Fig. 2: Ferrichrome abrogates M2 polarization of macrophages and promotes M1-like phenotype in vitro and in vivo.

a Representative scheme of in vitro polarization of RAW264.7 macrophages. M2 polarization of RAW264.7 cells were induced by culture with IL-4 (20 ng/mL) in the presence of ferrichrome or vehicle control (1× PBS) for 24 h. Control consisted of vehicle treated RAW264.7 cells. RNA was collected for qPCR analysis. b Differential gene expression of M1 and M2 biomarkers between IL-4-treated and ferrichrome (8 μM) + IL-4-treated RAW264.7 cells after 24 h of culture as evaluated by qPCR analysis. Graph is representative of two or more independent experiments with N = 2–4. c Differential gene expression of M1 and M2 markers in UN-KC-6141 tumors treated with vehicle or ferrichrome (pooled from n = 3 biological replicates). d Representative images and quantification of dual staining of macrophage markers F4/80 (green) and CD163 (red) in tumors treated with ferrichrome or vehicle as evaluated via immunofluorescence and imaged using confocal microscopy. Quantification was evaluated using ImageJ (n = 4). Nucleus was stained with DAPI (blue). Bar on micrographs indicates 50 µm. e Representative histogram of M2 marker CD206 Mean Fluorescence Intensity (MFI) in gated macrophages (CD45 + CD11b + Ly6C− Ly6G− F4/80+) of ferrichrome (blue) or vehicle-treated (red) tumor single cell suspensions as evaluated by flow cytometry analysis (n = 4 experiments). Fluorescence Minus One (FMO) (orange) was used as a negative control. Mean ± SEM shown **p < 0.05, ***p < 0.01, and ****p < 0.001, as determined by Student’s t test or two-way ANOVA.