Dissecting TGF-β-induced glioblastoma invasion with engineered hyaluronic acid hydrogels

Glioma stem cells (GSCs) contribute to rapid cellular invasion in glioblastoma (GBM). Transforming growth factor-β (TGF-β) has been strongly implicated in supporting key GSC functions, including stemness, immunosuppression, and resistance. Although TGF-β is well-known as a driver of cancer invasion, how TGF-β supports the invasion of GSCs is not well understood. Progress in understanding mechanisms of TGF-β-driven invasion in GSC-derived tumors has been limited by an absence of three-dimensional (3D) culture systems that support TGF-β-stimulated invasion. Here, we show that 3D hyaluronic acid (HA) matrices can address this need. We perform bioinformatic analysis of human glioma datasets, which reveals progressive enrichment of TGF-β-related gene expression with increasingly aggressive glioma grade and GBM subtype. We then experimentally screen the invasion of a panel of human GSC spheroids through a set of 3D matrix systems, including collagen I, Matrigel, and HA, and find that only HA recapitulates TGF-β-induced invasion. We then show that GSCs differ in their ability to invade HA in a way that can be predicted from TGF-β receptor 2 expression and SMAD2 phosphorylation. GSC spheroid invasion depends strongly on the presence of RGD peptides on the HA backbone but is surprisingly independent of matrix metalloprotease degradability. Finally, we demonstrate that TGF-β stimulates invasion through SMAD-dependent signaling, consistent with recent observations that TGF-β/SMAD signals drive tumor microtube formation and invasion. Our work supports further development of HA as a matrix platform for dissecting contributions of TGF-β and other cytokines to GBM invasion and screening of cytokine-dependent invasion in human tumors.