Melanoma stem cells drive macrophage reprogramming to a hybrid phenotype, modulating melanoma stemness and compromising NK cell-mediated immunity

Melanoma stem cells may contribute to tumor progression not only through intrinsic plasticity, but also by shaping the immune microenvironment. However, their interaction with the monocyte-macrophage compartment remains poorly understood. Melanosphere cultures derived from A375 and WM115 melanoma cell line were used as an in vitro model enriched for stemness-associated features. THP-1 monocytes were differentiated under a standardized low-dose PMA protocol and exposed to stem cell-conditioned media (SC-CM). Monocyte migration, macrophage transcriptomic and secretory profiling, effect of macrophage CM on NK cytotoxicity, melanoma stem cell phenotype and exploratory clinical relevance in the TCGA melanoma cohort were assessed. Both melanosphere models were enriched for stemness-associated programs and secreted high levels of CCL2. SC-CM promoted THP-1 migration, which was reduced by CCR2 inhibition. RNA-seq showed that SC-CM induced a shared but non-canonical macrophage phenotype enriched for inflammatory, interferon-related, pro-angiogenic, and immune-regulatory programs. Conditioned media from SC-educated macrophages impaired NK-cell cytotoxicity through heat-labile soluble mediators and induced cell line-dependent changes in melanoma stemness-associated transcriptional regulators. In TCGA, a high A375-educated macrophage score was associated with shorter overall survival, whereas the WM115-derived score showed a similar but non-significant trend. Melanoma stem cells actively shape the monocyte-macrophage compartment by promoting monocyte recruitment and inducing an inflammatory and immunomodulatory macrophage program with downstream effects on NK-cell function and melanoma cell-state regulation. These findings support a bidirectional melanoma stem cell-macrophage axis that warrants validation in more physiological systems.