising avenues for COVID-19 patient treatment. CM-derived EVs exhibit potential in mitigating SARS-CoV-2-induced cardiac damage, offering a hopeful therapeutic intervention for improved patient outcomes.

Msc Sarah Hilderink^{Physiology, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1118, Amsterdam, the Netherlands}, Rita Najor^{Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA}, Richard Goeij-de Haas^{Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Berend Gagestein, Jaco Knol^{Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Thang V Pham^{Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Kenneth C Bedi Jr^{Cardiovascular Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA}, Kenneth B Marguiles^{Cardiovascular Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA}, Michelle Michels^{Department of Cardiology, Erasmus MC, Rotterdam, The Netherlands}, Connie R Jimenez^{Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Asa Gustafsson^{Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA}, Jolanda van der Velden^{Physiology, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1118, Amsterdam, the Netherlands}, Diederik WD Kuster^{Physiology, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1118, Amsterdam, the Netherlands}

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac muscle disease with a prevalence of 1:500. HCM is most often caused by mutations in sarcomere-encoding genes (G+). In ∼40% of patients no mutation is identified (G-). Aside from cardiomyocyte dysfunction, cardiac microvascular changes are also evident in HCM. Extracellular vesicles (EVs) and their protein cargo are likely involved in cardiac intercellular communication of HCM pathophysiology, which is yet to be elucidated. To this end, a proteomics screen of HCM EVs was performed.

Methods: EVs were isolated from 196 ± 0.2 mg of frozen cardiac tissue of 24 HCM patients that underwent septal myectomy (G+ n = 16, G- n = 8) and non-failing donors (n = 8). EVs were obtained by ultracentrifuge at 100,000 x g followed by bead isolation for CD9, CD63, and CD81. Successful isolation of EVs was assessed by western blotting for EV markers CD81, flotillin-1, syntenin-1, and negative control Apolipoprotein A1. Isolated EV fractions were blotted against whole tissue lysate of the same samples. Total protein stain and protein quantification (≥1 mg/ml) were also performed, as well as electron microscopy on intact EVs. Subsequently, a proteomics screen based on data-independent acquisition was performed on the EVs, where HCM patients were screened against non-failing donors.

Results: The proteomics screen revealed 705 proteins to be upregulated in HCM and 377 proteins to be downregulated. HCM EVs contained increased levels of enzymes associated with metabolic processes, particularly pyruvate metabolism and fatty acid oxidation, whereas proteins involved in oxidative phosphorylation were downregulated. HCM EVs were also enriched for proteins associated with mitochondrial maturation. Altered levels of proteins contributing to microvascular integrity were also detected. Moreover, increased levels of Ldb3, a cardiac EV-associated protein, suggests increased EV release in HCM compared to non-failing donors. EV protein content did not differ between G+ and G- patients.

Conclusion: Metabolic processes are altered in HCM and transport of associated proteins through increased cardiac EV release may contribute to its pathophysiology. Moreover, proteins associated with microvascular integrity warrant further research to elucidate disease progression.

Informed consent was obtained for the use of human cardiac tissue in this study.

Research Specialist II Amber Eliason, Graduate Student Santiago Moreno, Assistant Professor David Marciano

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Endothelial Cell Derived Extracellular Vesicles Contribute to Laminar Shear Stress Adaptation

Herbert Wertheim College of Medicine

Center for Translational Science, Florida International University, Port Saint Lucie, FL, USA

Introduction:

Endothelial cells (EC) sense stimuli in the circulation and release responsive signaling molecules to coordinate the multicellular adaptations required to maintain vascular homeostasis. This includes sensing laminar shear stress (LSS), the frictional force of blood flow that imparts metabolic and gene expression changes required to maintain a quiescent EC phenotype. Here we investigate the effect of variable LSS on EV signaling, their molecular cargo, and the consequence of deficient EV release associated with endothelial cell dysfunction and vascular disease.

Methods:

Pulmonary arterial EC were exposed to variable levels of LSS in vitro for 10 days and EVs isolated from cultured media using differential centrifugation and high-resolution density gradient. RNA sequencing and proteomic analyses of EC and EV were performed to identify LSS dependent changes. Single particle EV analysis was performed using nanoparticle tracking analysis and flow cytometry. To determine the consequence of deficient EV release in response to LSS, Rab27a knockdown or GW4869 treatment of EC was performed.

Results:

EC released significantly more both large and small EV in response to LSS and this phenotype persisted over the duration of 10 days relative to static culture conditions. Rab27a knockdown and GW4869 treatment significantly reduced the number of LSS induced EV. Multiomic analysis of cells and EV identified significant changes in angiogenic and inflammatory factors consistent with LSS induced EC phenotypes.

Summary:

Our results demonstrate EC respond to LSS with a significant increase in EV release, and this response persists over a 10-day study. EC derived EV have demonstrated therapeutic potential in several models of vascular disease, and our findings suggest the application of LSS as an approach to produce sufficient material to support translational studies. Several vascular disease-associated genetic variants are reported to disrupt EV signaling and our findings demonstrate the consequence of deficient EV release contributing to endothelial dysfunction.

Miss Georgia Bateman, Professor Cliff Taggart

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Chronic obstructive pulmonary disease (COPD) is characterised by progressive and persistent inflammatory disease of the airway and/or alveoli, affecting approximately 300 million people globally. COPD is primarily caused by long-term inhalation of harmful particles, resulting in irreversible airflow limitations.

Extracellular vesicles (EVs) are lipid-membrane bound vesicles that are released from almost all cell types. Originally identified as disposal mechanisms for unneeded material within cells, these vesicles have since been linked to a diverse range of functions, both beneficial and detrimental, due to the active cargo they carry. They are able to transfer this cargo to other cells, altering the recipient cell's function.

As epithelial cells are the first line of defence against harmful particles in the airways, we aim to study COPD epithelial cell-derived EVs (Cep-EVs), and explore their role in the pathogenesis and progression of COPD.

Methods:

EVs were isolated from primary bronchial epithelial cells in air-liquid interface via ultracentrifugation. The EVs were then characterised, and mass spectrometry was used to assess EV protein content.

Results:

Proteomic analysis revealed 48 proteins significantly differentially enriched between healthy and Cep-EVs. Analysis of these proteins uncovered links to sustained inflammation, increased susceptibility to infection, and cellular senescence. Increased cellular senescence is an important risk factor in chronic inflammatory disease such as COPD, due in part to the release of an inflammatory secretome known as the senescence-associated secretory phenotype (SASP). Induction of senescence by Cep-EVs in healthy epithelial cells was then evaluated using known senescence markers.

Cep-EVs were also cultured with both THP-1 cells and primary macrophages to look at the effect of circulating EVs on other cell types. While senescence did increase in these cells, we saw greater increases in macrophage-derived inflammation, including TNF-α secretion and NF-κB signalling. Susceptibility to infection will also be evaluated in these cells.

Summary/Conclusion:

In conclusion, co-culturing Cep-EVs with healthy epithelial cells induces a senescent phenotype, providing a possible mechanism for the propagation of senescence seen in a COPD lung. We have also shown that the same EVs induce different responses depending on the recipient cell type, and that Cep-EVs elicit an inflammatory response in macrophages.

Dr Amy Baxter, Ms Caitlin Vella, Dr Pamali Fonseka, Dr Tien Nguyen, Dr Emma Grant, Prof Suresh Mathivanan, Prof Stephanie Gras, Prof Mark Hulett, A/Prof Ivan Poon

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Apoptotic bodies (ApoBDs) are small (∼1-5µm), membrane-bound extracellular vesicles (EVs) generated solely through apoptotic cell disassembly that are increasingly recognised as mediators of intercellular communication via the transfer of bioactive molecules to target cells. During the vascular inflammatory disease, atherosclerosis, environmental and physical stressors promote activation and apoptosis of endothelial cell (ECs) within vessel walls. EC apoptosis is considered an important early event that promotes plaque formation, while EC apoptosis in advanced plaques contributes to endothelial erosion and associated thrombosis. The ability of EC-derived ApoBDs formed within this highly inflammatory environment to modulate immune processes, remains poorly defined.

Methods:

The characteristics and functional properties of EC-derived ApoBDs generated in an atherogenic environment was determined in an in vitro model of inflammatory ApoBD formation. Human Umbilical Vein ECs (HUVECs) were stimulated +/- TNF-α for 24 h prior to apoptosis induction via BH3-mimetic cocktail and ApoBDs were isolated via differential centrifugation. ApoBDs formed by TNF-α-stimulated ECs were denoted ‘iApoBDs’. A non-biased proteomics analysis of lysates from ApoBDs vs iApoBDs was performed and functional validation studies were performed using cytometric bead array, chemotaxis and engulfment assays. The ability of iApoBDs to modulate an adaptive immune response was tested in a proof-of-concept model in which ApoBDs and iApoBDs generated from antigen-pulsed ECs were co-cultured with primed human patient T cells to determine IFNγ response.

Results:

Proteomics analysis revealed that iApoBDs display enrichment of inflammatory cytokines/chemokines, adhesion molecules and antigen presentation machinery. Functionally, iApoBDs released cytokines over time, correlating with membrane lysis and promoted monocyte chemotaxis. iApoBDs also promoted efferocytosis by macrophages in an ICAM-1-dependent manner. Human T cells co-cultured with antigen-pulsed iApoBDs displayed an enhanced IFNγ response compared with ApoBDs. Together, these data demonstrate that during inflammation, ECs generate ApoBDs capable of modulating innate immune signalling and influencing adaptive immune processes.

Conclusions:

These findings demonstrate a novel mode of intercellular communication by apoptotic ECs during inflammation. The ability of EC-derived ApoBDs to propagate inflammatory signalling may serve as a therapeutic target in the development of treatments for vascular inflammatory diseases.

Achala Moncy, Assistant Professor Sam Das

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Obesity induced cardiac dysfunction is growing alarmingly, dramatically increasing in global prevalence. During obesity, cardiac injury starts much before the onset of decreased cardiac contractility. Resting cells including adipocytes release small extracellular vesicles (sEVs) or exosomes to maintain regular cellular homeostasis. Increased adipocytes during obesity can increase the adipocyte-derived sEVs in the circulation. Transfer of cargo, including miRNAs, to other tissues such as heart, liver and skeletal muscle can alter the function of targeted organs.

We have identified (using RNA-seq), the top 20 most abundant miRNAs expressed in human tissue. We then validated that these 20 miRNAs are also expressed in adipocyte specific sEVs isolated from human plasma, implementing a novel immune-affinity based method, Immunocapture of Cell-Specific Exosomes (ICE), which we have developed and optimized in our lab. We observed a significant increase in these miRNAs in the adipocyte-specific sEVs isolated from the plasma collected from obese individuals BMI>30, compared to healthy individuals (BMI<25). In silico analysis showed that 8 out of 20 miRNAs can function in the cardiomyocytes. Furthermore, we have observed that human cardiomyocytes (AC16 cells) do not alter the expression of these 8 miRNAs under simulated obese conditions,25 mM glucose (high glucose) and 200 uM oleic acid (high lipid). Therefore, we hypothesize that the progressive transfer of miRNAs encapsulated in the sEVs from adipocytes to cardiomyocytes can perturb cardiomyocyte homeostasis and can ultimately lead to cardiac dysfunction during obesity. Preliminary data in adipocyte specific sEVs isolated from 18 week high-fat fed mouse serum showed elevated levels of miR-22-3p, miR-26a-5p and let-7c-5p, three of the miRNAs identified in our human study. Overexpression of miR-22-3p, miR-25a-5p and let-7c-5p independently or in a cocktail in cardiomyocytes showed a significant decrease in activity specifically for let-7c-5p and miRNA cocktail treated groups suggesting the cardiotoxic effect of let-7c-5p compared to other miRNAs studied. Hence transfer let-7c-5p from adipocytes to cardiomyocytes via sEVs may cause cardiac damage during obesity.

Dr Yu Fujita, Dr Shota Fujimoto, Dr Reika Kaneko, Dr Jun Araya

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Chronic Obstructive Pulmonary Disease (COPD) represents a prevalent respiratory ailment characterized by progressive and irreversible airflow constriction attributed to tissue degradation and compromised regenerative mechanisms. Emerging research has unveiled specific cellular and molecular aberrations in COPD, with a particular focus on alveolar type 2 (AT2) cells, which play a pivotal role in tissue regeneration. Augmenting AT2 cell stemness presents a promising avenue for the restoration of damaged lung tissue and the promotion of normal cellular differentiation. Lipofibroblasts (LipoFBs), stromal fibroblasts housing lipid droplets, have been identified in close proximity to AT2 cells and demonstrated to support AT2 function. Furthermore, it has been noted that LipoFBs can be induced in vitro through metabolic modulators. In this study, we explore the potential of extracellular vesicles (EVs) derived from induced LipoFBs to mitigate the pathological effects induced by cigarette smoke (CS) in COPD.

Methods: We characterized the LipoFB cell population in COPD lungs using scRNAseq. Subsequently, we induced LipoFBs from primary human lung fibroblasts using metformin or rosiglitazone and expanded the LipoFB population to collect conditioned medium. EVs were isolated using conventional ultracentrifugation and thoroughly characterized following the MISEV guideline. The therapeutic potential of LipoFB-derived EVs was evaluated in human bronchial epithelial cells (HBECs) exposed to cigarette smoke extract (CSE), AT2-derived organoids, and a smoking-exposed COPD mouse model.

Results: Induced LipoFBs exhibited significant lipid droplet accumulation and the activation of lipogenic genes. We discovered that LipoFB-derived EVs not only suppressed cellular senescence and inflammation in the CSE-treated HBEC model but also stimulated AT2 cell proliferation and differentiation. Furthermore, intratracheal administration of LipoFB-derived EVs curtailed the development of COPD pathology, encompassing inflammation, fibrosis, peripheral airway obstruction, and emphysematous changes in the bronchial wall in smoking-exposed COPD mice. The mechanistic insights identified that amino acid transport in HBECs and AT2 cells via LAT1 in LipoFB-derived EVs was linked to their therapeutic effects. Inhibition of LAT1 in LipoFB-derived EVs was found to abolish these phenotypes, including AT2 stem cell restoration.

Summary/Conclusion: The findings suggest the potential of LipoFB-derived EVs as a novel treatment for COPD, offering therapeutic benefits such as AT2 stem cell restoration.

Mr Brachyahu Kestecher

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

The association and co-isolation of low-density lipoprotein (LDL) and extracellular vesicles (EVs) have been shown in blood plasma, however, the biological significance of this relationship is not fully understood. Here we explore this relationship to better understand the role of EVs in atherogenesis.

Methods:

Wild-type (WT), PCSK9−/−, and LDLR−/− C57BL/6 mice were used in this study. 11-week-old male mice were fed high-fat diet (HFD) for 12 weeks or kept on normal diet for 22 months. Cardiac function was assessed by ultrasound. Circulating EVs were measured using flow cytometry. Plaques were analyzed post-mortem using Oil-Red-O staining of the aortic arch. EVs were also assessed in blood plasma samples of normocholesterolemic and hypercholesterolemic clinical patients.

Results

Based on annexin V and CD63 staining, we found a significant increase in EV levels in LDLR−/− and PCSK9−/− mice after HFD, and all mouse groups showed a significant reduction in EV levels compared to before commencing HFD, where cholesterol levels were increased in all groups. CD81+ EVs showed no significant change in any group either before or after HFD, or at 22 months of age. There was no significant change in plaque formation after HFD, however, a significant increase was observed in LDLR−/− mice at 22 months. PCSK9−/− mice had a significantly higher body mass (BM) at all given time points. After HFD, PCSK9−/− mice had a significantly improved cardiac function compared to WT while LDLR−/− showed an increased ejection fraction. At age 22 months, cardiovascular function was depleted in all groups, and CD63+ EV levels were depleted. Similar to mice, CD63+ EVs were significantly depleted in patients with hypercholesterolemia.

Conclusions:

PCSK9−/− mice show a prognostically favorable cardiovascular function during short-term HFD, however, these mice are also shown to be obesogenic. Circulating CD63+ EVs show an overall inverse relationship to cholesterol and increased CD63+ EV levels show lower risk for atherosclerotic cardiovascular disease. HFD causes reduced cardiac function, but atherosclerotic development is slow progressing even in hypercholesteraemic models. This study also highlights the importance of proper diet when undergoing PCSK9 inhibition treatment.

Associate Professor Vicky Yang, Dawn Meola, Nicole Moyer, Runzi Zhou, Sally Carnevale, Guoping Li, Saumya Das

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Mitral valve prolapse (MVP) is a common valvular disease, affecting 2-3% of the human population with myxomatous MVP as the most common form. 5-10% of those afflicted will develop severe mitral regurgitation, heart failure, and sudden death. MVP is a genetically complex disease, and the lack of mouse models that can accurately reflect the disease process has hindered therapeutic advances. Dogs develop MVP spontaneously, and their diseased valves exhibit histopathologic and molecular features that recapitulate human MVP. In both species, mitral valve interstitial cells (VICs) transition from a fibroblast (αSMA-low) to myofibroblast (αSMA-high) state in MVP, a process that promotes the spread of myxomatous pathology across the valve. We hypothesized that ncRNAs in VIC-derived small EVs (sEVs) are actively involved in the propagation of myxomatous pathology. Methods: VICs were isolated from normal and MVP-affected canine mitral valves collected through the tissue donation program of Tufts Foster Hospital for Small Animals. Passage 3 VICs were first cultured in αMEM with 10% FBS then transitioned to serum-free media for sEV collection. sEV were isolated by size exclusion chromatography (IZON qEV10/70nm) followed by Amicon 10kDa concentration. sEV was characterized by TEM, NTA, and immunoblotting. Fibroblastic VICs were treated with MVP or normal VIC-derived sEV (10×9/ml) to determine the effects of sEV signaling on VIC phenotype. RNAs in these VICs and sEV were isolated and sequenced, and the functions of differentially expressed miRNAs were studied. Results: miR-145 was upregulated in both MVP-associated VICs (padj = 0.029, fold change (FC) = 1.6) and their sEVs (padj = 0.019, FC = 1.8). miR-145 overexpression promoted the myofibroblast transition of VICs. In Silico prediction and experimental validations confirmed KLF4 as a direct target of miR-145 and was subsequently confirmed by luciferase assay. KLF4 overexpression led to a decrease in αSMA expression in VICs, while KLF4 knockdown had the opposite effect. Co-cultured of MVP-VIC sEV resulted in increased αSMA protein expression (p = 0.013, FC = 1.5). Conclusions: We confirmed the miR-145 – KLF4 – αSMA pathway in VICs in the context of MVP, and that the miR-145 signal carried in sEV can affect the VIC fibroblast-to-myofibroblast transition. This miR-145-KLF4-αSMA pathway may be a potential target for MVP therapy.

Naveed Akbar, Mr Daan Paget, Mr Lewis Timms, Dr Daniel Radford Smith, Ms Rebecca Rooney, Ms Heleah Soulati, Ms Carla De Villiers, Professor Paul Riley, Professor Robin Choudhury, Professor Daniel Anthony

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Myocardial infarction (MI) induces a peripheral inflammatory response in the liver, known as the acute-phase response (APR), which can influence exploratory behaviour in mice by supressing movement. How the very early APR is signalled between injured tissues and the liver remains unknown. Endothelial cell-derived extracellular vesicles (EC-EV) are enriched in the plasma following MI, but their role and the recognition molecules in APR remain to be elucidated.

Methods

MI was induced in adult wild-type mice using the left anterior artery ligation model (n = 10). Gene expression for Cxcl1 and Serum amyloid A (Saa1/2) was investigated in mouse livers 2 and 24 hours post-MI. EC-EV were generated in vitro using wild-type s.END1 or VCAM-1 knock-out (KO) cells and treated with recombinant mouse tumour necrosis factor-α (TNF-α) (10 ng/mL) for 16 hours (n = 8 per group). EC-EV were isolated by ultracentrifugation (120, 000 x g) with washing. EC-EV were characterized by Nanoparticle Tracking Analysis, Western blot, and cryo-transmission electron microscopy. EC-EV were injected intravenously into wild-type mice and livers harvested after 2, 6 and 24 hours (n = 6 per group). Mouse exploratory behaviour was assessed by AnyMaze tracking software as distance travelled in meters.

Results

MI livers showed an early induction of Cxcl1 (P<0.01) and Saa1/2 (P<0.01) post-AMI. Stimulation of sEND.1 cells with TNF-α resulted in a significant increase in EC-EV particles versus controls (P<0.01), which were ALIX, TSG101, CD9, eNOS and VCAM-1 positive and negative for mitochondrial ATP5A. Following intravenous injection of EC-EV, liver Cxcl1 gene expression at 2 (4.13±3.19-fold) and 6 (8.01±2.44-fold) hours was significantly increased versus control (P<0.01 both) and Saa1/2 was significantly increased at 6 hours (118.50±75.77-fold, p<0.01). VCAM-1 WT and KO EC-EV were positive for TSG101 and CD9. Both Cxcl1 and Saa1/2 induction was abrogated by injecting VCAM-1 KO EC-EV. VCAM-1 WT, but not VCAM-1 KO EC-EV lowered exploratory behaviour in mice 6 hours post-injection (P<0.05).

Conclusion

Our data show that EC-EV induce an early APR in the liver that leads to a reduction in exploratory behaviours in mice, dependent on EC-EV-VCAM-1. This study highlights a new signalling axis between the injured heart to mediate early peripheral inflammation.

Miss Rebecca Raven¹, Doctor Jessica Williams¹, Professor Keith Morris¹, Professor Philip James¹

¹Centre for Cardiovascular Health and Aging, Cardiff Metropolitan University, Cardiff, United Kingdom

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: A major hallmark of ischaemic stroke is tissue hypoxia and blood-brain barrier (BBB) dysfunction. The neurovascular unit (NVU) that comprises the BBB is crucial to maintaining homeostasis of the brain microenvironment. Two most integral cell types in the NVU are endothelial cells and astrocytes. The role of Extracellular vesicles (EVs) in this context has only recently become recognised. Disruption to the BBB causes leakage and it is plausible that EVs could not only travel across in a bidirectional manner, but also have a crucial role in this pathophysiology. Here, the aim was to determine whether EV production was altered in primary cells of the NVU following hypoxia and if barrier integrity was impacted using an in vitro BBB model.

Methods: Primary immortalised human brain endothelial cells (HBECs) and astrocytes (SVGs) were exposed to hypoxic (1%O2) and normoxic (21%O2) conditions for 24-hours. EVs were isolated from culture supernatant using differential ultracentrifugation and characterised, according to ISEV-guidelines, by Nanoparticle tracking analysis (NTA), flow cytometry (FC) and Time resolved fluorescence (TRF). HBECs and SVGs were cultured on trans-well inserts, creating an in vitro model of the BBB. Alterations in barrier integrity/permeability was measured using trans-endothelial electrical resistance (TEER) and FITC-dextran assays.

Results: These findings indicate that following 24-hours in hypoxia, EV concentration was increased in HBECs (5211±145EVs/Cell vs 3443±213EVs/Cell, p < 0.001), but not in astrocytes. No difference in the mean size of EVs produced was exhibited in both cell lines following hypoxia. The in-vitro barrier model exhibited a significant reduction in TEER after 24-hours exposure to hypoxia compared to normoxia (21.40±2.76 Ω.cm2 vs 76.35±2.38 Ω.cm2, p < 0.0001), signifying disruption to barrier integrity. Moreover, increased permeability of FITC-dextran through the barrier was identified following exposure to hypoxic conditions for 24-hours in comparison to normoxic conditions (10.34% ±038% vs 5.34%±0.22%, p < 0.001).

Summary/Conclusion: Hypoxia selectively increases EV in HBECs but not astrocytes, more research is needed to determine if the biocargo is altered. The reduction in TEER and increased permeability through the barrier suggests the potential for EV leakage in a bidirectional manner between the brain parenchyma and bloodstream following a hypoxic/ischaemic event.

Petra Ilvonen¹, Sanna Susila^1,2, Reetta Pusa¹, Ulla Impola¹, Tuukka Helin³, Lotta Joutsi-Korhonen³, Saara Laitinen¹, Jouni Lauronen¹, Minna Ilmankunnas^1,4,5

¹Finnish Red Cross Blood Service, Helsinki, Finland, ²Emergency Medical Service and Emergency Department, Päijät-Häme wellbeing services county, Lahti, Finland, ³Department of Clinical Chemistry, HUS Diagnostic Center, Helsinki University Hospital and University of Helsinki, Helsinki, Finland, ⁴Department of Anesthesiology and Intensive Care Medicine, Helsinki University Hospital and University of Helsinki, Helsinki, Finland, ⁵Meilahti Hospital Blood Bank, Department of Clinical Chemistry, HUS Diagnostic Center, Helsinki University Hospital and University of Helsinki, Helsinki, Finland

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) have procoagulative properties. As EVs are known to accumulate in stored blood products, we compared the EV content and coagulation capacity of leukoreduced cold-stored whole blood (CSWB) with current prehospital and in-hospital component therapies to understand the role of EVs in the hemostatic capacity of ageing CSWB.

Materials and methods: Blood was obtained from 12 O RhD positive male donors. Written informed consent was obtained from each participant for study participation and data publication and all study procedures were performed in accordance with the requirements of the World Medical Association's Declaration of Helsinki. CSWB was compared with in-hospital component therapy of red blood cells (RBCs), fresh frozen plasma and buffy-coat platelets and prehospital component therapy of RBC and lyophilized plasma. Samples were drawn on days 1 and 14 of CSWB and RBC cold storage. Blood count, hemolysis markers, rotational thromboelastometry, sonorheometry and thrombin generation were analyzed. EVs were analyzed using nanoparticle tracking analysis and cellular origin was determined using imaging flow cytometry.

Results: There was a trend towards increased production of both platelet and RBC derived EVs during CSWB storage. As the particle count increased, thrombin generation slowed down and in viscoleastic assays, clotting times prolonged, clot formation became impaired, and stiffness of the resulting clot decreased.

Conclusion: Both platelet and RBC derived EVs increased in number in CSWB during storage. This did not appear to compensate the in vitro decreasing hemostatic capacity of ageing CSWB, suggesting EVs produced during storage may not have active procoagulative effects, but rather reflect ageing of blood cells.

Ms, Ph.D. candidate Mahboubeh Shahrabi Farahani, Ph.D. Elham Hosseini-Beheshti, Ph.D. Mehdi Forouzandeh Moghadam, Ms, Ph.D. candidate Leila Darzi

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Among emerging cancer therapies, immunotherapy using T-cell engineering is one of the most promising. The CRISPR/Cas system is one of the most common gene editing tools. However, lacking an efficient and safe intracellular carrier is still the main challenge for in vivo delivery applications. Exosomes as natural, non-immunogenic nanoparticles can be an appropriate candidate for CRISPR/Cas system delivery.

Methods: In this study, targeted exosomes against LFA-1 on T cells were isolated from HEK293T modified cells by Exo-spin kit. The purified exosomes were characterized by DLS, TEM, and western blotting, and their uptake by T cells was evaluated in vitro using flow cytometry. Two gRNAs against the PD-1 gene were cloned into the pX-459 vector, and targeted exosomes were loaded by these CRISPR/Cas9 plasmids. Then, T cells were subjected to exosomes, and disruption in the PD-1 gene was examined by flow cytometry and sequencing. To evaluate the cytotoxicity and cytokine release of modified T cells, CFSE staining and ELISA were performed, respectively.

Results: Flow cytometry results indicated newly produced targeted exosomes could enter T cells more than non-targeted ones. pX-459 plasmids were loaded successfully into targeted exosomes and delivered to T cells functionally. CFSE staining and ELISA showed that knocking out the PD-1 gene in T cells, enhanced their cytotoxicity and cytokine release compared to non-treated T cells.

Conclusion: In the present study, targeted exosomes were applied to transfer the CRISPR / Cas9 system into T cells and were able to modify target cells genetically. This approach may improve and facilitate immunotherapy without needing to extract cells and perform ex vivo tests. To the best of our knowledge, this is the first study of genetic modification in T cells using targeted exosomes containing the CRISPR / Cas9 gene editing system.

Ms, Ph.D. candidate Mahboubeh Shahrabi Farahani, Ph.D. Elham Hosseini-Beheshti, Ph.D. Mehdi Forouzandeh Moghadam, Prof Seyed Mohammad Moazzeni, Ms Leila Darzi

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Background: Exosomes are natural nano-carriers that hold the required vital features of an ideal delivery system. However, using unmodified exosomes can have some limitations such as low accumulation in target sites. There are numerous studies have established that engineering exosomes against different cell surface markers can overcome most of these difficulties.

Methods: In this study, the newly designed fusion protein of ICAM-1/LAMP2b was initially modeled, and its stability and binding affinity to interact with LFA-1 were assessed by the MD simulation and the Z-dock server, respectively. HEK293T cells were then stably transduced by a lentiviral vector encoding ICAM-1/LAMP2b. The purified exosomes were characterized, and their interaction with recombinant LFA-1 was studied by ELISA and western blot analysis. The uptake of exosomes was also evaluated by imaging and flow cytometry. Lastly, to assess the ability of targeted exosomes to be applied as a safe carrier, pAAVS1-puro-DNR plasmids were encapsulated into exosomes by electroporation, and GFP expression in T cells was checked by imaging and flow cytometry.

Results: The MD simulation and docking results indicated that adding ICAM-1 I domain to LAMP2b did not significantly influence the binding affinity of ICAM-1 for LFA-1. The HEKT 293 cell line was successfully modified permanently by a lentiviral vector to express ICAM-1 on the surface of the derived exosomes. TEM imaging, DLS, and western blotting confirmed the intact nature of purified exosomes. Besides, the ELISA and western blot tests established the binding affinity of targeted exosomes for recombinant LFA-1 with a significant difference from non-targeted exosomes (1.85 OD difference). Furthermore, flow cytometry results revealed noteworthy differences in the binding of LFA-1-positive (56.7%), non-targeted exosomes (17.7%), and targeted exosomes to LFA-1-negative cells (24.2%). Finally, flow cytometry indicated that 21.5 % of T cells were GFP positive after treating them with loaded targeted exosomes.

Conclusion: Our study indicated that targeted exosomes expressing ICAM-1/LAMP2b fusion protein on their surfaces were able to efficiently interact with T cells as their recipient cells. These engineered exosomes can be applied as an ideal biological targeted delivery system to transfer various biomolecules to T cells, facilitating immunotherapies or other cell-based treatments.

Advanced Sirpα-enhanced Extracellular Vesicles: A Novel Approach In Fibrosis Treatment Minjeong Kwon, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Min Kyoung Jo, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Seohyun Kim, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Dong-U Shin, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Gi Beom Kim, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Gi-Hoon Nam, Advanced SIRPα-Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment In-San Kim

Poster Pitches (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:45 PM - 1:00 PM

Introduction

The pathological proliferation of fibroblasts is a key factor in the progression of fibrotic diseases. Overexpression of CD47 in these cells, which signals immune cells to avoid phagocytosis, impedes the clearance of diseased fibroblasts, exacerbating the condition. Here, we have engineered stem cell-derived extracellular vesicles enriched with SIRPα (SIRPα-EV), designed to counteract the CD47 ‘don’t eat me' signal, offering a new avenue for mitigating fibrotic disorders.

Method

To evaluate the effectiveness of SIRPα-EV against fibrosis, we employed two established animal models: the STAM™ model for Nonalcoholic Steatohepatitis (NASH) and a bleomycin-induced model for Idiopathic Pulmonary Fibrosis (IPF). We measured liver function through serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and conducted histological assessments. For lung fibrosis, we analyzed mRNA expression of fibrosis-related genes, performed histological examinations of lung tissue, and conducted immunohistochemical analyses to detect fibrotic markers.

Result

In the STAM™ model, SIRPα-EV treatment significantly reduced AST and ALT levels, indicating reduced liver toxicity. Histological examinations, including Hematoxylin and Eosin (H&E) and Sirius Red staining, demonstrated decreased liver tissue damage and a reduction in the non-alcoholic fatty liver disease activity score (NAS). In the IPF model, H&E and Masson's trichrome staining revealed notable restoration of alveolar structures and an improved Ashcroft score. Immunohistochemical analysis showed a marked decrease in α-SMA and CD47 expression, while mRNA analysis indicated lower levels of inflammatory cytokines and fibrosis-related factors, such as Collagen type 1 alpha 1 and TIMP1. These findings collectively underscore the potential of SIRPα-EV in promoting recovery from lung fibrosis.

Conclusion

This study highlights the therapeutic potential of SIRPα-EVs in reducing fibrosis in NASH and IPF models, paving the way for future clinical research and interventions in fibrotic disease management.

Student Miji Kim, Student Sujeong Park, Student Nayeong Lee, Student Dohyun Kim, Student Dongwoo Kim, Ph.D Seon-Jin Lee, Ph.D DVM Jung Joo Hong, Ph.D (Professor) Heedoo Lee

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction Alveolar macrophages (AMs), the primary sentinels against infections that trigger lung inflammation, exhibit a robust affinity for surfactant protein A (SP-A). Extracellular vesicles(EVs) have become a promising drug delivery because of their low cytotoxicity. Nonetheless, the accurate targeting of a specific cell type and the rapid lysosomal degradation of EVs in recipient cells remains an ongoing obstacle.

2) Methods We instilled 50µL of a 0.1mg/ml LPS solution intratracheally in C57BL/6 male(6 to 8 weeks old) mice to induce an acute lung injury model. We also collected BALF and incubated EVs with SP-A for isolation and surface engineering, and performed NTA and FACS to verify the surface engineering and the presence of SP-A receptor (SP-R210) on EVs. To confirm the AMs targeting pathway of EVs, we validated the fluorescence staining of TLR4 receptors and SP-A-associated EVs through confocal microscopy.

3) Results Remarkably, SP-A-EVs were efficiently taken up by AMs both in vitro and in vivo. Moreover, our study showed that SP-A-EVs were internalized through the TLR4-mediated endocytosis pathway, leading to a notable delay in their degradation compared to natural EVs, which predominantly underwent lysosomal degradation within AMs. In a functional study, Let-7b, an anti-inflammatory microRNA, was transfected into SPA-EVs to reduce LPS-induced lung inflammation and suppress AM activation.

4) Conclusion These results underscore the potential of SP-A-coated EVs as promising drug delivery systems for precise targeting of lung-related diseases, capitalizing on the robust interaction between SP-A and AMs.

Mr Madhusudhan Bobbili, Nuria Gimeno, Mr Stefan Vogt, Florian Rüker, Gordana Wozniak-Knopp, Johannes Grillari

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Despite recent advances with lipid nanoparticles (LNPs) for drug delivery, LNPs can display toxicity and can be limiting in targeted delivery. Thus ineffective drug delivery problem needs to be addressed urgently, which can be overcome by extracellular vesicles (EVs). EVs have innate therapeutic potential which can act as target-directed drug delivery vehicles, able to modulate proliferation, migration, differentiation, and other properties of the host recipient cell that are vital for health of the host organism. To enhance EV targeted drug-delivery, we employed an intrinsically over expressed protein, CD81, to serve for recognition of the desired target antigen.

Methods: Yeast libraries displaying mutant variants of the large extracellular loop of CD81 have been selected for binders to human placental laminin, EGFR and Her2 as an example target. Their specific interaction with laminin, EGFR and HER2 was confirmed in a mammalian display system. Derived sequences were reformatted to full-length CD81 and expressed in EVs produced by HeLa cells for laminin binders; by HEK293 cells for EGFR and HER2.

Results: To assess the novel functionality of antigen-binding CD81 LEL variants, internalization of such EVs into EGFR and HER2 over expressing cell lines was assessed. EVs derived from wild-type CD81 production cell line were included as controls. Indeed, we observed EVs targeted to EGFR were significantly internalized in EGFR over expressing cells compared EGFR negative cells. Similar results were observed with HER2 targeting EVs. Furthermore, compared to WT EVs, targeted EVs loaded with small molecules induce apoptosis when loaded with cytotoxic drugs.Currently, we are accessing the in vivo targeted drug delivery of our recombinant EVs.

Summary/Conclusion: To our knowledge, this is the first example of harnessing an EV membrane protein as mediator of de novo target antigen recognition via in vitro molecular evolution, opening horizons to a broad range of applications in various thera- peutic settings. The advantage of the method presented here is that it can rapidly deliver binders to any antigen of choice, which can simply be ‘clicked’ into the full-length CD81, recombinantly expressed on the EV surface, enabling specific EV-mediated delivery to a large variety of cells and tissues.

Dr. Ikuhiko Nakase

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Our research team focuses on developing therapeutic techniques based on isolated extracellular vesicles (EVs) and peptide chemistry to deliver therapeutic/diagnostic molecules into targeted cells. Because of pharmaceutical advantages of the EVs as carriers for intracellular delivery of therapeutic molecules, we are trying to develop “cassette-like” techniques to easily modify biofunctional peptides on EVs membranes for “on-demand” receptor targeting and enhanced cellular uptake of the EVs (PepEVs). In this presentation, modification techniques using biofunctional peptides such as arginine-rich cell-penetrating peptides (CPPs, macropinocytosis induction) [1], artificial coiled-coil peptides (receptor target) [2], membrane fusion peptides (cytosolic release) [3,4] will be introduced. And newly developed EVs decorated with cell-penetrating sC18 peptides, which are derived from cationic antimicrobial protein, CAP18 [5], and membrane curvature-recognition peptides (nFAAV5) will be also presented and discussed for application in EV-based future therapy.

An actin-dependent endocytosis pathway, macropinocytosis, has been shown to be very crucial for cellular EV uptake. Therefore, our research group developed the methods for modification of arginine-rich CPPs on EV membranes using chemical linkers or acylation technique, which can induce clustering of proteoglycans (e.g. syndecan-4) and macropinocytosis signal transduction [1]. In the research of artificial coiled-coil peptides, the artificial leucine zipper peptide-modified EVs recognize the counterpart peptide-tagged receptor expression on targeted cells [2]. Stearylation of branched sC18 peptides were easily modified on the EVs by their insertion of hydrophobic moiety in EV membranes, resulted in effective induction of macropinocytosis and cancer cellular uptake [5]. The modification system was also applied for boron neutron capture therapy (BNCT) [6]. These experimental techniques will further contribute to development for the EV-based “on-demand” intracellular delivery systems.

References: [1] Sci. Rep. 7, 1991 (2017), [2] Chem. Commun. 53, 317 (2017), [3] Sci. Rep. 5, 10112 (2015), [4] Nat. Chem. 9, 751 (2017), [5] Mol. Pharm. 18, 3290 (2021), [6] Mol. Pharm. 19, 1135 (2022)

Chia-Ling Hsieh, Doctor Anh Duy Do, Miss Mafewu Olga Raboshakga, Professor Shian-Ying Sung

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Small-interfering (si)RNAs allow specific gene silencing and, hence, a promising therapeutic tool for the treatment of various genetic disorders, including cancers. However, efficient delivery of siRNAs is facing multiple challenges of in vivo degradation, immunogenicity, and physiological barriers preventing the drug from penetrating into cells. To overcome such obstacles, siRNA encapsulation into extracellular vesicles (EVs) is emerging as a potential solution. We recently identified the pro-metastatic roles of L1CAM in prostate cancer as well as LOXL1-AS1, a long non-coding RNA, in pediatric medulloblastoma (MB). Here, we aimed to develop an EV-based siRNA delivery platform and assess its feasibility in cancer therapy using prostate cancer and MB as the models.

Methods: An immortalized human bone marrow-derived mesenchymal stem cell line (hMSC) was used for EV production. hMSC-EVs were isolated from serum-free conditioned medium using membrane-affinity columns. SiRNAs targeting L1CAM (siL1CAM), LOXL1-AS1 (siLOXL1-AS1), or non-targeting control (siNC-FITC) were synthesized and directly transfected into EVs, followed by RNase treatment and clean-up. Transfected hMSC-EVs were characterized by nanoparticle tracking, electron microscopy, qRT-PCR, and western blotting. For functional validation, cancer cells cocultured with siRNA-carrying EVs were subjected to cellular-uptake, wound-healing, transwell migration, and sphere formation assays. For comparison, small-hairpin (sh)RNAs with the same targeting sequences (shL1CAM and shLOXL1-AS1) were used for stable knockdowns in prostate cancer and MB cells, respectively.

Results: Isolated hMSC-EVs were smaller than 200 nm in diameter with intact membrane structure and enrichment of exosomal markers. Quantitative RT-PCR revealed an encapsulation of ∼400 siRNA copies per EV particle. Target cancer cells demonstrated high cellular uptake of siRNA-carrying EVs, a significant decrease in L1CAM or LOXL1-AS1 expression, and an inhibition in cell migration and cancer stemness, all comparable to stable knockdown cells. An orthotopic mice model transplanted with LOXL1-AS1 knockdown MB cells dramatically decreased tumor metastasis and improved mice survivals, suggesting the potential use of siRNA-based gene therapy for high-risk cancers. Currently, the in vivo study of siRNA-carrying EVs in anti-cancer efficacy, biodistribution and safety is under investigation.

Conclusions: Our study established an hMSC-EV production platform with the feature of in vivo siRNA delivery for various cancer treatments.

Ph.D. Student Sheng-Yun Hsu, Undergraduate Hsi-Ming Chiang-Hsieh, M.S. Chen-Guang Zhang, Ph.D. Chen-Yun Yeh, Ph.D. Pi-Hui Liang, Ph.D. Han-Chung Wu, Ph.D. Yungling Leo Lee

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

HEK293 cells, recognized for their ability to produce therapeutic exosomes, have demonstrated the safety of drug delivery, as confirmed by studies using HEK293-derived exosomes. Achieving precision in engineering exosomes with specific genetic or protein content requires effective material introduction methods. CD63, a crucial marker on exosome surfaces, significantly influences their structure and function. Manipulating CD63 has shown promise in improving drug delivery and stability, marking a significant advancement in medical science.

Targeting dendritic cells (DCs) strategically holds immense importance in biomedical research, particularly in initiating immune responses. Precision targeting of DCs offers extensive benefits in vaccine development, cancer treatment, autoimmune disease management, and personalized drug delivery, aiming to enhance immune responses and customize therapies.

Methods and Results:

Our methodology focused on precise modifications to CD63 between its 3rd and 4th transmembrane domains, incorporating proteins or targeting molecules. Using transmission electron microscopy (TEM), we visualized immune gold labeling on the surfaces of modified exosomes. Additionally, introducing the anti-CD11c peptide significantly improved cargo delivery efficiency, observed both in laboratory cultures and living organisms.

Moreover, integrating the Aryl hydrocarbon receptor inhibitor CH223191 into specific exosomes, combined with liposomes, had a significant impact on remodeling dendritic cells by enhancing co-stimulatory factors (CD80&CD86) and reducing immune regulators (IL10&IDO1) within the tumor microenvironment. These findings illustrate the potential of engineered exosomes with modified CD63 in enhancing cargo delivery and therapeutic effectiveness, observed in both cellular models and live subjects.

Summary and Conclusion:

In summary, HEK293-derived exosomes serve as a secure platform for therapeutic applications. Manipulating CD63 significantly improves exosome functionality, refining precision in drug delivery. Targeting dendritic cells holds significant biomedical promise. Our research highlights the potential of engineered exosomes for precise therapies, marking a substantial leap in advancing precision medicine and tailored treatments for various medical conditions.

Dr Saima Naz, Dharani bandi, farhan ahmed

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Abstract

A huge reservoir of latently infected TB individuals poses major threat to public health globally.so it is important to explore the development of therapeutic vaccine that can protect infected individuals against endogenous reactivation of TB. Here we have shown that when mice were vaccinated with exosomes isolated from immune dominant TB antigen pulsed Dendritic cells (Dexosome), enhanced antigen-specific T cell, high titre antibody response was observed, which is because Dexosome decorate itself with processed antigenic peptides along with MHC and costimulatory molecule, which are important for activation of naive T cells to elicit enhanced immune response. We have observed formation of germinal centres in secondary lymphoid organs of vaccinated mice which correlate with enhanced GC responses and may promote establishment of long-lived plasma cells that secrete antigen-specific, high-affinity antibodies. In this regard exosome could be postulated as effective adjuvant cum antigen delivery system.

Introduction

Exosomes are membranous nano structure secreted by a variety of cells including Antigen presenting cells (APC), known to play diverse function. It has been shown that Dendritic cells pulsed with antigen, it throw up huge repertoire of exosomes decorated with processed antigenic peptides along with MHC and costimulatory molecule which are required for the activation of naïve T cells.Considering several advantages of non-infectious, non-replicating exosomes, we tried to show that exosomes derived from latency associated immune dominant antigen pulsed DCs load the antigen peptide over MHCs which is capable of activating naïve T cells. Thereby suggesting that DCs derived exosome loaded with latency associated immune dominant antigen will serve as potential adjuvant and antigen delivery system, precondition the T and B cells to elicit better corelates of protective immunity that might provide effective protection against LTB.

Methodology

Potential DOSR encoded vaccine candidate identified using immunoinformatic. candidate was cloned, expressed and purified. Exosome were isolated by differential centrifugation, were characterized by DLS,TEM.Exosome markers were scored by immunoblot.Mice were vaccinated with exosome to study immunological parameters.

Result

DCs derived exosomes when immunized in mice, serve as a potential adjuvant and antigen delivery system which pre-condition T and B cells to elicit better correlates of protective immunity.

Miss Ayaulym Nurgozhina, Shynggys Sergazy, Madiyar Nurgaziyev, Laura Chulenbayeva, Mohamad Aljofan

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Hypothesis: Exosomes derived from mare's milk can effectively encapsulate antibiotics, exemplified by gentamicin, enabling penetration into intracellular environments. Exosomal gentamicin demonstrates bactericidal activity against intracellularly localized bacteria, such as S. aureus and E. coli, within macrophages.Objectives: The project aims to enhance antibiotic effectiveness against intracellular pathogens by utilizing exosomes from mare's milk to improve drug delivery into cells.Introduction: Intracellular bacterial survival poses challenges for treating chronic or recurrent infections, often impervious to antibiotics and host immune responses. Limited antibiotic efficacy results from poor penetration into intracellular spaces or rapid drug efflux, necessitating innovative transport systems. This study explores the use of exosomes from mare's milk, an untapped source free from significant casein contamination, to facilitate antibiotic delivery into cells.Methods:Modeling Intracellular Infection: A monolayer of THP-1 macrophages was established to simulate intracellular bacterial infection. Adherent macrophages were infected with S. aureus and E. coli in Mueller-Hinton bacterial broth.Exosome Isolation and Characterization: Exosomes containing gentamicin were extracted from mare's milk using isoelectric precipitation, size exclusion chromatography, and a total exosome isolation kit. Exosome properties were assessed, including size, zeta potential, protein content, and marker expression using Western blotting and transmission electron microscopy.Quantification of Gentamicin Penetration: Gentamicin release from exosomes into the intracellular environment was quantitatively analyzed using in vitro dialysis. High-performance liquid chromatography (HPLC) measured residual drug levels over 72 hours, demonstrating a 35% release of gentamicin from exosomes.Conclusion: This research provides insights into the potential of exosomes from mare's milk as a novel antibiotic delivery system, offering a promising avenue for combating intracellular infections more effectively. The findings contribute to the ongoing efforts to address challenges associated with antibiotic penetration into intracellular environments, opening new possibilities for therapeutic interventions against persistent bacterial infections. Further research may explore the broader applications of exosome-based drug delivery systems in combating various intracellular pathogens.

Dr. Mai Hazekawa, Dr. Dasuke Watase, Dr. Takuya Nishinakagawa, Dr. Masato Hosokawa, Dr. Daisuke Ishibashi

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: EVs are nano-sized extracellular vesicles that are known to carry various messages to distant cells or metastatic organs specifically. However, there are no reports on drug carrier development using autologous serum-derived small EVs (sEVs) in vivo. The purpose of this study was to deliver therapeutic siRNAs for melanoma lung metastases using autologous serum-derived sEVs. Methods: Spontaneous lung metastasis model mice were prepared by subcutaneously injecting melanoma cells into the hindlimbs of female C57BL/6 mice, and surgically removed the primary tumors. sEVs were isolated from serum collected from mice on days 0, 3, 7, 10, and 14 after primary tumor inoculation. After isolating serum sEVs, siRNA-loaded sEVs were prepared. siRNA-loaded sEVs were intravenously injected into the spontaneous lung metastasis mice model. The distribution of siRNA in the body after intravenous administration of siRNA-loaded sEVs was evaluated using an in vivo imaging device. For the drug efficacy evaluation, the number of lung metastasis colonies was counted after laparotomy and the survival period was observed. Results: In experiments using melanoma cells, this formulation significantly enhanced intracellular uptake of siRNA in flow cytometry analysis. In addition, knockdown effects in the target protein were also observed as an effect of siRNA delivered into the melanoma cell caused by this formulation. siRNA-loaded sEVs prepared with autologous serum-derived sEVs significantly decreased the number of metastatic lung colonies caused by EV's high organ accumulation. Notably, this formulation significantly prolonged survival period compared with standard therapy. Summary/Conclusion: This formulation was founded to be useful for melanoma treatment as a lung-directed siRNA carrier made from self-contained ingredients. Furthermore, this therapeutic concept can greatly contribute to the basic technology of target nucleic acid carriers with lower antigenicity.

Miss Leila Darzi, Dr Mehdi Forouzandeh Moghadam, Dr Mehdi Shamsara, Dr Elham Hosseini-Beheshti

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Finding a safe gene delivery system is crucial for gene therapy in a broad range of diseases. Exosomes as natural nanocarrier are considered as ideal delivery platforms for gene therapy that can efficiently and selectively deliver their cargo to the target cells. The significant potential of exosomes for being investigated and applied for gene therapy purposes is mainly derived from their high level of biocompatibility and low level of immunogenicity. The potential of using exosome for delivery of functional DNA to target cell should be further investigated.

Methods: HEK293T was engineered to express DARPin against HER2 and targeted exosomes were isolated from modified HEK293T. Purified exosomes were characterized by TEM, zeta sizer and exosomal CD markers. Purified pEGFP-C1 plasmid was introduced to DARPin/Exos using electroporation and termed to pEGFP-C1/Exos. PKH26-labeled pEGFP-C1/Exos were exposed to HER2-positive SKBR3 cell line and exosome uptake was validated by florescent microscopy and GFP expression was analyzed using real-time PCR, florescent microscopy and flow cytometry. Effect of increase in exosome quantity and GFP expression was measured.

Results: In this study, we demonstrated delivery of GFP-encoding plasmid to target cells by exosomes the quantity of pEGFP-C1 plasmid in pEGFP-C1/Exos was measured through Real-time PCR. Transferring of pEGFP-C1/Exos to the target cells and GPF expression in mRNA and protein level proved the sufficient delivery of the plasmids. PKH26 labeled-pEGFP-C1/Exos were uptaken by SKBR3 and expressed GFP. Increase in pEGFP-C1/Exos quantity caused more GFP expression in target cells.

Conclusion: Exosome as natural, nontoxic, and non-immunogenic carriers have potential for using in gene delivery system in vitro. In this study, through using proper electroporation protocol the 4 kbp GFP-encoding plasmid, pEGFP-C1were loaded into HER2-targeted exosomes successfully and delivered them to the target cells functionally. Based on these results, targeted exosomes may be promising vehicle for cancer therapeutics in the future. Broader assessments of this method using various types of cargoes and target cells can highlight the findings and guarantee their in-clinic applicability for gene delivery and gene therapy purposes.

Miss Leila Darzi, Dr Mehdi Forouzandeh Moghadam, Dr Mehdi Shamsara, Dr Elham Hosseini-Beheshti

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Gene editing tools in particular CRISPR/Cas9 system are promising technology as therapeutic agent for genetical disorders. Here we develop non-viral delivery platform using targeted exosome. In this study, we used designed exosome which (engineered exosome) express DARPin against Her2 on exosome surface that targets Her2-positive breast cancer cell lines such as SKBR3. Using reporter gene such as GFP, makes it easier to track the internalization of GFP-encoding vector to target cell and entry of the GFP gene into the SKBR3 genome in AAVS1 locus. Exosomes are specifically investigated as a promising targeted nucleic acid delivery system due to their encapsulation capacity and low immunogenicity. The ability of the exosome to transfer CRISPR system vectors into target cells for gene therapy opens up a new approach to improve therapeutics for genetic disorders.

Material and methods: Engineered HEK293T cells that express the DARPin against Her2 on their exosome surface were cultured. Exosome was extracted from HEK293/DARPin according to Exo-spin exosome extraction kit. Purified exosome characterized by TEM, zeta sizer and exosomal CD marker. CRISPR/Cas9 and donor vector, pCas9-guide-AAVS1 and pAAVS1-Puro-DNR/GFP vector were extracted and were loaded to exosomes using electroporation. Electroporation was carried out at 400 V and 125 µF and three pulses. Plasmids-loaded exosomes were termed Exo/pCas9- GFP-Puro. Exos/pCas9- GFP-Puro were exposed to SKBR3

Results: Internalization of PKH26-labeled Exo/GFP-Puro and GFP expression in SKBR3 was validated using flowcytometry and confocal microscopy.

Insertion of GFP gene along with puromycin into AAVS1 locus in SKBR3 cells validated using puromycin selection. GFP expression in resulting cells was measured by flowcytometry and almost all cells expressed GFP.

Conclusion:

Taken together, we demonstrate exosomes able to deliver CRISPR/Cas9 and donor vector functionally to target cells. Our results suggest that development of exosome-based delivery of CRISPR/Cas9 could be a viable way towards finding a promising therapeutic tool.

Doctoral Candidate Marc Liébana, Doctoral Candidate Silvia López, PhD Esperanza González, PhD Juan Manuel Falcón

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Nanotherapy is arising as more personalized and efficient cancer treatment approaches are pursued. Nanoencapsulation systems that allow decreasing drug dosages and a better control of the dose-response relation have been developed. Artificial systems are used with this aim, but the immunocompatibility and specificity problems that they usually exhibit lead to the exploration of alternative systems. Extracellular vesicles (EVs) are membranous vesicles released by virtually all cells, becoming a plausible biocompatible alternative. The objective of this work is the study of the use of EVs as pharmacological nanovehicles in cancer. The anthracycline doxorubicin has been used in this approach due to its use as first-line treatment for multiple malignant diseases and its feasibility to follow-up through different assays by its intrinsic fluorescence.

A major challenge is the isolation of pure EV preparations, especially relevant for their potential clinical use. With this goal, a size-exclusion chromatography platform developed in-house is being optimized for its daily use to isolate EVs from classical contaminants maintaining their integrity and functionality for their application as therapeutic vehicles in the clinical field.

Methods

MSCs and hepatic SK-Hep1 cells were used as EV producer cells. Sensitivity of SK-Hep1 cells to doxorubicin treatment was assessed by bright field microscopy and trypan blue staining. Doxorubicin uptake by producer cells was evaluated by fluorescence microscopy and flow cytometry, which was also used to estimate doxorubicin load into EVs. EV isolation has been conducted by in-house developed SMART-SEC. Finally, the functionality of doxorubicin-loaded EVs was evaluated by lactate dehydrogenase assay, confocal microscopy and flow cytometry on SH-SY5Y recipient cells.

Results

Doxorubicin – treated MSCs and SK-Hep1 cells can uptake drug and package it into EVs. These EVs are released into the extracellular medium, and can be separated from classical vesicular contaminants and unloaded doxorubicin through EV fractionation by a quicker in-house SMART-SEC. Moreover, fractions associated to doxorubicin-containing EVs display a cytotoxic effect on neuroblastoma SH-SY5Y recipient cells.

Summary/Conclusion

EVs could be loaded with doxorubicin and then separated from sample contaminants to obtain efficient and biocompatible anticancer drug – containing nano-vehicles.

Keywords: extracellular vesicles, doxorubicin, drug loading, EV isolation, in-house SMART-SEC, MSCs

Ms. Kasey Leung^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Miya Kang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Koushik Debnath^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Chun-Chieh Huang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Sadiq Umar^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Mrs. Yu Lu^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Sriram Ravindran^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) can be engineered to possess function-specific properties. Spatial and temporal control of these EVs can maximize therapeutic benefits. Our objectives were 1) engineer MSC-EVs that target the NLRP3 inflammasome pathway for enhanced anti-inflammatory function and 2) develop an EV release system from hydrogels meditated by protease activity for release during the inflammatory phase of healing.

Methods: MSCs were genetically modified to express hsa-miR-22-3p, an anti-inflammatory miRNA that targets NLRP3, specifically in EVs using an EV targeting sequence. Engineered EVs were recovered from conditioned media from MSCs every 48-72 hours using ExoQuick-TC, a precipitation polymer. EVs were characterized qualitatively and quantitatively by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot. Anti-inflammatory activity of the engineered EVs was evaluated in primary mouse macrophages with respect to appropriate controls using RT-qPCR, ELISA, and immunoblotting. A fusion peptide containing the MMP2 cleavage domain and an EV-binding RGD domain (MMP2-RGD) was conjugated to alginate for EV binding and enzymatic release. Release kinetics of EVs from MMP2-RGD alginate hydrogels in the presence/absence of MMP2 enzyme was evaluated quantitatively with respect to appropriate controls. Retention of EV anti-inflammatory function after release from hydrogels was evaluated on primary mouse macrophages. For all experiments, Student's t-test or ANOVA followed by Tukey's ad hoc test were used for statistics (CI = 95%).

Results: hsa-miR-22-3p expression was enhanced in engineered EVs. Engineered EVs had greater anti-inflammatory activity compared to control EVs. Faster EV release kinetics was observed from the MMP2-RGD hydrogels in the presence of MMP2 enzyme compared to controls. The released EVs retained their anti-inflammatory function.

Summary/Conclusions: MSC-EVs can be engineered to be anti-inflammatory in a pathway-specific manner by EV-targeted expression of hsa-miR-22-3p. Alginate hydrogels can be engineered to bind EVs, and using a fusion peptide recognizable by MMP2 enzyme, the EVs can be triggered for enzymatic release. Since MMP2 is present during the inflammatory and macrophage recruitment phase of healing, this system allows for spatial and temporal control of EV delivery at wound sites.

Professor Chun-Jen Huang, Doctor Chao-Yuan Chang, Doctor Ching-Wei Chuang

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction:

Aspiration pneumonia (refers to acute lung injury resulting from the aspiration of gastric contents) poses a serious threat, especially to frail and elderly individuals, with inflammation and oxidative stress as critical factors contributing to high mortality rates. The microRNA (miRNA) let-7i-5p exhibits potent anti-inflammatory and antioxidative properties. This study explores the use of engineered exosomes loaded with let-7i-5p miRNA to mitigate acute lung injury resulting from gastric content aspiration in a murine model.

2) Methods:

Engineered exosomes were generated from RAW264.7 cells transfected with a plasmid overexpressing hsa-let-7i-5p miRNA. A murine model of aspiration pneumonia was created using intra-tracheal administration of a gastric content mimic, a mixture comprising a xanthan gum-based thickener (12 mg/mL), pepsin (2 mg/mL), and lipopolysaccharide (2.5 mg/mL). Mice were divided into groups receiving the gastric content mimic alone (AP group) or with engineered exosomes (1x10⁹particles particles/mouse, also intra-tracheally administered; APEEXo group). Lung injury was assessed 48 hours later

3) Results:

Histological and lung injury score assays revealed significantly higher lung injury level in the AP group compared to the APEEXo group (p < 0.001). Total and differential cell counts in bronchoalveolar lavage fluids were also markedly elevated in the AP group compared to the APEEXo group (all p < 0.001). Lung function assessments showed significantly reduced inspiratory capacity in the AP group compared to the APEEXo (p = 0.006). Moreover, dynamic compliance was lower, and resistance was higher in the AP group compared to the APEEXo group (both p < 0.01). The wet/dry weight ratio was significantly higher in the AP group than in the APEEXo group (p < 0.001).

4) Conclusion:

Intra-tracheal administration of engineered exosomes loaded with let-7i-5p miRNA alleviates acute lung injury induced by gastric content aspiration in mice.

Dr. Wolf Holnthoner

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

The lymphatic vasculature has not received overwhelming scientific attention for many years, although it is fundamental for the development and homeostasis of higher organisms. To re-establish true tissue homeostasis after traumatic injuries and disease, therapeutic strategies need to consider the regeneration of our lymphatic system. Vascular endothelial growth factor C (VEGF-C) is a key mediator of lymphangiogenesis upon binding and activation with the lymph-specific vascular endothelial growth receptor 3 (VEGFR-3). It has mainly been investigated in settings such as lymphedema and cancer. Nevertheless, most tested applications of this receptor ligand did not consider its highly complex biosynthesis process. Different proteolytic processing gives rises to different variants showing varying levels of receptor specificity and affinity which influences all of its applications. To enhance the pro-lymphatic effects of VEGF-C and its systematic availability, the possibility of linking it to proteins such as tetraspanins (e.g. CD9, 63, 81), which are enriched in extracellular vesicles (EVs), has been proposed recently. In this project, we aim to establish stable adipose derived stromal/stem cell (ASC) lines which express different variants of VEGF-C linked to CD81. This fusion protein should exert a bimodal function of receptor activation and tissue-specific homing. Expression and subcellular localization of the fusion protein was confirmed via western blotting and immunofluorescence respectively. Using a BaF3 cell line expressing a VEGFR3/EpoR chimera, we tested different CD81-VEGFC variants for differences in their activation activity. In this study, we provide proof of principle that we can generate different ligand variants of fusion proteins on EV surfaces, test their bioactivity in subsequent receptor activation assay and pave the way for testing their therapeutic potential for enhancing lymphangiogenesis in various in vivo models of traumatic injuries as the next steps.

Distinguished professor KS Clifford Chao, Attending Physician Chi-Hsien Huang, Research Assistant Shi-Xuan Yan, Research Assistant Hsin-Yu Chang, Research Assistant Pei-Chen Yang, Associate Professor Kevin Chih-Yang Huang

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Radiation-induced lung injury (RILI) causes DNA damage and accumulation of reactive oxygen species (ROS), which induces an inflammatory response that causes alveolar epithelial cell damage. Exosomes from mesenchymal stem cells (MSC-Exos) have been distinguished by their low immunogenicity, lack of tumorigenicity, and high clinical safety, but their role in treating RILI and the mechanism involved need to be defined.

Methods: The transfer of engineered MSC-Exos to RT-treated lung epithelial BEAS-2B cells and its effect on mitochondrial function was evaluated. C57BL/6 mice were received consecutive irradiation (8Gy×3) on lung to induce RILI and engineered MSC-Exos (10 ug/mL) were administered via the tail vein 4 h after irradiation. Flow cytometry, H&E, quantitative real-time PCR, immunofluorescence (IF), seahorse bioanalyzer and confocal microscopy imaging was conducted to investigate the biodistribution of MSC-Exos and its impact on lung tissue inflammation and injury.

Results: In this study, we found that MSC-Exos contained mitochondrial components provides profound protection against radiation-induced lung injury in vitro and in vivo, accompanied by improvement of mitochondrial function in alveolar epithelial cell. Furthermore, engineered MSC-Exos can transfer stem cell-derived mitochondria components and ROS scavenger proteins to alleviate epithelial cell injury. Engineered MSC-Exos elevated the level of mtDNA, OXPHOS activity and ATP generation to relive RT-induced ROS stress in alveolar epithelial cell. Furthermore, restoring mitochondrial integrity via MSC-Exos treatment enhanced alveolar epithelial cell viability and decreased lung fibrosis in vivo, as featured with the down-regulation of TGF-b-mediated inflammation.

Summary: Taken together, our results showed that engineered MSC-Exo can effectively transfer mitochondria component to improve alveolar epithelial cell mitochondrial integrity and oxidative phosphorylation level, leading to the resumption of metabolic homeostasis to mitigate lung inflammatory pathology.

Mr Xin Zhou, Dr Jiancheng Wang, Dr Danyang Li

Poster Pitches (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:45 PM - 1:00 PM

Introduction: Psoriasis is an autoimmune disease that causes inflammation in skin. Recent intriguing reports implicate mesenchymal stem cell derived extracellular vesicles (MSC-EVs) as new therapeutic approaches due to their immune modulation functions. However, the therapeutic outcome of MSC-EVs alone is not satisfactory. Some studies uncover that arginase-1 overexpression induces accumulation of self-antigen that contribute to the epidermal hyperproliferation of psoriatic keratinocytes, which might be a potential therapeutic probability. Herein, we present an engineered MSC-EVs loaded with arginase-1 inhibitor nor-NOHA. We demonstrate the efficacy of the engineered MSC-EVs by modulating metabolic and immunological disorders via deactivating of arginase-1 mediated IL-23/Th 17 axis.

Methods: Immunohistochemistry (IHC) and high-performance liquid chromatography (HPLC) were applied for the analysis of skin sections and blood samples from patients with various erythematous scaly skin diseases to verify the specificity of arginase-1 expression in psoriasis. MSC-EVs were isolated from MSCs cultures using ultracentrifugation. Nor-NOHA was loaded into MSC-EVs via electroporation to form engineered nor@MSC-EVs. We evaluated the effect of nor@MSC-EVs for the deactivation of dendritic cell (DC) and Th1/17 cells by flow cytometry, as well as decreasing IL-23 level from DC cultures via ELISA assay. We examined the influence of nor@MSC-EVs on gene expression in keratinocytes via qPCR and Western blot. In vivo, nor@MSC-EVs were administered intravenously to IMQ-induced psoriasis mice. Then, we scored the condition of the mice before and after nor@MSC-EVs therapy, analyzed the proportion of activated DCs and Th1/17 cells in the skin, lymph nodes, and spleen via flow cytometry and measured polyamine concentrations in epidermis and plasma via HPLC.

Results: The analysis of clinical sample confirmed arginase1 was overexpressed and specific in psoriasis. Nor@MSC-EVs could reduce psoriasis related polyamine and gene expression in psoriaitic keratinocytes, Additionally, nor@MSC-EVs reduced the activated DCs and Th1/17 cells, and concentration of IL-23 from DC in vitro. In vivo study demonstrated nor@MSC-EVs could significantly restore skin lesions, modulate metabolic and immunological disorders.

Summary/Conclusion: Our results suggested that the engineered nor@MSC-EVs could effectively reduce self-antigen, modulate metabolic and immunological disorders via deactivating of arginase-1 mediated IL-23/Th 17 axis, which provided evidence and new insights for psoriasis therapy.

Professor Yiwen Chen, Seiner Engineer Kai-Wen Kan, Professor Ming-You Shie, Professor Shao-Chih Chiu, Superintendent Der-Yang Cho

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: The study discusses the potential of extracellular vesicles (EVs) as drug carriers for breast cancer therapy, emphasizing their low immunogenicity and effective tissue penetration due. A genetically engineered EV is created, featuring anti-human leukocyte antigen G (αHLA-G) antibody and encapsulated doxorubicin for precise drug delivery. The modified EVs, derived from Human Embryonic Kidney 293 cells with a mutant variant, aim for fewer disease-related pathways. While doxorubicin is widely used in chemotherapy, its cardiac toxicity limits its efficacy. The study introduces a natural nanomedicine, aiming to overcome these challenges and provide targeted drug delivery for effective breast cancer treatment.

Methods: The production of targeting EVs from genetically modified HEK293T cells expressing the αHLA-G nanobody is the critical method to create targeting carriers. The production process involves utilizing 3D cultured systems with microcarriers in dynamic bioreactors to enhance yield. Targeted EVs are isolated and purified through TFF, size exclusion, and immunoaffinity chromatography and characterized by TEM and NTA. Next, the EVs are resuspended in a sulfate solution and passed through a lipid extruder to establish a transmembrane gradient for doxorubicin active loading. Afterward, the loaded EVs undergo incubation with doxorubicin, and quantified the loading capacity by fluorescence calibration curve. The toxicity of the doxorubicin-loaded targeting EVs is assessed by treating MDA-MB-231 cells, and demonstrated the in vivo animal tumor toxicity and biodistribution test to prove the efficacy of the doxorubicin-loaded targeting EVs.

Results: The advantages of doxorubicin loaded in targeting EVs include: 1) The αHLA-G targeting EVs presented high specificity to tumor and demonstrate high efficacy and safety, 2) active drug loading can achieve up to 4-10 folds in vitro tumor cells cytotoxicity when carrying doxorubicin, and 3) a tumor suppression rate of 70-90% in animal experiments.

Summary/Conclusion: This study demonstrated the EVs containing anti-HLA-G nanobodies are targeted efficient carriers with low toxicity, low immunogenicity and low drug resistance. The targeting EVs can be mass production in 3D dynamic method and yield the high purity products. Doxorubicin loaded targeting EVs shows 4-10 folds in vitro cytotoxicity as well as significant tumor control and longer survival rates in in vivo testing.

Ms Meenakshi Mendiratta, Dr Sujata Mohanty

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Mesenchymal Stem cells-derived small extracellular vesicles (MSCs-sEVs) serve as a promising candidate for cell-free therapeutics in regenerative medicine. However, there are several challenges with the utility of sEVs, including non-specific packaging of cargo, loss in circulation, and target specificity. In an attempt to overcome these challenges, the current study employs surface as well as cargo modification strategies to enhance sEVs homing, target specificity, and specific cargo enrichment with respect to liver diseases.

Methods:

Tissue-specific MSCs (Bone Marrow, Wharton's Jelly) were isolated with due patient consent ((IC-SCR/140/23 (O)) and characterized as per ISCT guidelines. MSCs were cultured in serum-free media for the isolation of sEVs via ultracentrifugation, and characterized as per MISEV 2018 guidelines. The surface of tissue-specific sEVs was bioengineered with hepato-specific ligands using chemical conjugation methods, while the cargo of sEVs was enriched with miR125 essential for hepatoprotection, using lipofectamine RNAiMAX. Bioengineered sEVs were characterized as per MISEV 2018 guidelines. Surface modification was validated via the Lectin-induced aggregation method. Cargo enrichment was validated via qPCR, and the hepatoprotective functionality was validated via ROS in hepatic cells (HUH7) and non-hepatic cells (IHF cells used as a control) and macrophage polarization in THP1 cells.

Results:

It was observed that Bioengineered sEVs (BioEn-sEVs) maintained their characteristic features when compared to Naïve sEVs. However, BioEn-sEVs exhibited a slightly increased diameter (>200nm) compared to Naïve sEVs. The modification made the negative surface charge of Naive EVs become positive due to the presence of galactose on sEVs. Furthermore, the cellular uptake efficiency of the BioEn-sEVs was significantly greater than naïve sEVs in hepatic cells as compared to non-hepatic cells. BioEn-sEVs significantly reduced ROS production and enhanced the polarization of macrophages towards M2 macrophages as compared to Naïve sEVs. The significant hepato-regenerative capacity of cargo (miRNA) of BioEn-sEVs in hepatic cells further underscores their therapeutic promise.

Conclusion:

This study has demonstrated precise sEVs targeting through surface modification, enhancing anti-inflammatory effects via cargo modification. Bioengineered sEVs emerge as a promising avenue for targeted therapeutic interventions in diseases, prompting further research and clinical exploration in this innovative field.

Dr Daniel Humphrys

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Extracellular Vesicles (EV) offer a promising alternative to current methods of drug packaging and delivery, but engineering EV remains challenging. To enable more precise engineering of EV, we developed Enveloped Protein Nanocages (EPN): EV generated by designed self-assembling proteins which induce their own release from cells. In contrast to conventional passive pseudotyping of EV, we used the SpyCatcher-Tag system to introduce a covalent bond between the cytoplasmic tail of a TMP protein and the cage scaffold. EPN scaffold proteins provide a genetically addressable and modular platform to produce EV and this was leveraged to modify EPN size, recruit specific transmembrane proteins to EPN, target EPN to cell populations, deliver protein, and act as a platform for an mRNA-launched vaccine.

2) Use of the SpyCatcher-SpyTag system allowed for conjugation of the intravesicular EPN nanocage with the intravesicular tail of the designed TMPs, ensuring presentation of a particular TMP on the surface of released EPN. Western blots were used to probe for the presence and orientation of designed TMP, evaluate membrane integrity, and determine conjugation efficiency of the TMP-cage interaction. CryoEM was used to image particles and evaluate EPN morphology, and NTA was used to quantify EPN sizes. Flow cytometry was used to evaluate cell-specific EPN targeting efficiency and delivery was quantified by a HiBiT-LgBiT luminescence complementation assay. Efficacy as a vaccine platform was evaluated in a mouse model via LNP-delivered mRNA encoding the cage, antigen, and genetically-encoded adjuvant.

3) EPN cages were successfully conjugated to various TMP, which allows for better display of target TMP. Furthermore, conjugation changes EPN size which demonstrates some control of EV diameter. EPN were specifically targeted to EGFR+ cells, and EPN-mediated delivery of HiBiT to LgBiT+ recipient cells demonstrated protein delivery. Our preliminary vaccine study was successful in eliciting anti-CoV-2 binding titers in mice.

4) EPN leverage designed protein scaffolds for controllability unique in the engineered EV field, and here we show that recruited TMP are functional in this platform. This work shows the potential of the EPN platform, and generally highlights the utility of designed protein scaffolds in the EV field.

Juliette Suermondt, PhD Xiuming Liang, Guannan Zhou, Houze Zhou, Oskar Gustafsson, PhD H. Yesid Estupiñan, PhD Yang Liu, Professor Samir EL Andaloussi, Assistant professor Joel Nordin

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

CRISPR/Cas9 loaded EVs have previously been studied using in vitro reporter models based on cancer cell lines. Though easy to work with, editing of these cancer cell lines has limited therapeutic benefit. Ex vivo engineering of immune cells, on the other hand, has demonstrated great therapeutic benefit. The most well-known example of this is the development of ex vivo-generated CAR T cells, which have been successfully used in the treatment of haematological malignancies. Editing T cells ex vivo is therefore a valuable method to assess the therapeutic potential of a gene editing delivery platform, including engineered EVs. Here, the delivery of EVs carrying CRE to mouse T cells isolated from spleens of Ai9 mice is shown. I furthermore describe how to isolate and activate T cells from mouse spleens and how to adequately test EVs on these T cells. Ai9 mice contain a CRE reporter construct, and can therefore be utilized to study CRE delivery. The best-EV-loading scaffold used in my study was able to induce recombination in over 70% of activated T cells while maintaining viability as measured by flow cytometry, thereby reaching delivery levels close to that of lentiviral vectors. Lentiviral vectors are often used to edit activated T cells, however, they are unsuccessful at editing naïve T cells. On the other hand, when using the engineered EVs, almost 30% editing of naïve T cells was observed. Furthermore, we are currently extending our EV platform to include the delivery of CRISPR tools to target therapeutically relevant genes in human T cells. Previous data from our group has demonstrated higher editing results at the same EV dose when using CRISPR compared to CRE in all tested cell lines. Therefore, I anticipate similar or better results in T cells using Cas9-ribonucleoprotein-loaded EVs. Taken together, we describe ex vivo T cell editing as a useful method to evaluate EV editing capabilities in a therapeutically relevant model, and we demonstrate that our EVs can edit both activated and naïve T cells.

Ms Fui Teen Chong, Ms Hui Sun Leong, Ms Mengjie Ren, Dr Shen Yon Toh, Prof N Gopalakrishna Iyer

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Head and neck squamous cell carcinoma (HNSCC) is often an aggressive neoplasm with low overall survival and has poor response to current therapeutic treatment. Though epidermal growth factor receptor (EGFR) is usually defined as an established oncogene in HNSCC, the other members of the ErbB receptor family, namely ErbB2 (HER2), has also been reported in variable proportions of HNSCCs. Some studies have either demonstrated presence of HER2 isoforms in association with breast cancer tumor progression and development, or endosomal transportation of canonical HER2 receptor from the cell membrane to the nucleus. This study aims to examine a HER2 isoform that is secreted in extracellular vesicles (EVs) in tyrosine kinase inhibitor (TKI)-sensitive patient-derived cultures and its effects as a co-treatment on TKI-resistant tumor cells.

Methods

HNSCC cell lines were first cultured in T-175 tissue culture flasks till confluent and incubated with conditioning medium after a single washing step with 1 x PBS. EV fractions were then harvested after 72 hours and concentrated with a molecular cutoff filtration using a 50 kDa Amicon® Ultra-15 centrifugal filter unit at 4000 x g. The remaining adhered cells in T-175 flasks were harvested as well by trypsinisation and stored as a pellet for western blot as total cell lysate. To identify donor EVs from TKI-sensitive patient-derived cultures, western blot using HER2 antibody and HER2 TKIs treatment, such as Afatinib, Dacomatinib and Lapatinib, measured by a luminescent cell viability assay, CellTitre-Glo®, were used.

Results

Western blot of the patient-derived cell lines showed varying expressions of HER2 isoform in HNSCC. Of those identified as a possible donor EV, the HER2 isoform in these fractions compared to the total cellular lysates, were indeed secreted and concentrated as part of the secreted EV or exosomal fraction. The high expression of HER2 isoform correlated with sensitivity to HER2 TKIs.

Conclusion

With several HNSCC cell lines expressing higher levels of HER2 isoform, suggest that it may be a suitable therapeutic co-treatment to HER2 TKIs in HNSCC. However, further characterisation of the isolated EVs will be explored first.

Ms. Neona Lowe, Rachel Mizenko, Dr. Alyssa Panitch, Dr. Randy Carney

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Cell penetrating peptides (CPPs) are small (<30 amino acid residues) biomolecules that are capable of passively crossing biological membranes. There is interest in exploiting CPPs for the development of functional therapeutics. However, a limitation of CPP-based therapeutics is that most CPPs do not have targeting capabilities. We therefore propose using EVs as a drug delivery vehicle for CPP-based therapeutics. As CPP loading into EVs have not yet been studied, here we first investigated how physiochemical properties of CPP subclasses affect their interaction and loading into EVs.

Methods: Representative CPPs from each major category (primary amphiphilic, secondary amphiphilic, and cationic) with different physicochemical properties were synthesized and tagged with fluorescein isothiocyanate (FITC). EVs were collected from HEK293T cells via ultracentrifugation and non-specifically labeled with cell tracker far red (CTFR). EVs were incubated with CPPs for 0.5, 6, or 24 hours and isolated from free peptide via size exclusion chromatography. Samples were then analyzed by single particle flow cytometry to determine the location of CPP on the EV (i.e., EV lumen or outer membrane), by assessing fluorescence labeling with or without trypan blue, fluorescent quencher of FITC.

Results: After 6 hours of incubation, all three types of CPPs showed a significant difference between CTFR+/ FITC+ populations with and without trypan blue, which suggests that most of the CPP was not in the EV lumen and simply associated with the outer EV membrane. However, after 24 hours of incubation, no significant difference was found in CTFR+/ FITC+ populations with and without trypan blue samples for TAT (cationic CPP) and TP10 (primary amphiphilic CPP), which suggests that most of the CPP is located in the EV lumen. However, this was not observed with penetratin (secondary amphiphilic CPP), which suggest that most of the CPP was still located on the outer EV membrane.

Conclusion: Here we demonstrate an assay to assess surface vs lumen loading of cargo to report that the physiochemical properties of CPPs affect their interactions and loading into EVs in a time dependent manner, as well as their promise for loading into EVs for future applications in engineering EVs as therapeutics.

PhD Gang Han

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes, as natural biological nanocarriers with good bio-compatibility and low immunogenicity, show potential as ideal delivery vehicles. However, an affordable generalizable system for efficient loading and targeted delivery of oligonucleotides irrespective of chemistry on exosomes remain lacking. Here, we identified an exosome-binding DNA aptamer (Exosomal Anchor Aptamer-EAA) via SELEX against myotube-derived exosomes immobilized with our proprietary CP05 peptides. EAA shows high binding affinity to exosomes of different origins, and enables efficient loading of different nucleic acid drugs on exosomes. Serum stability of thrombin DNA aptamer inhibitor Nu172 was prolonged by exosome-loading, resulting in increased blood flow after injury in vivo. Importantly, Duchenne Muscular Dystrophy (DMD) phosphorodiamidate morpholino oligomers (PMOs) can be readily loaded on exosomes by incorporating a complementary sequence to EAA (EXOEAA-PMO). Compared to PMO, EXOEAA-PMO elicited significantly greater muscle cell uptake, tissue accumulation, and exon- skipping and dystrophin expression in vitro and in vivo. Systemic administration of EXOEAA-PMO at the PMO dose of 25mg/kg/week for 3 weeks elicited therapeutic levels of dystrophin restoration and significant functional improvements without any detectable adverse effect in mdx mice, indicating the potency of exosomes as viable delivery vehicles for PMOs. Simultaneous loading of PMO via EAA and muscle- targeting peptide via CP05 further enhanced targeted muscle delivery and therapeutic efficacy of PMO in vivo. Altogether, our study demonstrates that EAA enables efficient loading of different nucleic acid drugs on exosomes without conjugation or modification, thus providing an easy and generalizable strategy for loading nucleic acid therapeutics on exosomes and an orthogonal method of surface modifications by combining peptide targeting with oligonucleotide loading.

Ragnar Axel Adolfsson^{Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland}, Erna Jonsdottir^{Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland; Biomedical Center, University of Iceland, Iceland}, Dr. Jens Guðmundur Hjörleifsson^{Department of Biochemistry, Science Institue, University of Iceland}, Dr. Berglind Eva Benediktsdóttir^{Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland; Biomedical Center, University of Iceland, Iceland}

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Triple-negative breast cancer (TNBC) represents a heterogeneous group of malignancies and carries the worst prognosis among breast cancer patients, with limited targeted therapies. Subsets of TNBC exhibit Epidermal Growth Factor Receptor (EGFR) surface expression, opening avenues for targeted interventions. In this context, extracellular vesicles (EVs) have emerged as promising nano-drug delivery candidates due to their inherent stability compared to other lipid-based nanocarriers. Their potential for precise bioengineering enhances the feasibility of achieving specific targeting.

Methods

Suspension-adapted HEK293E (MEXi-HEK293) underwent PEI transfection with a plasmid encoding a novel ligand with high affinity for EGFR, along with GFP for transfection efficacy monitoring. Following puromycin selection, conditioned media from both original and transfected cell lines were collected. EVs were isolated via Strep-Tactin CD81 Fab-TACS® affinity chromatography (Iba-LifeScience) and concentrated using 100kDa ultrafiltration. EVs were characterized using nanoparticle-tracking-analysis (NanoSight-NS300) and capillary western blot (ProteinSimple JESS) for EV protein markers (ALIX, Synthenin-1, Calnexin) and GFP to confirm the generation of engineered EVs. Additionally, an Amplex Red Cholesterol assay (Thermo Fisher Scientific) was conducted to confirm the presence of EVs from the cell lysate (CL).

Results

With a transfection efficacy of 78% and a viability rate of 94%, engineered EVs were obtained from 25ml of media containing 4-8*10^6 cells with a mean particle concentration of 1.73±0.07*10^11 particles/ml (n = 15). These EVs displayed an average size of 140.7±1.6nm with a mode size of 117.4±4.3nm. Positive EV protein markers, ALIX and Synthenin-1, were consistently present in both the engineered and original EVs, as well as the cellular lysate (CL). Importantly, GFP presence was confirmed in both the engineered EVs and CL. The EVs exhibited a significantly higher cholesterol concentration (ug/ml protein) compared to CL (p<0.001), providing conclusive evidence of successful EV isolation.

Summary/Conclusions

These findings affirm the engineered cell line's efficacy in producing EVs with the EGFR ligand and GFP. Ongoing studies will evaluate their binding affinity to low and high EGFR-expressing breast cell lines, aiming to target TNBCs. The primary goal was to assess the viability of using these EVs as potential nanocarriers for therapeutic cargo, demonstrating their potential applicability in addressing challenges in TNBC therapy.

Ms Yeonju Lee, Mr Kyung-Min Kim, Mr Young-Pil Kim

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Despite the superior safety and therapeutic potential of proteins over mRNAs, their delivery into cells and extracellular vesicles (EVs) remains a formidable challenge due to their substantial size and intricate structure. Inspired by cyclic antimicrobial proteins found in diverse natural organisms that combat microbial infections, we designed N-C knotted cyclized proteins conjugated with cationic peptide tags for efficient delivery into cells and EVs. Through a structure-based computational design approach coupled with in silico membrane permeability simulations, we identified cyclized proteins with exceptionally enhanced membrane translocation capabilities. Upon engineering a panel of cyclized enhanced green fluorescent proteins (EGFPs) with various tags as a model using a split intein-mediated process, these proteins exhibited markedly increased permeability in cancer cells, liposomes, and EVs, even at low concentrations compared to their linear counterparts. Additionally, they demonstrated remarkable resistances to enzymatic degradation within living cells. These properties are likely attributed to structural constraints imposed on the proteins by the presence of short functional peptides, preserving their native functions. Consequently, photodynamic therapy using cyclized photoprotein-loaded vesicles in cancer cells showed significantly improved efficacy compared to that mediated by their non-cyclized counterparts. These findings collectively suggest that topologically stable cyclized proteins with tags holds universal applicability for EV-mediated intercellular delivery of therapeutic proteins

Researcher Hsiu-Jung Liao, Dr. Tai-Shan Cheng, Miss Yi-Shan Shen, Mr. Sin-Yu Chen, Professor Chih-Hung Chang, Professor Ly Lee, Professor Chi-Ying Huang

Poster Pitches (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:45 PM - 1:00 PM

Introduction

Osteoarthritis (OA) is a clinical condition marked by joint pain and the deterioration of cartilage encompassing the joints, impacting approximately 500 million adults globally. While specific measures are available to address OA symptoms, they frequently offer limited to moderate effectiveness for a significant number of patients. Hence, it is crucial to develop efficacious regenerative medicine treatments for OA.

Methods

This bioengineering solution comprises two primary components: source control and an engineered production platform. The high-potency mesenchymal stem cells (MSCs) screening and quality control platform leverage next-generation sequencing and big data analysis to establish a biomarker panel evaluating the regenerative potential of clinical-grade MSCs and their derived extracellular vesicles (EVs) as therapeutic agents. Additionally, an engineered EV production platform employs nano-electroporation (NEP) techniques to generate high-potency EVs loaded with therapeutic mRNA.

Results

This study introduces a therapeutic approach that involves delivering SOX9, a critical transcriptional factor in chondrogenesis, mRNA encapsulated in extracellular vesicles (EVs) utilizing allogeneic potent mesenchymal stem cells (MSCs) as a focal point in bioengineering efforts. Using this novel extracellular vesicle-based platform (NEP), we have developed the innovative osteoarthritis (OA) treatment agent “EXOS (EV-SOX9),” which encapsulates SOX9 mRNA in MSC-derived EVs. Our findings indicate a 100-fold increase in the EV production rate and the encapsulation of therapeutic SOX9 mRNA at a level 10,000 times higher than that of naive EVs, demonstrating significantly enhanced capabilities compared to other reported EV loading techniques. Furthermore, we have demonstrated the notable ability of EV-SOX9 to promote chondrocyte regeneration both in vitro and in vivo.

Summary

In conclusion, our bioengineering platform is dedicated to developing a safe and effective approach for treating OA through therapeutic mRNA-encapsulated allogeneic MSC-derived EVs. Additionally, we have successfully introduced clinical-grade EV products, including EV-SOX9, which is currently undergoing a clinical trial for canine OA. The ultimate objective is to position EV-SOX9 as an innovative pharmaceutical for treating OA.

Miss Patricia Wongsodirdjo, Dr Ya Hui Hung, Dr Fazel Shabanpoor, Dr Laura Vella, Dr Rebecca Nisbet

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Neurological disorders affect approximately one billion individuals worldwide and contribute to the 10 leading causes of death globally (WHO). Messenger RNA (mRNA) technology has revolutionised drug delivery in recent years. The advantage of mRNA is that it has versatile applications and an excellent safety profile. One of the main challenges facing central nervous system-targeted mRNA drug development, however, is the limited blood-brain barrier (BBB) permeability of conventional lipid nanoparticle formulations and their ability to trigger an inflammatory response in the brain. This project leverages our existing pre-clinical neurotherapeutics pipeline, our patented BBB-penetrant peptides and expertise in extracellular vesicles (EVs), to generate mRNA-containing EVs for enhanced brain delivery and optimised brain cell translation.

Zitong Yu, Huitao Zhang, PhD Rui Hao, PhD Candidate Shi Hu, Sihui Chen, Professor Hui Yang

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

INTRODUCTION

Small extracellular vesicles (sEVs) are nanometer-sized (30-200 nm) lipid-bilayer vesicles produced by all cell types. They have emerged as promising candidates for therapeutic drug delivery systems. Mounting evidence suggests that intracellular protein dysfunction underlies many diseases. Given their exceptional ability to protect cargo proteins, versatility for in vivo applications, biocompatibility, capability to cross biological barriers, and targeting behavior, sEVs have garnered increasing attention. However, their current applications are primarily restricted to delivering low molecular weight payloads, such as chemotherapeutic drugs and siRNAs. Attempts to genetically or chemically engineer sEVs for carrying biomacromolecules have been largely inefficient and challenging to scale up for clinical trials and commercial manufacturing. Therefore, developing a direct and efficient technology for loading biomacromolecules onto sEVs is imperative for expediting clinical validation, drug production, and driving industrial transformation.

METHODS

We present an exosome nanoengineering platform (ExoNP) for directly and efficiently packaging biomacromolecules into sEVs. Wild-type p53 tumour suppressor protein, a critical intracellular target linked to human cancers, is utilized as the payload. We assess the efficiency of intracellular delivery and endo/lysosomal escape capability of sEVs using confocal laser scanning microscopy. The loading capacity of the ExoNP is quantified by ELISA, and the CCK-8 assay evaluates the ability of the p53 protein delivered by sEVs to inhibit the growth of p53-nulled non-small cell lung cancer H1299 cells.

RESULTS

We have developed a nanofluidic constriction array to facilitate high-throughput sEVs treatment, leveraging a mechanoporation effect. Confocal microscopy and colocalisation analyses have revealed that most fluorescently labelled-sEVs are localised in the cytoplasm, suggesting efficient endocytic uptake and highly efficient endo/lysosomal escape. Furthermore, our quantitative results demonstrate that the ExoNP platform can successfully load p53 proteins into exosomes, achieving milligram quantities within a few minutes using a standard clinical dose of exosomes.

SUMMARY

In conclusion, we have developed a novel nanofluidic constriction-based technique for efficiently packaging biomacromolecules into sEVs for intracellular delivery. This innovative approach enhances the potential of biomacromolecules for intracellular delivery using engineered sEVs. Implementing this technology offers new opportunities to streamline the manufacturing practice of exosome-based therapeutic drugs for clinical applications.

Ph.D Candidate Liouba Le Roux, Ph.D. Adityas Purnianto, Ph.D. Laura Vella, Ph.D. Ya Hui Hung

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Niemann-Pick disease type C1 (NP-C1) is a childhood dementia. An mRNA-based gene therapy that restores the protein deficiency caused by mutations in the NPC1 gene offers a potential curative treatment. To restore neurological function in NP-C1 patients, therapeutic mRNA needs to enter the brain. However, naked mRNA is susceptible to digestion by circulating ribonucleases and incapable of crossing the blood-brain barrier (BBB). Extracellular vehicles (EVs) are natural nano-carriers of biological molecules, such as proteins and nucleic acids. Notably, they have a high biocompatibility with low immunogenicity, making them useful vehicles for ferrying therapeutic payloads in the body. Importantly, EVs have demonstrated the ability to cross the BBB. However, there is currently no robust method for loading mRNA into EVs.

Method

Therefore, we optimized existing techniques such as electroporation, freeze-thaw, sonication, and passive loading to improve the loading efficiency. Using passive loading as a reference control, we tested new modified methods to load NPC1-mRNA into milk-derived EVs.

Results

We found Method C achieved a 6-fold increase in loading efficiency compared with passive loading. However, high variability was observed across the assays. Therefore, we need to further characterize EV properties and optimize the loading method.

Conclusion

Despite the variability, we anticipate further optimization to yield an efficient mRNA loading method for all types of EVs.

Ms Melissa Tan, Dr Brenda Wan Shing Lam, Dr Harwin Sidik, Dr Tenzin Gocha, Dr Ronne Wee Yeh Yeo, Dr Minh TN Le, Dr Waqas Muhammad Usman

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Achieving effective DNA delivery without apparent toxicity remains a challenge. This is primarily due to constraints on vehicles to deliver sizable and multiple payloads to target cells without eliciting immune responses. While red blood cell-derived extracellular vesicles (RBCEVs) have demonstrated safe and efficient delivery of mRNA, their capability to deliver DNA payloads, notably larger constructs, remains unknown. This study investigates the potential of RBCEVs in delivering DNA constructs to specific cell types, while aiming for sustained transgene expression.

The size and polydispersity of RBCEVs loaded with different constructs were characterized by nanoparticle tracking analysis and loading efficiency determined by gel densitometry. Luciferase plasmid-loaded RBCEVs were delivered to Balb/c mice and sustained expression monitored by in-vivo imaging system. To express secreted proteins in hepatocytes, RBCEVs loaded with LSP-hFactor-IX plasmid were delivered systemically to mice. To explore the potential of RBCEVs to deliver two constructs simultaneously, RBCEVs co-loaded with light and heavy chain Herceptin plasmids were delivered to mice. To challenge the capacity of RBCEVs to carry different-sized plasmids, 3-32 kb GFP plasmids were loaded into RBCEVs and delivered to 293T cells.

The in vivo administration of luciferase plasmid-loaded RBCEVs led to sustained luciferase expression over a span of 6 months. Mice injected with a single bolus of Factor IX plasmid-loaded RBCEVs produced therapeutic levels of Factor IX protein in plasma, which corresponds to 6% of Factor IX levels in normal human plasma. Furthermore, RBCEVs showcase the ability to simultaneously encapsulate and transport two plasmids. The incorporation of heavy and light chain constructs into RBCEVs resulted in successful DNA delivery in mice, leading to sustained expression of Herceptin antibody in the bloodstream. RBCEVs also exhibit the capability to efficiently transport larger-sized plasmids to target cells without observable cytotoxicity.

The RBCEV drug delivery platform holds significant potential for safely and efficiently delivering nucleic acids for gene therapy. RBCEVs derived from mature RBCs are devoid of nucleic acids, mitigating the risk of horizontal gene transfer. Their substantial packing capacity allows for the accommodation of large transgenes and regulatory sequences, enhancing gene expression. RBCEVs have potential to address gene ablations, warranting investigation for clinical applications.

Professor Hongbo Chen, Xingyu Lu, Dr Fang Cheng

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Heart transplantation is intrinsically susceptible to ischemia-reperfusion injuries (IRI), which commonly result in immune cells-mediated allogeneic rejection. However, current approaches to relieve organ transplantation rejection mainly are simply broad immunosuppressive drugs targeting T and B cells, the long-term use of which increase the risks of infection, cancer and other serious disorders. Therefore, new therapies to alleviate sequential process of early graft rejection, especially those that could be combined with conventional immunosuppressive drugs, are urgently needed. Herein, we detail a reactive oxygen species (ROS)-responsive multifunctional fusion extracellular nanovesicles that can target heart graft sites in order to mitigate acute transplant rejection.

Methods

(1) In vitro screening and functional identification of plant-derived nanoparticles with anti-inflammatory and anti-oxidant activity;

(2) The construction and functional validation of MSC-derived extracellular nanovesicles expressing mCalreticulin (an “eat me” signaling);

(3) Construction and functional exploration of rapamycin-loaded fusion nanovesicles (FNVs@RAPA);

(4) Synthesis of ROS responsive Tetraacetated N-azide acetyl-D-mannosamine (ROS-N3), and then bioluminescence labeling studies in mouse heart transplantation model

(5) MASSON/HE/ Flow cytometry assays the efficacy of DBCO-FNVs@RAPA in mouse heart transplantation model.

Results

(1) We first discovered that the Exocarpium Citri grandis-derived extracellular nanovesicles (ENVs) possess notable anti-inflammatory and anti-oxidant properties, suitable for protecting myocardial tissue from oxidative damage.

(2) Secondly, we developed mesenchymal stem cell (MSC)-derived extracellular nanovesicles expressing mCalreticulin (an “eat me” signaling), fused with ENVs and loaded with rapamycin (RAPA) to form novel fusion nanovesicles (FNVs@RAPA) for diminishing the conversion of innate to adaptive immunity.

(3) To further enhance the targeting of FNVs@RAPA, we have also developed ROS-N3, a novel ROS-responsive derivative of established Ac4ManAz which can effectively label the graft. This is achieved through a sequence of environmentally-specific deprotection, substrate incorporation, and bioorthogonally azido-alkyne “click” reaction with dibenzylcyclooctyne-NHS-labelled FNVs@RAPA (DBCO-FNVs@RAPA).

(4) DBCO-FNVs@RAPA are able to achieve both the protection of myocardial tissues against oxidative IRI and promote the polarization to Ly6C- immunosuppressive macrophages to prevent subsequent immune-mediated rejection,

Conclusion

Taken together, this multifunctional fusion extracellular nanovesicle drug delivery system, which targets the sequential process of early graft rejection, exhibits promise as an immunosuppressant in organ transplantation.

Dr. Dokyung Jung, Sanghee Shin, Dr. Sung-Min Kang, Inseong Jung, Suyeon Ryu, Soojeong Noh, Dr. Sung-Jin Choi, Jongwon Jeong, Beom Yong Lee, Kwang-Soo Kim, Dr. Christine Seulki Kim, Dr. Jong Hyuk Yoon, Dr. Chan-Hyeong Lee, Dr. Felicitas Bucher, Dr. Yong-Nyun Kim, Prof. Sin-Hyeog Im, Dr. Byoung-Joon Song, Prof. Kyungmoo Yea, Prof. Moon-Chang Baek

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

T cell-derived small extracellular vesicles (sEVs) exhibit anti-cancer effects. However, their anti-cancer potential should be reinforced to enhance clinical applicability. Herein, we generated interleukin-2-tethered sEVs (IL2-sEVs) from engineered Jurkat T cells expressing IL2 at the plasma membrane via a flexible linker to induce an autocrine effect. IL2-sEVs increased the anti-cancer ability of CD8+ T cells without affecting regulatory T (Treg) cells and down-regulated cellular and exosomal PD-L1 expression in melanoma cells, causing their increased sensitivity to CD8+ T cell-mediated cytotoxicity. Its effect on CD8+ T and melanoma cells was mediated by several IL2-sEV-resident microRNAs (miRNAs), whose expressions were upregulated by the autocrine effects of IL2. Among the miRNAs, miR-181a-3p and miR-223-3p notably reduced the PD-L1 protein levels in melanoma cells. Interestingly, miR-181a-3p increased the activity of CD8+ T cells while suppressing Treg cell activity. IL2-sEVs inhibited tumour progression in melanoma-bearing immunocompetent mice, but not in immunodeficient mice. The combination of IL2-sEVs and existing anti-cancer drugs significantly improved anti-cancer efficacy by decreasing PD-L1 expression in vivo. Thus, IL2-sEVs are potential cancer immunotherapeutic agents that regulate both immune and cancer cells by reprogramming miRNA levels.

Bin Zeng, Duan Li

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Background:

The global number of patients with knee osteoarthritis (KOA) has exceeded 250 million. Due to the difficulty of conventional drugs penetrating dense, non-vascular, and negatively charged cartilage matrix, there is currently no effective treatment for osteoarthritis. Extracellular vesicles(EVs), as carriers mediating communication between cells and tissues, can efficiently penetrate the cartilage barrier and have unique advantages in drug delivery. This study used genetically modified extracellular vesicles to deliver CXCR7 (CXCR7-EVs) protein and investigated the therapeutic effect and mechanism of CXCR7-EVs on OA.

Method:

Genetic engineering was used to construct stable overexpression of CXCR7 in HEK293 cells; Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were used to characterize the size, number, and morphology of CXCR7-EVs; Transcriptome analysis, RT qPCR and WB analysis were used to investigate the mechanism of action of CXCR7-EVs on OA chondrocytes; and fluorescence imaging is used to evaluate the uptake efficacy of CXCR7-EVs in chondrocytes; Finally, the therapeutic effect of CXCR7-EVs on osteoarthritis was evaluated through in vitro and animal models.

Result:

The NTA results showed that the average particle size of CXCR7-EVs was 178.7 nm (± 3.0 nm), and the number of nanoparticles was 1.44 × 10 ^ 10 particles/ml, TEM shows that CXCR7 EVs exhibit a classic disc-shaped morphology; Fluorescence imaging shows that CXCR7-EVs can be effectively uptake by chondrocytes; The transcriptome analysis results indicate that CXCR7-EVs can exert cartilage protection by significantly increasing the expression of chi3l1 in chondrocytes. The results of WB, RT qPCR, and histological evaluation indicate that CXCR7-EVs can effectively alleviate the inflammatory response of chondrocytes after SDF1 treatment, promote cartilage tissue repair and regeneration in rat models of OA.

Conclusion:

Our study suggests that CXCR7-EVs can alleviate OA chondrocyte inflammation by upregulating Chil3l1. Intra-articular injection of overexpressed CXCR7-EVs can efficiently penetrate cartilage matrix and significantly promote cartilage repair in KOA rat models. The research results suggest that CXCR7-EVs can be a promising treatment method for osteoarthritis.

Ms Hui Sun Leong, Dr Shen Yon Toh, Ms Fui Teen Chong, Ms Mengjie Ren, Prof N. Gopalakrishna Iyer

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Targeting wild-type EGFR using tyrosine kinase inhibitors (TKIs) never achieve its purported success in most cancers, which are largely EGFR-dependent. We had previously shown that a novel lncRNA which results in over-expression of an EGFR splice-variant, isoform D (IsoD), can affect the intrinsic sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKI). We also demonstrated that this protein is secreted in extracellular vesicles (EVs) in TKI-sensitive patient-derived cultures, and the intrinsic sensitivity in EGFR-TKI sensitive cells was transferable to resistant lines, through these IsoD-expressing EV. Hence, this study aims to examine the exosomal EGFR IsoD as a co-drug to sensitise EGFR-driven cancers to EGFR-TKI targeting.

Methods

Full-length IsoD was cloned into a CMV-driven construct (pBob vector), which was then used as lentiviral particles to infect HEK293T. Stable IsoD expressing cell lines were selected with Zeocin, and 293T infected with the empty pBob vectors were used as controls (293T-pBob). The 293T-IsoD lines expressed high levels of IsoD which was packaged and secreted in exosomal fractions derived from these lines. The exosomes were purified using 50 kDa Amicon® Ultra-15 centrifugal filter units and transferred onto target resistant EGFR-driven cancer lines and co-treated with varying EGFR-TKIs. Cell viability will be assessed using CellTitre-Glo® Luminescent Assay. For consistency, we have validated the EV size and purity using Zetaview. IC50 value calculation in all experiments was based on dose-response curves using 10 different drug doses in each circumstance.

Results

By transferring purified exosomes from IsoD-overexpressing 293T cells, it was able to re-sensitise a panel of TKI-resistant cancer cells to EGFR-TKIs treatment. EGFR IsoD in the EV is necessary and sufficient to transfer the phenotype in multiple cancers and drugs.

Conclusion

Taken together, these results collectively support the sensitizing phenotype conferred by transferred exosomes enriched for EGFR IsoD. We propose a novel therapeutic strategy using EVs-containing EGFR IsoD as a co-drug to expand the use of TKI-therapy to EGFR-driven cancers.

Ms. Reese Wunsche, Dr. Morteza Jeyhani, Mr. Boyang Su, Dr. Hon Sing Leong, Dr. Scott Tsai

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

The transformative impact and therapeutic potential of extracellular vesicles (EVs) declines in vivo due to their rapid clearance and short half life [1]. Extracellular vesicles are lipid membrane-bound particles secreted into the extracellular environment that harbour lipids, proteins and nucleic acids for intercellular communication [2]. While there have been significant advancements in understanding the applications of EV based therapeutics, there remains a gap in the literature for an effective delivery system [3]. Current approaches face limitations such as poor EV stability, low retention in the target area and an uncontrolled EV release, which impedes the translation of these therapies from bench to bedside. Alginate hydrogel encapsulation nanotechnology facilitates EV protection during in vivo delivery, along with increased retention [4]. This study enzymatically degrades the alginate hydrogel EV scaffold with alginate lyase to achieve a controlled EV cargo delivery, as alginate lyase cleaves the covalent bonds in the alginate polymer chains [5]. By uncovering specific EV release profiles using alginate lyase-treated hydrogels, I aim to provide valuable insights for optimizing hydrogel formulations, and contribute to the advancements of EV-based therapies. By enhancing the delivery system, this strategy helps facilitate their integration into clinical applications.

Dr. Diem Nguyen, Thieu Nguyen, Nhan Vo, Dr. Lan N Tu

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Small extracellular vesicles (sEVs) are promising drug delivery vehicles. However, efficient and consistent drug loading into sEVs remains a challenge that limits their application in drug delivery. In this study, we proposed a new method to efficiently load ultrapure sEVs with cytotoxic drugs and significantly enhance their potency.

Methods: Wild-type (WT) and dual-peptide displaying (p32/EPCAM) human embryonic kidney cells (HEK293F) were grown in batch-refeed mode in a stirred-tank bioreactor. sEVs from conditioned media were purified through a quality-controlled pipeline involving tangential flow filtration, sucrose-cushion ultracentrifugation followed by bind-elute size-exclusion chromatography. The ultra-pure sEVs were loaded with doxorubicin (Dox) by different methods including incubation, sonication-incubation with and without the permeability enhancer Tween-20. The triple-negative breast cancer cell line, MDA-MB-231, was used for treatment with either free Dox, PEGylated liposomal Dox, Dox-loaded WT sEVs or Dox-loaded p32/EPCAM sEVs. The potency of Dox-loaded sEVs was compared with free Dox and PEGylated liposomal Dox using the cytotoxicity assay CCK-8.

Results: Our purification pipeline consistently achieved sEV purity of at least 1x10¹¹ particles/µg. The sEVs remained round and intact with an average size of 46 nm ± 10nm, were CD9-, CD81- and CD63- positive and free of biological contaminants. Using only the incubation method, we found that the Dox loading efficiency was inversely proportional to the purity of sEVs. A 20-fold increase in sEV purity resulted in a ∼87% decrease in the loading efficiency. For the ultrapure sEVs (minimum 1x10¹¹ particles/µg), the most optimal protocol to load Dox was using sonication-incubation with 0.2% Tween-20 that significantly improved the loading efficiency to 21.4% compared to incubation (4.9%) or sonication-incubation without Tween-20 (3.3%). The values of IC50 (half maximal inhibitory concentration) of free Dox, PEGylated liposomal Dox, Dox-loaded WT sEvs and Dox-loaded p32/EPCAM sEVs were 1.07, 2.20, 0.56 and 0.37 ng/µL respectively.

Conclusions: This study demonstrated an optimized protocol to improve drug loading into ultrapure sEVs and showed that sEVs could be a better nanocarrier than liposomes to enhance potency of cytotoxic drugs.

Ph.D. student Shota Shinagawa, Technical Staff Tamiko Minamisawa, Technical Staff Saki Matsumoto, Project Leader Kazuma Kiyotani, Kiyotaka Shiba

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction

One of the unresolved challenges in Extracellular Vesicles (EVs) research is the limited understanding of the numerous generation pathways and the specific biological functions involved by EV subtypes. This gap in knowledge complicates the engineering of EVs expressing specific epitopes for cellular immunity induction. Our study introduces the Craftgen@EV system, which utilizes a library of proteins with diverse physicochemical properties to select EVs that activate CD8+ T cells independently of antigen-presenting cells (APCs).

2) Methods

We engineered a library of 64 clones using the MolCraft system to express the HLA-A*24:02-specific SARS-CoV-2 S448-456 epitope peptide within various contexts, appending acylation and prenylation motifs, along with signal sequences, to their N and C termini. These clones were transfected into HEK293T HLA-A*24:02 cells to identify those that could specifically activate CD8+ T cells.

3) Results

Our observations support the premise that proteins with different physicochemical properties are processed through distinct intracellular pathways, as evidenced by full-length artificial proteins localizing to large and small EV fractions, depending on the clone. Clones residing in the large EV fraction with N-terminal acylation motifs exhibited significant activation potential for CD8+ T cells, even without APCs.

4) Summary/Conclusion

The incomplete understanding of EV heterogeneity underscores the potential of synthetic approaches, like our Craftgen@EV, for functionally screening and loading specific cargos onto EV subtypes. The discovery that EVs involved in HLA and T cell receptor interactions tend to be large EVs provides crucial insights for future research in this field.

Mr. Minghao Sun, Associate Director Mafalda Cacciottolo, Principal Scientist Yujia Li, Senior Scientist Mahrou Sadri, Senior Scientist Michael LeClaire, Research Associate David Tran, Chief Scientific Officer Kristi Elliott

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked disease affecting both skeletal and cardiac muscle with severely reduced life expectancy. The predominant strategy for treating DMD is employment of antisense oligonucleotides (ASOs) to exclude exons resulting in DMD proteins with partially restored function. In the last 6 years, 4 exon skippers have been approved by FDA. The main challenge of antisense drugs is their limited efficacy due to poor delivery to target tissues. It has been estimated that <1% of the ASO reaches the correct cellular compartment therefore limiting restoration of function. To overcome the targeting limitation, a muscle targeting moiety was engineered on the surface of exosomes using StealthXTM technology. Initially, the exosomes carrying the targeting moiety were stained with a far-red fluorescence dye and I.V. injected into wild-type Balb/c female mice with results showing that labeled exosomes were detected in the lower limbs 24 hours post-injection. Notably, the exosomes carrying the muscle targeting moiety were not detected in any other tissues except for the expected clearance pathway (kidney and liver). To further evaluate the possibility of using exosomes as a targeted delivery tool, a fluorescence labeled ASO was loaded into the exosomes carrying the muscle targeting moiety and I.V. injected into mice. Intriguingly, the labeled ASO was also detected in the lower limbs. Further dosing study suggested that repeated dosing with IV injection induced better targeting in muscle cells in the lower limb and accumulation of targeting effect observed after 3 doses. In general, Data collected here strongly suggests that StealthXTM technology developed at Capricor could potentially pair with current therapies for efficient delivery to muscle, improving restoration of function.

Dr. Shao-chih Chiu, Dr. Ming-You Shie, Dr. Shi-Wei Huang, Dr. Chih-Ming Pan, Dr. Der-Yang Cho

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction: Glioblastoma multiforme (GBM) is extremely malignant, mainly due to O6- methylguanine DNA Methyltransferase (MGMT). This gene is the main reason for the drug resistance of cancer cells. Exosomes are currently considered to be the most prospective and clinically applicable medical tools. Due to their high affinity, they are rich in protein and nucleic acid and have a nanometer size, which is very suitable for treatment as a drug carrier.

2) Methods: This study focuses on the design and functional verification of engineered targeted exosomes expressing nanobodies with affinity, specificity, and functional identification in a genetic engineering mode. It achieves a target-specific effect on the exocytic membrane's outer side. In addition, the engineering exosomes carry nucleic acid drugs/chemotherapy drugs it at the same time, and the nucleic acid drugs carried by exosomes reduce the drug resistance of GBM cells to TMZ by regulating the expression of MGMT.

3) Results: First, it is important to evaluate the efficiency of drug absorption using the exosome uptake assay. GBM cell line U87 and primary astrocyte cells were treated with engineering exosomes for different time points. It could be noted that there was increased uptake of engineering exosomes in the U87 as compared to astrocytes, thus indicating that engineering exosomes had target specificity for GBM cells. We further conducted a wavelength analysis to confirm the successful loading of TMZ into engineering exosomes. TMZ was noted to have a peak at 320 nm, whilst no peaks were noted in the engineering exosomes group. However, it could be seen that TMZ@engineering exosomes had slight elevations at the range of 320 nm, indicating that TMZ was successfully loaded into engineering exosomes.

4) Conclusion: These results indicated that engineering exosomes could be modified and further loaded with TMZ and increase its potential for future clinical applications as a target therapy.

Ms Rakshanya Sekar, Dr Yvette Wooff, Dr Adrian Cioanca, Associate Professor Riccardo Natoli

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Cells communicate by transferring molecular cargo using nanosized delivery vehicles called extracellular vesicles (EV). As we age, there is loss of EV-mediated communication, leading to the progression of neurological pathologies, including retinal degenerations such as Age-related Macular Degeneration (AMD). We hypothesise that if we can supplement the retina with essential cargo lost during degeneration, we can restore cellular communication and slow the progression of AMD. We propose to use autologous EV from red blood cells (RBC-EV) as delivery vehicles of these essential retinal cargo and develop a novel gene therapy for AMD.

Methods: Small-medium EV were isolated from RBC using differential ultracentrifugation and characterised using nanoparticle tracking analysis (NanoSight NS300), electron microscopy (CryoEM) and western blotting (Alix,TSG101).

RBC-EV were labelled with SYTO™ RNASelect™ and transfected in-vitro into immortalised cell lines of 4 key retinal cell types. Safety and uptake efficiency of RBC-EV was analysed using Incucyte® Live cell imaging, flow cytometry and CellTox™ Cytotoxicity Assay. SYTO-labelled RBC-EV were administered both locally and systemically in murine model of retinal degeneration. In-vivo safety and uptake efficacy was investigated using electroretinography (ERG), optical coherence tomography (OCT), live imaging (IVIS) and histology.

Therapeutic potential of native RBC-EV cargo was investigated using RNA sequencing and proteomic analysis.

Results: In-vitro, SYTO-labelled RBC-EV were safely and efficiently uptaken by all 4 retinal cell types with upto 90% transfection efficiency within 24 hours. Cytotoxicity assay showed no significant difference between RBC-EV treatment (1 x 10^9 EV/10^6 cells) and PBS control. In-vivo, both local and systemic administration of RBC-EV showed no significant functional or histological difference in dim-reared animals, but significant protection in murine model of retinal degeneration as recorded in both functional readouts (ERG) and histological analysis. Further, labelled RBC-EV were uptaken by cells in ganglion cell layer, inner nuclear layer, and external limiting membrane of the retina within 6 hours of injection.

Conclusion: This work not only supports use of RBC-EV as therapeutic delivery agents but shows their efficacy in native state as therapeutics. Further, RBC-EV loaded with healthy retinal cargo could be administered in AMD models to assess the therapeutic efficacy in restoring cell communication.

Phd Candidate Jonathan Lozano, Dr. Jarmon G Lees, Dr. Alin Rai, Dr. Kyah Grigolon, Dr. Helen Kiriazis, Ren Jie Phang, Jonathon Cross, Haoyun Fang, Dr. Daniel Donner, Shiang Y Lim, Dr. David W. Greening

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Background: Accumulating reports indicate that stem cell-originating extracellular vesicles (EVs) provide a potential strategy for cardiac tissue repair, including ischaemia-reperfusion injury. However, the limitations of natural EVs, such as scalable generation, and unknown biological mechanisms, impair their clinical application. Here we demonstrate that stem cell-derived nanovesicles (scNVs) offer a promising pathway towards customisable, reproducible, rapid, and scalable therapy for cardiac repair.

Methods: Herein, scNVs were rapidly generated from human-induced pluripotent stem cells through serial membrane-based extrusion strategy in large quantities (yield 900× natural EVs). The function and regulatory mechanism of scNVs in cardiac repair was explored using cell-based (2D), human cardiac organoid (3D), and murine model of ischaemia-reperfusion injury (IRI) to simulate myocardial infarction. Moreover, we customise scNVs cargo by engineering the parental cells and packaging key factors for tissue repair.

Results: scNVs isolated using density-gradient separation (1.13 g/mL) are spherical in shape and morphologically intact (∼100 nm) and readily internalised by human cardiomyocytes, primary cardiac fibroblasts, and endothelial cells. NVs captured the dynamic proteome of parental cells and include pluripotency markers (LIN28A, OCT4) and regulators of cardiac repair processes, including those identified on natural EVs. Functionally, single-dose NVs significantly promoted tubule formation of endothelial cells (angiogenesis) (p<0.05) and survival of cardiomyocytes exposed to low oxygen conditions (hypoxia)(p<0.0001), as well as attenuate activation of cardiac fibroblasts (p<0.0001). In human cardiac organoids (IRI), NVs preserve overall contractility function (p<0.05) and attenuated cardiac troponin release, a hallmark of cell death in the heart. In vivo, NVs regulate cardiac cellularity (cardiomyocytes, fibroblasts, and endothelial cells) and injury size. Quantitative proteome profiling of target cell, organoid and tissue following NV, treatment revealed their capacity to remodel intracellular signalling pathways including pro-survival network (MDH2, LRPPRC, NIPSNAP1), tissue repair/homeostasis (HSP70, CYFIP1), and cardiac function (XIRP1, MYH6, CTNNA1). Ontology analysis revealed that NVs regulate unfolded protein response and metabolism for treated cells to contend with the damage.

Discussion and conclusion: In summary, this study showcases a scalable approach to generating functional NVs, highlights their multimodal therapeutic potential, avenues for vesicle cargo customisation, and identifies key regulatory players and complex signalling involved in cell reprogramming for cardiac repair.

Ph.d Yaoyao Lu, Research assistant Nathalie Majeau, Ph.D Camile Bouchard, Professor Jacques-P Tremblay

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Duchenne muscular dystrophy (DMD) is a genetic disease that happens on the dystrophin gene, which abolishes the production of dystrophin in muscle. Muscles without dystrophin are more sensitive to damage, resulting in progressive loss of muscle tissue and function. The CRISPR-Cas9 system is a promising technology that revolutionized genome editing applications for the potential treatment of a variety of genetic diseases. However, efficiently delivering the CRISPR-Cas9 components to the target organ or cell remains a significant challenge. Extracellular vesicles (EVs) are endogenous membranous particles secreted spontaneously by all cells. They are key actors in cell-to-cell communication, allowing the exchange of molecules such as proteins, lipids, and RNAs to induce functional changes in the recipient cells. In our study, we loaded the components of CRISPR/Cas9 into mouse serum EVs coated with muscle-specific peptides to treat mdx mice, aiming to restore the dystrophin expression in vivo.

Methods:

We have purified EVs from mouse serum through size-exclusion chromatography. Using a protein transfectant reagent CRISPRMAX, we loaded these EVs with CRISPR ribonucleoproteins (RNPs) consisting of SpCas9 protein and single guide RNAs (gRNAs). The EVs were also subsequently incubated with different muscle-targeting peptides. The EVs carrying gRNAs targeting introns 22 and 24 of the dystrophin gene were injected into the TA muscles of mdx mice having a non-sense mutation in exon 23.

Results:

The DNA and protein extracted from EVs treated mdx mice muscle had the intended deletion of exons 23 and 24, allowing dystrophin expression in muscle fibers. The PCR result showed that the non-dense mutation point of the dystrophin gene was successfully removed by engineered EVs.The immunohistochemistry result showed that the number of positive muscle fibres was significantly higher when the EVs were modified with muscle-targeting peptides.

Conclusion:

Our study successfully used EVs to deliver the components of CRISPR/Cas9 to restore the dystrophin gene in vivo. This method opens new opportunities for the safe delivery of gene editing components to treat DMD.

PhD student Julia Anna Rädler, Giulia Corso, Antje Zickler, Noriyasu Kamei, Wenyi Zheng, Dhanu Gupta, Samir El Andaloussi

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Owing to their ability to target virtually any RNA within a cell, RNA interference (RNAi)-based drugs offer an immense therapeutic potential. However, the hydrophilic nature and low bioavailability of naked RNAi agents require a delivery strategy. Current strategies tend to suffer from toxic side effects of excipients and predominant hepatic accumulation. Here, we propose the use of extracellular vesicles (EVs) due to their immune privilege and ability to cross biological barriers. By applying state-of-the-art engineering approaches, we produced EVs harboring high amounts of short-hairpin RNA (shRNA) for potent delivery to recipient cells.

EV-producing cells were genetically modified to express (1) an shRNA of interest, (2) AGO2 as an RNA binding protein fused to an EV sorting domain, and (3) a fusogenic viral glycoprotein to ensure potent delivery to the cytosol of recipient cells. EVs were isolated from the conditioned media of producer cells and purified by size exclusion chromatography. shRNA copies in EVs were quantified by RT-qPCR and knockdown efficiency was evaluated on mRNA (RT-qPCR) and protein (Western blot) levels in recipient cells in vitro.

Here, we show that engineered EVs are potent delivery modalities for shRNA. Applying our approach to load shRNA targeting Gapdh (shGapdh), we detected up to 1 shRNA molecule per 2 EVs. Strikingly, our loading strategy boosted the number of shGapdh molecules per EV by 291-fold in comparison with stochastic loading. In an in vitro setting, these EVs achieved an average IC50 of 1.79 pM and Gapdh knockdown up to 95% on mRNA and 88% on protein levels thereby competing with state-of-the-art liposome-based transfection. Similarly, but with a therapeutic aim, we reduced the expression of neuroinflammation-related osteopontin (Spp1) by 90% in vitro, which demonstrates the versatility of our approach. Currently, we are evaluating engineered EVs for shRNA delivery in vivo.

Engineered EVs present a promising option for the delivery of RNAi agents. Importantly, we are confident that our strategy can be repurposed to deliver other small RNA therapeutics including siRNAs. Additionally, considering the modifiable nature of EVs, future studies will achieve cell- or tissue-specific targeting of EV-encapsulated RNAi-based drugs thereby facilitating therapeutic applications beyond the liver.

Doc. Pingping Wang¹, Professor Hang Zou¹, Prof. Lei Zheng¹

¹Department Of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Wounds with drug-resistant bacterial infection and impaired healing are recognized as preeminent threats to public health due to limited treatment strategies and severe clinical consequences, including amputation and death. Extracellular vesicles (EVs) from adipose tissue have been proposed as a possible solution to the current lack of therapeutic interventions for wound repair and skin regeneration, but it can not deal with the situation caused by drug-resistant bacterial infection. Herein, an aggregation-induced emission (AIE)-active photosensitizers with various reactive oxygen species (ROS)-generating efficiencies is synthesized and engineered adipose-derived EV/AIEgen hybrid nano-vesicles(EVA) are first obtained.

Methods

The EVA was characterized by Nanoparticle Tracking Analysis(NTA), scanning electron microscope, small animal imaging system in vivo. The biocompatibility of the EVA was investigated by blood biochemical safety evaluation experiments and cell experiments. Fluorescence confocal microscopy was used to investigate the ability of the EVA to visualizing bacteria. The antibacterial activity of the EVA was evaluated by its inhibition of bacterial growth in vitro and in vivo. To evaluate the effect of the EVA on wound repair, the system was applied to the mouse drug-resistant bacteria-infected full-thickness skin excisional model, the injury sites were observed and photographed at different time points to calculate the wound healing rate. Hematoxylin-eosin, massons trichrome, immunofluorescence stainings were performed to explore the effect of skin repair.

Results

EVA had a high binding affinity with the bacteria, and bright red fluorescence was observed on the surface of the bacteria and display distinctive photodynamic antibacterial effects under white light irradiation. In vitro and in vivo antibacterial experiment showed that it could effectively inhibit the growth of Gram-positive Staphylococcus aureus and drug-resistant MRSA. Histological results showed increased neovascularization, reduced wound scar, and more sebaceous gland and hair follicles formation, suggesting that the EVA could promotes wound closure, accelerates wound collagen deposition, and completely improves the microenvironment of infected wound regeneration.

Conclusions

This study demonstrates an effective novel therapy for drug-resistant bacterial infected wounds and providing a potential solution for the treatment of related diseases.

Dr. Ikuhiko Nakase¹, Kenta Morimoto¹, Jojiro Ishitobi¹, Dr. Kosuke Noguchi¹, Ryoichi Kira¹, Dr. Tomoka Takatani-Nakase², Dr. Ikuo Fujii¹, Dr. Shiroh Futaki³, Dr. Masamitsu Kanada⁴

¹Graduate school of Science, Osaka Metropolitan University, Sakai, Japan, ²School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan, ³Institute for Chemical Research, Kyoto University, Uji, Japan, ⁴Department of Pharmacology and Toxicology, Michigan State University, East Lansing, USA

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Microvesicles (MVs) are large extracellular vesicles (100-1,000 nm in a diameter) that bud from the plasma membrane and encapsulate bioactive molecules including genes and enzymes. In addition to elucidating the mechanisms of cell-to-cell communication, their applications as drug delivery carriers have been studied. In this research, we focus on macropinocytosis (actin-dependent cellular uptake pathway with surface membrane ruffles and large endocytic vacuoles larger than 1 µm in a diameter) for promoting internalization of the MVs. We also evaluated effects of octaarginine (R8), which is one of cell-penetrating peptides (CPPs) and induces macropinocytosis, on cellular uptake of the MVs by modification of the peptides.

We examined the effects of macropinocytosis induction on cellular MVs uptake. Activation of epidermal growth factor receptor (EGFR) on A431 cells for macropinocytosis induction resulted in enhanced cellular uptake of the MVs, suggesting critical involvement of cancer-associated macropinocytotic receptors and their activation in cellular MVs uptake and intercellular communication

Furthermore, modification of the R8 peptides on membrane of the MVs via stearyl groups (R8-MVs) markedly increased the internalization by various types of cells. Under the experimental conditions, the R8-MVs effectively enhanced cellular uptake of macropinocytosis marker, FITC-dextran (70 kDa), and uptake of the R8-MVs was decreased by treatment of 5-(N-ethyl-N-isopropyl)-amiloride, which is one of macropinocytosis inhibitors. Our developed experimental technique also achieved intracellular delivery of crucmin derivatives leading to anticancer biological activity.

We here successfully found that macropinocytosis is crucial for enhancing cellular uptake of MVs, and modification of the R8 peptides significantly increases MVs-based intracellular delivery. The fundamental findings and experimental techniques will contribute for further developing intracellular delivery system based on cellular membrane vesicles for future therapy.

Laboratory Director Gibeom Kim¹, Senior Researcher Inkyu Lee^1,3, CEO Gi-hoon Nam^1,2, Professor In-San Kim^3,4

¹Department of Research and Development, SHIFTBIO INC., Seoul, South Korea, ²Department of Biochemistry & Molecular Biology, Korea University College of Medicine, Seoul, South Korea, ³KU-KIST Graduate School of Converging Science and Technology. Korea University, Seoul, South Korea, ⁴Chemical and Biological Integrative Research Center, Korea Institute of Science and Technology, Seoul, South Korea

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

This study delves into the novel engineering of Extracellular vesicles (EVs), introducing an advanced EV sorting motif (ESM) that revolutionizes the incorporation of natural ligands and cargo molecules onto EVs. We discovered critical amino acid sequences in the transmembrane domain (TMD) of natural ligands essential for EV sorting, leading to the creation of an efficient ESM scaffold that outperforms existing technologies. Additionally, we demonstrate the adaptability of ESM with examples of its application in membrane proteins, secretory proteins, transcription factors, and nucleic acids.

Methods:

Our methodology included assessing EV cargo sorting efficiency through Western blot (WB), JESS system, and ELISA for expression levels. Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS) were used for EV characterization, alongside WB for EV/non-EV markers and Cryo-Transmission Electron Microscopy (Cryo-TEM) for nanoparticle analysis. AI-based in-silico analysis facilitated the understanding of the ESM cargo sorting mechanism, and specific bioassays were developed to evaluate the efficacy of multiplex cargo-loaded EVs.

Results:

In our study, we first elucidated the pivotal role of TMD in natural EV-associated ligands for EV sorting, surpassing the roles of ecto/cytoplasmic domains, and crafted a highly efficient scaffold by random-mutating these TMDs. Secondly, we highlighted key amino acid sequence features essential for EV cargo sorting, termed ESM. Thirdly, the binding sequences of ESM and their partners were identified via AI-based in-silico studies, and we further elucidated the sorting mechanism of ESM through interactions with CD9 and CD81. Finally, we applied ESM technology to engineer EVs for multiplex cargo loading of proteins and nucleic acids, demonstrating its potential to target previously undruggable pathways.

Conclusion:

The application of the ESM technology within the cell-free nanotherapy platform technologies mark a significant contribution to the field of cell and gene therapeutics. This research not only emphasizes the potential of engineered EVs in redefining the landscape of drug delivery and disease treatment but also reaffirms the importance of innovative approaches in advancing medical science. The specificity, versatility, and innovative application of the ESM platform allow for the development of sophisticated therapeutic strategies, marking a substantial progression in the field of targeted drug delivery.

Dr. Lisi Luo, Dr. Huijie Yang, Dr. Junfeng Huang, Dr. Shiyue Li

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Airway basal stem cell-derived extracellular vesicles: a promising strategy for fibroblasts regulation

Introduction

Airway or interstitial fibrosis represents common and important pathological changes in respiratory diseases, and its treatment remains a challenge. Airway basal stem cell (BSC) and fibroblasts serve critical roles in tissue repair and regeneration. Recent reports found that p16INK4a+ fibroblasts, acting as resident tissue-sentinels, contribute to these processes and may be involved in crosstalk with BSC. This study aimed to identify human basal stem cell-derived extracellular vesicles (BSC-EVs) and explore the dosage effect on fibroblasts.

Methods

Airway BSC was obtained by bronchoscopic brushing and differential centrifugation. Approximately 3×107 BSC was seeded onto a 5-layer cell factory with the exosome-depleted medium. Culture media was harvested for isolating EVs by ultracentrifugation and BSC-EVs presence was confirmed. We performed collagen contraction ability assay, senescence-associated β-galactosidase (SA-β-gal) staining assays, and western blot to explore the regulation of BSC-EVs on fibroblasts.

Results

BSC-EVs were successfully isolated, and NTA demonstrated their high sEV yield. In addition to exosomal markers, BSC-EVs expressed nucleus-feature marker (TP63) associated with BSC, without another specific BSC marker (KRT5). Moreover, the inhibitory effect of BSC-EVs on fibroblasts was confirmed through the suppression of collagen area contraction. The decrease in expression of α-sma and FAP was observed when fibroblasts were exposed to 10000 and 20000 particles per cell, respectively. The western blot result verified the expression of Collagen 1 was up-regulated in a dose-dependent manner upon exposure to BSC-EVs. Importantly, the senescence marker γ-H2AX was found to be transferred from BSC-EVs to fibroblasts, where it induced functional alterations and manifested signs of senescence.

Summary

This study represents the first extraction and identification of BSC-EVs that meet the MISEV2018 criteria, and carry feature of source cells (TP63). This finding also highlights the potential effect of BSC-EVs in inhibiting fibroblasts activation and promoting fibroblasts senescence. The evidence emphasized the regulatory effect of BSC-EVs on fibroblasts, supporting its promising role in the treatment of respiratory fibrotic diseases.

Miss Thitikan Jirakittisonthon, Dr. Orman Snyder, Dr. Hong He, Dr. Mark Weiss

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Prior work demonstrated enhanced neural uptake by the addition of the acetylcholine receptor ligand, Rabies Viral Glycoprotein (RVG), to the extracellular vesicle (EVs) membrane. RVG enhances EV uptake by neural tissues and increases EVs efficiency in crossing the blood-brain barrier. Here, we hypothesize that another ligand, GM-1 ganglioside, may enhance neural EV uptake. We will add a GM-1 ganglioside ligand to the EV membrane to test whether its addition enhances neural EVs uptake in vitro, and in vivo compared to naïve EVs.

Methods

MSC-EVs are isolated using size-exclusion chromatography from conditioned medium. Naïve and GM1-modified EVs will be characterized using nanoparticle tracking analysis, dynamic light scatter, Transmission Electron Microscopy, and Exoview™R-100. Naïve EVs and GM-1 modified EVs will be labeled with PKH 26 for tracking by confocal microscopy. For in vitro experiments, fluorescent EVs are added to cell culture medium of sarcoma cells or neural progenitor cells. The uptake of EVs will be quantitatively evaluated over sixteen hours. For animal experiments, EVs will be subcutaneously injected into a rat's hind paw. Fluoro-Gold, a fluorescent retrograde tracer, will be the positive control. Animals will be sacrificed 2, 3, and 5 days after injection, the retrogradely labeled dorsal root ganglion (DRG) neurons quantified by confocal microscopy. Thus, the efficiency of naïve and GM1-modified EVs to label the neural cells will be compared in vitro and in vivo.

Results

Pilot studies demonstrated uptake of naïve EVs in sarcoma and neural progenitor cells following 16 hours of incubation. GM1-modified EVs have not been generated yet. The in vitro and in vivo data from naïve and GM1-modified presented at the meeting.

Summary/Conclusion

Our study will compare naïve EVs and GM1-modified EVs in vitro and in vivo. We will establish the efficiency of neural uptake by comparing the dose-response relationship of naïve vs. GM1-modified EVs in vitro and the ability of naïve or modified EVs to retrogradely transport in neurons in vivo. Our hypothesis will be supported by observing neural specificity in vitro, and enhanced DRG labeling in vivo by GM1-modified EVs.

MSc Heikki Kyykallio, BSc Kirsti Härkönen, PhD Martina Hanzlíková, PhD Tatu Lajunen, Professor Tapani Viitala, PhD Kirsi Rilla

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Extracellular vesicles (EV) have great potential in targeted drug delivery due to their natural capability to transfer molecular cargo into their target cells. However, due to heterogeneity and abundance of EV in the body, EV with customized properties are required to enhance their targeting to diseased cells. It has been previously shown that cells with high levels of hyaluronan (HA) synthesis secrete HA-coated EV. HA is a negatively charged polysaccharide that binds with high affinity and specificity to the adhesion receptor CD44, which is overexpressed in variety of cancers. We utilized the unique properties of HA and its high affinity interaction with CD44 to enhance the targeting of EV to CD44 overexpressing cancer cells.

Methods

MCF7 breast cancer cell line with inducible GFP-Hyaluronan synthase 3 (GFP-HAS3) expression was used as a source of normal MCF7-EV and HA-coated GFP-HAS3-EV. Both EV types were isolated by ultracentrifugation and characterized by western blot, nanoparticle tracking analysis, and transmission electron microscopy. EV binding to target cells was investigated by treating CD44 overexpressing or -negative MKN74 gastric cancer cells, and their co-cultures with GFP-HAS3-EV and quantifying bound particles from confocal microscopy images. Additionally, the HA-CD44 interaction was disrupted by digesting HA using hyaluronidase, or by oversaturating the CD44 receptors with added HA. Multi-parametric surface plasmon resonance (MP-SPR) was used to quantitate interactions of MCF7-EV and GFP-HAS3-EV with the MKN74 cells. Additionally, formation of plasma-derived protein corona on MCF7-EV and GFP-HAS3-EV was determined using MP-SPR and mass spectrometry.

Results

We show that CD44-positive cells bind significantly more GFP-HAS3-EV, and the GFP-HAS3-EV targeting is dependent on HA on the EV surface and free CD44 on the cell surface. MP-SPR measurements indicate that both CD44-positive and -negative cells interact with MCF7-EV, but only CD44-positive cells have strong and rapid interactions with GFP-HAS3-EV.

Summary/Conclusion

HA-coat on EV increases their specificity in targeting CD44-positive target cells. This highlights the potential of HA-coated EV in development of novel target-specific delivery of drugs, especially applicable in targeting tumors with high CD44 expression.

Assistant Professor Wei-Ting Hung, Professor John S. Davis, Professor Lane Christenson

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

Well-orchestrated ovarian cycle is the key for generating a competent oocyte for fertilization in which the size of ovarian follicles significantly increases due to massive proliferation. Diverse cellular mediators contribute to the proliferation of granulosa cells including extracellular vesicles (EVs). Previously, we found EVs from small follicles caused bovine granulosa cells to proliferate greater than those EVs from large follicles. This difference was partially explained by their ability to enter recipient cells, with small follicle derived EVs entering cells 2-fold more than large follicle EVs. In this study, a proteomics approach was used to identify candidates responsible for the differential uptake of follicular EVs.

Methods

Bovine ovaries from the slaughterhouse were transported in sterile PBS. Follicular fluids were aspirated from bovine follicles of 3-5 and 9mm and processed through standard ultracentrifugation protocol. Follicular fluids were spined at 2000g followed by 12000g prior to a filtering step to remove cell debris and vesicles larger than 0.22µm. EVs were collected at 110000g and then a size exclusion column was used to remove potential soluble proteins before a final 110000g spin. EV samples were characterized according to MISEV2018. Finally, EV samples were trypsin-digested and ionized to quantify the total protein of three samples each by mass spectrometry.

Results

Morphology, size distribution, and immunostaining demonstrated our EV samples complying with MISEV2018 with minimal protein contamination. Concentration and CD81 peaked in small follicle EVs. Total protein analysis from mass spectrometry identified 422 proteins relatively enriched in small follicles and 128 proteins showed the opposite trend. CD81 was more abundant in EV from small follicles although it did not reach significance. Multiple membrane spanning proteins were identified as differentially enriched. The expression of the scavenger receptor class B type 1 receptor and lipoprotein receptor-related protein 8 have been known to increase with follicular growth and they were enriched in large follicle EVs.

Summary/Conclusion

We comprehensively analyzed the proteomics of EVs from small and large follicles and identified proteins which were preferentially present. The ongoing study will apply a high-throughput approach to screen for membrane proteins which serve as the key to enter recipient cells.

Ms Shinwon Chae, Mr Chul Won Seo, Ms Haekang Yang, Professor Yoon-Jin Lee, Professor Dongsic Choi

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: Extracellular vesicles (EVs) are endogenous vehicle to transfer their molecular contents to the proximal and distal tissues. Especially, cancer cell-derived EVs modulate their nascent tumor microenvironment but also regulate the metastatic niche via systemic circulation. However, biodistributions according to cancer type or oncogenic transformation are not well addressed. In this study, we revealed the distribution of EVs derived from colorectal and lung cancer cells with different RAS and RAF mutant status.

Methods: Following cell lines were used for EV isolation: colorectal cancer cell line Caco-2 (wildtype) WiDr, HT29 (BRAF mutant), LoVo, HCT116 (KRAS mutant); and lung cancer cell line H292, H1703 (wildtype), H358, A549 (KRAS mutant). Cells were cultured in EV-depleted FBS containing media for 3-days. Collected conditioned medium was concentrated with 100 kDa filtration and isolated by size exclusion chromatography. EVs were labeled by lipophilic fluorescent dye DiR and unlabeled dye was removed by size exclusion chromatography. DiR-EVs (5.0E+09 particles) were injected to BALB/c mouse via tail vein. Kinetic biodistribution of EVs are measured by IVIS imaging system at 10 min, 1-, 4-, 8- and 24-h. Mass spectrometry-based quantitative proteomics were conducted to reveal unique surface proteomic signature of EVs.

Results: Overall, all EVs were most abundantly detected in liver and spleen at 1-h time points and maximized at 24-h. Impressively, colorectal cancer cell-derived EVs were relatively enriched in intestine but lung cancer cell-derived EVs enriched in lung, implicating their homophilic interaction of EVs to sibling cells. From quantitative proteomic analyses, differential integrin distributions were identified according to cell origins and oncogenic mutations (RAS and RAF). Laminin-binding ITGB4 and ITGA6 were significantly enriched in EVs derived from mutant cell lines. Co-injection of EVs with integrin inhibitor peptide showed the differential in vivo distribution of EVs, implying integrin is key regulator of tissue specific EV destination of systemic circulation.

Summary/Conclusion: We first identified the differential kinetic distribution of EVs according to cellular origins and oncogenic mutant status. Our results suggest that EVs could destine in specific tissues affected by integrin decorations on their surface. Consequently, this information provides appreciated implications for EV-mediated biological effects, and drug delivery vehicle development.

Dr. Andrea Galisova, Dr. Jiri Zahradnik, Dr. Daniel Jirak

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Functionalized extracellular vesicles (EVs) targeted to specific cancer biomarkers can serve as multiplexed diagnostic tools or targeted drug delivery vehicles. We introduce Nanobody-Tag-Ligand surface system NaTaLi, in which nanobodies that specifically and with almost covalent affinity bind a tag are displayed on the surface of EVs. The EVs can then be coupled with any tagged ligand of interest, e.g.tumor penetrating peptides, by simple mixing. Additionally, the system uses various fluorescent markers allowing multicolor encoding. Crucially, NaTaLi allows one engineered cell line to produce EVs that can be coupled to many different targeting units, substantially reducing the amount of work and time to assay novel mechanisms.

Methods:

HEK293 cells stably expressing the anti-ALFA tag nanobody on the surface were used for isolation of EVs by a standard ultracentrifugation method (100,000g; 2h) combined with ultrafiltration (100kDa). Fluorescent proteins mNeonGreen/dTomato containing the ALFA tag were isolated and purified from bacteria. Tagged proteins were co-incubated (1h,RT) with cell/EV-nanobody to create a NaTaLi system. Binding of tags to nanobody-cells was examined by fluorescent microscopy and surface plasmon resonance (SPR). ALFA-nanobody-displaying EVs were characterized according to the MISEV guidelines (western blot and NTA). Affinity of the tagged proteins on the ALFA-Nanobody EVs was tested by a FIDA (Flow Induced Dispersion Analysis) device and the half maximal effective concentration (EC50) was assessed.

Results:

Cells displaying nanobodies on their surface specifically bind the tagged proteins as shown by mNeon/dTomato fluorescent signal localized on cell surface. Binding constant (Kd) measured on live cells was estimated to be 40nM (SPR). NaTaLi EVs show expected size (169.7±3.4nm) and expression of EV markers (CD81, Alix). EC50 of the tagged proteins (40nM) to EV-nanobody was assessed to be 3E09 EV/mL. Control tagged proteins did not show any binding to the EV-nanobody confirming specificity of the NaTaLi system. Several tumor targeting peptides were prepared for further multicolor cancer cell targeting.

Summary/Conclusions:

We show development of a highly versatile system for EV functionalization and targeting. Binding of tagged proteins to cell/EV-nanobody was shown to be strong and specific. The NaTaLi system opens new avenues for multiplexed cancer biomarker detection and delivery of cancer therapeutics.

Dr Yao Yue, Dr Xu Yi, Dr Judy Wai Ping Yam

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Background: Small extracellular vesicles (sEVs) mediate intercellular communication that contributes to hepatocellular carcinoma (HCC) progression via multifaceted pathways. The success of cell entry determines the effect of sEV on the recipient cells. Here, we aimed to delineate the mechanisms underlying the uptake of sEV in HCC.

Methods: Macropinocytosis was examined by the ability of cells to internalize dextran and sEV. Macropinocytosis was analyzed in Na(+)/H(+) exchanger 7 (NHE7) knockdown and overexpressing cells. The properties of cells were studied using functional assays. pH biosensor was used to evaluate the intracellular and endosomal pH. Expression of NHE7 in patients’ liver tissues was examined by immunofluorescent staining. Inducible silencing of NHE7 in established tumors was performed to reveal the therapeutic potential of targeting NHE7.

Results: The data revealed that macropinocytosis controlled the internalization of sEVs and their oncogenic effect on the recipient cells. It was found that metastatic HCC cells exhibited the highest efficiency of sEV uptake relative to normal liver cells and nonmetastatic HCC cells. Attenuation of macropinocytic activity by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) limited the entry of sEVs and compromised cell aggressiveness. Mechanistically, we delineated that high level of NHE7, a sodium-hydrogen exchanger, alkalized intracellular pH and acidized endosomal pH, leading to the maturation of macropinosomes. Inducible inhibition of NHE7 in established tumors developed in mice delayed tumor development and suppressed lung metastasis. Clinically, NHE7 expression was upregulated and linked to dismal prognosis of HCC.

Conclusions: This study advances the understanding of the sEV uptake mechanism facilitated by NHE7. Inhibition of sEV uptake via macropinocytosis can be exploited as a treatment alone or in combination with conventional therapeutic approaches for HCC.

Dr. Yosuke Tanaka, Dr. Ai Morozumi, Dr. Nobutaka Hirokawa

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Left-dominant [Ca2+]i elevation on the left margin of the ventral node furnishes the initial laterality of mouse embryos. It depends on cilia-generated leftward extracellular leftward fluid flow (nodal flow), fibroblast growth factor receptor (FGFR)/ sonic hedgehog (Shh) signaling, and the PKD1L1 polycystin subunit, of which interrelationship is still elusive.

Here, we show that leftward nodal flow directs PKD1L1-containing fibrous strands and facilitates Nodal-mediated [Ca2+]i elevation on the left margin. We generate KikGR-PKD1L1 knockin mice in order to monitor protein dynamics with a photoconvertible fluorescence protein tag. By imaging those embryos, we have identified extracellular fragile meshwork containing PKD1L1-EVs, being gradually transferred leftward. A portion of the meshwork finally bridges over the left nodal crown cells in an FGFR/Shh-dependent manner.

Consequently, PKD1L1 N-term is concentrated on the left margin, where it is predominantly associated with Nodal. Furthermore, PKD1L1/PKD2 overexpression significantly augmented cellular Nodal sensitivity.

Accordingly, we propose that leftward transfer of polycystin-containing fibrous strands triggers the first left-specific Ca signaling, which determines left-right asymmetry in developing embryos. (Y.T. and A.M.: equal contribution)

Dr. Daisuke Watase, Mai Hazekawa, Ayano Yamada, Mitsuhisa Koga

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction: It has been reported that small extracellular vesicles (sEVs) might regulate the targeting organ in the metastasis process of cancer cells. Therefore, in this study, we aimed to compare the distribution of serum-derived sEVs isolated from cancer mice with those isolated from normal mice in order to apply them as nucleic acid carriers for autologous transplant treatment using autologous ingredients. Methods: Spontaneous lung metastasis mice model were prepared by subcutaneously injecting melanoma cells into the hindlimbs of female C57BL/6 mice, and surgically removed the primary tumors. sEVs were isolated from serum collected from mice on days 0, 3, 7, 10, and 14 after primary tumor inoculation. Furthermore, sEVs were also extracted from normal mice after blood collection. After isolating serum sEVs, sEVs derived from cancer mice or sEVs derived from normal mice were intravenously injected into the spontaneous lung metastasis mice model. The distribution of sEVs in mice model were evaluated using in vivo molecular imaging device using ICG-loading sEVs. Results: sEVs derived from the serum of mice that had been tumor-bearing for a long time had higher amount accumulated in various organs as the tumor-bearing time was longer. Of particular note, it was revealed that sEVs from cancer mice were more likely to accumulate in the lung, which are metastatic organs, than sEVs from normal mice. Protein quantification using sEVs revealed that the sEVs isolated from cancer mice had higher expression levels of adhesion factors than those isolated from normal mice. Summary/Conclusion: Cancer-derived exosomes have been shown to be useful materials for natural targeting drug carriers. The knowledge obtained through this research can greatly contribute to the realization of personalized medicine made from self-derived ingredients in the future.

Dr James Rhodes, Dr Stefan Balint, Mr Andras Miklosi, Dr Nina Jajcanin-Jozic, Mr Andrei Traista, Dr Pradeep Kumar, Dr Grace DeSantis

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction

EVs are microscopic carriers that act as messengers by facilitating transfer of biomolecules between cells locally and systemically. The multifunctionality of EVs have spurred interest in their uptake mechanisms by recipient cells, which is the key to unraveling various physiological and pathological processes. Studying the interactions between recipient cells and EVs presents challenges rooted in their small size (30 - 1000 nm in diameter). Single-molecule localization microscopy (SMLM) emerges as a transformative solution to this challenge. Here we examined EV uptake and colocalization with LAMP-1 using a combination of live and dSTORM imaging.

Methods

Purified GFP-positive EVs were added to U2OS cells at 37°C. Cells were imaged live and were then fixed with strong fixative (PFA + glutaraldehyde) at different time points using the ONI Discovery Kit™: dSTORM in cells, followed by treatment with a quencher. After washing, cells were permeabilized and blocked to minimize nonspecific binding. Cells were labeled with a primary antibody against LAMP-1 and a secondary F(ab’)2 probe fluorescently labeled with AZ647. Cells were prepared for imaging, using ONI's BCubed imaging buffer, also a part of the Discovery Kit™: dSTORM in cells.

Results

After 30 minutes, ∼90% of all the cells had taken up EVs, with GFP-positive EVs showing colocalization with lysosomes at later time points. By using super-resolution dSTORM imaging, we then visualized the structure of the lysosomes with high resolution and used it to overlap and quantify the presence of GFP-EVs in the nanometer scale.

Summary/Conclusions

The colocalization of LAMP-1 with internalized EVs provides invaluable insights into the dynamics of endocytic processes and EV fate and shows a convergent molecular machinery for intracellular trafficking of endogenous cellular components and exogenous EVs. This offers researchers a platform to probe the underlying molecular mechanisms and signaling cascades that govern EV uptake, intracellular transport, and subsequent release of bioactive cargo.

Mr. Gaoge Sun¹, Zihan Liu¹, Ying Zhang¹, Hang Yin^1,2

¹School of Pharmaceutical Sciences, Tsinghua University, Beijing, China, ²Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

1) Introduction

Extracellular vesicles (EVs) play a crucial role in nucleic acids transport and intercellular communication. Tumor educated platelets, transient small anucleate circulating cells with a lifespan of 7-10 days, offer insight into tumor-driven functional phenotypes. Our study reveals a global mRNA degradation pattern affecting ribosomal protein mRNA (RP-mRNA) in both cancer patient plasma and platelets. Investigating further, we observed that EV cargoes from colorectal cancer cell lines stimulate the RNASEL pathway in megakaryocytes and platelets, leading to significant RP-mRNA decay. The subsequent platelet RP-mRNA degradation may result in platelet dysfunction, highlighting its potential as a valuable tumor biomarker for liquid biopsy.

2) Methods

EV collection: The HCT-116 cell culture medium containing 10% EV-depleted serum was subjected to gradient centrifugation. The validation of EVs was carried out according to MISEV2018 guidelines using NTA, TEM, and Western Blot.

Platelet RNA quantification: Platelet-like particles derived from Meg01 cells underwent purification using a 5-µm filter and subsequent centrifugation (3,000g, 15min), followed by RNA content analysis using RNA-seq and RT-qPCR with three biological replicates.

3) Results

Our research uncovered contrasting expression trends of RP-mRNAs between cancer patients’ blood and tumor tissue. We demonstrated that cancerous EVs activate the RNase pathway, leading to mRNA degradation in platelets. Specifically, stimulation with dsRNA from HCT116 cell-derived EVs resulted in a significant decline in RP-mRNA levels within platelets. EVs were found to trigger platelet RNASEL dimerization, facilitating mRNA degradation. Furthermore, we observed an interaction between mRNA quality control proteins and RNASEL, emphasizing the impact of tumor-educated platelets mediated by cancerous dsRNA carried by EVs.

4) Conclusion

Our study confirms that dsRNA in cancerous EVs activate the platelet RNASEL pathway and underscores the role of EV membrane structure in shielding RNA cargoes from extracellular RNase degradation. However, further exploration is necessary to identify the specific dsRNA structures. Our results suggest that cancerous EVs have the potential to modulate the platelet transcriptome, particularly the RP-mRNA content through RNASEL activation. The decay of plasma RP-mRNA emerges as a promising cancer-educated platelet biomarker for liquid biopsy.

Dr Pevindu Abeysinghe¹, Ms Breanna Humber², Mr Riccardo Cecchin², Prof. Kevin Morris¹

¹Centre for Genomics and Personalised Health, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia, ²Menzies Health Institute Queensland, School of Pharmacy and Medical Science, Griffith University, Gold Coast, Australia

Sponsor Exhibition, Poster Session and Lunch (Saturday), Exhibit hall-Doors 14-15, May 11, 2024, 12:00 PM - 2:00 PM

Introduction:

Exosomes, extracellular vesicles with pivotal roles in intercellular communication, are implicated in regulating gene expression through long non-coding RNAs (lncRNAs). This study delves into the regulatory influence of HNF4A-AS1, an antisense lncRNA, on Hepatocyte nuclear factor four alpha (HNF4A), and explores the potential involvement of exosomes and extracellular vesicles (EVs) in mediating these effects, especially on HNF4A isoforms crucial for hepatocyte function.

Methods:

HEPG2 cell culturing in Dulbecco's Modified Eagle Medium, supplemented with 10% Fetal Bovine Serum, preceded transfections in 12-well plates, validated through GFP plasmid transfections. RNA isolation utilized the Qiagen RNeasy Maxi Kit, and reverse transcription employed the Qiagen QuantiTect kit. For qRT-PCR, Promega GoTaq and Kappa Biosystems Sybr fast master mixes were used, adjusting cycling conditions accordingly. These methods facilitated targeted siRNA transfections, revealing the successful suppression of HNF4A-AS1 and providing insights into its regulatory impact on HNF4A isoforms.

Further experiments have planned to HNF4AS1 selective packaging into exosomes and/or EVs utilizing vector transfusion in EV producing stable cell lines, thus targeted therapeutics for inflammatory liver diseases.

Results:

Transfection of HepG2 cells resulted in the significant repression of HNF4A-AS1a and HNF4A-AS1b transcripts with individual and combined siRNA targeting, excluding the HNF4AS1_Pro2 siRNA for HNF4A-AS1b. Silencing HNF4A-AS1 isoforms consistently reduced P2-promoted HNF4a-1 transcription (38-60%), particularly pronounced with HNF4A-AS1a targeting. HNF4A-AS1b siRNA led to ∼30% reduction in spliced HNF4a mRNA transcription, while spliced HNF4a mRNA was maintained with HNF4A-AS1a-specific targeting (P>0.2).

Conclusion:

In conclusion, this study unveils HNF4A-AS1's transcriptional role and highlights the potential involvement of exosomes in modulating HNF4a isoform expression. Building on these insights, our future endeavors aim to selectively package HNF4A-AS1 into exosomes and extracellular vesicles (EVs) using vector transfusion in stable cell lines proficient in EV production. This strategic approach paves the way for targeted therapeutics tailored to address inflammatory liver diseases, emphasizing the promising intersection of lncRNA research, exosome biology, and therapeutic innovation.

Dr. Cao Dai Phung, Thi Tuyet Trinh Tran, Brendon Zhi Jie Yeo, Gao Chang, Rebecca Carissa Prajogo, Migara Kavishka Jayasinghe, Thi Thanh Xuan Dang, Yuan Ju, Mai Trinh Nguyen, Boya Peng, Hong Anh Le, Eric Yew Meng Yeo, Bonney Glenn, Boon Cher Goh, Dahai Luo, Wai Leong Tam, Minh TN Le

Introductory Talk and Oral Session: OS21 Cancer Immunotherapy, Plenary 1, May 11, 2024, 4:00 PM - 5:35 PM

Introduction:

Extracellular vesicles (EVs) provide multiple advantages over existing nucleic acid delivery systems due to their biocompatibility, stability, and low immunogenicity. In our previous studies, our group has demonstrated the safety, efficiency, and suitability of EVs derived from red blood cells (RBCEVs) for nucleic acid drug delivery in cancer treatment. In this study, we present a combination of RBCEVs loaded with antisense oligonucleotides (ASOs), and a RIG-I agonist immunomodulatory RNA (immRNA). This combination has the capacity to inhibit KRAS mutants and activate RIG-I, consequently inducing anti-tumor immunity, specifically type I interferon-mediated anti-tumor polarization, for the treatment of KRAS-dependent cancers.

Methods:

RBCEVs were loaded with anti-KRAS ASO or immRNA using REG-1. The biophysical and biochemical properties of loaded EVs, including the size, shape, surface charge, loading efficiency, and protein composition were characterized. The in vitro anticancer effect of the EV combination was assessed in multiple KRAS mutant-bearing cancer cell lines and patient-derived organoids. The in vivo antitumor effect of this combination was evaluated in syngeneic and genetically engineered mouse model of KRAS-mutated cancer.

Results:

The ASO and immRNA were loaded efficiently onto RBCEVs using REG-1 reagent without significant changes in particle size and EV morphology. These nucleic acids were effectively delivered to cancer cells using RBCEVs. RBCEVs loaded with KRAS ASO efficiently inhibit KRAS mutants, while sparing cells with wild-type KRAS gene, leading to specific tumor cell death. Additionally, we demonstrated that the combination of KRAS ASO and immRNA delivered by RBCEVs induced synergistic activation of RIG-I, leading to robust induction of tumor cell death and upregulation of type I-IFNs in KRAS-dependent cancers in both in vitro and in vivo settings without any observable adverse effects.

Conclusion:

These findings indicate that the RBCEVs loaded with KRAS ASO and immRNA could be a potential therapeutic solution for the treatment of KRAS-dependent cancers.

Postdoctoral Fellow Golnaz Morad, Brenda Melendez, Sarah Johnson, Manoj Chelvanambi, Matthew Wong, Ashish Damania, Nadim Ajami, Jennifer Wargo

Introductory Talk and Oral Session: OS21 Cancer Immunotherapy, Plenary 1, May 11, 2024, 4:00 PM - 5:35 PM

Introduction: Recent trials have demonstrated the benefits of fecal microbiota transplantation (FMT) in enhancing response to cancer immunotherapy, especially immune checkpoint blockade (ICB). However, the clinical application of FMT is limited by the rigorousness of screening criteria and the needed volume of fecal material. Our study aims to identify the active components in FMT material to develop scalable microbiome-based strategies to improve response to ICB. We hypothesized that microbial derivates within the stool (filtrate) and fecal extracellular vesicles (fEVs) can enhance response to ICB.

Methods: We prepared fecal filtrates from melanoma patients who were complete responders (CR) or non-responders (NR) to ICB. Filtrates were prepared via centrifugation and filtration of stool suspensions to remove bacteria, human cells, and debris. fEVs were subsequently isolated using density gradient ultracentrifugation and characterized through electron microscopy, nanoparticle tracking analysis, and marker evaluation. Germ-free C57BL/6 mice received oral gavages of FMT, filtrate, or fEVs from CR and NR patients. After a five-day period for engraftment, mice received subcutaneous injections of BP melanoma tumor cells and were treated with ICB. Tumor volume was measured using a caliper. The tumor and colon immune profiles were evaluated via digital spatial profiling (DSP). Mass spectrometry was used to characterize the components of filtrates and fEVs.

Results: Treatment with CR FMT, filtrate, and fEVs significantly improved response to ICB, suggesting that fecal filtrates and fEVs might be sufficient for inducing a response to ICB. In contrast, treatment with NR FMT, filtrate, and fEVs was associated with poor response. DSP of tumor and colon samples demonstrated an increase in innate immune cells and B cell lineages associated with CR filtrate treatment. Proteomic analysis of both CR filtrate and fEVs demonstrated enrichment of immunoglobulins and bacterial proteins from Bacteroides species, suggesting the involvement of specific bacterial species and B cell responses in the filtrate/fEV-induced enhancement of melanoma response to ICB.

Conclusion: Together, these studies suggest that the EV fraction of FMT may confer an improved response to ICB, though further studies are needed to derive optimal therapeutic targets and to gain mechanistic insights.

Miss Maria Angelica Rincon-Benavides, Miss Aarti Patel, Mrs. Tatiana Cuellar-Gaviria, Mr. Ethan Stamas, Mr. Jad Hussein, Mr. Diego Alzate-Correa, Miss Yuyan Yu, Miss Cintia Gomez, Mrs. Heather Powell, Mr. Daniel Gallego-Perez, Mrs. Natalia Higuita-Castro

Introductory Talk and Oral Session: OS21 Cancer Immunotherapy, Plenary 1, May 11, 2024, 4:00 PM - 5:35 PM

Introduction: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by the loss of function of neurofibromin protein due to mutations in the NF1 gene. The loss of function of Neurofibromin results in the over-activation of the RAS pathway, leading to uncontrolled cellular proliferation and tumor development. Patients with NF1 have a higher incidence of developing malignant peripheral nerve sheath tumors (MPNST), which are aggressive and highly invasive with a low survival rate. Gene therapies for NF1 using viral vectors are limited due to the large size of the NF1 gene. To overcome this limitation, we propose the implementation of engineered EVs (eEVs) specifically designed to drive effective non-viral delivery of NF1 cargo (e.g., mRNA, protein, and plasmid DNA) inducing restoration of the neurofibromin protein function in animal models of NF1.

Methods: Primary human dermal fibroblast cells (HDFs) were transfected with plasmids encoding for human NF1. Subsequently, eEVs were isolated from the culture media using size exclusion chromatography. EV loading with NF1 genetic material was confirmed using RT-PCR and conventional PCRs. eEV size distribution and concentration were analyzed via NanoFCM. The therapeutic efficacy of the NF1 eEVs was assessed using malignant tumor cells isolated from patients with NF1, where effective restoration of the neurofibromin protein and related pathways was characterized.

Results: Characterization of the eEVs showed significant packing of plasmid DNA and mRNA in the NF1 eEVs compared to control eEVs. NF1 eEVs were effectively captured and incorporated by malignant NF1-deficient cells, with effective transfection of these recipient cells with the NF1 cargo as confirmed via RT-PCR. NF1 eEV-treated cells showed robust NF1 overexpression. Proliferation pathways evaluated in NF1-deficient cells treated with NF1 eEVs revealed significant downregulation in their proliferative activity and effective rescue of neurofibromin protein function.

Conclusions: Overall, these findings reveal the potential of NF1 eEVs as a promising non-viral gene therapy strategy to rescue neurofibromin protein function for patients with neurofibromatosis and other types of cancer associated with NF1 gene mutations such as glioblastoma and melanoma.

Miss Jacqueline YT Yeo, Dr Yuganthini Vijayanathan, Miss Janice HY Tan, Mr Fikri Mohamad, Ms Rachel LY Ho, Miss Nurashikin Abdul Halim, Dr Tatsuya Kozaki, Mr Zhi Wei Zhang, Dr Hai Tao Tu, Dr Jann Sarkaria, Dr Florent Ginhoux, Dr Li Zeng, Dr Ivy Ho

Introductory Talk and Oral Session: OS21 Cancer Immunotherapy, Plenary 1, May 11, 2024, 4:00 PM - 5:35 PM

Jacqueline Yeo¹, Yuganthini Vijayanathan¹, Janice Tan¹, Fikri bin Mohamad¹, Rachel Ho¹, Nurashikin Bte Abdul Halim¹, Tatsuya Kozaki², Zhi Wei Zhang³, Hai Tao Tu³, Jann N. Sarkaria⁴, Florent Ginhoux²,⁵, Li Zeng³,⁶,⁷, Ivy Ho¹,⁷

¹Molecular Neurotherapeutics Laboratory, ³Neural Stem Cells Laboratory, National Neuroscience Institute, Singapore. ²Singapore Immunology Network, Singapore. ⁴Department of Radiology, Mayo Clinic, Rochester, USA. ⁵Institute Gustave Roussy, Paris, France, ⁶Lee Kong Chian School of Medicine, Singapore. ⁷Duke-NUS Medical School, Singapore.

Introduction: Extracellular vesicles (EVs) have been engineered to carry therapeutic agents for cancer therapy. Previously, we found that re-expression of the leucine-rich domain (LRD) of neurofibromin (NF1) inhibited glioblastoma (GBM) invasion and prevented the infiltration of microglia/macrophage into the tumor microenvironment (TME). Because GBM TME contains a high percentage of tumor-infiltrating microglia/macrophage (TAM) that affect tumor malignancy, we investigated whether EVs harboring the NF1-LRD (NF1-LRD-EVs) will mitigate GBM progression by influencing the tumor microenvironment.

Methods: NF1-LRD-EVs were isolated from glioma cells stably expressing the NF1-LRD domain using ultracentrifugation. These EVs were characterized according to MISEV2018 using western blot, Nanosight Tracking Analysis, and transmission electron microscopy. To assess the EVs, the primary mouse microglia and iPSC-derived macrophages (iMacs) were exposed to EVs for 24 h, and the morphological and molecular changes in the microglia/macrophages were analyzed using immunofluorescence staining, phagocytosis assay, tumor infiltration assay, and western blot analysis.

Results: NF1-LRD-EVs significantly reduced TAM recruitment and tumor infiltration in transwell migration assay and orthotopic mouse glioma model by at least 60-80% compared to the control EVs. The LRD-EVs-primed microglia/macrophages showed enhanced phagocytic activities similar to LPS-treated cells. The expression level of pro-inflammatory cytokines such as TNF-α and iNOS expression was elevated, while the anti-inflammatory cytokines Arginase and TGF- β1 were downregulated, suggesting that LRD-derived EVs may polarize microglia to a pro-inflammatory phenotype that discourages tumor growth.

Summary: Our results demonstrate that EVs harboring the NF1-LRD protein reprogram TAMs in the tumor microenvironment to heighten gene expression associated with sensing danger signals and dampened genes involved in cell migration and immunosuppression. Our data implicate the potential of using EVs as carriers for neuro-immuno-modulation.

Associate Professor Sharanjot Saini, Dr Sandip Nathani, Ms Diana Asante, Ms Amritha Sreekumar, Dr. Matthew Simmons

Introductory Talk and Oral Session: OS21 Cancer Immunotherapy, Plenary 1, May 11, 2024, 4:00 PM - 5:35 PM

Introduction: Therapy-induced neuroendocrine prostate cancer (NEPC) is an aggressive variant of castration-resistant prostate cancer (CRPC) (survival times < 1 year) that is increasing in incidence with the widespread use of second generation of androgen receptor (AR)-pathway inhibitors (APIs) such as Enzalutamide (ENZ). NEPC arises from CRPC-Adenocarcinomas (CRPC-Adeno) via a reversible trans-differentiation process, referred to as neuroendocrine differentiation (NED) wherein prostate cancer cells (PCa) undergo a lineage switch and express neuronal markers such as enolase 2 (ENO2), chromogranin A (CHGA) and synaptophysin (SYP). Current therapeutic options for NEPC are limited to highly toxic platinum drugs. The primary objective of our study is to evaluate novel, exosome-based therapy for NEPC.

Methods: Exosomes were isolated from HEK293T cells by ultracentrifugation. Isolated exosomes were characterized by Nanoparticle Tracking Analyses and electron microscopy. These exosomes were engineered to express CEACAM5 antibody on their cell surface. Exosomes were packaged with a combination of drugs (EZH2 inhibitor and AR inhibitor) by sonication. Engineered exosomes were tested in vitro using NCI-H660 cells and in vivo using LuCaP145.1 patient-derived xenograft model. Controls included LuCaP145.1 xenografts treated with IgG labelled exosomes.

Results: Engineered exosomes reduced the viability of NEPC cells. CEACAM5-labelled exosomes trafficked to tumor cells as CEACAM5 is a surface protein expressed by NEPC cells. Systemic administration of engineered exosomes induced tumor regression in NEPC PDX model.

Conclusions: Engineered-exosome based therapy may provide a new avenue as a targeted therapy for NEPC. Importantly, our exosome platform is versatile and can be used to target various surface antigens. Therefore, it is adaptable to target the heterogeneity of NEPC and CRPC tumors.

Acknowledgements: This work is supported by the US Army Medical Research Acquisition Activity (USAMRAA) Prostate Cancer Research Program Award No W81XWH-18-1-0303 and Augusta University startup funds.

Shiran Shapira, Prof., MD, MHA, CMO Nadir Arber

Introductory Talk and Oral Session: OS23 Applications of Engineered EVs, Room 105-106, May 11, 2024, 4:00 PM - 5:35 PM

Introduction: Every year, millions of people, world-wide, suffer from hyper inflammatory diseases that are prone to develop cytokine release syndrome (CRS), which is the common disastrous deterioration stage of acute respiratory distress syndrome (ARDS) with an urgent unmet need for medical therapy. In the last three decades there had been no improvement in the medical care of ARDS leading to 3M new cases annually and 1.2M deaths.

CD24 is a heavily glycosylated glycosylphosphatidylinositol GPI-anchored protein which functions as a biological immunomodulator. It crucially regulates cytokine and chemokine production by tight and selective regulation of the NFĸB pathway. CD24 discriminates between danger- and pathogen- associated molecular patterns (DAMPs and PAMPs). It acts upstream, reverting back the immune system to normal activity.

Exosomes are nano-sized (30-200nm) lipid vesicles secreted by most cell types. They are intraluminal vesicles which play an important role in intercellular communication. Exosomes are the ideal drug delivery carrier.

Methods:

Nano24 combined the two breakthrough technologies to create EXO-CD24, CD24-enriched exosomes derived from genetically manipulated cells. Nano24 has paved a unique road through in vitro, in vivo, GMP manufacturing, regulation and clinical trials in <180 patients. EXO-CD24 is given by inhalation.

Results: Efficacy and safety have been proven as a platform for ample of pulmonary and systemic diseases (sepsis/influenza/asthma/COPD/fibrosis) in mice models. Safety and promising efficacy were confirmed in >180 ARDS patients; phase Ib/IIa (NCT04747574) (35 patients, Israel) with COVID19-induced ARDS, phase IIb (NCT04902183) dose-finding study (91 patients, Greece), another phase IIb (37 patients), and compassionate use in severe ARDS (13pts). An international, multi-center, randomized, quadri-blind study versus placebo is on-going (13 patients, Israel)(NCT05947747).

Conclusions: EXO-CD24 is the medicine of tomorrow presenting a promising therapeutic approach for ample of hyper-inflammatory diseases with an urgent unmet need. It is a unique platform combining exosomes, as a carrier, and CD24, as the drug, smarter than steroids without side effects. EXO-CD24 administration by inhalation is an important clinical advantage in sever patients.

Samantha Roudi, Post-Doc Dhanu Gupta, Professor Samir El Andaloussi

Introductory Talk and Oral Session: OS23 Applications of Engineered EVs, Room 105-106, May 11, 2024, 4:00 PM - 5:35 PM

Introduction

In the era of new biological drugs, functional encapsulation and efficient delivery to target organs remains the major challenge. Furthermore, most of the delivery vectors have liver tropism. Lately, extracellular vesicles (EVs) have emerged as a next-generation drug delivery system owing to many well-known benefits. To expand the treatment possibilities to extrahepatic diseases and address challenges of exogenously produced EVs, such as cumbersome production and fast plasma clearance, we developed a novel platform, “in situ EV production”. We are utilizing gene therapy modalities to transform cells in vivo for endogenous secretion of EVs for improved availability of biotherapeutics in hard-to-reach organs.

Methods

We tagged an EV sorting domain with Nanoluciferase (NLuc) for highly sensitive tracking of in situ-produced EVs. The EV transgenes were administered hydrodynamically into mice as a plasmid DNA (pDNA), followed by biodistribution profiling 72h post-injection (PI). For transient production of EVs, we administered transgenes as mRNA-LNP and assessed the biodistribution 24h PI. To achieve long-term production of EVs, EV transgenes were administered incoroporated into adeno-associated viruses (AAVs) and analysed organs 4 weeks PI.

Results

pDNA and mRNA-LNP delivery strategies have liver, and in part spleen tropism, thus produced EVs are primarily of hepatic origin, as reflected in high Nluc signal in the liver. Importantly, NLuc-tagged EVs showed a body-wide distribution to all major organs, including CNS. We achieved sustained release of engineered EVs in circulation with drastic increase in pharmacokinetic profile over exogenous EVs. This approach, however, is only applicable for the transient production of EVs due to the short expression of LNP-mRNA forumlations.

With AAVs we observed increasing NLuc activity in serum over a tested period, proving long-term release of in situ-produced Nluc EVs to the bloodstream. At the endpoint, we detected NLuc EVs throughout the body, including the pancreas, brain, and intestine.

Summary

We have developed a platform for the endogenous production of engineered EVs using the liver as a biofactory. Considering its applicability for short and long-term EV release, as well as advanced EV engineering approaches, this innovative and versatile platform could be repurposed for the delivery of any desired biotherapeutic cargo.

Associate Professor Kyungmoo Yea, Professor Moon-Chang Baek

Introductory Talk and Oral Session: OS23 Applications of Engineered EVs, Room 105-106, May 11, 2024, 4:00 PM - 5:35 PM

Nonalcoholic steatohepatitis (NASH) is a progressing liver ailment with an unmet need for effective therapies. Given the complex pathogenesis of NASH, there is a consensus towards combination therapy, emphasizing the development of combinations rather than monotherapies. Small extracellular vesicles (sEVs) exhibit efficient liver delivery and can be engineered to carry diverse therapeutic substances. This study affirms the potential utility of engineered sEVs as a NASH treatment. In this study, sEVs were engineered to display fibroblast growth factor 21 (FGF21) on their surface and enclose miR-223. Human liver cell lines were exposed to PBS, control sEVs, or engineered sEVs. Effects were examined through ICC, qPCR, and immunoblot analysis. To assess in vivo effects, C57BL/6 mice were subjected to a choline-deficient, L-amino acid-defined, high-fat diet for 10 weeks. Subsequently, mice were randomly assigned to receive vehicle, control sEV, engineered sEV, or FGF21 mimetics. The analysis included IHC, qPCR, immunoblot analysis, ELISA, and µCT. Introduction of engineered sEVs into human liver cell lines led to a significant reduction in basal lipid storage and the expression of fibrosis and inflammation markers. This effect extended to significant improvements even under stimulating conditions such as palmitate or TGFβ1. Administering engineered sEVs with an FGF21-blocking antibody or miR-223 inhibitor effectively mitigated the impact on steatosis, fibrosis, and inflammation, thereby validating the crucial roles of FGF21 and miR-223 in these processes. In an in vivo model, intravenously administered sEVs exhibited liver-targeted delivery, resulting in a notable reduction in the NASH phenotype, as well as steatosis, inflammation, and fibrosis. Our study establishes the promising therapeutic role of engineered sEVs displaying FGF21 on their surface and encapsulating miR-223 for NASH treatment. These sEVs demonstrate a multifaceted impact on NASH development, reducing lipid storage and suppressing fibrosis and inflammation markers in both in vitro and in vivo models. The liver-specific delivery of engineered sEVs offers strategic advantages, providing a comprehensive approach to attenuate NASH progression through diverse pathways. This study lays a robust foundation for further exploration and development of engineered sEVs as a transformative therapeutic modality in NASH treatment.

Dr Alessia Brossa¹, Dr Michela Arena¹, Dr Alessandro Gori², Dr Marina Cretich², Dr Ilaria Giusti³, Prof Vincenza Dolo³, Benedetta Bussolati¹

¹Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy, ²Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Milano, Italy, ³Pathology Unit, Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy

Introductory Talk and Oral Session: OS23 Applications of Engineered EVs, Room 105-106, May 11, 2024, 4:00 PM - 5:35 PM

Introduction. The use of engineered extracellular vesicles (EVs) for clinical purposes represents a novel strategy in regenerative medicine. Red blood cell derived vesicles (RBC-EVs) represent a safe and abundant EV type, that could be used for large-scale production of engineered EVs with therapeutic potential, and for autologous therapy. We are evaluating different protocols of RBC-EVs engineering that would allow the loading of therapeutic RNAs and peptides. Methods. RBC-EVs are isolated from healthy donors using ultracentrifugation and tangential flow filtration, in order to scale-up EV production. RBC-EVs are characterized by nanoparticle tracking analysis, transmission electron microscopy (TEM), western blot and flow cytometry, in order to verify their number, dimension, purity and marker expression. RNA loading was performed using sonication, electroporation or incubation with cholesterol-modified miRNA, while click chemistry was used to engineer RBC-EVs with different peptides. Results. TEM analysis showed intact RBC-EVs, with a diameter around 50 nm. RBC-EVs express HBA, TSG101, the tetraspanins CD9, CD63, CD81, and the RBC marker CD47, as evaluated by western blot and flow cytometry. RBC-EVs were loaded with a peptide targeting KIM-1, specifically expressed in injured proximal tubular cells, to obtain specific cell targeting and increase the delivery efficiency, and with the anti-fibrotic Klotho peptide. Engineered RBC-EVs are tested on in vitro models of acute and chronic kidney damage using a temperature-sensitive human renal proximal tubule cell line. Conclusion. We aim to obtain an easy scalable protocol for RBC-EVs isolation and engineering that would allow the broadening of the therapeutic applications of extracellular vesicles in regenerative medicine.

To alleviate inflammation for acute lung injury

Mr. Sin-Yu Chen, Mr. Po-Chen Li, Dr. Tai-Shan Cheng, Ms. Hsin-Tung Chen, Ms. Wei-Ni Tsai, Dr. Hsiu-Jung Liao, Professor Ly James Lee, Professor Chi-Ying F. Huang

Introductory Talk and Oral Session: OS23 Applications of Engineered EVs, Room 105-106, May 11, 2024, 4:00 PM - 5:35 PM

1) Introduction

Acute Lung Injury (ALI) poses a life-threatening risk with severe inflammation and lung tissue damage, often resulting in swift respiratory failure and prolonged complications. The microRNA let-7a-5p has been implicated in the progression of lung injury, inflammation, and fibrosis by modulating cytokine production and immune cell activation. This study seeks to leverage an innovative cell electroporation platform to produce let-7a-5p-enriched extracellular vesicles (EVs) from transfected Wharton's jelly-mesenchymal stem cells (WJ-MSCs) as a potential therapeutic approach for ALI.

2) Methods

We utilized a Transwell®-based Asymmetric Cell Electroporation (TACE) platform to generate extracellular vesicles (EVs) enriched with let-7a-5p microRNA from WJ-MSCs transfected with the corresponding plasmid DNA. The engineered let-7a-5p-enriched EVs in conditioned medium were isolated through a tangential flow filtration (TFF) system. Characterization of the EVs adhered to the minimal consensus guidelines set by the International Society for Extracellular Vesicles (ISEV). Comprehensive therapeutic assessments were conducted, encompassing anti-fibrotic effects in a TGF-β-induced cell model, modulation effects on macrophage polarization, and the impact of let-7a-5p-enriched EVs in a rat model of hyperoxia-induced ALI.

3) Results

EV secretion from transfected WJ-MSCs saw a significant increase through the TACE platform. The encapsulated let-7a-5p in engineered EVs surpassed those from untreated WJ-MSCs. These let-7a-5p-enriched EVs demonstrated no impact on cell proliferation but effectively alleviated the TGF-β-induced fibrotic phenotype by down-regulating SMAD2/3 phosphorylation in LL29 cells. Moreover, let-7a-5p-enriched EVs regulated M2 macrophage activation in an inflammatory microenvironment, inducing notable IL-10 secretion. In the hyperoxia-induced ALI rat model, administration of naïve EVs from untreated WJ-MSCs showed a slight improvement in lung function and increased let-7a-5p expression in plasma. In contrast, let-7a-5p-enriched EVs significantly reduced macrophage infiltration, elevated IL-10 expression and led to a substantially improved lung function.

4) Summary/Conclusion

This study illustrates that employing the TACE platform to stimulate and transfect WJ-MSCs results in the production of abundant let-7a-5p-enriched EVs. These findings highlight the therapeutic potential in addressing inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. The discoveries present potential pathways for developing innovative therapeutic strategies for more effective interventions in ALI.

Sheng Yuan Leong, Ms. Wan Wei Lok, Ms Hui Min Tay, Mr. Hong Boon Ong, Dr. Poh Loong Soong, Dr. Roger Sik Yin Foo, Dr. Rinkoo Dalan, Dr. Han Wei Hou

Introductory Talk and Oral Session: OS24 EV Enrichment and Capture, Room 109-110, May 11, 2024, 4:00 PM - 5:35 PM

Introduction

Blood extracellular vesicles (EVs) are promising prognostics and diagnostics biomarkers in diseases but the clinical translation remains challenging due to the laborious and non-standardized EV isolation methods. EV detection is also confounded by various preanalytical variabilities in sample preparation such as delayed centrifugation or incomplete platelet depletion. Herein, we develop centrifuge-free microfluidic technology (ExoArc) for rapid platelet-free plasma (PFP) and EV isolation within 30 min. The automated and portable design enables on-site blood processing to minimize circulating EV losses and ex vivo cellular contamination in EV diagnostics.

Methods

PFP was separated from blood directly using ExoArc by depleting larger particles (> 500 nm), platelets (∼ 2 µm) and blood cells (> 5 µm) based on hydrodynamic focusing. Residual platelets in PFP were measured using flow cytometry. To characterize EV surface proteins, PFP were labelled with EV surface markers and analysed using fluorescent nanoparticle tracking analysis (fNTA). ExoArc-isolated PFP was processed using size exclusion chromatography (SEC) to deplete non-EV associated microRNAs prior microRNAs profiling using qPCR.

Results

Flow cytometry analysis showed complete cell removal and 99.99% platelet depletion in ExoArc-isolated PFP with residual platelets of < 1e4/mL, which is comparable to two-step centrifuged plasma processed according to ISTH guidelines. fNTA detected 2e9/mL CD9+ EVs and 3.9e8/mL CD81+ EVs in ExoArc-isolated PFP, with less particle concentration (∼ 5e7/mL) for platelet (CD41+), neutrophil (CD66b+), and monocyte (CD14+) EVs, thus suggesting minimal blood cell activation during blood processing. Coupling ExoArc with SEC further reduced background proteins to < 100 µg/mL with 10x higher EVs recovery as compared to ultracentrifugation. As a proof-of-concept for EV diagnostics, microRNAs in ExoArc+SEC-isolated EVs from type 2 diabetes mellitus (T2DM, n = 3) and healthy subjects (n = 3) were studied. Among 123 common microRNAs detected, upregulated miR-19a-3p, miR-129-5p, miR-21-5p and downregulated miR-141-3p were observed in T2DM subjects.

Conclusion

ExoArc provides a robust, cost-effective and user-friendly EV isolation workflow without requiring any centrifugation steps. This significantly reduces labour, processing time and preanalytical variabilities in EV isolation which will be key to increase clinical adoption of EV-based diagnostics.

Student Yicong Xue, doctor Zihao Ou, Professor Bo Situ, Professor Lei Zheng

Introductory Talk and Oral Session: OS24 EV Enrichment and Capture, Room 109-110, May 11, 2024, 4:00 PM - 5:35 PM

Introduction Colorectal cancer (CRC) poses a significant health threat, ranking high in both incidence and mortality. Recent studies have highlighted the crucial role of the gut microbiome in CRC development. Bacterial extracellular vesicles (BEVs), derived from gut bacteria, carrying proteins, lipids, and nucleic acids, actively engaging in inter-bacterial and bacteria-host communication, may potentially influence CRC pathology. Exploring BEVs derived from human feces is pivotal for understanding the heterogeneity of gut BEVs in CRC, demanding enhancement in isolation and purification strategies. Here, we optimized the isolation and purification process, applying this refined strategy to explore the diagnostic potential of BEVs in CRC.

Methods We employed BEVs from both gram-positive and gram-negative bacteria, as well as fecal BEVs (fBEVs), to refine isolation techniques using density gradient centrifugation (DGC) and size exclusion chromatography (SEC). Optimization involved evaluating separation strategies through assessments of particle morphology, size, concentration, protein content and specific markers (LPS, OmpA, LTA for BEVs). Advanced analytical methods, including TEM, NTA, NanoFCM, LC-MS/MS and WB, were utilized to determine optimal separation conditions. DNA from fecal bacteria and fBEVs in healthy individuals and CRC patients was PCR-amplified (16S rRNA gene v3-v4) and sequenced via Illumina MiSeq. A random forest model was constructed to gauge diagnostic efficacy via ROC analysis.

Results Our optimized approach revealed enriched BEVs distribution in DGC fractions 6-8 (F6-8), with F5 rich in eukaryotic EVs. A combined top-down DGC and SEC method proved most effective for clinical fBEVs isolation. Sequencing analysis of fecal bacteria and fBEVs from healthy individuals and CRC patients highlighted significant compositional differences. Notably, fBEVs displayed superior diagnostic potential over bacteria, with distinct variations at phylum and genus levels between healthy individuals and CRC patients.

Summary/Conclusion The refined methodology enables standardized isolation of BEVs, laying the groundwork for comprehensive multi-omics and functional analyses. The observed differences in fBEVs between healthy and CRC individuals suggest their potential as diagnostic markers, offering insights into the microbiome-cancer nexus.

Mr. Boyang Su, Dr. Morteza Jeyhani, Dr. Xiaojing Yang, Jina Nanayakkara, Reese Wunsche, Dr. Neil Renwick, Dr. Scott Tsai, Dr. Hon Leong

Introductory Talk and Oral Session: OS24 EV Enrichment and Capture, Room 109-110, May 11, 2024, 4:00 PM - 5:35 PM

Introduction: Extracellular vesicles (EVs) are cell fragments released by all cells, making them promising platforms for disease detection. Effective isolation of ultra-pure EVs for downstream analyses is key for EV-based liquid biopsy development. However, current EV isolation methods such as ultracentrifugation (UC) and other commercialized EV isolation kits are lackluster because of damage to EVs, length of time required, and low EV recovery rates. In response, we developed a “GET EV” kit based on a class of fluids called aqueous two-phase systems (ATPS), which can selectively partition EVs to particular fluid phases based on the surface properties of the lipids, to isolate EVs from any media.

Methods: EV concentration was measured by nanoscale flow cytometry (nFC) to compare EV enrichment and recovery capability of GET EV kit with other EV isolation methods. EV membrane biomarkers were determined by nFC after immunolabelling. Transmission electron microscopy (TEM) was performed to visualize EV morphology. Small RNA sequencing and proteomics were performed to determine EV molecular cargo profiles. In application, GET EV kit was used to isolate EVs from plasma samples of healthy volunteers and neuroendocrine neoplasm (NENs, a group of rare cancers) patients. RT-qPCR was performed to determine the expression of miRNA-375, a promising NEN biomarker, in patient plasma EVs.

Results: nFC revealed that ATPS has greater EV enrichment capability and higher EV recovery efficiency than UC. Other EV isolation kits were tested and were inferior to ATPS in terms of EV recovery (97.7%) and EV-RNA recovery (96.4%). EV subpopulations as determined by EV biomarkers (CD9, CD63, CD81, etc.) were confirmed by nFC. “Omics” studies also confirmed the presence of expected EV small RNAs and proteins. RT-qPCR demonstrated enriched expression of miR-375 in EVs isolated from lung and GI NEN patient plasma samples.

Summary/Conclusion: Our study establishes the use of GET EV kit to efficiently, rapidly, and gently isolate EVs from small amount of plasma samples (250 µL). This is a low-cost innovation that will finally enable the development of other RNA-based liquid biopsies for other disease sites with significant diagnostic and prognostic implications.

Colin Hisey, Xilal Rima, Colin Hisey, Chiranth Nagaraj, Sophia Mayone, Kim Nguyen, Sydney Wiggins, Chunyu Hu, Divya Patel, David Wood, Zachary Schultz, Derek Hansford, Eduardo Reategui

Introductory Talk and Oral Session: OS24 EV Enrichment and Capture, Room 109-110, May 11, 2024, 4:00 PM - 5:35 PM

Introduction: The role of extracellular vesicles (EVs) in human health and disease has garnered considerable attention over the past two decades. However, while several types of EVs are known to interact dynamically with the extracellular matrix (ECM), and there is great potential value in fabricating high-fidelity EV micropatterns beyond biomimicry, there are currently no label-free, high-resolution, scalable, and highly tunable platform technologies with this capability. We introduce Light-induced Extracellular Vesicle Adsorption (LEVA) as a disruptive and versatile EV micropatterning technique that will rapidly advance the study of ECM- and surface-bound EVs and other particles.

Methods: LEVA occurs when UV illumination is selectively applied to PLL/PEG-functionalized surfaces using a digital micromirror device (DMD), creating label-free regions with highly predictable EV binding affinity based on the grayscale values of input templates. LEVA's versatility is demonstrated using commercial GFP-EV standards, E. Coli outer membrane vesicles (OMVs), and EVs from U-87 MG glioblastoma bioreactors to create 2D micropatterns of the EVs on surfaces with different geometric shapes and gradients. The binding kinetics of LEVA were characterized using time-lapse total internal reflective fluorescence microscopy (TIRFM) and supercomputer-enabled COMSOL Multiphysics simulations for both small and large EVs. Following initial optimization, several applications were tested, including single EV characterization using TIRFM, tumor cell migration on migrasome-mimetic trails, and neutrophil activation and swarming by OMVs using time-lapse fluorescence microscopy. All EVs were characterized according to MISEV guidelines, including NTA, TEM, and immunoblotting.

Result: Time-lapse TIRFM and COMSOL simulations demonstrated slower adsorption kinetics of large EVs compared to small EVs. Successful LEVA patterning with consistent 2µm resolution enabled: (1) characterization of single EVs by TIRFM colocalization of fluorescent probes for CD63 and microRNA miR-21 (2) characterization of U-87 MG single cell migration behavior on “1D” U-87 MG migrasome-mimetic trails, and (3) characterization of OMV micropattern geometry-dependent influence on localized peripheral-blood derived neutrophil swarming.

Conclusion: LEVA's scalability, high-resolution, speed, and versatility will enable rapid advancements in the study of ECM- and surface-bound EVs and should encourage researchers from many disciplines to create novel diagnostic, biomimetic, immunoengineering, and therapeutic screening assays.

Mr. Jia Yi Voo, Dr. Sheng Yuan Leong, Dr. Rinkoo Dalan, Prof. Han Wei Hou

Introductory Talk and Oral Session: OS24 EV Enrichment and Capture, Room 109-110, May 11, 2024, 4:00 PM - 5:35 PM

1) Introduction

Extracellular vesicles (EVs) are nano-sized cell-derived particles that are key mediators of intercellular communication and emerging biomarkers for clinical diagnostics. However, the complexity of current EV isolation methods remains a huge challenge for clinical adoption which advocates an unmet need for more efficient EV isolation solutions. In this project, we report the development of a novel tangential flow filtration (µTFF) microfluidic device to isolate EVs from blood plasma.

2) Methods

The multi-layer polydimethylsiloxane (PDMS) microfluidic device is fabricated using soft lithography and a membrane layer (50 nm pore size) is sandwiched between the top and bottom microchannels. The top microchannel features a herringbone structure to induce mixing and enhance the protein filtration efficiency. Fluorescent imaging of polystyrene beads and bovine serum albumin (BSA) were used to characterize the working flow rates. Blood plasma samples were then tested to determine plasma protein depletion and EV separation performance using bicinchoninic acid (BCA) assay and nanoparticle tracking analysis (NTA), respectively.

3) Results

Fluorescent imaging showed that 50 nm polystyrene beads remained at the sample (top) channel while smaller plasma proteins were filtered through the membrane, thus indicating successful separation of EVs from proteins. The device achieved 20% EV recovery, 70% protein depletion and 70% volume reduction using diluted (1:20) platelet-poor plasma samples which could be further improved with channel geometry optimisation.

4) Summary/Conclusion

Overall, the µTFF microfluidic device facilitates high throughput (30 µL/min sample flow rate) EV isolation from blood plasma for on-chip fluorescent EV measurements due to the optical transparency of PDMS. Due to the large channel dimensions, the chip fabrication can be scaled up using plastic (e.g. PMMA or COC) materials to reduce production cost. We envision the µTFF module can be readily integrated with upstream microfluidic plasma or EV isolation modules, as well as downstream EV detection modules, towards developing a point-of-care EV isolation and sensing technology for clinical diagnostics.

Dr Ishmael Inocencio², Mr Naveen Kumar², A/Prof Rebecca Lim², Dr Tamara Yawno²

¹Hudson Institute Of Medical Research, Clayton, Australia, ²The Ritchie Centre, Clayton, Australia

Oral Session: Therapeutics (Late Breaking), Plenary 1, May 12, 2024, 10:30 AM - 11:30 AM

Introduction: Extracellular vesicles isolated from human amnion epithelial cells (hAEC-EVs) demonstrate low immunogenicity and potent anti-inflammatory and proangiogenic action. These properties have made hAEC-EVs an attractive potential fetal therapy as they address disease mechanisms that have no current therapies. A compromised fetus represents one of the most vulnerable human populations and determining the safety of potential therapeutic agents is of paramount importance for clinical translation. Chorioamnionitis describes bacterial infection of the fetal membranes and causes inflammatory organ injury, greatly increasing the risk of long-term disease. Several small animal studies demonstrate the therapeutic potential for chorioamnionitis-driven injury. However, physiological parameters such as blood pressure, heart rate and oxygen saturation, which are important indices of safety, cannot be easily measured in rodents.

Aim: Using sheep as a large animal model we aimed to evaluate the impact of intravenous hAEC-EV administration on fetal physiology and organ structure in an experimental setting of chorioamnionitis.

Methods: hAECs were cultured in chemically defined media. hAEC-EVs were isolated via tangential flow filtration and concentrated via size exclusion chromatography from conditioned media. Arterial, venous and amniotic catheters were surgically implanted in fetal sheep at 99/152 days of gestation (dGA) and connected to pressure transducers for continuous in-utero physiological recordings. Fetuses were randomly allocated to three groups: 1) control, 2) injury or 3) injury + treatment. Chorioamnionitis was induced in groups 2 and 3 via fetal exposure to IV lipopolysaccharide (LPS). Group 3 received IV hAEC-EVs. Group 1 (control) received saline. Fetuses were continuously monitored until 113 dGA. Fetuses were euthanised and brains and lungs were collected for histological analysis.

Results:

No significant differences were seen in blood pressure, heart rate and arterial oxygen saturation following hAEC-EV administration. Alveolar thickness and epithelial sloughing (indices of lung injury) and microglial activation (indices of neuroinflammation and injury) increased following the LPS challenge but decreased following hAEC-EV administration.

Conclusion: The stability of physiological parameters suggests acute fetal exposure to hAEC-EVs is safe. Decreased indices of brain and lung injury suggests therapeutic potential in the setting of experimental chorioamnionitis. Future studies will focus on the optimisation of dosage and timing of hAEC-EV administration.

Lecturer Cong He^1,2, Guangxin Shao², Dr. Yumin Li⁴, Dr. Xiao Yun⁵, Dr. Bo Sun⁴, Prof. Zhongdang Xiao⁴, Prof. Beicheng Sun³

¹Jiangsu Key Laboratory for Biofunctional Molecules, College of Life Science and Chemistry, Jiangsu Second Normal University, Nanjing, China, ²Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China, ³Department of Hepatobiliary Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, China, ⁴State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China, ⁵Department of General Surgery, The Affiliated Changzhou No. 2 People's Hospital of Nanjing Medical University, Changzhou, China

Oral Session: Therapeutics (Late Breaking), Plenary 1, May 12, 2024, 10:30 AM - 11:30 AM

Introduction: The immune-suppressive tumor microenvironment (TME) attenuates the effector functions of tumor infiltrating lymphocytes, which poses challenge in immunotherapy in HCC. As functional cell-to cell communicators, extracellular vesicles (EVs) are capable of inducing immune response by presenting antigen and co-stimulators.

Methods: In the study, we engineered EVs to deliver mRNA encoding co-stimulator OX40L specifically to GPC3 overexpressed HCC cancer cells via HN3-GPC3 axis for in situ regulation of TME and enhanced tumor killing efficacy. In our previous study, HN3 scFv has been fused with LAMP2b protein(N-terminal) to get specific targeting efficiency. Here we conjugated RNA binding protein LA7e to the C-terminus of LAMP2b, and inserted a Box C/D element into 3’-UTR of the OX40L gene, hypothesizing that transcripts encoding the OX40L could be incorporated into EVs via the interaction between the LA7e RNA binding protein and Box C/D element.

Results: Our data indicated the successfully construction of engineered EVs carrying OX40L mRNA with GPC3+ HCC cancer cell targeting ability while the expression of OX40L in targeted HCC cells was verified as well. Further, the expression of OX40L in targeted HCC cells promoted the secretion of cytokines in CD8+/CD4+T cells respectively and enhanced CD8+T-cell activation and proliferation in a co-culture model in vitro. Furthermore, engineered EVs treatment significantly reduced tumor growth in HCC tumor bearing mice by increasing the proportion of Teff and decreasing Treg.

Conclusion: Taken together, our findings confirmed engineered EVs could deliver costimulatory mRNA to modulate tumor microenvironment and hence increase the antitumor immune response, which foreshadows a natural and novel delivery system for immunotherapy in HCC in the future.

Dr. Feng Chen^1,2, Dr Tao-Tao Tang², Dr. Zhi-qing Chen², Prof. Zhong Wang¹, Dr Bi-Cheng Liu²

¹Tsinghua University, Beijing, China, ²Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, China

Oral Session: Therapeutics (Late Breaking), Plenary 1, May 12, 2024, 10:30 AM - 11:30 AM

1) Introduction: Sepsis is a fatal disease with high morbidity and mortality, and acute kidney injury is a common complication. Recent evidence suggests that inflammation, metabolic reprogramming and microcirculation dysfunction are the three basic mechanisms underlying the development of septic acute kidney injury (SAKI). Extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hucMSC-EVs) have the capacity for intercellular signaling communication, making them a novel therapeutic strategy for various diseases. However, the use of hucMSC-EVs in SAKI has been rarely reported. The aim of this study was to investigate the renal protective effects and mechanisms of hucMSC-EVs in sepsis.

2) Methods: hucMSC-EVs were isolated and purified by differential ultracentfugation and size exclusion chromatography, and then characterized by Nanoparticle Tracer Analysis, Transmission Electron Microscopy, Western blot and NanoFlow Cytometry. Sepsis mouse model was established by cecal ligation and puncture (CLP). hucMSC-EVs were injected into tail vein of mice at 0h, 24h and 48h after CLP, respectively. Mice were euthanized at 72h for sampling. The body weight and survival rate of mice were analyzed statistically. The organ distribution of hucMSC-EVs was observed by IVIS. The renal function, inflammatory response, glycolysis and activation of related signal pathways were evaluated by RNA sequencing, ELISA, Western blot, qRT-PCR, and immunohistochemistry/fluorescence etc.

3) Results: We successfully isolated the hucMSC-EVs and confirmed their characteristics. hucMSC-EVs improved SAKI and reduced mortality by delivering miR-125a-5p and miR-125b-5p, highly enriched miRNAs in the hucMSC-EVs. miR-125a-5p and miR-125b-5p collectively suppressed TNFR2 expression, inhibiting NF-κB signaling activation, resulting in downregulation of IL-1β, IL-6, and TNF-α, thus alleviating the inflammatory response. Additionally, hucMSC-EVs also exerted renal protective effects by inhibiting HK-2 expression to alleviate glycolysis in renal tubular epithelial cells.

4) Conclusion: Our study reveals the potential molecular mechanisms by which hucMSC-EVs improve SAKI, providing new insights for the prevention and treatment of SAKI.

Dr Lien Van Hoecke¹, Yanis Mouloud², Tobias Tertel², Prof Bernd Giebel², Prof Roosmarijn E Vandenbroucke¹

¹VIB-UGent, Gent (Zwijnaarde), Belgium, ²University Hospital Essen, University Duisburg-Essen, Essen, Germany

Oral Session: Therapeutics (Late Breaking), Plenary 1, May 12, 2024, 10:30 AM - 11:30 AM

Alzheimer's disease (AD) represents a profound neurodegenerative condition with a troubling increase in global prevalence. While recent FDA approval of monoclonal antibodies targeting Aβ offer some clinical benefits, the demand for improved therapies is urgent. Recognizing the inflammatory component in AD, and having demonstrated that mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) can suppress neuroinflammation in various preclinical disease models, we decided to explore the therapeutic impact of potent MSC-EV products to suppress symptoms in AppNL-G-F mice, a second generation murine model for AD. Aiming to increase the consistency of product activities and reduce impacts of inter and intra-donor heterogeneities as well as of aging processes of the EV releasing MSCs, we recently have immortalized human bone marrow-derived MSCs and expanded them at the monoclonal level. As confirmed in a neonatal murine hypoxia-induced neuroinflammation model, EVs prepared from conditioned media of such clonal immortalized MSCs (ciMSCs) retain neuroinflammation suppressive activities. Building on these findings, we now show the potential of these ciMSC-EVs to mitigate neuroinflammation in AD mice. We observed an improved performance on complex cognitive tasks after intranasal administration of ciMSC-EVs. Notably, this observation is pathologically associated with a reduction in amyloid-β pathology, a decrease in glial cell activation, and an alleviation of neuronal dysfunction. Additionally, we observed diminished infiltration of macrophage, neutrophil, and CD8+ T cells into the brain parenchyma. Next, we demonstrate that through intranasal administration of CRE-loaded ciMSC-EVs, they can deliver their cargo to various brain regions pivotal for AD pathology. This implies a direct impact of ciMSC-EVs on AD pathology within the brain. Overall, our study underscores the ability of appropriate ciMSC-EV products to mitigate neuroinflammation in the AD model, offering a promising therapeutic intervention for AD and potentially other neuroinflammation-associated neurodegenerative disorders.

Dr Ebony Monson¹, Miss Irumi Amarasinghe¹, Mr William Phillips², Dr Amy Baxter², Ms Camille Braganca¹, Ms Abbey Milligan², Dr Donna Whelan², Dr Eduard Willms², Professor Andrew Hill², Professor Karla Helbig¹

¹Department of Microbiology, Anatomy, Physiology & Pharmacology, La Trobe University, Melbourne, Australia, ²La Trobe Institute for Molecular Sciences, La Trobe University, Melbourne, Australia, ³Institute for Health and Sport, Victoria University, Melbourne, Australia

Oral Session: Biology and Pathology (Late Breaking), Eureka, May 12, 2024, 10:30 AM - 11:30 AM

Introduction

Lipid droplets (LDs) are intracellular lipid-filled vesicles. We have previously demonstrated that LDs play vital roles in the production of effective host immune responses against viral infection, and now have evidence that they transfer cargo between cells, impacting immune responses. LDs share characteristics with extracellular vesicles (EVs) such as their size, density, and also protein and lipid cargo. Here, we explore a novel role for LDs, as extracellular communicators that interact with EVs during viral infection.

Methods

Primary immortalised astrocyte cell lines were created to fluorescently express PALM-GFP (tagging EVs) coupled with PLIN-2-mCherry (tagging the main LD protein). EV isolations were performed from the media of astrocyte cells infected with zika virus, using size exclusion chromatography. Confocal, super-resolution microscopy, and molecular techniques were used to evaluate antiviral effects.

Results

Using the PALM-GFP/ PLIN-2-mCherry astrocyte cells, we were able to demonstrate that LDs can move between cells. The presence of LDs was also observed in EV isolates, which was further quantified using a BD FACS Symphony A3 analyser with a small particle module to define that 1-3% of total particles were found to have LD markers. Additional confocal experiments demonstrated that a small population of LDs were also packaged inside small EVs, as well as being present within apoptotic bodies following apoptosis of THP-1 monocytes. Proteomics, western blotting, and single-molecule localization microscopy identified multiple ESCRT pathway proteins (TSG-101 and Alix) present on the surface of LDs. To understand if LDs impact surrounding cells following secretion, fluorescent LDs isolated from cells that had activated innate immune responses (via dsRNA viral mimic stimulation) were placed onto naïve cells prior to ZIKV infection. This treatment of LDs significantly increased type I and III interferon responses resulting in a 50% decrease in ZIKV replication.

Conclusion

Collectively this data may suggest that viral infection drives the secretion of LDs, facilitated by EVs, to act as biocommunicators between cells. A better understanding of this relationship and the role LDs and EVs collectively play in viral infection may offer novel biological insights into how cells communicate to effectively control viruses.

Dr Luize Lima¹, Dr Sunyoung Ham^1,2,3, Professor Andreas Möller^1,2,3

¹Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, Australia, ²Department of Otorhinolaryngology, Head and Neck Surgery, Chinese University of Hong Kong, Shatin, Hong Kong, ³School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia

Oral Session: Biology and Pathology (Late Breaking), Eureka, May 12, 2024, 10:30 AM - 11:30 AM

Introduction: Tumour-derived small extracellular vesicles (sEVs) are implicated in the guidance of cancer cell metastatic dissemination, with their specific surface composition determining some aspects of organotropism. However, whether and how the tumour microenvironment modulates cancer sEV biodistribution has yet to be understood.

Methods: sEVs were isolated from the conditioned media of breast cancer cell cultures in vitro, as well as from the plasma of breast cancer patients. Cytokines were either purchased as recombinant proteins or obtained as a milieu from sEV-depleted tumour interstitial fluids (TIF), prepared from syngeneic orthotopic cancer masses. TIF showed a range of cytokines and growth factors, including CCL2, mimicking the complexity of the tumour microenvironment. To assess the role cytokines play in cancer sEV organ-specific localization and function, cytokine-conjugated, fluorescently labelled cancer cell-derived sEVs were intravenously injected into syngeneic mice. Further injection of syngeneic breast cancer cells allowed for the assessment of metastatic burden.

Results: Cytokines present in the tumour microenvironment bound to breast cancer sEV surfaces via proteoglycans. These CCL2+ sEVs were selectively taken up by cells that express the CCL2 receptor CCR2 in specific tissues such as the lungs, demonstrating that sEV-bound cytokines are critical for sEV-target cell interactions. sEV accumulation resulted in changes in the immune landscape within these organs, which was associated with an increase in metastasis.

Summary: This study describes a novel mechanism of organotropic metastasis mediated by the interaction between tumour microenvironment cytokines and cancer sEVs, and their consequent uptake in specific organs and cell lineages. Understanding these mechanisms is vital for deciphering how cancer cells manipulate their microenvironment and how they communicate with distant cells, which is critical for developing targeted therapies and diagnostic tools, potentially impacting cancer treatment strategies.

Phd Anran Shen^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}, Phd Xin Zhong^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}, Phd Ning Li^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}, Phd Yuqi Fu^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}, Professor Linli Lv^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}

¹Medical School Of Southeast University, Nanjing, China

Oral Session: Biology and Pathology (Late Breaking), Eureka, May 12, 2024, 10:30 AM - 11:30 AM

Introduction: Chronic kidney disease (CKD) is characterized with progressive fibrosis caused by excessive extracellular matrix (ECM) deposition. Tubular epithelial cells secret increasing exosomes into extracellular space under pathological conditions. This study aims to investigate the movement of tubular-derived exosomes in the ECM microenvironment, exploring the potential mechanism of exosomal integrin in the activation of latent TGF-β1.

Methods: A unilateral ureteral obstruction (UUO) fibrosis mouse model and an ITGβ6 overexpression cell line were established to observe the effect of ITGβ6+ exosomes in vitro. Elastic or stress-relaxing hydrogels based on Polymethyl Methacrylate (PMMA) and acrylic acid-WGG(KA)3-heparin were constructed to mimic ECM under normal and fibrotic conditions with different stiffness (2kPa, 50kPa). Exosomes mobility and retention capability in hydrogels were tracked by single particle tracking (SPT). Exosomal integrin β6 and exosome secretion inhibitor, GW4869, was applied to clarify their role in latent TGF-β1 activation and renal fibrosis.

Results: In UUO model, immunofluorescence staining showed latent TGF-β1 localization in the renal tubular interstitial which was activated with fibrosis progression. Meanwhile, we observed increasing integrin β6 expression in tubule and interstitial area with the progression of renal fibrosis. Impressively, tubular exosomes carried higher amount of integrin β6 molecules per vesicle, as demonstrated via single exosome analysis by Nanoimaging(Oxford). Interestingly, exosomes diffused through hydrogels, while maintaining their morphology and size. Moreover, lower stiffness stress-relaxing hydrogels exhibited better retention of exosomes while diffusion rate increased in ECM with higher stiffness as demonstrated in transwell experiments analyzed by SPT. Considering the critical role of integrin in latent TGFβ activation, we propose integrin β6 exosome may exert such effect through residing inside ECM microenvironment. Impressively, purified ITGB6+ exosomes significantly increased TGF-β1 activation in TGF-β1 overexpressed HK-2 cell, while GW4869 treatment reversed such activation remarkably.

Conclusion: We demonstrated that tubular epithelial cell derived exosomal integrin β6 diffused in ECM actively influenced by the stiffness of ECM which promoted latent TGF-β1 activation. Exosomal integrin β6 may represent a novel manner of TGF-β1 activation which may become a new therapeutic target for renal fibrosis.

Dr Yiyao Huang^1,2, Ahmed Abdelgawad³, Dr Olesia Gololobova¹, Zhaohao Liao¹, Xinyu Cong⁴, Associate Professor Mona Batish³, Prof. Lei Zheng², Dr Kenneth Witwer^1,5,6

¹Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, United States, ²Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China, ³Department of Medical and Molecular Sciences, University of Delaware, Newark, United States, ⁴Division of Biostatistics and Bioinformatics, Department of Environmental and Public Health Sciences, University of Cincinnati, Cincinnati, United States, ⁵Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, United States, ⁶Richman Family Precision Medicine Center of Excellence in Alzheimer's Disease, Johns Hopkins University School of Medicine, Baltimore, United States

Oral Session: Biology and Pathology (Late Breaking), Eureka, May 12, 2024, 10:30 AM - 11:30 AM

1) Introduction: Similar to retroviruses, EVs can transport molecules like RNAs between cells. HIV manipulates host RNA splicing to control its gene expression, altering RNA levels in host cells and EVs. In the macaque model of HIV disease, we found an unreported association of plasma EV U6 small nuclear RNA (snRNA) with virus peak in acute infection. As a spliceosome component primarily located in the nucleus, U6 snRNAs only transiently assemble with ribonucleoproteins in the cytoplasm. There is limited knowledge of U6 snRNA shuttling between the nucleus, cytoplasm, and EVs, especially during SIV infection. We thus aimed to explore the association of U6 snRNA and its related protein machinery in cellular fractions and EVs.

2) Methods: SIV B670 and SIV 17E-Fr were used to infect CEMx174 cells. Cell culture medium was collected for EV separation by size exclusion chromatography and ultrafiltration 3 days post-infection. Medium processing controls were included in parallel to evaluate possible contamination originating from culture media components. EVs and MCs were thoroughly characterized per MISEV2018. SIV-infected and -uninfected CEMx174 cells were collected and fractionated into cytoplasmic and nuclear fractions. RT-qPCR was used to evaluate U6 and U1 snRNA levels in EVs, cytoplasm, and nucleus. Protein analytes of EVs, cytoplasm, and nucleus were detected by nanoflow, proteomics, and Western blot.

3) Results: During HIV infection, we observed a distinct pattern of U6 snRNA distribution, characterized by a decrease in the nucleus and an increase in EVs. Distinct protein profile patterns were also seen in these three fractions during SIV infection. Moreover, alterations were identified in proteins associated with spliceosome pathways within both the SIV-infected nucleus and EVs. U6 machinery-related proteins were also changed in EVs and cells after SIV infection. Intriguingly, these alterations occur in a contrasting manner within the nucleus and EVs.

4) Summary: The apparent translocation of U6 snRNA from the nucleus to EVs during HIV infection appears to be influenced by both intracellular U6 snRNA concentration and proteins associated with the splicing machinery. The alterations of U6 snRNA and RNA splicing-related proteins point towards potential regulatory roles, possibly also establishing distinctive signatures for retroviral infection.

Master's degree Kim Seungmin, Doctor of Philosophy (Ph.D.) ByeongHyeon Choi, Hyunku Shin, Master's degree Kihun Kwon, Doctor of Philosophy (Ph.D.) Sung Yong Lee, Doctor of Philosophy (Ph.D.) Hyun Koo Kim, Doctor of Philosophy (Ph.D.) Yeonho Choi

¹Department of Biomedical Engineering, Korea University, Seoul, South Korea, ²Korea Artificial Organ Center, Korea University, Guro, Republic of Korea, ³Department of Thoracic and Cardiovascular Surgery, Korea University, Guro, Republic of Korea, ⁴Exopert Corporation, Seoul, Republic of Korea, ⁵Department of Internal Medicine, Korea University, Guro, Republic of Korea, ⁶School of Biomedical Engineering, Korea University, Seoul, Republic of Korea

Oral Session: Disease Biomarkers (Late Breaking), Room 105-106, May 12, 2024, 10:30 AM - 11:30 AM

This abstract is about a novel technique for detecting protein mutations using liquid biopsy, by analyzing plasma exosomes through nanoplasmonic spectra and deep learning

Introduction: Exosomes, which are abundant in plasma and carry intact proteins from their cells of origin, serve as a promising biomarker. This technique proved particularly effective in distinguishing mutations in the epidermal growth factor receptor (EGFR) among healthy controls (n = 17), non-small cell lung cancer (NSCLC) patients with wildtype EGFR (n = 14) and mutated EGFR (n = 20). It aims to monitor the mutation status, including major EGFR mutations such as L858R, E19del, L858R + T790M, and E19del + T790M.

Methods: The research team acquired molecular information about the structural changes in

Mutated proteins from exosomes using Raman spectra. To extract unique features of the mutated proteins from the complex Raman spectra, a classification algorithm was developed, incorporating two deep learning models. The algorithm was built with two steps for the differential diagnosis of wild-type vs mutations and primary mutation (E19del, L858R) vs secondary mutation (+T790M).

Results: Our method was validated using three different steps involving cells, patients’ plasma, and monitoring of patients. First, the model was used to classify the wild-type protein and mutated protein from cell-derived exosomes with high accuracy (AUC 0.99). Second, the patients with mutated proteins (n = 23) were identified from non-mutated individuals with AUC 0.85. Lastly, we clearly confirmed the potential of early detection by performing periodic monitoring tests. We followed up the patients with primary mutation to detect the differences when secondary mutation occurs and found that the mutation value was 2.47-fold higher when secondary mutation occurs.

Summary and Conclusions: This research proposes a new method to enhance the diagnostic accuracy of protein mutation detection through liquid biopsy. Combining nanoplasmonic spectra and deep learning, this technique is expected to be particularly useful for the diagnosis and treatment monitoring of NSCLC patients with EGFR mutations. Being non-invasive, this method represents a significant advancement in overcoming the current limitations of liquid biopsy-based mutation diagnostics, offering a novel approach for companion diagnostics.

Dr Samantha Emery-Corbin^{1. Division of Advanced Technology and Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia 2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia}, Dr Jumana Yousef^{1. Division of Advanced Technology and Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia 2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia}, Professor Yugeesh R Lankadeva^{3. The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia 4. Department of Critical Care, University of Melbourne, Parkville, Victoria, Australia}, Professor Rinaldo Bellomo^{4. Department of Critical Care, University of Melbourne, Parkville, Victoria, Australia 5. Department of Intensive Care, Austin Hospital, Melbourne, Victoria, Australia 6. Australian and New Zealand Intensive Care Research Centre (ANZIC-RC), Monash University, Melbourne, Victoria, Australia 7. Department of Intensive Care, Royal Melbourne Hospital, Parkville, Victoria, Australia 8. Data Analytics Research and Evaluation Centre, Austin Hospital, Melbourne, Victoria, Australia}, Dr Fumitaka Yanase^{5. Department of Intensive Care, Austin Hospital, Melbourne, Victoria, Australia 6. Australian and New Zealand Intensive Care Research Centre (ANZIC-RC), Monash University, Melbourne, Victoria, Australia}, Associate Professor Mark P Plummer^{9. Department of Intensive Care, Royal Adelaide Hospital, Adelaide, South Australia, Australia}, Professor Clive N May^{3. The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia 4. Department of Critical Care, University of Melbourne, Parkville, Victoria, Australia}, Dr Laura F Dagley^{1. Division of Advanced Technology and Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia 2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia}

¹Division of Advanced Technology and Biology Division, Walter And Eliza Hall Institute Of Medical Research, Melbourne, Australia, ²Department of Medical Biology, University of Melbourne, Melbourne, Australia, ³The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Australia, ⁴Department of Critical Care, University of Melbourne, Melbourne, Australia, ⁵Department of Intensive Care, Austin Hospital, Melbourne, Australia, ⁶Australian and New Zealand Intensive Care Research Centre (ANZIC-RC), Monash University, Melbourne, Australia, ⁷Department of Intensive Care, Royal Melbourne Hospital, Melbourne, Australia, ⁸Data Analytics Research and Evaluation Centre, Austin Hospital, Melbourne, Australia, ⁹Department of Intensive Care, Royal Adelaide Hospital, Adelaide, Australia

Oral Session: Disease Biomarkers (Late Breaking), Room 105-106, May 12, 2024, 10:30 AM - 11:30 AM

Introduction: Sepsis causes 30-50% of all in-hospital deaths (∼11 million deaths annually). Current care is palliative, therefore, new, effective treatments are required. Trials of megadose sodium ascorbate (NaAsorbate) in pre-clinical ovine models reduced vasopressor support, restored renal function, and reversed acute kidney injury (1), but its molecular mechanism remains unknown. To investigate this, we evaluated circulating extracellular vesicles (cEVs) from a Phase 1A, single-dose, double-blind, randomised controlled trial (2) of a single intravenous megadose NaAsorbate (60g) compared to placebo in patients with septic shock (n = 18).

Methods: Plasma was collected pre-treatment (0 hr), and 1, 4, 6 and 24 hrs post-treatment or placebo (n = 90). We evaluated two high-throughput, magnetic-bead workflows for plasma-derived, cEVs, including size-exclusion and antibody-based mag-beads (ExoNet, INOVIQ) and SAX-based mag-beads (MagNet, ReSynBio (3)), alongside neat plasma. Technical replicates and pooled controls were included for plasma, whereas cohort pools from patients across timepoints based on sepsis source and treatment/placebo were used for cEV controls. Neat plasma and cEV fractions were processed for LC-MS/MS analysis via on-bead enzymatic digestion (4), analysed on a timsTOFpro mass spectrometry (Bruker) with diaPASEF on a 30-min analytical gradient, and data were searched library-free in DIA-NN (5). We performed pairwise (limma) and continuous (maSigPro, moanin) statistical analyses to evaluate molecular progression of sepsis between placebo and NaAscorbate.

Results: After pre-processing and data filtration, a non-redundant 2231 total proteins were identified across all methods (599 Plasma, 762 MagNet, 1921 ExoNet), of which 320 (14%) were common across methods and 1254 (56%) uniquely identified in ExoNet. Patient heterogeneity, including source of sepsis infection, contributed to inter-patient and intra-patient (timepoint) variation in cEV data, although cEV technical controls (cohort/patient pools) shared Pearson correlations > 0.9. After normalisation, principal component analysis (PCA) separated placebo and treatment on the PC1. Evaluation of EV-specific markers demonstrated reproducible identification in ExoNet, whereas key markers had been filtered in MagNet (CD63, CD81) owed to differences in missingness between the two cEV datasets.

Summary/Conclusions: Together, these data will provide a molecular reference for high-throughput cEV methods and human sepsis pathology, and will critically inform approaches for upcoming Phase 1B clinical trials.

Senior Staff Scientist Dmitry Ter-Ovanesyan¹, Sara Whiteman, Tal Gilboa, Siddharth Iyer, Bogdan Budnik, Aviv Regev, George Church, David Walt

¹Wyss Institute, Harvard University, Boston, United States

Oral Session: Disease Biomarkers (Late Breaking), Room 105-106, May 12, 2024, 10:30 AM - 11:30 AM

Isolating neuron-specific EVs from human biofluids such as plasma and measuring their cargo represents an exciting approach for understanding the state of neurons and diagnosing neurological disease. Achieving this goal, however, is incredibly challenging. First, a neuron-specific marker is needed for immuno-isolation and it is important to verify this marker is truly on EVs and not present as a soluble protein. Second, it is important to verify that the protein cargo measured after immuno-isolation is truly in EVs. We are working on developing methods to address both of these challenges. Most previous attempts to isolate neuron-derived EVs have relied on immuno-isolation of L1CAM, which we have previously found to be a free protein in CSF and plasma. Thus, new candidate markers are needed. We developed an unbiased pipeline for the identification of cell-type specific EV markers based on gene expression and EV proteomics data and applied it to prioritizing novel handles for the isolation of neuron EVs. Additionally, we developed methods to differentiate whether a protein is in plasma EVs and quantify the ratio of a protein in EVs to total plasma. To achieve this, we combined a high-yield size exclusion chromatography (SEC) protocol with an optimized protease protection assay and Single Molecule Array (Simoa) digital ELISA assays for ultrasensitive measurement of proteins inside EVs. We applied these methods to analyze key proteins involved in neurodegenerative diseases: α-synuclein, Tau, Aβ40, and Aβ42. Using immunoassays against several of the predicted neuron-specific proteins, we confirmed one marker as present on EVs in CSF and plasma by size exclusion chromatography (SEC) and density gradient centrifugation (DGC). Finally, we developed efficient EV immuno-isolation methods and applied them to isolate neuron EVs using this marker. Our work provides a framework for identifying neuron-specific EV markers and determining the levels of proteins in both total and neuron EVs, which we hope will enable the use of EVs as biomarkers for neurological disease.

Dapi Menglin Chiang, Dr. Christina Ludwig, Dr. Chen Meng, PD Dr. Marlene Reithmair, Laura Benecke, Yannik da Silva, PD Dr. Laurent Müller, Prof. Dr. Michael W. Pfaffl

Oral Session: Disease Biomarkers (Late Breaking), Room 105-106, May 12, 2024, 10:30 AM - 11:30 AM

Head and neck squamous cell carcinoma (HNSCC) significantly impacts patient's quality of life, especially in advanced stages. Although surgery is the primary method for removing HNSCC from the oral cavity, it is crucial to determine the presence of tumor-related biomarkers in the local area or circulation. Therefore, the discovery of HNSCC related proteins in liquid biopsy is desirable and their potential as prognostic or diagnostic biomarkers needs to be examined. Since extracellular vesicles (EVs) contain mostly abundant, cellular information in the circulation, this study aims to investigate intraoperatively the EV protein biomarker signature before (BO) and after operation (AO) in HNSCC patients.

Therefore in-house developed and optimized galectin-based glycan recognition particles (EXÖBead) isolation, high-resolution liquid chromatography mass spectrometry-based proteomics (HRLC-MS/MS) and functional assays were applied to discover a valid EV biomarker signature. Patients platelet-poor plasma (PP) was collected from peripheral venous blood, before (BOPP) and one hour after tumor removal surgery (AOPP). Comparably local tumor venous plasma (TV) was collected before (BOTV) and after tumor removal surgery (AOTV) in parallel.

HRLC-MS/MS proteomics result showed that 14 proteins, including platelet derived growth factor subunit B (PDGFB), were significantly higher in BOPP compared to AOPP. Furthermore, PDGFB derived from BOTV plasma EVs showed a significant increment compared to AOTV. Gene ontology enrichment analysis showed that the identified proteins were significantly related to ‘cancer pathways’, ‘positive regulation of cell proliferation’ and ‘cell migration’. EV sizes and morphology, determined by NTA and Cryo-TEM, were comparable in all 4 groups, but showed no differences. Treatment with BOTV plasma EVs in HNSCC cell line (UPCI-SCC-016) showed a significantly increment in cell proliferation compared to no treatment.

These results suggest that cancer ablative surgery removed majority of tumor-related EVs in peripheral and local biopsies measurable intraoperatively at the end of the surgery. PDGFB derived from plasma EVs may benefit the tumor growth and its decrease at the end surgery indicates the removal of cancer tissue and point to a successful oncological treatment. Overall PDGFB in blood EVs could be a powerful biomarker during cancer ablation and guide the extent of surgery.

Section Chief Ryuichi Ono¹, Mie Naruse², Makiko Kuwagata¹, Yusuke Yoshioka³, Yoko Hirabayashi¹, Takahiro Ochiya³, Masahito Ikawa⁴, Satoshi Kitajima¹

¹National Institute Of Health Sciences, Japan, Kawasaki, Japan, ²National Cancer Center Research Institute, Tokyo, Japan, ³Tokyo Medical University, Tokyo, Japan, ⁴Osaka University, Osaka, Japan

Oral Session: Techniques (Late Breaking), Room 109-110, May 12, 2024, 10:30 AM - 11:30 AM

1) Introduction

While extracellular vesicles (EVs) are known to circulate in the bloodstream, originating from various organs, the proportion of EVs derived from the liver remains unclear. In this study, we created mice expressing human CD9-mEmerald specifically in hepatocytes by generating Cre recombinase-inducible human CD9-mEmerald knock-in mice at the Rosa26 locus and mating them with Alb-Cre transgenic mice. We then administered hepatotoxic substances, carbon tetrachloride, and a vehicle control to induce liver injury. By detecting human CD9 in the bloodstream, we aimed to observe the dynamics of liver-derived EVs.

2) Methods

Male Cre-inducible human CD9-mEmerald knock-in mice (flox) were crossed with female Alb-Cre mice to generate flox/Alb-Cre mice and WT/Alb-Cre mice. At 10 weeks of age, a single dose of carbon tetrachloride (70 mg/kg) or vehicle control (corn oil) was administered. Serum was collected 24 hours later for blood biochemical analysis using AST and ALT. Additionally, serum was analyzed for human CD9-positive EVs using the EXOVIEW IMAGER with human CD9 antibodies. EVs were isolated from serum using ultracentrifugation, and particle number and size analysis were performed using Nanosight. Western blot analysis using human CD9 and mouse CD9 antibodies was also conducted.

3) Results

Elevated levels of AST and ALT were observed only in the carbon tetrachloride-treated group. In the vehicle control group, less than 1% of EVs in the serum were positive for human CD9, whereas in the carbon tetrachloride-treated group, the proportion of human CD9-positive EVs increased to 7%. The increase in human CD9-positive EVs was also confirmed by Western blot analysis using human CD9 antibodies.

4) Summary/Conclusion

Even in the liver, the largest organ, liver-derived EVs in the bloodstream comprise less than 1% of circulating EVs. However, liver injury induced by carbon tetrachloride administration increased the proportion of liver-derived EVs in the bloodstream to 7%. This suggests a significant alteration in the presence of liver-derived EVs in response to liver damage. Further investigations are planned to determine when the increase in EVs occurs after carbon tetrachloride administration and whether similar results are obtained in other organ toxicities.

Dr Alin Rai¹, Prof David Greening

¹Baker Heart and Diabetes Institute, MELBOURNE, Australia

Oral Session: Techniques (Late Breaking), Room 109-110, May 12, 2024, 10:30 AM - 11:30 AM

Introduction: Extracellular vesicles (EVs) are now being increasingly recognized as an essential signalling entity in human plasma, linking them to health and various diseases. Still, their core protein and lipid componentry, which epicentres EV form and function, remains poorly defined. Achieving this unmet milestone remains greatly hindered by abundant non-EV components in mass spectrometry-based analyses.

Method: Here, we employed high-resolution density gradient separation of over 150 human plasma samples to isolate a major EV sub-type called small EVs. We verify EV enrichment and identity using various biochemical and biophysical characterization, ensuring high degree of separation of EVs and non-EV particles. We then systematically construct their high-confident quantitative proteome (>5000 proteins) and lipidome (829 lipids) maps using mass spectrometry.

Results: We discovered a highly conserved panel of 182 proteins (ADAM10/STEAP23/STX7) and 52 lipids (PS/PIPs/Hex2Cer/PAs), providing a deep survey of hallmark features and biological pathways of circulating EVs, and their conservation in different EV sub-populations (CD81+/CD63+/CD9+) in the circulatory system. We also map the surfaceome diversity, identifying 126 proteins conserved on EV surface. This multi-omics investigation also unravels nanoscale lipid-raft assemblies of core proteins and lipids conserved in circulating EVs. We further establish a set of 42 proteins and 114 lipids features that serve as hallmark features of non-EV particles; importantly, using ensemble machine learning, we submit ADAM10 and PS(36:1) as conserved EV biological markers that enable precise differentiation between EV and non-EV particles, and can be used to directly track EV isolation in plasma and assess their level of purity. Our pipeline also provides extensive coverage of low-abundant disease proteins in circulating EVs in a disease cohort, revealing novel insights into coronary artery pathology.

Conclusion: We provide a first high-confident proteome and lipidome drafts of circulating EVs in human, identifying universal protein and lipid features. These findings can be explored via open-source, freely available Shiny web tool, and serve as a valuable repository to the research community for a clearer understanding of circulating EV biology.

Dr Hema Saranya Ilamathi^1,2, Dr Doste Mamand^1,2, Anna Maria Zimbo³, Dr Samir El Andaloussi^1,2, Dr Oscar Wiklander^1,2

¹Division of Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden, ²Center for Cell Therapy and Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital, Stockholm, Sweden, ³Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Italy

Oral Session: Techniques (Late Breaking), Room 109-110, May 12, 2024, 10:30 AM - 11:30 AM

Introduction

Extracellular vesicles (EVs) are nanovesicles with potential disease diagnosis and treatment applications. There are two challenges to using EVs for therapeutic applications: targeting them to the point of action and efficient delivery of drug load inside the cell. Our group recently showed that efficient cancer targeting can be achieved by engineering EVs with Fc fragments to decorate their surface with antibodies. EVs are generally taken up by the cell through the endocytic pathway. However, the endocytic system limits the successful delivery of drugs or other cargo inside the cell cytosol. Viral fusogens are commonly used to facilitate endosomal escape, but they are associated with an increased risk of the immune response. This work aims to improve the endocytic escape of engineered therapeutic-carrying EVs.

Methods

Customized vectors carrying tetraspanin fused to positive or amphipathic-charged amino acid sequences are designed by recombinant technology. We transfected HEK cells with different customized vectors and isolated EVs from these cells by tangential flow filtration. The net charge of EVs was measured by dynamic light scattering. EV uptake was then measured by flow cytometry based on the mNeonGreen signal. To monitor endosomal escape, we used luciferase-based HEK reporter cells. Engineered EVs were electroporated with splice-switching oligonucleotides to restore luciferase expression in reporter cells.

Results

Our data demonstrates that cellular EV uptake was increased with amphipathic amino acid sequence compared to EVs carrying positively charged amino acids. Furthermore, we show amphipathic-charged EVs efficiently escape endosomes. Amphipathic-charged EVs demonstrated over 15- and 10-fold efficiency over unmodified and positively charged EVs respectively. Our work demonstrates that altering the charge and lipophilicity could facilitate efficient endosomal escape and successful delivery of cargo molecules.

Conclusion

Our work demonstrates that designing EVs with higher positive charge and lipophilicity promotes efficient escape from the endosomal compartment. We predict that the acidic environment of endosomal further enhances the net positive charge of EVs and the lipophilic region of EVs fuses with the endosomal membrane and releases cargoes in the cytosol. In the future, we aim to address this mechanism and test the efficacy of amphipathic EVs for the targeted delivery with antibodies.

Dr. Andrea Capuano¹, Meia Numan², Prof. Thomas Hankemeier¹

¹University of Leiden, Leiden, The Netherlands, ²EXIT071 B.V., Leiden, The Netherlands

Oral Session: Techniques (Late Breaking), Room 109-110, May 12, 2024, 10:30 AM - 11:30 AM

Introduction

While techniques like ultracentrifugation and microfiltration are commonly used to ensure high purity for extracellular vesicles (EVs), separation strategies based on electrophoretic mobility, particularly depletion-zone isotachophoresis, can offer superior purity. This is because EVs possess a unique electrophoretic mobility that is based on charge and size, a characteristic that can be leveraged to separate them from charged contaminants in sample preparation.

Methods

By leveraging the depletion-zone isotachophoresis (dzITP) principle, we employ a method that replaces the trailing electrolyte commonly used in isotachophoresis with an ion-depleted zone, creating a barrier that anions cannot cross. When an external electric field is applied, negatively charged analytes trapped between the ion-depleted zone and the leading electrolyte are, therefore, concentrated, depending on their electrophoretic mobility, in bands that occupy different positions in the microfluidic channel, thus mirroring the capabilities of classical isotachophoresis. DzITP is implemented on a novel silicon-and-glass-based microchip that is manufactured by our partner SINTEF (Oslo, Norway).

Results

We demonstrate the simultaneous concentration and separation of exosomes derived from cell cultures (HEK 293 and human neuroblastoma cell lines) and blood plasma. Interestingly, although the EV samples used in this study are pre-purified by density- and size-based techniques (e.g., tangential flow filtration and ultracentrifugation), dzITP is capable of further separating them from contaminants such as proteins or residues of cleaved EVs. In fact, after about 70 minutes, the EVs in the microfluidic channel are concentrated in a separate zone from the sample contaminants. Moreover, following the assessment (fluorescence-based) of the bandwidth of the EVs while being separated, around a million-fold concentration is achieved in the device, in line with comparable devices described in the literature.

Conclusion

To summarize, in this work, using dzITP, we show that even when pretreated with ultracentrifugation and tangential flow filtration, EV samples (from both cell cultures and blood plasma) still contain contaminant residues, which have higher electrophoretic mobility than EVs, and that our technique can spatially separate them. Therefore, our device can further purify EVs with respect to state-of-the-art methods while concentrating the sample by a million-fold factor.