PROJECT SUMMARY Glioblastoma (GBM) is a deadly disease with no effective therapy and is associated with one of the worst 5-year survival rates of all human cancers. Current treatments, which include aggressive surgical care, radiotherapy, and chemotherapy, are ineffective in part because of the highly adaptable nature of GBM, which facilitates therapy evasion and a persistent evolution of disease. Recent work has revealed the crucial role of the tumor microenvironment (TME) in regulating tumor cell plasticity, leading to efforts to create targeted TME-dependent treatments to combat GBM progression. However, due to the complex nature of the TME and a limited understanding of the relevant promoters of disease embedded within this milieu, progress in identifying promising therapeutic targets remains challenging. Within in the TME, biophysical cues including matrix stiffness and composition, have emerged as significant regulators of GBM cell aggressiveness. Focused investigations on the biophysical mechanisms regulating GBM cell aggressiveness represent a novel approach to develop effective GBM TME targeting therapies. The emergence of biophysical alterations in the TME remains poorly understood with the process of TME tissue remodeling being an underappreciated aspect of GBM progression. Current investigations indicate that alterations in hyaluronic acid (HA) secretion and digestion may affect tumor progression through two major processes: 1) shape the biophysical characteristics of the evolving TME, and 2) induce mesenchymal shifts that leads to increased dysregulations in ECM secretion, increased invasiveness, and increased proliferation. This proposal seeks to clarify the contributions and mechanisms of ECM remodeling by quantifying ECM alterations in tumor core and rim and by dissecting the mechanotransductive mechanisms underlying HA-dependent tumorigenic control. To do this we will test the following hypotheses: 1) Core ECM remodeling leads to elevated HA content that increases tissue stiffening due to increased HAS2 and HYAL2 activity in mesenchymal tumor regions, 2) Increased mechanical stiffness and HA presentation will synergistically promote mesenchymal transitions that will lead to increased HASes and decreased HYALs, leading to accumulation of HA with varying molecular weights and subsequent biophysical alterations, and 3) Increased HA stiffness will increase CD44 mechanotransduction through CD44 clustering and ERM activity, leading to upregulated pro-mesenchymal signaling via STAT3-NF-ΚB and LOX-Twist1 and LMW HA secretion. The proposed studies will be the first to systematically dissect the ECM components of various patient matched regions of GBM tumors and study their contributions to biophysical characteristics, mesenchymal transitions, and HA-CD44 mechanotransduction.