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Technical Report

Subtyping Circulating Tumor Cells in Breast Cancer for Personalized Therapeutics

Lori M. Millner, Kevin Goudy, Mark W. Linder, Roland Valdes, Jr. Department of Pathology and Laboratory Medicine, School of Medicine, University of Louisville, Louisville, Kentucky Enumeration of circulating tumor cells (CTCs) in blood is used in breast cancer patients as an independent predictor of outcome.  Present methods do not distinguish subtypes and only detect epithelial-type CTCs. This is significant because CTCs experience epithelial to mesenchymal transition (EMT), a process that increases motility, disease progression, and decreases epithelial marker expression. These mesenchymal CTCs likely pose a greater threat to the patient than epithelial CTCs due to their highly aggressive and motile nature. 

The goal of this project was to establish the feasibility of enriching, detecting, isolating and performing molecular analysis on single cells after being spiked into whole blood. We did this using a model composed of 4 heterogeneous breast cancer cell lines and a method for capturing and characterizing distinct CTC subsets regardless of EMT status. The 4 breast cancer molecular subtypes were used: luminal (MCF-7), HER2 (SK-Br-3), basal-like (HCC1954), and claudin-low (MDA-MB-231) and were chosen to represent patient CTC heterogeneity. 25,000 or 2,500 cells of each cell line were combined and then identified using a combination of antibodies including HER2, EpCAM, and CD44.

The specificity (separation efficiency) for each subtype was 67.3% ± 7.1 (±SE) (HCC1954), 91.7% ± 9.7 (MCF-7), 57.3% ± 8.7 (MDA-MB-231), and 100% ± 19.0 (MCF-7). The overall separation efficiency was 79.4 ± 5.9% (± SE). Spiking experiments of a single mesenchymal-like cell line that does not express EpCAM were conducted in whole human blood, and a sensitivity (percent recovery) of 84.9 ± 14.6% (±SE) was achieved.

Single cells were captured using the DEPArray, a novel separation technology that relies upon capturing cells in individual dielectrophoretic cages. These cages are then individually manipulated to sort single cells with 100% purity. A viewer allows the cells selected to be positively identified and cell identify and integrity is visually monitored throughout the sorting process. These single cells were then interrogated to analyze RNA expression of multiple genes. We achieved 100% sorting efficiency and 100% amplification of all sorted cells.  Expression of a house-keeping gene and at least one target was observed for 100% of the cells sorted. 

High percent recovery of a spiked mesenchymal-like breast cancer cell line into whole blood was achieved. The combination of antibodies has high separation efficiency with 2 of the 4 cell lines. Enrichment processes and antibody selection are being optimized to improve specificity of all 4 subtypes. The single cell sorting performance of the DEPArray was 100% and molecular analysis on the single cell level was conducted. Single cells from all 4 subtypes were analyzed and expression of a house-keeping gene and at least one target gene was observed for each cell. This data indicates that phenotypically diverse CTCs are capable of being subtyped and characterized on a single cell level. This information with improve prognostic capabilities and will allow therapies to be individually tailored to address each patient's CTCs.

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