Methods in cell biology最新文献

The use of single-cell technologies in characterizing the interactions between immune and cancer cells is in continuous expansion. Indeed, the combination of different single-cell approaches enables the definition of novel phenotypic and functional aspects of the immune cells infiltrating the tumor microenvironment (TME). This approach is promoting the discovery of relevant and reliable predictive biomarkers, along with the development of new promising treatments. In this chapter, we describe the main subsets of tumor-infiltrating lymphocytes from a phenotypical and functional point of view and discuss the use of single-cell technologies used to characterize these cell populations within TME.

The Programmed Cell Death Protein 1/Programmed Cell Death Protein Ligand 1 (PD-1/PD-L1) axis stands as one of the most widely acknowledged targets for cancer immunotherapy. This ligand is considered a therapeutic target for this disease as it might play an important role in tumor immune evasion and drug resistance. In multiple myeloma (MM), PD-L1 is overexpressed in abnormal plasma cells and Myeloid-Derived Suppressor Cells (MDSCs). In MDSCs, unlike tumoral cells or derived cell lines, the PD-L1 protein is presented in a conformation not recognized by the monoclonal antibody. In contrast, when stimulating the sample with PMA, the PD-L1 molecule undergoes a conformational change that enables its recognition. Hence, we have developed a flow cytometric screening assay to determine PD-L1 conformational changes in MDSCs based on a minimal manipulation of the sample, to preserve the structure and functionality of the ligand. In this chapter, we provide detailed protocols to assess PD-L1 levels in MDSCs together with the representative results obtained in multiple myeloma patients. The obtained results enable the classification of MM patients based on the different PD-L1 detection after stimulation, which increases compared with unstimulated samples. We also provide protocols to assess kinetic analysis of PD-L1 expression over time and to compare PD-L1 cell surface expression with cytoplasmic expression. Finally, competitive experiments in the presence of durvalumab are also described to study its interaction with PD-L1. This approach can also be used to study the contribution of potential conformational changes in other proteins.

Alkaline phosphatase (ALP) is a membrane-associated hydrolase enzyme with dimeric structure that catalyzes phosphate esters, optimally at alkaline pH. ALP has a focus of interest, since this enzyme is highly expressed in primitive stem cells, such as progenitor cells, non-differentiating cells, and primordial cells. We previously adapted a fluorescent microscopy-based assay for quantifying ALP^high and ALP^low cells by flow cytometry in combination with immunophenotyping. Our method uses a minimal sample perturbation approach, avoiding the use of erythrocyte lysing solutions and washing steps, and offering opportunities to combine live cell response and functional assessment with cell immunophenotyping, while minimizing sample preparation effects on the cell biology. Here we provide a detailed experiment protocol to determine alkaline phosphatase activity in CD34⁺ hematopoietic stem cells from blood and apheresis products obtained from patients involved in a stem cell mobilization process for allo- or auto-transplant. This study may provide the early detection of progenitors at different levels of differentiation and therefore, relate this information to long-term engraftment in hematopoietic stem cell transplants.

Tyro-3, Axl, and Mertk (abbreviated TAM receptors or TAMs), have well established functions in efferocytosis, the process by which apoptotic cells are engulfed by phagocytic cells such as macrophages and dendritic cells. In addition to their roles in efferocytosis, TAMs are also pleiotropic immune modulators that dampen inflammation and promote immune resolution and tolerance. Mice lacking one or more of the TAM receptors in various murine models leads to chronic inflammation and in some cases autoimmunity and chronic disease. In recent years, TAMs have emerged as important contributors in cancer, functioning both as oncogenic tyrosine kinases as well as immune modulators. Many recent studies indicate that TAM inhibitors, including monoclonal antibodies, kinase inhibitors, decoy receptors and ligands, and small molecular wedge inhibitors have therapeutic potential in cancer biology and immunotherapy. Here, we report the development and characterization of two type of TAM reporter lines that can be adapted to screen a wide range of TAM inhibitor types. The first involves TAM-IFN-γR1 chimeric CHO lines, where the extracellular domain of human TAM receptors is fused with the transmembrane and intracellular signaling domains of the human IFN-γ receptor chain. The second type of TAM reporter line described is the EGFR-TAM chimeric CHO lines, which involves fusing the extracellular domain of the EGFR receptor with the transmembrane and intracellular tyrosine kinase domains and cytosolic tail of TAM receptors. With minimal adaptation, both lines can be adopted for high throughput screening with immune-oncology applications.

Neoplastic cells are characterized by alterations in metabolic pathways, typically leading to an aberrant use of glycolysis even under aerobic conditions - a phenomenon known as the Warburg effect. One consequence of this metabolic shift is the production of lactate, an oncometabolite often found at elevated levels in tumors. Lactate not only fuels the growth of cancer cells but also promotes angiogenesis, immune escape, and metastasis, thereby contributing to tumor progression and resistance to therapy. This highlights the importance of lactate in cancer metabolism and underscores the need for methods to measure it. In this study, we describe various centrifugation and elution protocols to isolate interstitial fluid and measure lactate in experimental tumors. These tumors were generated in immunocompetent mice using the MC38 colon cancer cell line. We propose that, with minor modifications, the methods here described could be successfully adapted for use with tumors originating from other human or murine cell lines. Furthermore, these methods could potentially enable the detection of other oncometabolites in the tumor microenvironment, which could have significant implications for both basic research and therapeutic strategies.

Binge drinking (BD) is a widespread pattern of excessive alcohol consumption among adolescents and young adults with detrimental consequences for brain development. Animal models are essential for investigating the neurobiological mechanisms underlying BD, but selecting an appropriate model is critical to ensure relevance to human behavior. This study aims to validate a murine model of (BD) using Swiss Webster mice. To achieve this, both adolescent and adult mice were exposed to either a single binge (SB) or multiple binge (MB) of BD through intraperitoneal ethanol injections. The findings reveal that the SB protocol produces high blood alcohol concentrations (BACs) (150-400 mg/dL) sustained for several hours, with no significant differences based on age or episode repetition. However, the neurotoxic effects vary, showing that in adolescents, a single episode of BD reduces brain cell survival by 25 %, whereas in adults, multiple episodes are required to observe a 17 % decrease. This murine model of BD in Swiss Webster mice fulfills the main validation criteria identified in the literature. It presents valuable opportunities for studying individual variability and the neurobiological mechanisms of BD in adolescents, in order to identify potential therapeutic targets.

Cystic fibrosis (CF) is a genetic disorder primarily known for its severe impact on lung function, but it also significantly affects the digestive system, leading to complications such as intestinal blockages, malabsorption, inflammation, and microbial dysbiosis. The study of CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) effects on intestinal physiology is critical for developing new effective treatments. This work highlights the use of the mouse intestine as a valuable model for analyzing cellular electrophysiology and CFTR function. The method reviews the molecular mechanisms underlying intestinal absorption in CF and the role of CFTR in maintaining gut homeostasis. The transgenic mouse models mimicking CF features allow researchers to explore drug responses, and potential therapeutic interventions by studying the molecular mechanisms involved, such as inflammation, autophagy or immunity. The Ussing chamber assay is a versatile tool for evaluating ion transport, barrier function, and the effects of drugs on intestinal epithelial integrity. This assay provides insights into CFTR functionality and the influence of various compounds on intestinal absorption and permeability. This protocol underscores the importance of understanding CF's gastrointestinal implications emphasizing the potential for mouse models to advance therapeutic strategies not only for CF but also for other gastrointestinal disorders.

Breast cancer (BC) represents a major socio-economic challenge worldwide due to its high morbidity and mortality rates. Despite various therapeutic strategies, the heterogeneity of breast cancer and the resistance of tumour cells often lead to treatment failure. Consequently, the use of animal models of BC is crucial for understanding the cellular and molecular mechanisms involved in the different stages of carcinogenesis and for screening new drugs to assess their efficacy, potential safety and side effects. The accuracy, advantages and limitations of these models are the subject of ongoing debate among researchers, as no single model is standardised for all types of BC due to their unique genetic and phenotypic characteristics and the diversity of responses to treatment. This chapter focuses on the use of xenograft animal models by directly transplanting tumour cells into the BC microenvironment. It encompasses cell and animal preparation procedures, as well as transplantation methods. This technique provides a model of stage IV triple-negative BC in immune-competent animals, characterised by a short, non-surgical induction period. This model is particularly valuable for the study of carcinoma in situ and metastasis.

Chronic kidney disease (CKD) is currently a serious global health problem, due to its high risk of progression, prevalence and mortality. It not only affects the kidneys but also causes multi-organ damage. Moreover, there is no effective pharmacological treatment, and the only available alternatives are dialysis or transplantation, both of which impose a significant financial burden on healthcare systems. Given these challenges, a better understanding of CKD is essential for early diagnosis, identifying the pathological events involved, and assessing is systemic consequences. To achieve this, experimental animals models play a crucial role. Within the range of possibilities, the Unilateral Ureteral Obstruction (UUO) model is one of the most commonly options for studying CKD. This is because it is a simple, fast, inexpensive and easily reproducible animal model that, effectively simulates key events observed in human CKD, such as tubular cell death, fibrosis and interstitial inflammation, among others. This chapter describes in detail the UUO technique in a mouse model which provides a tool for the study of CKD, the molecular mechanism of damage underlying it, and the opportunity to develop future pharmacological alternatives.

With rising incidence of autoimmune disease and increasing number of patients being treated with immunotherapies that have the potential for autoimmune side effects, it is critical that we evaluate autoimmunity in preclinical models. Rheumatic diseases constitute some of the most common autoimmune conditions and to this end, we describe here several common murine models of joint inflammation. The K/BxN T cell transgenic model is a rapidly developing, synchronous and highly penetrant model of autoimmune arthritis. Arthritis can be induced (1) spontaneously; (2) by serum transfer of autoantibodies; or (3) by adoptive transfer of KRN T cells into an appropriate recipient. A second common model of arthritis, the collagen-induced arthritis model, is also described. Both models are useful for testing autoimmune effects of various therapies, and the K/BxN model has the advantage of a C57BL/6 background, making it amenable to genetic studies. Together, these models may be helpful for testing the autoimmune effects of monoclonal antibodies, nanotherapies, and small molecules as well as genetic contributions to autoimmune disease.