(P 304) A COMPARATIVE ANALYSIS OF METABOLIC FLEXIBILITY IN OSTEOSARCOMA CELLS

Objective: Naturally occurring osteosarcoma (OS) in pet dogs and experimental mouse models provide a translational platform for human OS research. To better understand cellular metabolic changes during OS initiation and progression, we conducted a study to investigate the metabolic signature of canine and murine OS cells, specifically their utilization of glucose versus alternative fuels in central carbon metabolism. We hypothesized that metabolic flexibility post-sarcomagenesis would reveal unique, targetable signatures in the OS landscape.

Methods: We mapped the requirements of primary and metastatic canine, murine and human OS cell lines in comparison with species-specific control mesenchymal stromal cell (MSC) lines. Measurements of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were obtained with Seahorse respirometry. Cell viability was evaluated after supplementation with glucose, glutamine, pyruvate, or lactate. Canine and murine cells were grown on biomimetic hydrogel cultures with matrix stiffness recapitulative of lung and bone microenvironments, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) of select metabolites was performed.

Results: Canine and murine metastatic OS cells were highly energetic (high OCR/high ECAR) compared to primary OS cell lines and MSCs (low OCR/high ECAR), and preferred glutamine more than glucose or pyruvate, but also consumed the major stereoisomer L-lactate, suggesting a high degree of metabolic flexibility. From LC-MS/MS, the carbon intermediates lactate and malate were heavily co-upregulated in metastatic OS lines, especially in soft lung-like environments (2 kPa), while succinate was comparatively decreased. Citrate release was significantly upregulated in OS cell lines. We later tested the effects of lactate transport inhibition with monocarboxylate transporters MCT1 +/- MCT4 inhibitors AZD3965 and syrosingopine in combination with carboplatin chemotherapy, and found that syrosingopine, but not AZD3965 significantly reduced cell viability.

Conclusion: Collectively, our results suggest that coordinate regulation of lactate metabolism in OS fulfills a metabolic gap in respiration to assist in aerobic cancer survival, and that targeted metabolic inhibitors may synergise with chemotherapy. Furthermore, metastatic OS cells appear to be more energetic and energetically flexible than primary OS cells and mesenchymal cells. Together, these and future investigations into the metabolic landscape of OS may lead to future clinical applications for OS patients.