Development and optimisation of 3d in vitro models to study the role of the microenvironment in osteosarcoma

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. Despite advances in surgery and chemotherapy, prognosis remains poor for patients with metastatic or recurrent disease. Increasing evidence highlights the critical role of the tumor microenvironment (TME), particularly human mesenchymal stromal cells (hMSCs), in promoting OS progression, metastasis, and therapy resistance. In this context, three distinct three-dimensional (3D) culture models were optimized to address specific biological questions and to better elucidate the influence of the microenvironment on osteosarcoma behaviour. Osteosarcoma cell lines were first characterized for their ability to form spheroids. Saos-2 cells did not generate compact aggregates; however, when co-cultured with hMSCs, stable and viable 3D structures were formed, underscoring the contribution of stromal cells to tissue organization and tumor cohesion, likely through structural support and paracrine signaling. The established 3D co-culture system was subsequently employed to investigate tumor–stromal interactions, with particular focus on vascular organization and extracellular matrix features. Complementary tube formation assay and Chorioallantoic Membrane (CAM) assay, combined with histological analysis and Synchrotron X-ray phase-contrast microtomography, demonstrated that the presence of hMSCs enhances structural complexity and promotes the formation of more organized vascular networks. The study further explored osteogenic differentiation within complex 3D environments, including hydrogel-based and bioprinted systems designed to mimic key aspects of the native extracellular matrix. These models allowed the evaluation of how differentiation influences tumor cell activity, proliferation, and invasion potential. Finally, the integration of a bicompartmental microfluidic setup enabled the establishment of spatially controlled co-cultures, providing a versatile platform to study tumor–stromal interactions. Taken together, these results show that combining different 3D culture models captures fundamental aspects of the osteosarcoma microenvironment, providing a platform for further mechanistic investigations and novel preclinical and translational applications.