Renal cell carcinoma (RCC) is an immunogenic malignancy responsive to immunotherapy, but durable responses and predictive biomarkers remain insufficient. The gut microbiota influences cancer progression and immunotherapy efficacy, yet its role in RCC is unclear. This study explores microbiome-based insights to enhance treatment prediction and overcome resistance in advanced RCC (aRCC) by profiling gut microbiota after immunotherapy, analyzing its correlation with outcomes, and identifying key microbes influencing treatment response.

Study Cohort We conducted a baseline follow-up of 28 aRCC patients receiving immunotherapy combined with targeted therapy and collected their fecal samples. Treatment response was assessed at 12 months using iRECIST. Tumor Model SPF mice pretreated with antibiotics received a cocktail (metronidazole, vancomycin, ampicillin, neomycin) in drinking water for one week. Renca cells were subcutaneously injected into BALB/c mice, followed by intraperitoneal PD-1 blockade or isotype control twice weekly. For targeted therapy experiments, axitinib or sunitinib was administered daily. Bacteroides ovatus (B.o, ATCC-8384) was orally administered (1×10⁸ CFU) twice weekly.

We categorized 28 patients receiving immunotherapy combined with targeted therapy into responders (n=20) and non-responders (n=8) based on iRECIST criteria, and analyzed their post-treatment fecal metagenomes. LEfSe identified B.o as enriched in responders. Cox regression revealed a significant negative correlation between high B.o abundance and post-treatment progression; patients with higher B.o abundance had improved PFS. Oral B.o administration enhanced anti-PD-1 mAb efficacy in tumor-bearing mice, an effect lost upon B.o clearance with metronidazole. However, B.o failed to improve targeted therapy efficacy. Only live B.o and conditioned media treatments showed comparable immunotherapeutic efficacy. Untargeted LC-MS/MS identified arginine as a key B.o-derived metabolite influencing immunotherapy response, further validated by colorimetric assay and HPLC. Previous studies suggest that B.o enhances immunotherapy by increasing arginine levels and CD8+ T cell activity. Flow cytometry of tumor tissues from PD-1 mAb-treated mice confirmed this, showing higher CD8+ T cell numbers and increased GZMB and TNFα expression in the B.o-treated group.

Our study reveals a strong correlation between increased gut B.o and enhanced immunotherapy response in aRCC. B.o promotes progression-free survival by modulating immune responses through arginine secretion. Mouse models confirm its role in augmenting PD-1 blockade efficacy, with elevated arginine levels enhancing CD8+ T cell function. These findings highlight B.o and its metabolites as potential adjuncts to improve immunotherapy outcomes in aRCC, supporting their clinical application.