Journal
ONCOGENE
Volume 36, Issue 23, Pages 3223-3231Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/onc.2016.480
Keywords
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Funding
- Dusseldorf School of Oncology (Comprehensive Cancer Center Dusseldorf/Deutsche Krebshilfe)
- EU grant reference EU FP7 project CTCTrap [305341]
- Deutsche Krebshilfe [109600]
- Innovative Medicines Initiative Joint Undertaking (IMI JU)
- CANCER-ID [115749]
- Dusseldorf School of Oncology (Medical Faculty HHU Dusseldorf)
- Deutsche Krebshilfe (NHS)
- Medical Research Council [G0601308] Funding Source: researchfish
- MRC [G0601308] Funding Source: UKRI
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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 agaroseembedded bisulfite treatment, which allows investigating DNA methylation of multiple loci via a multiplex PCR (multiplexedscAEBS). 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 multiplexedscAEBS to 159 single CTCs from 11 patients with metastatic breast and six with metastatic castration-resistant prostate cancer, isolated via CellSearch (EpCAM(pos)/CKpos/CD45(neg)/DAPI(pos)) and subsequent FACS sorting. In contrast to CD45(pos) 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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