Profiling the 3D interaction between germ cell tumors and microenvironmental cells at the transcriptome and secretome level

The tumor microenvironment (TM), consisting of the extracellular matrix (ECM), fibroblasts, endothelial cells and immune cells, might affect tumor invasiveness and the outcome of standard chemotherapy. This study investigated the cross-talk between germ cell tumors (GCT) and surrounding TM cells (macrophages, T-lymphocytes, endothelial cells, fibroblasts) at the transcriptome and secretome level. Using high-throughput approaches of three-dimensional (3D) co-cultured cellular aggregates, this study offers newly identified pathways to be studied with regard to sensitivity towards cisplatin-based chemotherapy or tumor invasiveness as a consequence of the cross-talk between tumor cells and TM components. Mass-spectrometry-based secretome analyses revealed that TM cells secreted factors involved in ECM organization, cell adhesion, angiogenesis and regulation of insulin-like growth factor (IGF) transport. To evaluate direct cell–cell contacts, green fluorescent protein (GFP)-expressing GCT cells and mCherry-expressing TM cells were co-cultured in 3D. Afterwards, cell populations were separated by flow cytometry and analyzed by RNA-sequencing. Correlating the secretome with transcriptome data indicated molecular processes such as cell adhesion and components of the ECM being enriched in most cell populations. Re-analyses of secretome data with regard tolysine- and proline-hydroxylated peptides revealed a gain in proteins, such as collagens and fibronectin.Cultivation of GCT cells on collagen I/IV- or fibronectin-coated plates significantly elevated adhesive and migratory capacity, while decreasing cisplatin sensitivity of GCT cells. Correspondingly, cisplatin sensitivity was significantly reduced in GCT cells under the influence of conditioned medium from fibroblasts and endothelial cells. This study sheds light on the cross-talk between GCT cells and their circumjacent TM, which results in deposition of the ECM and eventually promotes a pro-tumorigenic environment through enhanced migratory and adhesive capacity, as well as decreased cisplatin sensitivity. Hence, our observations indicate that targeting the ECM and its cellular components might be a novel therapeutic option in combination with cisplatin-based chemotherapy for GCT patients.肿瘤微环境（tumor microenvironment, TM）由细胞外基质（extracellular matrix, ECM）、成纤维细胞、内皮细胞与免疫细胞构成，可影响肿瘤侵袭性及标准化疗的疗效。本研究从转录组与分泌组层面，探究了生殖细胞肿瘤（germ cell tumors, GCT）与其周围肿瘤微环境细胞（巨噬细胞、T淋巴细胞、内皮细胞、成纤维细胞）之间的细胞交互。本研究采用三维（3D）共培养细胞聚集体的高通量分析方法，新发现了多条可用于探究顺铂基化疗敏感性或肿瘤细胞与肿瘤微环境组分交互所引发的肿瘤侵袭性的通路。基于质谱的分泌组分析显示，肿瘤微环境细胞可分泌参与细胞外基质组织、细胞黏附、血管生成以及胰岛素样生长因子（insulin-like growth factor, IGF）转运调控的因子。为评估直接细胞-细胞接触情况，本研究将表达绿色荧光蛋白（green fluorescent protein, GFP）的生殖细胞肿瘤细胞与表达mCherry的肿瘤微环境细胞进行三维共培养。随后通过流式细胞术分离细胞群，并借助RNA测序（RNA-sequencing）进行分析。将分泌组数据与转录组数据关联分析后发现，多数细胞群中富集了细胞黏附及细胞外基质组分相关的分子过程。针对赖氨酸与脯氨酸羟化肽的分泌组数据再分析显示，胶原蛋白、纤连蛋白等蛋白的丰度出现上调。在I/IV型胶原蛋白或纤连蛋白包被的培养板中培养生殖细胞肿瘤细胞，可显著提升其黏附与迁移能力，同时降低其对顺铂的敏感性。相应地，在成纤维细胞与内皮细胞的条件培养基影响下，生殖细胞肿瘤细胞对顺铂的敏感性也显著降低。本研究阐明了生殖细胞肿瘤细胞与其周围肿瘤微环境之间的细胞交互——这种交互可促使细胞外基质沉积，并最终通过增强细胞黏附与迁移能力、降低顺铂敏感性，构建促肿瘤生长的微环境。因此，本研究结果提示，靶向细胞外基质及其细胞组分，或许可作为联合顺铂基化疗治疗生殖细胞肿瘤患者的新型治疗策略。