Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Minhang District, Shanghai, 200240, China.
Shanghai Bone Tumor Institute, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai, 201620, China.
Anal Bioanal Chem. 2019 Aug;411(21):5373-5382. doi: 10.1007/s00216-019-01666-9. Epub 2019 Feb 28.
Circulating tumor cells (CTCs) are extraordinarily rare in blood samples and represent a real-time "liquid biopsy" of tumors. Although genetic and transcriptional sequencing of single CTCs has been reported, these methods fail to provide phenotypic and functional information of CTCs such as protein levels of surface proteins. Studies of single-cell proteomic assays of CTCs have been rare because of a lack of single-cell proteomic methods to handle and analyze rare cells in a high background of non-target cells with high sensitivity, throughput, and multiplexing capacity. Here, we develop a microchip-assisted single-cell proteomic method for profiling surface proteins of CTCs based on antibody and cellular DNA barcoding strategy. We combine DNA-encoded antibody tags and cell indexes to profile 15 proteins in ~ 100 single rare cells simultaneously, and use high-throughput sequencing as the readout to generate surface protein profiles of CTCs according to their cell indexes and antibody-derived protein barcodes. A 6400-well microchip and the automated puncher are used to rapidly retrieve single CTCs from enriched CTC population with minimal cell loss (~ 10%). This technological platform integrates reliable isolation and proteomic analysis of single CTCs and can be extendable to ~ 100 proteins in hundreds of rare cells with single-cell precision.
循环肿瘤细胞 (CTC) 在血液样本中极为罕见，代表了肿瘤的实时“液体活检”。虽然已经有报道称可以对单个 CTC 进行遗传和转录测序，但这些方法无法提供 CTC 的表型和功能信息，如表面蛋白的蛋白质水平。由于缺乏能够处理和分析高背景中非靶细胞中稀有细胞的单细胞蛋白质组学方法，因此单细胞 CTC 蛋白质组学研究很少见，这些方法需要具有高灵敏度、高通量和多重检测能力。在这里，我们开发了一种基于抗体和细胞 DNA 条码策略的微芯片辅助单细胞蛋白质组学方法，用于分析 CTC 的表面蛋白。我们结合 DNA 编码的抗体标签和细胞索引，同时对大约 100 个稀有细胞中的 15 种蛋白质进行分析，并使用高通量测序作为读出，根据细胞索引和抗体衍生的蛋白质条码生成 CTC 的表面蛋白图谱。使用 6400 孔微孔板和自动打孔器从富集的 CTC 群体中快速提取单细胞 CTC，细胞损失最小 (~10%)。该技术平台集成了单细胞 CTC 的可靠分离和蛋白质组学分析，可扩展至数百个稀有细胞中的大约 100 种蛋白质，具有单细胞精度。
