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Analysis of DNA methylation in single circulating tumor cells

Oncogene volume 36, pages 3223–3231 (2017)Cite this article

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Abstract

Direct analysis of circulating tumor cells (CTCs) can inform on molecular mechanisms underlying systemic spread. Here we investigated promoter methylation of three genes regulating epithelial-to-mesenchymal transition (EMT), a key mechanism enabling epithelial tumor cells to disseminate and metastasize. For this, we developed a single-cell protocol based on agarose-embedded bisulfite treatment, which allows investigating DNA methylation of multiple loci via a multiplex PCR (multiplexed-scAEBS). We established our assay for the simultaneous analysis of three EMT-associated genes miR-200c/141, miR-200b/a/429 and CDH1 in single cells. The assay was validated in solitary cells of GM14667, MDA-MB-231 and MCF-7 cell lines, achieving a DNA amplification efficiency of 70% with methylation patterns identical to the respective bulk DNA. Then we applied multiplexed-scAEBS to 159 single CTCs from 11 patients with metastatic breast and six with metastatic castration-resistant prostate cancer, isolated via CellSearch (EpCAMpos/CKpos/CD45neg/DAPIpos) and subsequent FACS sorting. In contrast to CD45pos white blood cells isolated and processed by the identical approach, we observed in the isolated CTCs methylation patterns resembling more those of epithelial-like cells. Methylation at the promoter of microRNA-200 family was significantly higher in prostate CTCs. Data from our single-cell analysis revealed an epigenetic heterogeneity among CTCs and indicates tumor-specific active epigenetic regulation of EMT-associated genes during blood-borne dissemination.

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Acknowledgements

KR, BB, EH, DN, HN, TF, WTK, CS, NHS and RPLN are members of the Disseminated Cancer Cell Network (DCC Net) Düsseldorf. This work was supported by the Düsseldorf School of Oncology (funded by the Comprehensive Cancer Center Düsseldorf/Deutsche Krebshilfe and the Medical Faculty HHU Düsseldorf) (to CFP), by an EU grant reference EU FP7 project CTCTrap (Grant #305341 to JSDB), the Deutsche Krebshilfe (Grant #109600 to CS and NHS) and the Innovative Medicines Initiative Joint Undertaking (IMI JU) in conjunction with CANCER-ID (Grant Agreement #115749 to NHS).

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  1. N H Stoecklein and R P L Neves: These two authors contributed equally to this work

Authors and Affiliations

  1. Department of General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany

    C F Pixberg, F Müller, B Behrens, W T Knoefel, N H Stoecklein & R P L Neves

  2. Institute for Transplantation Diagnostics and Cell Therapeutics, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany

    K Raba

  3. Department of Obstetrics and Gynecology, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany

    E Honisch, D Niederacher, H Neubauer & T Fehm

  4. Department of Urology, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany

    W Goering & W A Schulz

  5. Division of Cancer Therapeutics and Division of Clinical Studies, The Institute of Cancer Research, London, UK

    P Flohr, G Boysen, M Lambros & J S De Bono

  6. Drug Development Unit, The Royal Marsden NHS Foundation Trust, London, UK

    P Flohr, G Boysen, M Lambros & J S De Bono

  7. Department of Oral, Maxillo- and Plastic Facial Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany

    C Sproll

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  1. C F Pixberg
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Pixberg, C., Raba, K., Müller, F. et al. Analysis of DNA methylation in single circulating tumor cells. Oncogene 36, 3223–3231 (2017). https://doi.org/10.1038/onc.2016.480

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