Abstract
Despite genomic heterogeneity, most high-grade gliomas (HGG), including IDH wild-type glioblastoma, display diffusely infiltrative growth, which impedes complete surgical resection and leads to inevitable recurrence. Understanding of HGG biology comes predominantly from studies using resected "core" tissue. Paradoxically, chemoradiation targets residual disease at the resection margin, which remains poorly defined. To address this, we generated a high-throughput single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) multiomic dataset from matching "core" and "margin" dissections in four distinct grade 4 HGG (36,811 snRNA-seq and 30,705 snATAC-seq nuclei after filtering from EGFR amplified, NF1 mutant, FGFR3-TACC3 fused, and IDH1 mutant HGG) and combined it with new spatial transcriptomics data from two additional HGG (EGFR amplified and CDK4 amplifed) to evaluate "core-to-margin" transition. Computational analyses included functional enrichment, comparison with prior HGG datasets, differential analyses in core versus margin cell types or regions of interest for genes, chromatin accessibility peaks, cell-cell interactions, transcription factor motif activity and associated regulon targets, and reconstruction of core-to-margin transition using RNA velocity and pseudotime. Contrasting tumor-specific biology in matching core and margin dissections defined a unique, shared "glioma infiltration" signature near the margin. EGFR was prioritized as a top differentially expressed and accessible tumor margin marker across HGG subtypes that showed dynamic expression along a core-to-margin infiltration trajectory. CRISPR/Cas9-mediated deletion of EGFR in two patient-derived models validated its role in migration, and combined snATAC-seq with chromatin immunoprecipitation sequencing studies suggested a role for TEAD1 as a transcriptional regulator of EGFR at the margin. This multiomic resource will enable further studies into residual disease biology of tumors and the microenvironment at the infiltrative margin. Significance: Characterization of infiltrating tumor-margin cells across human high-grade gliomas advances the understanding of residual disease biology at the surgical margin, identifies mechanisms of therapy resistance and recurrence, and elucidates targetable molecular features.