BACKGROUND: Multiple myeloma is a hematologic malignancy characterized by clonal plasma cell proliferation, with recent therapeutic advances focusing on immunotherapies targeting cell surface antigens. While several surf...BACKGROUND: Multiple myeloma is a hematologic malignancy characterized by clonal plasma cell proliferation, with recent therapeutic advances focusing on immunotherapies targeting cell surface antigens. While several surface markers are well-characterized, there remains a critical need to identify additional specific targets for relapsed cases. Comprehensive surface proteome analysis is challenging due to the low abundance of surface proteins and limited cell numbers available from patient samples. METHODS: We developed an optimized cell surface capture (CSC) protocol coupled with data-independent acquisition (DIA) mass spectrometry for comprehensive surfaceome profiling of multiple myeloma cell lines AMO1 and MM1.S. The method utilizes periodate-based oxidation of surface glycoproteins followed by biocytin hydrazide labeling and NeutrAvidin enrichment. Surface-labeled proteins were analyzed using DIA mode on an Orbitrap Eclipse Tribrid mass spectrometer with optimized parameters for low-input samples. RESULTS: Our approach successfully identified and quantified 989 high-confidence surface proteins from minimal sample inputs (10-fold less than standard protocols), demonstrating excellent reproducibility between biological replicates (R=0.907-0.916). Comparative analysis revealed 790 shared proteins between cell lines, with 93 and 106 proteins uniquely expressed in AMO1 and MM1.S, respectively. Principal component analysis showed clear separation between cell lines (PC1=84.6% variance), highlighting distinct surface protein expression profiles. The method detected known myeloma markers including BCMA and identified additional potential therapeutic targets. CONCLUSIONS: CSC-DIA methodology enables comprehensive surface proteome analysis from limited cell numbers, making it suitable for rare patient samples. This provides a robust platform for discovering novel surface antigens to advance personalized myeloma immunotherapy.
Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of proteins has been challenging i...Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of proteins has been challenging in vivo. A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP system in fruit flies. This approach depends on the reconstitution of the GFP and GFP fragments, creating a fluorescence signal. We insert the GFP fragment into a specific genomic locus while expressing its counterpart, GFP, through an available Gal4 driver line. The unique aspect of this system is that neither GFP nor GFP alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells expressing the GFP tagged endogenous protein. We illustrate this technique using the adhesion molecule gene teneurin-m (Ten-m) as a model, highlighting the generation and validation of GFP protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.
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...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.
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 limita...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 de...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.
The use of antibodies as research tools has contributed to the characterization of specific biomolecules and the understanding of their role in biological processes and disease. However, the usefulness of antibodies is o...The use of antibodies as research tools has contributed to the characterization of specific biomolecules and the understanding of their role in biological processes and disease. However, the usefulness of antibodies is only enhanced by their specificity for the target molecule because off-target and non-specific reactivity confounds results. Using accessible websites and standard hybridoma technology, we describe the development of targeted antibodies with high specificity and antibody validation using genetically deficient animals.
Mouse blood extraction is a crucial technique in a wide range of scientific research, including immunological studies. Various blood sampling methods have been outlined and categorized based on factors such as sample vol...Mouse blood extraction is a crucial technique in a wide range of scientific research, including immunological studies. Various blood sampling methods have been outlined and categorized based on factors such as sample volume, sampling frequency, and the necessity of anesthesia. However, the complexity of these techniques arises from the anatomy and size of the mouse, potentially impacting sample quality, endangering the mouse's well-being, and influencing its behavior and the accuracy of analytical results. In this context, the protocol for blood extraction from the saphenous vein of the leg is presented with the goal of minimizing stress in animals and ensuring the safe collection of samples of suitable quality and quantity for different immunological analyses. Thus, this presented technique aims to reduce analytical variations resulting from distress, inflammation, or the use of anesthesia, which might affect subsequent immunological studies.
The tumor microenvironment (TME) plays a pivotal role in tumor development, influencing interactions with immune cells, tumor progression, and responses to treatment. Understanding the heterogeneity of the TME is essenti...The tumor microenvironment (TME) plays a pivotal role in tumor development, influencing interactions with immune cells, tumor progression, and responses to treatment. Understanding the heterogeneity of the TME is essential for uncovering the mechanisms underlying cell-to-cell interactions and their contribution to tumor dynamics. Recent advancements in high-dimensional imaging technologies, such as multiplex immunofluorescence imaging, imaging mass cytometry (IMC), and multiplexed ion beam imaging (MIBI), have provided powerful tools to investigate the complexity of the TME. These technologies allow for the simultaneous analysis of multiple cellular markers and spatial organization of cells within tissue samples, offering detailed insights into the composition and dynamics of the TME. Specifically, IMC offers a unique advantage by enabling the detection of over 40 proteins in a single tissue slide, facilitating a deeper understanding of cellular interactions in situ, at high resolution, and with minimal interchannel crosstalk. Here, we present the analysis approaches and tools developed for imaging mass cytometry data to unravel cellular composition, spatial organization, and interactions within the TME. These methods aim to enhance our understanding of the intricate interplay between cancer cells, immune cells, and stromal components, ultimately supporting the development of more effective therapeutic strategies.
Extracellular ATP (eATP) serves as a crucial signaling molecule in diverse physiological and pathological processes, including neurotransmission, inflammation, and cancer. Despite its importance, accurate measuring eATP...Extracellular ATP (eATP) serves as a crucial signaling molecule in diverse physiological and pathological processes, including neurotransmission, inflammation, and cancer. Despite its importance, accurate measuring eATP dynamics in vivo has remained a significant technical challenge. Traditional methods, such as soluble luciferase systems, fluorescent probes, microelectrode biosensors, and high-performance liquid chromatography (HPLC), exhibit limitations in spatial resolution, tissue permeability, and real-time monitoring. Fluorescent probes offer high spatial resolution but are hindered by poor tissue penetration and the need for excitation light. Microelectrode biosensors provide localized detection but require invasive procedures, while HPLC, though highly sensitive, is restricted to ex vivo applications.To address these limitations, the plasma-membrane-targeted luciferase (pmeLUC) probe was developed. This bioluminescent system allows real-time, quantitative monitoring of eATP levels in living cells and animal models without the need for external excitation light. The pmeLUC is anchored on the outer surface of the plasma membrane, positioning its catalytic site extracellularly for direct eATP sensing. Its high sensitivity, tissue permeability, and adaptability for both in vitro and in vivo studies have enabled significant advancements in understanding eATP dynamics across different pathological contexts, including tumor microenvironments, immune responses, and brain injury models. Furthermore, the creation of pmeLUC-transgenic mice and of AAV-mediated delivery systems, has expanded the applications of this tool for longitudinal and systemic monitoring of eATP in living organisms. This review highlights the rationale behind choosing pmeLUC over other methodologies, emphasizing its superior capabilities in overcoming existing technical barriers and advancing eATP research in both basic and translational sciences.
Methods Cell Biol
· 2025 · PMID 41106940
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Full text
Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that are correlated with improved patient outcomes in several solid cancers, including melanoma. Multiplex immunofluorescent histology (mIFH) has been us...Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that are correlated with improved patient outcomes in several solid cancers, including melanoma. Multiplex immunofluorescent histology (mIFH) has been used in numerous studies to identify and characterize TLS. However, detailed studies evaluating immune cell subsets and markers of immune activity at TLS sites have been limited. Here, we introduce multiplex immunofluorescence histology methods to identify TLS, their associated immune cell components, and markers of immune activity. We outline two mIFH panels for evaluating and quantifying TLS, and markers of immune activity, offering methodologies that can be used to gain a more nuanced understanding of the role and immunological activity of TLS in cancer prognosis and therapy.
High-throughput screening (HTS) is a cornerstone of modern drug discovery and particularly important for the development of novel anticancer therapeutics as it allows to identify agents with specific biological activitie...High-throughput screening (HTS) is a cornerstone of modern drug discovery and particularly important for the development of novel anticancer therapeutics as it allows to identify agents with specific biological activities among a large number of chemical compounds. So far, most anticancer HTS approaches focused either on specifically targeting pathways critical for tumor development or on identifying drugs capable of increasing the immunogenicity of cancer cells, thus directly decreasing tumor cell viability or rendering them detectable to the immune system, respectively. Here, we employ an in vitro antigen presentation system for HTS with the purpose to identify agents that exhibit immunostimulatory potential in the context of dendritic cell (DC)-mediated antigen presentation. To this aim DCs derived from immortalized precursors are treated with an array of compounds and are then confronted with the model antigen ovalbumin (OVA). Coculture of these DCs with T-cell hybridoma cells expressing an OVA-specific T cell receptor (TCR) that triggers the secretion of IL-2 then allows the ELISA-quantifiable assessment of antigen presentation. Altogether, this system can be used to identify immunomodulatory drugs that affect the intricate crosstalk between innate and adaptive immunity.
Eosinophils are a rare immune cell subset with important roles in Th2 immunity and cancer. Interleukin IL-33 (IL-33) plays important functions in the survival and activation of eosinophils. Recent evidence unraveled the...Eosinophils are a rare immune cell subset with important roles in Th2 immunity and cancer. Interleukin IL-33 (IL-33) plays important functions in the survival and activation of eosinophils. Recent evidence unraveled the role of IL-33 in activating the anti-tumor activities of eosinophils enhancing their degranulation and tumor cell killing in a contact-dependent manner. We propose a dual approach methodology to extrapolate the physical interactions of eosinophils with tumor cells as a result of eosinophil stimulation. Human eosinophils isolated from the blood of healthy donors by dextran sedimentation followed by magnetic sorting are exposed to either IL-33 (Eos33) or IL-5 (Eos5) for 18 h. These pre-conditioned cells are then co-cultured with A375P melanoma cells to monitor cell-cell interactions. Acoustic focusing flow cytometry analysis is employed to evaluate the presence of eosinophil-tumor cell conjugates after 1 h incubation of human eosinophils and A375P melanoma cells. Moreover, a 24 h time-lapse video recording approach is employed to obtain single cell tracking eosinophil profiles. This allows to quantitatively determine the interaction extent of Eos33, as opposed to Eos5, with tumor cells. In conclusion, our protocols easily and quickly allow the extrapolation of relevant kinematic and biologically relevant parameters for tumor reactive eosinophils. Furthermore, these methods are adaptable to various models with other types of immune cell subsets and cancer cells and can be implemented on different video microscopy platforms and advanced flow cytometry systems.
Strategies to enhance the recruitment of immune cells in brain tumors to restore its sensitivity to immunotherapy is an area of active investigation. In that context, flow cytometry analysis that assesses the brain immun...Strategies to enhance the recruitment of immune cells in brain tumors to restore its sensitivity to immunotherapy is an area of active investigation. In that context, flow cytometry analysis that assesses the brain immune contexture before and after treatment is critical for developing approaches that foster an inflammatory phenotype of brain tumors. In this chapter, we present a flow cytometry protocol that can be utilized to determine the immune landscape of brain tumors before and after treatment in preclinical models.
Cryotherapy is a device-based modality used to treat certain localized cancers, including cancers of the breast, prostate, liver and skin. This therapeutic approach is based on image-guided placement into a tumor of a ne...Cryotherapy is a device-based modality used to treat certain localized cancers, including cancers of the breast, prostate, liver and skin. This therapeutic approach is based on image-guided placement into a tumor of a needle-like probe that generates ultra-cold conditions at the probe tip to destroy tumor tissue and grossly inflame the local tissue microenvironment. Necrotic and osmotic cell deaths triggered by this local freeze-thaw process enable immune exposure of non-denatured tumor neoantigens released as a result of the tumor ablation. Notably, there is evidence that cryotherapy can exert vaccine-like effects, triggering antitumor immune signals beyond the locally treated tumor that are analogous to the abscopal effects of cancer radiotherapy. Accordingly, there is growing interest in device/drug studies to capture and leverage cryotherapy in combination with immune checkpoint antibodies and/or other immunostimulatory agents for cancer immunotherapy. Here we describe the development of a preclinical mouse model and methods to help elucidate the immunogenic effects of tumor cryoablation, based on an adaptation of the orthotopic metastatic breast cancer model 4T1 to focus on mechanisms of cryo-immunology and their potential implications for treating localized or systemic malignancy.
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,...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 intestinal epithelium consists of a single layer of cells that includes enterocytes, enteroendocrine and goblet cells. This monolayer is in close contact with the underlying lamina propria and spatially segregates th...The intestinal epithelium consists of a single layer of cells that includes enterocytes, enteroendocrine and goblet cells. This monolayer is in close contact with the underlying lamina propria and spatially segregates the gut microbiota and other environmental stimuli from the mucosal immune system. Under certain circumstances, however, gut bacterial components and/or metabolites can perturb tissue homeostasis resulting in abnormal immune responses and tissue damage. In this context, some bacterial toxins, besides stimulating an inflammatory response, may contribute to the activation of pathways related to carcinogenesis. Since immunosurveillance represents a critical barrier to an emerging tumor, characterizing tissue resident immune cells can prove a relevant tool for translational research. The protocol described herein describes the required steps for the isolation and flow cytometry phenotype analysis of tissue resident immune cells from mouse colon following repeated exposure to a pathogenic E. coli toxin as model stimulus.
T cell engagers (TCE) are a class of bispecific antibodies that simultaneously target a tumor antigen and CD3, acting as a bridge between T cells and tumor cells. They promote the formation of T cell receptor (TCR)-indep...T cell engagers (TCE) are a class of bispecific antibodies that simultaneously target a tumor antigen and CD3, acting as a bridge between T cells and tumor cells. They promote the formation of T cell receptor (TCR)-independent canonical immune synapses and potent tumor-specific cytotoxic responses at extremely low concentrations. TCE-based immunotherapeutic have revolutionized the treatment landscape for hematological cancers. Structurally, TCE can be divided into non-IgG-like formats consisting of antibody fragments fused to an Fc-free polypeptide, and IgG-like formats containing an Fc region for construct dimerization. For the former, tandem single-chain variable fragment (scFv) are the most common design. They are potent and small molecules with high tissue penetration and improved tumor penetration, but also have a short serum half-life. To maintain effective serum antibody levels, they require continuous infusion or other strategies such as engineering T cells to secrete the TCE in situ. On the other hand, to enable monovalent CD3 interaction, the binding domains of IgG-like TCEs are usually heterodimerized by incorporating complementary docking mutations in the CH3 domains. In addition, most IgG-like TCEs contain engineered silent Fc to eliminate unwanted Fc interactions and mitigate potential toxicity, while retaining the potential for half-life extension. Here we present detailed protocols for the design, generation and validation of both non-IgG-like and IgG-like TCE, as well as their recombinant production and characterization. The methodology covers construct generation, antibody expression in mammalian systems and purification by chromatography. Finally, structural and functional characterization includes biophysical studies including in vitro and in vivo studies.
Detection of tumor antigen-specific T cells at the tumor site and in the periphery is pivotal to study and monitor T cells directly involved in the antitumor immune response. Through their T-cell receptor (TCR), T cells...Detection of tumor antigen-specific T cells at the tumor site and in the periphery is pivotal to study and monitor T cells directly involved in the antitumor immune response. Through their T-cell receptor (TCR), T cells recognize tumor cells presenting their antigenic peptides by major histocompatibility complex (MHC) molecules. Analyses of antigen-specific CD8 and CD4 T cells can be realized by flow cytometry, using fluorochrome-conjugated multiplexed peptide-MHC molecules. These peptide-MHC multimers, in conjunction with continuing advances in flow cytometry, have transformed the characterization of antigen-specific T cells. They enable the visualization, enumeration, tracking, phenotype characterization and isolation of T cells during infection, autoimmunity and cancer. They can be useful to monitor specific adaptive responses during various type of therapies. Here we detail a simple and accurate protocol of multimer staining of tumor antigen-specific CD8 T cells isolated from human and mouse tumors and Peripheral Blood Mononuclear Cells (PBMCs).
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 precli...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.
Spatial technologies have been revolutionary in the analysis of intact tissue samples as they allow for in depth investigation of cellular spatial interactions that cannot be elucidated through conventional single-cell m...Spatial technologies have been revolutionary in the analysis of intact tissue samples as they allow for in depth investigation of cellular spatial interactions that cannot be elucidated through conventional single-cell methodologies. Imaging mass cytometry (IMC) is a unique and novel spatial proteomics platform that utilizes metal-conjugated antibodies to generate multi-plex images where spatial information of single cells can be extracted from intact tissue samples, allowing for in depth spatial analyses. However, extracting single cells data and spatial information from IMC multi-plex images can be technically challenging for basic scientists without experience in bioinformatics or computational sciences. In this chapter, we describe how steps on how to extract single cell data and spatial information from multi-plex IMC images of intact tissue samples and tips on performing cell clustering and spatial analyses.