Deciphering the interaction between osteosarcoma and mesenchymal stem cells in a 3D bone-mimetic co-culture model

Osteosarcoma (OS) is a highly heterogeneous and aggressive bone malignancy whose complexity is strongly influenced by its Tumor Microenvironment (TME). Within this niche, Mesenchymal Stem Cells (MSCs) play a pivotal role in tumor progression by undergoing phenotypic and functional reprogramming under tumor-derived cues, acquiring Cancer-Associated Fibroblast (CAF)-like features that promote proliferation, invasion, and immune evasion. The Extracellular Matrix (ECM), once regarded as a passive structural element, is now recognized as an active regulator of tumor behavior, acting as a reservoir of signaling molecules and a modulator of cell fate. However, the molecular crosstalk between OS cells, MSCs, and the ECM remains poorly understood, largely due to the limitations of conventional two-dimensional models. In this study, we established a three-dimensional (3D) bone-mimetic model of osteosarcoma (mOS-3D) by co-culturing human OS cells and MSCs within a hydroxyapatite-collagen (MgHA/Coll) scaffold that recapitulates the biochemical and structural features of native bone ECM. This in vitro platform reproduces key aspects of the OS microenvironment, enabling the investigation of tumor-stroma interactions and their impact on stemness, stromal activation, and ECM remodeling. The mOS-3D model provides a physiologically relevant and tunable system for studying the cellular mechanisms driving OS progression and offers a promising preclinical tool to explore therapeutic strategies targeting the TME.