Tumor-derived sphingosine-1-phosphate shapes angiogenesis in the acidic microenvironment of osteosarcoma via paracrine and autocrine signaling.

INTRODUCTION: Tumor-associated angiogenesis is a critical driver of tumor progression and is frequently characterized by excessive branching and structural disorganization. These abnormalities arise from dynamic interactions between tumor cells and the microenvironment, where metabolic stressors such as hypoxia and extracellular acidosis promote the release of pro-angiogenic factors. Among these, sphingosine-1-phosphate (S1P) has emerged as a key bioactive lipid involved in vascular development. We previously demonstrated that acidosis promotes sphingomyelin turnover and S1P secretion in osteosarcoma cells, enhancing tumor cell survival and migration. In this study, we investigated the role of S1P in osteosarcoma-associated angiogenesis and the contribution of tumor acidosis to this process.

METHODS: Matrigel®-based angiogenesis assays, HUVEC cultures, 3D osteosarcoma spheroids, and microfluidic systems were employed to evaluate endothelial sprouting and tubulogenesis. S1P signaling was pharmacologically inhibited using the FDA-approved S1P modulator FTY720 (Fingolimod). Conditioned media from osteosarcoma spheroids cultured under neutral or acidic conditions were analyzed for their pro-angiogenic activity. Soluble and extracellular vesicle-associated angiogenic mediators were also assessed.

RESULTS: S1P dose-dependently impaired endothelial tubulogenesis while strongly promoting endothelial sprouting. Conditioned media derived from acid-stimulated osteosarcoma spheroids significantly increased endothelial tubule length and branching compared with conditioned media from spheroids maintained at neutral pH. These effects were markedly reduced by FTY720 treatment. Furthermore, tumor-derived S1P activated autocrine signaling in osteosarcoma cells, enhancing the secretion of soluble and extracellular vesicle-associated pro-angiogenic mediators, including bFGF and the TGF-β co-receptor Endoglin (CD105).

DISCUSSION: These findings identify a previously unrecognized acidosis-S1P axis that contributes to angiogenic remodeling in osteosarcoma. Our results highlight the multifaceted role of S1P in regulating endothelial behavior and suggest that targeting S1P signaling may represent a promising strategy to disrupt pathological neoangiogenesis in osteosarcoma.