Supplementary Figure 6: Neutrophil inhibition enhances intratumoral granzyme B expression and improves the synergistic anti-cancer effect of cisplatin/anti-CSF-1R in K14cre;Cdh1^F/F;Trp53^F/F mice.

(a-c) Quantification of CD8⁺ T cells (a), CD4⁺ T cells (b) and FoxP3⁺ regulatory T cells (c) in viable areas of mammary tumors of time point-sacrificed KEP mice (CD8: cisplatin/control ab n=7 animals, cisplatin/anti-CSF-1R n=7 animals, cisplatin/anti-CSF-1R/anti-Ly6G n=7 animals; CD4: cisplatin/control ab n=7 animals, cisplatin/anti-CSF-1R n=6 animals, cisplatin/anti-CSF-1R/anti-Ly6G n=6 animals; FoxP3: cisplatin/control ab n=7 animals, cisplatin/anti-CSF-1R n=7 animals, cisplatin/anti-CSF-1R/anti-Ly6G n=6 animals). (d) Proportion of granzyme B⁺ CD45⁺ lymphocytes (lymphocyte gate was based on SSC and FSC) determined by flow cytometry in the tumor of time point-sacrificed KEP mice treated as indicated (cisplatin/control ab n=3 animals, cisplatin/anti-CSF-1R n=4 animals, cisplatin/anti-CSF-1R/anti-Ly6G n=5 animals). (e-f) Quantification of S100A9⁺ cells in viable areas of mammary tumors (e) and lung (f) of end-stage KEP mice treated with cisplatin/anti-CSF-1R (n=8 animals) or cisplatin/anti-CSF-1R/anti-Ly6G (n=6 animals). (g-h) Representative tumor growth graphs of six individual KEP mice treated with cisplatin/anti-CSF-1R (g) and cisplatin/anti-CSF-1R/anti-Ly6G (h). Data are representative of 16 cisplatin/anti-CSF-1R-treated mice and 10 cisplatin/anti-CSF-1R/anti-Ly6G-treated mice. Red arrows indicate cisplatin injections. In pink, growth curve of a secondary tumor that developed in another mammary gland during the treatment. (i) Kaplan-Meier tumor-specific survival curves of KEP mice treated with control ab, anti-CSF-1R (same groups as Fig. 1f) or anti-CSF-1R/anti-Ly6G (n=10 animals). (j-n) Quantification of BrdU⁺ (j) γH2AX⁺ cells (k), cleaved caspase 3⁺ cells (l), CD31⁺ vessels (m) and cisplatin adducts⁺ cells (n) in viable areas of mammary tumors of time point-sacrificed KEP mice treated with cisplatin/anti-CSF-1R (same as Fig. 1h and Supplementary Fig. 2) and cisplatin/anti-CSF-1R/anti-Ly6G (BrdU n=6 animals; γH2AX n=5 animals; cCasp3 n=8 animals; CD31 n=5 animals; CIS adducts n=5 animals). Values presented in a-c, e-f, j-l and n represent average number of positive cells per field of view (FOV) as determined by IHC quantified by counting five high-power microscopic fields per tumor. Values presented in m represent average number of positive cells per FOV as determined by immunofluorescence by counting six field per tumor. Data presented in a-f and j-n are mean values ± SEM. Statistical analysis was performed using two-tailed Mann–Whitney test. CIS, cisplatin.