Figure 4 from Tumoral Interferon Beta Induces an Immune-Stimulatory Phenotype in Tumor-Associated Macrophages in Melanoma Brain Metastases

Transcriptomic analyses of IFNβ-treated bone marrow-derived macrophages reveal an immune-stimulatory phenotype characterized by the expression of a proinflammatory type I IFN-response signature. A, Principal component analysis of bulk RNA sequencing gene expression profiles of M2-like polarized BMDM treated with supernatant from Dox-treated YUMM5.2 eGFP control cells (gray), supernatant from Dox-treated YUMM5.2 Ifnb1eGFP cells (dark red), rIFNβ (pink) or untreated control (light pink; n = 3). B, Spearman correlation analysis of log₂-transformed mean gene counts in M2-like polarized BMDM after indicated treatments. sn, supernatant collected after Dox pre-treatment (n = 3). C, GSEA of a curated M1 phenotypic marker gene set in M2-like polarized BMDM after treatment with supernatant from Dox-treated YUMM5.2 Ifnb1eGFP cells compared to treatment with supernatant from Dox-treated YUMM5.2 eGFP control cells. Enrichment Score = 0.62, NES = 2.38, adjusted P value = 1.00e–10 (n = 3). D, GSEA of the Molecular Signature Database Hallmark Gene Sets in M2-like polarized BMDM after treatment as in C. Top activated and suppressed Hallmark gene sets are displayed according to their gene ratio (n = 3). E, Heatmap of normalized gene counts of upregulated immune-related genes in M2-like polarized BMDM after indicated treatments (n = 3). F and G, mRNA levels of CD69 (F) and H2-Q6 (G) measured by qPCR in F4/80+ cells isolated from intracranial YUMM5.2 Ifnb1eGFP tumors from mice receiving control diet compared to Dox diet. Displayed as log₂ fold change (Log₂FC; n = 4). H, Immunohistochemistry staining of F4/80 (red) and CD69 (H) or MHCI (I; yellow) on tissue sections from mice with intracranial YUMM5.2 Ifnb1eGFP tumors receiving control diet compared to Dox diet. Left, representative images. Scale bars, 50 μm. Right, quantitative analysis of CD69 (H) or MHCI (I) expression in F4/80+ cells displayed as MFI (n = 3, 2 fields of view per animal). For the M1 phenotypic marker gene set see Supplementary Table S3. Data are expressed as individual values for A, B, D, and E and median + IQR and individual values for F–I. Statistical significance was determined by Mann–Whitney U test for F–I. ES, effect size.