Figure 3 from Tumoral Interferon Beta Induces an Immune-Stimulatory Phenotype in Tumor-Associated Macrophages in Melanoma Brain Metastases

B16-F10 Ifnb1eGFP-derived IFNβ repolarizes macrophages and reduces tumor growth. A, B16-F10 Ifnb1eGFP cells were treated with 1 μg/mL Dox and cell culture supernatant was harvested after 24, 48 and 72 hours. IFNβ concentrations were analyzed by ELISA and are displayed in pg/mL (n = 3). B–G, C57Bl/6J mice received control diet or Dox diet from day 1 on following intracranial B16-F10 Ifnb1eGFP tumor cell injection. Tumor growth was monitored by MRI on day 14, tumors were excised and blood was drawn on day 16. B, Intratumoral IFNβ concentrations were analyzed by ELISA and are displayed in pg/mL of original tumor volume (n = 6, control diet in light pink; n = 7, Dox diet in dark red). C, Heatmap of quantitative flow cytometry analysis of B16-F10 Ifnb1eGFP-associated macrophages displayed as relative MFI (n = 6 and n = 7). D–F, Quantitative flow cytometry analysis of B16-F10 Ifnb1eGFP-infiltrating T cells displayed as percentage of parental population (n = 6 and n = 7). G, B16-F10 Ifnb1eGFP tumor volume as measured by MRI is displayed in μL (T1 sequence; n = 8). H and I, Quantitative flow cytometry analysis of CD8⁺ T cells after a 1:1 CTFR-stained T cell to M2-polarized BMDM allo-co-culture after indicated BMDM pretreatment (sn, supernatant collected after Dox pre-treatment; n = 3) Displayed as percentage (H) or relative expression (I) of CD8⁺ T cells. For gating strategies see Supplementary Figs. S2 and S3G. Data are expressed as median + IQR and individual values for A, B, and D–H and individual scaled values for C and I. Statistical significance was determined by Mann–Whitney U test for A, B, and G, by paired t test for D–F and by one-way analysis of variance (ANOVA) with Tukey post hoc testing for H. ES, effect size.