Combination of cetuximab and U3-1287 treatment of CtxRtumors leads to growth delay in vivo. (A) Growth-delay effects of U3-1287 in CtxR tumors in vivo. The black arrow designates the starting time point of U3-1287 treatment. The average tumor volume of mice treated with IgG is included in all groups for comparison purposes. (B) Combination treatment with cetuximab and U3-1287 inhibited HER3 expression and HER2 activation in vivo. Total and phosphorylation levels of HER2 and HER3 proteins in CtxR xenograft tumors were determined by immunoblot analysis after cetuximab, U3-1287 or the combination of cetuximab and U3-1287 treatments. (C) The inhibition of phospho-HER3 and phospho-HER2 expression in CtxR tumors after combinatorial treatment corresponds with reduced proliferation and increased apoptosis. CtxR tumor samples after cetuximab, U3-1287 or the combination of cetuximab and U3-1287 treatment in vivo were prepared and analyzed for Ki67 and cleaved caspase 3 by immunohistochemistry. Images were quantified via taking the average staining intensity measured from 3 tumors per treatment group (3 images/tumor, n = 9). Magnification 100X.