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

Introduction: 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. Methods: Melanosphere cultures derived from A375 and WM115 melanoma cell lines 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. Monocyte migration, macrophage transcriptomic and secretory profiling, the effect of macrophage- conditioned medium on NK cytotoxicity and melanoma stem cell phenotype and exploratory clinical relevance in a melanoma patients’ cohort were assessed. Results: Both melanosphere cell models were enriched for stemness-associated programs. Stem cell-conditioned media promoted THP-1 monocyte migration, which was reduced by CCR2 inhibition. RNA-seq showed that stem cell- conditioned media induced a shared but non-canonical macrophage phenotype enriched for inflammatory, interferon-related, pro-angiogenic, and immune-regulatory programs. Conditioned media from stem cell-educated macrophages impaired NK-cell cytotoxicity through heat-labile soluble mediators and induced cell line-dependent changes in melanoma stemness- associated transcriptional regulators. In an independent melanoma cohort, a shared stem cell-educated macrophage score was associated with inflammatory macrophage programs and with shorter overall survival. Conclusions: Melanoma stem cells actively shape the monocyte-macrophage compartment by promoting monocyte recruitment and inducing an inflammatory and immunomodulatory macrophage program coupled 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.