Methods in cell biology最新文献

Glioblastomas (GBMs) are the most common and aggressive brain tumors, with a poor prognosis. Effective preclinical models are crucial to investigate GBM biology and develop novel treatments. Syngeneic models, which consist in injecting murine GBM cells into mice with a similar genetic background, offer reproducibility, cost-effectiveness, and an intact immune system, making them ideal for immunotherapy research. This chapter presents a comprehensive protocol for stereotactic injection of murine GBM cells into immunocompetent mice to induce intracranial GBM. The protocol covers cell culture, anesthesia, surgical procedures, and post-operative care, allowing the reliable induction of orthotopic brain tumors. This method can be used to study anti-GBM therapies, including immunotherapies, and has the potential to accelerate the development of effective treatments.

Understanding the immunological synapse formation and dynamics can be enriched by measuring cell-cell interaction forces and their kinetics. Microscopy imaging reveals structural organization of the synapse, while physical methods detail its mechanical construction. Various techniques have been reported for measuring forces needed to rupture the interface between a T lymphocyte and its target cell but most of them measure one pair at a time. We describe here a laminar shear flow-based method that exerts dragging forces on T cell-target cells pairs immobilized on the surface of a flow chamber. Increasing flow rate allows us to observe the detachment of hundreds of cell conjugates on the wide field of a light transmission microscope. Monitoring precisely the flow rate gradient exerted on T cells readily yields synapse rupture measurements. Dragging forces are measured at the point of rupture as a linear function of the flow speed in minutes from 10pN to 20nN for each cell pair among a statistically representative cell population in the whole field of view of a single experiment. The output cells can be collected in multi-well plate sorted in the increasing order of rupture forces. We used this approach to unveil the involvement of the cytoskeleton regulator adenomatous polyposis coli (APC) in the stability of immunological synapses formed between human cytotoxic T cell and tumor target cells. APC is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Reduced APC expression impairs T cell adhesion with tumor target cells suggesting an impact of APC mutation in anti-tumor immune defense.

Natural Killer cells (NK) are cytotoxic lymphocytes from the innate immune system that recognize and eliminate virally infected and tumor cells. Accordingly, manipulation of NK cells has been the focus of several immunotherapy protocols aimed at eradicating cancer cells. Allogeneic NK cell therapy was initially described over two decades ago, emphasizing KIR-mismatch's importance in preventing NK cell inhibition and promoting cytotoxicity and tumor elimination without inducing graft-versus-host disease (GvHD). While unstimulated NK cells have shown limited antitumoral activity in adoptive cell therapy, various activation and expansion protocols have been proposed to enhance their cytotoxic potential. Activated and expanded allogeneic NK cells, especially with the rise of chimeric antigen receptor (CAR) therapies, have attracted significant attention from academic and commercial sectors. Protocols typically involve using cytokines and stimulatory cells, such as Epstein-Barr virus (EBV)-transformed lymphoblastoid B cell lines (LCLs) or K562 leukemic cells, before or after NK cell enrichment. Here we present two different standardized protocols for NK cell activation and expansion, offering insights into NK cell-based immunotherapies for cancer treatment. We also present a comprehensive methodology for assessing NK cell-mediated cytotoxicity against Neuroblastoma cell lines in both 2D and 3D cultures. The comprehensive methodology presented here lays the foundation for further research in the field, driving advancements in NK cell-based therapies against malignancies.

High endothelial venules (HEVs) are specialized blood vessels that act as the entry point for lymphocytes into the lymph node. These vessels have a unique cuboidal morphology and modified sugar coating, which favors the entry process. A major challenge in studying these vessels is that isolating and culturing them leads to the loss of their phenotype, making in vivo assessment the only reliable method for their examination. Moreover, there is a current need for consensus guidelines to assess alterations in HEVs. Therefore, we have developed a manual for the assessment of HEV alterations under stress, such as during inflammation or lymph node metastasis, at the protein level via immunofluorescence staining and at the RNA level via single-cell RNA sequencing.

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.

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.

T cell activation and its maintenance have been a focus of research within groups that study immunotherapy approaches for cancer treatment. Therefore, agents that regulate T cell activity are often tested in vitro and in vivo. In this chapter, we describe a technique to directly answer the question of whether an external agent employed at the time of T cell activation can impact the anti-tumor efficacy of activated T cells. We have used this technique to understand the timing of administering immune checkpoint inhibitors, to understand the effects of activation agents for professional antigen presenting cells, and similarly to understand the effects of vaccine adjuvants.

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.

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.

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.