Project Summary Despite standard treatment with surgery, radiation and chemotherapy, glioblastomas (GBMs) inevitably recur with fatal consequences. GBMs diffusely infiltrate the brain, making complete removal by surgical resection impossible. The mixtures of neoplastic and non-neoplastic cells that remain in the infiltrated brain after surgical debulking form the biological context for both adjuvant therapeutic intervention and recurrence. Furthermore, radiation therapy, while beneficial for extending survival, may compromise the efficacy of targeted therapy and induce treatment resistance. The goal of this study is to identify the molecular signatures in gliomas responsible for recurrence and treatment resistance. Four innovative features will be incorporated: 1) a methodology for radiographically -localized sampling of GBM and surrounding brain tissue; 2) new methods to acquire and analyze Large-Scale Single Cell RNA-Seq data from mri-localized biopsies 3) a Ribotag mouse model of GBM to experimentally track cell type-specific alterations associated with progression and recurrence. In this proposal, tumor heterogeneity is approached from the perspective of CNS lineages, specifically looking at the mixture of cell-types and the alterations in cell phenotypes that populate the tumor and surrounding infiltrated brain tissue. Aim1 will determine the cellular and molecular composition of GBM at infiltrative margins to demonstrate that glioma cells and reactive glia in the infiltrating margins differ from those in the tumor core. We will also determine changes in cellular and molecular composition that occur between initial GBM resection and post-treatment tumor recurrence. In Aim 2 we will use the RiboTag mouse model of proneural glioma to determine if radiation induces mesenchymal transformation with a resultant resistance to targeted therapy. By understanding the cellular and molecular composition of GBM and infiltrated brain tissue and comparing the patterns of cell type-specific alterations that occur during glioma progression and following radiation therapy, this proposal will reveal putative targets for glioma treatment and determine how specific glioma phenotypes respond to current therapies.