Characterization of DNA Methylation in Circulating Tumor Cells

Abstract

Epigenetics contributes to molecular mechanisms leading to tumor cell transformation and systemic progression of cancer. However, the dynamics of epigenetic remodeling during metastasis remains unexplored. In this context, circulating tumor cells (CTCs) might enable a direct insight into epigenetic mechanisms relevant for metastasis by providing direct access to systemic cancer. CTCs can be used as prognostic markers in cancer patients and are regarded as potential metastatic precursor cells. However, despite substantial technical progress, the detection and molecular characterization of CTCs remain challenging, in particular the analysis of DNA methylation. As recent studies have started to address the epigenetic state of CTCs, we discuss here the potential of such investigations to elucidate mechanisms of metastasis and to develop tumor biomarkers.

Keywords: CTC; DNA methylation; circulating tumor cells; epigenetics; molecular analyses.

Figure 1

Bisulfite conversion of DNA and methods used for high resolution mapping of methylcytosines. (Upper panel) DNA conversion upon treatment with bisulfite. Bisulfite reacts with non-methylated cytosine residues converting them to uracils by initial sulfonation, hydrolytic deamination and final desulfonation. Methylcytosines essentially do not react with bisulfite. After conversion, uracil residues indicate non-methylated cytosines, while cytosines indicate methylcytosines in the original DNA sequence; (Lower panel) Converted DNA can be analyzed by multiple techniques discriminating between cytosines and uracils to detect or precisely map methylcytosines. These include clone sequencing (Clone Seq.), pyrosequencing (Pyro Seq.), MALDI-TOF mass spectroscopy (Mass Spec.), bead arrays, bisulfite padlock probes (BSPP), solution hybrid selection (SHS), reduced-representation bisulfite sequencing (RRBS), and whole genome sequencing (MethylC-Seq). These methods differ in the size of the target sequence, the number of CpGs covered, as well as in their cost.

Figure 2

Potential clinical and research applications of epigenetic analyses of CTCs. Standard blood samples or diagnostic leukapheresis products obtained from patients with cancer can be used for CTC enrichment, detection and isolation by different workflows. Isolated CTCs may be analyzed for DNA methylation by different technologies. The information collected from these analyses can be used to address basic molecular mechanisms of metastasis and, in the future, may also prove to be of value for clinical applications.