Fig. 6

Effects of combined treatment of MEK inhibitor, EGFR inhibitor and PD-L1 inhibitor on syngeneic colorectal cancer tumor xenograft models with acquired resistance to MEKi. a. C57BL/6 and BALB/c immune-competent mice were injected subcutaneously in the right flank with MC38-MR and CT26-MR cells as described in the Materials and Methods. After two weeks (average tumor size 200–300 mm³) mice were treated with: PBS (phosphate-buffered saline) as control, MEKi (BAY86–9766 25 mg/kg every day for 5 days a week, by oral gavage), EGFRi (erlotinib 10 mg/Kg every day for 5 days a week by oral gavage), mouse PD-L1i (clone 10F.9G2) injected twice a week i.p. at total dose of 200 μg/mouse, MEKi+PD-L1i at doses described above and MEKi+EGFRi+PD-L1i at doses described above. The treatment was continued for 3 weeks. Mice were followed for additional 5 weeks. Each group consisted of 10 mice. The mean data are present. Tumor growth curves were calculated based on twice weekly times tumor measurements during the treatment period and after 5 weeks of observation after termination of therapy. Animals were sacrificed when tumors achieved 2.000 mm³ in size. b. At the end of treatment one mouse per group treated with MEKi, EGFRi, PD-L1i or with their combination was sacrificed. As control, we used one mouse engraft with MC38-MR and CT26-MR respectively that has not undergone to any type of treatment. Tumor samples were collected and total cell protein extracts were subjected to immunoblotting with the indicated antibodies, as described in Materials and Methods. Anti-tubulin antibody was used for normalization of protein extract content