Icariin Enhances the Enzymatic Activity of N‐acetylgalactosaminidase to Augment Akkermansia Abundance in Gut Microbiota for Improved PD‐1 Blockade Efficacy in Tumor Suppression

ABSTRACT Akkermansia (Akk) is a commensal bacterium in gut microbiota known to enhance anti‐tumor immunity and improve responses to immunotherapy. However, the clinical translation of Akk‐based therapies still faces critical challenges, including intestinal colonization difficulties, inter‐patient variability, and safety risks. Here, we identified the flavonoid icariin as a novel prebiotic candidate that could specifically enrich intestinal Akk abundance under various conditions both in vivo and in vitro. Oral administration of icariin selectively increased intestinal Akk abundance in tumor‐bearing and non‐tumor mice, regardless of their immunocompetent or immunodeficient status. Moreover, in vitro assays confirmed that icariin promoted Akk growth in a mucin‐dependent manner. Transcriptomic and metabolomic analyses revealed that icariin enhanced mucin‐associated metabolic pathways to support Akk growth. Mechanistically, we found that icariin increased the enzymatic activity of N‐acetylgalactosaminidase Amuc_0920 by stabilizing key residues at the binding sites for the substrate GalNAc, which enhanced mucin catabolism and promoted Akk growth. Functionally, we further found that this Akk enrichment enhanced intratumoral CD8+ T cell functions by single‐cell RNA sequencing. Consequently, icariin‐induced Akk enrichment significantly enhanced the efficacy of anti‐PD‐1 immunotherapy in tumor mouse models. These findings establish icariin as a promising Akk‐targeting prebiotic that selectively enriches Akk to improve cancer immunotherapy.