Simple SummaryMelanoma, one of the deadliest cancers, presents challenges due to the incomplete understanding of the key mechanisms driving its aggressive behavior. High numbers of macrophages in the melanoma microenvironment are associated with poor outcomes. Macrophages are immune cells that act as a double-edged sword, supporting tumor growth but holding the potential to re-activate the anti-tumor response. Previous studies on other cancer models have linked the tumor-supporting roles of tumor-associated macrophages with their ontogeny. In this report, we investigated macrophage infiltration in a melanoma model to understand their origin, evolution, activation profile, and association with the immunosuppressive microenvironment. Unlike other models, we found a common origin in the two main macrophage subsets, which varied over time and differed in their functional and metabolic profiles. These findings shed light on the features and evolution of melanoma-supportive macrophage subsets, which could be used to discover new ways of targeting these cells therapeutically.AbstractTumor-associated macrophages (TAMs) can be widely heterogeneous, based on their ontogeny and function, and driven by the tissue-specific niche. TAMs are highly abundant in the melanoma tumor microenvironment (TME), usually correlating with worse prognoses. However, the understanding of their diversity may be harnessed for therapeutic purposes. Here, we used the clinically relevant YUMM1.7 model to study melanoma TAM origin and dynamics during tumor progression. In i.d. YUMM1.7 tumors, we identified distinct TAM subsets based on F4/80 expression, with the F4/80highfraction increasing over time and displaying a tissue-resident-like phenotype. While skin-resident macrophages showed mixed ontogeny, F4/80+TAM subsets in the melanoma TME originated almost exclusively from bone-marrow precursors. A multiparametric analysis of the macrophage phenotype showed a temporal divergence of the F4/80+TAM subpopulations, which also differed from the skin-resident subsets and their monocytic precursors. Overall, the F4/80+TAMs displayed co-expressions of M1- and M2-like canonical markers, while RNA sequencing showed differential immunosuppressive and metabolic profiles. Gene-set enrichment analysis (GSEA) revealed F4/80highTAMs to rely on oxidative phosphorylation, with increased proliferation and protein secretion, while F4/80lowcells had high pro-inflammatory and intracellular signaling pathways, with lipid and polyamine metabolism. Overall, we provide an in-depth characterization of and compelling evidence for the BM-dependency of melanoma TAMs. Interestingly, the transcriptomic analysis of these BM-derived TAMs matched macrophage subsets with mixed ontogeny, which have been observed in other tumor models. Our findings may serve as a guide for identifying potential ways of targeting specific immunosuppressive TAMs in melanoma.