Supplementary Figure 2: CSF-1R blockade synergizes with platinum-based chemotherapy drugs, and not with docetaxel.

(a-b) Proportion of CD45⁺ immune cells gated on live cells (a) and F4/80⁺Ly6G^- macrophages gated on CD11b⁺ cells (b) determined by flow cytometry in tumors of time point-sacrificed KEP mice treated as indicated (n=5 animals/group). (c) Kaplan Meier tumor-specific survival curves of KEP mice treated with control ab, anti-CSF-1R (same groups as Fig. 1f), docetaxel/control ab (n=10 animals) or docetaxel/anti-CSF-1R (n=10 animals). Docetaxel/control ab versus Control ab, p=0.0021; Docetaxel/control ab versus docetaxel/anti-CSF-1R, p=0.329 (two-tailed log-rank test). (d) Quantification of cleaved caspase 3⁺ cells in viable areas of mammary tumors of time point-sacrificed KEP mice treated with control ab (n=6 animals), anti-CSF-1R (n=5 animals), cisplatin/control ab (n=7 animals) and cisplatin/anti-CSF-1R (n=9 animals) as determined by IHC. (e) Quantification of CD31⁺ vessels in viable areas of mammary tumors of time point-sacrificed KEP mice treated with control ab (n=5 animals), anti-CSF-1R (n=5 animals), cisplatin/control ab (n=5 animals) and cisplatin/anti-CSF-1R (n=4 animals) as determined by immunofluorescence. Values represent average number of positive cells per FOV quantified by counting six fields per tumor. (f) Percentage of vessels covered by alpha-SMA⁺ pericytes in viable areas of mammary tumors as determined by immunofluorescence. Same mice as e. Percentage was determined by counting alpha-SMA⁺CD31⁺ cells and total CD31⁺ cells in six high-power microscopic fields per tumor. (g-h) Quantification of γH2AX⁺ cells (g) and cisplatin adducts⁺ cells (h) in viable areas of mammary tumors of time point-sacrificed KEP mice treated as indicated (γH2AX: control ab n=6 animals, anti-CSF-1R n=4 animals, cisplatin/control ab n=7 animals, cisplatin/anti-CSF-1R n=8 animals; CIS-adducts: cisplatin/control ab n=5 animals, cisplatin/anti-CSF-1R n=6 animals). (i) Percentage of non-viable area per tumor section of time point-sacrificed KEP mice quantified by digital area analysis of H&E stained sections (n=5 animals/group). (j-m) Quantification of CD34⁺ cells (j), cleaved caspase 3⁺ cells (k), BrdU⁺ cells (l), γH2AX⁺ cells (m) in viable areas of mammary tumors of time point-sacrificed KEP mice treated as indicated (CD34: docetaxel/control ab n=5 animals, docetaxel/anti-CSF-1R n=3 animals; cCasp3: n=4 animals/group; BrdU: n=5 animals/group; γH2AX: n=4 animals/group). (n) Proportion of CD11b⁺F4/80⁺ macrophages gated on CD45⁺ cells as determined by flow cytometry in tumors of end-stage KEP mice treated as indicated (oxaliplatin/Control ab treatment n=6 animals; oxaliplatin/anti-CSF-1R treatment n=5 animals). Data presented in d, g-h, j-m show average number of positive cells per field of view (FOV) quantified by counting five high-power microscopic fields per tumor. Data presented in a-b and d-n are mean values ± SEM and statistical analysis was performed using two-tailed Mann–Whitney test. DOCE, docetaxel, CIS, cisplatin, OX, oxaliplatin.