Fig 1: Anti-CSF-1R treatment promotes metastasis in the 4T1 model. (A) Growth curves of 4T1-luc mammary tumors treated with anti-CSF-1R (n = 10) or IgG control (n = 9) (both 200 µg/mouse every second day), two-way ANOVA. (B) On the day of sacrifice, the lymph nodes, lung and liver were dissected from the treated mice 10 min after a luciferin substrate injection and were imaged for metastatic burden using IVIS. The table shows metastasis positive organs on a yes/no basis, based on the IVIS signal and are shown as metastasis positive/total number of mice. The lower graphs show the radiance signal for each organ. Significant outliers were excluded from statistical analyses based on Grubbs' test and have been marked with a cross. Statistical significance was analyzed using two-tailed Student's t-test with Welch's correction. (C) ELISA analysis of serum M-CSF levels from mice treated either with IgG or anti-CSF-1R, two-tailed Student's t-test. (D) End point hematological analysis of monocytes analyzed with Sysmex XT-2000iV in IgG and anti-CSF-1R-treated mice. (E) Immunofluorescence staining of tumor sections (peritumoral area) with anti-F4/80 (green) and anti-MR antibodies (red) in IgG and anti-CSF-1R-treated mice. Quantification of double positive cells was done using ImageJ of three to five microscopic fields/mouse/treatment and analyzed using two-tailed Student's t-test. (F) Flow cytometric analysis of cell surface CSF-1R and G-CSFR expression on peripheral blood CD11b+Ly6C+ myeloid cells in C57BL/6 wild type (WT) and G-CSFR knockout (KO) mice where gate Q1 represents the monocytic population (Ly6G-) and gate Q3 the granulocytic population (Ly6G+). Because we used human recombinant G-CSF conjugated with AlexaFluor488 (G-CSF-488) for the detection of G-CSFR, the G-CSFR KO mice served as a control for specific binding of the protein. The histograms show binding of G-CSF-488 to the monocytic and granulocytic populations in WT (black line) and KO (gray-filled line) mice. (G) Flow cytometric analysis of cell surface CSF-1R and G-CSFR expression on peripheral blood CD11b+Ly6C+ myeloid cells in Balb/c mice without tumors or carrying a 4T1 tumor (size 5 × 5 mm). As in (F), the Q3 gate served as a positive cell population for G-CSFR expression, and is shown as the gray-filled histograms. Note that the 4T1 tumor induced a granulocytic cell population double positive for CSF-1R and G-CSFR (Q2).