ABSTRACT
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.
Subject(s)
Antigens, Neoplasm/genetics , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Cell Adhesion Molecules/genetics , Claudins/genetics , Flow Cytometry , Gene Expression Profiling , Immunomagnetic Separation , Neoplastic Cells, Circulating/metabolism , RNA, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Adhesion Molecules/metabolism , Claudins/metabolism , Epithelial Cell Adhesion Molecule , Feasibility Studies , Female , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Humans , MCF-7 Cells , Neoplastic Cells, Circulating/pathology , Phenotype , Sequence Analysis, RNA , Time FactorsABSTRACT
When proteins are conjugated to fluorescent organic dyes, fluorescence emission of the dye molecules is usually decreased, sometimes up to 50-70%. This quenching phenomenon has been acknowledged for decades, but as yet, there are no simple, practical methods to control the fluorescence of dyes conjugated to proteins, especially for dyes conjugated to immunoglobulins. Here, we report that the addition of (2-hydroxypropyl)-ß-cyclodextrin (HPßCD) to dye-antibody conjugates can increase fluorescence up to 2.5-fold in cell imaging and flow analysis. This method may be an effective way to increase the sensitivity of detection of fluorescent organic labels used in immunology, histochemistry, and cell biology.