Abstract 224: Enrichment and isolation of uncontaminated breast cancer cells from human blood samples
Authors: Fernandez Sandra V. , Wagner Christopher , Parveen Zahida , Aburto Lucy , Paolillo Carmela , Hvichia George , Mu Zhaomei , Austin Laura , Cristofanilli Massimo 
Published: August 2015

Cancer cells in peripheral blood, known as circulating tumor cells (CTCs), play an important role in tumor dissemination. CTCs are shed from either the primary tumor or its metastases and circulate in the peripheral blood of patients; thus they can be regarded as “liquid biopsies” of metastasizing cells. Although the exact origin and physiology of CTCs is unknown, a fraction of these cells are thought to be viable metastatic precursors capable of initiating a clonal metastatic lesion. The molecular characterization of CTCs is important because it may provide insights into the molecular biology of metastasis, the association of their molecular profiles with treatment outcomes, and reveal the presence of potential therapeutic targets. The process of CTC enrichment represents a significant step toward the isolation of CTCs from whole blood. Given that there are approximately 5×109 erythrocytes and 107 leukocytes per ml of whole blood, purification must represent a reduction of many orders of magnitude. In this study, we show a method to isolate CTCs which combines a microfluidic cell separation device (Parsortix system) as an enrichment method followed by the DEPArray selection system. The Parsortix system is versatile in its ability to separate, capture and harvest CTCs in an enriched form compatible with the needs of downstream processes such as DEPArray. Here we demonstrate enrichment of CTCs via the Parsortix system using both breast cancer cell spiked healthy blood donor samples and blood from metastatic breast cancer patients. For these experiments, two milliliters of blood were processed. The cells were harvested and stained to identify CTCs in the enriched harvest product. Cells were stained with fluorescently labeled monoclonal antibodies specific for pan cytokeratin (CK-8/18/19-PE), leukocyte common antigen (CD45-APC), and nuclear stained with [4′,6-diamidino-2-phenylindole (DAPI)]. With spiked samples, tumor cells were isolated by DEPArray selection, which yielded a pure population of tumor cells for molecular characterization. Tumor cells were defined by presence of a clear DAPI-stained nucleus, CK-PE-positive cytoplasm and CD-45-APC negative. We demonstrated that by using a combination of enrichment and isolation/selection methods, we are able to isolate single, uncontaminated tumor cells to achieve single cell molecular analysis. The use of single cells is emerging as a powerful approach to molecular analysis in oncology, and this study demonstrates its potential application with circulating tumor cells.

Citation Format: Sandra V. Fernandez, Christopher Wagner, Zahida Parveen, Lucy Aburto, Carmela Paolillo, George Hvichia, Zhaomei Mu, Laura Austin, Massimo Cristofanilli. Enrichment and isolation of uncontaminated breast cancer cells from human blood samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 224. doi:10.1158/1538-7445.AM2015-224