循环肿瘤细胞时空异质性及其在肝癌转移复发中相关机制研究

We previously reported that circulating tumor cells (CTCs) were significantly correlated with postoperative recurrence and metastasis of hepatocellular carcinoma (HCC). Our recently research identified that the phenotype of CTC showed remarkable variation during tumor evolution and among different location of dissemination route(temporal and spatial heterogeneity). However, the clinical significance and underlying molecular mechanism of CTC's spatial and temporal heterogeneity is still unclear. In this study, we plan to (1) determine the distribution of EpCAM+, CD133+, CD90+, CD24+ CTC subsets in different vascular compartments and their dynamic changes during anti-HCC treatment, analyzing their relationship with clinical prognosis and treatment responsiveness. (2) Depict the expression profile of CTC using single-cell RNA-Seq technology in different vascular compartments and their dynamic changes during anti-HCC treatment; Develop a molecular characterization system of CTC that is highly correlated with CTC survival, HCC recurrence, organ specific metastasis and treatment responsiveness/resistance. (3) Investigate metastatsis/recurrence and drug resistance related key genes and signal pathways, revealing the underlying molecular mechanism. (4) Prospectively evaluate the clinical utility of CTC molecular characterization system in predicting postoperative recurrence/metastasis, and monitoring treatment efficacy in a real-time manner. In summary, the mechanism of CTC's spatial and temporal heterogeneity and its potential clinical significance are expectable from this project.

我们前期研究表明循环肿瘤细胞（Circulating tumor cell, CTC）数量与肝癌术后转移、复发关系密切，进一步研究提示CTC在肿瘤演进关键节点和转移关键部位CTC存在显著差异（即时空异质性）。目前CTC时空异质性在肝癌转移复发中的作用及分子机制不明。本课题在大量前期工作基础上：1）检测关键治疗节点和部位EpCAM+、CD133+、CD90+或CD24+ CTC亚群比例和分布情况，探讨其与肝癌疗效及预后的关系；2）利用单细胞RNA-Seq技术，比较CTC表达谱差异，绘制CTC特征性基因表达谱；3）基于CTC表达谱，筛选肝癌转移复发和耐药相关基因及通路并明确鉴定；4）验证决定CTC异质性的特征性表达谱和关键分子在肝癌转移复发预测和疗效监测中的作用。通过本研究进一步明确肝癌CTC的时空异质性，初步阐明其在转移复发和耐药中作用和机制，为临床提供新的肝癌转移复发预测和干预策略。

肝细胞癌（Hepatocellular carcinoma，HCC）（简称肝癌）是最常见的恶性肿瘤之一，肝癌术后极易出现肿瘤复发和转移。循环肿瘤细胞（Circulating Tumor Cell，CTC）被认为是肿瘤转移复发具体实施者。本课题原定研究目标为：1）揭示CTC 在肝癌演进过程中和转移路径上的异质性（时空异质性）；2）探讨CTC 时空异质性参与肝癌转移复发的相关分子机制；3）阐明CTC 时空异质性在肝癌转移复发及疗效监测中的作用。课题执行情况顺利，原定目标全部完成。主要结果及关键数据如下：1）通过自主研发的CTC微流控芯片富集、检测肝癌患者关键治疗节点中EpCAM+、CD133+、CD90+和CD24+ 四类CTC 亚群的数量及比例。这四类CTC亚群在肝癌患者中检出比例呈现异质性，且CTC亚群比例随疾病进展和治疗干预表现出特征性的动态变化。CTC单细胞数字PCR检测结果表明这四类CTC基因表达谱存在显著差异；2）联合CellSearch系统和共聚焦扫描技术检测73 例肝癌患者血行转移路径上5个关键血管部位内的CTC 及其微瘤栓。不同血管部位内的CTC在数量、形态学等方面存在与检测部位相关的特征性差异，CTC 和CTC微瘤栓的数量及分布差异与肝癌术后患者预后显著相关，表达谱分析表明CTC在不同血管部位的上皮-间质特征呈现异质性，初步提示CTC在肝癌血行播散路径上存在空间异质性；3）进一步对10名HCC患者4个血管部位的113个CTC进行单细胞转录组测序，分析发现CTC与对应的原发肿瘤相比具有特征性的转录组模式；来自不同血管部位的CTC，在其转录谱中显示出血管内和血管间异质性；差异表达分析提示细胞生长/增殖、血小板活化、趋化因子信号传导和缺氧等关键通路的改变可能参与CTC与血流微环境中的生物物理因素的相互作用；无监督的聚类分析揭示了CTC的休眠子集，休眠CTC可能更具侵略性和存活优势。本项目通过对肝癌患者关键治疗节点和不同部位的CTC进行检测分析，在单细胞水平上揭示了肝癌CTC的时空异质性及其在肝癌转移复发中的分子机制，初步阐明了肝癌CTC时空异质性的特征性变化在肝癌预后判断、靶向转移预测、疗效实时监测及个体化治疗中的价值。