A vascularized 3D-bioprinted model of the osteosarcoma microenvironment reveals a proliferation-to-invasion switch that confers chemoresistance

The tumor microenvironment (TME) is a major driver of osteosarcoma progression, metastasis, and therapeutic resistance, yet conventional models fail to effectively recapitulate the multicellular interactions and biomechanical cues of the bone niche. In this study, we developed a vascularized 3D-bioprinted osteosarcoma model using a biomimetic hydrogel composed of decellularized extracellular matrix, chondroitin sulfate, and hydroxyapatite. U2OS osteosarcoma cells, human mesenchymal stem cells (HMSCs), and human umbilical vein endothelial cells were co-cultured within the printed constructs. Confocal imaging, RNA sequencing, xenograft validation, and integration with clinical single-cell RNA-sequencing data were used to define TME-driven changes in tumor behavior. The engineered TME induced a proliferation-to-invasion switch characterized by G0/G1 arrest, reduced proliferation activity, and enhanced invasiveness, angiogenic potential, and extracellular-matrix remodeling. Transcriptomic profiling showed downregulation of cell-cycle programs, including E2F and MYC targets, together with activation of TGF-β signaling, epithelial–mesenchymal transition (EMT), and hypoxia-related pathways. Mechanistically, HMSCs were identified as key regulatory cells that promoted migratory programs in tumor cells, partly through CXCL chemokine signaling engaging CXCR2. The TME-induced state also conferred robust chemoresistance to paclitaxel, associated with cell-cycle quiescence and pro-survival signaling, such as NF-κB activation. Critically, the transcriptional signature of these quiescent-invasive cells closely mirrored that of a clinically observed osteosarcoma subpopulation with low proliferation and high EMT activity. This high-fidelity vascularized 3D-bioprinted model recapitulates major TME-dependent features of osteosarcoma and provides a biologically relevant platform for studying tumor plasticity and evaluating therapies targeting microenvironment-driven treatment resistance.