Supplementary Figure 1: CSF-1R blockade does not influence spontaneous metastasis formation.

(a) Presence of CD68⁺ macrophages in untreated human invasive lobular carcinomas and in adjacent normal breast tissue. Influx of CD68⁺ macrophages was scored based on immunohistochemistry (normal breast tissue, n=5 patients; invasive lobular carcinomas, n=14 patients). Representative images are shown. Scale bar=25μm. (b) Representative IHC staining of F4/80⁺ macrophages in a mammary tumor of a KEP mouse and in a normal mammary gland of an age-matched WT mouse. Scale bar=20μm. Percentage of CD11b⁺F4/80⁺ macrophages gated on CD45⁺ cells in KEP mammary tumors (n=20 animals) and in normal mammary glands of age-matched WT mice (n=4 animals). (c) Representative flow cytometry histogram and plot showing CD206 and MHCII expression, respectively, on F4/80⁺ macrophages in a KEP mammary tumor. Data are representative of 5 independent KEP mammary tumors. (d) Flow cytometry histograms showing CSF-1R expression levels (white) and Fluorescence minus one (FMO) control (grey) in 3 independent KEP mammary tumors. (e) Proportion of CD45⁺ immune cells of total live cells in tumors of time point-sacrificed KEP mice treated with control ab (n=6 animals) or anti-CSF-1R (n=5 animals) as determined by flow cytometry. (f) Organs collected from KEP mice bearing end-stage mammary tumors treated with control ab (n=20 animals) or anti-CSF-1R (n=22 animals) were microscopically analyzed for the presence of metastases. Percentage of tumor-bearing KEP mice with metastases is displayed. p=0.1 by Fisher’s exact test (Two-sided). (g) Schematic overview of continuous and adjuvant antibody treatment in the KEP-based spontaneous metastasis model as described in Methods. (h) Kaplan-Meier metastasis-specific survival curves of recipient mice orthotopically transplanted with tumor fragments from KEP mice and treated either continuously (control ab n=13 animals, anti-CSF-1R n=14 animals) or in an adjuvant setting (control ab n=13 animals, anti-CSF-1R n=11 animals). An event is defined as an animal that was sacrificed because of clinical signs of metastatic disease. (i) Quantification of the number of spontaneous pulmonary metastases in mice treated either continuously (control ab n=9 animals; anti-CSF-1R n=8 animals) or in an adjuvant setting (Control ab n=12 animals; anti-CSF-1R n=9 animals). Data presented in b, e and i are mean ± SEM and statistical analysis was performed using two-tailed Mann–Whitney test.