Methods in cell biology最新文献_第5页

Preparation and analysis of monotypic and organotypic tumor spheroids. 单型和器官型肿瘤球体的制备与分析。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-12-14 DOI: 10.1016/bs.mcb.2024.11.003

Ana Carolina M Domingues, Claire Palin, Yi Sun, Hongyan Xie, Elliot C Woods, Russell W Jenkins, Or-Yam Revach

{"title":"Preparation and analysis of monotypic and organotypic tumor spheroids.","authors":"Ana Carolina M Domingues, Claire Palin, Yi Sun, Hongyan Xie, Elliot C Woods, Russell W Jenkins, Or-Yam Revach","doi":"10.1016/bs.mcb.2024.11.003","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.11.003","url":null,"abstract":"Patient-derived tumor models are emerging as promising tools to explore patient-specific tumor behavior and to address a central gap in tumor immunology and immunotherapy drug development - the need for clinically relevant tumor models that recapitulate the complexity of the human tumor ecosystem, in which cancer cells are interacting with various immune and stromal elements. Patient-derived organotypic tumor spheroids (PDOTS), a biomimetic 3D-patient tumor avatar (3D-PTA), are comprised of cancer cells and autologous tumor-infiltrating immune and stromal cells that are grown within collagen hydrogels embedded in a 3D microfluidic culture device to model physiologic conditions and enable the study of tumor-immune dynamics. PDOTS and their murine counterparts (MDOTS, murine-derived organotypic tumor spheroids) are responsive to immune checkpoint blockade (ICB) and mirror in vivo response dynamics. We have also confirmed the utility of MDOTS/PDOTS in examining novel therapeutic strategies to overcome ICB resistance and testing the efficiency of T cell-based immunotherapies, demonstrating the utility of PDOTS profiling in examining the tumor-immune dynamics of immunotherapy response and resistance. Here, we provide a detailed protocol for processing, ex vivo culture, and analysis of patient-derived tumor organotypic tumor spheroids (PDOTS) in 3D microfluidic culture for immuno-oncology applications. The protocol can be readily adapted for ex vivo profiling of murine-derived organotypic tumor spheroids (MDOTS) and cancer cell line-derived monotypic tumor spheroids (MTS) for robust and iterative testing of immuno-oncology targets.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"139-159"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667976","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}

引用次数: 0

Assessing chromosomal abnormalities in leukemias by imaging flow cytometry. 利用成像流式细胞术评估白血病染色体异常。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2023-05-18 DOI: 10.1016/bs.mcb.2023.04.001

Stephanie J Lam, Henry Y L Hui, Kathy A Fuller, Wendy N Erber

{"title":"Assessing chromosomal abnormalities in leukemias by imaging flow cytometry.","authors":"Stephanie J Lam, Henry Y L Hui, Kathy A Fuller, Wendy N Erber","doi":"10.1016/bs.mcb.2023.04.001","DOIUrl":"10.1016/bs.mcb.2023.04.001","url":null,"abstract":"Chromosome analysis assists in the diagnostic classification and prognostication of leukemias. It is typically performed by karyotyping or fluorescent in situ hybridization (FISH) on glass slides. Flow cytometry offers an alternative high throughput automated methodology to analyze chromosomal content. With the advent of imaging flow cytometers, specific chromosomes and regions of interest can be identified and enumerated within specific cell types. The inclusion of immunophenotyping increases the specificity of this technique to ensure only the leukemic cell is analyzed. With many thousands of cells acquired, and neoplastic cells of interest identified by antigen expression, this technology has expanded the role of flow cytometry for cytogenomics in oncology. Applications to date have focused on hematological malignancies to detect aneuploidy (chromosome gains and losses) and structural defects (e.g., deletions; translocations) of diagnostic or prognostic significance at the time of diagnosis. With limits of detection of 1 cytogenetically abnormal cell in 100,000, also makes this new flow cytometry protocol eminently suitable for monitoring low level disease, detecting clonal evolution after therapy and identifying circulating tumor cells. The technique is equally applicable to solid tumors, many of which have chromosomal aberrations, with selection of appropriate immunophenotypic markers and FISH probes.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"195 ","pages":"71-100"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780451","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}

引用次数: 0

Evaluation of agents that affect anti-tumor function of CD8 + T cells when employed at the time of T-cell activation. 评价在T细胞活化时使用的影响CD8 + T细胞抗肿瘤功能的药物。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2025-03-12 DOI: 10.1016/bs.mcb.2025.02.016

Ichwaku Rastogi, Jena E Moseman, Donghwan Jeon, Anusha Muralidhar, Douglas G McNeel

引用次数: 0

Analysis of single-cell TCR repertoires and gene expression from multi-modal scRNA-seq data. 基于多模态scRNA-seq数据的单细胞TCR谱和基因表达分析。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2025-04-10 DOI: 10.1016/bs.mcb.2025.03.007

Christina Plattner, Gregor Sturm, Dietmar Rieder

{"title":"Analysis of single-cell TCR repertoires and gene expression from multi-modal scRNA-seq data.","authors":"Christina Plattner, Gregor Sturm, Dietmar Rieder","doi":"10.1016/bs.mcb.2025.03.007","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.03.007","url":null,"abstract":"Single-cell RNA and T-cell receptor (TCR) sequencing are powerful tools for dissecting T-cell diversity and function with unprecedented resolution. Analyzing transcripts and TCR sequences expressed by individual T-cells, enables comprehensive characterization of T-cell repertoires, antigen specificity and clonal dynamics which is fundamental in understanding the adaptive immune responses in various physiological and pathological conditions, including cancer, autoimmune diseases, and infectious diseases. To perform integrative analyses of multi-modal data from single-cell RNA and TCR sequencing experiments specialized bioinformatic tools are required. Here we exemplify the application of Scirpy, a versatile Python package specifically designed for single-cell TCR sequencing analysis, which streamlines the processing and analysis of TCR sequencing data. Scirpy offers a user-friendly framework for tasks like repertoire characterization, visualization, and clonotype identification. Moreover, Scirpy integrates seamlessly with other single-cell analysis tools from the scverse ecosystem, enabling comprehensive multi-modal data integration and downstream analyses.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"199 ","pages":"191-220"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145313297","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}

引用次数: 0

Bacterial predators and BALOs: Growth protocol and relation with mitochondria. 细菌捕食者和BALOs：生长方案和与线粒体的关系。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-08-24 DOI: 10.1016/bs.mcb.2024.07.003

Valerio Iebba

{"title":"Bacterial predators and BALOs: Growth protocol and relation with mitochondria.","authors":"Valerio Iebba","doi":"10.1016/bs.mcb.2024.07.003","DOIUrl":"10.1016/bs.mcb.2024.07.003","url":null,"abstract":"The microbial world is characterized by mechanisms of competition and predation, akin to the animal world. However, while predation's ecological role is well-established in animals, it's less understood in bacteria due to fewer known predators and unclear phylogenetic affiliations. Nevertheless, microorganisms can prey on bacterial cells, including Bacteriophages, Protists, and Predatory Prokaryotes. These predators inhabit various habitats and may play vital roles in bacterial ecology and ecosystem regulation. Predatory interactions between host and parasite are common in nature. Predatory bacteria, such as Bdellovibrio and like organisms (BALOs), employ various strategies, including epibiotic predation and direct invasion. BALOs, which thrive in the periplasmic space of Gram-negative bacterial cells, modulate bacterial populations and could serve as preventive or therapeutic agents against Gram-negative infections. While primarily active against extracellular prey, BALOs may also target mitochondria, which are crucial for cellular processes. The relationship between intracellular bacteria and host mitochondria, including morphology, function, and apoptosis, warrants further exploration. Protocols for growing, propagating, and detecting predatory activities of BALOs, particularly Bdellovibrio bacteriovorus, are provided to assess their presence and activities against potential prey.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"194 ","pages":"151-167"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586227","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}

引用次数: 0

A comprehensive guide to study the immunological synapse using imaging flow cytometry. 利用成像流式细胞术研究免疫突触的综合指南。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-04-03 DOI: 10.1016/bs.mcb.2024.03.001

Andrea Michela Biolato, Liza Filali, Max Krecke, Clément Thomas, Céline Hoffmann

{"title":"A comprehensive guide to study the immunological synapse using imaging flow cytometry.","authors":"Andrea Michela Biolato, Liza Filali, Max Krecke, Clément Thomas, Céline Hoffmann","doi":"10.1016/bs.mcb.2024.03.001","DOIUrl":"10.1016/bs.mcb.2024.03.001","url":null,"abstract":"Cytotoxic lymphocytes, such as cytotoxic T cells and natural killer (NK) cells, are instrumental in the recognition and eradication of pathogenic cells, notably those undergoing malignant transformation. Cytotoxic lymphocytes establish direct contact with cancer cells via the formation of a specialized cell-cell junction known as the lytic immunological synapse. This structure serves as a critical platform for lymphocytes to integrate surface signals from potential cancer cells and to direct their cytolytic apparatus toward the confirmed targets. Conversely, cancer cells evolve synaptic defense strategies to evade lymphocyte cytotoxicity. This chapter delineates protocols using imaging flow cytometry to examine and quantify important subcellular processes occurring within cytotoxic lymphocytes and cancer cells engaged into an immunological synapse. These processes encompass the spatial redistribution of cytoskeletal components, vesicles, organelles and cell surface molecules. We specifically describe methods to generate and select conjugates between MDA-MB-231 breast cancer cells or K-562 leukemic cells and either the NK-92MI cell line or primary human NK cells. In addition, we detail procedures to evaluate the synaptic polarization of the actin cytoskeleton, CD63-positive vesicular compartments, MHC class I molecules, as well as the microtubule-organizing center in effector cells.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"69-97"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370849","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}

引用次数: 0

Simplified acid extraction and quantification of histones in human tumor cells. 人肿瘤细胞组蛋白的简化酸提取和定量。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-18 DOI: 10.1016/bs.mcb.2024.10.002

Lourdes Hontecillas-Prieto, Daniel J García-Domínguez, Rocío Flores-Campos, Juan Antonio Flores, Antonio Pérez-Pérez, Luis de la Cruz-Merino, Víctor Sánchez-Margalet, Nabil Hajji

{"title":"Simplified acid extraction and quantification of histones in human tumor cells.","authors":"Lourdes Hontecillas-Prieto, Daniel J García-Domínguez, Rocío Flores-Campos, Juan Antonio Flores, Antonio Pérez-Pérez, Luis de la Cruz-Merino, Víctor Sánchez-Margalet, Nabil Hajji","doi":"10.1016/bs.mcb.2024.10.002","DOIUrl":"10.1016/bs.mcb.2024.10.002","url":null,"abstract":"Histones are essential nuclear proteins that package eukaryotic DNA into chromosomes, play a vital role in gene regulation, DNA replication, DNA repair and chromosome condensation. Understanding histone modifications is crucial for grasping biological and disease-related processes. Specific alterations in histone modifications serve as sensitive and selective biomarkers for conditions like cancer, impacting both tumor and immune cells and affecting their interactions. Indeed, the interest in histone modifications is growing in the field of tumor immunology and immunotherapy. Different techniques have been developed to characterize histone proteins and their modifications. Here, we present a simple acid extraction protocol to identify and quantify histones. The workflow described here can be used to detect and measure histone proteins or specific residues of histone, even capturing changes resulting from treatment with epigenetic drugs (Epi-drugs) or other drugs in in different human cancer cell line models.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"1-14"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008163","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}

引用次数: 0

Generation of human and murine exhausted CD8⁺ T cells in vitro. 体外培养人和小鼠耗竭CD8+ T细胞。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-13 DOI: 10.1016/bs.mcb.2024.10.007

Rosa Ana Lacalle, Raquel Blanco, Rebeca García-Lucena, Santos Mañes

{"title":"Generation of human and murine exhausted CD8+ T cells in vitro.","authors":"Rosa Ana Lacalle, Raquel Blanco, Rebeca García-Lucena, Santos Mañes","doi":"10.1016/bs.mcb.2024.10.007","DOIUrl":"10.1016/bs.mcb.2024.10.007","url":null,"abstract":"T cell exhaustion is a state of dysfunction that can occur due to persistent exposure to antigens, such as in the tumor microenvironment. The progressive loss of effector functions in exhausted T cells can lead to resistance to immune checkpoint inhibitors and adoptive cell immunotherapies. Improving our understanding of the exhaustion process is thus crucial for optimizing the clinical outcomes of immunotherapy. A significant hurdle in this area is obtaining an adequate quantity of exhausted T cells. One solution could be the in vitro production of exhausted T cells by mimicking exhaustion-induced conditions. We present a simple, repeatable, flow cytometry-assisted method for generating exhausted CD8+ T cells from both human and mouse sources. This flexible protocol works with various cell sources and activation methods. Our results confirm the production of dysfunctional CD8+ T cells, akin to those in mouse tumor models and patient tumor samples. This methodology could help identify genes involved in the exhaustion process and serve as a platform for finding agents capable of altering, reversing, or accelerating this dysfunctional state. By using both mouse and human models, we increase the adaptability of the method, making it a powerful instrument for assessing potential substances with immunotherapeutic utility.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"93-114"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008126","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}

引用次数: 0

Characterizing tumor-infiltrating group 1 innate lymphoid cells in PyMT breast tumors. PyMT乳腺肿瘤浸润性1组先天淋巴样细胞的特征。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-04-24 DOI: 10.1016/bs.mcb.2024.03.008

Douglas C Chung, Alisha R Elford, Nicolas Jacquelot

引用次数: 0

Deciphering neutrophil heterogeneity in human blood and tumors: Methods for isolating neutrophils and assessing their effect on T-cell proliferation. 解读人类血液和肿瘤中中性粒细胞的异质性：分离中性粒细胞和评估其对t细胞增殖影响的方法。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-25 DOI: 10.1016/bs.mcb.2024.10.010

Nicolas Delhez, Frank Aboubakar Nana, Camille Houbion, Alexandre Bayard, Annika Bruger, Christophe Vanhaver, Sven Brandau, Pierre van der Bruggen, Thibault Hirsch

{"title":"Deciphering neutrophil heterogeneity in human blood and tumors: Methods for isolating neutrophils and assessing their effect on T-cell proliferation.","authors":"Nicolas Delhez, Frank Aboubakar Nana, Camille Houbion, Alexandre Bayard, Annika Bruger, Christophe Vanhaver, Sven Brandau, Pierre van der Bruggen, Thibault Hirsch","doi":"10.1016/bs.mcb.2024.10.010","DOIUrl":"10.1016/bs.mcb.2024.10.010","url":null,"abstract":"Neutrophils were historically considered a homogenous population of cells with functions limited to innate immunity against external threats. However, with the rise of immunotherapy, recent works have shown that neutrophils are also important actors in immuno-oncology. In this context, neutrophils appear as a more heterogenous population of cells. However, many reported neutrophil subpopulations, or neutrophils with various transcriptional states, lack functional characterization to confirm their suspected roles. Thus, we believe that functional assays remain essential to define the role of neutrophils in cancer. In this chapter, we present a T-cell proliferation assay based on the use of allogeneic T-cells to assess the suppressive capabilities of neutrophils isolated from human blood or tumor samples. Allogeneic T-cells are isolated in large quantities from the blood of non-cancerous donors and frozen in aliquots to be used in several experiments. This reduces variability by excluding other cancer-derived factors, which would be present if autologous T-cell were used and allows to isolate the effect of neutrophils on T-cell proliferation. Thawed T-cells have poor proliferative capacities and to initiate proliferation they require co-culture with mature dendritic cells that we generate from monocytes isolated from the same blood sample. Initially developed for lung cancer patients, our method to isolate low-density neutrophils (LDN) and normal-density neutrophils (NDN) can be used with any patient and adapted to other kind of samples (e.g., ascites, urine, …).","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"151-196"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008070","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}

引用次数: 0