Figure 4: Single injection of probiotics expressing checkpoint inhibitors demonstrates robustness.
(A-C) BALB/c mice were implanted subcutaneously with 5×106 A20 cells on both hind flanks. When tumors reached ~150–200 mm3, mice received one intratumoral injection of EcN-lux, SLIC-2, or a combination of anti-PD-L1 and anti-CTLA-4 mAbs at 100 μg/mouse and 200 μg/mouse, respectively. (A) Mean absolute tumor trajectories (n = 8–10 tumors per group, 2-way ANOVA with Bonferroni post-test, ****P < 0.0001, error bars represent SEM of biological replicates); individual tumor trajectories shown in fig. S5A. (B) Serum concentrations of TNFα ( n = 3 mice per group, ordinary 1-way ANOVA with Holm-Sidak post-test, *P = 0.0385, data are represented as means +/− SEM. of biological replicates). (C) Rate of body weight change in g/day (n = 4–5 mice per group, ordinary 1-way ANOVA with Tukey’s post-test, *P = 0.0387, error bars represent SEM of biological replicates; ns, not significant). (D) Scatter plot showing each tumor’s final volume plotted against its initial tumor volume. Black line (y=x) represents the threshold where points below the line indicate tumor regression and points above the line indicate tumor growth. (E) Representative IVIS images of mice from the experimental groups described above, where mice were dosed once with nonlysing EcN-lux or SLIC-2. (F) Heatmaps quantifying total flux (photons/second) of luminescent bacteria populations over time, corresponding to IVIS images. (G) Plate reader experiment showing the oscillations of plated colonies from tumors harvested on days 3 and 14 after treatment and a grid showing the number of successful lysis events. (H-J) A20-bearing mice were grafted as stated above, and mice received a single intraveneous injection of either 5×106 EcN-lux or SLIC-2 via tail vein. (H) Mean absolute tumor trajectories (n = 9–11 tumors per group, 2-way ANOVA with Bonferroni post-test, ****P < 0.0001, error bars represent SEM of biological replicates); individual tumor trajectories shown in fig. S5D. (I) Representative IVIS images from mice treated with SLIC-2 and a heatmap quantifying the total flux (photons/second) of luminescent bacterial populations over time. (J) Plate reader experiment showing the oscillations of colonies plated from tumors harvested on day 14 after treatment and a grid of the number of successul lysis events. (K) Biodistribution of bacterial populations in the tumor and peripheral organs (liver, lung, spleen, and kidney) calculated as colony-forming units per gram of tissue (CFU/g).