Genome-Wide DNA Methylation Enhances Stemness in the Mechanical Selection of Tumor-Repopulating Cells

Background: DNA methylation plays essential roles in tumor occurrence and stemness maintenance. Tumor-repopulating cells (TRCs) are cancer stem cell (CSC)-like cells with highly tumorigenic and self-renewing abilities, which were selected from tumor cells in soft three-dimensional (3D) fibrin gels. Methods: Here, we presented a genome-wide map of methylated cytosines for time-series samples in TRC selection, in a 3D culture using whole-genome bisulfite sequencing (WGBS). Results: A comparative analysis revealed that the methylation degrees of many differentially methylated genes (DMGs) were increased by the mechanical environment and changed from 2D rigid to 3D soft. DMGs were significantly enriched in stemness-related terms. In 1-day, TRCs had the highest non-CG methylation rate indicating its strong stemness. We found that genes with continuously increasing or decreasing methylation like CREB5/ADAMTS6/LMX1A may also affect the TRC screening process. Furthermore, results showed that stage-specific/common CSCs markers were biased toward changing their methylation in non-CG (CHG and CHH, where H corresponds to A, T, or C) methylation and enriched in gene body region. Conclusions: WGBS provides DNA methylome in TRC screening. It was confirmed that non-CG DNA methylation plays an important role in TRC selection, which indicates that it is more sensitive to mechanical microenvironments and affects TRCs by regulating the expression of stemness genes in tumor cells.