基于tmTNF-α mAb磁分离微流控技术的乳腺癌转移超早期预警研究

Recent studies indicate that micrometastasis emerges in early phase of breast cancer, then detection of circulating tumor cells (CTCs) becomes a good indicator for monitoring relapse and early warning tumor metastasis. As a result of the heterogeneity of tumor, EMT, ultra-early occult metastasis, precancerous inflammatory lesion and failure to specifically distinguish normal-like CTCs, the application of Cellsearch assay was restricted, which was the only method approved by FDA. Researches showed that the development of breast cancer was closely related with inflammatory process, and that TNF-α, cooperating with other molecules, acted as an important role in the process of tumorigenesis and metastasis. Hence, TNF-α became a molecular target for ultra-early warn and intervention. Our research group turned out in previous works: 1) TNF-α was highly expressed in many tumors, but rarely in normal and peritumoral tissues; 2) It was also expressed in different subtypes of breast cancer and all grades of breast hyperplasia, which was positive correlated with histological grades; 3) The tmTNF-α monoclonal antibody that we developed independently could efficiently suppress breast cancer growth and metastasis. So, in this project, by means of a combination of triple steps-targeted recognition by tmTNF-α mAb, specific capture by magnetic microfluidic chip and precisely labeling by quantum dots for sensitive imaging and spectrum analysis, we plan to establish a new diagnostic approach for detecting CTCs with High sensitivity, high-throughput, and high specificity on the surface of the chip, and then conduct clinical validation. The cells we captured will be cultured in vitro for the further identity of their molecular expression profiling. As a result, our work will provide strong support for developing antibodies-based multiplex magnetic microfluidic, as well as provide experimental evidence for identity of molecular expression profiling and being "liquid biopsy" of real time CTCs in vivo.

循环肿瘤细胞（CTCs）是监测肿瘤复发和早期转移预警的良好指标。由于肿瘤的异质性、EMT、超早期隐匿性转移、癌前炎性改变等因素, 使Cellsearch 检测CTCs临床应用受限。新近研究表明：TNF-α 协同其他分子在乳腺癌的成瘤和转移中发挥作重要用，成为超早期预警和干预的分子靶点。本课题组前期证实：1）TNF-α在多种肿瘤中高表达，而正常组织和癌旁鲜见；2）在不同类型乳腺癌和癌前各级增生中表达并与组织学分级正相关；3）自主研发的tmTNF-α mAb 能高效抑制乳腺癌生长和转移。本项目拟以tmTNF-α mAb 靶向识别、磁性微流控芯片细胞捕获和量子点荧光标记成像并进行光谱分析，建立一种磁性微流控芯片高灵敏、高通量、高特异的检测CTCs的新方法并进行临床验证，捕获后的细胞鉴定分子表征。为开发抗体的多元磁控技术提供佐证，为分子表征谱的鉴定以及实时CTCs的"液体活检"提供实验依据。

CTCs能提供方便和非侵入性的方法在致命肿瘤转移发生之前检测肿瘤细胞。目前应用于检测乳腺癌CTCs的方法有： CTCs 富集技术； CTCs 分析技术；CTCs 富集与分析相结合的技术。然而有研究表明：利用细胞表面的EpCAM进行CTC分离的Cellsearch系统尤其不能识别normal-like型乳腺癌，而此种细胞表现有侵袭性。人们急需新的检测方法，利用能特异性识别normal-like型乳腺癌细胞而不能识别血细胞的抗体。同时，由于肿瘤的异质性、上皮细胞间质化、超早期隐匿性转移、血细胞捕获效率低等因素，使得CTCs的分离和计数成为了技术性的难题。.本项研究首先以前期制备的tmTNF-α单克隆抗体为循环乳腺癌细胞超早期预警识别的靶向分子，制备单抗并检测鉴定其在乳腺癌发生发展中的表达情况；再次，制备了磁性纳米抗体柱阵列的微流控芯片为循环乳腺癌细胞捕获载体，此芯片的捕获效能我们进行了细胞系以及临床病人标本的比对试验，并与不同的肿瘤也进行了横向比较；然后我们对于纳米量子点耦联单抗的复合物为CTCs分子识别荧光成像，检测其发光的效能是否被检测并做定量和定性分析；最后，我们应用CTCs的单细胞分析体系结合临床病例进行了FISH和单细胞测序验证，其间我们发现了病人外周血循环乳腺癌细胞团CTM(CTC-clusters)内部有异质性，且预示乳腺癌病人预后较差。我们对于捕获的外周血模拟环境中乳腺癌CTCs，可体外培养、裸鼠体内成瘤；捕获的临床乳腺癌病人外周血CTCs，体外培养、裸鼠体内成瘤还在进展过程中。本研究结合我国乳腺癌早诊早治的防治需要，以乳腺癌CTCs为研究对象，建立基于自主知识产权的tmTNF-α mAb靶向识别、微流控芯片捕获及量子点多色成像的循环乳腺癌细胞检测新方法，集合了免疫分析的高特异性，磁场的独特分离能力以及微流控芯片的集成化和自动化功能，为临床的乳腺癌早期转移预警提供有力的诊断标志物。.