基于微球-抗体条形码芯片技术的循环肿瘤细胞功能蛋白质组定量检测研究

Circulating tumor cells (CTCs) shed from tumor sites and represent the molecular characteristics of the primary tumor. Besides genetic and transcriptional characterization, it is important to profile functional proteins expressed by CTCs with single-cell precision. Functional proteins include phosphoprotein kinases, associated effector proteins and transcriptional factors that comprise the intracellular signaling cascades, as well as secreted cytokines and growth factors that are commonly associated with immune cell functions and cell-to-cell communications. Such proteins are often hyperactivated in cancer cells with functional consequences that can be associated with various hallmarks of cancer, and are consequently recognized as therapeutic targets. Therefore, multiplexed functional proteomics is becoming an integral part of cancer research to identify diagnostic biomarkers, monitor drug response profiles, understand mechanisms of cancer pathogenesis, and design new therapeutics. However, the intratumoral heterogeneity in human cancer, originating from the complex interplay between genetic instability and non-genetic plasticity induced by immunological, hormonal and environmental factors, emphasizes the need for analyzing functional proteins expressed by circulating tumor cells at single cell resolution.. Unfortunately, current single-cell proteomics tools limited by the low multiplexing capacity and the low limit of detection can normally sample around 20 proteins in regular practice and likely reach a limit between 50 to 100 proteins per cell, which represent sampling only a small part of the proteome.. To address these challenges, we propose to develop a global scale single-cell functional proteomics platform by combining highly flexible and scalable bead-based protein detection scheme with a highly versatile microfluidics-based single-cell barcode chip. The unprecedented level of multiplexing of this platform can be achieved by using innovative bead-antibody barcode structure that offers multi-dimensional protein identification by assigning three independent identifiers (the bead size, color and its location) to each protein. This bead-antibody barcode chip platform will offer high throughput and simultaneous detection of 180 functional proteins from a few hundred to ten thousand single circulating tumor cells, thus providing useful information for early diagnosis of tumor, carcinoma prognosis and the development of targeted drugs.

循环肿瘤细胞携带了原发肿瘤的全部生物学信息，在单细胞水平上定量检测循环肿瘤细胞的功能蛋白质组，将为鉴别诊断标记物、监测药物反应、理解癌症发病机理和设计新治疗方法等提供重要信息。然而现有的单细胞蛋白质检测技术受其多路复用程度和灵敏度的限制，只能对功能蛋白质组中的小部分蛋白质（90种）进行抽样研究。针对上述问题，本项目拟开发一种全新的微球-抗体条形码芯片技术。该技术将在单细胞条形码芯片的基础上，结合一种高度灵活可扩展的微球-蛋白质检测方案，通过微球的尺寸、颜色和位置进行编码，实现在单细胞水平上大规模定量检测循环肿瘤细胞的功能蛋白质组（180种），为肿瘤的早期诊断、预后评估以及靶向药物的开发等提供信息。

在常规癌症诊断中，组织活检具有一定的局限性，并常常会给患者带来一定程度的创伤和痛苦。循环肿瘤细胞液体活检技术为癌症诊断提供了新思路。目前，针对CTC单细胞基因组和转录组的研究多有被报道，但基因组和转录组并不能很好地反应细胞的表型和功能信息。而蛋白质作为细胞功能的主要执行者，能够直接反应细胞的表型和功能状态。因此，在单细胞水平上对CTC蛋白质组进行定量检测研究可为肿瘤细胞的鉴定、分类、追踪用药和实时疗效监测等提供重要信息。但是由于技术受限，目前并没有能真正意义上实现稀有肿瘤细胞单细胞多重蛋白高通量检测的方法。为此，我们借助微孔芯片辅助稀有肿瘤细胞的分离与鉴定，并以寡核苷酸单链取代传统荧光物质标记抗体，从而通过测序结果实现在单细胞水平上同时检测稀有肿瘤细胞的多种蛋白质。然后，我们以肺癌患者外周血为样本，实现了对五个肺癌患者共90个CTC单细胞的15种蛋白质的同步检测，为临床稀有肿瘤细胞蛋白质组定量检测提供了新的技术手段。