Modeling glioblastoma in 3D hydrogels enables investigation of Zika virus targeting and immune modulation in oncolytic virotherapy

Glioblastoma (GBM) is a highly aggressive brain tumor with limited therapeutic options. Zika virus (ZIKV) has emerged as a potential oncolytic agent due to its selective tropism for tumor cells. To investigate how the extracellular matrix (ECM) modulates ZIKV oncolytic efficacy, we developed a three-dimensional (3D) GBM model using photocrosslinked Gelatin methacryloyl (GelMA) hydrogel supplemented with fibronectin. This platform recapitulates the biomechanical and structural features of solid tumors, enabling analysis of ZIKV–cell–ECM interactions. ZIKV efficiently penetrated the 3D hydrogel, replicated within GBM cells, and induced pronounced cytotoxicity. In comparison to 2D cultures, cells in stiff 3D scaffolds exhibited elevated expression of Nestin, GFAP, ABCC1, COL1A1, and COL5A1. Notably, ZIKV infection elicited stronger and more complex immune responses in 3D cultures, with increased pro-inflammatory cytokines and interferon signaling. While 2D monolayers enriched TNF/NF-κB pathways, 3D cultures preferentially activated T cell–related, chemotactic, and JAK–STAT pathways, highlighting the critical influence of spatial context on innate and adaptive immune activation. These results establish the 3D GelMA-based GBM model as a robust preclinical platform and underscore ZIKV's potential as a multifaceted oncolytic virotherapy capable of both direct tumor lysis and immune modulation.