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Diagnostic Leukapheresis Enables Reliable Transcriptomic Profiling of Single Circulating Tumor Cells to Characterize Inter-Cellular Heterogeneity in Terms of Endocrine Resistance

1
Department of Obstetrics and Gynecology, University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, 40225 Duesseldorf, Germany
2
Department of General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, 40225 Duesseldorf, Germany
3
Institute for Transplantation Diagnostics and Cell Therapeutics, University Hospital and Medical Faculty of the Heinrich-Heine University Duesseldorf, 40225 Duesseldorf, Germany
*
Authors to whom correspondence should be addressed.
Cancers 2019, 11(7), 903; https://doi.org/10.3390/cancers11070903
Received: 20 May 2019 / Revised: 11 June 2019 / Accepted: 24 June 2019 / Published: 28 June 2019
(This article belongs to the Special Issue Liquid Biopsy for Cancer)
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Abstract

Circulating tumor cells (CTCs) hold great promise with regard to prognosis, treatment optimization, and monitoring of breast cancer patients. Single CTC transcriptome profiling might help reveal valuable information concerning intra-patient heterogeneity relevant to therapeutic interventions. In this study, we combined Diagnostic Leukapheresis (DLA), which is a microfluidic enrichment using the ParsortixTM system, micromanipulation with CellCelectorTM and subsequent single cell multi-marker transcriptome profiling. First, a PCR panel consisting of 30 different endocrine resistance and phenotypic marker genes was validated for single cell profiling by using different breast cancer cell lines. Second, this panel was applied to characterize uncultured and cultured CTCs, which were enriched from a cryopreserved DLA product obtained from a patient suffering from metastatic breast cancer resistant to endocrine therapy. Gene expression profiles of both CTC populations uncovered inter CTC heterogeneity for transcripts, which are associated with response or resistance to endocrine therapy (e.g., ESR1, HER2, FGFR1). Hierarchical clustering revealed CTC subpopulations with different expressions of transcripts regarding the CTCs’ differential phenotypes (EpCAM, CD44, CD24, MYC, MUC1) and of transcripts involved in endocrine signaling pathways (FOXO, PTEN). Moreover, ER-positive CTCs exhibited significant higher expression of Cyclin D1, which might be relevant for CDK4/6 inhibitor therapies. Overall, gene expression profiles of uncultured and cultured CTCs resulted in a partly combined grouping. Our findings demonstrate that multi-marker RNA profiling of enriched single uncultured CTCs and cultured CTCs form cryopreserved DLA samples may provide important insights into intra-patient heterogeneity relevant for targeted therapies and therapy resistance. View Full-Text
Keywords: breast cancer; liquid biopsy; diagnostic leukapheresis; DLA; circulating tumor cells; CTC; single cell profiling; transcriptomic profiling; RT-qPCR breast cancer; liquid biopsy; diagnostic leukapheresis; DLA; circulating tumor cells; CTC; single cell profiling; transcriptomic profiling; RT-qPCR
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Reinhardt, F.; Franken, A.; Meier-Stiegen, F.; Driemel, C.; Stoecklein, N.H.; Fischer, J.C.; Niederacher, D.; Ruckhaeberle, E.; Fehm, T.; Neubauer, H. Diagnostic Leukapheresis Enables Reliable Transcriptomic Profiling of Single Circulating Tumor Cells to Characterize Inter-Cellular Heterogeneity in Terms of Endocrine Resistance. Cancers 2019, 11, 903.

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