手术活检无法监测癌细胞亚克隆动态演化进程，不能实时反应肿瘤异质性。“CTC液相活检”为靶向肿瘤异质性检测提供新策略。循环肿瘤细胞（CTC）基因型、表型和功能高度异质，单细胞分析更有意义。我们已建立核酸适体功能化纳米材料的肝癌CTC捕获，鉴定出不同分子表型的CTC亚克隆。本项目拟EpCAM/核酸适体LY1双靶标捕获CTC及单细胞分析，研究肝癌CTC分子表型时空异质性和单个CTC基因组拷贝数变异，鉴定出驱动CTC转移的拷贝数变异、基因突变及分子表型谱，评估CTC拷贝数变异和分子表型谱临床意义。体外培养CTC和构建CTC源移植瘤CDXs模型，解析CTC功能异质性。建立基于“CTC液相活检”的靶向肝癌异质性检测。该研究双靶标捕获，避免EpCAM阴性CTC漏检；单个CTC拷贝数变异、分子表型异质性及CDXs模型，进一步揭示肝癌异质性；基于CTC异质性的实时“液相活检”可提高靶向肝癌异质性治疗效果。

Tumor heterogeneity, derived from the prevailing view of cancer as a process of clonal evolution, has important implications for personalized medicine approaches as it can limit therapeutic efficacy and lead to resistance to therapy. In tumor progression, tumor colonies are highly dynamic and heterogeneous, which make extremely difficult for conventional approaches, such as classic surgical biopsy, to monitor the dynamic evolutionary process of tumors. Circulating tumor cells (CTCs) are cells shed from primary or metastatic tumors into the bloodstream. Similar to original tumor cells, CTCs are highly heterogeneous in genotype, phenotype and functional activities. Thus, liquid biopsy targeting single CTC analysis shall be a promising detective approach for evaluation of tumor heterogeneity. Based on our previous studies, we have established aptamer-based functional nanomaterial that can capture hepatocellular carcinoma CTCs, in which different CTC subclones were determined according to their molecular phenotype. In the present application, we propose to combine EpCAM and aptamer LY1 to capture CTC for single cell analysis. We hope that we can determine the spatiotemporal heterogeneity of CTCs in hepatocellular carcinoma patients mainly by detecting genomic copy number variations (CNVs) of CTCs in association with single-cell analysis. Next, the clinical significance of metastasis-related CNVs/mutation/aberrant expression in CTCs will be evaluated and calculated by bioinformatics algorithms analysis. Simultaneously, functional heterogeneity of CTCs from hepatocellular carcinoma patients will be determined in vitro culture assays and in vivo CTCs-derived xenograft (CDXs) model. Therefore, tumor heterogeneity will be revealed by liquid biopsy in combination with single CTC analysis. Taken together, by fulfill the proposed studies in this application, we hope to achieve the following goals: 1. Dual EpCAM/aptamer LY1 capture system will enhance the efficiency to capture CTCs as missed detection of EpCAM negative CTCs will be avoided; 2. Single cell analysis will advance our understanding of heterogenetic property of hepatocellular carcinoma; 3. Finally, our proposed CTC analysis will provide a novel approach for diagnosis of heterogeneity in hepatocellular carcinoma, and provide molecular targets for precision and personalized therapies, and enhance the efficacy of precise therapeutic technology that specifically target the heterogeneity of hepatocellular carcinoma.