A spatially resolved single-cell atlas identifies blood-brain barrier integrity as a central regulator of macrophages and microglia localization and functional states in gliomas.

Given that glioma-associated macrophages and microglia (GAMs) critically shape tumor evolution, we aimed to define their spatial distribution and functional reprogramming in relation to MRI-defined BBB integrity. Twelve patients underwent MRI-guided sampling, yielding 65 biopsies from 40 enhancing and 25 non-enhancing areas. Single-cell RNA-seq generated a high-resolution cellular atlas, and we analyzed cellular composition, transcriptional programs, macrophage polarization, and transcriptional subtype distribution relative to anatomical region and enhancement status. We profiled 5445 cells and identified 9 major cell populations. Macrophages were enriched in tumoral and enhancing regions, whereas microglia predominated in peritumoral and non-enhancing niches. Tumor cells in enhancing regions exhibited a growth-dominant anabolic state, while non-enhancing and peritumoral tumor cells retained neuronal development and synapse organization programs. GAMs displayed region-specific functional polarization: peritumoral and non-enhancing macrophages were enriched for neural development and tissue remodeling programs, whereas tumoral and enhancing macrophages activated chemotactic, inflammatory, and immunoregulatory pathways. Integration with transcriptional subtypes demonstrated spatial coupling of Mesenchymal/Classical programs with enhancing regions and Proneural features with non-enhancing regions, underscoring substantial intra-tumor spatial mosaicism. MRI-defined BBB permeability spatially organizes a glioma ecosystem where tumor metabolism and macrophage states are tightly coupled, refining understanding of the immune microenvironment and rationale for spatially informed immunotherapy.