FIGURE 4.
Effect of therapeutic anti-EGFR or -HER2 mAbs and TKIs on EGFR/HER2 heterodimer concentration in SKOV-3 cells. 105 cells/well were treated with increasing concentrations (from 0.1 to 100 μg/ml) of Px (irrelevant antibody), Cetuximab, Trastuzumab, Cetuximab + Trastuzumab (ratio 1:1), and Pertuzumab (A and B); or with increasing concentrations (from 0.01 to 10 μm) of the TKIs Lapatinib and Erlotinib (C). Cells were then fixed with formalin for 2 min, and EGFR/HER2 heterodimers were quantified using the antibody-based TR-FRET assay with anti-EGFR (d2-labeled m425) and anti-HER2 (Lumi4 Tb-labeled FRP5) antibodies. After extensive washes, fluorescence was measured and the TR-FRET signal was expressed as ΔF665 (%) = ΔF665/F665Terbium (see “Experimental Procedures”). ΔF665 (%) represents the concentration of EGFR/HER2 dimers normalized to HER2 expression. Data are the mean ± S.E. of three independent experiments performed in triplicate. p, ***<0.001; **<0.01, n.s. non-significant, by one- and two-way analysis of variance (B and A, respectively), with Bonferroni's multiple comparison post-tests. D, Western blot analysis of SKOV-3 cells treated with increasing concentrations of TKIs without prior induction by EGF. Antibodies against total EGFR and HER2, phosphorylated EGFR and HER2, AKT and phosphorylated AKT and GAPDH were used to detect variations in EGFR and HER2 expression/phosphorylation and AKT phosphorylation.