Tumor-targeted aptamer-conjugated engineered bacteria for CXCL9 cytokine delivery in non-small cell lung cancer immunotherapy

Abstract Background Bacterial cancer therapies have regained attention as a strategy to remodel the immunosuppressive tumor microenvironment (TME). Engineered bacteria equipped with tumor-targeting moieties can enhance intratumoral specificity; however, safety concerns and the need for repeat dosing limit their translational potential. Methods Here, we developed an aptamer-conjugated engineered bacterial strain (ApCB) carrying CXCL9, and evaluated its tumor-targeting colonization and immunomodulatory effects in a subcutaneous LLC tumor model. After determining the in vitro minimum inhibitory concentration (MIC) of kanamycin and extrapolating an equivalent in vivo dose based on mouse blood volume, we implemented a tail-vein antibiotic administration strategy to precisely regulate intratumoral bacterial burden. Results Antibiotic treatment substantially lowered peak bacterial abundance in tumors while retaining a viable intratumoral bacterial reservoir, allowing sustained bacterial proliferation and periodic CXCL9 release without repeated re-administration of engineered bacteria. In vivo, ApCB–CXCL9 treatment significantly inhibited tumor growth, induced extensive tumor necrosis, decreased Ki67 expression, and increased intratumoral CD8⁺ T-cell infiltration together with elevated effector cytokines (IFN-γ, TNF-α). Conclusion These findings indicate that aptamer-guided engineered bacteria combined with antibiotic-mediated population control can safely and controllably remodel the tumor immune microenvironment, offering a practicable approach for sustained delivery and clinical translation of bacterial immunotherapies.