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Int. J. Mol. Sci. 2017, 18(9), 1885; doi:10.3390/ijms18091885

A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer

Department of Obstetrics and Gynecology, Heinrich Heine University of Duesseldorf, Life Science Center, Merowingerplatz 1A, Moorenstr. 5, 40225 Duesseldorf, Germany
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Received: 14 July 2017 / Revised: 21 August 2017 / Accepted: 25 August 2017 / Published: 31 August 2017
(This article belongs to the Special Issue Single Cell Technology)
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Abstract

Circulating tumor cells (CTCs), potential precursors of most epithelial solid tumors, are mainly enriched by epithelial cell adhesion molecule (EpCAM)-dependent technologies. Hence, these approaches may overlook mesenchymal CTCs, considered highly malignant. Our aim was to establish a workflow to enrich and isolate patient-matched EpCAMhigh and EpCAMlow/negative CTCs within the same blood samples, and to investigate the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutational status within single CTCs. We sequentially processed metastatic breast cancer (MBC) blood samples via CellSearch® (EpCAM-based) and via Parsortix™ (size-based) systems. After enrichment, cells captured in Parsortix™ cassettes were stained in situ for nuclei, cytokeratins, EpCAM and CD45. Afterwards, sorted cells were isolated via CellCelector™ micromanipulator and their genomes were amplified. Lastly, PIK3CA mutational status was analyzed by combining an amplicon-based approach with Sanger sequencing. In 54% of patients′ blood samples both EpCAMhigh and EpCAMlow/negative cells were identified and successfully isolated. High genomic integrity was observed in 8% of amplified genomes of EpCAMlow/negative cells vs. 28% of EpCAMhigh cells suggesting an increased apoptosis in the first CTC-subpopulation. Furthermore, PIK3CA hotspot mutations were detected in both EpCAMhigh and EpCAMlow/negative CTCs. Our workflow is suitable for single CTC analysis, permitting—for the first time—assessment of the heterogeneity of PIK3CA mutational status within patient-matched EpCAMhigh and EpCAMlow/negative CTCs. View Full-Text
Keywords: circulating tumor cell; epithelial cell adhesion moleculeow/negative; CellSearch; Parsortix; CellCelector; phosphatidylinositol 3-kinase catalytic subunit alpha circulating tumor cell; epithelial cell adhesion moleculeow/negative; CellSearch; Parsortix; CellCelector; phosphatidylinositol 3-kinase catalytic subunit alpha
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Lampignano, R.; Yang, L.; Neumann, M.H.D.; Franken, A.; Fehm, T.; Niederacher, D.; Neubauer, H. A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer. Int. J. Mol. Sci. 2017, 18, 1885.

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