How to isolate and characterize EpCAMnegative circulating tumor cells in metastatic breast cancer patients

Circulating tumor cells (CTCs) are epithelial cells identified in the peripheral blood stream of solid tumors and are believed to be their metastatic precursors. CTCs are mostly detected by EpCAM-based systems that miss those cells which acquired mesenchymal features and believed to be the most malignant ones. In this project we established a workflow combining the Parsortix© system and the CellCelector™micromanipulator to enrich for EpCAM-negative CTCs by size and to isolate them for further molecular characterizations. A cohort of 21 metastatic breast cancer blood samples depleted for EpCAM-positive CTCs was processed to enrich for EpCAM-negative cells (Parsortix©). Captured cells were stained in situ for EpCAM, cytokeratins and CD45 to identify epithelial cells and were then harvested. CTCs were detected by immune fluorescence microscopy and isolated as single cells (CellCelector™). Whole genome amplification (Ampli1™) and array-based comparative genomic hybridization (aCGH) were used to confirm their malignant origin. In 18 out of 21 samples EpCAM-positive (467 cells) and in 16 samples EpCAM-negative CTCs (260 cells) were found. Hundredeleven of EpCAM-negative and comparable numbers of EpCAM-positive cells and leucocytes were successfully isolated. A successful genome-wide amplification was observed 7% of EpCAM-negative cells vs. 30% of EpCAM-positive cells indicating a lower genome quality in the first group. Six out of 24 EpCAM-positive and 5 out of 8 EpCAM-negative cells with high quality genomic amplification were processed for aGCH which confirmed their tumoral provenience. Our workflow allows to successfully isolate EpCAMneg CTCs from EpCAM depleted samples and to further process them for molecular characterizations.