Osteopontin-induced M2 macrophage polarization via PI3K/AKT/mTOR signaling in melanoma and its inhibition by andrographolide

Melanoma progression is critically shaped by the tumor microenvironment, where tumor- associated macrophages predominantly acquire an immunosuppressive M2-like phenotype. Osteopontin (OPN), a secreted glycoprotein overexpressed in melanoma, has been implicated in immune modulation; however, its role in macrophage polarization and therapeutic targeting remains inadequately defined. In this study, we investigated OPN- driven macrophage polarization and evaluated the effect of andrographolide as a potential inhibitor of this process.

THP-1–derived macrophages were treated with recombinant OPN (rOPN, 15 nM), A375 melanoma-conditioned medium (CM), and OPN-neutralized A375 CM, with or without andrographolide (20 μM). Macrophage polarization was assessed by flow cytometry using single and dual staining of M2 markers CD163 and CD206. rOPN and A375 CM significantly increased CD163 and CD206 expression, including a distinct CD163⁺CD206⁺ double- positive macrophage population. Neutralization of OPN in A375 CM markedly reduced M2 marker expression, confirming OPN dependency. Notably, andrographolide treatment significantly suppressed CD163 and CD206 expression both individually and in the dual-positive population across all treatment conditions.

Mechanistic investigations revealed that OPN-induced macrophage polarization was mediated through activation of the PI3K/AKT/mTOR signaling pathway, as evidenced by increased expression of PI3K p85, phosphorylated AKT, and phosphorylated mTOR. Andrographolide treatment effectively attenuated activation of this pathway, correlating with reduced M2 marker expression.

Collectively, these findings identify OPN as a key mediator of melanoma-induced macrophage polarization via the PI3K/AKT/mTOR axis and demonstrate that andrographolide effectively inhibits this process. Targeting OPN-driven macrophage reprogramming using andrographolide or related compounds may represent a promising therapeutic strategy to remodel the melanoma tumor microenvironment and limit disease progression.