Methods in cell biology最新文献

Inter-organ communication, including the release of secreted proteins, plays a key role in synchronized physiological responses and organismal homeostasis. Recent studies have emphasized functions of muscle-secreted proteins (i.e., myokines), in regulating metabolic pathways and improving metabolic dysfunction distally in the liver. Thus, experimental workflows to study myokines and their impact on target cell types are of scientific value. Here, we describe a cell culture-based method to investigate communication from muscle to liver mediated by secreted proteins. Briefly, C2C12 myoblasts are differentiated into myotubes, myotube-conditioned media is collected, and myotube-secreted proteins are isolated and stored. To demonstrate the utility of this method, AML12 hepatocytes were treated with myotube-secreted proteins and effects on bioenergetics were assessed. This method can be useful as a proof of principle tool, for mechanistic studies, or paired with proteomic or biochemical analyses to identify novel myokines. We also envision it is adaptable in terms of cell type, downstream application, and signaling direction.

In vitro tumor models might be useful to study tumor growth, invasion and therapy resistance, but also extracellular matrix (ECM) remodeling. This protocol will provide information for the generation of a 3D tumor cell cultures for high content analysis and describes a novel method to monitor the ECM morphology and remodeling as well as therapy responses using live 3D tumor cell spheroids. Using 3D spheroids of thyroid carcinoma (TC) cells as a model, our method involves the treatment of TC cells with inhibitory agents followed by subsequent analysis of ECM components to assess the influence of these drugs on the structural integrity of the EMT using fluorescence labeled antibodies (Ab) and confocal microscopy. Employing this method, the morphology of the formed spheroids under different conditions and co-cultures as well as the distribution of ECM components can be assessed, such as e.g. fibronectin 1 (FN1). The results will also provide valuable insights into the tumor microenvironment (TME) and potential interactions of viable spheroids with the components of the TME during the ECM remodeling process. The implementation of 3D spheroids for studying EMT in TC as a model has been shown to provide more accurate and representative results compared to traditional 2D monolayer cell cultures.

Novel therapeutic approaches highlight the need for advanced ex vivo cell culture models that more closely resemble the physiological and genetic properties of the primary tumor. Patient-derived models could serve as an attractive strategy to investigate the crosstalk between cancer cells and its microenvironment and to test potential therapeutic targets, paving the way for precision oncology. In this chapter, we provide a detailed step-by-step protocol for enabling a direct co-culture system of patient-derived colorectal cancer (CRC) organoids with autologous tumor-infiltrating lymphocytes (TILs). The present protocol provides a methodology to gain direct access to the apical side of the epithelial cells forming the organoids. This method can be used to investigate patient-specific cell-to-cell interactions, T cell functionality and efficacy and provides a robust platform to validate potential immunogenic neoantigens.

Extracellular vesicles (EVs) are released by all cell types in the bodily fluids. EVs represent key players of inter-cellular communications, including maternal-fetal cross-talk and mother-infant information transmission. Here, we will focus on EVs derived by amniotic liquid and maternal milk. We will report methodologies of EV isolation from these maternal fluids.

Immunohistochemistry (IHC) is a powerful technique that utilizes specific antibodies to visualize distinct cell populations within tissues. However, this method has some limitations, in particular the specificity and detection of low expressed markers including soluble factors, like e.g., cytokines. Other methods, such as in-situ hybridization (ISH), are a suitable alternative to visualize these factors in the tissue. Recent advances have opened exciting opportunities for quantitative data acquisition related to both protein and mRNA expression. Moreover, these techniques allow the analysis of their spatial distribution within a tissue and in a cell-specific context. Moreover, improvements in tissue preparation, fluorescent dyes, tissue imaging and downstream analysis have addressed challenges related to quantitative precision. Nowadays, researchers can obtain more accurately measurements of protein and mRNA expression levels of multiple targets within one sample. This technique gives us the opportunity to visualize and record the spatial relationship between different cells in formalin-fixed, paraffin-embedded samples. This chapter summarizes a protocol developed for cytokine expression analysis in the bone marrow of myeloid neoplasms. It provides an overview of the workflow that can be adapted to other tissues and other disease specific contexts.

Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of proteins has been challenging in vivo. A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP_1-10/11) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP_1-10/11 system in fruit flies. This approach depends on the reconstitution of the GFP_1-10 and GFP₁₁ fragments, creating a fluorescence signal. We insert the GFP₁₁ fragment into a specific genomic locus while expressing its counterpart, GFP_1-10, through an available Gal4 driver line. The unique aspect of this system is that neither GFP_1-10 nor GFP₁₁ alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells expressing the GFP₁₁ tagged endogenous protein. We illustrate this technique using the adhesion molecule gene teneurin-m (Ten-m) as a model, highlighting the generation and validation of GFP₁₁ protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.

Colorectal cancer (CRC) development is initiated in the colon-rectum sections of the gut, by the emergence of a primary tumor. CRC slowly progresses to a multiple locations metastatic disease, involving secondary tumors arising in various organs, such as the liver. Mouse models have been developed to investigate the immune response, locally generated in primary tumors. Classically, tumors are implanted under the skin, for practical reasons and simplicity of monitoring. However, the skin location does not necessarily recapitulate the tumor immune microenvironment (TME) that would normally be generated in the gut. The orthotopic CRC mouse model, that we describe hereafter, was generated to investigate the colon-local mechanisms driving the establishment and the polarization of primary tumors TME. In this chapter, we detail the procedures used to implant syngeneic colon tumor cells in the cecum of immunocompetent mice and to monitor the progression of visceral tumors in live mice. The same procedure can be implemented using other tumor cell lines and mouse genetic backgrounds.

Lung cancer remains a leading cause of cancer-related deaths, highlighting the importance of understanding the lung's immune landscape, which plays a key role in tumor progression and response to therapy. As an organ constantly exposed to environmental and microbial challenges, the lung's unique tumor microenvironment is shaped by these additional stimuli, influencing immune responses. Here, we present a standardized protocol to examine dynamic changes in the lung immune cell compartment in response to bacterial challenges in a genetically engineered mouse model of lung cancer. This method ensures efficient tissue processing and dissociation while preserving cell viability for high-throughput flow cytometry. Additionally, we describe an integrative analysis approach to systematically analyze data across tissues and conditions, providing a comprehensive framework for studying immune cell dynamics in lung carcinoma.

In the era of T cell-mediated immunotherapies, a central and growing problem is the recurrence of tumors lacking target antigen (Ag). Strategies that can prevent outgrowth of antigen-loss cells may improve response to therapy more effectively than those that rely on identifying multiple Ag targets after resistance arises. In addition to a T cell's direct killing response to binding cognate Ag, upregulation of death-receptor ligands and secretion of pro-inflammatory cytokines contribute to the indirect killing of surrounding, antigen-negative (bystander) cell populations, in a process termed "bystander killing". To investigate the mechanism and scope of T cell bystander killing, we describe methods of in vitro killing assays with flow cytometry and live microwell imaging, as well as in vivo tumor models with bioluminescent imaging and multiphoton live imaging, to observe this process in real time. The approaches can be easily adapted to investigate many other tumor types, T cell therapies, and targeting strategies.