Single-cell/spatial integration reveals an MES2-like glioblastoma program orchestrated by immune communication and...

Glioblastoma (GBM) exhibits marked plasticity and intense microenvironmental crosstalk. We aimed to delineate mesenchymal programs with spatial resolution, clinical relevance, and mechanistic anchors.

We integrated single-cell RNA-seq, bulk transcriptomes, and Visium spatial data. After rigorous QC and Harmony integration, we annotated 12 cell states using canonical markers, decoupler-based ORA, and AUCell. Tumor boundaries were defined by inferCNV/CopyKAT; developmental potential by CytoTRACE2 and PHATE. Post-translational modification (PTM) axes were scored from curated gene sets. A cell type-aware GNN linked bulk expression to a patient-similarity graph for survival modeling and gene-level hazard attribution. Network convergence combined bulk WGCNA (TCGA/CGGA), single-cell hdWGCNA, BayesPrism deconvolution, and external GEO validation. Ligand-receptor (LR) signaling was inferred with LIANA+, embedded in a signed causal network, and mapped spatially. ARRDC3 expression was assessed in GBM tissues; U251 gain- and loss-of-function assays evaluated proliferation and migration.

We resolved major GBM states, including two mesenchymal programs (MES1-like, MES2-like). CNV-high regions marked malignant cores, and CytoTRACE2 identified high-potency niches within MES2-like and Proliferation states along non-linear trajectories. PTM landscapes segregated by state; S-nitrosylation, glycosylation, and lactylation were enriched in mesenchymal programs. A GNN risk score stratified overall survival in TCGA (n=157) and generalized to CGGA-325 (n=85) and CGGA-693 (n=140). MES2-like abundance remained an independent adverse predictor (HR = 2.31; 95% CI, 1.04-5.10). MES2-high tumors upregulated EMT, TNFα/NF-κB, JAK/STAT, hypoxia, angiogenesis, and glycolysis; S-nitrosylation associated with increased hazard. Cross-modal convergence defined a conservative MES2 core enriched for ECM remodeling, collagen modification, focal adhesion, and TGF-β regulation. LR analysis prioritized a TAM-to-MES2 axis (e.g., GRN-TNFRSF1A, ADAM9/10/17-ITGB1, TGFB1-ITGB1/EGFR) converging on a CEBPD-centered module. Spatial mapping localized MES2 hotspots within CNV-defined territories and revealed a TNFRSF1A-CEBPD-ARRDC3 focus at an infiltrative rim. ARRDC3 was upregulated in GBM tissues; in U251 cells, knockdown promoted and overexpression suppressed proliferation and migration, indicating context-dependent roles.

MES2-like GBM is an ECM-driven, stress-adapted state with strong prognostic impact. We nominate CEBPD and TNFRSF1A/ITGB1 as actionable nodes and identify ARRDC3 as a spatially restricted effector with context-dependent tumor-modulatory functions warranting therapeutic exploration.