Adhesion assays

For measurements of cell surface integrin levels wells were coated with mouse monoclonal antibodies against integrin extracellular domains (Chemicon- Millipore) QCM™, ECM535). Monoclonal antibodies against α1, α2, α3, α4, α5, αV, αVβ3, β1, β2, β3, β4, β6, αVβ5, α5β1 integrins and a goat anti-mouse IgG as negative control were immobilised. Cells were cultured for 24 h, in the presence or absence of the IGF-IR inhibitor (1 μM AG1024). Cells were washed three times with a serum free medium and then suspended in serum free medium and 1.5 × 10⁵ cells were placed in the wells. After 2 h incubation the cells were washed three times with serum free DMEM so that non-adherent cells were removed. Afterwards the attached cells were lysed and incubated with CyQUANT ™ GR Dye. Measurements were made on a fluorimeter (Infinite^®, Tecan) with excitation at 485 nm and emission at 530 nm.

In order to evaluate the adhesive potential of breast cancer cells the following adhesion protocol was conducted as described previously⁶⁰. Briefly, 40 μg/ml of collagen type I in PBS and 0.1% BSA solution in serum free medium were prepared. 96-well plate coated for 12 h at 4 °C with collagen type I solution (30 μg/ml). Then, the solution was removed and the plate was air-dried. Cancer cells were deprived of serum for 8 h prior to the adhesion assay and in the case of blocking with P1E6 antibody or anti-mouse IgG, a 30 min incubation before the beginning of the assay in final concentration of 20 μg/mL for both antibodies was used. Then the cells were detached with PBS-EDTA 1x, re-suspended with 0.1% BSA and seeded at a density of 2 × 10⁴ cells/well. Cells were incubated for 30 min in order to be allowed to adhere to the surface. Non-adherent cells were removed with serum free medium and then cells were incubated with medium supplemented with 10% FBS for 4 h for recovery. After incubation period, Premix WST-1 (water-soluble tetrazolium salt) Cell Proliferation Assay System (Takara Bio Inc., Japan) was added at a ratio 1:10 and the absorbance at 450 nm was measured (reference wavelength at 650 nm).