TMIC-08. Multiplex immunofluorescent imaging and VINE-seq demonstrates the temporospatial complexity of the blood-tumor-barrier in pediatric-type diffuse high grade glioma

Abstract Pediatric-type high-grade gliomas (HGGs) are aggressive brain tumors characterized by rapid growth, infiltrative behavior, and significant therapy resistance. One major challenge in treating HGGs involves the interplay of tumor cells to their microenvironment; specifically the blood-tumor barrier (BTB). Our studies combined histologic and transcriptomic evaluations of immunodeficient rodents intracortically injected with H3K27-altered and H3 WT glioma cells. Multiplex immunohistochemical staining provided temporospatial analysis of known cellular markers involved in BBB formation, including pericytes, endothelial tight-junction proteins, and myeloid cell markers. Regions of interest included the tumor, peritumor (+250µM), and distant (>250µM) areas. Analysis was conducted with the use of QuPath and cell segmentation performed using Mesmer; with expression proportions quantified for each cell/protein marker. Utilization of vessel isolation and nuclei extraction for sc-RNAseq (VINE-seq) allowed us to specifically explore the molecular landscape of vasculature within the tumor microenvironment. All rodent models were untreated and evaluated early to late post-intracranial injection (D20-50). Preliminary results in H3-K27-altered glioma models reveal elevated tight junctions within distal regions compared to the tumor (p<0.0001); demonstrating an intact BTB furthest from tumor bulk. Within the tumor, high Iba1+ macrophages (46%) and low neuronal (1%) expression was present (p<0.0001), while the intratumoral vasculature demonstrated the highest localization of glioma stem cells, and PDGFRβ+ during late disease progression (p<0.0001). VINE-seq identified unique cell-clusters in the tumor bearing vs. contralateral hemisphere at middle and late timepoints (D40-50). Ongoing VINE-seq studies will identify single cell expression changes both regionally and temporally with correlation to multiplex staining. These findings of blood-tumor barrier expression profiles among glioma cells offer a glimpse of changes for both the cellular and molecular landscape with both region and time specificity. Our research underscores the importance of understanding these interactions related to restrictions of CNS treatment and pediatric-type HGG progression.