title: Analysis of DNA methylation in single circulating tumor cells.
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 CD45poswhite 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.
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: C F
authors.initials: CF
authors.lastname: Pixberg
authors.affiliation: Institute for Transplantation Diagnostics and Cell Therapeutics, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Raba
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Müller
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Behrens
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Honisch
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Niederacher
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Neubauer
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Fehm
authors.affiliation: Department of Urology, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Goering
authors.affiliation: Department of Urology, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: W A
authors.initials: WA
authors.lastname: Schulz
authors.affiliation: Division of Cancer Therapeutics and Division of Clinical Studies, The Institute of Cancer Research, London, UK.|Drug Development Unit, The Royal Marsden NHS Foundation Trust, London, UK.
authors.forename: P
authors.initials: P
authors.lastname: Flohr
authors.affiliation: Division of Cancer Therapeutics and Division of Clinical Studies, The Institute of Cancer Research, London, UK.|Drug Development Unit, The Royal Marsden NHS Foundation Trust, London, UK.
authors.forename: G
authors.initials: G
authors.lastname: Boysen
authors.affiliation: Division of Cancer Therapeutics and Division of Clinical Studies, The Institute of Cancer Research, London, UK.|Drug Development Unit, The Royal Marsden NHS Foundation Trust, London, UK.
authors.forename: M
authors.initials: M
authors.lastname: Lambros
authors.affiliation: Division of Cancer Therapeutics and Division of Clinical Studies, The Institute of Cancer Research, London, UK.|Drug Development Unit, The Royal Marsden NHS Foundation Trust, London, UK.
authors.forename: J S
authors.initials: JS
authors.lastname: De Bono
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: W T
authors.initials: WT
authors.lastname: Knoefel
authors.affiliation: Department of Oral, Maxillo- and Plastic Facial Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Sproll
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: N H
authors.initials: NH
authors.lastname: Stoecklein
authors.affiliation: Department of General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Düsseldorf, Germany.
authors.forename: R P L
authors.initials: RPL
authors.lastname: Neves
mesh_terms: D015703:Antigens, CD; D014408:Biomarkers, Tumor / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D015820:Cadherins / Q000235:genetics; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000235:genetics; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D064129:Prostatic Neoplasms, Castration-Resistant / Q000235:genetics* / Q000473:pathology; D059010:Single-Cell Analysis / Q000379:methods*; D014407:Tumor Cells, Cultured
chemical_list: D015703:Antigens, CD; D014408:Biomarkers, Tumor; C513384:CDH1 protein, human; D015820:Cadherins; D035683:MicroRNAs