Abstract Background Pediatric high grade glioma (PHGG) comprises several aggressive tumor subtypes with five-year survival rates of less than 2% to 20%. PHGG is spatially heterogeneous, posing challenges in designing therapies. Intratumoral interactions among tumor and immune cells have been inferred from single-cell RNA sequencing data and verified using protein staining and RNA in-situ hybridization but have not been comprehensively investigated at the single-cell transcriptional level. We investigated spatial heterogeneity and intratumoral interactions in PHGG using spatial transcriptomics to probe expression of 480 genes relevant to pediatric brain tumors at sub-cellular resolution. Methods We acquired spatial RNA expression data from six PHGG patient samples. We designed a custom 480-gene expression panel focused on genes differentially expressed in pediatric brain tumors and common immune cell markers. Frozen samples were embedded in OCT and cut into 10 mm thick sections. RNA expression data and immunofluorescence images for cell segmentation were acquired using the Xenium Analyzer (10X Genomics). Results were analyzed to identify cell types, cell-cell interactions and proximity using Xenium Explorer and Xenium Ranger as well as customized work flows in R. Results At a multi-cellular scale, clumps of specific tumor cell types (e.g., oligodendrocytic or astrocytic) occurred. Vascularization and areas of hypoxia were readily identifiable. Infiltrating and tumor resident macrophages tended to occur in closer proximity to astrocytic or mesenchymal cell groups than to oligodendrocytic groups. Spatial juxtaposition of TMEM119+macrophages and tumor cells suggest macrophage infiltration may play multiple roles in PHGG tumorigenesis, including oligodendrocytic tumor cell transitions to a mesenchymal phenotype and tumorigenic behavior of astrocytic tumor cells. Conclusions Sub-cellular spatial transcriptomics provides a detailed view of in situ RNA expression in PHGG. Our data suggest cellular interactions within the tumor microenvironment that are significant for tumorigenesis and likely important to consider for therapeutic development.
DeSisto et al. (Fri,) studied this question.