To maintain homeostasis, cells undergo a tightly regulated process called programmed cell death. Some forms of programmed cell death, such as pyroptosis, can elicit a strong inflammatory response by releasing cytokines t...To maintain homeostasis, cells undergo a tightly regulated process called programmed cell death. Some forms of programmed cell death, such as pyroptosis, can elicit a strong inflammatory response by releasing cytokines through small protein pores. The terminal event of pyroptosis in most cells is plasma membrane rupture (PMR), which breaks down large sections of the plasma membrane and emits intracellular contents that can further amplify the inflammatory signal. In opposition to the previous dogma that PMR is a passive event, it was recently discovered that the transmembrane protein, Ninjurin-1 (NINJ1), is the key executor of PMR. Two models of NINJ1-mediated PMR predict that NINJ1 oligomerizes into filaments or ring-like structures to either open large pores or to excise sections of the membrane. Both models underpin how NINJ1 must oligomerize to execute PMR. When at rest, NINJ1 will autoinhibit oligomerization and activation by forming face-to-face dimer-dimer structures on the plasma membrane. Follow-up studies have shown that NINJ1 executes PMR for other forms of programmed cell death, including apoptosis, ferroptosis, and PANoptosis, as well as mechanical cell death. Thus, assessing NINJ1 function directly through quantification of NINJ1 oligomerization is important to expanding our understanding of both programmed and mechanistic cell death. Here, we describe methods to visualize and quantify NINJ1 oligomerization via immunofluorescence imaging of NINJ1 puncta. This protocol enables more precise and accurate measurement of NINJ1 function during PMR, surpassing conventional methods that just quantify PMR by-products.
Gasdermin-family proteins initiate the lytic cell death program pyroptosis upon proteolytic removal of an autoinhibitory domain. For each gasdermin, proteases capable of mediating an activating cleavage event have been c...Gasdermin-family proteins initiate the lytic cell death program pyroptosis upon proteolytic removal of an autoinhibitory domain. For each gasdermin, proteases capable of mediating an activating cleavage event have been clearly defined. However, for new proteases, there are unique challenges in evaluating whether a specific cleavage event results in an active or inactive gasdermin. This chapter outlines methods and key controls suitable for the initial discovery and evaluation of protease activation of gasdermins that are particularly suitable for the study of alternative pathways for activation, such as by the proteases of bacteria and viruses.
Acute Myeloid Leukemia (AML) is a hematopoietic malignancy characterized by the uncontrolled proliferation of aberrant myeloid blasts within the bone marrow, resulting in disrupted hematopoiesis and severe clinical conse...Acute Myeloid Leukemia (AML) is a hematopoietic malignancy characterized by the uncontrolled proliferation of aberrant myeloid blasts within the bone marrow, resulting in disrupted hematopoiesis and severe clinical consequences. Drug resistance represents a major barrier in AML treatment, frequently manifesting as relapse following initial remission with conventional chemotherapeutic agents such as cytarabine and venetoclax. The underlying mechanisms of drug resistance include enhanced drug efflux, altered drug metabolism, and activation of pro-survival signaling pathways, necessitating the elucidation of specific genetic determinants to enable the development of effective therapeutic strategies. The advent of CRISPR/Cas9 system has facilitated precise genomic modifications, permitting the generation of cell libraries with targeted gene knockouts in AML cells. This approach can identify genes whose disruption alters drug sensitivity, implicating their involvement in survival and resistance to cell death. This protocol outlines a systematic strategy to uncover genes associated with drug resistance in AML cells by leveraging CRISPR/Cas9-mediated functional genomic screening. By employing this methodology, genes conferring drug susceptibility upon knockout are noted as potential drivers of drug resistance, offering valuable insights for the rational design of targeted therapies.
The inflammasome is a multiprotein cytosolic signalling platform that initiates inflammatory responses upon detection of microbial and danger-associated stimuli. Inflammasome assembly occurs due to the involvement of spe...The inflammasome is a multiprotein cytosolic signalling platform that initiates inflammatory responses upon detection of microbial and danger-associated stimuli. Inflammasome assembly occurs due to the involvement of specific cytosolic pattern recognition receptors (PRRs) in response to pathogens and danger signals in host cells. This causes activation of inflammatory caspases that results in cytokine release and pyroptosis. A central player in inflammasome formation is the adaptor protein ASC (Apoptosis-associated speck-like protein containing a CARD), which bridges activated PRRs to pro-Caspase-1, leading to the formation of the inflammasome complex. This chapter provides detailed protocols for analysing inflammasome assembly by monitoring ASC speck formation using confocal microscopy and biochemically by detecting ASC oligomerisation using DSS-mediated crosslinking. Together, these methods offer complementary insights into spatial organisation and activity of inflammasomes in response to pathogens or cellular damage.
Programmed cell death (PCD) serves as a fundamental biological mechanism essential for maintaining cellular homeostasis and orchestrating physiological processes, while its dysregulation constitutes a hallmark of diverse...Programmed cell death (PCD) serves as a fundamental biological mechanism essential for maintaining cellular homeostasis and orchestrating physiological processes, while its dysregulation constitutes a hallmark of diverse pathological conditions. Substantial evidence has implicated aberrant PCD pathways in the pathogenesis of inflammatory diseases, autoimmune diseases and neurodegenerative disorders. Despite the various in vitro systems for cell death investigation, in vivo models retain significant advantages. The current research proposal specifically delineates two well-characterized in vivo models: (1) lipopolysaccharide (LPS)-induced sepsis model and (2) tumor necrosis factor-alpha (TNF-α)-mediated systemic inflammatory response syndrome (SIRS) model. These established in vivo experimental systems enable more convincing evidence for cell death research.
Programmed cell death (PCD) represents an essential biological mechanism for preserving tissue homeostasis and removing compromised cells. The most extensively characterized forms of programmed cell death - apoptosis, py...Programmed cell death (PCD) represents an essential biological mechanism for preserving tissue homeostasis and removing compromised cells. The most extensively characterized forms of programmed cell death - apoptosis, pyroptosis, and necroptosis - are each initiated through unique signaling mechanisms, encompassing TNF-TNFR1 signaling, Toll-like receptor activation, interferon-mediated responses, along with both the canonical NLRP3-GSDMD and non-canonical Caspase-11-GSDMD pyroptotic pathways. In this protocol, we systematically review combinatorial pharmacological strategies employing small-molecule drugs and cognate ligands to selectively induce specific PCD modalities, with parallel discussion of established detection platforms for monitoring cell death progression.
The programmed cell death process of necroptosis is activated when Receptor Interacting protein Kinase (RIPK)3 phosphorylates its substrate Mixed Lineage Kinase Like protein (MLKL). Phosphorylated MLKL oligomers then tra...The programmed cell death process of necroptosis is activated when Receptor Interacting protein Kinase (RIPK)3 phosphorylates its substrate Mixed Lineage Kinase Like protein (MLKL). Phosphorylated MLKL oligomers then traffic to cellular membranes, including the plasma membrane, and perforate these membranes. MLKL-driven pore formation in the plasma membrane alters the cell osmolarity, causing it to swell and undergo necrotic death. Phosphorylation of MLKL by RIPK3 is essential for the execution of necroptosis, and is thus a widely accepted biomarker for this mode of cell death. In this chapter, we describe our procedure for detecting phosphorylated murine MLKL in influenza A virus (IAV) infected lung tissues, and within tumors following treatment with the compound CBL0137.
Apoptosis and other regulated cell death pathways display complex, dynamic, and heterogeneous behaviors that are frequently masked by conventional endpoint assays relying on bulk measurements. Here, we describe a live-ce...Apoptosis and other regulated cell death pathways display complex, dynamic, and heterogeneous behaviors that are frequently masked by conventional endpoint assays relying on bulk measurements. Here, we describe a live-cell imaging workflow for temporally resolved, single-cell quantification of both apoptotic and non-apoptotic cell death in cell cultures. In colorectal carcinoma cell lines, cell death was triggered by agents including Staurosporine, RSL3, and Cisplatin, and continuously tracked using a live-cell imaging platform with fluorescent markers such as Annexin V-FITC, Caspase-3/7 activity dye, and propidium iodide. Parameters of cell death were monitored for 24-48 h, allowing precise discrimination between early and late apoptotic events as well as necrosis. This methodology enables reproducible, quantitative profiling of cell death kinetics at single-cell resolution, reduces artifacts from manual handling, and offers valuable insights into the temporal dynamics and interplay of different cell death modalities.
Acute tubular necrosis (ATN) is a central driver of nephron loss and progression to kidney failure. Dissecting the cellular pathways that govern tubular necrosis requires reliable systems to model and quantify cell death...Acute tubular necrosis (ATN) is a central driver of nephron loss and progression to kidney failure. Dissecting the cellular pathways that govern tubular necrosis requires reliable systems to model and quantify cell death. This chapter introduces methodologies for isolating murine renal tubules ex vivo and describes a panel of assays-including biochemical, molecular, and imaging approaches-that enable the detection of regulated necrosis in kidney tubules. We provide theoretical background, practical workflows, examples of applications.
Alkaliptosis is a pH-dependent form of regulated cell death induced by the small molecule JTC801, characterized by disruption of lysosomal and cytoplasmic pH homeostasis. Unlike classical forms of cell death such as apop...Alkaliptosis is a pH-dependent form of regulated cell death induced by the small molecule JTC801, characterized by disruption of lysosomal and cytoplasmic pH homeostasis. Unlike classical forms of cell death such as apoptosis, necroptosis, or ferroptosis, alkaliptosis is driven by dysregulation of intracellular pH-regulatory proteins, leading to aberrant alkalinization and metabolic dysfunction. This mechanism is particularly effective against apoptosis-resistant cancers, including pancreatic ductal adenocarcinoma, and represents a promising strategy for overcoming therapeutic resistance. Monitoring alkaliptosis remains challenging, as it is not detected by conventional cell death markers such as caspases or pore-forming proteins. This chapter introduces methodologies for assessing alkaliptosis, including dynamic pH measurement, analysis of alkaliptosis-related protein expression, and evaluation of lysosomal function. Improved detection strategies will advance the understanding of non-canonical cell death pathways and support the development of novel therapeutic interventions targeting alkaliptosis.
CD95/Fas belongs to the tumor necrosis factor (TNF) receptor superfamily and plays a pivotal role in immune homeostasis. Its ligand, CD95L (FasL or CD178) belongs to the TNF superfamily. CD95 contains three cysteine-rich...CD95/Fas belongs to the tumor necrosis factor (TNF) receptor superfamily and plays a pivotal role in immune homeostasis. Its ligand, CD95L (FasL or CD178) belongs to the TNF superfamily. CD95 contains three cysteine-rich domains (CRDs) in its extracellular (EC) region numbered 1-3 from the N-terminal region to the plasma membrane. While CRD2 and CRD3 interact with CD95L, CRD1 has been designed the pre-ligand assembly domain (PLAD) since it promotes the receptor self-aggregation in a ligand-independent manner. Upon CD95L binding, CD95 recruits Fas-associated death domain (FADD) at the level of the CD95 death domain (DD). FADD in turn aggregates caspase-8 to form the "death-inducing signalling complex" (DISC). Herein, we describe a method, called protein-fragment complementation assay (PCA) to identify PPIs and use it to validate interaction of CD95 with itself and with FADD. PCA is performed with the reporter enzyme Renilla reniformis luciferase (Rluc). RLuc has been divided in two inactive fragments, fused to different proteins of interest (POIs). If the two POIs interact, they can bring closer the two unfolded RLuc fragments fostering their folding and the quantification of luciferase activity.
Assessing cellular viability is a fundamental step for high-throughput screening (HTS) in drug development, particularly for evaluating the unintended adverse toxicity of standard medications or, conversely, the therapeu...Assessing cellular viability is a fundamental step for high-throughput screening (HTS) in drug development, particularly for evaluating the unintended adverse toxicity of standard medications or, conversely, the therapeutic efficacy of oncological agents, in which inducing cell death is the desired outcome. Conventional methods typically rely on ATP-dependent enzymatic substrate conversion assays to measure cellular metabolic activity as a proxy for viability. While effective, these plate reader-based methods provide only bulk measurements, limiting the level of detail in the analysis. Recent advancements in convolutional neural networks for biological image analysis have introduced promising alternatives, allowing the assessment of cellular viability at single-cell resolution. This approach provides more precise and detailed viability assessments than bulk assays. In this study, we present a methodology employing a trained YOLOv8n detection model, developed by Ultralytics, for endpoint and kinetic assays. This method offers rapid and precise toxicity predictions, making it a valuable tool in drug discovery.
Three-dimensional (3D) models more closely represent the in vivo situation and are therefore more relevant models for drug screening. Among the more than twelve regulated cell death (RCD) modalities, immunogenic cell dea...Three-dimensional (3D) models more closely represent the in vivo situation and are therefore more relevant models for drug screening. Among the more than twelve regulated cell death (RCD) modalities, immunogenic cell death (ICD) stands out for its ability to initiate efficient anti-tumor immune response. For example, ferroptosis, which can be induced via the inhibition of GPX4 leading to uncontrolled lipid peroxidation, might be immunogenic under certain conditions. It is crucial to identify which cell death modality is induced, as certain cancer types exhibit resistance to specific forms of cell death. However, a major limitation of 3D models is the lack of high-throughput assays, which often require dissociation of the 3D models, potentially leading to misinterpretation of results. Here, we describe a protocol for identifying and quantifying the induction of RCD modalities in 3D models, such as spheroids. This method eliminates the need for tumor spheroid dissociation and is compatible with other screening techniques, including confocal microscopy. This protocol enables high-throughput screening of various cell death inducers in intact 3D models, serving as a crucial first step in the identification of novel inducers and their specificity for particular ICD and RCD types.
Microfluidic devices are becoming more prevalent in fundamental and pre-clinical cancer research for their versatility and ease to culture, treat and analyze 3D tumor models of increased complexity and human/in vivo cont...Microfluidic devices are becoming more prevalent in fundamental and pre-clinical cancer research for their versatility and ease to culture, treat and analyze 3D tumor models of increased complexity and human/in vivo contextualization, paired with their overall user-friendliness and low footprint. Although mainly used for drug development, microfluidic devices and on-chip 3D models are compatible with irradiation, whether with laboratory irradiators or clinical radiotherapy equipment. As such, methodologies for analyzing cellular response to irradiation must be adapted for microfluidic devices (i.e. chips) and associated on-chip 3D tumor models. Here, we present a protocol for the generation of 3D cell culture models on-chip, along with three gold-standard radiobioassays protocols for quantifying their response to irradiation: clonogenic assay, neutral comet assay, and immunofluorescence staining for various markers. Moreover, examples of media-based assays are provided. These methods all offer different insights into response to radiotherapy, from the individual cell to that of the whole sample, and take advantage of the simplified generation of 3D cell culture models on-chip.
Tumoroids, which replicate patient tumor genetics and drug responses, have revolutionized oncology by enabling personalized treatment development and reducing reliance on animal testing. Despite remarkable advances in re...Tumoroids, which replicate patient tumor genetics and drug responses, have revolutionized oncology by enabling personalized treatment development and reducing reliance on animal testing. Despite remarkable advances in reproducing ever more sophisticated biology in advanced organoid platforms, there remain foundational challenges in standardizing and scaling-up tumour organoid development. Moreover, recreating key aspects of the tumour niche, namely its biomechanical properties is still overlooked in drug screening. To address these shortcomings, we introduce a technology for high-throughput derivation and high-content screening of tumoroids. Our technology relies on biofabrication of microengineered cell culture devices, which are scalable and tailored for standard automated lab-setups and real-time analysis of thousands of individual tumoroids trapped in microcavity arrays, within a polymer-hydrogel substrate. We validated our system using patient-derived urothelial cancer tumoroids, as they represent a good example of one of the most frequent cancer types with a debilitating clinical entity and a high financial burden related to the disease recurrency. Our scalable-culture technology should open new avenues toward more patient-tailored treatments or even new drugs targeting the extracellular matrix. Ultimately, the economic burden of the disease can be decreased by increasing treatment efficiency through larger, faster and more reliable screening.
Lysosomes are involved in the transport, degradation, and recycling of macromolecules through the autophagy and endocytosis pathways. Cholesterol is taken up by cells through the internalization of low-density lipoprotei...Lysosomes are involved in the transport, degradation, and recycling of macromolecules through the autophagy and endocytosis pathways. Cholesterol is taken up by cells through the internalization of low-density lipoprotein (LDL) via LDL receptor-mediated endocytosis or micropinocytosis. Free cholesterol generated by the action of acid lipases contained in lysosomes can be transferred to other organelles. Dysfunctions in either cholesterol uptake or release from lysosomes can compromise the function and integrity of these organelles, thereby contributing to the pathogenesis of lysosomal storage disorders. We previously showed that some cationic amphiphilic drugs (CADs) mimic the phenotype of lysosomal storage disorders by inducing lysosomal cholesterol accumulation followed by lysosomal damage. Here, we describe two fluorescence microscopic methods for the visualization of cholesterol accumulation in lysosomes in response to the CAD leelamine. In the first method, the cell-permeable cholesterol analog labeled with the fluorophore BODIPY is used. In the second method, endogenous cholesterol-rich microdomains are labeled with filipin complex. Both methods imply the additional visualization of the lysosomal associated membrane protein 2 (LAMP2) by immunofluorescence. Finally, the role of lysosomal cholesterol accumulation in the induction of lysosomal membrane permeabilization (LMP) was assessed through a method based on the recruitment of Galectin-3 on damaged lysosomes.
Cancer cell killing by cytotoxic T cells is a dynamic and multi-step interaction. Currently, most in vitro assays either provide detailed measurements on a small number of samples, or high throughput but with limited res...Cancer cell killing by cytotoxic T cells is a dynamic and multi-step interaction. Currently, most in vitro assays either provide detailed measurements on a small number of samples, or high throughput but with limited resolution of cellular interactions. Here, we present a high throughput microfluidic platform that enables co-culture of 3D cancer spheroids with immune cells in droplets, allowing precise monitoring of their interactions. Using murine melanoma cells co-cultured with cytotoxic T cells as a model system, we provide experimental details from chip fabrication and loading to time-lapse microscopy and endpoint killing efficiency measurements. The anchored-droplet format enables up to 85 realizations to be performed in parallel on a single chip, at pre-determined spatial locations. As a result, automated time-lapse microscopy yields measurements with high spatiotemporal resolution of many parallel realizations, thus providing both high throughput and highly resolved measurements. Beyond immuno-oncology, the system described here enables quantification of diverse dynamic cellular interactions with high statistical sensitivity.
Nanoparticles (NPs), the most common physical form of drug or therapeutic delivery systems enhance treatment safety and efficacy thanks to protection and vehiculation of their payload into the targeted tissue. Recently,...Nanoparticles (NPs), the most common physical form of drug or therapeutic delivery systems enhance treatment safety and efficacy thanks to protection and vehiculation of their payload into the targeted tissue. Recently, nanomedicines have received increasing interest in the area of cancer therapeutics. However, the failure to overcome biological barriers at the tissue and cellular levels prevents NP distribution and delivery, which carries unsuccess in clinical trials. With the introduction of advanced three-dimensional (3D) tumor replicas such as cancer spheroids and patient-derived organoids (PDOs) that provide a physiological 3D tissue-like context, the intrinsic NP-cell interaction features such as cellular uptake and intratumor behavior can be directly tested on a translational and preclinical basis. High-content screening (HCS) plays a pivotal role for predicting the efficacy of each NP design in an automated and unbiassed manner. This chapter presents in detail some protocols developed for confocal microscopy HCS on miniaturized cultures of tumor spheroids and PDOs, quantitative profiling of NP intra-spheroid transport and RNA-interference screening to unveil NP penetrance cellular effectors. In perspective, these modules put together, envision a core for a nano-PDO HCS workflow to advance cancer nanomedicine development.
Vascularized organoids-on-chip aim at assessing systemic drug delivery, but also drug screening, toxicity testing, pharmacokinetics and dynamics, and disease modeling. Here, we provide a detailed experiment protocol to v...Vascularized organoids-on-chip aim at assessing systemic drug delivery, but also drug screening, toxicity testing, pharmacokinetics and dynamics, and disease modeling. Here, we provide a detailed experiment protocol to vascularize organoids-on-chip, in regards to pancreatic islet spheroids and hydrogel preparation towards the modelisation of autoimmune diseases, inflammation, immunotherapies.