Objective: Non-alcoholic fatty liver disease (NAFLD)-associated hepatocellular carcinoma (HCC) is an increasing healthcare burden worldwide. We examined the role of dietary cholesterol in driving NAFLD–HCC through modulating gut microbiota and its metabolites. Design: High-fat/high-cholesterol (HFHC), high-fat/low-cholesterol or normal chow diet was fed to C57BL/6 male littermates for 14 months. Cholesterol-lowering drug atorvastatin was administered to HFHC-fed mice. Germ-free mice were transplanted with stools from mice fed different diets to determine the direct role of cholesterol modulated-microbiota in NAFLD–HCC. Gut microbiota was analysed by 16S rRNA sequencing and serum metabolites by liquid chromatography–mass spectrometry (LC–MS) metabolomic analysis. Faecal microbial compositions were examined in 59 hypercholesterolemia patients and 39 healthy controls. Results: High dietary cholesterol led to the sequential progression of steatosis, steatohepatitis, fibrosis and eventually HCC in mice, concomitant with insulin resistance. Cholesterol-induced NAFLD–HCC formation was associated with gut microbiota dysbiosis. The microbiota composition clustered distinctly along stages of steatosis, steatohepatitis and HCC. Mucispirillum, Desulfovibrio, Anaerotruncus and Desulfovibrionaceae increased sequentially; while Bifidobacterium and Bacteroides were depleted in HFHC-fed mice, which was corroborated in human hypercholesteremia patients. Dietary cholesterol induced gut bacterial metabolites alteration including increased taurocholic acid and decreased 3-indolepropionic acid. Germ-free mice gavaged with stools from mice fed HFHC manifested hepatic lipid accumulation, inflammation and cell proliferation. Moreover, atorvastatin restored cholesterol-induced gut microbiota dysbiosis and completely prevented NAFLD–HCC development. Conclusions: Dietary cholesterol drives NAFLD–HCC formation by inducing alteration of gut microbiota and metabolites in mice. Cholesterol inhibitory therapy and gut microbiota manipulation may be effective strategies for NAFLD–HCC prevention.

Objective: Roseburia intestinalis is a probiotic species that can suppress intestinal inflammation by producing metabolites. We aimed to study the role of R. intestinalis in colorectal tumourigenesis and immunotherapy. Design: R. intestinalis abundance was evaluated in stools of patients with colorectal cancer (CRC) (n=444) and healthy controls (n=575). The effects of R. intestinalis were studied in Apc ^Min/+ or azoxymethane (AOM)-induced CRC mouse models, and in syngeneic mouse xenograft models of CT26 (microsatellite instability (MSI)-low) or MC38 (MSI-high). The change of immune landscape was evaluated by multicolour flow cytometry and immunohistochemistry staining. Metabolites were profiled by metabolomic profiling. Results: R. intestinalis was significantly depleted in stools of patients with CRC compared with healthy controls. R. intestinalis administration significantly inhibited tumour formation in Apc ^Min/+ mice, which was confirmed in mice with AOM-induced CRC. R. intestinalis restored gut barrier function as indicated by improved intestinal permeability and enhanced expression of tight junction proteins. Butyrate was identified as the functional metabolite generated by R. intestinalis. R. intestinalis or butyrate suppressed tumour growth by inducing cytotoxic granzyme B ⁺ , interferon (IFN)-γ ⁺ and tumour necrosis factor (TNF)-α ⁺ CD8 ⁺ T cells in orthotopic mouse models of MC38 or CT26. R. intestinalis or butyrate also significantly improved antiprogrammed cell death protein 1 (anti-PD-1) efficacy in mice bearing MSI-low CT26 tumours. Mechanistically, butyrate directly bound to toll-like receptor 5 (TLR5) receptor on CD8 ⁺ T cells to induce its activity through activating nuclear factor kappa B (NF-κB) signalling. Conclusion: R. intestinalis protects against colorectal tumourigenesis by producing butyrate, which could also improve anti-PD-1 efficacy by inducing functional CD8 ⁺ T cells. R. intestinalis is a potential adjuvant to augment anti-PD-1 efficacy against CRC.

Objective: Gut microbiota is a key player in dictating immunotherapy response. We aimed to explore the immunomodulatory effect of probiotic Lactobacillus gallinarum and its role in improving anti-programmed cell death protein 1 (PD1) efficacy against colorectal cancer (CRC). Design: The effects of L. gallinarum in anti-PD1 response were assessed in syngeneic mouse models and azoxymethane/dextran sulfate sodium-induced CRC model. The change of immune landscape was identified by multicolour flow cytometry and validated by immunohistochemistry staining and in vitro functional assays. Liquid chromatography-mass spectrometry was performed to identify the functional metabolites. Results: L. gallinarum significantly improved anti-PD1 efficacy in two syngeneic mouse models with different microsatellite instability (MSI) statuses (MSI-high for MC38, MSI-low for CT26). Such effect was confirmed in CRC tumourigenesis model. L. gallinarum synergised with anti-PD1 therapy by reducing Foxp3⁺ CD25⁺ regulatory T cell (Treg) intratumoural infiltration, and enhancing effector function of CD8⁺ T cells. L. gallinarum-derived indole-3-carboxylic acid (ICA) was identified as the functional metabolite. Mechanistically, ICA inhibited indoleamine 2,3-dioxygenase (IDO1) expression, therefore suppressing kynurenine (Kyn) production in tumours. ICA also competed with Kyn for binding site on aryl hydrocarbon receptor (AHR) and antagonised Kyn binding on CD4⁺ T cells, thereby inhibiting Treg differentiation in vitro. ICA phenocopied L. gallinarum effect and significantly improved anti-PD1 efficacy in vivo, which could be reversed by Kyn supplementation. Conclusion: L. gallinarum-derived ICA improved anti-PD1 efficacy in CRC through suppressing CD4+Treg differentiation and enhancing CD8+T cell function by modulating the IDO1/Kyn/AHR axis. L. gallinarum is a potential adjuvant to augment anti-PD1 efficacy against CRC.

Objective: Using faecal shotgun metagenomic sequencing, we identified the depletion of Lactobacillus gallinarum in patients with colorectal cancer (CRC). We aimed to determine the potential antitumourigenic role of L. gallinarum in colorectal tumourigenesis. Design: The tumor-suppressive effect of L. gallinarum was assessed in murine models of CRC. CRC cell lines and organoids derived from patients with CRC were cultured with L. gallinarum or Escherichia coli MG1655 culture-supernatant to evaluate cell proliferation, apoptosis and cell cycle distribution. Gut microbiota was assessed by 16S ribosomal DNA sequencing. Antitumour molecule produced from L. gallinarum was identified by liquid chromatography mass spectrometry (LC-MS/MS) and targeted mass spectrometry. Results: L. gallinarum significantly reduced intestinal tumour number and size compared with E. coli MG1655 and phosphate-buffered saline in both male and female murine intestinal tumourigenesis models. Faecal microbial profiling revealed enrichment of probiotics and depletion of pathogenic bacteria in L. gallinarum -treated mice. Culturing CRC cells with L. gallinarum culture-supernatant (5%, 10% and 20%) concentration-dependently suppressed cell proliferation and colony formation. L. gallinarum culture-supernatant significantly promoted apoptosis in CRC cells and patient-derived CRC organoids, but not in normal colon epithelial cells. Only L. gallinarum culture-supernatant with fraction size <3 kDa suppressed proliferation in CRC cells. Using LC-MS/MS, enrichments of indole-3-lactic acid (ILA) was identified in both L. gallinarum culture-supernatant and the gut of L. gallinarum -treated mice. ILA displayed anti-CRC growth in vitro and inhibited intestinal tumourigenesis in vivo . Conclusion: L. gallinarum protects against intestinal tumourigenesis by producing protective metabolites that can promote apoptosis of CRC cells.

Despite the promising advances in novel cancer therapy such as immune checkpoint inhibitors (ICIs), limitations including therapeutic resistance and toxicity remain. In recent years, the relationship between gut microbiota and cancer has been extensively studied. Accumulating evidence reveals the role of microbiota in defining cancer therapeutic efficacy and toxicity. Unlike host genetics, microbiota can be easily modified via multiple strategies, including faecal microbiota transplantation (FMT), probiotics and antibiotics. Preclinical studies have identified the mechanisms on how microbes influence cancer treatment outcomes. Clinical trials have also demonstrated the potential of microbiota modulation in cancer treatments. Herein, we review the mechanistic insights of gut microbial interactions with chemotherapy and ICIs, particularly focusing on the interplay between gut bacteria and the pharmacokinetics (eg, metabolism, enzymatic degradation) or pharmacodynamics (eg, immunomodulation) of cancer treatment. The translational potential of basic findings in clinical settings is then explored, including using microbes as predictive biomarkers and microbial modulation by antibiotics, probiotics, prebiotics, dietary modulations and FMT. We further discuss the current limitations of gut microbiota modulation in patients with cancer and suggest essential directions for future study. In the era of personalised medicine, it is crucial to understand the microbiota and its interactions with cancer. Manipulating the gut microbiota to augment cancer therapeutic responses can provide new insights into cancer treatment.

Objective: Helicobacter pylori is associated with gastric inflammation, precancerous gastric atrophy (GA) and intestinal metaplasia (IM). We aimed to identify microbes that are associated with progressive inflammation, GA and IM 1 year after H. pylori eradication. Design: A total of 587 H. pylori –positive patients were randomised to receive H. pylori eradication therapy (295 patients) or placebo (292 patients). Bacterial taxonomy was analysed on 404 gastric biopsy samples comprising 102 pairs before and after 1 year H. pylori eradication and 100 pairs before and after 1 year placebo by 16S rRNA sequencing. Results: Analysis of microbial sequences confirmed the eradication of H. pylori in treated group after 1 year. Principal component analysis revealed distinct microbial clusters reflected by increase in bacterial diversity (p<0.00001) after H. pylori eradication. While microbial interactions remained largely unchanged after placebo treatment, microbial co-occurrence was less in treated group. Acinetobacter lwoffii , Streptococcus anginosus and Ralstonia were enriched while Roseburia and Sphingomonas were depleted in patients with persistent inflammation 1 year after H. pylori eradication. A distinct cluster of oral bacteria comprising Peptostreptococcus , Streptococcus , Parvimonas , Prevotella, Rothia and Granulicatella were associated with emergence and persistence of GA and IM. Probiotic Faecalibacterium praustznii was depleted in subjects who developed GA following H. pylori eradication. Functional pathways including amino acid metabolism and inositol phosphate metabolism were enriched while folate biosynthesis and NOD-like receptor signalling decreased in atrophy/IM-associated gastric microbiota. Conclusion: This study identified that gastric microbes contribute to the progression of gastric carcinogenesis after H. pylori eradication.