TMIC-84. MULTIREGIONAL ANALYSIS OF GLIOBLASTOMA REVEALS TOPOLOGICAL DIVERGENCE OF CANCER CELLS AND THEIR SURROUNDING MICROENVIRONMENT

Tissue organization plays an important role in tumor development and evolution. Systematic investigation of the differences in the local composition of the tumor microenvironment (TME) in glioblastoma (GBM) in distinct regions of the tumor is needed for identification of traits driving tumor recurrence. Thus, we performed an analysis of three tumors, six spatially distant biopsies each, representing tumor surface, core, and deep margin. GBM cell fluorescence generated by the 5-aminolevulinic acid (5-ALA) metabolic labeling prior to surgery allowed us to enrich for the tumor cell fractions and the cells of the microenvironment, respectively, from each biopsy. Using RNA-seq and scRNA-seq we compared the transcriptomic profiles of GBM and tumor microenvironment (TME) at distinct locations within the tumor. We observed a gradient of GBM subtypes across the tumor regions, with a molecular identity progressively less defined from the tumor core towards the margins. Interestingly, the single-cell based copy number analysis revealed a subset of GBM cells that appeared to evade the 5-ALA labelling. The high prevalence of these 5-ALA-negative GBM cells within the deep tumor margins points to a metabolically distinct subpopulation that could be responsible for local tumor recurrence. The phenotypic gradient of cancer cells from tumor core towards the margins was also mirrored by tumor microenvironment-enriched fractions. Deep margin samples from the profiled tumors were transcriptionally more divergent than biopsies from other regions. Moreover, they were depleted from major populations of TME cells. In situ analysis revealed that TERT promoter mutant cells are not equally distributed across the tumor indicating the coexistence of GBM sub-clones with distinct evolutionary potential. Altogether, our results provide a novel insight into co-evolution of tumor microenvironment and GBM clonal heterogeneity within the context of the tumor tissue.