TMIC-92. Longitudinal assessment of tumor immune microenvironment changes during DMG progression using flow cytometry

Abstract Diffuse GK1 Midline Glioma (DMG) accounts for 80% of brain stem tumors in children and adolescents. Standard combinatorial therapies, radiotherapy, immune checkpoint inhibitors, and chemotherapy have not proved effective against DMG, and surgical resections are usually difficult considering their location and diffuse state. With no effective treatment, children with DMG survive only 9-11 months post-diagnosis. Unique composition and biology of the DMG tumor immune microenvironment (TIME) and its changes over time pose a significant challenge. Thus, its understanding is vital for designing new therapies to effectively treat DMG. To recapitulate the TIME of DMG, we injected murine tumor cells generated from an IUE model of DMG (H3.3K27M, DNp53, PDGFRAD842V) into C57BL/6 mice to study longitudinal changes. At days 7, 14, 18, 22, and 28 post-tumor implantation, animals were euthanized and whole brains were isolated (3 mice at each time point). CD45+ cells from the whole brain were magnetically sorted to enrich for only immune cells and TIME cellular compositions were assessed using flow cytometry. We used 17 parameters to identify myeloid and lymphoid subtypes, as well as microglia. By day 14, there was a marked enrichment of the myeloid population and a reduction in microglia compared to baseline and day 7 (when tumor burden was low). Both lymphoid and myeloid populations showed a steady increase over time. Additionally, we observed a shift within the myeloid compartment, with a decrease in monocytes and an increase in macrophages. Notably, normoxic and inflammatory macrophages increased, while hypoxic macrophages declined, consistent with the creation of a more inflammatory milieu supporting tumor growth/progression. Together, here we report an optimized flow-based technique for the longitudinal assessment of the DMG TIME. We show that the DMG TIME is highly dynamic, and we have established a baseline for determining additional changes induced by DMG-directed therapies. Specifically, we will use this technique to next interrogate the changes in the DMG TIME following standard-of-care radiotherapy.