Melanoma stem cells drive macrophage reprogramming to a hybrid phenotype, modulating melanoma stemness and...

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 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.

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.

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.