Fig. 5

Effective combinatorial strategy targeting heterogeneous subclones in mRCC. a, b Combined effects of afatinib and dasatinib on viability of mRCC cells were analyzed 6 days after treatment under 2D non-adherent culture conditions a or 3D ECM scaffold culture system b. c Effects of afatinib and dasatinib combination therapy on EGFR and Src downstream pathways were validated by immunoblotting. Cells were incubated with 0.5 μM afatinib and/or dasatinib for 1 h. GAPDH = loading control. Cells that were mock-treated with 0.5 μM DMSO served as negative controls. Error bar = SEM (n = 3 for each group). **P <0.01, ***P <0.001. d–i Superior antitumor efficacy of combinations of afatinib and dasatinib in mRCC subcutaneous xenografts. d Mice bearing mRCC tumors (=5 mice/group) were p.o. dosed with afatinib (each at 20 mg/kg) or i.p. administered dasatinib (each at 15 mg/kg), either alone or in combination, on a daily dosing regimen for up to 15 days. Growth curves based on tumor volumes are shown as the mean ± SEM for each time point. *P <0.05, **P <0.01. e Changes in body weight of mice treated with afatinib and/or dasatinib. Body weight was measured on the indicated days. Data show mean ± SEM. Tumor tissues from each group were harvested on day 15 and subjected to immunoblot analysis with the indicated antibodies to detect p-EGFR, p-Src, and p-AKT (f; GAPDH = loading control), or immunostained with anti-p-ERK antibody g. Proliferation and apoptotic rates in each group were determined by Ki-67 immunostaining h and TUNEL assay i. Results are presented as mean values ± SEM. *P <0.05, ***P <0.001. Scale bars = 100 μm