Tmic-15. genetic and spatial heterogeneity of pediatric high-grade gliomas confer unique inflammatory microenvironments

Abstract Significant progress has been made molecularly defining pediatric high-grade gliomas (pHGG), including diffuse midline gliomas (DMG), yet little progress has been made with respect to delineating the inflammatory microenvironment. We utilize molecularly defined human samples and immunocompetent genetic mouse models to study how tumor location and genetic driver mutations influence the tumor microenvironment (TME). We report human DMGs have a significant enrichment of Elane+ neutrophils compared to their hemispheric counterparts. We validate this utilizing the RCAS-Tva mouse model, which histologically and genetically recapitulates human pHGGs. Using this model we demonstrate each distinct pHGG/DMG entity confers unique transcriptional identities as made evident by NanoString RNA expression profiles and whole-tumor single-cell RNA sequencing. H3.3WT DMGs and H3.3K27M DMGs cluster together and have high expression of inflammatory genes such as Ptprc, Trem2, Lag3, and Cd274 while H3.3WT and H3.3G34R hemispheric tumors, and H3.1K27M DMGs cluster together with low expression of these genes. Several genes were found to significantly correlate with median survival in human bulk RNA sequencing data including IL13RA2. Flow cytometry and immunohistochemistry demonstrate H3WT DMGs are enriched for monocytes and lymphocytes while H3.3K27M DMGs are enriched for microglia. Genetic perturbations were made to prevent TAM or neutrophil infiltration, including targeting Ccl3, Cxcl1, and Ccl8/12. Only Ccl8/12 knockout resulted in significant extension of survival in H3K27M DMGs, which was met with an increase in CD4+ and CD8+ T-cells and decreased neutrophil infiltration. CD4+ T-cell depletion and anti-PD1 therapy was performed to further study the role of lymphocyte infiltration in DMGs. Lastly, CCR1/CCR5 inhibitors were utilized to abrogate TAM infiltration in DMGs, resulting in decreased microglia infiltration and significant survival extension comparable to radiation therapy. Together, this work provides the foundation for developing or improving immunotherapies designed at specific subgroups of pHGG and DMGs, such as CAR-T-cell, oncolytic viral therapy, and checkpoint blockade.