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.

Biomedical Center Faculty of Medicine in Pilsen Charles University Pilsen Czech Republic

Department of Biomedical Engineering and Aerospace and Mechanical Engineering Viterbi School of Engineering University of Southern California Los Angeles CA USA

Department of Medicine Keck School of Medicine University of Southern California Los Angeles CA USA

Department of Urology Keck School of Medicine University of Southern California Los Angeles CA USA

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

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Ashworth TR (1869) A case of cancer in which cells similar to those in the tumours were seen in the blood after death. Med J Aust 14:146–147

Salsbury AJ (1975) The significance of the circulating cancer cell. Cancer Treat Rev 2:55–72 PubMed

Chang YS, Tomaso E d, McDonald DM et al. (2000) Mosaic blood vessels in tumors: frequency of cancer cells in contact with flowing blood. Proc Natl Acad Sci U S A 97:14608–14613 PubMed PMC

Massagué J, Obenauf AC (2016) Metastatic colonization by circulating tumour cells. Nature 529:298–306 PubMed PMC

Cristofanilli M, Budd GT, Ellis MJ et al. (2004) Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351:781–791 PubMed

Miller MC, Doyle GV, Terstappen LWMM (2010) Significance of circulating tumor cells detected by the cellsearch system in patients with metastatic breast colorectal and prostate cancer. J Oncol 2010:617421. PubMed PMC

Aktas B, Kasimir-Bauer S, Heubner M et al. (2011) Molecular profiling and prognostic relevance of circulating tumor cells in the blood of ovarian cancer patients at primary diagnosis and after platinum-based chemotherapy. Int J Gynecol Cancer 21:822–830 PubMed

Minciacchi VR, Zijlstra A, Rubin MA et al. (2017) Extracellular vesicles for liquid biopsy in prostate cancer: where are we and where are we headed? Prostate Cancer Prostatic Dis 20:251–258 PubMed PMC

Thiele J-A, Bethel K, Králĺčková M et al. (2017) Circulating tumor cells: fluid surrogates of solid tumors. Annu Rev Pathol 12:419–447 PubMed PMC

Hesari A, Moghadam SAG, Siasi A et al. (2017) Tumor-derived exosomes: potential biomarker or therapeutic target in breast cancer?: exosomes in breast cancer. J Cell Biochem 119 (6):4236–4240 PubMed

Alix-Panabieres C, Pantel K (2016) Clinical applications of circulating tumor cells and circulating tumor DNA as liquid biopsy. Cancer Discov 6(5):479–491 PubMed

Cho WCS (2014) Emerging techniques in molecular detection of circulating tumor cells. Expert Rev Mol Diagn 14:131–134 PubMed

Alix-Panabières C (2012) EPISPOT assay: detection of viable DTCs/CTCs in solid tumor patients. Recent Results Cancer Res 195:69–76 PubMed

Tewes M, Kasimir-Bauer S, Welt A et al. (2015) Detection of disseminated tumor cells in bone marrow and circulating tumor cells in blood of patients with early-stage male breast cancer. J Cancer Res Clin Oncol 141:87–92 PubMed

Nagrath S, Sequist LV, Maheswaran S et al. (2007) Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450:1235–1239 PubMed PMC

Marrinucci D, Bethel K, Kolatkar A et al. (2012) Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers. Phys Biol 9:016003. PubMed PMC

Carlsson A, Nair VS, Luttgen MS et al. (2014) Circulating tumor microemboli diagnostics for patients with non–small-cell lung cancer. J Thorac Oncol 9:1111–1119 PubMed PMC

Newton PK, Mason J, Bethel K et al. (2013) Spreaders and sponges define metastasis in lung cancer: a Markov chain Monte Carlo mathematical model. Cancer Res 73:2760–2769 PubMed PMC

Malihi PD, Morikado M, Welter L et al. (2018) Clonal diversity revealed by morphoproteomic and copy number profiles of single prostate cancer cells at diagnosis. Converg Sci Phys Oncol 4:015003. PubMed PMC

Ruiz C, Li J, Luttgen MS et al. (2015) Limited genomic heterogeneity of circulating melanoma cells in advanced stage patients. Phys Biol 12:016008. PubMed PMC

Bethel K, Luttgen MS, Damani S et al. (2014) Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction. Phys Biol 11:016002. PubMed PMC

Xuan J, Yu Y, Qing T et al. (2013) Next-generation sequencing in the clinic: Promises and challenges. Cancer Lett 340:284–295 PubMed PMC

Ranek L (1976) Cytophotometric studies of the DNA, nucleic acid and protein content of human liver cell nuclei. Acta Cytol 20:151–157 PubMed

Baslan T, Kendall J, Rodgers L et al. (2012) Genome-wide copy number analysis of single cells. Nat Protoc 7:1024–1041 PubMed PMC

Dago AE, Stepansky A, Carlsson A et al. (2014) Rapid phenotypic and genomic change in response to therapeutic pressure in prostate cancer inferred by high content analysis of single circulating tumor cells. PLoS One 9: e101777. PubMed PMC

Ni X, Zhuo M, Su Z et al. (2013) Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients. Proc Natl Acad Sci U S A 110:21083–21088 PubMed PMC

Navin N, Hicks J (2011) Future medical applications of single-cell sequencing in cancer. Genome Med 3:31. PubMed PMC

Navin N, Kendall J, Troge J et al. (2011) Tumour evolution inferred by single-cell sequencing. Nature 472:90–94 PubMed PMC

Baslan T, Kendall J, Ward B et al. (2015) Optimizing sparse sequencing of single cells for highly multiplex copy number profiling. Genome Res 25:714–724 PubMed PMC

Venkatraman ES, Olshen AB (2007) A faster circular binary segmentation algorithm for the analysis of array CGH data. Bioinformatics 23:657–663 PubMed

Krzywinski M, Schein J, Birol I et al. (2009) Circos: An information aesthetic for comparative genomics. Genome Res 19:1639–1645 PubMed PMC

Liquid Biopsy in Colorectal Carcinoma: Clinical Applications and Challenges