Division of Tumour Biology and Immunology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Mouse Clinic for Cancer and Aging (MCCA), Imaging Unit, Netherlands Cancer Institute, Amsterdam, The Netherlands.
Curr Protoc. 2021 Jun;1(6):e147. doi: 10.1002/cpz1.147.
The rising incidence and increasing mortality of hepatocellular carcinoma (HCC), combined with its high tumor heterogeneity, lack of druggable targets, and tendency to develop resistance to chemotherapeutics, make the development of better models for this cancer an urgent challenge. To better mimic the high diversity within the HCC genetic landscape, versatile somatic murine models have recently been developed using the hydrodynamic tail vein injection (HDTVi) system. These represent novel in vivo tools to interrogate HCC phenotype and response to therapy, and importantly, allow further analyses of the associated tumor microenvironment (TME) shaped by distinct genetic backgrounds. Here, we describe several optimized protocols to generate, collect, and experimentally utilize various samples obtained from HCC somatic mouse models generated by HDTVi. More specifically, we focus on techniques relevant to ex vivo analyses of the complex liver TME using multiparameter flow cytometric analyses of over 21 markers, immunohistochemistry, immunofluorescence, and histochemistry. We describe the transcriptional assessment of whole tissue, or of isolated immune subsets by flow-cytometry-based cell sorting, and other protein-oriented analyses. Together, these streamlined protocols allow the optimal use of each HCC murine model of interest and will assist researchers in deciphering the relations between cancer cell genetics and systemic and local changes in immune cell landscapes in the context of HCC progression. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of HCC mouse models by hydrodynamic tail vein injection Basic Protocol 2: Assessment of HCC tumor progression by magnetic resonance imaging Basic Protocol 3: Mouse sacrifice and sample collection in HCC mouse models Support Protocol 1: Preparation of serum or plasma from blood Basic Protocol 4: Single-cell preparation and HCC immune landscape phenotyping by flow cytometry Alternate Protocol 1: Flow cytometric analysis of circulating immune cells Support Protocol 2: Generation, maintenance, and characterization of HCC cell lines Support Protocol 3: Fluorescence-activated cell sorting of liver single-cell preparation Basic Protocol 5: Preparation and immunohistochemical analysis of tumor tissues from HCC-bearing liver Alternate Protocol 2: Preparation and analyses for immunofluorescence staining of HCC-bearing liver Support Protocol 4: Liver-specific phenotypic analyses of liver sections Support Protocol 5: Immunohistochemical quantification in liver sections Basic Protocol 6: Preparation of snap-frozen tumor tissue from extracted liver and transcriptional analyses of bulk tumor or sorted cells Alternate Protocol 3: Protein analyses from HCC samples and serum or plasma.
肝细胞癌 (HCC) 的发病率不断上升，死亡率不断增加，加上其肿瘤异质性高、缺乏可靶向的治疗靶点以及对化疗药物的耐药倾向，使得开发更好的 HCC 模型成为当务之急。为了更好地模拟 HCC 遗传背景中的高度多样性，最近使用流体动力学尾静脉注射 (HDTVi) 系统开发了多功能的体细胞小鼠模型。这些模型是一种新颖的体内工具，可以用于研究 HCC 表型和对治疗的反应，并且重要的是，它们可以进一步分析由不同遗传背景塑造的相关肿瘤微环境 (TME)。在这里，我们描述了几种优化的方案，用于生成、收集和实验利用通过 HDTVi 生成的 HCC 体细胞小鼠模型获得的各种样本。更具体地说，我们专注于使用超过 21 种标记物的多参数流式细胞术分析、免疫组织化学、免疫荧光和组织化学对复杂的肝 TME 进行离体分析的相关技术。我们描述了通过流式细胞术基于细胞分选对整个组织或分离的免疫亚群进行转录评估的方法，以及其他面向蛋白质的分析方法。这些简化的方案共同允许最佳地使用每个有兴趣的 HCC 小鼠模型，并将帮助研究人员在 HCC 进展的背景下，解析癌症细胞遗传学与系统和局部免疫细胞景观变化之间的关系。 © 2021 作者。当前方案由 Wiley 期刊出版公司出版。基本方案 1：通过流体动力学尾静脉注射生成 HCC 小鼠模型基本方案 2：通过磁共振成像评估 HCC 肿瘤进展基本方案 3：在 HCC 小鼠模型中处死小鼠和收集样本支持方案 1：从血液中制备血清或血浆基本方案 4：通过流式细胞术对 HCC 免疫图谱进行单细胞制备和表型分析替代方案 1：循环免疫细胞的流式细胞术分析支持方案 2：HCC 细胞系的生成、维持和鉴定支持方案 3：肝单细胞制备的荧光激活细胞分选基本方案 5：准备 HCC 阳性肝组织并进行免疫组织化学分析替代方案 2：准备并分析 HCC 阳性肝组织的免疫荧光染色支持方案 4：肝组织切片的肝特异性表型分析支持方案 5：免疫组化定量分析肝组织切片基本方案 6：从提取的肝脏中制备冷冻肿瘤组织，并对大块肿瘤或分选细胞进行转录分析替代方案 3：HCC 样本和血清或血浆中的蛋白质分析。
