Gut-targeted antibiotic modulation enhances radiotherapy response in NSCLC and uncovers epigenetic and microbiome-driven immune programs enabling live biotherapeutic design

Stereotactic body radiotherapy (SBRT) can induce immunogenic cell death and stimulate systemic antitumor immunity, yet clinical responses remain heterogeneous. Preclinical studies indicate that the gut microbiome modulates host immune tone and influences responsiveness to therapy. We previously demonstrated in lung cancer and melanoma models that oral vancomycin enhances the antitumor effects of radiotherapy by depleting Gram-positive taxa responsible for generating short-chain fatty acids (SCFAs) and other immunosuppressive metabolites. Based on these observations, we conducted the first randomized clinical pilot study evaluating SBRT with or without oral vancomycin in early-stage non-small cell lung cancer (NSCLC). Patients eligible for SBRT (50 Gy in 4-5 fractions) were randomized to SBRT alone or SBRT plus vancomycin administered for one week before and five weeks after SBRT. Serial stool, serum, and PBMC samples were collected at five predefined time points. Primary endpoints were feasibility and safety, while immune activation; progression-free survival; overall survival; and changes in stool SCFAs served as exploratory endpoints. Seventeen patients enrolled; seven completed vancomycin treatment. No grade ≥3 toxicities occurred, and gastrointestinal symptoms were the most frequent adverse events. Cancer recurrence occurred in four patients, three of whom received SBRT alone, with nodal failure as the predominant pattern. Microbiome sequencing revealed marked reductions in specific Gram-positive taxa and compensatory expansion of vancomycin-resistant organisms. Stool metabolomics demonstrated substantial decreases in SCFAs in the vancomycin arm. RNA sequencing of PBMCs showed robust enrichment of antigen-processing and presentation pathways, together with interferon-related immune activation signatures. To extend these findings, we performed epigenetic and functional validation studies. PBMC chromatin profiling confirmed increased accessibility at interferon- and antigen-presentation-associated regulatory regions in vancomycin-treated subjects. Guided by these results, we evaluated whether the vancomycin effect was microbiome-transferable by engrafting stool from SBRT-alone or SBRT+vancomycin patients into antibiotic-conditioned mice. Mice colonized with post-vancomycin human stool reproduced the enhanced radiotherapy response, demonstrating that the immune effects are mediated by the gut ecosystem. These results provide the mechanistic rationale for developing ViRTuE-LTP (RT-enhancing live biotherapeutic) candidates. Consistent with the clinical observations, a defined 3-strain ViRTuE-LTP consortium enhanced APC activation and potentiated RT responsiveness in complementary murine studies.