Abstract: MPI R01- Hambardzumyan and Becher Pediatric high-grade gliomas (pHGG) account for the most cancer-related deaths in children and have a median survival of 12-15 months. One promising avenue of research is developing novel therapies targeting the properties of non-neoplastic cell types within the tumor, such as myeloid cells, including tumor-associated macrophages (TAMs) and neutrophils. Much is known about TAMs in adult high-grade gliomas. However, very little is known about TAMs and neutrophils in pHGGs. pHGGs more commonly arise in infratentorial locations like the brainstem, beyond the cerebral hemispheres as seen in adults. pHGGs also harbor distinct histone mutations not found in adults. This raises the question of whether pHGGs possess a distinct constituency of TAMs due to their unique genetic landscapes and locations. Using human pHGG tissue samples and NanoString RNA sequencing, we demonstrate brainstem/midline pHGGs (DMG) and murine diffuse intrinsic pontine glioma (DIPG) possess higher inflammatory scores and increased neutrophil scores compared to hemispheric pHGGs, which are associated with shorter patient survival. When examining only human hemispheric pHGGs, our results revealed two patient subsets with high and low inflammatory scores. Patients with a high inflammatory score had significantly shorter survival times compared to those with low inflammatory scores. We also show that human pHGGs possess high infiltration of IBA1+ TAMs, which are the most abundant non-neoplastic component of the pHGG tumor microenvironment (TME). Our preliminary data utilizing mouse models to recapitulate pHGG in newborn immunocompetent mice combined with various histone mutations in biologically relevant locations demonstrate murine tumors are strikingly similar to their human counterparts with regard to their inflammatory immune profile and myeloid cell infiltration. Using PDGFB and PDGFA as driver mutations to generate hemispheric pHGG in mice, we were able to recapitulate human pHGGs with high and low inflammatory scores. We showed that in comparison to PDGFA-driven tumors, PDGFB tumors have a higher inflammatory score, increased infiltration of monocytes from the blood, and shorter survival time of tumor-bearing mice. We identify CCL3 as a potential key chemokine for CCR1/CCR5-positive monocytes and CXCL1 for CXCR2-positive neutrophil recruitment in pHGG. Together, these results provide strong rationale to extend our studies to understand how specific histone mutations and tumor locations influence myeloid cell infiltration and how these cells promote pHGG growth. The outcome of these studies will: (i) reveal the molecular and functional diversity of the myeloid compartment of pHGG; (ii) determine how distinct myeloid subsets and myeloid-specific genes influence tumor growth and the TME; (iii) provide insight into how myeloid subsets influence, and are affected by driver mutations, pediatric-specific histone mutations, and tumor location...