Fig. 5.

Combined CB-839/anti-EGFR mAb therapy overcomes acquired cetuximab-resistance in HCA-7 CC-CR xenografts in vivo. (A) Mice with HCA-7 CC-CR cell line xenografts, a model of acquired cetuximab-resistance, were treated with either vehicle (black), CB-839 (red), cetuximab (blue) or cetuximab and CB-839 (purple) for 21 days. At this time point, treatment was stopped (dotted line). Tumor volumes were monitored to day 57 (n = 8 mice/group). Mice progressed on single agent CB-839 or cetuximab yet exhibit reduced tumor volume with combination cetuximab and CB-839. (B) IHC was performed on tumors from each of the treatment groups to evaluate Ki67 and pS6 expression. Shown are representative images (40×). The number of positive cells were counted in regions of interest on images from Ki67 IHC (C) and pS6 IHC (D) (n = 3–15 fields of view, at least 3 animals/treatment group). Reduced Ki67 and pS6 were observed when treated with CB-839 in combination with cetuximab compared to cetuximab or CB-839 alone. (E) Representative H&E images (0.46×) of tumor tissues from each of the treatment groups. (F) IHC was performed on tumors from each of the treatment groups to evaluate cleaved caspase-3 expression. Shown are representative images (20×). (G) The number of positive cells were counted in regions of interest on images from cleaved caspase-3 IHC (n = 3–15 fields of view, at least 3 animals/treatment group). Error bars represent ± SD. Statistical significance is defined as follows: ^#p = 0.0574, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)