Osteocyte-like differentiation of osteosarcoma by inorganic phosphate

Osteosarcoma (OS) cells that deviate from the normal osteogenic differentiation pathway from mesenchymal stem cells (MSCs) to less migratory osteocytes are in an undifferentiated and highly malignant state. β-glycerophosphate (β-gp) is commonly used to induce the osteogenic differentiation of MSCs, inorganic phosphate (Pi) is also widely used to promote MSC differentiation into osteocytes. Recently, OS cells were found to differentiate into an osteocyte-like state by culturing in osteocyte differentiation medium containing β-gp, dexamethasone, and ascorbate. However, whether Pi can induce the osteogenic differentiation of OS cells and its underlying mechanisms remain unclear. In this study, we evaluated the ability of two types of Pi (i.e., disodium phosphate [Na₂HPO₄] and monosodium phosphate [NaH₂PO₄]) to promote the osteogenic differentiation of OS cells. Culturing OS cells in Pi-supplemented medium resulted in increased osteogenic marker gene expression and calcium deposition and reduced cell motility. Notably, Na₂HPO₄ exhibited particularly strong differentiation-inducing effects. Furthermore, our data suggest that WNT5b, a key factor of the noncanonical Wnt signaling pathway, is involved in the Na₂HPO₄-induced osteogenic differentiation of OS cells. These findings suggest that above 3 mM of Na₂HPO₄ function as an inducer of osteocyte-like differentiation in OS cells and that targeting this pathway may offer new therapeutic strategies to suppress OS metastasis.