Macrophage and osteosarcoma cell crosstalk is dependent on oxygen tension and 3D culture

Osteosarcoma (OS), the most common form of primary bone cancer in young adults, has had no improvements in clinical outcomes in 50 years. This highlights a critical need to advance mechanistic understanding of OS to further therapeutic discovery, which will only be possible with accurate models of the disease. Compared to traditional monolayer studies and preclinical models, in vitro models that better replicate the three-dimensional (3D) bone marrow microenvironment will facilitate methodical investigations of the events and factors that drive OS progression. Herein, we use fibrin-alginate interpenetrating network (FA IPN) hydrogels to model the hematological bone marrow environment. We interrogated the effects of oxygen tension, 3D culture, and macrophage phenotype on OS behavior and specifically examine the immunomodulatory crosstalk between OS and macrophages. We observe that OS is more sensitive to oxygen tension when cultured in 3D. Specifically, both highly and less metastatic OS exhibit decreased changes in DNA content over time in 3D, but then demonstrate diverging behaviors in heterotypic culture with macrophages. OS response to macrophages differs as a function of metastatic potential, where highly metastatic OS shows increased immunosuppression that varies with oxygen tension but relies on direct coculture conditions. To our knowledge, this is among the first work to report the effects of 3D culture on the interplay between OS and macrophages in a coculture microenvironment. Together, these data introduce FA IPNs as a promising platform for cancer research and emphasize the importance of novel models for the mechanistic study of OS. © 2024 The Authors