Effects of vancomycin on the gut microbiota and metabolic profile to sensitize microsatellite-stable colorectal cancer to anti-PD-1 immunotherapy.

Abstract

e15580 Background: Microsatellite-stable (MSS) colorectal cancer (CRC) remains largely refractory to immune checkpoint blockade (ICB). While the gut microbiota regulates the tumor immune microenvironment, mechanisms linking antibiotic-induced dysbiosis to ICB efficacy remain unclear. We investigated whether vancomycin sensitizes MSS CRC to anti-PD-1 therapy and elucidated underlying mechanisms involving microbial metabolites and dendritic cell (DC) function. Methods: MSS CRC subcutaneous syngeneic models were established and treated with vancomycin, anti-PD-1, or their combination. Tumor growth was monitored longitudinally. Integrated multi-omics analysis (16S rRNA sequencing, metagenomics, and metabolomics) profiled gut microbiota and metabolic alterations. Molecular mechanisms were validated in bone marrow-derived DCs (BMDCs) and tumor-draining lymph nodes (TDLNs) using RNA sequencing, molecular docking, co-immunoprecipitation, and ubiquitination assays. Results: Compared with monotherapy, the combination of vancomycin and anti-PD-1 significantly suppressed tumor growth and prolonged survival. Microbiome analysis revealed that vancomycin dramatically altered gut microbiota composition, enriching beneficial bacterial taxa. Metabolomics identified marked downregulation of systemic and intratumoral asparagine levels in the combination group. Mechanistically, high asparagine levels impaired DC function specifically within TDLNs. Asparagine supplementation upregulated the E3 ubiquitin ligase FBXO21 in DCs. FBXO21 directly interacted with p100, promoting K48-linked ubiquitination and processing to p52, thereby aberrantly activating the non-canonical NF-κB pathway. This activation caused defective DC maturation and impaired antigen cross-presentation in TDLNs, hindering T cell priming. Conversely, vancomycin-induced asparagine depletion downregulated FBXO21, inhibited p100-to-p52 processing, and suppressed non-canonical NF-κB signaling, effectively restoring DC antigen-presenting capacity in TDLNs and promoting effector CD8+ T cell infiltration into tumors. Conclusions: Vancomycin enhances anti-PD-1 efficacy in MSS CRC by remodeling the gut microbiota and depleting asparagine. We identified a novel axis wherein asparagine downregulation inhibits FBXO21-mediated p100 processing and non-canonical NF-κB activation, restoring DC function in TDLNs. Targeting the microbiota-asparagine-FBXO21 axis offers a promising therapeutic strategy to overcome ICB resistance in MSS CRC.