Methods in cell biology最新文献

Extracellular ATP (eATP) serves as a crucial signaling molecule in diverse physiological and pathological processes, including neurotransmission, inflammation, and cancer. Despite its importance, accurate measuring eATP dynamics in vivo has remained a significant technical challenge. Traditional methods, such as soluble luciferase systems, fluorescent probes, microelectrode biosensors, and high-performance liquid chromatography (HPLC), exhibit limitations in spatial resolution, tissue permeability, and real-time monitoring. Fluorescent probes offer high spatial resolution but are hindered by poor tissue penetration and the need for excitation light. Microelectrode biosensors provide localized detection but require invasive procedures, while HPLC, though highly sensitive, is restricted to ex vivo applications.To address these limitations, the plasma-membrane-targeted luciferase (pmeLUC) probe was developed. This bioluminescent system allows real-time, quantitative monitoring of eATP levels in living cells and animal models without the need for external excitation light. The pmeLUC is anchored on the outer surface of the plasma membrane, positioning its catalytic site extracellularly for direct eATP sensing. Its high sensitivity, tissue permeability, and adaptability for both in vitro and in vivo studies have enabled significant advancements in understanding eATP dynamics across different pathological contexts, including tumor microenvironments, immune responses, and brain injury models. Furthermore, the creation of pmeLUC-transgenic mice and of AAV-mediated delivery systems, has expanded the applications of this tool for longitudinal and systemic monitoring of eATP in living organisms. This review highlights the rationale behind choosing pmeLUC over other methodologies, emphasizing its superior capabilities in overcoming existing technical barriers and advancing eATP research in both basic and translational sciences.

Immunotherapy has revolutionized cancer treatment by harnessing the immune system to target tumor cells expressing neoantigens. Neoantigens are peptides arising from tumor-specific aberrations that are presented by cancer cells and recognized by T cells. The computational prediction of cancer neoantigens from somatic mutations and other tumor-specific aberrations using patients' sequencing data is key for the investigation of anticancer immune responses and for the design of personalized immunotherapies. However, neoantigen prediction requires the implementation of complex computational pipelines to distill large-scale information from RNA and DNA sequencing data and derive neoantigen candidates together with associated features for their prioritization and selection. We previously developed nextNEOpi, a comprehensive and stand-alone bioinformatics pipeline that not only predicts class-I and -II neoantigens and fusion neoantigens, but also sheds light onto the tumor-immune cell interface, quantifying neoantigen clonality, immunogenicity, and tumor-specific metrics like tumor mutational burden and immune-cell receptor repertoire diversity. In this chapter, we showcase the main capabilities of the nextNEOpi pipeline by analyzing genomic and transcriptomic data generated from multiple biopsies collected from patients with lung cancer.

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.

Adenosine (ADO), an anti-inflammatory and immunosuppressive metabolite, plays a crucial role in mediating purinergic signaling alongside adenosine triphosphate (ATP) and adenosine monophosphate (AMP) within the tumor microenvironment. Dysregulated ADO signaling has been implicated in tumor immune evasion and progression, highlighting the importance of measuring ADO production. This method chapter presents a protocol for assessing ADO levels in both two- and three- dimensional tumor cell culture conditions. The protocol employs a competitive AMP blockade strategy, where excessive AMP is introduced to inhibit CD73-mediated conversion of AMP to ADO, enabling the quantification of relative ADO production. Given ADO's potent immunosuppressive properties and its influence on various immune responses, accurate measurement of ADO production is crucial for understanding its role in tumor immune evasion and for guiding the development of targeted immunotherapeutic strategies.

Cancer is a major global health concern marked by uncontrolled cellular proliferation and genetic modifications leading to malignancy. The disease's complexity encompasses various forms of cancer, increased rates of diagnosis and prognosis and numerous treatment modalities, including surgery, chemotherapy, and radiation, each confronting problems such as medication resistance and side effects. Solid tumors, comprising approximately 85 % of malignancies, provide significant treatment challenges due to their uneven vascular supply and interstitial pressure, resulting in inadequate medication distribution and therapeutic failure. The tumor microenvironment (TME) comprises cancer cells and diverse supportive cells such as immune cells, endothelial cells and fibroblasts, which interact to facilitate tumor growth and progression. T lymphocytes, B lymphocytes, natural killer cells, and macrophages are only a few types of immune cells that can aid or impede cancer progression, which makes treatment more complicated. In this chapter we will explore the TME in solid cancers, focusing on its role in cancer biology and therapeutics strategies. In the future, advancing therapies that more precisely target TME components will minimize treatment resistance and improve patient outcomes.

The field of molecular biology has undergone tremendous advancements in recent years, with the development of powerful techniques that allow for in-depth exploration of cellular processes at the molecular level. This chapter, "Advanced Molecular Biology Techniques," provides a detailed protocol of the molecular techniques. We begin with CRISPR-Cas9 genome editing, a transformative tool for precise and efficient gene manipulation, enabling targeted mutations and gene knockouts in various organisms. Gene amplification via Real-Time PCR is then discussed, highlighting its ability to quantify gene expression and detect rare genetic variants with high sensitivity. Flowcytometry follows, offering a robust platform for analyzing cellular populations based on specific markers, enabling the study of immune cells, cancer diagnostics, and cell cycle analysis. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) is explored as a method for mapping protein-DNA interactions, providing insights into gene regulation and epigenetic modifications. The chapter also covers Single-cell RNA sequencing (scRNA-Seq), a groundbreaking technique for profiling gene expression at the single-cell level, allowing for the discovery of cell heterogeneity and complex biological processes. Next, we explore into proteomics through Mass Spectrometry-Based Analysis, which offers detailed proteome characterization and biomarker discovery by identifying and quantifying proteins in complex samples. Finally, Fluorescence In Situ Hybridization (FISH) is discussed as a method for visualizing the spatial localization of specific nucleic acid sequences within intact cells or tissues. Together, these advanced molecular biology techniques offer unparalleled precision and insight into the molecular mechanisms underlying health, disease, and cellular function.

Combined blockade of the immune checkpoints PD-1 and CTLA-4 has shown remarkable efficacy in patients with melanoma, renal cell carcinoma, non-small-cell lung cancer and mesothelioma, among other tumor types. However, a proportion of patients suffer from serious immune-related adverse events (irAEs). In severe cases, a reduction of the doses or the complete cessation of the treatment is required, limiting the antitumor efficacy of these treatments. Colitis is among the most frequent and problematic irAE associated with immune checkpoint blockade. In this context, animal models that recapitulate the pathophysiological features of immunotherapy-related colitis are needed. In this manuscript, we describe our experience with a mouse model in which the combined CTLA-4 and PD-1 blockade exacerbates the deleterious effects of dextran sulfate sodium (DSS)-induced colitis. This model may constitute a valuable tool for the study of immunotherapy-related colitis.

Currently, Ovarian Cancer (OC) is the most lethal gynecological malignancy. In most patients, it progresses without clinical signs or symptoms, leading to a late diagnosis when it has already spread in the peritoneal cavity as peritoneal carcinomatosis (PC). To date, OC PC management is based on cytoreductive surgery to remove the macroscopic disease, followed by chemotherapy. Many patients respond to this treatment, but disease recurs in 70-90% of them. Therefore, new therapeutic approaches are needed. The field of targeted radionuclide therapy (TRT) has witnessed considerable progress and several radiopharmaceuticals have been approved in the last decade. In TRT, radiolabeled molecules are injected to specifically recognize, irradiate, and kill tumor cells. TRT is a multisite radiotherapy that delivers dose to all malignant lesions. Therefore, TRT could be an alternative approach for OC PC because conventional external beam radiotherapy cannot be used at curative dose due to toxicity to healthy tissues. Here, we describe an OC PC model based on grafting human SK-OV-3 OC cells in the peritoneal cavity of immunodeficient mice. We also explain how to label trastuzumab with lutetium-177 to specifically target and irradiate SK-OV-3 cell nodules in these mice, and how to monitor the response to this TRT in vivo. With minor variations, the same technique can be conveniently applied to a variety of human (or mouse) tumors.

The foremost cause of dementia is Alzheimer's disease (AD). The vital pathological hallmarks of AD are amyloid beta (Aβ) peptide and hyperphosphorylated tau (p-tau) protein. The current animal models used in AD research do not precisely replicate disease pathophysiology, making it difficult for researchers to quickly and effectively gather data or screen potential therapy possibilities. Several transgenic animals are used as models for AD; however, they have cost and time concerns. Zebrafish (Danio rerio) has become a suitable model organism for high-throughput pharmacological screening of neuroactive substances and neurodegenerative research. The past few decades have seen a significant increase in research on AD. The fight against amyloidosis has, however, been unexpectedly unsuccessful. It may be due to a need for more relevant in vivo models for high throughput screening, which emphasizes the need to find other anti-AD models. Alternative animal models, including zebrafish, have developed into a potentially useful research tool that must be employed for AD research to be effective. Only a few comprehensive zebrafish models exhibiting AD-like pathogenesis have been reported in the literature, and this book chapter describes these models.

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_35-55 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.