Methods in cell biology最新文献

Cancer immunotherapies leverage the immune response to target cancer cells with T cells playing a pivotal role. However, tumor microenvironments often harbor immune suppressive elements hindering T cell function. This chapter describes in vitro T cell stimulation assays analyzing proliferation, inhibitory marker expression, and effector functions to assess the impact of immune suppression on T cell responses. These assays also evaluate the efficacy of immunotherapeutic interventions in overcoming immune suppression and enhancing anti-tumor immunity, thereby unraveling the intricate T cell-tumor microenvironment dynamics for more effective cancer immunotherapies.

T cells are central mediators of anti-tumor immune responses, with their activation critically depending on finely tuned, and kinetically controlled signaling through the T cell receptor (TCR)-CD3 complex. Early TCR-proximal phosphorylation events shape downstream pathways that govern T-cell proliferation, differentiation, and effector functions, including responses to cancer immunotherapies, thus playing a pivotal role for effective tumor surveillance and eradication. Despite their importance, accurately capturing these early signaling events, at the single-cell level, remains technically challenging. Here, we present an optimized flow cytometry-based phospho-profiling protocol to assess early TCR signaling dynamics, using ERK phosphorylation as a sensitive readout. By overcoming the challenges of traditional approaches, this protocol provides an easy, yet powerful tool to evaluate T cell functionality both systemically and within the tumor immune microenvironment (TIME). Its application holds significant promise for advancing our understanding of T cell biology and for guiding the development and likely refinement of next-generation cancer immunotherapies.

The tumor microenvironment (TME) represents a complex ecosystem composed of tumor cells and various non-cancerous cell types, embedded within an altered extracellular matrix (ECM). In solid tumors, the ECM plays multiple roles: it provides mechanical support, delivers signaling molecules and transmits biophysical stimuli that influence cellular functions. Various cell types, primarily cancer-associated fibroblasts (CAFs) and immune cells such as macrophages, actively participate in the secretion and remodeling of ECM. However, whether the ECM directly instructs or educates immune cells, particularly macrophages within the TME, remains poorly understood. Here, we present a protocol to investigate the impact of ECM derived from non-small cell lung cancer (NSCLC) CAFs on macrophage state.

Several studies have shown that extracellular vesicles (EVs) are biosynthesized in all cells and then transported to other cells in an endocrine or paracrine manner through the bloodstream. They are considered to be taken up via the EV receptor in the destination cell membrane, resulting in a subsequent intracellular signal transduction cascade induced by the contents inside EV particles (e.g., DNA, RNA, miRNA protein, etc.). Since EVs are synthesized and secreted by all cells, it is important to "classify" them for EV research. However, challenges such as the numerous types and large number of EVs in biological samples and the complexity of target antigens for classification necessitate the development of more effective experimental methods. This chapter discusses a novel strategy to identify specific EV surface markers for classifying EVs using the "proximity labeling" analysis, which labels proximal molecular groups typically used for protein-protein interaction analysis in recent years.

Combining MetRS*-based cell-selective protein labeling with mass spectrometry-based proteomics is a powerful approach for investigating intercellular communication within tissues. Cell-selective labeling overcomes limitations of cell sorting techniques and facilitates cell type-specific proteome and secretome analyses in vivo. Our recent work has showcased the application of this method for the comprehensive proteomic characterization of cellular proteins in tissues, as well as released proteins in the bloodstream. Here, we present experimental guidelines for MetRS*-based cell-selective proteomics experiments in vivo and a detailed sample preparation protocol for tissues and body fluids.

Serum is a complex mixture of proteins that originate from a wide range of cells and tissues. At present, it is difficult to know what set of proteins any given tissue contributes to the circulating proteome. Here, we describe a method of using proximity biotinylation of proteins that normally transit through the endoplasmic reticulum to profile secreted proteins from cells in culture. We also describe a mouse model that enables the elucidation of the in vivo tissue-specific secretome. As examples, we demonstrate how we can readily identify in vivo endothelial-specific secretion, and how this model allows for the characterization of muscle-derived serum proteins that either increase or decrease with exercise. Our "Secretome Mouse" model is broadly applicable to other cell and tissue types.

Cell type-specific proteome labeling provides enhanced understanding of cellular function and structure within tissues by tagging proteins during translation, while cells and tissues are in their "native" states. TurboID is a methodology to enable rapid and efficient biotinylation of proteins. New TurboID viral constructs and a Cre-mediated TurboID transgenic mouse line enable cell type-specific proteomic investigations in cell culture and in vivo settings. Together, these new tools enable diverse studies designed to interrogate individual cell type contributions within complex multi-cellular systems. While biotin-based labeling enables enrichment of the labeled proteome via immunoprecipitation, it is also compatible with biotin/streptavidin-based immunoassays, including the Luminex xMAP multiplexed immunoassay platform. Here, we detail protocols to utilize existing commercially available Luminex kits to directly detect TurboID-biotinylated (and therefore cell type-specific) proteins of interest from cell culture and bulk tissue samples. Luminex immunoassays have multiple advantages compared to other methodologies, including (1) requiring small sample volumes/masses, (2) direct immuno-reaction-based quantification from a target sample without intermediate processing steps, (3) reduced costs and (4) direct readout. Below, we describe an adapted Luminex xMAP protocol to quantify phospho-proteins and cytokines from TurboID-labeled cells or tissues with cell type-specificity.

Communication between tissues or different cells within a tissue is often a result of secreted molecules such as metabolites, lipids, nucleic acids, or proteins (referred to as the secretome). These enter the extracellular space and may subsequently pass into the circulation. Depending on their nature, concentration and context, these molecules initiate specific responses in their target cells. Environmental stimuli such as exercise and cold exposure, but also different diseases, are known to significantly alter the secretome and thereby affect whole body homeostasis. Thus, identifying these factors is of great interest. The analysis of secreted proteins, however, represents a unique challenge for the field. This is mainly because mass spectrometry can be limited by the dynamic range problem, whereby the detection of low abundance polypeptides can be masked by the presence of high abundance proteins. Plasma, muscle, and fat all contain specific proteins of very high abundance, making it tremendously challenging to detect low abundance proteins in these biological samples. Thus, secreted, hormone-like polypeptides frequently remain undetected. Because muscle and fat are known to communicate by secretion of myokines and adipokines, respectively, we have sought to develop methods that can circumvent these issues through the isolation of extracellular fluids (EF) which surround these tissues. EFs had previously been isolated for analysis of metabolites; however, whether this method could be made useful for in depth proteomics analysis was not known. Recently, we have developed a method that modifies these procedures and makes it applicable for the study of EF proteins. We have applied this to muscle and fat EFs, but in principle, it can be used to study secreted proteins from almost any tissue in any species, including humans. A step-by-step protocol and methods of quality control are given below.

Gene expression is frequently regulated with cell type specificity and at multiple levels, including through tightly-controlled protein synthesis. While existing methods of protein synthesis profiling with non-canonical amino acid or isotope-containing amino acid labeling are effective and efficient for use in cultured cells, they are often costly to carry out in vivo. We previously developed a method to visualize and capture nascent proteomes in a cell type-specific manner in Drosophila brain explants using phenylacetyl-OPP (PhAc-OPP), a modified form of the puromycin analog O-propargyl-puromycin (OPP) that can incorporate into growing polypeptide chains. Targeted expression of Penicillin G acylase (PGA) in a cell population of interest removes an enzyme-labile blocking group on PhAc-OPP, thus permitting OPP-dependent labeling of nascent proteins in that cell population. This method, which we call POPPi (PGA-dependent OPP incorporation), provides a versatile approach to visualize or identify proteins synthesized in cell populations of interest in vivo. Here, we provide detailed protocols for labeling newly-synthesized protein in Drosophila brain cell populations by POPPi followed by detection via immunofluorescence or by capture and protein identification.

STAb-T therapy is an emerging cancer immunotherapy strategy that combines the advantages of adoptive cell therapies and bispecific antibodies. STAb (Secreting T cell engager bispecific Antibodies) T cells are T lymphocytes genetically engineered to secrete bispecific T cell engagers (TCEs) that recruit T cells to target and destroy tumor cells. Adoptive transfer of STAb-T cells has demonstrated potent anti-tumor activity in preclinical models and evaluation in human patients is forthcoming. Here, we provide comprehensive guidelines for generating STAb-T cells and assessing their functionality and efficacy in vitro and in relevant in vivo models of hematological malignancies.