TMIC-71. SPATIAL ANALYSIS OF GENETIC HETEROGENEITY AND TUMOR MICROENVIRONMENT INTERACTIONS IN GLIOBLASTOMA

Abstract

Abstract Genetic variation can be considered a driver of somatic evolution in glioblastoma (GBM); however, the tumor microenvironment (TME) is a key contributor to the selection process and evolutionary potential of cancer cells. Although diverse ecosystems exist in GBM which impact tumor cell fitness and characterize an aggressive pathophysiology, little is known about how genetically distinct subpopulations of GBM cells interact with the surrounding TME. Therefore, we sought to investigate how the varying clonal genotypes of GBM cells are influenced by different types of selective pressures within the TME. Our imaging-based strategy is centered around Specific-to-Allele PCR Florescence In Situ Hybridization (STAR-FISH), an in situ PCR based probe hybridization method for detection of point mutations and copy number variations at the single-cell level. Using STAR-FISH to illuminate the spatial distribution of clones with mutations in the TERT promoter, we integrated multicolor immunofluorescent staining, DNA-FISH, reflectance imaging, and computational cellular analysis methods to achieve highly-multiplexed single cell spatial profiling of intact GBM specimens. With this strategy we are able to simultaneously evaluate tumor tissues for the presence of hypoxic, angiogenic, and inflammatory niches, extracellular matrix organization, nuclear morphology, mutational status, and several oncogenic amplifications on the same slide and location. We analyzed 20 matched primary and recurrent GBM samples, imaging several distinct regions of the same tumor. Our initial results have revealed interesting and potentially novel distributions of subclones residing within specific microenvironments. These results include TERT mutation positive clones enriched in areas possessing more structured extracellular matrix, as well as unique arrangements of infiltrating immune cells in the angiogenic tumor niche. This research underscores the importance of the TME in selecting for certain phenotypes that characterize somatic evolution, highlighting novel genetic and spatial dependencies which sustain heterogeneity in GBM.