Abstract Glioblastoma multiforme (GBM) is a malignant glioma whose progression is associated with rampant extracellular matrix (ECM) remodeling. We recently found that GBM ECM stiffness predicts reduced survival in human patients. Instead of collagen fibrosis, which is common in many solid tumors, we showed that GBM stiffening involves increased production of extracellular glycoproteins, glycosaminoglycans, and sugar-binding proteins. Using bioinformatics, we revealed that genes of the glycocalyx (transmembrane glycoproteins and their interacting partners) are disproportionately upregulated in GBM relative to lower grade gliomas. Further, these genes are overexpressed within GBM in the mesenchymal (MES) relative to the proneural (PRO) subtype, the former of which is associated with treatment resistance and relapse. Using mouse models of human GBM, we showed that MES tumors are more lethal than PRO, and present with elevated ECM stiffness and mechanical signaling. To test our hypothesis that mechanical signaling can drive the MES phenotype, we engineered PRO GBM cells with constitutively-elevated integrin signaling. Compared to control PRO cells, these undergo a robust MES-like transition, upregulate bulky glycoprotein expression, and result in stiffer and more lethal tumors. This phenotype was reversed by the inhibition of focal adhesion kinase in MES cells. To test whether an enhanced glycocalyx can directly elevate mechanical signaling, we decorated GBM cells with synthetic glycoprotein polymers. Indeed, this resulted in enhanced integrin-focal adhesion signaling and more aggressive tumor progression. The invasive properties and therapy resistance observed in mesenchymal tumor cells are often associated with elevated stem cell-like features. To investigate a link between the glycocalyx, tissue mechanics, and the mesenchymal-stem cell phenotype, we interfered with components of the gylcocalyx or mechanical signaling machinery and found a reduction in stem cell genes and surface proteins, as well as increased sensitivity to chemotherapy. These data support a model in which glycoprotein-mediated tissue stiffening drives GBM aggression through promotion of a mesenchymal phenotype. This abstract is also being presented as Poster A39. Citation Format: J. Matthew Barnes, Elliot C. Woods, Russell O. Bainer, Yekaterina A. Miroshnikova, Kan Lu, Gabriele Bergers, Carolyn Bertozzi, Valerie M. Weaver. Glycoprotein-mediated tissue mechanics regulate glioblastoma aggression. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr PR05.