W62 - Osteosarcoma Tumorigenesis is Affected by Macrophage Behavior in 3D Engineered Bone Marrow Model

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents, yet treatment has remained unchanged for 4 decades and offers less than a 25% 5-year survival rate for those with metastatic disease. This underscores a critical lack of understanding of OS progression and necessitates the study of this disease in a novel system. Here, we adapt an engineered bone marrow (eBM) construct for use as an in vitro model for OS research. We first optimized eBM synthesis by testing the influence of the anatomical implantation site on the quality of generated tissue. Compared to constructs formed subcutaneously, eBM formed subfascially better recapitulated native mouse bone marrow. Next, homotypic cultures of highly metastatic K7M2 and less metastatic K12 OS cell lines were loaded into the eBM. eBM was also loaded with heterotypic cultures comprised of each cell line and macrophages at an 85:15 ratio and cultured under 21% and 5% O₂. Flow cytometry for OS cell number and macrophage polarization revealed that both K7M2s and K12s under heterotypic culture exhibited significantly decreased cell number only under 21% O₂. However, M1 and M2 macrophage behavior were not consistent between K7M2s and K12s, suggesting that the inflammatory status of the immune microenvironment does not dictate OS behavior as previous studies often conclude. The eBM in this project will advance the treatment of patients afflicted by OS by mimicking the complex environment in which OS arises, thereby surpassing other in vitro models that fail to account for these key parameters.