Methods in cell biologyPub Date : 2025-01-01Epub Date: 2024-06-17DOI: 10.1016/bs.mcb.2024.05.006
Raquel Ibáñez-Pérez, Alberto Anel
{"title":"Human cancer cells xenografts to assess the efficacy of granulysin-based therapeutics.","authors":"Raquel Ibáñez-Pérez, Alberto Anel","doi":"10.1016/bs.mcb.2024.05.006","DOIUrl":"10.1016/bs.mcb.2024.05.006","url":null,"abstract":"<p><p>9-kDa Granulysin is a protein present in the granules of human activated cytotoxic T lymphocytes and natural killer cells. It has been shown to exert cytolytic activity against a wide variety of microbes: bacteria, fungi, yeast and protozoa. Recombinant isolated granulysin is also capable of inducing tumor cell death, so it could be used as an anti-tumor therapy. Our group has developed granulysin-based immunotoxins in order to target granulysin to tumor cells in vivo. We describe in this chapter the suitable animal model used for testing these immunotoxins against human tumor cells in preclinical assays. This method consists in the xenotransplantation of a given number of human tumor cells subcutaneously in nude mice of the Swiss nu/nu strain or homozygous for the nude gene. Nude mice are immune-deficient, with a very reduced number of T cells, being unable to reject efficiently allo- or xeno-transplanted tissues. Using this approach we follow tumor growth in the different experimental conditions assayed, in the presence or absence of treatment with granulysin alone or with the tumor-directed immunotoxins. We also estimate in this model the possible adverse effects of the treatment in the absence of tumor development. Finally, after sacrifice of the experimentation animals, we use several immunohistochemical assays to study the effect of the treatment on the tumors and the presence of apoptotic cell death markers in the tumor tissue.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"192 ","pages":"83-99"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039853","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2025-01-01Epub Date: 2025-02-06DOI: 10.1016/bs.mcb.2025.01.007
Markus Ausserhofer, Dietmar Rieder, Francesca Finotello
{"title":"Comprehensive prediction of tumor neoantigens with nextNEOpi.","authors":"Markus Ausserhofer, Dietmar Rieder, Francesca Finotello","doi":"10.1016/bs.mcb.2025.01.007","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.01.007","url":null,"abstract":"<p><p>Immunotherapy has revolutionized cancer treatment by harnessing the immune system to target tumor cells expressing neoantigens. Neoantigens are peptides arising from tumor-specific aberrations that are presented by cancer cells and recognized by T cells. The computational prediction of cancer neoantigens from somatic mutations and other tumor-specific aberrations using patients' sequencing data is key for the investigation of anticancer immune responses and for the design of personalized immunotherapies. However, neoantigen prediction requires the implementation of complex computational pipelines to distill large-scale information from RNA and DNA sequencing data and derive neoantigen candidates together with associated features for their prioritization and selection. We previously developed nextNEOpi, a comprehensive and stand-alone bioinformatics pipeline that not only predicts class-I and -II neoantigens and fusion neoantigens, but also sheds light onto the tumor-immune cell interface, quantifying neoantigen clonality, immunogenicity, and tumor-specific metrics like tumor mutational burden and immune-cell receptor repertoire diversity. In this chapter, we showcase the main capabilities of the nextNEOpi pipeline by analyzing genomic and transcriptomic data generated from multiple biopsies collected from patients with lung cancer.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"113-137"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667964","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2025-01-01Epub Date: 2025-02-13DOI: 10.1016/bs.mcb.2025.01.010
Shih-Chun Shen, James B DuHadaway, Arpital Mondal, Souvik Dey, Alexander J Muller
{"title":"Matrigel implants embedded with IDVCs (IDO1-dependent vascularizing cells) to study inflammatory neovascularization.","authors":"Shih-Chun Shen, James B DuHadaway, Arpital Mondal, Souvik Dey, Alexander J Muller","doi":"10.1016/bs.mcb.2025.01.010","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.01.010","url":null,"abstract":"<p><p>The pathological expansion of immature blood vessels through neovascularization contributes to the development of a variety of diseases. In cancer, neovascularization supports tumor outgrowth and influences how tumors respond to therapy. Our studies have revealed that a defined cell population termed IDVCs (IDO1-dependent vascularizing cells) expressing the tryptophan catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) can foster a local inflammatory environment that promotes neovascularization. A powerful tool for investigating the biological role of isolated IDVCs in this inflammatory neovascularization process has been the Matrigel plug assay. In this assay, isolated cells are incorporated into a subcutaneously implanted Matrigel plug which is subsequently evaluated by confocal immunofluorescence microscopy for blood vessel density. We have employed this assay to demonstrate that isolated IDVCs are capable of promoting local neovascularization in an IDO1-dependent manner. Furthermore, the use of genetically engineered mouse strains and pharmacological interventions has enabled us to carry out in-depth investigations into IDO1's function as a nodal modifier of the local inflammatory environment responsible for eliciting a shift in the cytokine milieu from a neovasculature-restrictive to a neovasculature-sustaining status. Here we present a detailed methodology describing the reagents and procedures developed to isolate IDVCs and perform quantitative neovascularization studies. This assay should have great utility as a means for conducting investigative studies delving into the cellular and molecular processes involved in the complex interplay between inflammation and neovascularization.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"251-270"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667972","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2025-01-01Epub Date: 2025-02-18DOI: 10.1016/bs.mcb.2025.01.006
Apple Hui Min Tay, Andreas Lundqvist
{"title":"Relative adenosine production assay suitable for 2D and 3D tumor cell culture.","authors":"Apple Hui Min Tay, Andreas Lundqvist","doi":"10.1016/bs.mcb.2025.01.006","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.01.006","url":null,"abstract":"<p><p>Adenosine (ADO), an anti-inflammatory and immunosuppressive metabolite, plays a crucial role in mediating purinergic signaling alongside adenosine triphosphate (ATP) and adenosine monophosphate (AMP) within the tumor microenvironment. Dysregulated ADO signaling has been implicated in tumor immune evasion and progression, highlighting the importance of measuring ADO production. This method chapter presents a protocol for assessing ADO levels in both two- and three- dimensional tumor cell culture conditions. The protocol employs a competitive AMP blockade strategy, where excessive AMP is introduced to inhibit CD73-mediated conversion of AMP to ADO, enabling the quantification of relative ADO production. Given ADO's potent immunosuppressive properties and its influence on various immune responses, accurate measurement of ADO production is crucial for understanding its role in tumor immune evasion and for guiding the development of targeted immunotherapeutic strategies.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"171-176"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667977","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2025-01-01Epub Date: 2025-06-21DOI: 10.1016/bs.mcb.2025.02.020
Zubair Ahmed, Muneeb Ullah, Danish Zeshan, Shahid Ullah Khan, Fawad Ali, Abdul Wahab
{"title":"Exploring the tumor microenvironment in solid cancer: From biology to therapy.","authors":"Zubair Ahmed, Muneeb Ullah, Danish Zeshan, Shahid Ullah Khan, Fawad Ali, Abdul Wahab","doi":"10.1016/bs.mcb.2025.02.020","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.02.020","url":null,"abstract":"<p><p>Cancer is a major global health concern marked by uncontrolled cellular proliferation and genetic modifications leading to malignancy. The disease's complexity encompasses various forms of cancer, increased rates of diagnosis and prognosis and numerous treatment modalities, including surgery, chemotherapy, and radiation, each confronting problems such as medication resistance and side effects. Solid tumors, comprising approximately 85 % of malignancies, provide significant treatment challenges due to their uneven vascular supply and interstitial pressure, resulting in inadequate medication distribution and therapeutic failure. The tumor microenvironment (TME) comprises cancer cells and diverse supportive cells such as immune cells, endothelial cells and fibroblasts, which interact to facilitate tumor growth and progression. T lymphocytes, B lymphocytes, natural killer cells, and macrophages are only a few types of immune cells that can aid or impede cancer progression, which makes treatment more complicated. In this chapter we will explore the TME in solid cancers, focusing on its role in cancer biology and therapeutics strategies. In the future, advancing therapies that more precisely target TME components will minimize treatment resistance and improve patient outcomes.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"198 ","pages":"359-385"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145206761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2025-01-01Epub Date: 2025-03-13DOI: 10.1016/bs.mcb.2025.02.022
Yakhlesh Gupta, Kunzang Chosdol
{"title":"Practical approaches to advanced molecular biology techniques.","authors":"Yakhlesh Gupta, Kunzang Chosdol","doi":"10.1016/bs.mcb.2025.02.022","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.02.022","url":null,"abstract":"<p><p>The field of molecular biology has undergone tremendous advancements in recent years, with the development of powerful techniques that allow for in-depth exploration of cellular processes at the molecular level. This chapter, \"Advanced Molecular Biology Techniques,\" provides a detailed protocol of the molecular techniques. We begin with CRISPR-Cas9 genome editing, a transformative tool for precise and efficient gene manipulation, enabling targeted mutations and gene knockouts in various organisms. Gene amplification via Real-Time PCR is then discussed, highlighting its ability to quantify gene expression and detect rare genetic variants with high sensitivity. Flowcytometry follows, offering a robust platform for analyzing cellular populations based on specific markers, enabling the study of immune cells, cancer diagnostics, and cell cycle analysis. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) is explored as a method for mapping protein-DNA interactions, providing insights into gene regulation and epigenetic modifications. The chapter also covers Single-cell RNA sequencing (scRNA-Seq), a groundbreaking technique for profiling gene expression at the single-cell level, allowing for the discovery of cell heterogeneity and complex biological processes. Next, we explore into proteomics through Mass Spectrometry-Based Analysis, which offers detailed proteome characterization and biomarker discovery by identifying and quantifying proteins in complex samples. Finally, Fluorescence In Situ Hybridization (FISH) is discussed as a method for visualizing the spatial localization of specific nucleic acid sequences within intact cells or tissues. Together, these advanced molecular biology techniques offer unparalleled precision and insight into the molecular mechanisms underlying health, disease, and cellular function.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"198 ","pages":"73-101"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145206916","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2025-01-01Epub Date: 2025-05-03DOI: 10.1016/bs.mcb.2025.03.025
Abolaji Samson Olagunju, Isau Henrique Noronha, José Ronnie C Vasconcelos, Gustavo P Amarante-Mendes
{"title":"Evaluation of in vivo target cell elimination by antigen-specific cytotoxic T lymphocytes.","authors":"Abolaji Samson Olagunju, Isau Henrique Noronha, José Ronnie C Vasconcelos, Gustavo P Amarante-Mendes","doi":"10.1016/bs.mcb.2025.03.025","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.03.025","url":null,"abstract":"<p><p>The pivotal role of cytotoxic T lymphocyte (CTL) killing of target cells in vivo continues to be underscored by emerging research. CTLs are antigen-specific effector CD8 + T lymphocytes that serve as adaptive defenders against a myriad of threats, including viral infections, cancerous cells, and other pathogenic invaders. In vivo CTL killing assays contemplate the interaction of effector and target cells in the context of a proper microenvironment, making the analysis biologically more relevant than in vitro assays. They play a crucial role in advancing our understanding of immune responses, disease mechanisms, and therapeutic strategies in physiologically relevant settings, with implications for both basic research and clinical practice in immunology and related fields. Here, we describe and discuss in detail a method to explore the in vivo elimination of target cells by antigen-specific CTLs using immunized/vaccinated mice as our experimental model.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"197 ","pages":"141-155"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145033786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Methods for experimentally increasing circulating acyl-CoA-binding protein (ACBP) levels in mice under chronic restraint stress.","authors":"Hui Chen, Yanbing Dong, Yan Rong, Flavia Lambertucci, Sijing Li, Guido Kroemer, Isabelle Martins","doi":"10.1016/bs.mcb.2025.04.002","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.04.002","url":null,"abstract":"<p><p>Chronic restraint stress (CRS) is a widely recognized model to study stress-induced anorexia and metabolic dysregulation in mice. Acyl-coenzyme A-binding protein (ACBP) has emerged as a critical player in metabolic regulation, with potential implications for stress-related disorders. This study presents two complementary methodologies to artificially elevate circulating Acyl-CoA-binding protein (ACBP) levels in mice under CRS. The first approach involves the continuous delivery of recombinant ACBP via subcutaneously implanted osmotic pumps. The second approach utilizes the retention using selective hooks (RUSH) system, a chemical-genetic platform enabling controlled secretion of ACBP through a biotin-activated mechanism. These methodologies aim to counteract the metabolic and behavioral impacts of CRS, offering a framework for investigating ACBP's therapeutic potential in mitigating anorexia and restoring metabolic homeostasis. The integration of these delivery systems provides a robust tool for advancing research on stress-related disorders.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"197 ","pages":"253-263"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145033799","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2025-01-01Epub Date: 2024-11-21DOI: 10.1016/bs.mcb.2024.10.003
Varsha Gadiyar, David C Calianese, Rachael Pulica, Christopher Varsanyi, Ziren Wang, Ahmed Aquib, Alok Choudhary, Raymond B Birge
{"title":"Expression, purification and characterization of phosphatidylserine-targeting antibodies for biochemical and therapeutic applications.","authors":"Varsha Gadiyar, David C Calianese, Rachael Pulica, Christopher Varsanyi, Ziren Wang, Ahmed Aquib, Alok Choudhary, Raymond B Birge","doi":"10.1016/bs.mcb.2024.10.003","DOIUrl":"10.1016/bs.mcb.2024.10.003","url":null,"abstract":"<p><p>The externalization of Phosphatidylserine (PS) from the inner surface of the plasma membrane to the outer surface of the plasma membrane is an emblematic event during apoptosis and serves as a potent \"eat-me\" signal for the efferocytosis of apoptotic cells. Although less well understood, PS is also externalized on live cells in the tumor microenvironment and on live virus-infected cells whereby it serves as an immune modulatory signal that drives tolerance and immune escape. Given the importance of PS in cancer immunology and immune escape, PS-targeting monoclonal antibodies have been characterized with promising immunotherapeutic potential. Here, we describe the cloning and characterization of a series of PS targeting antibodies and their potential use and utility in immuno-oncology.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"15-40"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008118","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Methods in cell biologyPub Date : 2025-01-01Epub Date: 2024-04-18DOI: 10.1016/bs.mcb.2024.03.010
Yaiza Senent, Ana Remírez, Beatriz Tavira, Daniel Ajona
{"title":"A mouse model to assess immunotherapy-related colitis.","authors":"Yaiza Senent, Ana Remírez, Beatriz Tavira, Daniel Ajona","doi":"10.1016/bs.mcb.2024.03.010","DOIUrl":"10.1016/bs.mcb.2024.03.010","url":null,"abstract":"<p><p>Combined blockade of the immune checkpoints PD-1 and CTLA-4 has shown remarkable efficacy in patients with melanoma, renal cell carcinoma, non-small-cell lung cancer and mesothelioma, among other tumor types. However, a proportion of patients suffer from serious immune-related adverse events (irAEs). In severe cases, a reduction of the doses or the complete cessation of the treatment is required, limiting the antitumor efficacy of these treatments. Colitis is among the most frequent and problematic irAE associated with immune checkpoint blockade. In this context, animal models that recapitulate the pathophysiological features of immunotherapy-related colitis are needed. In this manuscript, we describe our experience with a mouse model in which the combined CTLA-4 and PD-1 blockade exacerbates the deleterious effects of dextran sulfate sodium (DSS)-induced colitis. This model may constitute a valuable tool for the study of immunotherapy-related colitis.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"192 ","pages":"33-38"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039665","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}