肝细胞癌“液相活检”：基于核酸适配体功能化纳米材料和单细胞分析的CTC异质性研究

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.

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。

靶向肿瘤异质性，开启癌症分层靶向治疗新篇章。但组织活检无法监测癌细胞亚克隆动态演化进程，不能实时呈现肿瘤异质性。面向靶向肿瘤异质性检测的困境，“CTC液相活检”为肿瘤异质性检测提供了一种新视角。循环肿瘤细胞CTC反应肿瘤实时进程，作为一个完整的活力细胞，包含了基因、蛋白、功能等生物学信息，可用于肿瘤表型、基因组/转录组、肿瘤异质性、生物学功能等方面研究。本项目从CTC新型检测技术、CTC基因型/表型/生物功能异质性、CTC单细胞分析技术、CTC体外培养等方面开展了系列研究。.建立了以EpCAM /靶向肝癌核酸适体LY1双靶标识别、功能化微纳米材料捕获和释放CTCs及量子点多色成像的循环肝癌细胞检测新方法。该方法可动态监测CTC数量变化，CTCs数量与肿瘤负荷相关。同时，鉴定出循环肝癌细胞中具有肿瘤干性样细胞和 EMT 双表型的亚群以及肝癌 CTCs 关联性分子表征谱，联合检测 CTCs 中 CK-19/PD-L1、AFP/lncRNA-PVT1这四种表型分子可为肿瘤伴随诊断提供循证依据。.开展了基于纳米材料基底和声表面波器件相结合的工作，利用声表面波装置产生液滴将单个CTC包覆，解决单个目标细胞无法有效提取的问题。同时，基于数字PCR的单个CTC分子突变谱TP53/CTNNB1和表达谱lncRNA HULC/PD-L1联合检测，揭示了 CTCs 基因型和表型存在高度多样性，在 CTC 群体之间、CTC 群体内个体之间以及疾病不同进展期和不同解剖位置均高度异质。.筛选和优化了CTCs培养三维纳米基底材料和形貌特征。不同基底材料及其形貌特征可影响细胞的存活、增殖等生物功能。为了能够筛选出适合肝癌 CTCs培养基底材料和形貌，测试了纤连蛋白、胶原蛋白、纤维素、水凝胶等材料三维结构和形貌特征。 .发现了CTC和循环免疫细胞亚群的综合分析被认为比单独CTC具有更多的癌症预后价值，干性样CTC是预测低分化HCC重要标志物，联合外周血免疫抑制细胞和血小板有助于HCC病理分级。.本研究聚焦CTC，从捕获体系构建、CTCs计数、表型分析，到CTC分子表征谱、单细胞分析，再到‘CTC +’研究，如免疫细胞（Treg、 MDSC、NK等）、成纤维细胞、血小板等。围绕“CTC液相活检”深入研究，为肿瘤个体化诊疗提供新的无创策略，为发现肿瘤治疗新靶点提供思路。