400 Modeling osteosarcoma progression and therapeutic response using engineered induced pluripotent stem cell-derived 3D organoids

Objectives/Goals: Our objective is to develop and validate a human iPSC-derived 3D osteosarcoma (3D-iOSA) model that replicates chromosomal instability (CIN), metastasis, and chemoresistance. The model will serve as a platform to evaluate chemotherapy and immune cell therapeutics, offering improved predictive fidelity compared to 2D and spheroid systems. Methods/Study Population: Human iPSCs were engineered by CRISPR/Cas9-mediated knock out of TP53 and RB1, differentiated to mesenchymal stem cells with overexpression of cMYC ± hRAS. Cells were embedded in hyaluronan and osteogenically differentiated to generate 3D organoids (3D-iOSA), with osteoblastic commitment confirmed by RUNX2 and BGLAP immunohistochemistry (IHC). Tumorigenic and metastatic potential was assessed via subcutaneous implantation into immunodeficient mice, followed by histopathology, IHC, RNA sequencing, and 5X whole genome sequencing (WGS). MAP (methotrexate, doxorubicin, and cisplatin) chemotherapy response and chimeric antigen receptor (CAR)-immune cell cytotoxicity were compared across 2D and 3D systems to evaluate chemoresistance, immune evasion, and translational fidelity of the 3D-iOSA model. Results/Anticipated Results: 3D-iOSA exhibited osteogenic lineage commitment with RUNX2 and BGLAP expression and disorganized, anaplastic morphology correlated with mutational burden. Tumors displayed greater histologic complexity than 2D-derived tumors, with pulmonary metastases in 50% of primary and 70% of secondary recipients. WGS revealed recurrent copy number alterations and subclonal variation, indicating ongoing CIN. MAP half-maximal inhibitory concentration values were 106-260× higher in 3D-iOSA organoids than 2D cultures, increasing with genetic complexity. B7H3 CAR-iPSC NK-cell and B7H3/HER2 dual-CAR γδ T-cell screening showed system-dependent efficacy, with reduced cytotoxicity in 3D-iOSA organoids versus 2D and spheroid cultures, highlighting 3D-iOSA superior predictive power for immunotherapy evaluation. Discussion/Significance of Impact: 3D-iOSA is the first human OSA model to replicate CIN-driven evolution, chemoresistance, and metastasis. It distinguishes differential immune therapy responses with greater predictive power than 2D or spheroid models, providing a scalable, physiologically relevant platform to accelerate targeted therapy development.