Abstract Paediatric-type diffuse high-grade gliomas (pDHGG) are a group of molecularly and clinically defined tumor entities of the central nervous system. The investigation of the spatial organization of pDHGG tissues may provide new insights to decipher their heterogeneous nature and identify vulnerabilities in their aggressive behaviour. Here we exploit Imaging Mass Cytometry (IMC), a spatial biology approach enabling multiplex tissue phenotyping at single-cell resolution. We used 16 total formalin-fixed paraffin-embedded (FFPE) tissues from 4 patients including 1 Diffuse hemispheric glioma H3G34-mutant (DHG-H3G34), 1 Diffuse pediatric-type high-grade glioma H3-wildtype and IDH-wildtype (DHGG-WT) and 2 Diffuse midline glioma H3K27-altered (DMG-H3K27, 1 H3.3K27M and 1 H3.1K27M). Classical pathology assessment and DNA methylation profile were also performed. For IMC staining, 2 metal-tag custom-designed antibody panels (tumour and immune) of 31 antibodies were used. The panels include antibodies for differentiated and stem-cell markers, extracellular matrices, immune markers and H3K27M and H3.3G34R variants. An IMC analysis pipeline was developed: images are extracted in tiff format from the proprietary MCD files produced by Hyperion instrument, filtered to remove hot pixels via top-hat and OpenCV in Python, segmented using steinbock and analysed using the IMC Data Analysis toolbox in R, using docker. A total of 209 mm2 of tissue were analysed. IMC analysis showed that DHG-H3G34 displays a higher heterogeneity in terms of cell phenotypes compared to the other tumor entities. Moreover, immune infiltrate of T, B cells, NK and monocytes was observed in the DHG-H3G34 and DHGG-WT tissues, while the DMG-H3K27 had low/no immune infiltrate with the H3.3K27M showing higher PDL-1 expression. Analysis of longitudinal and multi-region samples highlighted trajectories of tumor evolution and identification of extracellular matrix niches in tumor core versus the infiltrative front. Our study provides novel insights into pDHGG complex biology by investigating their tissue architecture, spatial organization and cell-cell interactions.

HGG-47. SPATIAL SINGLE-CELL PROTEOMIC ANALYSIS MAPS THE DISTINCT PEDIATRIC-TYPE DIFFUSE HIGH-GRADE GLIOMA TUMOR ENTITIES AND DEFINES THEIR TUMOR AND IMMUNE MICROENVIRONMENTS