Journal: Human Mutation

Article Title: A Macrophage/Monocyte‐Related Four‐Gene Signature for Prognostic Assessment of Uveal Melanoma: BTBD6 , C2CD4B , CCL24 , and S100A4

doi: 10.1155/humu/4978880

Figure Lengend Snippet: Role of four prognostically relevant genes. (A) Quantitative reverse transcription PCR for the calculated levels of four prognostically relevant genes in human epidermal melanocytes. (B) Validation of the knockdown efficiency of C2CD4B. (C, D) Scratch and Transwell assays exploring the migration and invasion of A375 cells in vitro. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, p > 0.05.

Article Snippet: Melanoma cell line A375 (CRL‐1619) and human epidermal melanocytes (PCS‐200‐013) were purchased from American Type Culture Collection (ATCC, Manassas, Virginia) and cultured as follows.

Techniques: Reverse Transcription, Biomarker Discovery, Knockdown, Migration, In Vitro

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: Transcriptomic profiling of melanoma SCs identifies stemness-associated and cell line-specific molecular programs. (A) PCA of RNA-seq data from A375 and WM115 cells cultured under adherent conditions (2D) or as melanospheres (3D). Each dot represents one sample. (B) ssGSEA enrichment scores for the Melanoma stemness, WNT/β-catenin signaling, Hedgehog signaling, and Notch signaling gene sets in A375 and WM115 cells cultured in 2D or 3D conditions. Data are presented as the mean ± SEM. Each dot represents one sample. *p<0.05 by Welch’s t-test, followed by Benjamini-Hochberg correction for multiple testing across signatures. (C) Left, number of up-regulated and down-regulated DEGs identified in melanospheres compared to matched adherent cultures in A375 and WM115 cells (padj < 0.05). Right, filtered volcano plots showing DEGs (padj < 0.05, |log2FC| > 1, and baseMean > 50) in A375 and WM115 melanospheres relative to 2D cultures. The 10 most upregulated and 10 most downregulated genes are labelled. (D) Bubble plots of the top 10 most statistically significant (padj < 0.05) Reactome pathways enriched among genes up-regulated in A375 and WM115 melanospheres relative to matched 2D cultures determined by GSEA analysis. The X-axis represents the Gene Ratio and Y-axis indicates enriched pathway terms. Bubble area represents the gene count and bubble color indicates the adjusted p value. (E) VIPER analysis showing the top 15 inferred transcription factor activity in melanospheres relative to matched 2D cultures in A375 and WM115 cells. Red bars indicate transcription factors inferred to be activated in 3D cultures, whereas blue bars indicate transcription factors inferred to be activated in 2D cultures.

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: RNA Sequencing, Cell Culture, Activity Assay

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: Distinct secretory profiles and monocyte chemoattractant capacity of A375 and WM115 SCs. (A) PCA of multiplex ELISA assay data obtained from CM of A375 and WM115 melanospheres. Each dot represents one sample. (B) Multiplex ELISA assay quantification of soluble factors in CM from A375 and WM115 SCs. Data are presented as the mean ± SEM. Each dot represents one sample. (C) Transwell migration assay of THP-1 cells exposed to control medium, A375 SC-CM, or WM115 SC-CM, in the absence or presence of the CCR2 antagonist RS504393. Data are presented as mean ± SEM. Each dot represents one sample. Statistical analysis was performed by two-way ANOVA followed by Bonferroni-adjusted post hoc comparisons. Asterisks indicate comparisons between inhibitor-treated and untreated conditions within each medium. **p < 0.01, ***p < 0.001.

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: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Transwell Migration Assay, Control

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: Transcriptomic characterization of THP-1-derived macrophages exposed to melanoma SC-CM. (A) PCA of RNA-seq data from M0 macrophages and macrophages exposed to CM from A375 or WM115 melanospheres. (B) Number of upregulated and downregulated DEGs (padj<0.05) in A375 SC-CM-educated and WM115 SC-CM-educated macrophages compared with M0 macrophages. (C) Venn diagrams showing the overlap of upregulated and downregulated DEGs between A375 SC-CM-educated and WM115 SC-CM-educated macrophages, each compared with M0 macrophages. (D) Filtered volcano plots showing DEGs (padj < 0.05, |log2FC| > 1, and baseMean > 50) in A375 SC-CM-educated versus M0 macrophages and WM115 SC-CM-educated versus M0 macrophages. The 10 most upregulated and 10 most downregulated genes are labelled. (E) Bubble plots of the top 10 most statistically significant (padj < 0.05) Reactome pathways enriched among genes upregulated in A375 SC-CM-educated macrophages and WM115 SC-CM-educated macrophages determined by GSEA analysis. The X-axis represents the Gene Ratio and Y-axis indicates enriched pathway terms. Bubble area represents the gene count, and bubble color indicates the adjusted p value. (F) VIPER analysis showing the top 15 inferred transcription factor activity in A375 SC-educated and WM115 SC-educated macrophages compared to M0 macrophages. Red bars indicate transcription factors inferred to be activated in melanoma SC-educated macrophages, whereas blue bars indicate transcription factors inferred to be activated in M0 macrophages.

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: Derivative Assay, RNA Sequencing, Activity Assay

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: ssGSEA-based inference of macrophage phenotypes induced by melanoma SC-CM. (A) Bar plots showing normalized ssGSEA scores for literature-derived TAM signatures and curated macrophage-state signatures in M0 macrophages and in macrophages exposed to A375 SC-CM or WM115 SC-CM. Curated signatures were derived from GSE115978 . Data are presented as the mean ± SEM. Each dot represents one sample. **p<0.01, ***p<0.001 by one-way ANOVA with Benjamini-Hochberg correction across signatures followed by Tukey’s multiple-comparison test. (B) Integrated matrix showing pairwise relationships among the signatures significantly modulated in vitro . The lower triangle reports Spearman correlation coefficients calculated from ssGSEA scores across samples, with asterisks indicating Benjamini-Hochberg-adjusted significance levels (*p<0.05, **p<0.01, ***p<0.001). The upper triangle reports overlap coefficients based on gene-set composition. Abbreviations: IFN, interferon-primed TAMs; Inflamm, inflammatory cytokine-enriched TAMs; Angio, pro-angiogenic TAMs; Reg, immune regulatory TAMs. (C) Bar plots representing MPI (macrophage polarization index) and AMDI (activation-induced macrophage differentiation index) values from MacSpectrum analysis of M0 macrophages and macrophages exposed to CM from A375 or WM115 melanospheres. Data are presented as the mean ± SEM. Each dot represents one sample. *p<0.05, **p<0.01 by Kruskal-Wallis test with Holm correction across the two endpoints, followed by Dunn’s post hoc test with Holm correction.

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: Derivative Assay, Comparison, In Vitro, Activation Assay

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