Single-cell DNA methylome analysis of circulating tumor cells

Objective: Previous investigations of circulating tumor cells (CTCs) have mainly focused on their genomic or transcriptomic features, leaving their epigenetic landscape relatively uncharacterized. Here, we investigated the genome-wide DNA methylome of CTCs with a view to understanding the epigenetic regulatory mechanisms underlying cancer metastasis. Methods: We evaluated single-cell DNA methylome and copy number alteration (CNA) in 196 single cells, including 107 CTCs collected from 17 cancer patients covering six different cancer types. Our single-cell bisulfite sequencing (scBS-seq) covered on average 11.78% of all CpG dinucleotides and accurately deduced the CNA patterns at 500 kb resolution. Results: We report distinct subclonal structures and different evolutionary histories of CTCs inferred from CNA and DNA methylation profiles. Furthermore, we demonstrate potential tumor origin classification based on the tissue-specific DNA methylation profiles of CTCs. Conclusions: Our work provides a comprehensive survey of genome-wide DNA methylome in single CTCs and reveals 5-methylcytosine (5-mC) heterogeneity in CTCs, addressing the potential epigenetic regulatory mechanisms underlying cancer metastasis and facilitating the future clinical application of CTCs.