3D bioprinted silk fibroin- and hyaluronic acid-based biomimetic extracellular matrices with tunable stiffness regulate glioblastoma invasion and macrophage polarization.

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by rapid proliferation and extensive infiltration, severely limiting the efficacy of surgical resection. Biomimetic in vitro models that recapitulate the tumor extracellular matrix (ECM), particularly the hyaluronic acid (HA)-rich ECM and stiffness-associated remodeling, are therefore essential for studying GBM progression and developing physiologically relevant therapeutic strategies. In this study, silk glycidyl methacrylate (Silk-GMA) and methacrylated hyaluronic acid (HAMA) were used to construct a hybrid hydrogel system. Silk-GMA served as the structural backbone to provide mechanical stability and printability, while HAMA was incorporated to mimic the HA-rich brain ECM and modulate matrix stiffness. By varying the composition ratios, bioinks with tunable mechanical properties were developed and applied to 3D bioprinting of heterogeneous GBM-macrophage spheroids, thereby establishing a biomimetic brain tumor microenvironment model. The results demonstrated that increasing HAMA content increased hydrogel stiffness and significantly influenced both tumor and immune cell behavior. GBM cells exhibited enhanced migratory activity in the presence of macrophages, particularly within stiffer hydrogel constructs. Moreover, higher HAMA concentrations were associated with increased expression of M2-associated macrophage markers, suggesting polarization toward a tumor-associated macrophage (TAM)-like phenotype. These cellular responses were accompanied by enhanced tumor spreading within the 3D constructs, indicating that HA-associated ECM stiffening promotes a tumor-supportive microenvironment. Overall, this study presents a 3D bioprinted GBM model with tunable ECM stiffness that recapitulates key features of glioblastoma progression. The platform provides a physiologically relevant system for investigating tumor-immune interactions and evaluating microenvironment-targeted therapeutic strategies.