Figure 3. Characterization of breast epithelial cells transfected with oncogenic PIK3CA with MET.

MCF-10A–derived cells co-transfected with various MET genotypes and PIK3CA-E545K (A) or PIK3CA-H1047R (B) were seeded in 12-well plates in triplicate with a density of 2 × 10⁴ per well in 2.5% horse serum with withdrawn EGF, insulin, and hydrocortisone, supplemented with HGF (40 ng/ml) for 3 days. Cells were counted each day. Data are mean ± standard deviation of triplicates, representative of two independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control cells, ANOVA). C. Effects of MET aberrations on mammary acinar morphogenesis were tested as described in material and Methods. MCF-10A cells transfected with PIK3CA-H1047R and various MET genes were resuspended in modified growth medium (2.5% horse serum and 5 ng/ml EGF) containing 2% Matrigel supplemented with HGF (40 ng/ml). Representative field images of acini were taken on day 8; original magnification, ×40. The clonogenic assay was analyzed as described in Materials and Methods. The MCF-10A–derived cells (D, F) were seeded in triplicate at a density of 1000 cells in 60-mm dishes in DMEM/F12 medium supplemented with 2.5% horse serum and 40 ng/ml HGF. The dishes were scanned on day 10. Quantitative analysis of the total number of clones was performed with AlphaVIEW SA software (E, G). Data are mean ± standard deviation of triplicates, representative of two independent experiments (**, P < 0.01; ***, P < 0.001, vs. cells transfected with mutant PIK3CA alone, ANOVA). Cell invasion in vitro was analyzed as indicated in Materials and Methods. The MCF-10A–derived cells were induced to invade with HGF (40 ng/ml) and fibronectin (5 μg/ml). H. Cells invading through Matrigel were photographed at × 100 magnification. I. Data are mean ± standard deviation of triplicates, representative of two independent experiments (***, P < 0.001; ****, p < 0.0001 vs. vector, ANOVA).