肝星状细胞促进循环肝癌细胞在肝内定植形成转移复发的研究

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China, and generally develops on a background of cirrhosis. At present surgical resection is the main treatment for HCC. But the high rates of postoperative recurrence seriously limit the effectiveness. According to reports, many factors contribute to HCC recurrence. Many clinical retrospective studies showed that the cirrhotic liver is an independent risk factor for HCC recurrence. The dissemination of circulating tumor cells (CTCs) is now recognized as the main cause of recurrence after curative resection. A growing number of studies have suggested that the activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis, and there is a close relationship between HSCs and HCC. So why are circulating HCC cells easier to colonize a cirrhotic liver? Do HSCs in the cirrhotic liver play a key role in promoting circulating HCC cells intrahepatic colonization and metastasis? What is the effective method of preventing and treating recurrence and metastasis? In order to resolve these scientific problems the subject will carry out the research in the following three aspects: (1) To establish mouse models of circulating HCC cell metastasis in cirrhotic livers, and mouse models of circulating HCC cell metastasis in HSC-rich livers. By using these models, we will investigate the effects of a background of cirrhosis and HSCs on circulating HCC cell intrahepatic metastasis. (2) On the basis of these in vivo models, we will use a known HSC-specific inhibitor icaritin to treat these two mouse models for the sake of studying the inhibitory effect of icaritin on circulating HCC cell intrahepatic metastasis in cirrhotic livers and HSC-rich livers. (3) A novel three-dimensional cell co-culture system will be used to study the role of HSCs in promoting HCC cells directional migration, colonization and growth, providing in vitro experimental evidence that HSCs initiate the pre-metastatic niche to promote HCC cells metastasis and recurrence. In summary, the expected accomplishments of the project would reveal a key role for HSCs in promoting circulating HCC cells intrahepatic colonization and metastasis, and identify HSCs as a target cells for prevention and treatment of HCC metastasis and recurrence.

肝癌术后复发率很高，循环肿瘤细胞是术后复发的根源，而肝硬化背景是一个危险因素。然而，肝硬化影响循环肿瘤细胞在肝内定植形成转移的机制并不清楚。鉴于肝星状细胞活化是肝硬化发生的核心环节，我们推测肝硬化中的活化肝星状细胞通过旁分泌机制帮助循环肝癌细胞在肝内定植，刺激其形成转移性克隆。为了验证这一推测，本课题将构建小鼠硬化肝循环肝癌细胞肝转移模型和肝星状细胞种植肝循环肝癌细胞肝转移模型，并用肝星状细胞抑制剂淫羊藿素处理，研究肝硬化背景和肝星状细胞促进循环肝癌细胞在肝内形成转移复发的作用。同时应用三维细胞培养系统研究肝星状细胞诱导肝癌细胞定向迁移和生长的过程。通过研究，预期能获取肝星状细胞作为一个转移小生境促进循环肝癌细胞在肝内定植形成复发的体内外实验证据，评价淫羊藿素通过靶向杀伤肝星状细胞，干预循环肝癌细胞在肝硬化背景上转移复发的潜能，从而为深入探索肝癌转移复发诊治新策略提供实验依据和新视野。

肝硬化被广泛认为是肝癌转移复发的一个关键因素。既往研究多重视肿瘤本身的侵袭特性对肝细胞癌（hepatocellular carcinoma，HCC，以下简称肝癌）术后复发的影响，而忽略了肝硬化背景对手术切除疗效的影响及其在肝癌复发中的作用。已有的研究表明，肝硬化与肝癌复发率呈现明显的相关性，但是肝硬化环境中具体影响肝癌转移复发的因素、影响的环节及其确切的作用机制尚不清楚。.本项目研究工作以阐明这一基本问题为出发点，将肝星状细胞（stellate cells， HSCs）、循环肿瘤细胞（circulating tumor cells，CTCs）与肝癌复发转移的关系作为研究重点。首先我们从人肝癌组织成功分离HSCs，并且证明其能够诱导肝癌细胞发生上皮间质转化（epithelial-mesenchymal transition，EMT），促进肝癌细胞增殖、侵袭和转移。肝癌组织中HSCs的表达水平与肝癌患者术后的复发和预后有关，可以作为一个独立因素预测肝癌患者预后。在肝硬化小鼠模型基础上，通过尾静脉水流动力学细胞传递法，成功建立了肝癌细胞经血行途径形成肝内复发转移的小鼠模型。通过深入研究发现，肝硬化可促进肝癌CTCs在肝脏形成转移灶，但不影响肝癌CTCs在肺、肾、脾脏转移灶的形成。针对肝硬化背景中的关键因素HSCs和肝癌CTCs，我们研究发现，淫羊藿素（icaritin）和sorafenib分别对上述两个关键因素具有很好的靶向抑制作用。临床资料研究结果显示，肝硬化和外周血中CTCs数目是肝癌患者术后复发的关键因素。肝癌患者术前外周血中CTCs个数为18是预测肝癌根治术后复发的最适临界值。肝硬化是CTCs个数≥18的肝癌患者根治术后复发的一个危险因素，而CTCs个数＜18的肝癌患者与术后复发无显著相关性。进一步研究发现，肝癌患者外周血中的CTCs能够通过体外三维培养方式存活并扩增成CTCs球，并且CTCs球对于肝癌患者术后早期复发有一定的预测作用。这一研究结果不仅有助于加深人们对肝硬化背景在肝癌转移复发机制中所起作用的认识，而且为探索肝癌转移复发诊治新策略的研究提供新的视野，为指导制定肝癌个体化治疗策略提供新的理论基础。