Figure 2.
Integrin αvβ6 and HER2-blockade and breast cancer cell invasion. A) Expression of integrin αvβ6 and HER2 in a breast cancer cell line panel assessed by flow cytometry. Isotype controls all showed lower expression nearer the y-axis side, while integrin αvβ6 and HER2 expression shift the curves to the right (see Supplementary Table 4, available online, for full panel of cell lines analyzed). B) Transwell invasion assay of breast cancer cell lines expressing varying levels of integrin αvβ6 and HER2. 5 x 104 cells/well were seeded and the number of cells that invaded was counted after 72 hours. C and D) Breast cancer cell-line invasion is integrin αvβ6 dependent. Cells were subjected to either 30 minutes of incubation with IgG or αvβ6 blocking antibody (β6 Ab) (10 μg/mL) (C) or 72 hours of transfection with control or β6 siRNA (20μM) (D) and subjected to a Transwell invasion assay as before. E and F) Breast cancer cell-line invasion is HER2 dependent. Cells were pretreated for 30 minutes with IgG or Trastuzumab (TRA) (10 μg/mL) (E) or transfected for 72 hours with control or HER2 siRNA (20μM) (F) and subjected to a Transwell invasion assay. G) Cells were pretreated for 30 minutes with IgG, β6 Ab, TRA (all 10 μg/mL), or a combination of the blocking antibodies, and subjected to a Transwell invasion assay. All experiments were performed in triplicate, representative experiments shown (n = 6, error bars represent 95% confidence interval). *P = .05, **P = .01, ***P < .001, relative to IgG or control-treated cells. C-F; Student’s t-test, B and G; one-way analysis of variance with Bonferroni’s Multiple Comparison Test. All tests were two sided. Please also see Supplementary Figure 2, available online. HER2 = Human Epidermal Growth Factor Receptor 2; IgG = immunoglobulin; TRA = trastuzumab.