Supplementary Figure 3: Impact of CSF-1R inhibition on the intratumoral presence of diverse myeloid immune cell types.

(a) Representative dot plots of a KEP mammary tumor illustrating the gating strategy for the identification of cell populations. Antibody panel used: “tumor panel I” (see supplementary Table 1). Arrows indicate directionality of sub-gates. (b-d) tdTomato⁺ (Lineage^-SiglecF^-cKIT^-CD11b^intLy6G^-Ly6C⁺) monocytes were isolated from the bone marrow of mTmG mice and adoptively transferred into tumor-bearing KEP mice that had previously received either control ab or anti-CSF-1R. 4 days after the monocyte transfer, the presence and phenotype of tdTomato⁺ cells in tumors were analyzed. (b) Gating strategy showing intratumoral tdTomato⁺ cells that express F4/80 in control ab- or anti-CSF-1R-treated recipient KEP mice. (c) Representative flow cytometry histograms showing CX3CR1, PD-L1, CCR2 and CD80 expression in tdTomato⁺ and tdTomato^- macrophages in KEP tumors. (d) Overlay of representative dot plots showing Ly6C expression in tdTomato⁺ and tdTomato^- macrophages in control ab- and anti-CSF-1R-treated KEP mice. Data presented in b-d are representative of 2 (control ab treatment) and 3 (anti-CSF-1R treatment) independent experiments. (e) Quantification of Ly6G⁺ neutrophils in viable areas of mammary tumors of time point-sacrificed KEP mice treated with control ab (n=6 animals), anti-CSF-1R (n=6 animals), cisplatin/control ab (n=7 animals) or cisplatin/anti-CSF-1R (n=7 animals). (f) Proportion of Ly6C⁺Ly6G^- monocytes determined by flow cytometry in KEP mammary tumors treated with control ab (n=4 animals) or anti-CSF-1R (n=5 animals). (g) Quantification of Major Basic Protein (MBP)⁺ cells in viable areas of mammary tumors of time point-sacrificed KEP mice as determined by IHC (control ab n=5 animals, anti-CSF-1R n=6 animals, cisplatin/control ab n=7 animals, cisplatin/anti-CSF-1R n=7 animals) and proportion of Siglec F⁺ eosinophils gated on intratumoral CD45⁺ cells of time point-sacrificed KEP mice as determined by flow cytometry (cisplatin/control ab n=5 animals, cisplatin/anti-CSF-1R n=6 animals). (h) Quantification of Toluidine Blue⁺ mast cells in viable areas of mammary tumors of time point-sacrificed KEP mice (control ab n=4 animals, anti-CSF-1R n=6 animals, cisplatin/control ab n=7 animals, cisplatin/anti-CSF-1R n=7 animals) as determined by histochemistry. Values in e, g and h represent average number of positive cells per field of view (FOV) quantified by counting five high-power microscopic fields per tumor. Data presented in e-h are mean values ± SEM. Statistical analysis was performed using two-tailed Mann–Whitney test.