Abstract BACKGROUND Diffuse midline glioma (DMG) is a lethal pediatric tumor, and new treatment strategies are urgently needed. To design therapies that can cross the blood-tumor barrier, we designed a pilot study to investigate protein expression on DMG tumor vasculature using spatial profiling, with attention to targetable surface proteins. METHODS We identified 5 patients and 4 controls consented to institutional tissue banking protocols with adequate autopsy tissue from multiple anatomical regions within the brainstem. Portions of formalin-fixed, paraffin-embedded tissue were arranged into tissue microarrays profiled using the nanoString GeoMX platform. Briefly, a multiplexed cocktail of 51 primary antibodies tagged with UV-photocleavable oligonucleotide barcodes and fluorescent morphology markers were applied to tissues and 10-14 regions of interest (ROIs) were segmented by vessel area for each patient. ROIs were processed using UV light and barcodes were quantified using ex situ digital counting. RESULTS Five patients were included in the pilot study with average age 9.8 years (range 8-13), and overall survival was 9.5 months (range 3-18 months). Three tumors were classified as DMG, H3K27-mutant, WHO grade 4; one as high grade glioma without identified histone alteration but with recurrent DMG drivers (ACVR1 and PIK3CA mutations) and one as infiltrating atypical glioma with high proliferative rate and no DMG driver mutations. Controls ranged from 4-15 years and did not have pathological changes in the brain at death. Protein abundance was compared between cases and control and within cases where tumor-adjacent normal tissue was identified. Several proteins were significantly more abundant on tumor vasculature compared to non-tumor adjacent or control brainstem vessels. Validation studies in primary tissue are ongoing. CONCLUSIONS Digital spatial profiling is a promising technology for investigating tumor vasculature and is feasible in autopsy specimens. Work is ongoing to validate candidate biomarkers and expand the cohort to develop a vascular atlas for DMG.