Integration of biomimetic organoid-on-chip and 2D models advances the mechanistic understanding of STEAP3-mediated regulation in intestinal viral infection

Traditional investigations of viral infection mechanisms have predominantly relied on two-dimensional (2D) cell culture models, which lack the structural organization and physiological relevance of native tissues. These systems often fail to capture key features such as spatial cell–cell interactions, tissue-specific heterogeneity, and microenvironmental complexity that govern virus–host dynamics in vivo. To address these limitations, we established an integrative platform that combines the strengths of both 2D and three-dimensional (3D) models to investigate the role of six-transmembrane epithelial antigen of prostate 3 (STEAP3), a membrane ferrireductase, in regulating viral infection in human intestinal epithelium. The 2D system enabled high-resolution mechanistic interrogation of STEAP3-dependent viral entry processes, while the patient-derived 3D colon organoid model recapitulated the architectural and cellular complexity of intestinal tissue, allowing spatially resolved assessment of infection patterns. Using this integrated approach, we found that STEAP3 knockdown significantly increased viral entry and infection, particularly in enterocytes and enteroendocrine cells. To further mimic physiological conditions in human body, we developed a vascularized organoid-on-chip model, in which increased viral signals were observed within vascular lumens upon STEAP3 depletion, suggesting a protective role of STEAP3 in limiting viral dissemination. For efficient and multiplexed screening of antiviral mechanisms, we also fabricated a 3D-printed 27-well chip tailored for organoid culture. By leveraging the complementary advantages of both 2D and 3D systems, this study demonstrates the power of integrated biomimetic modeling platforms to investigate antiviral defense mechanisms and underscores their value for engineering physiologically relevant infection models.