Methods in cell biology最新文献

{"title":"Evaluating transcriptional tumour microenvironment response to immunotherapy using microarray analysis: From harvesting tumours to data analysis.","authors":"Olga Cormier, Maria Eugenia Davola, Susan Collins, Karen Mossman","doi":"10.1016/bs.mcb.2025.01.004","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.01.004","url":null,"abstract":"<p><p>In the recent years, a better understanding of the cellular and molecular processes that shape the dynamic balance of the immune system and the tumour has been gained. Accumulating evidence shows that cellular and acellular components in the tumour microenvironment (TME) can reprogram the response to immunotherapies. Transcriptome analyses of immunotherapy treatments have been invaluable for studying signalling pathways in cancer. Transcriptional characterization of tumours has allowed researchers to both predict the likely response to therapy as well as to evaluate the effect of therapy on tumours before any phenotypic changes are observed. Despite the rise of single-cell RNA sequencing (scRNAseq) it remains costly and technically challenging and, in many situations, unnecessarily detailed. Presented here is a protocol describing processing of tumours for microarray transcriptome analysis to evaluate the responses to therapy. This method has proven to be cost-effective in cases when broad transcriptional responses are being investigated.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"225-236"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667967","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":"Next-generation deconvolution of the tumor microenvironment with omnideconv.","authors":"Lorenzo Merotto, Alexander Dietrich, Markus List, Francesca Finotello","doi":"10.1016/bs.mcb.2025.01.003","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.01.003","url":null,"abstract":"<p><p>The tumor microenvironment and, particularly, tumor-infiltrating immune cells can profoundly influence tumor progression and response to therapy. Deconvolution is a powerful computational technique to estimate cell-type fractions from bulk RNA sequencing (RNA-seq) data leveraging expression signatures specific to the cell types of interest. Recently, a new generation of deconvolution algorithms has emerged, making it possible to directly learn cell-type-specific signatures to be used for deconvolution from annotated single-cell RNA-seq (scRNA-seq) datasets. Thanks to their flexibility, these next-generation methods can extend deconvolution to any cell type, tissue, and organism for which a suitable single-cell reference is available. However, these methodologies are highly diverse in terms of programming languages, computational workflows, and input/output data, which complicate their usage and comparison. To overcome these challenges, we developed omnideconv, an R package that integrates several deconvolution methods, streamlining their usage and unifying their semantics. In this chapter, we demonstrate how omnideconv can be integrated with an annotated scRNA-seq dataset, comprising both malignant and normal cells from the breast cancer microenvironment, to quantify the cellular composition of bulk RNA-seq data from a cohort of breast cancer patients.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"87-112"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667973","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":"Quantification of mitochondrial reactive oxygen species in macrophages during sepsis.","authors":"Kanmani Suganya, Paulraj Kanmani, Guochang Hu","doi":"10.1016/bs.mcb.2024.01.005","DOIUrl":"10.1016/bs.mcb.2024.01.005","url":null,"abstract":"<p><p>Sepsis is the leading causes of death globally, arising from an imbalanced host response to severe infection. It leads to multi-organ failure and poor outcomes in septic patients due to compromised glucose and lipid oxidation, reduced oxygen consumption, elevated levels of circulating substrates, and impaired mitochondrial function. Mitochondria, essential cellular organelles, play a vital role in regulating various cellular activities and the host immune response to infection. Pathogens, particularly bacteria, often disrupt mitochondrial functions to dysregulate host immunity. Additionally, the mitochondrial function is closely associated with most host immune responses, making mitochondria crucial in maintaining host homeostasis during infection. The intrinsic inflammatory response triggered by pathogens in sepsis impairs mitochondrial function, resulting in excessive production of mitochondrial reactive oxygen species (ROS) and subsequently damage to multiple organs. Here, we present a simple protocol for assessing mitochondrial ROS levels in bone marrow-derived macrophages (BMDMs) isolated from mice. We observed a higher level of ROS generation in lipopolysaccharide (LPS)-treated BMDMs, indicating the effectiveness and efficiency of our designed protocol for assessing mitochondrial ROS generation in vitro.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"194 ","pages":"59-75"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586249","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":"Visualizing mitochondrial electron transport chain complexes and super-complexes during infection of human macrophages with Legionella pneumophila.","authors":"Mariatou Dramé, Daniel Schator, Carmen Buchrieser, Pedro Escoll","doi":"10.1016/bs.mcb.2024.01.003","DOIUrl":"10.1016/bs.mcb.2024.01.003","url":null,"abstract":"<p><p>The ultrastructure of mitochondria is pivotal for their respiratory activity. Thus, the regulation of the assembly of the super-complexes (SCs) of the mitochondrial electron transport chain (ETC) might be a core aspect of macrophage immunometabolism during bacterial infection. In order to study the impact of infection by Legionella pneumophila on the configuration of mitochondrial complexes and SCs in human macrophages, we have adapted and combined different methods such as cell sorting of infected cells, magnetic isolation of highly pure and functional mitochondria, quality control of mitochondrial purity by flow cytometry, and BN-PAGE (Blue-Native Polyacrylamide Gel Electrophoresis) coupled to Western Blot using near-infrared (NIR) fluorescence. The here presented protocol uses infected and non-infected human macrophage-like THP-1 cells and GFP-expressing L. pneumophila, but the method can be used to analyze the configuration of ETC complexes and SCs also in other mammalian cells and infected with different intracellular bacteria expressing a fluorescent protein.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"194 ","pages":"19-42"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586259","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":"Monitoring cellular dynamics upon infection using a holotomography-based approach.","authors":"Ilaria Nunzi, Gloria D'Achille, Nada Dhaouadi, Fabio Marcheggiani, Caterina Licini, Mariangela Di Vincenzo, Monia Orciani, Gianluca Morroni, Saverio Marchi","doi":"10.1016/bs.mcb.2024.12.003","DOIUrl":"10.1016/bs.mcb.2024.12.003","url":null,"abstract":"<p><p>Many intracellular bacteria interfere with mitochondrial dynamics or target other organelles, thereby inducing a specific cellular response that could emerge as a strategy of the pathogen to ensure its survival, or as a form of defense employed by the host cell to restrict dissemination. In this context, the concomitant monitoring of both pathogen migration and (intra)cellular dynamics in live cells emerges as a pivotal aspect for the comprehension of the infection sequence and to visualize the pathogen-mediated remodeling that could occur to the entire cellular system. Holotomographic microscopy can be used to achieve this goal, allowing the simultaneous analysis of both bacterial movement and intracellular alteration for extended periods of time, with high spatial resolution and avoiding side-effects due to phototoxicity. Here we provide a holotomography-based approach to detect Listeria monocytogenes dynamics and its effects on the entire cellular system at morphological level.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"194 ","pages":"109-118"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586233","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":"A screening system to determine the effect of bacterial metabolites on MAdCAM-1 expression by transformed endothelial sinusoidal cells.","authors":"Ai-Ling Tian, Marion Leduc, Marine Fidelle, Laurence Zitvogel, Guido Kroemer, Oliver Kepp","doi":"10.1016/bs.mcb.2024.01.007","DOIUrl":"10.1016/bs.mcb.2024.01.007","url":null,"abstract":"<p><p>Mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expression in high endothelial venules is regulated by bacterial metabolites emanating from the gut and the interaction of MAdCAM-1 with α4β7 integrin mediates lymphocyte diapedesis into gut-associated secondary lymphoid tissues. MAdCAM-1 thus controls the abundance of circulating immunosuppressive T cells that can reach malignant tissue and compromise the therapeutic efficacy of anticancer immunotherapy. Here we describe a biosensor-based phenotypic assessment that facilitates the high throughput screening (HTS)-compatible assessment of MAdCAM-1 regulation in response to exposure to bacterial metabolites. This screening routine encompasses high endothelial venule cells expressing green fluorescent protein (GFP) under the control of the MAdCAM-1 promoter combined with robot-assisted bioimaging and a multistep image analysis pipeline. Altogether this system facilitates the discovery of bacterial composites that control anticancer immunity via the sequestration of Th17-specific regulatory T cells (Treg17) in the gut.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"194 ","pages":"119-133"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586222","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":"Quantitative analysis of the B cell immune synapse using imaging techniques.","authors":"Oreste Corrales Vázquez, Teemly Contreras, Martina Alamo Rollandi, Felipe Del Valle Batalla, Maria-Isabel Yuseff","doi":"10.1016/bs.mcb.2023.08.002","DOIUrl":"10.1016/bs.mcb.2023.08.002","url":null,"abstract":"<p><p>This chapter presents a series of quantitative analyses that can be used to study the formation of the immune synapse (IS) in B cells. The methods described are automated, consistent, and compatible with open-source platforms. The IS is a crucial structure involved in B cell activation and function, and the spatiotemporal organization of this structure is analyzed to provide a better understanding of its mechanisms. The analyses presented here can be applied to other immune cells and are accessible to researchers of diverse fields. In addition, the raw data derived from the results can be further explored to perform quantitative measurements of protein recruitment and tracking of intracellular vesicles. These techniques have the potential to enhance not only our understanding of the IS in B cells but also in other cell models.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"229-252"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370865","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":"Assessment of adhering and invading properties of Escherichia coli strains.","authors":"Valerio Iebba","doi":"10.1016/bs.mcb.2024.08.011","DOIUrl":"10.1016/bs.mcb.2024.08.011","url":null,"abstract":"<p><p>Gastrointestinal infections, caused by Enterobacteriaceae, pose a major global health challenge, resulting in significant morbidity and mortality. Enhanced adherence and invasion properties are widespread among enteric pathogenic species, particularly those linked to invasive infections such as some pathovars of Escherichia coli or pathogens like Shigella and Salmonella. Pathogenic E. coli strains are categorized into various pathotypes, including diarrheagenic E. coli (DEC) and extraintestinal pathogenic E. coli (ExPEC). Notably, Enteroinvasive E. coli (EIEC) and Adherent-invasive E. coli (AIEC) demonstrate significant invasive properties. EIEC, similar to Shigella, invades intestinal epithelial cells causing dysentery-like illness, while AIEC persists in the gut epithelium, potentially contributing to chronic inflammatory bowel diseases (IBD). Techniques like cell culture assays are vital for assessing E. coli's adherence and invasion capabilities, with specific virulence factors such as fimbriae and type III secretion systems (T3SS) playing crucial roles. Comparatively, Shigella and Salmonella also utilize T3SS for epithelial cell invasion, but with distinct effector proteins and mechanisms. Understanding these differences is crucial for diagnosis and treatment, as advanced molecular diagnostics improve the identification of invasive E. coli strains. Potential therapeutic interventions targeting fimbrial adherence, T3SS and effector proteins offer promising avenues for developing antivirulence drugs. Here are provided protocols for studying the adherence and invasion properties of E. coli and other Enterobacteriaceae to enhance diagnostic methods, ultimately improving the management of enteric infections.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"194 ","pages":"169-190"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586224","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":"Gauging antigen recognition by human primary T-cells featuring orthotopically exchanged TCRs of choice.","authors":"Vanessa Mühlgrabner, Angelika Plach, Johannes Holler, Judith Leitner, Peter Steinberger, Loïc Dupré, Janett Göhring, Johannes B Huppa","doi":"10.1016/bs.mcb.2024.03.003","DOIUrl":"10.1016/bs.mcb.2024.03.003","url":null,"abstract":"<p><p>Understanding human T-cell antigen recognition in health and disease is becoming increasingly instrumental for monitoring T-cell responses to pathogen challenge and for the rational design of T-cell-based therapies targeting cancer, autoimmunity and organ transplant rejection. Here we showcase a quantitative imaging platform which is based on the use of planar glass-supported lipid bilayers (SLBs). The latter are functionalized with antigen (peptide-loaded HLA) as adhesion and costimulatory molecules (ICAM-1, B7-1) to serve as surrogate antigen presenting cell for antigen recognition by T-cells, which are equipped with T-cell antigen receptors (TCRs) sequenced from antigen-specific patient T-cells. We outline in detail, how the experimental use of SLBs supports recoding and analysis of synaptic antigen engagement and calcium signaling at the single cell level in response to user-defined antigen densities for quantitative comparison.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"127-154"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370853","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":"Measuring interaction kinetics between T cells and their target tumor cells with optical tweezers.","authors":"Edison Gerena, Sophie Goyard, Nicolas Inacio, Jerko Ljubetic, Amandine Schneider, Sinan Haliyo, Thierry Rose","doi":"10.1016/bs.mcb.2024.09.002","DOIUrl":"10.1016/bs.mcb.2024.09.002","url":null,"abstract":"<p><p>T cell adhesion kinetics are a powerful indicator of target cell recognition during the cell-cell exploration process and formation of the immunological synapse facilitating cell communication and activation through specific intercellular molecular interactions. Various techniques have been used to document these binding kinetics, which foreshadow the dynamics of immunological synapse formation. Here, optical tweezers were used for studying at the level of single cells, the adhesion kinetics of human leukemia T lymphocyte cell line (CEM) to mouse mast cell line (P815) used as a tumor cell model. The P815 FcγRII receptors were saturated with the mouse anti-human CD3ɛ immunoglobulin G (OKT3) for initiating the T cell-P815 interaction through the engagement of the T cell CD3 nucleating the TCR complex formation structuring the synapse. Methods were developed to assess the time required to turn a contact between a T cell and a tumor cell into a stable interaction, and thus initiate the synapse formation. Single T cells were manipulated with the optical tweezers while the tumor cells were adhered to the glass surface under culture conditions. Three adhesions scenario were investigated by exerting either repetitive contacts engaging the same area of the two cells, repetitive contacts engaging the same area of the T cell but different areas on the tumor cell surface, or rolling the T cell over the tumor cell surface. With these methods, we observed that the median time of contact of CEM on P815 decreased in the presence of anti-CD3 OKT3 from 46s to 1.3s and the median rolling distance decreased from 50μm to 1.8μm prior the T cell immobilization. T cell adhesion speed assays can be used for measuring their lack of early response, identifying molecules involved in cell adhesion, or screening potential modulators. The techniques and quantitative methods, described here for studying T cell/target cell interaction based on manipulations using optical tweezers, can be generalized to all types of immunological or virological synapses as between T cell/dendritic cell, cytotoxic T cell/target, T cell/macrophage, T cell/B cell, NK cell/target, immune cell/infected cell and others.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"155-174"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370863","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}