Spatial multi-omics defines a shared glioblastoma infiltrative signature at the resection margin [snATAC-seq]

Glioblastoma (GBM) remains an untreatable disease. Understanding of GBM's infiltrative biology at the resection margin is limited, despite causing disease recurrence and progression. To address this, we generated a high-throughput single-nucleus (sn)RNA-seq and snATAC-seq multi-omic dataset from six tumors with distinct genomic drivers and combined it with spatial transcriptomics to characterize the unique molecular phenotype of GBM near the margin. By contrasting GBM-specific biology in matching “Core” vs. “Margin” dissections, we define unique, shared “GBM infiltration” and chromatin accessibility signatures near the margin. We prioritize EGFR as a top differentially expressed and accessible “Margin” marker across GBM subtypes, show its dynamic expression along a core-to-margin infiltration trajectory, and validate its role in migration through CRISPR/Cas9 deletion in two patient-derived models. ChIP-seq studies furthermore corroborate preferential TEAD1 binding at EGFR's accessible regulatory elements. This validated multi-omic dataset enables further studies into tumor and microenvironment biology in the context of residual GBM disease. Overall design: Surgical glioma tissue was collected from 6 patients with confirmed diagnosis of glioblastoma, WHO grade 4 (2016 and 2021 diagnostic criteria), representing diverse genetic backgrounds and all major mutational drivers. Tissue procurement was coordinated with neurosurgery to include contrast-enhancing tumor “Core” (C) and FLAIR-hyperintense infiltrative “Margin” (M) representing the distal extent of gross supratotal resection and having a “normal brain” appearance on fresh tissue inspection. Experienced neuropathologist confirmed the presence of necrosis in “Core” and the presence of infiltrative brain parenchyma without necrosis in “Margin” tissues, during frozen section dissection. Sections were snap-frozen, stored at -80°C for snRNA-seq (n=6).