Fig. 2: FRβ⁺ TAMs display an M2-like profile.

TAMs obtained from ID8 ascites were defined as live, CD45⁺CD11b⁺F4/80⁺. a Immunofluorescence of flow-sorted FRβ⁻ (upper panel) and FRβ⁺ (lower panel) TAMs stained for actin with phalloidin-rhodamine (red) and for cell nuclei with DAPI (blue). Scale bar, 10 µm. Representative images from one experiment (n = 3 mice). b Differential gene expression profile between FRβ⁻ and FRβ⁺ TAMs as measured by RNA-seq (n = 4 per group). Log 2 of fold change (FC) vs. log 2 of counts per million (CPM) is represented. Red dots represent genes upregulated in FRβ⁺ TAMs, black dots represent downregulated genes, gray dots represent not differentially expressed genes, and open dots represent genes expressed at low levels. Relevant genes are highlighted. Statistics were calculated by EdgeR (cutoff:log 2(FC) = ±0.26, FDR < 0.1). c Heatmap of normalized mean-centered mRNA expression of genes associated to M1 or M2 macrophages in flow-sorted FRβ⁻ and FRβ⁺ TAMs (n = 4 per group). d Representative flow plots showing coexpression of mFRβ (X-axis) and CD204 (left panel) or CD206 (middle panel, Y-axis) in FRβ⁺ TAMs (red) and FRβ⁻ TAMs (black). Mean frequencies of CD204⁺ (n = 4 mice) and CD206⁺ (n = 8 mice) cells ± SD are shown (right panel). Expression of e PD-L1 (n = 4 mice) or f Arg1 (n = 5 mice) and Egr2 (n = 8 mice) in FRβ⁻ and FRβ⁺ TAMs. Representative histograms (gray–isotype, black—FRβ⁻ TAMs, red—FRβ⁺ TAMs, left panel) and mean frequencies (right panel) ± SD are shown. g Production of cytokines by flow-sorted FRβ⁻ and FRβ⁺ TAMs as quantified by a cytokine bead-based assay after 24 h of incubation unstimulated or in the presence of 5 ng/mL of LPS. Mean concentrations of six replicates ± SD are represented. h Oxygen consumption rates (OCR) of flow-sorted FRβ⁻ and FRβ⁺ TAMs after one day in culture in response to mitochondrial inhibitors added at indicated times as detected by a Seahorse assay. Mean OCR of six replicates ± SD is represented. Data are representative of g two and h three independent experiments. P values by a d–h two-way ANOVA with Sidak’s multiple comparison test are indicated. OM oligomycin, FCCP phenylhydrazone, Rot rotenone, AA antimycin. Source data are provided in the Source Data file.