Coupling culturomics and metagenomics sequencing to characterize the gut microbiome of patients with cancer treated with immune checkpoint inhibitors.

Background: The gut microbiome represents a novel biomarker for melanoma and non-small cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors (ICI). Gut microbiome metagenomics profiling studies of patients treated with immunotherapy identified bacteria associated with ICI efficacy, while others have been linked to resistance. However, limitations of metagenomics sequencing, such as complex bioinformatic processing requirements, necessity of a threshold for positive detection, and the inability to detect live organisms, have hindered our ability to fully characterize the gut microbiome. Therefore, combining metagenomics with high-throughput culture-based techniques (culturomics) represents an ideal strategy to fully characterize microbiome composition to more robustly position the microbiome as a biomarker of response to ICI.

Methods: We performed culturomics using fecal samples from 22 patients from two academic centres in Canada and the United Kingdom with NSCLC and cutaneous melanoma treated with ICI (cancer group), comparing their microbiome composition to that of 7 healthy volunteers (HV), along with matching shotgun metagenomics sequencing.

Results: For culturomics results, 221 distinct species were isolated. Among these 221 distinct species, 182 were identified in the cancer group and 110 in the HV group. In the HV group, the mean species richness was higher compared to the cancer group (34 vs. 18, respectively, p = 0.002). Beta diversity revealed separate clusters between groups (p = 0.004). Bifidobacterium spp. and Bacteroides spp. were enriched in HV, while cancer patients showed an overrepresentation of Enterocloster species, as well as Veillonella parvula. Next, comparing cancer patients' clinical outcomes to ICI, we observed that among the 20 most abundant bacteria present in non-responder patients, 2 belonged to the genus Enterocloster, along with an enrichment of Hungatella hathewayi and Cutibacterium acnes. In contrast, responders to ICI exhibited a predominance of Bacteroides spp. In NSCLC patients, metagenomics analysis revealed that of the 154 bacteria species isolated through culturomics, 61/154 (39%) were also identified by metagenomics sequencing. Importantly, 94 individual species were uniquely detected by culturomics.

Conclusion: These findings highlight that culturomics and metagenomics can serve as complementary tools to characterize the microbiome in patients with cancer. This integrated approach uncovers specific microbiome signatures that differentiate HV from cancer patients, and identifies specific species associated with therapy response and resistance.