Analysis of 4T1 tumors from mice treated with AEE788 + dovitinib. (A) Frozen sections from 4T1 tumors harvested after 14 days of treatment as described in Figure 5A were stained for proliferation (phospho-histone H3; blue), apoptosis (cleaved caspase 3; red) and endothelial cells (CD31; green). Scale bars, 100 μm. (B) Phosphorylated histone-3 (PH3)-positive cells were manually counted and expressed as the average number of cells per field. Proportion of tumor area expressing cleaved Caspase-3 and CD31 immunoreactivity were quantified using ImageJ. N = 3 to 4 tumors for each group with five fields analyzed per tumor (15 to 20 images from each treatment). Bars represent mean ± SD in each treatment group. (C) 2.5 × 105 4T1 cells were injected into tail veins and indicated treatments were started 7 days later for a total of 11 days. Lungs were harvested and metastases were quantified. N = 5, representative of two separate experiments. *P < 0.05; **P < 0.01 (Mann-Whitney U-test).