Integrated genomic and transcriptomic profiling of glioblastoma reveals ecDNA-driven heterogeneity and microenvironmental reprogramming.

Glioblastoma (GB) is an aggressive brain tumor with limited treatment options, making it crucial to integrate genomic and transcriptomic profiling to identify genetic alterations and cellular functional states. We perform short- and long-read whole-genome sequencing (WGS) and single-nucleus RNA sequencing on 42 Chinese GB patients to characterize the intra- and extrachromosomal mutation landscape, cell-type composition, and pathway activity. Our analysis identifies amplified oncogenes, including EGFR, MYC, CDK4, PDGFRA, and PPARGC1A, localized on extrachromosomal DNA (ecDNA). Notably, EGFR ecDNA harbors distinct structures that correlate with patient survival and exhibit a unique DNA methylation pattern that influences gene expression, driving malignant cell differentiation toward MES-like and AC-like subtypes. Specifically, EGFR ecDNA stabilizes tumor-associated macrophages in a hypoxia- and metabolism-driven state, reinforcing a reciprocal AREG-EGFR signaling loop with mesenchymal-like tumor cells. Together, these findings uncover a mechanistic link between ecDNA architecture, transcriptional subtypes, and microenvironmental remodeling, offering critical insights for advancing precision oncology in GB.