Figure 1.

The antitumor responses of CD47 blockade rely on gut microbiota accumulated outside the GI tract. (A–D) C57BL/6 mice were injected subcutaneously with 5 × 10⁵ MC38 cells and treated i.t. with 50 µg anti-CD47 antibody (Ab) or rat IgG on days 10 and 14 after tumor inoculation. Tumor volume was measured at indicated time points. (A) MC38 tumor growth kinetics in newly arrived Jax and Tac mice treated with anti-CD47 Ab or rat IgG. (B) Jax and Tac mice were cohoused for 3 wk before MC38 tumor inoculation and anti-CD47 Ab treatments. (C) Jax and Tac mice were given an oral antibiotic cocktail solution (0.5 mg/ml ampicillin, 0.5 mg/ml gentamicin, 0.5 mg/ml metronidazole, 0.5 mg/ml neomycin, and 0.25 mg/ml vancomycin) 3 wk before MC38 tumor inoculation and anti-CD47 Ab treatment and the oral feeding of antibiotics was stopped at the end of the experiments. (D) MC38 tumor growth kinetics in germ-free mice treated with anti-CD47 Ab or rat IgG. (E) Newly arrived Jax C57BL/6 mice were s.c. injected with 5 × 10⁵ MC38 cells. Each mouse was i.t. injected with 70 µg anti-CD47 Ab or 70 µg rat IgG on day 9 or day 13, respectively. The mice in antibiotics groups were i.t. injected with 100 µl antibiotic cocktail solution every other day. Tumor volume was measured at indicated time points. One representative experiment (A andD) is depicted from at least two experiments yielding similar results. Each group contains at least four mice. Results in C were pooled based on two experiments yielding similar results. Each group contains ≥10 mice. Results in B and E were from one experiment; each group contains at least six (B) and four (E) samples. Presented as mean ± SEM. Two-way ANOVA was used to analyze data in A–E. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.