核酸适配体在非小细胞肺癌循环肿瘤细胞异质性分析中的作用及机制研究

The detection of Circulating Tumor Cells (CTC) plays significant rolls in the personalized therapy of Non-Small-Cell Lung Cancer (NSCLC) patients. However, due to the problem in tumor heterogeneity, the detection results of the technology that relies on single antibody were not satisfied. In contrast, DNA aptamer, also named as chemical antibody, are now drawing more and more attention for its application in CTC analysis. In our previous research, for the first as we known, the aptamer cocktails were successfully used in CTC capturing of NSCLC patients, which not only improved the effect of CTC enrichment, but also reflect the heterogeneity of the CTC population. However, the exact mechanism that worked in this process is not clarified yet. In this continued research, we aim to illustrate this mechanism by finding these aptamers’ binding target on CTC and analyzing these binding targets’ characteristics in structure and function. Further, by comparing the difference of patient’s CTCs that captured with different aptamer in aspects such as proliferation, migration, tumorigenic potential, gene expression and prognosis prediction, we are intending to interpret the significance of this mechanism for patient’s therapy. This research will bring helps in broadening our recognition for CTC’s characteristics and their migration mechanism in NSCLC patients, directing the decision making for patient’s individualized therapy. Also, it could provide valuable clues for future drug development. Therefore, our research would have important academic value and good prospect in clinical application.

循环肿瘤细胞（CTC）检测对非小细胞肺癌（NSCLC）患者的个体化治疗有重要指导意义，但由于肿瘤异质性的问题，依赖单一抗体技术的检测效果并不理想。核酸适配体又称化学抗体，近年来在CTC检测中的应用受到了广泛关注。在前期工作中，我们首次将核酸适配体的组合成功应用于NSCLC患者的CTC检测，不仅大幅提高了检出效果，而且还能反映CTC群体的异质性，但其具体机制还有待进一步研究。在本课题中，我们拟进一步探索这些核酸适配体所对应的细胞作用靶点，了解这些靶点的结构和功能特点，分析它们能够区分CTC异质性的作用机制，通过对比不同适配体所捕获的CTC在增殖、迁移、成瘤、基因表达以及提示患者预后等方面的差别，分析上述机制对临床治疗的意义。本课题将有助于我们深入认识NSCLC患者CTC的特性及其转移机制，指导个体化治疗策略的制定，也可为未来的药物研发提供有益线索，具有重要的科学意义和良好的临床应用前景。

循环肿瘤细胞（CTC）检测对非小细胞肺癌（NSCLC）患者的个体化治疗有重要指导意义，但是其检测技术并不足以满足临床的需求。在本课题中，我们首先在既往适配体工作的基础之上，尝试采用磁珠代替纳米芯片作为适配体的固相载体进行CTC的检测，克服了适配体结合位点不足的限制，有利于细胞的释放和后续分析。其次是对三个商业化的CTC检测体系进行了测试，这三个体系分别在CTC的单细胞检测、体外培养以及自动化过滤检测等方面具有优势，各完成21例（CTC检出率95.2%，数值范围2-21个/3ml），6例（连续培养成功率83.3%）及21例（CTC检出率100%，数值范围2-493/4ml）患者的验证，证实这三个体系均能够有效的检出CTC并适合进行下游的功能和分子分析，未来在临床中具有一定的应用前景。第三，针对体液标本中肿瘤来源材料（CTC及ctDNA）相对较少的特点，我们尝试采用了数字PCR方法，证实其在胸水（与测序相比对为75.4% vs. 43.8%，P < 0.0001，与ARMS相比为61.3% vs. 38.7%，P=0.016）和血浆标本（相比于Super ARMS方法可以在EGFR 19del或L858R突变和T790M突变上分别提高13%和22.1%的检出率，P值分别为0.002和<0.001）之中具有良好的EGFR突变检出，优于临床目前使用的常规方法。更重要的是，数字PCR具有绝对定量的优势，能够反映EGFR突变丰度的动态变化，这在某种程度上反映了患者体内肿瘤克隆的异质性变化，对于患者TKI药物的使用具有重要的指导价值。最后，我们还对非小细胞肺癌患者ALK融合基因的血液检测以及乳腺癌患者的精确分型进行了尝试。以上工作使我们对于肿瘤液体活检工作有了更一步的认识和理解，为今后在临床工作用应用这些技术解决患者的实际问题奠定了基础。