EpCAM based capture detects and recovers circulating tumor cells from all subtypes of breast cancer except claudin-low

Abstract

PurposeThe 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.ResultsCancer 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.Materials and MethodsTen 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.ConclusionsEpCAM 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.

Circulating tumor cells in breast cancer

“…The impact of EMT-associated EpCAM downregulation on CTC detection by EpCAM-dependent techniques, such as the CellSearch Ò system, has been repeatedly reported. Although most of BC subtypes can be efficiently captured by EpCAMbased assays (Ring et al, 2015), several EMT-related antigens have been successfully investigated in addition to EpCAMbased immunoselection as an effective way to increase CTC detection (Mostert et al, 2012Onstenk et al, 2015a;Satelli et al, 2015).…”

Liquid Biopsy Based on Circulating Cancer‐Associated Cells: Bridging the Gap from an Emerging Concept to a Mainstream Tool in Precision Medicine

“…In fact, relatively high numbers of apoptotic CTCs have been reported in blood samples from peripheral blood, evidencing their fragility. It has been reported that cancer cell numbers in blood samples rapidly decline over a time span of 5 h, revealing that the time from blood draw to cell harvest is critical . Other reports have shown that a significant RNA degradation occurs within a period of 2–4 h .…”

Microfluidics cell sample preparation for analysis: Advances in efficient cell enrichment and precise single cell capture

“…Anti-EpCAM antibody coating has been demonstrated to have an outstanding cell capture efficiency in both static and dynamic systems. [98][99][100][101] In the case of CTCs, most positive enrichment is grounded on the EpCAM antibodies. For example, He et al proposed a biocompatible and surface roughness controllable nano-film which is composed of TiO 2 nanoparticles for highly efficient CTC capture and in-situ identification by immunocytochemistry ( Fig.…”