Garcia-Martinez, E.; Nieto-Valle, A.; Barrio-Alonso, C.; Lopez-Navarro, B.; Aviles-Izquierdo, J. A.; Parra-Blanco, V.; Gutierrez-Seijo, A.; Sanchez-Mateos, P.; Samaniego Garcia, R.

Tumor-associated macrophages (TAMs) represent a major immune population within the tumor microenvironment, influencing cancer progression and immune responses. Our group previously identified a subset of pro-inflammatory TAMs associated with poor prognosis in human melanoma. GM-CSF, a myeloid-priming cytokine, exhibits context-dependent effects on tumor growth and, despite its clinical use, its role in human melanoma remains undefined. In this study, we demonstrate that GM-CSF is significantly enriched in primary cutaneous melanoma samples from patients who subsequently developed metastasis, compared with non-metastasizing ones. By quantifying GM-CSF expression in TAMs and tumor cells (TCs), we found that high levels, in both TAMs and TCs, correlated with reduced disease-free and overall survival (p< 0.0001). Although GM-CSF receptor subunits were present in TCs and TAMs, their expression did not correlate with clinical outcomes. To explore the pro-metastatic role of GM-CSF, we performed in vitro assays and found that it activates non-canonical signaling pathways, such as STAT3 and PI3K/AKT, and promotes melanoma cell invasion. Consistently, its administration enhanced lung colonization by melanoma cells in a murine model, an effect reversed by CD116 receptor blockade. Furthermore, GM-CSF-primed macrophages secreted higher levels of inflammatory cytokines upon interaction with melanoma cells than those unprimed or primed with the counterpart cytokine M-CSF. Reciprocally, RNA-seq analyses revealed a broader transcriptional reprogramming in melanoma cells exposed to GM-CSF-primed macrophages, which displayed enhanced expression of inflammatory-response genes, suggesting a feedforward loop. Altogether, our findings highlight a potentially pro-tumorigenic GM-CSF-driven paracrine axis in patients with poor-prognosis melanoma, supporting therapeutic strategies aimed at disrupting this signaling network.