A375 melanoma-derived lactate controls A375 melanoma phenotypes by inducing macrophage M2 polarization via TCA cycle and TGF-β signaling.

Abstract

Introduction: Macrophage phenotypes have been linked to progression and prognosis of cutaneous melanoma. However, the association between Warburg effect in A375 melanoma and macrophages polarization, as well as the underlying mechanisms, remains less well documented.

Objective: The present study aimed to investigate the effect of lactate derived from A375 melanoma on macrophage polarization, melanoma phenotype responses and the underlying mechanisms.

Methods: Flow cytometry was performed to evaluate the expression of M1 and M2 markers, cell cycle and apoptosis. Levels of transforming growth factor β (TGF-β) and tumor necrosis factor α (TNF-α) were determined with enzyme-linked immunosorbent assay (ELISA) kit. Proliferation and invasion were assessed by CCK8 and transwell assays, respectively. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were analyzed using an XF96 extracellular flux analyzer. Protein expressions were determined by Western blotting.

Results: Our results revealed that melanoma A375 conditioned medium (A375-CM) induced peripheral blood mononuclear cells (PBMCs) to polarize toward anti-inflammatory M2 macrophages. M2 markers CD206 and ARG1 expression increased, as did TGF-β secretion. Conversely, M1 marker CD68 expression decreased. Furthermore, hypoxia promoted macrophage M2 polarization induced by A375-CM. Elevated lactate level in PIG1-conditioned medium (PIG1-CM) induced M2 polarization, whereas the lactate transport inhibitor AZD3965 suppressed this effect in PBMCs cultured with A375-CM. Additionally, lactate derived from melanoma regulated M1/M2 polarization by the tricarboxylic acid (TCA) cycle instead of glycolysis. Significantly, polarized macrophages altered melanoma phenotypes including proliferation, clone formation, cell cycle, apoptosis, migration and invasion via TCA cycle and TGF-β.

Conclusion: Our data collectively demonstrate that lactate derived from melanoma facilitates polarization of M2 macrophages, which subsequently leads to modifications in melanoma phenotypes via TCA cycle and TGF-β signaling.