EpCAM based capture detects and recovers circulating tumor cells from subtypes of breast cancer

The potential utility of circulating tumor cells (CTCs) as liquid biopsies is of great interest. We hypothesized that CTC capture using EpCAM based gating is feasible for most breast cancer subtypes.

Cancer cells could be recovered from all intrinsic subtypes of breast cancer with IE/FACS, however, claudin-low cell lines showed very low capture rates compared to the four other groups (p = 0.03). IE/FACS detection of CTC mimic cells was time sensitive, emphasizing controlling for pre-analytic variables in CTC studies. Median fluorescent intensity for flow cytometry and RNA flow cell type characterization were highly correlated, predicting for CTC isolation across molecular subtypes. RNA-Seq of IE/FACS sorted single cell equivalents showed high correlation compared to bulk cell lines, and distinct gene expression signatures compared to PB.

Ten cell lines representing all major subtypes of breast cancer were spiked (as CTC mimics) into and recovered from peripheral blood (PB) using immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). Flow cytometry and RNA flow were used to quantify the expression of multiple breast cancer related markers of interest. Two different RNA-Seq technologies were used to analyze global gene expression of recovered sorted cells compared to bulk cell lines and PB.

Characterization of breast cancer subtypes. A–C. RNA flow. A. Density plots representing mesenchymal (FITC, x-axis) and epithelial (PE-YG, y-axis) marker RNA expression (top row), as well as ER (APC, x-axis) and HER2 (BV421, y-axis) RNA expression in 10 breast cancer cell lines. B. The number of positive cells (panel A) correlated with subtype classification for all probe sets, except for epithelial markers. C. MFI revealed bright subpopulations within each cell line, independent of subtype. Cells expressing high levels of mesenchymal markers were present in luminal subtypes (T47D, BT474), while the basal like cell line SUM149 showed high expression of epithelial markers. D. FACS. Epithelial cell (EpCAM, CDH1) and breast cancer marker-expression (HER2, EGFR) correlated with the clinical subtype classification. Staining with the benzothiazole thioflavin (TF) successfully captured nucleated cells. Expression of the white blood cell marker CD45 was equally low in all cell lines.

EpCAM based IE/FACS detected and captured a portion of spiked cells from each of the 10 cell lines representing all breast cancer subtypes, including basal-like but not claudin-low cancers. The assay allows for the isolation of high quality RNA suitable for accurate RNA-Seq of heterogeneous rare cell populations.