Modeling Phenotypic Heterogeneity Towards Evolutionary Inspired Osteosarcoma Therapy
Osteosarcoma is the most common bone sarcoma in children and young adults. While chemotherapy is universally delivered, benefit from chemotherapy is limited to roughly half of localized patients.  Increasingly, intratumoral heterogeneity is being appreciated as a source of therapeutic resistance.  In this study we developed and evaluated an in vitro model of osteosarcoma heterogeneity, characterizing phenotype (growth in varying environments, sensitivity to chemotherapy) and genotype. We present the genotypic and phenotypic characterization of an osteosarcoma cell line panel with a focus on coculture of the most phenotypically divergent cell lines, 143B and SAOS2. The extent of phenotypic heterogeneity can be altered with relatively modest environmental (pH, glutamine) or chemical perturbations.  We demonstrate that in nutrient rich in vitro culture conditions 143B outcompetes SAOS2, but with selection pressure from nutrient variations or conventional chemotherapy, SAOS2 growth can be favored in spheroids.  Importantly, perturbations that affect the faster growing cell line have only a modest effect on final spheroid size when the simplest heterogeneity state (a two-cell line coculture) is evaluated, and thus the only evaluated therapies to eliminate the spheroids were by switching therapies from a first strike to a second strike. This extensively characterized, widely available system can be modeled and scaled to allow for improved strategies to anticipate resistance in osteosarcoma due to phenotypic heterogeneity. Overall design: In this study we evaluated 6 established osteosarcoma cell lines (U2OS, MG-63, OS252, SAOS2, 143B, MNNG).  U2OS, MG-63, SAOS2, 143B, and MNNG were obtained from ATCC. OS252 was a gift from Richard Gorlick at MD Anderson{Wang, 2022 #5303}. The identities of the lines were confirmed using STR analysis. Cells were maintained in DMEM+15% FBS and antibiotics. Basic growth characteristics and kinetics including doubling time and log-phase were determined at several starting cell concentrations. Cell lines at 80% confluence were subjected to RNA-sequencing for gene expression analysis.