3D organoids are vital for biomedical research, offering complex tissue models superior to 2D cultures for studying development, disease, and drug responses. High-throughput imaging of these structures is challenging. Th...3D organoids are vital for biomedical research, offering complex tissue models superior to 2D cultures for studying development, disease, and drug responses. High-throughput imaging of these structures is challenging. This report details a method for screening 3D organoids using the Leica Thunder microscope, featuring Computational Clearing for enhanced deep imaging. The protocol covers sample preparation, optimized image acquisition using LAS X, and analysis with FiJi/ImageJ for quantification. Applications include drug discovery and disease modeling. The Leica Thunder facilitates efficient 3D organoid screening.
Immunotherapy has revolutionized cancer treatment by enhancing the immune system's ability to target malignant cells. Despite its success, significant challenges remain, particularly treatment-induced toxicities such as...Immunotherapy has revolutionized cancer treatment by enhancing the immune system's ability to target malignant cells. Despite its success, significant challenges remain, particularly treatment-induced toxicities such as "on-target, off-tumor" (OTOT) effects and immune-related adverse events (irAEs). CAR-T cells, bispecific T-cell engagers (BiTEs), and monoclonal antibodies can target antigens shared by both tumors and normal tissues, leading to severe organ-specific toxicities. Similarly, immune checkpoint inhibitors (ICIs) disrupt immune self-tolerance, causing autoimmune-like complications in various organs. Current preclinical models fail to fully predict these toxicities due to their limited ability to replicate human immune-tissue interactions. To address this, we developed an ex vivo human tissue slice model that preserves native immune microenvironments and enables the study of T-cell-mediated toxicity. Using a superantigen to induce broad T-cell activation, our model assesses immunotherapy-induced cytotoxicity through cytokine secretion assays and apoptosis detection (cleaved caspase-3). This approach provides a powerful tool for predicting immunotherapy-induced toxicities and optimizing treatment strategies.
Lysosomotropism refers to the ability of certain basic lipophilic compounds to accumulate in lysosomes via pH partitioning. Various drugs including anticancer agents are trapped in lysosomes, and this process can prevent...Lysosomotropism refers to the ability of certain basic lipophilic compounds to accumulate in lysosomes via pH partitioning. Various drugs including anticancer agents are trapped in lysosomes, and this process can prevent such drugs from reaching their primary target, thereby limiting their effectiveness. Strategies aimed at preventing drug sequestration or inducing drug release from lysosomes have garnered considerable interest. Chloroquine is a widely used anti-malarial drug that triggers lysosome membrane permeabilization (LMP) to liberate sequestered drugs from these organelles. In this study, we first evaluate the lysosomotropism of various fluorescent anticancer agents in silico. Next, we outline a simple, fast and robust method for the visualization and quantification of their lysosomal sequestration and release by fluorescence microscopy. The method is used on live cells and consists of two steps: (i) visualization of the compounds in lysosomes by analyzing their colocalization with a specific fluorescent lysosomal marker, and (ii) assessment of drug release from lysosomes. Furthermore, we present fluorescence microscopy protocols for monitoring LMP by analyzing the subcellular localization of LGALS3 (Galectin-3), which normally distributes diffusely in the cytoplasm but translocates into lysosomes upon LMP. This can be achieved on fixed cells by detecting endogenous LGALS3 with immunostaining or by the visualization of a transgenic LGALS3-mCherry fusion protein on live cells. Altogether, these methods facilitate qualitative and quantitative fluorescence imaging of lysosomal sequestration and liberation of lysosomotropic drugs.
Here we detail a method for the exploration of patient-derived xenografts (PDX) based on organoids (PDX-O), using antibodies validated for immunohistochemistry. Importantly this approach can be used to characterize the r...Here we detail a method for the exploration of patient-derived xenografts (PDX) based on organoids (PDX-O), using antibodies validated for immunohistochemistry. Importantly this approach can be used to characterize the response of malignant cells to antineoplastic treatments. Cancer progression and metastasis reflect tumor heterogeneity and plasticity. Here we analyzed cells in PDX grown as spheroids or tumoroids embedded in collagen/basement membrane extract (BME) matrix. The use of 3D cellular models instead of 2D allows the inclusion in the study of non-adherent tumors. Even if the cells are adherent the spheroid and tumoroids conformation or organization in space and cellular polarity differ for 2D. This difference can change the response to treatment. Adding a matrix component is an important addition to the tumoral microenvironment that can change also the cellular response to treatment. We combined this tridimensional model with a multiplexed staining protocol to analyze tumor cell differentiation and responses to drugs. We analyzed paraffin embedded organoid sections, allowing to analyze the core of cell aggregates, and analyzed cell plasticity, differentiation and proliferation with and without treatment with palbociclib. Spheroids included an outer layer of luminal cytokeratin (CK) 8-positive cells surrounding a mixed population of cells expressing both CK5 and CK8. Palbociclib treatment resulted in a proliferation arrest, linked to dedifferentiation of the cancer cells that switched to a mostly basal phenotype, supporting a hybrid CK5/CK8 phenotype. In summary, this method offers a simple and versatile strategy to analyze tumor cell responses to drugs, using routine antibodies validated for immunohistochemistry and multiplex analysis.
In nearly all pathophysiological processes, mitochondrial membrane potential serves as a crucial indicator of mitochondrial function and activity. However, there remains a need for high-content imaging techniques that in...In nearly all pathophysiological processes, mitochondrial membrane potential serves as a crucial indicator of mitochondrial function and activity. However, there remains a need for high-content imaging techniques that incorporate multiparametric measurements for comprehensive mitochondrial assessment. This paper introduces a novel unbiased approach for quantifying mitochondrial membrane potential in vitro, applicable to both two-dimensional and three-dimensional experimental systems. Furthermore, the incorporation of automated image analysis with machine learning algorithms enabled precise identification and segregation of distinct cell types within complex co-culture systems, allowing for targeted evaluation of individual subpopulations. Here, we provide a protocol for large-scale profiling of mitochondrial activity across various experimental contexts.
Phenotypic two-dimensional (2D) high-throughput screening (HTS) is a well-established approach extensively employed in oncological drug discovery by both Academia and the pharmaceutical industry. This methodology has pla...Phenotypic two-dimensional (2D) high-throughput screening (HTS) is a well-established approach extensively employed in oncological drug discovery by both Academia and the pharmaceutical industry. This methodology has played a pivotal role in the development of a wide range of systemic and targeted therapeutic anticancer agents for clinical use. Recent advances in automation, imaging technologies, and labware design have paved the way for image-based HTS in three-dimensional (3D) cell culture systems. These 3D systems enable the analysis of more physiologically relevant models that closely replicate the characteristics of tumors and their microenvironment. In this study, we present an image-based phenotypic 3D HTS assay utilizing imaging-compatible labware specifically designed to support spheroid formation.
Rare diseases, characterized by their low prevalence, cumulatively affect millions of people around the world and place significant burden on the healthcare system. With limited clinical expertise and infrastructure in t...Rare diseases, characterized by their low prevalence, cumulatively affect millions of people around the world and place significant burden on the healthcare system. With limited clinical expertise and infrastructure in this field, patients encounter barriers in obtaining an accurate diagnosis and accessing treatment. Rare diseases are commonly attributable to genetic alterations; thus, we can optimize modern genetic technologies to pinpoint pertinent genes and molecular pathways involved in disease phenotypes. In this article, we discuss rare diseases in context of multi-omics, an integrative approach combining data from various sources, including genomics, transcriptomics, and epigenomics. Advancements in multi-omics have facilitated the collection of more high-dimensional data, particularly useful for rare diseases comprising limited sample sizes. Artificial intelligence (AI) and machine learning (ML) are powerful tools for extracting disease-relevant patterns from complex datasets and unraveling causative markers underlying disease. Together, these tools are invaluable for incorporating precision medicine in rare diseases through guiding therapeutic strategies aimed at modifying the structure and functionality of specific genes to address the root cause of disease. Specifically, we curate a list of twenty-three rare diseases, prioritized by the medical community based on unmet medical needs and prevalence. To illustrate the current landscape of precision medicine for these diseases, we summarize advancements in genomic sequencing and computational methods for their diagnosis, and utilization of gene-editing technologies for personalized treatment. Overall, the various bioinformatic strategies discussed in this paper help formulate an end-to-end workflow of the integration of gene testing, multi-omics, and AI/ML to guide effective rare disease management.
Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy for which no approved targeted therapies are available. Standard therapies as of now lack the ability to eradicate leukemic clones nor can th...Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy for which no approved targeted therapies are available. Standard therapies as of now lack the ability to eradicate leukemic clones nor can they modify disease outcome. To address this therapeutic gap, we conducted a high-throughput chemical drug screen using a robotized platform on four genetically engineered hematopoietic murine cell lines that model recurrent CMML mutations. Our approach aimed to identify genotype-dependent therapeutic vulnerabilities through a drug repurposing strategy, leveraging the rapid clinical translation potential of pre-approved compounds. Cell viability was assessed using the CellTiter-Glo® luminescent cell viability assay, a robust method widely recognized for its accuracy and efficiency in measuring cell proliferation and cytotoxicity. The luminescent output, directly proportional to viable cell numbers, allows for high-throughput screening. The protocol detailed in this article enables the testing of a large number of compounds at multiple doses, with a 48-hour readout. This method provides a reliable, time-efficient, and flexible approach that can be adapted for different cell types and research contexts beyond oncology.
Neurodegenerative diseases, such as Alzheimer's disease (AD), pose significant socioeconomic and personal burdens due to progressive cognitive and motor decline. AD is characterized by the accumulation of amyloid-beta (A...Neurodegenerative diseases, such as Alzheimer's disease (AD), pose significant socioeconomic and personal burdens due to progressive cognitive and motor decline. AD is characterized by the accumulation of amyloid-beta (Aβ) plaques and tau tangles, alongside with emerging evidence linking metabolic dysfunction to its early disease pathogenesis. Impaired mitochondrial selective autophagy (known as mitophagy) and excessive mitochondrial dysfunction have been implicated as key contributors to disease progression. To uncover the mechanistic underpinnings of AD, Caenorhabditis elegans offers a powerful model system providing a fully mapped nervous system, transparency for live imaging, and evolutionary conserved pathways mirroring human pathophysiology. Here, we employ a pan-neuronal Aβ -expressing C. elegans strain to phenocopy early metabolic disturbances characteristic of AD. Our methodology integrates automated motility tracking with confocal microscopy, utilizing the mitochondria-targeted Rosella biosensor to assess mitophagy dynamics in vivo. This platform enables quantitative assessment of locomotion deficits and spatiotemporal monitoring of mitophagy alterations driven by Aβ-induced toxicity. Our method provides a robust tool for screening genetic and pharmacological interventions aimed at mitigating AD-associated mitochondrial dysfunction and neurodegeneration.
Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is a rare autosomal dominant disorder that is characterized by the development of multiple cutaneous and uterine leiomyomas and predisposes individuals to an aggres...Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is a rare autosomal dominant disorder that is characterized by the development of multiple cutaneous and uterine leiomyomas and predisposes individuals to an aggressive and highly metastatic form of Renal Cell Cancer (RCC).
For decades, transgenic mouse models have been developed and utilized to study tumorigenesis in vivo, offering the ability to manipulate oncogene and tumor suppression gene expression systemically or within entire organs...For decades, transgenic mouse models have been developed and utilized to study tumorigenesis in vivo, offering the ability to manipulate oncogene and tumor suppression gene expression systemically or within entire organs and tissue compartments. However, the induction of tumorigenesis in many of these experimental models contrasts sharply with the development of most human cancers, where mutations affecting gene expression occur in a spatially restricted manner and lesions generally originates from the clonal expansion of one single mutated cell. This discrepancy raises critical questions regarding the relevance of the existing transgenic mouse models in accurately replicating the mechanisms of tumor initiation observed in humans. To overcome this limitation and study tumor initiation in vivo, I developed an innovative mouse model to induce early tumorigenesis through light-targeted mutagenesis of single cells, achieving unprecedented spatio-temporal resolution. This model provides a more accurate representation of tumor initiation processes, thus enhancing our understanding of cancer mechanisms at its inception.
Hearing loss is the most common sensory impairment in the population, with more than 400 million affected worldwide. Over half the cases of congenital hearing loss have a genetic cause, ∼80 % of which are autosomal reces...Hearing loss is the most common sensory impairment in the population, with more than 400 million affected worldwide. Over half the cases of congenital hearing loss have a genetic cause, ∼80 % of which are autosomal recessive with a prevalence of about 1-2 every 1000 births. Gene therapy would represent a curative option for these patients, and adeno-associated viral vectors (AAVs) have become the flagship delivery vehicle for in vivo gene therapy, especially for local delivery and small organ targeting. However, their preclinical development for hearing loss is slower than for other indications due to the inaccessible nature of the cochlea and the limited availability of mouse models that accurately reproduce the phenotype observed in humans. In this book chapter we detail the basic methodology for gene therapy administration to the inner ear in a mouse model of hereditary hearing loss, as well as cellular and functional analysis of cochlear functions.
Allergic dermatitis (AD) is a skin disease characterized by chronic inflammation. In the early stage, swollen, rush, hyperemia, and so on, are observed and moreover thickening and lichenification of the skin can occur at...Allergic dermatitis (AD) is a skin disease characterized by chronic inflammation. In the early stage, swollen, rush, hyperemia, and so on, are observed and moreover thickening and lichenification of the skin can occur at chronic stage. Most case of ADs are type Ⅰ allergy and immunoglobulin E and chemical such as histamine are involved. Type Ⅰ allergy is considered to be Th2 immunoreaction. Furthermore, lesions of AD contain eosinophil that play a role in Th2 immunoreaction. Clinically, lichenification lesions are refractory and dermal fibrosis and epidermal thickening are visible histopathologically. Mast cells and eosinophils are considered to be involved in dermal fibrosis in AD however the mechanism is not uncovered. There are two accessible preexisting models of AD, chemically induced models using BALB/c mice and naturally occurring model of NC/Nga mice, although their fibrosis of derms weak unfortunately that make them unsuitable for analyzing refractory AD. Yama mouse, novel inbred mouse strain, demonstrates eosinophilia with elevated eosinophil in peripheral blood and bone marrow. Naive Yama mouse has no organ failure and pathological change. When the mouse induced AD chemically, the lesions contain severe infiltration of eosinophil and severe dermal fibrosis. This novel mouse model can develop research area of AD. Here, we aim to describe the detailed procedure of developing AD mouse model with two preexisting and the novel mouse models with focusing of the difference in three models.
Immune cell intravasation into the tumor is an absolute requirement for the immune system to exert control on tumor growth. Its regulation directly influences immune surveillance and therapeutic response to immunotherapi...Immune cell intravasation into the tumor is an absolute requirement for the immune system to exert control on tumor growth. Its regulation directly influences immune surveillance and therapeutic response to immunotherapies and other immune cell-dependent cancer therapies. Despite its significance, investigation of this process remains a major challenge due to technical limitations. Intravital microscopy (IVM) has emerged as an essential tool for visualizing dynamic cellular interactions within the tumor microenvironment. This study presents an optimized IVM-based technique to track immune cell interactions with tumor endothelium in a preclinical model. Our approach combines high-resolution imaging with fluorescent labeling strategies to capture real-time interactions between immune and endothelial cells. By providing an enhanced method for studying immune infiltration in tumors, this technique could contribute to a better understanding of the tumor microenvironment and the development of novel therapeutic strategies.
Proteinopathies are a type of disorders characterized by the intracellular or extracellular accumulation of misfolded proteins that disrupt cellular proteostasis and exert toxic effects. These proteotoxic effects are a c...Proteinopathies are a type of disorders characterized by the intracellular or extracellular accumulation of misfolded proteins that disrupt cellular proteostasis and exert toxic effects. These proteotoxic effects are a common hallmark of various age-related neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and polyglutamine disorders such as Huntington's disease (HD). Misfolded protein accumulation can impair numerous cellular processes, including mitochondrial function, protein degradation pathways, the endoplasmic reticulum stress response, and redox homeostasis, ultimately compromising cell viability. The nematode Caenorhabditis elegans (C. elegans) has emerged as a powerful model for studying proteotoxic stress due to its genetic tractability and the high degree of conservation of key cellular pathways when compared to mammals. These include mitochondrial dynamics and function, regulation of reactive oxygen species (ROS), and the cellular capacity to manage protein aggregates in terms of number, size, and clearance efficiency. The integration of these conserved stress response pathways together with C. elegans experimental versatility positioned this nematode as an ideal system to investigate the molecular mechanisms underlying proteinopathy-induced toxicity. In this chapter, we describe a set of complementary methodologies to evaluate proteotoxic stress in C. elegans models of protein misfolding. These include assays to measure mitochondrial reactive oxygen species (ROS) and membrane potential (Δψm), analyses of mitochondrial morphology and oxygen consumption, protein extraction protocols, and in vivo staining and semi-automated quantification of protein aggregates.
Breast cancer remains one of the most prevalent and deadly malignancies in women. Metastasis remains an unmet clinical need specially in some subtypes such as triple negative breast cancer. Among non-metastatic patients,...Breast cancer remains one of the most prevalent and deadly malignancies in women. Metastasis remains an unmet clinical need specially in some subtypes such as triple negative breast cancer. Among non-metastatic patients, more than 40 % fail to achieve a complete pathological response during the course of treatment, facing high rates of local failure post-resection and distant metastases. Recent studies suggest an association between the presence of circulating tumor cells (CTCs) and the aggressiveness of the disease. Herein, we describe an orthotopic syngeneic mouse model in which tumor cells are directly inoculated into the exposed mammary gland. This model allows mechanistic studies on tumor progression and the evaluation of novel therapeutic strategies. Furthermore, the inclusion of surgical resection of established tumor closely mimics current clinical approaches in breast cancer treatment and enables the investigation of mechanisms underlying tumor relapse and metastasis. This chapter outlines the complete protocol, including all necessary steps and materials, for establishing a metastatic breast cancer model in mice. The model is based on CTCs that persist in the bloodstream and subsequently colonize distant organs (primarily the lungs) following surgical resection of the primary tumor.
Lung cancer is the leading cause of cancer-related mortality globally, primarily due to the high rate of diagnoses at advanced stages when metastasis has already occurred. The metastatic process is complex, making its st...Lung cancer is the leading cause of cancer-related mortality globally, primarily due to the high rate of diagnoses at advanced stages when metastasis has already occurred. The metastatic process is complex, making its study challenging. Experimental models that replicate metastasis are crucial for understanding the underlying biological mechanisms. Among these, intracardiac injection in mice is a valuable method for studying metastasis, as it releases tumor cells directly into the systemic circulation, promoting the colonization of secondary organs like the brain, bones, liver, and adrenal glands. This model is particularly significant in lung cancer research, as it enables the evaluation of tumor cell's ability to survive in circulation, adapt to distant microenvironments and grow there. This chapter outlines the protocol for intracardiac tumor cell injection in mice, focusing on technical considerations providing an essential tool for studying metastatic lung cancer.
Methods Cell Biol
· 2026 · PMID 41724562
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Regulatory T (Treg) cells are a subset of CD4 lymphocytes broadly recognized for their critical role in maintaining peripheral tolerance. More recent findings have highlighted their growing importance in maintaining home...Regulatory T (Treg) cells are a subset of CD4 lymphocytes broadly recognized for their critical role in maintaining peripheral tolerance. More recent findings have highlighted their growing importance in maintaining homeostasis in several non-lymphoid tissues. These functions have been characterized in a number of tissues, but Treg cell physiologic function in mammary glands remains unexplored. Due to their low abundance in this tissue, Treg cells can be challenging to isolate for study; thus, here we describe a protocol for isolating Treg cells from murine mammary glands through fluorescence-activated cell sorting (FACS).
The tumour microenvironment (TME) represents a heterogenous and dynamic niche, comprising cancer cells, extracellular matrix, stromal cells, vasculature and immune cells. The composition of the immune infiltrate varies b...The tumour microenvironment (TME) represents a heterogenous and dynamic niche, comprising cancer cells, extracellular matrix, stromal cells, vasculature and immune cells. The composition of the immune infiltrate varies between patient and tumour type, with the overall number, spatial localisation and functional status constituting the immune contexture. The presence of certain immune cells, including specific subsets of CD8 and CD4 T cells, natural killer cells, B cells and dendritic cells has been linked with favourable prognosis and response to treatment, including immunotherapy. In contrast, subsets of immune cells including macrophages, myeloid cells and neutrophils are frequently associated with poor prognosis, treatment resistance and tumour growth. Therefore, assessment of the immune contexture can be used to evaluate prognostic and predictive immune biomarkers, with CD8 cytolytic and memory T cells emerging as key immune effectors associated with clinical benefit across multiple solid malignancies. Here, we illustrate a straightforward automated immunohistochemistry (IHC) protocol for the detection and characterization of different subtypes of T-cells within the microenvironment of murine tumours. We also briefly illustrate the downstream process of visualization of these specific lymphocytic populations, ultimately providing both qualitative and quantitative analysis. The general protocol can readily be adapted to study a wide range of murine tumours and normal tissue (including secondary lymphoid organs), as well as profiling of other immune cell populations.