ID #379 Radiotherapy accelerates myeloid-driven immunosuppression in diffuse midline gliomas through spatial reprogramming of the tumor immune microenvironment

Abstract

Diffuse midline gliomas (DMG) are universally fatal pediatric brain tumors, for which no effective treatment exists. These tumors are known for their “cold” non-inflammatory tumor-immune microenvironment (TIME), which is thought to be a major hurdle for immunotherapy efficacy. To investigate how this environment evolves and how it is shaped by therapy, we allografted immunocompetent mice with Histone 3.3 Lysine 27 (H3.3K27M) and 3.1 Lysine 27 (H3.1K27M) mutant murine DMG tumors generated by intra-uterine electroporation and performed longitudinal single-cell analyses of the untreated TIME. Using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), we mapped the RNA and protein expression profiles of tumor-associated immune cells in DMG and healthy pontine tissue, with a dominant focus on the myeloid compartment. Differential expression and pathway enrichment analyses revealed that tumor-associated microglia progressively adopt anti-inflammatory programs and are replaced over time by infiltrating, highly immunosuppressive macrophages. We confirmed these dynamics in single-cell sequencing data from DMG patients at diagnosis and late-stage disease, where we noticed that radiotherapy was associated with a more pronounced immunosuppressive myeloid phenotype. To mechanistically investigate this observation, we treated immunocompetent DMG-bearing mice with image-guided precision radiotherapy (SARRP) and applied high-dimensional spatial proteomics using the MACSima platform with an 80-plex antibody panel. Single-cell spatial mapping uncovered distinct microglial and macrophage niches with location-dependent interaction patterns, and demonstrated that radiotherapy profoundly reshapes this architecture by accelerating macrophage accumulation in perivascular and invasive tumor regions, coinciding with enhanced tumor dissemination and reinforcement of an immunosuppressive TIME. Together, these data indicate that radiotherapy is not immunologically neutral in DMG but actively drives spatial and functional reprogramming of the myeloid DMG compartment towards enhanced immunosuppression. This underscores that optimizing the timing of radiotherapy relative to immunotherapies, such as CAR-T cell therapy, may be essential to achieve durable disease control in these devastating pediatric tumors.