Liquid biopsy is a non-invasive molecular test that uses biofluid to detect cancer. This is a rapidly growing field that encompasses a diverse class of assays that rely on circulating nucleic acids, proteins, and cells t...Liquid biopsy is a non-invasive molecular test that uses biofluid to detect cancer. This is a rapidly growing field that encompasses a diverse class of assays that rely on circulating nucleic acids, proteins, and cells to identify cancer. Recently, there is particular interest in using circulating tumor DNA (ctDNA) to screen for cancer, to monitor treatment response, and to help guide therapeutic decisions. Here, we will review the procedural aspects of ctDNA assays, as well as the various applications of ctDNA assays in cancer care. The utility of various biofluids to conduct ctDNA assays, the technical considerations for performing the assays, and several possible available assay formats that can be used for ctDNA analysis are reviewed. Several protocols that can be used for ctDNA isolation, detection, and quantification are presented, including targeted ctDNA qPCR.
Extracellular vesicles (EVs) are released by all cell types in the bodily fluids. EVs represent key players of inter-cellular communications, including maternal-fetal cross-talk and mother-infant information transmission...Extracellular vesicles (EVs) are released by all cell types in the bodily fluids. EVs represent key players of inter-cellular communications, including maternal-fetal cross-talk and mother-infant information transmission. Here, we will focus on EVs derived by amniotic liquid and maternal milk. We will report methodologies of EV isolation from these maternal fluids.
Colorectal cancer (CRC) development is initiated in the colon-rectum sections of the gut, by the emergence of a primary tumor. CRC slowly progresses to a multiple locations metastatic disease, involving secondary tumors...Colorectal cancer (CRC) development is initiated in the colon-rectum sections of the gut, by the emergence of a primary tumor. CRC slowly progresses to a multiple locations metastatic disease, involving secondary tumors arising in various organs, such as the liver. Mouse models have been developed to investigate the immune response, locally generated in primary tumors. Classically, tumors are implanted under the skin, for practical reasons and simplicity of monitoring. However, the skin location does not necessarily recapitulate the tumor immune microenvironment (TME) that would normally be generated in the gut. The orthotopic CRC mouse model, that we describe hereafter, was generated to investigate the colon-local mechanisms driving the establishment and the polarization of primary tumors TME. In this chapter, we detail the procedures used to implant syngeneic colon tumor cells in the cecum of immunocompetent mice and to monitor the progression of visceral tumors in live mice. The same procedure can be implemented using other tumor cell lines and mouse genetic backgrounds.
Myeloid-derived suppressor cells (MDSC) are heterogenous group of immature and mature myeloid cells that accumulate in chronic inflammatory conditions and contribute to the immune suppression. Their ability to inhibit im...Myeloid-derived suppressor cells (MDSC) are heterogenous group of immature and mature myeloid cells that accumulate in chronic inflammatory conditions and contribute to the immune suppression. Their ability to inhibit immune responses makes them critical targets for therapeutic interventions. However, working with patient-derived MDSC poses challenges. Variability in cell yields and the complexity of their isolation procedures significantly hinder the conduct of in vitro experiments aimed at investigating potential targeting strategies. In this chapter, we introduce a method to generate human CD14 MDSC-like cells in vitro from healthy donor monocytes. This method provides a consistent and reproducible framework for studying MDSC functions and testing potential inhibitors of their activity.
Novel therapeutic approaches highlight the need for advanced ex vivo cell culture models that more closely resemble the physiological and genetic properties of the primary tumor. Patient-derived models could serve as an...Novel therapeutic approaches highlight the need for advanced ex vivo cell culture models that more closely resemble the physiological and genetic properties of the primary tumor. Patient-derived models could serve as an attractive strategy to investigate the crosstalk between cancer cells and its microenvironment and to test potential therapeutic targets, paving the way for precision oncology. In this chapter, we provide a detailed step-by-step protocol for enabling a direct co-culture system of patient-derived colorectal cancer (CRC) organoids with autologous tumor-infiltrating lymphocytes (TILs). The present protocol provides a methodology to gain direct access to the apical side of the epithelial cells forming the organoids. This method can be used to investigate patient-specific cell-to-cell interactions, T cell functionality and efficacy and provides a robust platform to validate potential immunogenic neoantigens.
In vitro tumor models might be useful to study tumor growth, invasion and therapy resistance, but also extracellular matrix (ECM) remodeling. This protocol will provide information for the generation of a 3D tumor cell c...In vitro tumor models might be useful to study tumor growth, invasion and therapy resistance, but also extracellular matrix (ECM) remodeling. This protocol will provide information for the generation of a 3D tumor cell cultures for high content analysis and describes a novel method to monitor the ECM morphology and remodeling as well as therapy responses using live 3D tumor cell spheroids. Using 3D spheroids of thyroid carcinoma (TC) cells as a model, our method involves the treatment of TC cells with inhibitory agents followed by subsequent analysis of ECM components to assess the influence of these drugs on the structural integrity of the EMT using fluorescence labeled antibodies (Ab) and confocal microscopy. Employing this method, the morphology of the formed spheroids under different conditions and co-cultures as well as the distribution of ECM components can be assessed, such as e.g. fibronectin 1 (FN1). The results will also provide valuable insights into the tumor microenvironment (TME) and potential interactions of viable spheroids with the components of the TME during the ECM remodeling process. The implementation of 3D spheroids for studying EMT in TC as a model has been shown to provide more accurate and representative results compared to traditional 2D monolayer cell cultures.
Immunohistochemistry (IHC) is a powerful technique that utilizes specific antibodies to visualize distinct cell populations within tissues. However, this method has some limitations, in particular the specificity and det...Immunohistochemistry (IHC) is a powerful technique that utilizes specific antibodies to visualize distinct cell populations within tissues. However, this method has some limitations, in particular the specificity and detection of low expressed markers including soluble factors, like e.g., cytokines. Other methods, such as in-situ hybridization (ISH), are a suitable alternative to visualize these factors in the tissue. Recent advances have opened exciting opportunities for quantitative data acquisition related to both protein and mRNA expression. Moreover, these techniques allow the analysis of their spatial distribution within a tissue and in a cell-specific context. Moreover, improvements in tissue preparation, fluorescent dyes, tissue imaging and downstream analysis have addressed challenges related to quantitative precision. Nowadays, researchers can obtain more accurately measurements of protein and mRNA expression levels of multiple targets within one sample. This technique gives us the opportunity to visualize and record the spatial relationship between different cells in formalin-fixed, paraffin-embedded samples. This chapter summarizes a protocol developed for cytokine expression analysis in the bone marrow of myeloid neoplasms. It provides an overview of the workflow that can be adapted to other tissues and other disease specific contexts.
Lung cancer remains a leading cause of cancer-related deaths, highlighting the importance of understanding the lung's immune landscape, which plays a key role in tumor progression and response to therapy. As an organ con...Lung cancer remains a leading cause of cancer-related deaths, highlighting the importance of understanding the lung's immune landscape, which plays a key role in tumor progression and response to therapy. As an organ constantly exposed to environmental and microbial challenges, the lung's unique tumor microenvironment is shaped by these additional stimuli, influencing immune responses. Here, we present a standardized protocol to examine dynamic changes in the lung immune cell compartment in response to bacterial challenges in a genetically engineered mouse model of lung cancer. This method ensures efficient tissue processing and dissociation while preserving cell viability for high-throughput flow cytometry. Additionally, we describe an integrative analysis approach to systematically analyze data across tissues and conditions, providing a comprehensive framework for studying immune cell dynamics in lung carcinoma.
Methods Cell Biol
· 2026 · PMID 41620272
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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 ch...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.
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 lymphocyt...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.
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...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.
Proteome-level investigations of distinct cell types while retaining their native states in tissue can provide key insights into disease mechanisms that may not be captured by transcriptomic studies. We describe protocol...Proteome-level investigations of distinct cell types while retaining their native states in tissue can provide key insights into disease mechanisms that may not be captured by transcriptomic studies. We describe protocols to achieve cell type-specific in vivo biotinylation of proteins (CIBOP) in mouse brain. CIBOP uses a proximity labeling approach in which biotin ligase TurboID is expressed in specific cell types, leading to broad proteomic biotinylation. Subsequently, biotinylated proteins can be enriched from bulk tissue homogenates without requiring cell type isolation, followed by mass spectrometry-based quantitative proteomics of biotinylated proteins, yielding cell type-specific proteomes. We showcase CIBOP to label neurons and astrocytes, using adenovirus-based as well as transgenic approaches. This versatile pipeline may be readily applicable to various cell types as well as to neurological and non-neurological disease model systems.
In the heart, interaction among cells is essential for homeostasis and response to injury. Identifying paracrine/autocrine factors released by cardiomyocytes is fundamental to understand this complex network. However, li...In the heart, interaction among cells is essential for homeostasis and response to injury. Identifying paracrine/autocrine factors released by cardiomyocytes is fundamental to understand this complex network. However, limitations in the tools available to culture and manipulate isolated adult cardiomyocytes have hindered the study of these released factors. Here, we outline a protocol for profiling the secretome from isolated and in vitro cultured adult cardiomyocytes, using the well-established proximity ligation system.
One important strategy of cells to communicate with their environment is the release of proteins which can serve as signals for other cells nearby or at distant places in a complex organism. A first step in the character...One important strategy of cells to communicate with their environment is the release of proteins which can serve as signals for other cells nearby or at distant places in a complex organism. A first step in the characterization and investigation of cell-cell communication in this context is to figure out which proteins are released from cells under defined experimental conditions. Here, we present an approach that will give rise to a high-quality secretome and detects proteins that will be confidently released by cultured cells. This approach is based on the separate preparation of proteins from conditioned medium and corresponding cell lysates. After protein digestion and quantitative mass spectrometric analysis, protein abundances are compared and proteins showing a significantly higher abundance in the secretome are identified. We assume that these proteins have a higher probability of being released by well-directed processes and not simply by contamination of (dead) cells. We show an optimized protocol in which samples from primary human normal dermal fibroblasts (NHDF) are prepared with the single-pot solid-phase-enhanced sample preparation (SP3) method from only 450μL conditioned medium along with one-hour gradient separations and data-independent mass spectrometric data acquisition.
S-palmitoylation of cysteine residues is the only lipid-based posttranslational modification of proteins that is reversible and therefore has important implications in cellular function. S-palmitoylation has been associa...S-palmitoylation of cysteine residues is the only lipid-based posttranslational modification of proteins that is reversible and therefore has important implications in cellular function. S-palmitoylation has been associated with several cellular processes (e.g., cell signaling, protein transport, cell cycle, immune response, lipid metabolism, host-pathogen interaction) and human diseases, including neurological disorders, cancer, and infectious diseases. However, S-palmitoylation research has been hampered by the cumbersome experimental protocols necessary for its study. Currently, there are two main methodologies that, coupled with mass spectrometry (MS), allow the study of S-palmitoylated proteins proteome-wide. They mainly differ in the way of labeling palmitoylated proteins: one relies on "metabolic labeling" with a palmitic acid analog in living cells, while the other is based on "chemical labeling" of thiol groups derived from palmitoylated sites in extracted proteins. Although metabolic labeling is restricted to cultured cells, we will focus on this technique as it is more sensitive and specific than others. Here, we describe the protocol to measure palmitoylation in cancer cells using metabolic labeling coupled to SILAC-based mass spectrometry quantification, which can be applied to other mammalian cell models. Facilitating the use of this methodology will extend the knowledge of palmitoylation signaling and unravel potential therapeutic avenues for diseases in which this unexplored modification is implicated.
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 take...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.
Methods Cell Biol
· 2026 · PMID 41276349
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Inter-organ communication, including the release of secreted proteins, plays a key role in synchronized physiological responses and organismal homeostasis. Recent studies have emphasized functions of muscle-secreted prot...Inter-organ communication, including the release of secreted proteins, plays a key role in synchronized physiological responses and organismal homeostasis. Recent studies have emphasized functions of muscle-secreted proteins (i.e., myokines), in regulating metabolic pathways and improving metabolic dysfunction distally in the liver. Thus, experimental workflows to study myokines and their impact on target cell types are of scientific value. Here, we describe a cell culture-based method to investigate communication from muscle to liver mediated by secreted proteins. Briefly, C2C12 myoblasts are differentiated into myotubes, myotube-conditioned media is collected, and myotube-secreted proteins are isolated and stored. To demonstrate the utility of this method, AML12 hepatocytes were treated with myotube-secreted proteins and effects on bioenergetics were assessed. This method can be useful as a proof of principle tool, for mechanistic studies, or paired with proteomic or biochemical analyses to identify novel myokines. We also envision it is adaptable in terms of cell type, downstream application, and signaling direction.
Methods Cell Biol
· 2026 · PMID 41276348
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Full text
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 extracellul...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.