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The identification of "trained immunity/tolerance" in myeloid cells has changed our perception of the performance of monocytes and macrophages during inflammatory and immune responses. Pemetrexed (PMX) and methotrexate (MTX) are blockers of the one-carbon metabolism (OCM) and commonly used therapeutic agents in cancer and rheumatoid arthritis (RA). We have previously showed that MTX promotes trained immunity in human macrophages. In the present manuscript, we have assessed the anti-inflammatory effects of PMX and MTX and found that OCM blockers alter the functional and gene expression profile of human macrophages and that OCM blockade reprograms macrophages towards a state of lipopolysaccharide (LPS) tolerance at the signaling and functional levels. Moreover, OCM blockade reduced macrophage LPS responsiveness by impairing the expression of membrane-bound and soluble CD14 (sCD14). The therapeutic relevance of these results was later confirmed in early RA patients, as MTX-responder RA patients exhibit lower sCD14 serum levels, with baseline sCD14 levels predicting MTX response. As a whole, our results demonstrate that OCM is a metabolic circuit that critically mediates the acquisition of innate immune tolerance and positions sCD14 as a valuable tool to predict MTX response in RA patients., Competing Interests: IG-A reports personal fees from Lilly and Sanofi; personal fees and non-financial support from BMS; personal fees and non-financial support from AbbVie; research support, personal fees, and non-financial support from Roche Laboratories; and non-financial support from MSD, Pfizer, and Novartis, not related to the submitted work. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fuentelsaz-Romero, Barrio-Alonso, García Campos, Torres Torresano, Muller, Triguero-Martínez, Nuño, Villalba, García-Vicuña, Jansen, Miranda-Carús, González-Álvaro and Puig-Kröger.)

During cancer evolution, tumor cells attract and dynamically interact with monocytes/macrophages. To find biomarkers of disease progression in human melanoma, we used unbiased RNA sequencing and secretome analyses of tumor–macrophage co‐cultures. Pathway analysis of genes differentially modulated in human macrophages exposed to melanoma cells revealed a general upregulation of inflammatory hallmark gene sets, particularly chemokines. A selective group of chemokines, including CCL8, CCL15, and CCL20, was actively secreted upon melanoma–macrophage co‐culture. Because we previously described the role of CCL20 in melanoma, we focused our study on CCL8 and CCL15 and confirmed that in vitro both chemokines contributed to melanoma survival, proliferation, and 3D invasion through CCR1 signaling. In vivo, both chemokines enhanced primary tumor growth, spontaneous lung metastasis, and circulating tumor cell survival and lung colonization in mouse xenograft models. Finally, we explored the clinical significance of CCL8 and CCL15 expression in human skin melanoma, screening a collection of 67 primary melanoma samples, using multicolor fluorescence and quantitative image analysis of chemokine–chemokine receptor content at the single‐cell level. Primary skin melanomas displayed high CCR1 expression, but there was no difference in its level of expression between metastatic and nonmetastatic cases. By contrast, comparative analysis of these two clinically divergent groups showed a highly significant difference in the cancer cell content of CCL8 (p = 0.025) and CCL15 (p < 0.0001). Kaplan–Meier curves showed that a high content of CCL8 or CCL15 in cancer cells correlated with shorter disease‐free and overall survival (log‐rank test, p < 0.001). Our results highlight the role of CCL8 and CCL15, which are highly induced by melanoma–macrophage interactions in biologically aggressive primary melanomas and could be clinically applicable biomarkers for patient profiling. © 2024 The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

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. 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. [ABSTRACT FROM AUTHOR]