Analysis of DNA methylation in single circulating tumor cells

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 metastas...

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Bibliographic Details
Published in:Oncogene 2017-06, Vol.36 (23), p.3223-3231
Main Authors: Pixberg, C F, Raba, K, Müller, F, Behrens, B, Honisch, E, Niederacher, D, Neubauer, H, Fehm, T, Goering, W, Schulz, W A, Flohr, P, Boysen, G, Lambros, M, De Bono, J S, Knoefel, W T, Sproll, C, Stoecklein, N H, Neves, R P L
Format: Article
Language:English
Subjects:
Antigens, CD
Biomarkers, Tumor - genetics
Bisulfite
Breast
Breast Neoplasms - genetics
Breast Neoplasms - pathology
Cadherins - genetics
Cancer
Cancer cells
Cancer metastasis
Castration
Deoxyribonucleic acid
DNA
DNA Methylation
E-cadherin
Epigenesis, Genetic
Epigenetics
Epithelial-Mesenchymal Transition
Female
Flow cytometry
Gene expression
Gene regulation
Genes
Genetic aspects
Health aspects
Humans
Leukocytes
Male
Mesenchyme
Metastases
Metastasis
MicroRNAs - genetics
miRNA
Molecular modelling
Neoplastic Cells, Circulating - pathology
Prostate
Prostate cancer
Prostatic Neoplasms, Castration-Resistant - genetics
Prostatic Neoplasms, Castration-Resistant - pathology
Risk factors
Single-Cell Analysis - methods
Surgery
Tumor cells
Tumor Cells, Cultured
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Summary: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 (EpCAM /CK /CD45 /DAPI ) and subsequent FACS sorting. In contrast to CD45 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.
ISSN:0950-9232
1476-5594
DOI:10.1038/onc.2016.480