CCL20/TNF/VEGFA Cytokine Secretory Phenotype of Tumor-Associated Macrophages Is a Negative Prognostic Factor in Cutaneous Melanoma

SIMPLE SUMMARY: Cutaneous melanoma is characterized by its heterogeneous metastatic behavior and robust biomarkers are still needed to identify those patients with increased risk for distant metastasis, to guide new adjuvant treatments. We aimed to assess the prognostic role of different features of tumor-associated macrophages (TAMs) using multicolor immunofluorescence microscopy and single-cell analysis. Rather than the number, size, or location of TAM, quantitative assessment of CCL20, TNF, and VEGFA cytokine content was associated with strong prognostic significance in primary melanoma. This novel TAM cytokine signature serves as a readout of TAM prometastatic activation and provides independent information to traditional TNM melanoma staging. In addition, we show that this particular cytokine profile is coregulated by p53 and NF-κB, suggesting that therapies targeting both pathways may modulate the prometastatic deviation of TAMs in melanoma. ABSTRACT: TAMs constitute a large fraction of infiltrating immune cells in melanoma tissues, but their significance for clinical outcomes remains unclear. We explored diverse TAM parameters in clinically relevant primary cutaneous melanoma samples, including density, location, size, and polarization marker expression; in addition, because cytokine production is a hallmark of macrophages function, we measured CCL20, TNF, and VEGFA intracellular cytokines by single-cell multiparametric confocal microscopy. The Kaplan–Meier method was used to analyze correlation with melanoma-specific disease-free survival and overall survival. No significant correlations with clinical parameters were observed for TAM density, morphology, or location. Significantly, higher contents of the intracellular cytokines CCL20, TNF, and VEGFA were quantified in TAMs infiltrating metastasizing compared to non-metastasizing skin primary melanomas (p < 0.001). To mechanistically explore cytokine up-regulation, we performed in vitro studies with melanoma-conditioned macrophages, using RNA-seq to explore involved pathways and specific inhibitors. We show that p53 and NF-κB coregulate CCL20, TNF, and VEGFA in melanoma-conditioned macrophages. These results delineate a clinically relevant pro-oncogenic cytokine profile of TAMs with prognostic significance in primary melanomas and point to the combined therapeutic targeting of NF-kB/p53 pathways to control the deviation of TAMs in melanoma.