Analyses of 4T1 tumors and lung metastases in mice treated with dovitinib, NVP-BEZ235 or the combination. (A) 4T1 tumors from mice treated for 14 days with the inhibitors described in Figure 1C were harvested, and frozen sections 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 was quantified using ImageJ. N = 3 to 4 tumors for each group with 5 fields analyzed per tumor (15 to 20 images from each treatment). (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. (D) After 14 days treatment with the combination of dovitinib (TKI, 20 mg/kg) and NVP-BEZ235 (10 mg/kg) or with vehicle, 4T1-tumor bearing mice were sacrificed, and blood was collected and plated. The graph shows the number and SD of tumor cell colonies growing/mouse/ml of blood; N = 5 to 9 animals per group. *P < 0.05 **P < 0.01 (Mann-Whitney U-test).