Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma.

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.

Activin A Sustains the Metastatic Phenotype of Tumor-Associated Macrophages and Is a Prognostic Marker in Human Cutaneous Melanoma.

Tumor cells attract and dynamically interact with monocytes/macrophages to subvert their differentiation into tumor-associated macrophages (TAMs), which mainly promote immune suppression and neoplastic progression, but the pathways and microenvironmental cues governing their protumoral deviation are not completely understood. To identify the molecular pathways responsible for TAM differentiation, we screened the biomarkers secreted during melanoma‒macrophage interactions using Quantibody microarrays and RNA sequencing of macrophages. We found that activin A, a member of the transforming GF family, plays an instrumental role in the cross-talk between melanoma cells and monocytes/macrophages, which results in the upregulation of distinct tumor-sustaining genes and the achievement of proinvasive and immunosuppressive functions of TAMs. Blockade of activin reduces the upregulation of part of these genes and prevents the acquisition of protumoral functions, facilitating human melanoma rejection by transferred human lymphocytes in a xenograft mouse model. Remarkably, screening of two independent cutaneous primary melanoma collections showed that activin A is enriched in TAMs and melanoma cells from patients with worse outcomes and constitutes a new and independent prognostic marker. Thus, we identify activin A as a key intermediary in the protumoral and immunosuppressive functions of TAMs, with significant potential as a disease biomarker as well as an immunotherapeutic target.

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

CCL20 Expression by Tumor-Associated Macrophages Predicts Progression of Human Primary Cutaneous Melanoma.

The chemokine axis CCR6/CCL20 is involved in cancer progression in a variety of tumors. Here, we show that CCR6 is expressed by melanoma cells. The CCR6 ligand, CCL20, induces migration and proliferation in vitro, and enhances tumor growth and metastasis in vivo Confocal analysis of melanoma tissues showed that CCR6 is expressed by tumor cells, whereas CCL20 is preferentially expressed by nontumoral cells in the stroma of certain tumors. Stromal CCL20, but not tumoral CCR6, predicted poor survival in a cohort of 40 primary melanoma patients. Tumor-associated macrophages (TAM), independently of their M1/M2 polarization profile, were identified as the main source of CCL20 in primary melanomas that developed metastasis. In addition to CCL20, TAMs expressed TNF and VEGF-A protumoral cytokines, suggesting that melanoma progression is supported by macrophages with a differential activation state. Our data highlight the synergistic interaction between melanoma tumor cells and prometastatic macrophages through a CCR6/CCL20 paracrine loop. Stromal levels of CCL20 in primary melanomas may be a clinically useful marker for assessing patient risk, making treatment decisions, and planning or analyzing clinical trials. Cancer Immunol Res; 6(3); 267-75. ©2018 AACR.