Abstract P1-04-07: Isolation and expression profiling of EpCAM-negative circulating tumor cells in human and xenograft models of breast cancer
Authors: Lang JE , Mineyev N , Ring A , Kahn M , Punj V , Tripathy D , Barsky LW , Zhu W 
Published: August 2014

Abstract
Background: While several assays currently exist to enumerate circulating tumor cells (CTCs), limitations to existing assays include reliance on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM) and difficulty isolating pure populations of rare CTC RNA from peripheral blood without background subtraction of leukocytes. We evaluated the biomarkers CD146 and CD49f as a strategy to isolate CTCs that underwent epithelial to mesenchymal transition (EMT). We hypothesized that using immunomagnetic separation and fluorescence activated cell sorting (IE/FACS) it is possible to isolate RNA derived from EpCAM-negative CTCs.
Methods: 10 breast cancer cell lines were acquired from the ATCC, authenticated by short tandem repeat profiling, and stratified according to subtype: HER2 positive (SKBR3, MDA-MB-453, SUM190), luminal A (T47D, MCF7, ZR751), luminal B (BT474) and basal-like (SUM149, MDA-MB-231, Hs578T). IE/FACS with EMT gates was performed using CD146 ferrofluid particles via a magnetic separator followed by incubation with Thioflavin T, CD 31 and CD45 PE-Cy7 (for human or mouse blood), CD146-V450, and CD49f-A647. FACS sorting was performed using a FACS Aria II (BD Biosciences). Known quantities of cells were spiked into PBS or peripheral blood (PB) from healthy female donors and subjected to IE/FACS with EMT gates. Absolute cell counts and recovery rates were determined using the TruCOUNT method (BD Biosciences) with acquisition of 35,000 beads. MDA-MB-231 xenografts were established in 4 male NOD/SCID/IL2 gamma chain deficient mice; cardiac punctures were performed to acquire a complete xenograft PB volume for IE/FACS. Sorted xenograft CTCs, human PB and MDA-MB-231 were used to isolate RNA, amplify and profile via Affymetrix human gene ST 1.0 arrays. The effect of sample processing with IE/FACS was assessed using our EpCAM gates (EpCAM ferrofluid, EpCAM-PE, thioflavin, CD45 PE-Cy7) to sort BT474 and profiled via next generation sequencing (NGS) using the Helicos Single Molecule sequencing approach.
Results: MDA-MB-231 spiked into PB from an unaffected mouse and negative mouse PB controls were used to establish gating thresholds. This gating strategy recovered 14% of MDA-MB-231 cells from spiked human PB via the TruCOUNT method, which represented a 200% yield versus EpCAM based IE/FACS. CTCs were successfully isolated from 4/4 mice bearing xenografts (ranging from 193-6352 cells captured per ∼1mL mouse blood). Principal component analysis demonstrated that gene expression may differentiate xenograft CTCs from PB and MDA-MB-231. Recovery rates using the EMT gates for 10 cell lines spiked into PBS and human PB will be reported and compared to our typical EpCAM gating strategy. Pearson's correlation coefficient of BT474 spiked into PB and recovered via IE/FACS using an EpCAM gating strategy was 92% by NGS.
Conclusions: We have established a method for the isolation and expression profiling of EpCAM-negative CTCs as well as EpCAM-positive CTCs. Future studies are required to determine if expression profiling of CTCs can be used as prognostic, predictive and discovery tools.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-07.