Adenosine (ADO), an anti-inflammatory and immunosuppressive metabolite, plays a crucial role in mediating purinergic signaling alongside adenosine triphosphate (ATP) and adenosine monophosphate (AMP) within the tumor mic...Adenosine (ADO), an anti-inflammatory and immunosuppressive metabolite, plays a crucial role in mediating purinergic signaling alongside adenosine triphosphate (ATP) and adenosine monophosphate (AMP) within the tumor microenvironment. Dysregulated ADO signaling has been implicated in tumor immune evasion and progression, highlighting the importance of measuring ADO production. This method chapter presents a protocol for assessing ADO levels in both two- and three- dimensional tumor cell culture conditions. The protocol employs a competitive AMP blockade strategy, where excessive AMP is introduced to inhibit CD73-mediated conversion of AMP to ADO, enabling the quantification of relative ADO production. Given ADO's potent immunosuppressive properties and its influence on various immune responses, accurate measurement of ADO production is crucial for understanding its role in tumor immune evasion and for guiding the development of targeted immunotherapeutic strategies.
Aquib A, Wang Z, Gadiyar V
… +9 more, Pulica R, Varsanyi C, Frederick T, Tsou WI, Kimani SG, Smirnov S, De Lorenzo MS, Kotenko SV, Birge RB
Methods Cell Biol
· 2025 · PMID 40683675
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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....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.
Immune surveillance by T cells is a key determinant of cancer progression, and T cell-based immunotherapies have shown great promise as a treatment modality for cancer patients. As such, physiologically relevant methods...Immune surveillance by T cells is a key determinant of cancer progression, and T cell-based immunotherapies have shown great promise as a treatment modality for cancer patients. As such, physiologically relevant methods to evaluate interactions between T cells and tumor cells are of particular interest. In vitro systems that enable the culture patient-derived tumor cells in the presence of autologous tumor-infiltrating lymphocytes (TIL) serve as an invaluable tool to understand the basic biological role of T cells in cancer and how their functioning might be modulated to gain therapeutic benefit. Hence, this chapter describes a flow cytometry-based approach to assess TIL activation by exposure to autologous tumor cells in culture. In particular, the chapter will focus on ways to assess the capacity of cytotoxic lymphocytes (CTL) to degranulate and secrete pro-inflammatory cytokines in such culture systems.
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 releva...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.
Immunotherapy has revolutionized cancer treatment by harnessing the immune system to target tumor cells expressing neoantigens. Neoantigens are peptides arising from tumor-specific aberrations that are presented by cance...Immunotherapy has revolutionized cancer treatment by harnessing the immune system to target tumor cells expressing neoantigens. Neoantigens are peptides arising from tumor-specific aberrations that are presented by cancer cells and recognized by T cells. The computational prediction of cancer neoantigens from somatic mutations and other tumor-specific aberrations using patients' sequencing data is key for the investigation of anticancer immune responses and for the design of personalized immunotherapies. However, neoantigen prediction requires the implementation of complex computational pipelines to distill large-scale information from RNA and DNA sequencing data and derive neoantigen candidates together with associated features for their prioritization and selection. We previously developed nextNEOpi, a comprehensive and stand-alone bioinformatics pipeline that not only predicts class-I and -II neoantigens and fusion neoantigens, but also sheds light onto the tumor-immune cell interface, quantifying neoantigen clonality, immunogenicity, and tumor-specific metrics like tumor mutational burden and immune-cell receptor repertoire diversity. In this chapter, we showcase the main capabilities of the nextNEOpi pipeline by analyzing genomic and transcriptomic data generated from multiple biopsies collected from patients with lung cancer.
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...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.
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 a...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.
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 nov...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.
Cancer immunotherapy has been a real revolution and has given many survival chances to several patients. However, the understanding of resistance to immunotherapy is still an unmet need in clinical practice. Monitoring o...Cancer immunotherapy has been a real revolution and has given many survival chances to several patients. However, the understanding of resistance to immunotherapy is still an unmet need in clinical practice. Monitoring of immune mechanisms could be a tool to better understand this phenomenon. FCM and CyTOF could be used in this field, since they allow the simultaneous analysis of several protein expressions pattern, thus possibly understanding the functions of several immune cell populations, such as T cells, and their interactions with tumor cells and tumor microenvironment. Furthermore, automated cytometry could be used to understand the interaction of drugs with their target through the analysis of receptor occupancy. Spectral overlap, however, could be a limit for multiple simultaneous analyses. Other possible limitations of these techniques are a low number of cells in samples and the need for viable cells (with the possible interference of cell debris). The lack of standardized protocols, and thus the difficult reproducibility, have been the major limit to their application in clinical practice, so international efforts have been made to get to shared guidelines. Ongoing trials are to answer to the possibility of clinical application of these techniques.
Schares HAM, Hayes MJ, Balsamo JA
… +3 more, Thirman HL, Bachmann BO, Irish JM
Methods Cell Biol
· 2025 · PMID 40180452
·
Full text
Flow cytometry has great potential for screening in translational research areas due to its deep quantification of cellular features, ability to collect millions of cells in minutes, and consistently expanding suite of v...Flow cytometry has great potential for screening in translational research areas due to its deep quantification of cellular features, ability to collect millions of cells in minutes, and consistently expanding suite of validated antibodies that detect cell identity and functions. However, cytometry remains under-utilized in discovery chemical biology due to the differences in expertise between chemistry groups developing chemical libraries and cell biologists developing single cell assays. This chapter is designed to bridge this gap by providing a detailed protocol aimed at both chemistry and biology audiences with the goal of helping train novice researchers. Assay users select from three elements: a small molecule input, a target cell type, and a module of cytometry readouts. For each, we explore basic and advanced examples of inputs, including screening fractionated microbial extracts and pure compounds, and target cells, including primary human blood cells, mouse cells, and cancer cell lines. One such module of cytometry readouts focuses on cell function and measures DNA damage response (γH2AX), growth (phosphorylated S6), DNA content, apoptosis (cleaved Caspase3), cell cycle M phase (phosphorylated Histone H3), and viability (membrane permeabilization). The protocol can also be adapted to measure different functional readouts, such as cell identity or differentiation and contrasting cell injury mechanisms. The protocol is designed to be used in 96-well plate format with fluorescent cell barcoding and the debarcodeR algorithm. Ultimately, the goal is to encourage the next generation of chemical biologists to use functional cell-based cytometry assays in discovery and translational research.
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...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...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 and ALP 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.
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the principal human pathogens, causing severe infections in skin wounds. MRSA infection triggers a cell response mainly by mitochondrial-mediated pathway, resu...Methicillin-resistant Staphylococcus aureus (MRSA) is one of the principal human pathogens, causing severe infections in skin wounds. MRSA infection triggers a cell response mainly by mitochondrial-mediated pathway, resulting in mitochondrial outer membrane permeabilization, extrusion of the mitochondrial inner membrane into the cytoplasm, and then spillage of mitochondrial DNA (mtDNA) into the cytoplasm. The cell recognizes the discharged cytosolic mtDNA (cmtDNA) as "not-itself" because of mtDNA properties and triggers cascade events, such as the activation of inflammasomes. Here, we detail a method to detect and measure the mtDNA release into the cytoplasm in immortalized keratinocytes (HaCaT cells), after the infection with MRSA at different time points after the infection.
Sepsis, a condition characterized by systemic infection that becomes aggravated and dysregulated, is a significant cause of mortality in critically ill patients. Emerging evidence suggests that severe sepsis is often acc...Sepsis, a condition characterized by systemic infection that becomes aggravated and dysregulated, is a significant cause of mortality in critically ill patients. Emerging evidence suggests that severe sepsis is often accompanied by alterations in cell metabolism, particularly mitochondrial dysfunction, resulting in multiorgan failure. Normally, metabolically active cells or tissues exhibit higher levels of mitochondrial turnover, respiration, and adenosine triphosphate (ATP) synthesis. However, during sepsis, these processes become overwhelmed or dysregulated, leading to impaired ATP production in mitochondria. Here, we present two straightforward protocols for quantifying ATP production from mitochondria in bone marrow-derived macrophages (BMDMs). Our workflow facilitates the easy isolation of BMDMs and mitochondria from BMDMs treated with lipopolysaccharide (LPS), the major cell wall component of Gram-negative bacteria. We quantified intracellular and mitochondrial ATP production in macrophages in vitro using this protocol. The results indicated a decrease in mitochondrial ATP content in BMDMs in response to LPS. With minimal adjustments, this method can be adapted for use with various human and mouse primary cells and cell lines.
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 oxid...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.
Sepsis, a life-threatening condition characterized by dysregulated host response to infection, poses a significant public healthcare challenge. Excessive inflammatory responses during sepsis can lead to mitochondrial dys...Sepsis, a life-threatening condition characterized by dysregulated host response to infection, poses a significant public healthcare challenge. Excessive inflammatory responses during sepsis can lead to mitochondrial dysfunctions, resulting in organ damage. One hallmark of mitochondrial dysfunction is the reduction of mitochondrial membrane potential, which disrupts cellular metabolism, bioenergetics, and decreases the production of high-energy ATP through oxidative phosphorylation. In human sepsis, the mitochondrial membrane potential in peripheral blood monocytes has been identified as a marker of disease severity. Here, we present a detailed and widely accepted protocol for the detection of mitochondrial membrane potential using the JC-1 fluorescent dye in murine bone marrow-derived macrophages and J774A.1 macrophages following stimulation with lipopolysaccharides. This protocol is routinely employed and can be easily adapted for various cell types, intact tissues, and isolated mitochondria with minimal modifications. By utilizing this technique, researchers can gain valuable insights into mitochondrial function in different experimental contexts, potentially advancing our understanding of the pathogenesis and treatment of sepsis-related mitochondrial dysfunction.
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 o...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.
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 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.
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 t...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.