Fig. 3
The suppression of colorectal tumor growth targeting ATG7 is contingent upon the presence of CD8+ T cells. (A) Image of isolated tumors derived from immunodeficient nude mice (n = 5). 6-week-old male BALB/c nude mice were randomly divided into four groups: shNC group, shATG7 group, vehicle group and ATG7-IN-1 group. Mice were subcutaneously injected with 1 × 107 MC38 cells transfected with lentiviral vectors or control cells. When the tumor volumes reached 100 mm3, vehicle group and ATG7-IN-1 group were treated with ATG7-IN-1 (10 mg/kg) or vehicle solution once a day. Tumor tissues were harvested for weight measurement at day 21. (B-C) Tumor volumes were monitored every 3 days. (D) Weight of tumors was measured at time of sacrificed. (E) Schematic representation of the CD8 depletion strategy in shNC, shATG7-treated MC38 tumor-bearing mice or using ATG7-IN-1 to treat. Mice were injected intraperitoneally (i.p.) with 100 µg of either control isotype or αCD8 antibody on the indicated days (black arrows). Then mice were randomly divided into four groups: shNC group, shATG7 group, vehicle group and ATG7-IN-1 group. 1 × 107 MC38 cells were injected into syngeneic C57BL/6 mice and palpable tumors appeared on day 7. Tumor bearing mice were treated with either vehicle or ATG7-IN-1 daily from day 8 to day 20. At day 8, the efficiency of the CD8 depletion was determined by flow cytometry analysis on blood samples of mice chosen randomly. (F) Flow cytometry analysis of CD8+ T cells in tumors treated with shNC, shATG7 or anti-CD8. (G) CD8 protein expression was examined by immunohistochemical analysis (400×magnification). (H) Photograph of xenograft shNC, shATG7-treated MC38 tumors from mice treated anti-CD8. (I) Photograph of xenograft tumors from mice treated with ATG7-IN-1 or vehicle or anti-CD8. (J-M) The curve of tumor growth monitored at different time points and isolated tumor weight. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001