Thiele Jana- A, Pitule Pavel, Hicks James, Kuhn Peter
Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic.
USC Michelson Center for Convergent Biosciences, CSI-Cancer, Department of Biological Sciences, Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA, USA.
Methods Mol Biol. 2019;1908:243-264. doi: 10.1007/978-1-4939-9004-7_17.
Circulating tumor cells (CTCs) are rare cells that can be found in the peripheral blood of cancer patients. They have been demonstrated to be useful prognostic markers in many cancer types. Within the last decade various methods have been developed to detect rare cells within a liquid biopsy from a cancer patient. These methods have revealed the phenotypic diversity of CTCs and how they can represent the complement of cells that are found in a tumor. Single-cell proteogenomics has emerged as an all-encompassing next-generation technological approach for CTC research. This allows for the deconstruction of cellular heterogeneity, dynamics of metastatic initiation and progression, and response or resistance to therapeutics in the clinical settings. We take advantage of this opportunity to investigate CTC heterogeneity and understand their full potential in precision medicine.The high-definition single-cell analysis (HD-SCA) workflow combines detection of the entire population of CTCs and rare cancer related cells with single-cell genomic analysis and may therefore provide insight into their subpopulations based on molecular as well as morphological data. In this chapter we describe in detail the protocols from isolation of a candidate cell from a microscopy slide, through whole-genome amplification and library preparation, to CNV analysis of identified cells from the HD-SCA workflow. This process may also be applicable to any platform starting with a standard microscopy slide or isolated cell of interest.
循环肿瘤细胞（CTCs）是可在癌症患者外周血中发现的稀有细胞。它们已被证明在许多癌症类型中是有用的预后标志物。在过去十年中，已开发出各种方法来检测癌症患者液体活检中的稀有细胞。这些方法揭示了循环肿瘤细胞的表型多样性以及它们如何代表肿瘤中发现的细胞互补情况。单细胞蛋白质基因组学已成为一种用于循环肿瘤细胞研究的全方位下一代技术方法。这使得在临床环境中能够解构细胞异质性、转移起始和进展的动态以及对治疗的反应或抗性。我们利用这个机会来研究循环肿瘤细胞的异质性，并了解它们在精准医学中的全部潜力。高清单细胞分析（HD-SCA）工作流程将对所有循环肿瘤细胞和罕见癌症相关细胞群体的检测与单细胞基因组分析相结合，因此可能基于分子和形态学数据深入了解它们的亚群。在本章中，我们详细描述了从显微镜载玻片上分离候选细胞、通过全基因组扩增和文库制备，到对高清单细胞分析工作流程中鉴定的细胞进行拷贝数变异（CNV）分析的方案。这个过程也可能适用于任何以标准显微镜载玻片或感兴趣的分离细胞为起点的平台。
