Figure 2.

Administration of Bifidobacterium, a tumor-targeting member of the microbiota, sufficiently recovers the antitumor efficacy of anti-CD47 immunotherapy in nonresponders. (A) Statistical analysis of the percentage of HIF1α-positive area on different-volume tumor tissue slides. (B) Detection of Bifidobacterium in tumor tissues and lung sites after treatment by anaerobic culture. MC38 tumor–bearing Tac mice were intravenously injected with 1.5 × 10⁷ CFU activated Bifidobacterium three times when tumor sizes approached 200 mm³. 7 d after the final injection, tumor tissues were collected and homogenized for anaerobic culture of Bifidobacterium. N.D., not detected. (C and D) MC38 tumor–bearing Tac mice were i.t. injected with 70 µg anti-CD47 antibody (Ab) or 70 µg rat IgG on days 9 and 13, respectively. (C) Tumor growth kinetics of intravenous injection of Bifidobacterium. MC38 tumor-bearing Tac mice were treated with 70 µg anti-CD47 Ab or 70 µg rat IgG on days 9 and 13. For i.v. injection of Bifidobacterium, mice were intravenously injected with 1.5 × 10⁷ CFU Bifidobacterium or PBS on the same days on days 6, 9, and 13. (D) For i.t. injection of Bifidobacterium, mice were i.t. injected with 2.5 × 10⁶ CFU Bifidobacterium or PBS on the same days as i.v. injection of Bifidobacterium. The i.t. Bif + anti-CD47 group were compared with the IgG group. (E) EG7 tumor–bearing TAC mice were treated with 70 µg anti-CD47 Ab or 70 µg rat IgG on days 11 and 13. I.t. injection of 2.5 × 10⁶ CFU Bifidobacterium or PBS was administered on days 8, 11, and 13. Tumor volume was measured at the indicated time points. (F–H) Newly arrived Tac C57BL/6 mice (F and H) and germ-free (GF) mice (G) 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 days 9 and 13, respectively. For oral administration of Bifidobacterium (Bif), mice were gavaged with 5 × 10⁸ CFU Bifidobacterium or PBS on days 6, 9, and 13, respectively. The Tac Bif gavage + anti-CD47 group was compared with the Tac IgG group. (H) Mice were i.t. injected with 100 µl antibiotic cocktail solution or PBS every other day. Tumor volume was measured at the indicated time points. One representative experiment from at least two experiments yielding similar results is depicted in A–H. Each group in A and B contains at least three samples. The number of mice in each group is depicted in the panels C–H. Presented as mean ± SEM. *, P < 0.05 (nonpaired Student’s t test; A); *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (two-way ANOVA; C–H).