Figure 7. CSF1R inhibition and cisplatin target distinct macrophage populations in murine KP tumors.

(A) Left: representative images of F4/80 (white) immunostaining and DAPI nuclear staining (blue) of KP tumors treated as indicated. Scale bar, 50 μm. Bottom right: quantification of the data. Dots represent individual tumors, whereas bars indicate mean values ± SEM. Statistical analysis by one-way ANOVA followed by Tukey’s multiple comparison test. (B) Left: pie chart representation of the mean frequency of macrophage/monocyte subsets in total Ly6G^–F4/80⁺ cells. Right: frequencies of MO-TAMs and AM-TAMs within CD45⁺ cells in KP tumors 4 weeks after treatment initiation. Dots represent individual tumors, whereas bars indicate mean values ± SEM. Statistical analysis by one-way ANOVA followed by Tukey’s multiple comparison test (AM-TAMs) or Kruskal-Wallis test followed by Dunn’s multiple comparison test (MO-TAMs). (C) Expression of Csf1r and Mki67 mRNA by RNAseq in enriched MO-TAMs (Ly6G^–Ly6C^–F4/80⁺CD11b⁺CD11c^+/lowCD206^+/low), AM-TAMs (Ly6G^–Ly6C^–F4/80⁺CD11b^- CD11c⁺CD206⁺) and live tumor-derived cells (Total) from KPM tumors, analyzed 1 week after treatment initiation. Dots represent individual mice, whereas bars indicate mean values; several tumors were pooled from each mouse. Statistical analysis by two-way ANOVA followed by Tukey’s multiple comparison test. (D) Number of CD4⁺Foxp3⁺ Tregs per mm² of DAPI⁺ KP tumor area quantified by immunostaining. Dots represent individual tumors, whereas bars indicate mean values ± SEM. Statistical analysis by Kruskal-Wallis test followed by Dunn’s multiple comparison test. (E) qPCR analysis of Foxp3, Ccl17, Il10 and Tgfb1 mRNA expression in KP tumors treated as indicated, normalized to the expression in IgG-treated mice. Gapdh and Hprt were used as housekeeping genes. Dots represent individual tumors, whereas bars indicate mean values ± SEM. Statistical analysis on non-normalized ΔCt values by one-way ANOVA followed by Tukey’s multiple comparison test. (F) Distance of individual Tregs from the tumor center (core) normalized to the tumor area. A normalized distance equal to 1 is the mean distance between the core and edge of the tumor. Dots represent individual cells, whereas bars indicate mean values. Statistical analysis by Mann Whitney test.

P values are coded as *: P < 0.05; **: P < 0.01; ***: P < 0.001; and ****: P < 0.0001. Exact P values and the number of mice, tumors or samples analyzed, are reported in table S1. Numerical values are reported in table S2.