Development and characterization of 3D osteosarcoma models recapitulating tumor heterogeneity and bone microenvironment

Introduction: Osteosarcoma is the most common primary bone cancer in adolescents and young adults. It is characterized by high heterogeneity and a hostile bone microenvironment shaped by osteoclasts, endothelial cells, and immunosuppressive myeloid populations, underscoring the need for innovative models to evaluate novel therapeutic strategies. Objective: This study aims to develop and characterize novel three-dimensional (3D) culture models that recapitulate the complexity and the microenvironment of osteosarcoma. Methods: Tumor samples from patient-derived xenografts (PDXs) established from patients with osteosarcoma at relapse or treatment failure were processed to generate 3D tumor cultures. Tumor tissues were mechanically and enzymatically dissociated into single-cell suspensions and seeded into round-bottom plates with very low adhesion to promote spontaneous formation of 3D tumoroids. Culture conditions were optimized using medium supplemented with specific growth factors to support cell viability and proliferation. Morphology and growth of the tumoroids were monitored over time. The resulting 3D models were characterized by histology (H&E and specific immunostaining like SATB2, SPP1, SOX9⋯), and by RNA sequencing and Whole exome sequencing (WES) to assess transcriptional stability relative to the parental primary/PDX tumors. In addition, these 3D tumoroid models were used for drug testing to evaluate therapeutic responses and identify potential treatment sensitivities. Results: We established five 3D osteosarcoma models from PDX with an establishment rate of 83%. The tumoroids formed spontaneously under low-adhesion conditions within approximately 7 to 30 days, (depending on the derived sample) and remained viable and morphologically stable during this culture period. Histological analysis revealed that the 3D tumoroids recapitulated key features of the parental PDX tumors and the patient tumors, including cellular heterogeneity, hypoxic regions, and osteoid-like matrix deposition. RNA sequencing and drug testing studies to evaluate therapeutic responses are currently ongoing. Conclusions: Patient-derived 3D osteosarcoma models faithfully preserve tumor heterogeneity and microenvironmental features, providing a robust and scalable system for preclinical drug testing. This approach holds a strong potential to accelerate the development of personalized combination therapies targeting both tumor cells and their supportive bone microenvironment in osteosarcoma.