Attenuated Salmonella secreting IFN-beta via the flagellar secretion system enhances antitumor immunity and therapeutic efficacy

Purpose: Bacterial cancer therapy offers a unique approach to target tumor tissues and modulate the immune microenvironment. Here, we aimed to establish an attenuated Salmonella platform capable of secreting interferon-beta(IFN-beta) through the flagellar type III secretion system(FT3SS) to potentiate antitumor immunity, and to evaluate its therapeutic efficacy as monotherapy and in combination with PD-L1 blockade. Methods: The attenuated Salmonella Typhimurium strain was constructed by deleting the rcsB gene from the parental BRD509 background and engineered to secrete IFN-beta through the FT3SS under L-arabinose induction. IFN-beta secretion was verified by Western blot and ELISA. The biodistribution of the strain was evaluated using IVIS bioluminescence imaging. The antitumor efficacy and immune activation induced by the IFN-beta secreting Salmonella were assessed in CT26 tumor-bearing mice by evaluating tumor growth inhibition, apoptosis, and immune cell activation. Combination therapy with an anti-PD-L1 antibody was performed to potentiate the therapeutic effect. In addition, the in vivo safety of the strain was confirmed by monitoring serum liver and kidney function markers, body weight, and H&E histology of major organs. Results: In this study, we developed an attenuated Salmonella strain capable of secreting IFN-beta through the FT3SS. IFN-beta secretion was regulated by L-arabinose induction without affecting the viability of the Salmonella strain. In vivo bioluminescence imaging of Lux-expressing IFN-beta secreting Salmonella demonstrated rapid tumor colonization and selective intratumoral proliferation, confirming its tumor-targeting capability. In CT26 tumor-bearing mice, treatment with the IFN-beta secreting strain followed by L-arabinose induction from day 3 post-injection inhibited tumor growth and induced tumor cell apoptosis. The therapy also promoted N1-type neutrophil infiltration and activation of antitumor immune responses, and its safety was confirmed in vivo . However, IFN-beta monotherapy did not completely eradicate tumors, and PD-L1 expression was upregulated in the tumor tissues after bacterial injection. Therefore, combination therapy with the IFN-beta secreting Salmonella and an anti PD-L1 antibody resulted in synergistic antitumor effects, including markedly enhanced tumor regression and prolonged survival compared to either monotherapy. These results highlight the clinical potential of this controllable bacterial platform for next-generation cancer immunotherapy. Conclusion: Overall, these findings demonstrate that the FT3SS-based IFN-beta secreting Salmonella represents a safe, potent, and versatile therapeutic platform that enhances antitumor immunity and synergizes with immune checkpoint blockade.