6B12 antibody suppresses the formation of the pre-metastatic niche in the lungs. Unless otherwise indicated, the primary tumor in mice was induced by s.c. injection of CSML100 cells followed by i.v. administration of S100A4(+/+) MEFs. (A) Immunohistochemical staining of pre-metastatic lung tissue sections from tumor-bearing mice boosted with S100A4(+/+) and S100A4(-/-) MEFs using antibodies against CD3 (red) and FN (green) (400x magnification). (B) qRT-PCR analysis of FN expression in lungs of tumor-bearing mice non-boosted or boosted with S100A4(+/+) MEFs. Mice only boosted with S100A4 (+/+) MEFs served as control. (C) qRT-PCR analysis of FN and G-CSF expression in the pre-metastatic lungs after treatment with the 6B12 antibodies. (D) Quantification of the FN protein expression level in pre-metastatic lungs of mice treated with the 6B12 antibodies (left panel). Right panel: Western-blot analysis of FN expression in pre-metastatic lungs of mice treated with 6B12 or IgG control (right panel). (E) Quantification of CD3+ T-cells at the site of blood vessels in pre-metastatic lungs of CSML100 tumor-bearing mice, co-injected with S100A4(+/+) or S100A4(-/-) MEFs and treated with 6B12 or IgG control (left panel). Representative images of immunofluorescence staining of pre-metastatic lungs using antibodies against CD3 (green) to visualize T-cells and smooth muscle actin (red) to visualize blood-vessels. The nucleus is stained by 4′,6-diamidino-2-phenylindole (DAPI, blue). (*, P < 0.05; **, P < 0.01; ***, P < 0.001, 400x magnification, right panel).