Next, functional gene families associated with R-enriched bacteria were investigated. A total of 189 Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologies (KOs) were identified to be enriched in R (Kruskal-Wallis rank sum test, P < 0.05). Significant positive correlations were identified between 123 R-enriched KOs and 18 R-enriched species (Spearman’s correlation, rho > 0.5, Additional file 6: Table S1). Pathway identification of KOs verified the potential bioactivities of R-enriched species (Fig. 2c and Additional file 5: Figure S5). In detail, the cellobiose-transport system (ko02010) was significantly correlated with B. dentium; the pectin lyase (K01732), which may be involved in pectin metabolism, was correlated with A. muciniphila. Both cellulose and pectin have been highlighted for their prebiotic and anti-inflammatory potential as dietary fibers [11, 12]. Methanogenesis pathway (ko00680) was found to be correlated with R. obeum and the four Lactobacillus species. Methane generated in human gastrointestinal tract has been reported to ameliorate oxidative stress injury and suppress the host inflammatory response [13]. Other pathways with potential benefits included sulfate reduction (ko00920) and carbon fixation (ko00720) functions that were correlated with R. obeum, carotenoid biosynthesis (ko00906) correlated with B. cellulosilyticus and A. colihominis, and unsaturated fatty acid metabolism (ko00590) associated with C. comes (Additional file 7: Table S2). Such findings further illustrated the potential underlying mechanisms of gut microbiome influencing the anti-PD-1 immunotherapy efficacy in HCC patients.

It is well recognized that factors including age, genetics, and diet may influence microbiome composition [14]. However, the long-term stability of the human gut microbiota has been demonstrated in previous studies. A study demonstrated that an individual’s microbiota could be remarkably stable, with 60% of strains remaining over the course of five years; this finding highlighted that such stability and responsiveness to physiologic change confirmed the potential of the gut microbiota as a diagnostic tool and therapeutic target [15]. In the present study, patients were enrolled with strict criteria to minimize potential dietary or geographical influences during the whole course of treatment, as well as to ensure that the dynamic changes of gut microbiota in both R and NR were attributed to the therapeutic intervention instead of any daily factors.

Although our present microbiome-wide association study highlighted the association between changes in the gut microbiome and host-immune responses to drugs, any causal relationship of these correlative associations remains unknown. To further elucidate this relationship, several strategies have been developed. Firstly, ‘meta-omic’ analysis combining sequencing and multiple biochemical techniques can greatly advance the knowledge of the human microbiome and its specific role in governing disease states. For example, Gopalakrishnan et al. compared the tumor-associated immune infiltrates via multi-parameter immunohistochemistry (IHC) and found a statistically significant positive correlation between CD8+ T cell infiltrate in the tumor and the Faecalibacterium genus as well as the Ruminococcaceae family, suggesting a possible mechanism through which the gut microbiome may modulate anti-tumor immune responses [7]. More importantly, in vitro or in vivo experimental models are required in order to allow the systematic manipulation of variables and, thus, allow experimental testing and validation of results derived from meta-omics [16]. In our previous study, we had demonstrated that Behcet’s disease (BD), a kind of autoimmune disorder, was associated with considerable gut microbiome changes [17]. To determine whether the gut microbiome contributes to development of this disease, fecal microbiota transplantation (FMT) was performed in mice undergoing autoimmune uveitis. We demonstrated that mice colonized with whole-gut microbiomes from BD patients showed an exacerbation of disease activity and an excessive production of pro-inflammatory cytokines. These results may confirm the hypothesis that specific bacterial patterns contributed to the development of intraocular inflammatory disease. In our future studies, similar meta-omics strategies, as well as the use of mouse FMT models, will also be incorporated in order to deeply explore the interaction between the gut microbiome and host responses to immunotherapies among HCC patients.

Gopalakrishnan et al. also assessed the landscape of the oral and gut microbiomes in patients with metastatic melanoma receiving anti-PD-1 immunotherapy and demonstrated a high abundance of Lactobacillales in the oral microbiomes compared with that of fecal microbiomes in all subjects [7]. Galloway-Pena et al. further reported a high degree of intra-patient temporal instability of both stool and oral microbial diversity in patients with acute myeloid leukemia undergoing induction chemotherapy [18]. Both studies showed the possibility of using oral microbiome, rather than fecal microbiome, as an indicator for specific clinical outcomes. In our future longitudinal studies, both fecal and oral microbiome will be taken into consideration for a more comprehensive visualization of the host-microbe interactions.

In conclusion, by metagenomic sequencing of periodic fecal samples, we illustrated that the dynamic-variation characteristics of the gut microbiome might be used for early prediction of the six-month outcomes of anti-PD-1 immunotherapy in HCC at 3–6 weeks after treatment initiation, which is critical for disease monitoring and treatment decision-making. To our knowledge, this is the first study to focus on the association between the gut microbiome and the response to anti-PD-1 immunotherapy for HCC. Furthermore, while previous studies had mostly only provided cross-sectional comparisons, the present study showed the different trajectories of microbiome shifts between R and NR and revealed a stronger stability of the gut microbiome in R during the whole course of treatment. We believe that the potential of the gut microbiota as a therapeutic target is indicated in this study, and that the relevant bacterial species and metabolic pathways revealed here could be developed as a modulation strategy for better treatment options for HCC patients.