Dynamic assessment of a humanized bone tumour microenvironment reveals insights into osteosarcoma primary tumour remodelling and lung metastases

Abstract Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents, with poor prognosis linked to metastasis and recurrence. Preclinical models that replicate human disease progression and the tumour microenvironment (TME) are critical for advancing therapies. We developed a novel orthotopic and humanised bone tumour microenvironment model in highly immunocompromised Il2rg and Rag2 double knockout rats. A humanised bone niche (HN) was established by implanting orthotopic humanised bone constructs (ohTEBCs) around the rat femur, followed by SaOS-2-luc cell injection. Longitudinal µCT and bioluminescent imaging tracked HN formation, tumour growth, and metastasis. Histological and immunohistochemical analyses revealed pathological mineralisation, extensive collagen deposition, tumour-specific ECM production, and neovascularisation within primary tumours and lung metastases. This model supports consistent engraftment of SaOS-2-luc tumours with robust metastatic spread, recapitulating key human OS features. It offers a translatable preclinical platform for studying OS progression and testing chemotherapy and surgical interventions.