Matrix type influences embedded patient-derived osteosarcoma organoid invasion and response to treatment.

Osteosarcoma remains a lethal paediatric malignancy when it metastasises to lung, yet therapeutic progress has stalled for decades, in part because preclinical models poorly capture patient specific tumor microenvironments. Here, patient- derived osteosarcoma (PDOS) cells are assembled into uniform, matrix-free organoids and then embedded into photocrosslinkable extracellular matrices with independently tuneable stiffness and composition: collagen-rich gelatin methacryloyl (GelMA) or basement membrane protein-enriched lung decellularized extracellular matrix (dECM) methacryloyl (LungMA), each tuneable to soft (approximately 1 kPa) or stiff (approximately 4 kPa) conditions. Embedding converts otherwise compact organoids into invasive, expanding structures and increases resistance to doxorubicin relative to both 2D cultures and matrix-free organoids. Matrix identity influenced early invasion kinetics and treatment response, with LungMA promoting more aggressive invasion shortly after embedding and greater chemoresistance than GelMA, particularly under stiffer conditions. Image-based segmentation of core and invasive compartments revealed a critical divergence between metabolic viability readouts and functional invasion inhibition under chemotherapy, exposing limitations of conventional screening endpoints. These findings establish stiffness-controlled, tissue-derived extracellular matrix (ECM) organoid systems as a tuneable platform to interrogate microenvironment-driven osteosarcoma aggressiveness and to advance patient-specific assessment of therapeutic vulnerability in metastatic niche-like contexts.