Journal: Frontiers in Immunology

Article Title: Melanoma stem cells drive macrophage reprogramming to a hybrid phenotype, modulating melanoma stemness and compromising NK cell-mediated immunity

doi: 10.3389/fimmu.2026.1698412

Figure Lengend Snippet: Functional characterization of THP-1-derived macrophages exposed to melanoma SC-CM. (A) PCA performed on multiplex ELISA assay of soluble mediator profiles from M0 macrophages and macrophages exposed to CM from A375 or WM115 melanospheres. Each dot represents one sample. (B) Multiplex ELISA assay quantification of soluble mediators secreted by M0 macrophages and by macrophages previously exposed to CM from A375 or WM115 melanospheres. Data are presented as the mean ± SEM. Each dot represents one sample. *p<0.05 by Kruskal-Wallis test with Benjamini-Hochberg correction across analytes, followed by Dunn’s post hoc test with Holm correction; pairwise p values were additionally Benjamini-Hochberg-corrected across analytes within each contrast. (C) Percentage of lysis of NK-92 cells against K562 target cells after pre-exposure to CM from M0 macrophages or from macrophages educated with A375 or WM115 SC-CM, assessed by Calcein-AM release cytotoxicity assay at the indicated E:T ratios. Data are presented as the mean ± SEM. Each dot represents one sample. The results are representative of one of at least three independent experiments showing similar results. **p<0.01, ***p<0.001 by Tukey-adjusted comparisons among experimental groups within each E:T ratio following two-way ANOVA. (D) Percentage of lysis of NK-92 cells against K562 target cells after pre-exposure to untreated or heat-treated CM from M0 macrophages or from macrophages educated with A375 or WM115 melanoma SC-CM assessed by Calcein-AM release cytotoxicity assay. Data are presented as the mean ± SEM. Each dot represents one sample. The results are representative of one of at least three independent experiments showing similar results. ***p<0.001 by Tukey-adjusted comparisons between treatment conditions within each experimental group following two-way ANOVA. (E) RT-qPCR analysis of KLF4, SOX10, POU5F1, SOX9, SOX2 in A375 and WM115 SC-enriched cultures after exposure to CM from M0 macrophages or from macrophages previously educated with melanoma SC-CM. Bar plots show mean ± SEM of 2 −ΔCt RT-qPCR values. The results are representative of one of at least three independent experiments showing similar results. *p<0.05, **p<0.01, ***p<0.001 by Welch’s t-test, followed by Holm correction for multiple testing across genes within each cell line.

Article Snippet: A375 human melanoma cells (CRL-1619TM), THP-1 human acute monocytic leukemia cells (TIB-202TM), NK-92 human natural killer cell line (CRL-2407TM), and K562 human chronic myelogenous leukemia cells (CCL-243TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Functional Assay, Derivative Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Lysis, Cytotoxicity Assay, Quantitative RT-PCR