Abstract
DNA methylation plays a pivotal role in regulating cellular processes, and altered DNA methylation pattern is a general hallmark of cancer. However, DNA methylome in circulating tumor cells (CTCs) is still a mystery due to the lack of proper analytical techniques. We introduced an efficient workflow, LCM–µWGBS, which can efficiently profile the DNA methylation of microdissected CTC samples. LCM–µWGBS combines the laser capture microdissection (LCM)-based CTC capture method and whole-genome bisulfite sequencing in very small CTC population (µWGBS) to gain insight into the DNA methylation landscape of CTCs. We herein profiled the DNA methylome of CTCs from lung cancer patients. Deriving from a comprehensive analysis of CTC methylome, a unique “CTC DNA methylation signature” that is distinct from primary lung cancer tissues was identified. Further analysis showed that promoter hypermethylation of epithelial genes is a hallmark of stable epithelial–mesenchymal transition process. Moreover, it has been suggested that CTCs are endowed with a stemness-related feature during dissemination and metastasis. This work constitutes a unique DNA methylation analysis of CTCs at single base-pair resolution, which might facilitate to propose noninvasive CTC DNA methylation biomarkers contributing to clinical diagnosis.
Highlights
Circulating tumor cells (CTCs) are cells that have shed into the bloodstream from primary or metastatic tumors andThese authors contributed : Lei Zhao, Xiaohong Wu
The enriched CTC samples were encapsulated in a hydrogel matrix, which made them to be isolated by laser capture microdissection (LCM) and compatible with downstream analysis. μWGBS is an established DNA methylation analysis method of a small number of cells based on the post-bisulfite adapter tagging (PBAT) assay
With the attempt to robustly and efficiently identify the whole-genome DNA methylation profiles of CTCs, we have developed a LCM–μWGBS workflow by combining LCM-based CTC capture and μWGBS method (Fig. 1)
Summary
Circulating tumor cells (CTCs) are cells that have shed into the bloodstream from primary or metastatic tumors and. With the emerging of high-throughput technologies, such as comprehensive DNA methylation microarrays and genome-wide bisulfite sequencing (WGBS), large amount of DNA methylation profiling data have been generated [12, 17]. These data provided us great importance to improve the prediction of cancer diagnosis and prognosis. CTC DNA methylation data were generated by using WGBS method from lung cancer CTCs. Our work comprehensively addressed the epigenetic state of CTCs, and provided an epigenetic picture to elucidate mechanisms during dissemination and metastasis and to develop tumor biomarkers
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