Abstract 6496: Humanized tridimensional model of osteosarcoma to test immunotherapeutic strategies

Abstract Osteosarcoma (OS) is the most common primary malignant bone cancer, affecting both young adults (ages 10-30) and older adults (over 60). Despite progress in cancer research, survival rates have remained largely unchanged for the past 40 years, particularly for patients with metastatic disease, where 5-year survival rate is about 20%. This lack of progress is partly due to inadequate preclinical models that replicate the complex tumor microenvironment, hindering the development of effective new treatments. Traditional 2D cell culture models fail to replicate the intricate interactions between tumor cells, the extracellular matrix (ECM), and stromal cells, which are key factors in cancer behavior and therapy response. In contrast, three-dimensional (3D) models offer a more accurate representation of the tumor’s architecture, improving predictions of drug efficacy and tumor progression. Moreover, one of the most critical factors influencing OS treatment outcomes is the presence of a subpopulation of cells known as cancer stem cells (CSCs). CSCs contribute to tumor recurrence and metastasis due to their self-renewal and differentiation ability. Furthermore, CSCs are resistant to conventional therapies, such as chemotherapy and radiotherapy. This, coupled with the fact that OS is considered a 'cold tumor, ' makes it particularly challenging to treat with immunotherapy. Our lab is dedicated to developing humanized 3D OS models that closely mimic the tumor microenvironment, integrating OS cells, immune components, and ECM elements such as collagen. These advanced models address the limitations of animal studies by offering a physiologically relevant platform to investigate CSC behavior, drug resistance mechanisms, and tumor-stroma interactions specific to OS. Utilizing patient-derived xenografts (PDX), we generated 3D tumor sarcospheres that recapitulate key features of OS, including high CSC enrichment and resistance to conventional therapies. To characterize these 3D sarcospheres, we employed CellTiter Glo assays and Live/Dead staining to evaluate viability, flow cytometry for stemness markers (CD44, CD117, and CD133), and PCR analysis for CSC-associated genes (OCT4, Nanog, and SOX2). Our findings consistently demonstrate enhanced CSC populations and distinct tumorigenic features in 3D cultures compared to traditional 2D models. Additionally, we incorporated immune components in the 3D model by co-culturing OS PDX cells with immune cells (THP-1), further refining the tumor microenvironment. This integrated approach facilitates a comprehensive understanding of tumor cell-immune cell interactions and provides a robust, scalable platform for testing immunotherapeutic strategies. Ultimately, these humanized 3D models aim to address a significant gap between preclinical studies and clinical applications, thus improving the development of novel and effective therapies for OS. Citation Format: Bobby Dow, Federica Banche-Niclot, Lorenzo Apolloni, Giada Bassi, Jason Yustein, Monica Montesi, Francesca Taraballi. Humanized tridimensional model of osteosarcoma to test immunotherapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6496.