A Novel Workflow to Enrich and Isolate Patient-Matched EpCAM
| Autor: | Rita, Lampignano, Liwen, Yang, Martin H D, Neumann, André, Franken, Tanja, Fehm, Dieter, Niederacher, Hans, Neubauer |
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| Rok vydání: | 2017 |
| Předmět: |
CellSearch
Class I Phosphatidylinositol 3-Kinases phosphatidylinositol 3-kinase catalytic subunit alpha Breast Neoplasms CellCelector Epithelial Cell Adhesion Molecule Flow Cytometry Neoplastic Cells Circulating circulating tumor cell Article Neoplasm Proteins Workflow epithelial cell adhesion moleculelow/negative Mutation MCF-7 Cells Humans Parsortix Female |
| Zdroj: | International Journal of Molecular Sciences |
| ISSN: | 1422-0067 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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