Spatial Multi-omics Defines a Shared Tumor Infiltrative Signature at the Resection Margin in High-Grade Gliomas

Abstract Despite genomic heterogeneity, most high-grade gliomas (HGG), including IDH-wildtype 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 (sn)RNA-seq and snATAC-seq multi-omic dataset from matching “core” and “margin” dissections in four distinct grade 4 HGG (EGFR-amplified, NF1-mutant, FGFR3-TACC3 fused, IDH1-mutant; n= 36,811 snRNA-seq and 30,705 snATAC-seq nuclei after filtering) and combined it with new spatial transcriptomics data from two additional HGG (EGFR-amplified, CDK4-amplifed) to evaluate “core-to-margin” transition. Computational analyses included functional enrichment, comparison to prior HGG datasets, differential analyses in core vs. 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 ChIP-seq studies suggested a role for TEAD1 as a transcriptional regulator of EGFR at the margin. This multi-omic resource will enable further studies into residual disease biology of tumors and the microenvironment at the infiltrative margin.