Fig. 6.

Blockade of ICAM-1 using a neutralizing antibody can suppress breast cancer metastasis. A, B Western blotting (A) and phospho EGFR ELISA (B) of MDA-MB-231 cells treated with various doses of ICAM-1 neutralizing antibody for 24 h. C, D Transwell migration, invasion assay (C) and (D) western blotting of MDA-MB-231 cells treated with ICAM-1 neutralizing antibody (500 ng/ml, 24 h). E Co-IP with EGFR antibody and western blotting to evaluate the interaction between ICAM-1 and EGFR in MDA-MB-231 cells treated with ICAM-1 neutralizing antibody. F Co-IP with EGFR antibody and western blotting to evaluate the interaction between EGFR and EGF in MDA-MB-231 cells treated with ICAM-1 neutralizing antibody. rh-EGF was added in cell lysates. G Schematic illustration of animal experiments. H, I H&E staining (H) and the number of metastatic nodules (I) of lung in MDA-MB-231 orthotopic xenograft mice treated with ICAM-1 neutralizing antibody. Scale bar = 200 μm. J-M Phospho EGFR ELISA (J), IHC (K), western blotting (L), and in situ PLA (M) of in MDA-MB-231 orthotopic xenograft tumors mice treated with ICAM-1 neutralizing antibody. Scale bar = 100 μm. N Schematic illustration of the mechanism underlying ICAM-1-mediated EGFR activation, leading to cancer metastasis in breast cancer. Highlighted arrows indicate the pathways suggested by our study. Data are presented as mean ± SD and analyzed using one-way ANOVA or Student’s t-tests. n.s, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001