Supplementary Figure 5: CSF-1R blockade increases intratumoral Ifna expression.

(a) Transcripts of Ifna and Ifnb in KEP mammary tumors were determined by qPCR and normalized to β-actin (n=5 animals/group). Mice were analyzed one day after the second docetaxel injection. Graphs show the mean ± SEM in ΔCt values. (b-c) Transcripts of Isg15 and Oas1a in orthotopically transplanted K14cre;Trp53^F/F (KP) tumors (Isg15: control ab n=7 animals, anti-CSF-1R n=8 animals; Oas1a: n=8 animals/group) (b) and subcutaneous MC38 tumors (n=8 animals/group) (c) treated as indicated were determined by qPCR and normalized to β-actin. Mice were analysed at a tumor size of 100mm² (KP) or after 12 days from the start of the treatment (MC38). Graphs show the mean ± SEM in ΔCt values. (d) Representative dot plots of a KEP tumor illustrating the gating strategy for cell sorting by flow cytometry. After cell separation based on CD11b expression by magnetic columns, the CD11b⁺ and CD11b^- fractions were stained as described in Methods followed by flow cytometry-based sorting of intratumoral cell populations. (e) Representative dot plots of a KEP tumor illustrating the gating strategy for the identification of pDCs. Antibody panel “tumor panel II” was used. Arrows indicate directionality of sub-gates. (f) Proportion of plasmacytoid dendritic cells (pDCs) in mammary tumors of end-stage KEP mice as determined by flow cytometry (control ab n=5 animals, anti-CSF-1R n=3 animals, cisplatin/control ab n=5 animals, cisplatin/anti-CSF-1R n=4 animals). (g-h) Transcripts of Ifna and Ifnb in CD11b⁺F4/80^-Ly6G^-Ly6C⁺ monocytes (Ifna and Ifnb: n=4 animals/group), CD45⁺CD11b^-CD11c^- lymphocytes (Ifna: n=4 animals/group; Ifnb: cisplatin/control ab n=3 animals, cisplatin/anti-CSF-1R n=4 animals), CD11b⁺F4/80^-Ly6G⁺Ly6C^low neutrophils (Ifna and Ifnb: cisplatin/control ab n=2 animals, cisplatin/anti-CSF-1R n=4 animals) and CD45^-CD11b^-CD31^- tumor cells/fibroblasts (Ifna: n=4 animals/group; Ifnb: cisplatin/control ab n=4 animals, cisplatin/anti-CSF-1R n=3 animals) isolated from end-stage KEP tumors were determined by qPCR and normalized to β-actin. (i) Transcript of Ifna in cultured bone marrow-derived macrophages treated for 24h with either control antibody or anti-CSF-1R in the presence of KEP cancer cell line-derived conditioned medium. Data are representative of 4 independent experiments. Data presented in a-c and f-h are mean values ± SEM and statistical analysis was performed using two-tailed Mann–Whitney test. CIS, cisplatin; DOCE, docetaxel.