Spatiotemporal Regulation of Cholangiocarcinoma Growth and Dissemination by Peritumoral Myofibroblasts in a Vcam1-dependent Manner. Spatiotemporal Regulation of Cholangiocarcinoma Growth and Dissemination by Peritumoral Myofibroblasts in a Vcam1-dependent Manner

Intrahepatic cholangiocarcinoma (iCCA) is characterized by its highly desmoplastic stroma. Myofibroblasts (MFs) are present both within the tumor mass (intratumoral MFs, iMFs) and at the tumor border (peritumoral MFs, pMFs). Using a spheroid-based coculture system, we show that the initial iCCA-pMF contact is growth suppressive to the tumor cells. However, prolonged iCCA-pMF interaction elicits significant tumor cell invasion and dissemination. We find that vascular cell adhesion molecule-1 (Vcam1) level is elevated in tumor cells in contact with pMFs but low in disseminated tumor cells both in vitro and in vivo. A gene regulatory network analysis of mouse and patient iCCA tumors and Vcam1 knockout (Vcam1KO) demonstrate a heavy involvement of Vcam1 in epithelial-to-mesenchymal transition. While Vcam1KO has only a limited impact on tumor cell growth in their monoculture, Vcam1KO spheroids exhibit instant dissemination and a severe growth defect when cocultured with pMFs. When transplanted into the liver, Vcam1KO iCCA cells show a similar increase in dissemination but a significant defect in establishing primary and metastatic tumors. Incomplete blocking of Vcam1 in vivo reduces the size but increase the number of metastatic lesions. Overall, our study shows a spatiotemporal regulation of iCCA growth and dissemination by pMFs in a Vcam1-dependent manner. Overall design: Two Vcam1KO single-cell clones were generated (Vcam1KO1 and Vcam1KO2) to identify the direct downstream targets of Vcam1 by RNA-seq.

肝内胆管癌（intrahepatic cholangiocarcinoma, iCCA）以高度促结缔组织增生的间质为典型特征。肌成纤维细胞（myofibroblasts, MFs）既可分布于肿瘤实体内部（瘤内肌成纤维细胞，intratumoral MFs, iMFs），也可存在于肿瘤边缘区域（瘤旁肌成纤维细胞，peritumoral MFs, pMFs）。本研究借助基于球体的共培养体系证实：肝内胆管癌与瘤旁肌成纤维细胞的初始接触会抑制肿瘤细胞增殖；但二者长时间相互作用，则会显著诱导肿瘤细胞发生侵袭与播散。研究发现，在与瘤旁肌成纤维细胞接触的肿瘤细胞中，血管细胞黏附分子1（vascular cell adhesion molecule-1, Vcam1）的表达水平显著升高，而在体外及体内的播散性肿瘤细胞中，该分子的表达均处于较低水平。对小鼠及患者来源的肝内胆管癌组织与Vcam1敲除（Vcam1 knockout, Vcam1KO）样本开展的基因调控网络分析显示，Vcam1广泛参与上皮间质转化（epithelial-to-mesenchymal transition）过程。尽管Vcam1敲除对单培养状态下的肿瘤细胞增殖仅存在有限影响，但当与瘤旁肌成纤维细胞共培养时，Vcam1敲除的肿瘤球体可快速发生播散，并出现严重的生长缺陷。将Vcam1敲除的肝内胆管癌细胞原位移植入肝脏后，其播散能力虽有所增强，但在形成原发肿瘤与转移瘤方面存在显著缺陷。体内不完全阻断Vcam1，可减小转移灶的体积，却增加其数量。综上，本研究揭示了瘤旁肌成纤维细胞以Vcam1依赖的方式，对肝内胆管癌的生长与播散实施时空调控。总体实验设计：本研究构建了两株Vcam1敲除单细胞克隆（Vcam1KO1与Vcam1KO2），通过RNA测序（RNA-seq）鉴定Vcam1的直接下游靶标。