Nanoprodrug targeting tumor-associated intracellular bacteria enhances colorectal cancer immunotherapy.

The tumor-associated intracellular microbiota influences cancer progression by fostering an immunosuppressive tumor microenvironment. Targeting pro-tumor intracellular bacteria may represent a promising strategy for cancer immunotherapy. Herein, we reveal that Fusobacterium nucleatum (F. nucleatum) is preferentially enriched within M2-polarized macrophages in both human colorectal cancer (CRC) specimens and cellular models. Notably, its intracellular presence correlates significantly with reduced CD8 T cell infiltration, highlighting its immunosuppressive function. To disarm this microbial shield, we develop infection-specific ciprofloxacin (CIP) prodrug nanoparticles (MTCP-NPs) conjugated to an M2 macrophage-targeting peptide (IL4Rpep-1) via a cathepsin B (CTSB)-cleavable linker (FRRG) to selectively kill intramacrophage F. nucleatum. MTCP-NPs undergo IL4 receptor-mediated endocytosis in F. nucleatum-infected macrophages, where intracellular CTSB triggers CIP release to eliminate F. nucleatum and suppress indoleamine 2,3-dioxygenase (IDO) secretion. In F. nucleatum-infected CRC models, MTCP-NPs clear intracellular bacteria, alleviate immunosuppression by downregulating IDO and reducing MDSCs to mobilize CD8 T cell immunity. Furthermore, this treatment modulates the gut microbiota by reprogramming tryptophan metabolism. When combined with anti-PD-L1 therapy, MTCP-NPs amplify anti-tumor efficacy and prolong survival while promoting the generation of memory-like T cells associated with sustained immune responses. This strategy establishes a unique microbiome-immunotherapy framework by targeting tumor-associated intracellular bacteria to unleash T cell responses.