Metagenomics and culturomics reveal the dual role of the gut microbiome in the development of immune-related toxicities and the efficacy of immune checkpoint inhibitors in cancer

Abstract Background Despite their major impact on cancer treatment, immune checkpoint inhibitors (ICI) are frequently associated with immune-related adverse events (irAE). Growing evidence suggests that the occurrence of irAE may be correlated with enhanced ICI efficacy, although the underlying mechanisms remain unknown. Most studies investigating the role of the gut microbiome in oncology have relied on sequencing approaches, particularly shotgun metagenomics. Although microbiome profiling revealed strong associations between specific bacterial taxa and clinical outcomes, it has limitations, including an inability to detect low-abundance bacteria and to recover live cultivable bacteria. To overcome these limitations, we combined shotgun metagenomics and culturomics on fecal samples collected from patients with melanoma and non-small cell lung cancer (NSCLC), at baseline and at the onset of immune related (ir)-colitis. Results We first validated across three independent cohorts of 589 patients with melanoma or NSCLC treated with ICI that grade ≥ 2 irAE were associated with significantly longer overall survival (OS) and progression-free survival (PFS). Complementary analysis using shotgun metagenomics and culturomics revealed that patients who developed grade ≥ 2 irAE had a lower alpha diversity compared to those who did not develop grade ≥ 2 irAE. Metagenomics results showed enrichment of Ruminococcus gnavus and Streptococcus vestibularis at baseline in grade ≥ 2 irAE patients, while Clostridium paraputrificum and Streptococcus spp. were isolated by culturomics from baseline stool samples from ir-colitis patients. Longitudinal analysis of paired stool samples revealed a shift in microbiome composition with enrichment of Paraclostridium bifermentans and Clostridium paraputrificum , lower lipopolysaccharide and higher flagellin concentrations at baseline compared with the time of ir-colitis. Fecal microbiome transplantation from a patient with ir-colitis into mice induced surrogate markers of colonic inflammation and enhanced the anti-tumor activity of combined anti-PD-1/CTLA-4. P. bifermentans isolated from this patient sample demonstrated direct epithelial barrier disruption in Caco-2 monolayers, characterized by decreased ZO-1 and Occludin immunofluorescence signal and increased TNF-α and IL-1β expression. Moreover, in the dextran sodium sulfate (DSS) colitis model, P. bifermentans worsened weight loss. In a separate tumor model, it amplified the anti-tumor effect of dual ICI. This beneficial effect was also maintained after treatment with P. bifermentans < 3 kDa filtered supernatant. Conclusion Altogether, our results suggest that P. bifermentans promotes subclinical colitis while increasing the efficacy of dual ICI. This provides a potential microbiome-derived link between irAE and improved anti-tumor responses.