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