7P - Detection and Quantitation of Circulating Tumor Cells, Cancer Stem Cells and Endothelial Progenitor Cells Using Multiparameter Flow Cytometry

Abstract

Advances in medical technology and an increased understanding of tumor cell biology have led to the discovery of promising cellular biomarkers including circulating tumor cells (CTCs), cancer stem cells (CSCs) and endothelial progenitor cells (EPCs). CTCs and CSCs provide an early, reliable indication of disease progression and survival for patients on systemic therapy for metastatic breast cancer, whereas levels of EPCs are reported to correlate with tumor stage and have been evaluated as biomarkers of the efficacy of anticancer/ antiangiogenic treatment. The aim of this study was to establish practical methods for detection and quantitation of CTCs, CSCs and EPCs using multiparameter flow cytometry. In brief, immediately after enrichment by density gradient centrifugation, cells were stained with 2 sets of fluorescently conjugated antibodies; against CD34, VEGFR2 and CD45 for EPC and against EpCAM, CD44, CD24 and CD45 for CTCs and CSCs. The labeled cells were analyzed on a flow cytometer by collecting 1,000,000 events. For CTCs detection and quantitation, spiking experiments with breast cancer cell line MCF-7 demonstrated a linear performance with an accepted reproducibility. CTCs and CSCs were defined as CD45-EpCAM+ and CD45-CD44 + CD24-, respectively. These protocols could be used for detection of cancer stem cells in enzyme digested tissues from breast cancer patients. EPCs (CD45dim CD34 + VEGFR2+) were significant higher in breast cancer patients compared with the cancer-free controls. These simple and sensitive multiparameter flow cytometry protocols could be used for detection and quantitation of CTCs, CSCs and EPCs in breast cancer patients. However, clinical validation studies should be performed before their implementation as potential surrogate biomarkers of clinical response and prognostic markers. Disclosure: All authors have declared no conflicts of interest.