Metabolic Competition-Driven Antitumor Immunity of glpX-Deficient Engineered Salmonella via Glucose Deprivation and STING Activation in Bladder Cancer.

Background: Bacteria-mediated cancer therapy is emerging as a promising approach due to its low cost, scalability, and sustained efficacy. While bacterial immunomodulation has been extensively studied, the role of bacterial metabolic competition, particularly for glucose, in tumor suppression remains unclear. We aimed to investigate how engineered Salmonella strains exploit metabolic competition to trigger antitumor immunity and improve therapeutic outcomes in bladder cancer models. Methods: Using genetic screening, we generated a gluconeogenesis-deficient Salmonella strain, the AISIH strain with the glpX gene knocked out (GLA1), with enhanced glucose uptake. In vitro and in vivo assays were performed to assess tumor cell glucose deprivation, mitochondrial stress, cytosolic mitochondrial DNA (mtDNA) release, and activation of the cGAS/STING pathway. An engineered strain, GLA2, was generated from the AISIH strain by knockout of the glpX gene and was subsequently further engineered to locally deliver an anti-tumor necrosis factor-α (TNF-α) nanobody under quorum-sensing control. Efficacy was evaluated in subcutaneous and orthotopic bladder cancer models, with analysis of tumor-infiltrating immune cells, dendritic cell (DC) maturation, and T cell priming. Results: GLA1 competitively depleted glucose in the bladder tumor microenvironment, inducing energy stress, mitochondrial damage, and mtDNA release, which noncanonically activated the STING pathway. This promoted type I interferon production, DC maturation, and antitumor immune responses. However, TNF-α-mediated vascular abnormalities limited efficacy. GLA2 normalized tumor vasculature via local anti-TNF-α delivery, enhancing nutrient competition and immune activation. Both strains markedly suppressed tumor growth, with GLA2 showing superior efficacy due to synergistic metabolic and immune reprogramming. Conclusions: This study demonstrates that bacterial competition for glucose is a pivotal mechanism of antitumor activity in bladder cancer, linking metabolic stress to STING-dependent immune activation. Engineered Salmonella strains that combine enhanced glucose uptake with local vascular normalization offer a robust platform for bladder cancer. These findings highlight a novel strategy to integrate microbial metabolic engineering with immune modulation, providing a foundation for precision bacteriotherapy in oncology.