Detection and characterization of circulating tumor cells (CTCs) with an epithelial-to-mesenchymal transition (EMT) phenotype is very important as it can contribute to the identification of high-risk for relapse and death patients. However, most of the methods are underestimating CTC numbers, due to their dependence on epithelial markers. In the current study, we evaluated the EMT phenotype in CTCs isolated from breast cancer (BC) patients, using the CellSearch system. Spiking experiments for the evaluation of the specificity and sensitivity of our method were performed using HeLa cells. Sixty-five breast cancer (BC) patients (47 early and 18 metastatic) were enrolled in the study. Vimentin is a mesenchymal marker which indicates tumoral cells acquiring invasive and malignant properties. We studied the vimentin (VIM) expression using the extra channel of the CellSearch system and an anti-vimentin antibody conjugated with FITC. In our present results, we reported the percentage of circulating tumor cells that expressed vimentin in early and in metastatic breast cancer patients. Interestingly, the incidence of cells with a CK-VIM+CD45- phenotype was detected in both settings. These cells were detected in 31.4% of CK-negative (11/35) and 82.3% of CK-positive (10/12) early BC patients. The corresponding numbers for metastatic disease were 15.4% (2/13) and 100% (5/5), respectively. Our results suggest that in CTC-negative patients, potentially undetectable tumor cells could be identified using the FDA-approved CellSearch system, based on the (CK-VIM+CD45-)-phenotype, offering additional information regarding the metastatic dissemination in cancer patients. Further experiments evaluating more biomarkers are necessary to elucidate the mechanisms that regulate tumorigenesis and metastasis.
EpCAM-independent isolation of circulating tumor cells with epithelial-to-mesenchymal transition and cancer stem cell phenotypes using ApoStream® in patients with breast cancer treated with primary systemic therapy
