Isolation and characterization of circulating tumor cells from patients with metastatic breast cancer

Introduction: Circulating tumor cells (CTCs) are cells originating from primary and/or metastatic tumor and circulating freely in the peripheral blood of cancer patients. CTCs can serve as a liquid biopsy for real time monitoring of disease progression and therapy efficacy. Due to CTCs rarity in the blood and the lack of strategies to isolate CTCs entire heterogeneous population, CTCs phenotype has not been fully defined. Commonly used epithelial markers-based isolation approaches failed in detecting epithelial mesenchymal (EMT) transitioned CTCs. The purpose of this study was to isolate and characterize the entire population of viable CTCs from metastatic breast cancer (MBC) patients. Methods: Peripheral blood samples from 44 locally advanced and MBC patients and 4 healthy donors (HD) were collected in this study. In the first part of the study we isolated CTCs from blood samples of 14 MBC patients through an antibody-mediated enrichment procedure (RosetteSepTM). Then, freshly isolated CTCs from 8 samples were analyzed by immunofluorescence (IF), whereas isolated cells from 6 samples were seeded on cover glass. In the second part of the study we isolated circulating cells from 30 MBC samples and 4 HD by using Ficoll density gradient followed by the seeding of free labeled PBMCs on cover glass. In 6/30 samples mRNA expression levels of CD45, ERα, HER2, CK19 and Vimentin were evaluated by qRT-PCR for both PBMCs and adherent cells. Furthermore, IF experiments were performed on adherent isolated cells, and on samples collected after three months of therapy, for testing the presence of ERα, HER2 and different EMT markers. Results: CTCs isolated by RosetteSepTM protocol stained positive only for the mesenchymal marker Vimentin, and RosetteSepTM seemed to affect the adherence ability of circulating cells. Interestingly, adherent cells isolated by Ficoll stratification showed reduced mRNA levels of leukocyte marker CD45 and higher mRNA levels of CK19 and Vimentin than PBMCs. Also, Vimentin and CK19 proteins were the most representative markers in adherent cells fraction. Furthermore, therapy reduced the number of CD45- cells and affected the number of different adherent cells subpopulations. Conclusions: We established a label-free method for the isolation and characterization of circulating cells in MBC, which allowed the depletion of most contaminating leukocytes and the enrichment of circulating cells expressing epithelial and mesenchymal markers. Heterogeneity of adherent cells underlined the importance of further characterize these cells to understand more about their role in metastatic disease and to improve personalized therapeutic approaches.