EpCAM based capture detects and recovers circulating tumor cells from all subtypes of breast cancer except claudin-low
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Alexander Ring1,2, Neal Mineyev3, Weizhu Zhu1,2, Emily Park4, Chip Lomas5, Vasu Punj2,6, Min Yu2,7, Dany Barrak1,2, Victoria Forte2, Tania Porras1,2, Debu Tripathy8, Julie E. Lang1,2
1Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
2Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA
3Department of Surgery, Lenox Hospital New York, New York, NY 10065, USA
4Advanced Cell Diagnostics, Research and Development, Hayward, CA 94545, USA
5BD Biosciences, Research and Development, San Jose, CA 95131, USA
6Division of Hematology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
7Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
8Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
Julie E. Lang, e-mail: [email protected]
Keywords: breast cancer, circulating tumor cells, IE/FACS, EpCAM
Received: July 10, 2015 Accepted: October 09, 2015 Published: October 19, 2015
Purpose: 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.
Results: 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.
Materials and Methods: 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.
Conclusions: 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.
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