面向乳腺癌精准诊疗的循环肿瘤细胞单细胞分析芯片研究

Utilizing microfluidic technology to enumerate and genetically sequence circulating tumor cells (CTC) at single cell level is likely to remarkably benefit the tumor precision medicine. Existing CTC technologies are still with defects of relying on manually interpreting immune-fluorescent images, as well as lacking of molecular diagnosis on single cells. This proposal is aiming at developing a multi-functional microfluidic chip to simultaneously accomplish blood filtering, CTC isolating, identifying, enumerating and single cell gene amplifying. By furtherly integrating unique image interpreting algorithm and automatic control, a multifunctional automatic CTC single cell analysis system will be developed to avoid off-chip cell transfer and manually interpreting, therefore eliminating the loss of rare cells and the human bias. We intend to realize that by directly processing whole blood, the precise CTC numbers and its protein expression status will be acquired in 2 hours while the gene amplification products of each and every CTC will be provided in 4 hours. By employing such technology, we will compare the coverage and consistency of molecular information acquired from CTC and tumor biopsy, respectively. Furtherly, we will explore if CTC can be used to predict the tumor progression and therapy effect, laying both theoretical and technical foundations for applying CTC single cell analysis in the frontier of cancer precise diagnosis and treatment.

利用微流控技术检测循环肿瘤细胞（CTC）并进行单细胞基因分析对癌症精准诊疗有重要意义。现有技术对CTC鉴定及计数仍依赖于主观判读免疫荧光，也无法单细胞CTC分子诊断。本项目提出用单块微流控芯片完成血液过滤、CTC分离、精准鉴定及计数、单细胞基因扩增的策略，进一步整合计算机图像分析和自动控制，形成多功能自动化CTC单细胞分析系统，避免了在芯片外转移细胞和人工鉴定免疫荧光，从根本上消除稀有CTC的实验损失及检验者的主观误差。采用该策略，有望实现：向芯片内直接输入全血，在2小时内获得CTC精确数量和蛋白表达情况，4小时内获得每个CTC的单细胞基因扩增产物。基于上述CTC单细胞分析技术，本项目将通过循证医学研究，探索CTC单细胞分析对病理组织主要分子诊断信息的覆盖度和一致性，并研究基于CTC的分子诊断与临床疗效及疾病进展的拟合程度，从而为将CTC单细胞分子诊断用于癌症精准诊疗奠定理论和技术基础。

本项目针对循环肿瘤细胞的单细胞基因分析等方面的关键问题，研制出了一套基于微流控芯片的单细胞基因分析方法，产生了一系列科研成果。首先，该芯片集成了单细胞捕获、鉴定、裂解和原位MDA扩增的功能，并可检测单细胞突变丰度变化情况。此外，基于N-钙粘蛋白靶向多肽构建了一种新型的间质型CTC捕获的方法，并在单细胞转录组水平获得了异质型CTC的基因表达谱。其次，构建了一个集血液过滤、CTC富集、CTC分离鉴定、CTC裂解和全基因组扩增为一体的自动化微流控芯片SCIGA-Chip，实现了“全血输入，单细胞全基因组扩增产物输出”的单细胞分析策略。最终，实现了CTC用于乳腺癌患者新辅助治疗疗效及预后预测的临床探索性研究。该项目通过CTC的单细胞分析获得每个患者的肿瘤分型、药物靶点和耐药基因等信息，为肿瘤早期诊断、制定精准诊疗方案及预后评估等临床研究提供新的思路与新的方法。