Extended Data Fig. 4: Comparative profile of intratumoral treatment with engineered microbial neoantigen vaccines.

a–d, BALB/c mice with established hind-flank CT26 tumors were treated when average tumor volumes were 150–200mm³. a,b, Mice received intratumoral injection of wildtype EcN, EcNc^Δlon/ΔompT or EcNc^{Δlon/ΔompT/LLO+} either without neoantigen expression (NC) or the strain mixture nAg¹⁹ on day 0. a, Relative body weight of CT26 tumor-bearing mice (n = 5 mice for PBS, EcN NC, and EcN nAg¹⁹, 7 for other groups, **P = 0.0034, ****P < 0.0001, ns = P > 0.05, two-way ANOVA with Tukey’s multiple comparisons test). b–d, Mice received intratumoral injection on day 0 and 8 (n = 7 mice for EcNc^Δlon/ΔompT NC and EcNc^{Δlon/ΔompT/LLO+} nAg¹⁹, 8 for other groups). b, Tumor growth curves (**P = 0.0020, ****P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test). c, Kaplan-Meier survival curves for CT26 tumor-bearing mice (**P = 0.0061, **P = 0.0076, Log-rank Mantel-Cox test). d, Individual tumor trajectories after intratumoral treatment with indicated microbial strain. e,f, BALB/c mice were implanted with CT26 tumors on both hind flanks. When average tumor volumes were 100–150mm³ mice received an intratumoral injection of PBS, EcNc^{Δlon/ΔompT/LLO+} (NC), or EcNc^{Δlon/ΔompT/LLO+} nAg¹⁹ into a single tumor. e, Tumor growth curves (n = 6 mice for EcNc^{Δlon/ΔompT/LLO+} nAg¹⁹, 5 for other groups, **P = 0.0014, ****P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test). f, CFU g⁻¹ of tumor (n = 4 mice for EcNc^{Δlon/ΔompT/LLO+} nAg¹⁹, 5 for all other groups), LOD 1 ×103 CFU g⁻¹. a,b,e,f, Data are mean ± s.e.m.

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