Fig. 6: Anti-tumor effects of PD-1 blockade on Nedd8 deficient breast tumors.

The Nedd8 gene was deleted using CRISPR/Cas9 in EO771 cells. a NEDD8 protein expression was tested at different passages by Western Blotting and cell proliferation was monitored using a live-cell imaging system. Representative experiment of 3 independent repeats was shown. b Four hundred thousand control (Ctrl) or Nedd8 knock-out (KO) EO771 cells were injected subcutaneously (s.c.) in 100 μl medium in 6–10 weeks old female C57BL/6NTac mice. When tumors were palpable, 50 μg of an αPD-1 antibody (RMP1-14) or the Rat IgG2a isotype control (2A3) were injected intraperitoneally (i.p.) in 100 μl PBS on day 5, 8 and 11 (8 mice per group). Tumor volumes were compared on day 24. Representative experiment of 3 repeats was shown. c Ctrl or Nedd8 KO EO771 cells were injected s.c. as above and treatment began when average tumor volume reached 50 mm³ on day 14, 17 and 20 (at least 5 mice per group). Tumor growth was followed in all mice until the study endpoint. Survival of the mice was demonstrated in a Kaplan–Meier curve. Representative experiment of 2 independent repeats was shown. d Six hundred thousand ctrl or Nedd8 KO EO771 cells were injected s.c. as above. A depletion antibody against CD8+ T cells (2.43) or the Rat IgG2b isotype control (LTF-2) was injected i.p. in 100 μl PBS every 3 days from day 4 (200 μg per mouse, 7 mice per group). Tumor growth was compared on day 17 and survival of the mice was demonstrated in a Kaplan–Meier curve. Representative experiment of 2 independent repeats was shown. Data were shown as mean ± SEM. Statistical differences on the tumor volumes were determined using unpaired two-tailed T-test and survival differences were calculated using Kaplan–Meier curves and a log-rank test (Mantel–Cox). *p < 0.05; **p < 0.01; ****p < 0.0001. Source data are provided as a source data file.