An epigenetically stabilized regulatory state underlies stable glioblastoma invasion

Glioblastoma is a highly invasive brain tumor in which diffuse infiltration into the surrounding brain parenchyma severely limits therapeutic outcome. While invasion is often described as a plastic and reversible process driven by microenvironmental cues, it remains unclear whether invasive capacity can also be maintained as stable, cell-intrinsic regulatory state. Using a serial in vitro selection approach in patient-derived glioma stem-like cultures, we identified a subpopulation with persistently increased invasive potential that was retained under non-selective conditions. Comparative transcriptomic and epigenomic profiling by RNA sequencing and genome-wide DNA methylation analysis using the EPIC array revealed coordinated transcriptional and DNA methylation remodeling associated with the invasive phenotype. Integration of transcription factor activity inference, motif enrichment analysis, and publicly available ChIP-seq chromatin datasets identified convergent invasion-associated regulatory networks and suggested altered accessibility of methylation-sensitive transcription factor binding sites. Representative methylation changes were further recapitulated across an independent panel of patient-derived glioma stem-like cultures individually classified according to their invasive capacity by transwell invasion assays, supporting the broader relevance of the identified epigenetic alterations. Functional perturbation of the transcription factor networks centered on ATF4 and MYC/MAX in established glioblastoma cell lines and patient-derived glioma stem-like cultures demonstrated their contribution to invasive behavior. Finally, invasion-associated transcriptional signatures and a 41-CpG methylation signature significantly stratified overall survival in TCGA IDH-wildtype glioblastoma patients, highlighting the clinical relevance of invasion-associated epigenetic and transcriptional remodeling. Together, these findings support a model that shapes transcription factor activity and maintains invasion-associated gene expression programs.