基于核酸适体的循环肿瘤细胞检测及应用

Circulating tumor cells (CTC) are cancer cells that shed from solid tumor and circulate in bloodstream. The detection of CTC remains a great challenge because of their extremely rare population. Cell-SELEX technology offers a potential solution to enrich CTC using aptamers obtained through in vitro selection. Though the capture yield is promising in cell line studies, there has been no clinical success reported using aptamers so far. One major encumbrance is that the attempt to capture CTC using one single kind of aptamer might encounter cell losses since the tumor cells are highly heterogeneous, which indicates that real CTC and the cell lines used for aptamer selection could be remarkably different in their surface components. Herein, the applicant proposes to solve this problem by applying a multivalence effect. Using aptamer cocktails, it is possible to target multiple surface molecules at the same time and benefit the capture efficiency of patient CTC correspondently. In this project, Non-small cell lung cancer is chosen as the model disease, and the aptamer combinations will be engineered based on the NSCLC-specific sequences we already have. After systematic optimization, the optimal aptamer combination will be grafted into a microfluidic channel and eventually used for clinical assay. Besides enumeration, CTC molecular analysis (such as EGFR mutations), is also included to explore its application potential in personalized treatment of cancer.

循环肿瘤细胞（CTC）是从实体肿瘤脱落进入血管，并在外周血中循环的肿瘤细胞。由于数量太低检测难度极大。SELEX技术提供了一个以肿瘤细胞株为靶标进行核酸适体（Aptamer）筛选并最终用于CTC检测的方法。然而由于细胞异质性，利用体外筛选得到的核酸适体对患者CTC进行富集的工作目前尚未见报道。本项目旨在建立一个基于核酸适体的普适性检测方法，通过优化组合利用多价增强效应克服细胞异质性造成的漏检，实现对临床样本的检测。项目拟选择非小细胞肺腺癌（NSCLC）为模型，以本课题组已有的核酸适体为基础，通过亲和力测定及竞争关系的研究，选出最可能用于CTC检测的若干种组合，再结合细胞捕获实验优化工作条件，并最终将该方法用于NSCLC患者的CTC检测。除对患者CTC实现计数以外，本项目还计划对捕获的CTC进行分子分析，以EGFR突变检测为切入点，探索CTC分子分析在肿瘤个性化治疗中的应用可行性。

肿瘤的远端转移是造成肿瘤患者死亡的主要原因。本课题以循环肿瘤细胞（CTC）为研究对象，开发了一种基于核酸适配体（aptamer）的、具有高度普适性的CTC检测方法。使用该方法，即便对于表面标志物不明的癌种，也可以通过体外筛选获得若干能与靶细胞特异性结合的适配体组合。在该项目的资助下，我们以非小细胞肺癌为模型，通过体外实验得到与肺癌细胞结合力较强核酸适配体组合，并实现了患者样本的CTC检测，以及疗效监测。结果显示，基于适配体的纳米芯片具有较高的检出率。同时我们发现随着治疗的进行，患者CTC的异质性会不断发生变化，而单一的CTC检测方法可能无法对患者的病情进行全面的反映。此外，我们结合磁珠与微阵列芯片，进一步提高了CTC产物的纯度，为之后的分子诊断奠定了基础。