EPCO-15. SPATIAL-TRANSCRIPTOMICS REVEALS UNIQUE MOLECULAR FEATURES OF FLUORESCENCE-SORTED 5-AMINOLEVULINIC ACID+ INFILTRATIVE TUMOR CELLS ASSOCIATED WITH RECURRENCE AND POOR SURVIVAL IN GLIOBLASTOMA

Abstract

Abstract Spatiotemporal heterogeneity of glioblastoma (GBM) originating from genomic and transcriptional variation in spatially distinct sites, may contribute to subtype switching in GBM prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) has enabled the isolation of infiltrative margin tumor cells (5ALA+ cells) from a background of non-neoplastic cells. To falsify the hypothesis that the 5ALA+ subpopulation(s) is defined by a unique cellular state, we have explored the spatial-transcriptomic (ST) landscape to interrogate molecular signatures unique to infiltrating 5ALA+ cells in comparison to GBM core, rim, and invasive margin non-neoplastic cells. ST analysis reveals that GBM molecular subtype plasticity is not restricted to recurrence, but manifests regionally in a cell-type-specific manner. Whilst GBM core and rim are highly enriched with Classical and Proneural subtypes, 5ALA+ cells are uniquely enriched with the Mesenchymal subtype (MES). Upregulation of the wound response pathway in 5ALA+ cells implicate hijacking of the wound healing pathway of neural cells to promote tumor growth. Exon-intron split analysis revealed an upregulation of exonic counts for MES and wound-response genes in 5ALA+ cells, implying that these genes are under active post-transcriptional control. Network analysis suggests that wound response genes, including chemokine CCL2 that recruits regulatory T-cells and monocytic myeloid-derived suppressor cells, are controlled by an IRF8-mediated transcriptional program in 5ALA+ cells. A higher stemness signature both in 5ALA+ cells and non-neoplastic cells of the invasive margin emphasizes the role of this microenvironment in stemness acquisition and defines 5ALA+ cells as a rare sub-population of GBM stem cells. Finally, we establish a link between the unique molecular-signatures of 5ALA+ cells, and poor survival and GBM recurrence. Characterization of the 5ALA+ infiltrative sub-population offers an opportunity to develop more effective GBM treatments and urges focus away from the GBM proliferative core, upon which failed targeted therapies have been predicated.