Development of a Protocol for Single-Cell Analysis of Circulating Tumor Cells in Patients with Solid Tumors

  • Carolina Reduzzi
  • Rosita Motta
  • Giulia Bertolini
  • Patrizia Miodini
  • Antonia Martinetti
  • Elisa Sottotetti
  • Maria Grazia Daidone
  • Vera CappellettiEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 994)


Genomic characterization of circulating tumor cells (CTCs) enables the monitoring of tumor progression and of adaption occurring during treatment. CTC molecular characterization represents indeed a precious tool to implement in the clinical practice for better dealing with acquired resistance to systemic treatment and tumor evolution. Unfortunately CTCs are very rare and enrichments from blood samples and subsequent identification of these cells are technically very challenging. We describe here the main steps leading to the development of a technical protocol for visualization, enumeration and recovery of single CTCs exploiting the recently developed DEPArray™platform. Our description of the technical workflow starts with evaluation of pre-analytical aspects related to blood sample collection warning about the possible effects on immunoreactivity profiles which may bias the interpretation. Subsequently, other CTC-enrichment approaches are critically discussed and compared in relation to their performances with the DEPArray™. Identification of CTCs represents another critical point due to their heterogeneity and due to the still-to-be clarified role of different subpopulations, typically epithelial, mesenchymal or mixed. Finally, issues related to single cell analysis are illustrated. The chapter ends with an overview of results obtained on real clinical samples which support the reliability of the protocol and its transferability to the daily clinical routine.


Circulating tumor cells CTC enrichment and identification Single-cell analysis Whole genome amplification DEPArray™ 



The study was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), grant IG 16900 to M.G.D and grant IG 15792 for supporting the fellowship of CR, from the Italian Ministry of Health (grant RF2013-02359692 to MGD) and from the European Commission under the 7th Framework Program, grant agreement n° 260791 Eurocan Platform.

RM, PM, MGD and VC are paid with funds from the Italian Ministry of Health.


  1. Abonnenc M, Manaresi N, Borgatti M et al (2013) Programmable interactions of functionalized single bioparticles in a dielectrophoresis-based microarray chip. Anal Chem 85(17):8219–8224CrossRefPubMedGoogle Scholar
  2. Bidard FC, Proudhon C, Pierga JY (2016) Circulating tumor cells in breast cancer. Mol Oncol 10(3):418–430CrossRefPubMedGoogle Scholar
  3. Bulfoni M, Gerratana L, Del Ben F et al (2016) In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis. Breast Cancer Res 18(1):30CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chudziak J, Burt DJ, Mohan S et al (2016) Clinical evaluation of a novel microfluidic device for epitope-independent enrichment of circulating tumour cells in patients with small cell lung cancer. Analyst 141(2):669–678CrossRefPubMedGoogle Scholar
  5. 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(8):e101777CrossRefPubMedPubMedCentralGoogle Scholar
  6. de Bourcy CF, De Vlaminck I, Kanbar JN et al (2014) A quantitative comparison of single-cell whole genome amplification methods. PLoS One 9(8):e105585CrossRefPubMedPubMedCentralGoogle Scholar
  7. Elliott P, Peakman TC, Biobank UK (2008) The UK Biobank sample handling and storage protocol for the collection, processing and archiving of human blood and urine. Int J Epidemiol 37(2):234–244CrossRefPubMedGoogle Scholar
  8. Fina E, Reduzzi C, Motta R et al (2015) Did circulating tumor cells tell us all they could? The missed circulating tumor cell message in breast cancer. Int J Biol Markers 30(4):e429–e433CrossRefPubMedGoogle Scholar
  9. Fuchs AB, Romani A, Freida D et al (2006) Electronic sorting and recovery of single live cells from microlitre sized samples. Lab Chip 6(1):121–126CrossRefPubMedGoogle Scholar
  10. Heitzer E, Auer M, Gasch C et al (2013) Complex tumor genomes inferred from single circulating tumor cells by array-CGH and next-generation sequencing. Cancer Res 73(10):2965–2975CrossRefPubMedGoogle Scholar
  11. Holland NT, Smith MT, Eskenazi B et al (2003) Biological sample collection and processing for molecular epidemiological studies. Mutat Res 543(3):217–234CrossRefPubMedGoogle Scholar
  12. Hou Y, Wu K, Shi X et al (2015) Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing. GigaScience 4:37CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hvichia GE, Parveen Z, Wagner C et al (2016) A novel microfluidic platform for size and deformability based separation and the subsequent molecular characterization of viable circulating tumor cells. Int J Cancer 138(12):2894–2904CrossRefPubMedPubMedCentralGoogle Scholar
  14. Joosse SA, Gorges TM, Pantel K (2014) Biology, detection, and clinical implications of circulating tumor cells. EMBO Mol Med 7(1):1–11CrossRefPubMedCentralGoogle Scholar
  15. Kang Q, Henry NL, Paoletti C et al (2016) Comparative analysis of circulating tumor DNA stability In K3EDTA, Streck, and CellSave blood collection tubes. Clin Biochem. S0009-9120(16)30040-6Google Scholar
  16. Klein CA, Schmidt-Kittler O, Schardt JA (1999) Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proc Natl Acad Sci USA 96(8):4494–4499CrossRefPubMedPubMedCentralGoogle Scholar
  17. Lohr JG, Adalsteinsson VA, Cibulskis K et al (2014) Whole-exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer. Nat Biotechnol 32(5):479–484CrossRefPubMedPubMedCentralGoogle Scholar
  18. Navin NE (2014) Cancer genomics: one cell at a time. Genome Biol 15(8):452CrossRefPubMedPubMedCentralGoogle Scholar
  19. 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 USA 110(52):21083–21088CrossRefPubMedPubMedCentralGoogle Scholar
  20. Paoletti C, Hayes DF (2016) Circulating tumor cells. Adv Exp Med Biol 882:235–258CrossRefPubMedGoogle Scholar
  21. Polzer B, Medoro G, Pasch S et al (2014) Molecular profiling of single circulating tumor cells with diagnostic intention. EMBO Mol Med 6(11):1371–1386CrossRefPubMedPubMedCentralGoogle Scholar
  22. Qin J, Alt JR, Hunsley BA et al (2014) Stabilization of circulating tumor cells in blood using a collection device with a preservative reagent. Cancer Cell Int 14(1):23CrossRefPubMedPubMedCentralGoogle Scholar
  23. Van Loo P, Voet T (2014) Single cell analysis of cancer genomes. Curr Opin Genet Dev 24:82–91CrossRefPubMedGoogle Scholar
  24. Wang Y, Navin NE (2015) Advances and applications of single-cell sequencing technologies. Mol Cell 58(4):598–609CrossRefPubMedPubMedCentralGoogle Scholar
  25. Xu L, Mao X, Imrali A et al (2015) Optimization and evaluation of a novel size based circulating tumor cell isolation system. PLoS One 10(9):e0138032CrossRefPubMedPubMedCentralGoogle Scholar
  26. Yu M, Ting DT, Stott SL et al (2012) RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis. Nature 487(7408):510–513CrossRefPubMedPubMedCentralGoogle Scholar
  27. Yu M, Bardia A, Wittner BS et al (2013) Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science 339(6119):580–584CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Carolina Reduzzi
    • 1
  • Rosita Motta
    • 1
  • Giulia Bertolini
    • 2
  • Patrizia Miodini
    • 1
  • Antonia Martinetti
    • 3
  • Elisa Sottotetti
    • 3
  • Maria Grazia Daidone
    • 1
  • Vera Cappelletti
    • 1
    Email author
  1. 1.Biomarker Unit, Department of Experimental Oncology and Molecular MedicineFondazione IRCCS Istituto Nazionale dei TumoriMilanoItaly
  2. 2.Tumor Genomics Unit, Department of Experimental Oncology and Molecular MedicineFondazione IRCCS Istituto Nazionale dei TumoriMilanoItaly
  3. 3.Medical Oncology UnitFondazione IRCCS Istituto Nazionale dei TumoriMilanoItaly

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