MMP3-PKM2 axis controls loading of the glycolytic system in extracellular vesicles and macrophage polarity in oral cancer

Extracellular vesicles (EVs) mediate intercellular communication and microenvironmental remodeling in oral squamous cell carcinoma (OSCC). Our proteomic analysis revealed that OSCC EVs contain the complete suite of anaerobic glycolytic enzymes, with pyruvate kinase M2 (PKM2) and GAPDH significantly enriched. Moreover, matrix metalloproteinase-3 (MMP-3) regulated EV secretion and glycolytic cargo loading. MMP3 knockout in tumoroids markedly reduced EV release and impaired the loading of glycolytic enzymes, including PKM2. Co-culture assays showed that cancer EVs transfer PKM2 and MMP3 to recipient macrophages in an MMP3-dependent manner. EV subtypes exerted distinct effects on macrophage polarity: MMP3-loaded large EVs (LEVs) selectively induced immunosuppressive M2 macrophage polarization (elevated MRC1, decreased NOS2), whereas MMP3-loaded small EVs (SEVs) modulated both M1 and M2 phenotypes. Clinical database analysis confirmed that high PKM expression correlates with poor overall survival in head and neck cancer. Pharmacological PKM2 inhibition with shikonin suppressed EV release in a concentration-dependent manner and reduced OSCC cell viability in 2D and 3D cultures. Together, the MMP3–PKM2 axis controls EV glycolytic cargo loading and dictates subtype-dependent macrophage polarization.