Obesity is a multifactorial disease characterized by excessive accumulation of adipose tissue, resulting from an imbalance between energy intake and expenditure. Mouse models have emerged as invaluable tools for elucidat...Obesity is a multifactorial disease characterized by excessive accumulation of adipose tissue, resulting from an imbalance between energy intake and expenditure. Mouse models have emerged as invaluable tools for elucidating the complex genetic, environmental, and physiological mechanisms driving to obesity. This chapter provides an overview of the methodologies employed to establish and study obesity in mice, highlighting their relevance to human disease. We described diet-induced obesity (DIO) protocols, which replicate environmental factors contributing to weight gain. Additionally, we discussed the use of genetic models, including whole-body knockouts such as leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice, as well as conditional knockout models targeting the hypothalamus, a critical brain region for maintaining body homeostasis. Key considerations in experimental design, such as strain selection, sex differences, and the impact of housing conditions, are discussed to ensure reproducibility and translational relevance. This chapter serves as a practical guide for researchers aiming to leverage mouse models in obesity studies, fostering a better understanding of this global health challenge.
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, ther...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.
Head and neck squamous cell carcinoma (HNSCC) is among the most prevalent human malignancies globally, with approximately 887,000 new cases diagnosed each year. Currently, the standard treatment for HNSCC involves surger...Head and neck squamous cell carcinoma (HNSCC) is among the most prevalent human malignancies globally, with approximately 887,000 new cases diagnosed each year. Currently, the standard treatment for HNSCC involves surgery, followed by radiotherapy, chemotherapy and immunotherapy. However, despite these available treatments, the survival rate of patients with HNSCC remains low. A key factor in the development of HNSCC is genomic instability, which significantly influences several phases of the disease such as initiation and progression. Given the complexity of HNSCC, preclinical models are essential for exploring the mechanisms driving HNSCC progression, at the (epi)genetic (e.g., DNA mutations, histone modifications), cellular (e.g., resistance to cell death, autophagy) and histological (e.g., cancer-immunity cycle) levels. Moreover, such a model is precious for developing new therapies. In this context, we provide a comprehensive protocol outlining the steps required to establish a syngeneic orthotopic tongue mouse model, including cell preparation and injection steps. By implanting cells in their natural anatomical location within immunocompetent mice, this model allows to investigate interactions between the tumor and surrounding tissues, which can significantly influence tumor growth.
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 neuro...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.
Mesentery is a crucial part of an animal's digestive system since it holds the intestine in place, while also contains the specialized lymph nodes and immune cells that help protect the intestines from infections and sup...Mesentery is a crucial part of an animal's digestive system since it holds the intestine in place, while also contains the specialized lymph nodes and immune cells that help protect the intestines from infections and support the body's immune response in the abdominal cavity. Analyzing mesenteric lymph can help better understand the transport mechanisms and potential implications for various conditions, such as lymphatic disorders or underlying infections. The first step towards this is the precise collection of the mesenteric lymph. In this chapter, we describe the optimized protocol of cannulation to collect afferent and efferent mesenteric lymphatic fluid.
Cushing's syndrome is caused by chronic exposure to excessive levels of glucocorticoids. It is characterized by significant phenotypic alterations including increased visceral adiposity and fat deposits on the cheeks, le...Cushing's syndrome is caused by chronic exposure to excessive levels of glucocorticoids. It is characterized by significant phenotypic alterations including increased visceral adiposity and fat deposits on the cheeks, leading to a characteristic 'moon face' appearance. Although glucocorticoid therapy is widespread, its associated side effects are of significant clinical concern. Early diagnosis and treatment of Cushing's syndrome can reduce its morbidity and mortality. The use of mouse models is crucial for the discovery and exploration of treatment options against Cushing's syndrome. This article describes a methodology to assess phenotypic determinants of Cushing's syndrome in mice through the combined measurement of the elevated surface area of the animal, which is a proxy of weight gain, together with the increased angle from the nasal tip to the cheeks, which is a proxy of the 'moon face'.
The pivotal role of cytotoxic T lymphocyte (CTL) killing of target cells in vivo continues to be underscored by emerging research. CTLs are antigen-specific effector CD8 + T lymphocytes that serve as adaptive defenders a...The pivotal role of cytotoxic T lymphocyte (CTL) killing of target cells in vivo continues to be underscored by emerging research. CTLs are antigen-specific effector CD8 + T lymphocytes that serve as adaptive defenders against a myriad of threats, including viral infections, cancerous cells, and other pathogenic invaders. In vivo CTL killing assays contemplate the interaction of effector and target cells in the context of a proper microenvironment, making the analysis biologically more relevant than in vitro assays. They play a crucial role in advancing our understanding of immune responses, disease mechanisms, and therapeutic strategies in physiologically relevant settings, with implications for both basic research and clinical practice in immunology and related fields. Here, we describe and discuss in detail a method to explore the in vivo elimination of target cells by antigen-specific CTLs using immunized/vaccinated mice as our experimental model.
Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder characterized by progressive degeneration of motor neurons, leading to muscle weakness, paralysis, and death. While there is a plethora of studie...Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder characterized by progressive degeneration of motor neurons, leading to muscle weakness, paralysis, and death. While there is a plethora of studies focusing on many aspects of ALS, the pathogenesis of this disease is not well understood, and effective treatments are scarce. Drosophila melanogaster is a powerful model organism for studying ALS due to its genetic tractability and its evolutionarily conserved cellular and molecular processes which are also shared between the fly and human. Here, we introduce two simple and cost-effective methodologies for assessing motor neuron dysfunction in Drosophila: (1) Fast Inexpensive Climbing Test (FICT), and (2) Economical Leg Fluorescence Imaging (ELFI). These methods are established based on using basic equipment and straightforward procedures, making them accessible and applicable in various research and educational settings. FICT provides a non-invasive and high-throughput measure of motor dysfunction, while ELFI allows for direct visualization of fluorescently labeled cells in the Drosophila leg, facilitating the study of cell-cell communications in vivo. Our approach emphasizes the importance of both neuronal and glial contributions to ALS pathogenesis, offering valuable insights for the development of novel therapeutic strategies. These methods democratize access to ALS research tools, promoting global scientific collaboration and advancing our understanding of this devastating disease.
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that is characterized by a severe and progressive demyelinating process. It is considered a neurodegenerative autoimmune disor...Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that is characterized by a severe and progressive demyelinating process. It is considered a neurodegenerative autoimmune disorder driven by immune cell infiltration, overproduction of cytokines and reactive oxygen species (ROS) accumulation that leads to axonal and neuronal injury. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used pre-clinical model of multiple sclerosis (MS), since it resembles many aspects of the human disease. EAE can be induced in a variety of species and strains (rodents and monkeys), providing models of acute monophasic, relapsing-remitting and chronic progressive CNS inflammation. Thus, the pathology of the lesions varies according to the animal model used. We herein describe in detail a protocol for induction of EAE in C57BL/6 mice by immunization with MOG in CFA, which induces a monophasic, chronic and sustained form of EAE. In addition, we also describe approaches to evaluate disease induction and a technique for pathological examination of CNS tissues to assess ROS accumulation. This animal model could be useful for acute and chronic studies and to assess the effectiveness of different treatments.
The present study focuses on the phenotypic characterization of several mutants of Flavobacterium psychrophilum, obtained from a transposon mutant library. This Gram-negative bacterium is the etiological agent of the "co...The present study focuses on the phenotypic characterization of several mutants of Flavobacterium psychrophilum, obtained from a transposon mutant library. This Gram-negative bacterium is the etiological agent of the "cold water disease", pathology that usually affects salmonids, mainly Oncorhynchus mykiss. This microorganism is considered a "fastidious bacterium" due to the difficulty to isolate it. Multiple secreted molecules have been described as contributors to virulence, such as proteases, toxins, and/or adhesins. In addition, F. psychrophilum has a particular way to move across wet surfaces, a physiological process known as gliding. In this research, four mutants were selected from a F. psychrophilum transposition library: (i) an ATPase chaperone activity (ClpB); (ii) a secretion system regulatory protein (PorY); (iii) a protein involved in exopolysaccharide synthesis (Wzc); (iv) a fungalysin with proteolytic activity (FpfB). Mutant characterization in some parameters (such as growth and colony spreading) showed that there were not significant differences between the mutants and the parental strain (except for the clpB mutant). Further analysis of the clpB mutant revealed differences in LD and response to HO and thermal shock compared with the parental strain.
The tumor microenvironment and, particularly, tumor-infiltrating immune cells can profoundly influence tumor progression and response to therapy. Deconvolution is a powerful computational technique to estimate cell-type...The tumor microenvironment and, particularly, tumor-infiltrating immune cells can profoundly influence tumor progression and response to therapy. Deconvolution is a powerful computational technique to estimate cell-type fractions from bulk RNA sequencing (RNA-seq) data leveraging expression signatures specific to the cell types of interest. Recently, a new generation of deconvolution algorithms has emerged, making it possible to directly learn cell-type-specific signatures to be used for deconvolution from annotated single-cell RNA-seq (scRNA-seq) datasets. Thanks to their flexibility, these next-generation methods can extend deconvolution to any cell type, tissue, and organism for which a suitable single-cell reference is available. However, these methodologies are highly diverse in terms of programming languages, computational workflows, and input/output data, which complicate their usage and comparison. To overcome these challenges, we developed omnideconv, an R package that integrates several deconvolution methods, streamlining their usage and unifying their semantics. In this chapter, we demonstrate how omnideconv can be integrated with an annotated scRNA-seq dataset, comprising both malignant and normal cells from the breast cancer microenvironment, to quantify the cellular composition of bulk RNA-seq data from a cohort of breast cancer patients.
Invariant natural killer T (iNKT) cells are a non-conventional T-cell population characterized by the expression of a conserved semi-invariant T-cell receptor (TCR) with specificity towards self or microbial lipid antige...Invariant natural killer T (iNKT) cells are a non-conventional T-cell population characterized by the expression of a conserved semi-invariant T-cell receptor (TCR) with specificity towards self or microbial lipid antigens, presented by the non-polymorphic MHC class I-related molecule CD1d. iNKT cells play a pivotal role in tumor immunosurveillance and serve as a potent tool for anti-cancer therapies. Notably, iNKT cells can be effectively redirected against both hematological and solid malignancies through genetic engineering using either Chimeric Antigen Receptors (CARs) or TCRs targeted to tumor antigens. However, due to their low frequency, iNKT cell expansion in vitro is an essential step to obtain suitable cell number for adoptive cell therapy (ACT). Here we describe two robust methods for efficiently isolating primary mouse and human iNKT cells that can be easily genetically modified and expanded. iNKT cells are isolated from the spleens of iVα14-Jα18 transgenic mice or from human buffy coats resulting in highly enriched populations. Both mouse and human iNKT cells are activated with anti-CD3/CD28 beads, IL-2 and IL-7, and subsequently transduced with tumor-specific receptors, yielding millions of ready-to-use, highly pure, and stably transduced tumor-redirected iNKT cells. The final cell product is suitable for in vitro investigation of iNKT cell activation and function mechanisms, as well as for pre-clinical ACT studies.
It is well established that reciprocal communication between cancer cells and other cells in the tumor microenvironment plays a crucial role in cancer progression and therapy response. There are multiple ways by which ce...It is well established that reciprocal communication between cancer cells and other cells in the tumor microenvironment plays a crucial role in cancer progression and therapy response. There are multiple ways by which cells communicate, including direct cell-cell contact and the secretion of soluble mediators. The secretome of cancer cells contains valuable information to disentangle the complex conversation that is happening between cancer cells and neighboring or distant cells such as immune cells, fibroblasts and endothelial cells. Here, we provide a workflow of mapping the cancer cell secretome in an unbiased way using amino acid-analog labeling in combination with mass spectrometry. The generation of single cells from fresh tumors, isolation of primary cancer cells from a complex multi-cellular pool, and the detection of newly synthesized proteins that are secreted into the medium is described in detailed protocols. Using this experimental pipeline the secretome of cancer cells across different tumors can be determined, paving the way to unravel cell-cell communication networks in the tumor microenvironment, which may uncover novel therapeutic targets.
Dendritic cells (DCs) are professional antigen-presenting cells that are pivotal in operating tumor immunosurveillance and orchestrating anticancer immune responses. Endowed with phagocytic and migratory capacities, DCs...Dendritic cells (DCs) are professional antigen-presenting cells that are pivotal in operating tumor immunosurveillance and orchestrating anticancer immune responses. Endowed with phagocytic and migratory capacities, DCs can capture and process tumor antigens, travel to lymphoid organs and prime naïve T cells, altogether leading to the clonal expansion of cytotoxic T lymphocytes (CTLs) that can specifically target and lyse cancer cells. Additionally, DCs contribute to the formation of immunological memory, ensuring durable therapeutic effects and long-term surveillance against tumor recurrence. Upon antigen engagement, DCs undergo a maturation process characterized by the production of specific cytokines as well as the increased expression of costimulatory molecules and chemokine receptors on their surface. Here we propose a panel of custom sandwich enzyme linked immunosorbent assays (ELISAs) for assessing DC maturation via the precise quantification of cytokines. This economic approach achieves high precision and reproducibility and can be readily applied in labs equipped with basic molecular cell biology facilities. With appropriate automatization, this protocol can be employed for high-throughput screening campaigns for the discovery of DC maturation modulators.
The pathological expansion of immature blood vessels through neovascularization contributes to the development of a variety of diseases. In cancer, neovascularization supports tumor outgrowth and influences how tumors re...The pathological expansion of immature blood vessels through neovascularization contributes to the development of a variety of diseases. In cancer, neovascularization supports tumor outgrowth and influences how tumors respond to therapy. Our studies have revealed that a defined cell population termed IDVCs (IDO1-dependent vascularizing cells) expressing the tryptophan catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) can foster a local inflammatory environment that promotes neovascularization. A powerful tool for investigating the biological role of isolated IDVCs in this inflammatory neovascularization process has been the Matrigel plug assay. In this assay, isolated cells are incorporated into a subcutaneously implanted Matrigel plug which is subsequently evaluated by confocal immunofluorescence microscopy for blood vessel density. We have employed this assay to demonstrate that isolated IDVCs are capable of promoting local neovascularization in an IDO1-dependent manner. Furthermore, the use of genetically engineered mouse strains and pharmacological interventions has enabled us to carry out in-depth investigations into IDO1's function as a nodal modifier of the local inflammatory environment responsible for eliciting a shift in the cytokine milieu from a neovasculature-restrictive to a neovasculature-sustaining status. Here we present a detailed methodology describing the reagents and procedures developed to isolate IDVCs and perform quantitative neovascularization studies. This assay should have great utility as a means for conducting investigative studies delving into the cellular and molecular processes involved in the complex interplay between inflammation and neovascularization.
Natural Killer cells (NK) are cytotoxic lymphocytes from the innate immune system that recognize and eliminate virally infected and tumor cells. Accordingly, manipulation of NK cells has been the focus of several immunot...Natural Killer cells (NK) are cytotoxic lymphocytes from the innate immune system that recognize and eliminate virally infected and tumor cells. Accordingly, manipulation of NK cells has been the focus of several immunotherapy protocols aimed at eradicating cancer cells. Allogeneic NK cell therapy was initially described over two decades ago, emphasizing KIR-mismatch's importance in preventing NK cell inhibition and promoting cytotoxicity and tumor elimination without inducing graft-versus-host disease (GvHD). While unstimulated NK cells have shown limited antitumoral activity in adoptive cell therapy, various activation and expansion protocols have been proposed to enhance their cytotoxic potential. Activated and expanded allogeneic NK cells, especially with the rise of chimeric antigen receptor (CAR) therapies, have attracted significant attention from academic and commercial sectors. Protocols typically involve using cytokines and stimulatory cells, such as Epstein-Barr virus (EBV)-transformed lymphoblastoid B cell lines (LCLs) or K562 leukemic cells, before or after NK cell enrichment. Here we present two different standardized protocols for NK cell activation and expansion, offering insights into NK cell-based immunotherapies for cancer treatment. We also present a comprehensive methodology for assessing NK cell-mediated cytotoxicity against Neuroblastoma cell lines in both 2D and 3D cultures. The comprehensive methodology presented here lays the foundation for further research in the field, driving advancements in NK cell-based therapies against malignancies.
In the recent years, a better understanding of the cellular and molecular processes that shape the dynamic balance of the immune system and the tumour has been gained. Accumulating evidence shows that cellular and acellu...In the recent years, a better understanding of the cellular and molecular processes that shape the dynamic balance of the immune system and the tumour has been gained. Accumulating evidence shows that cellular and acellular components in the tumour microenvironment (TME) can reprogram the response to immunotherapies. Transcriptome analyses of immunotherapy treatments have been invaluable for studying signalling pathways in cancer. Transcriptional characterization of tumours has allowed researchers to both predict the likely response to therapy as well as to evaluate the effect of therapy on tumours before any phenotypic changes are observed. Despite the rise of single-cell RNA sequencing (scRNAseq) it remains costly and technically challenging and, in many situations, unnecessarily detailed. Presented here is a protocol describing processing of tumours for microarray transcriptome analysis to evaluate the responses to therapy. This method has proven to be cost-effective in cases when broad transcriptional responses are being investigated.
Epigenetic deregulation is implied in cancer initiation and resistance to antitumor drugs. In melanoma, aberrant DNA hypermethylation is frequently observed, resulting in the silencing of several genes involved in cell c...Epigenetic deregulation is implied in cancer initiation and resistance to antitumor drugs. In melanoma, aberrant DNA hypermethylation is frequently observed, resulting in the silencing of several genes involved in cell cycle regulation, apoptosis, tumor growth and drug resistance. DNA hypomethylating agents have been recently evaluated in both preclinical and clinical studies as a strategy to restore tumor suppressor genes and to increase immune recognition by tumors, highlighting their potential in pre-clinical models of melanoma. Advanced microfluidic system for the culture of complex three-dimensional cell, tissue and organ models have proven utility for oncoimmunology studies and drug testing. Here we present a protocol employing ad hoc fabricated microfluidic devices to reproduce a three-dimensional (3D) tumor microenvironment (TME) to study two aspects of the crosstalk between immune and cancerous cells under the effect of Decitabine (DAC), a DNA methyl transferase inhibitor (DNMTi). First, we evaluated the preferential migration of immune cells towards treated and non-treated melanoma cells inside the chip. Next, we identified a specific subpopulation of migrated immune cells, with an on-chip immunostaining protocol resulting in the acquisition and evaluation of 3D images on a Laser-Scanning Confocal Microscopy (LSCM) station for in-depth characterization of tumor-immune interactions. This protocol may find broad application for pre-clinical drug testing in cancer studies.
Basophils constitute a rare population of granulocytes with key functions in allergies, immunodeficiencies and cancer. The scarcity of basophils in human blood and tissues constitutes a considerable limit for the study o...Basophils constitute a rare population of granulocytes with key functions in allergies, immunodeficiencies and cancer. The scarcity of basophils in human blood and tissues constitutes a considerable limit for the study of these cells. Interleukin-3 (IL-3) stimulates both the differentiation and the expansion of basophils from bone marrow (BM) precursors by positively regulating the expression of the IL3Ra receptor. We have standardized an in vitro differentiation protocol of mouse basophils (mBaso) from BM precursors through culture in presence of IL-3 for 10 days followed by cell sorting. At the end of the 10-day differentiation, a considerable number of mBaso can be obtained and cell sorting procedures further improved the isolation of an extraordinarily pure (>98 %) and vital FcεR1 CD11c c-kit mBaso population. Phenotypic analysis of terminally differentiated (day 10) unsorted mBaso cultured for 24 h showed a decrease in basophilic lineage (c-kit) and an increase of mastocytic lineage (c-kit) and reduced the expression of basophil markers FcεRI, CD49b and CD200R either in absence of stimuli or following activation with the alarmin IL-33, indicating cell dedifferentiation. In contrast, terminally differentiated and FcεR1 CD11c c-kit sorted mBaso do not dedifferentiate in mast cells when placed in culture, and responded to IL-33 stimulation by up-regulating the activation marker CD63 without down-modulation of FcεRI and CD200R3. These evidences highlight that in vitro differentiation followed by cell sorting is a useful method to obtain elevated numbers of highly pure mBaso that preserve their lineage markers and thus are suitable for conducting the desired functional studies.
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 thi...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.