Gut microbiota regulates the ALK5/NOX1 axis by altering glutamine metabolism to inhibit ferroptosis of intrahepatic cholangiocarcinoma cells.

Intrahepatic cholangiocarcinoma (ICC) is a kind of hepatobiliary tumor that is increasing in incidence and mortality. The gut microbiota plays a role in the onset and progression of cancer, however, the specific mechanism by which the gut microbiota acts on ICC remains unclear. In this study, feces and plasma from healthy controls and ICC patients were collected for 16S rRNA sequencing or metabolomics analysis. Gut microbiota analysis showed that gut microbiota abundance and biodiversity were altered in ICC patients compared with controls. Plasma metabolism analysis showed that the metabolite glutamine content of the ICC patient was significantly higher than that of the controls. KEGG pathway analysis showed that glutamine plays a vital role in ICC. In addition, the use of antibiotics in ICC animals further confirmed that changes in gut microbiota affect changes in glutamine. Further experiments showed that supplementation with glutamine inhibited ferroptosis and downregulated ALK5 and NOX1 expression in HuCCT1 cells. ALK5 overexpression or NOX1 overexpression increased NOX1, p53, PTGS2, ACSL4, LPCAT3, ROS, MDA and Fe2+ and decreased FTH1, SLC7A11 and GSH. Knockdown of NOX1 suppressed FIN56-induced ferroptosis. In vivo, supplementation with glutamine promoted tumor growth. Overexpression of ALK5 repressed tumor growth and induced ferroptosis in nude mice, which could be reversed by the addition of glutamine. Our results suggested that the gut microbiota altered glutamine metabolism to inhibit ferroptosis in ICC by regulating the ALK5/NOX1 axis.

PI3K-CCL2-CCR2-MDSCs axis: A potential pathway for tumor Clostridia-promoted CD 8+ T lymphocyte infiltration in bile tract cancers.

BACKGROUND: Most patients with resected bile tract cancers (BTCs) survive for less than 5 years; however, some achieve better prognosis. The tumor microbiome can improve survival by regulating the tumor immune microenvironment. However, whether the tumor microbiome promotes immune cell infiltration in BTCs is unknown. This study aimed to determine the association between CD8+ T lymphocyte infiltration and the tumor microbiome in patients with resected BTCs. METHODS: Archived formalin-fixed paraffin-embedded tumor specimens were collected from patients with resected BTCs and analyzed using 16S rRNA gene sequencing to identify that prognosis-related and significantly differentially enriched taxa. Gene ontology (GO) analysis of the differentially enriched taxa was used to assess how CD8+ T lymphocyte infiltration is affected by the tumor microbiome of BTCs. RESULTS: We enrolled 32 patients with resected BTCs. The high CD8+ lymphocyte-infiltration (CD8hi) group had four significantly enriched taxa, and in the low CD8+ lymphocyte-infiltration (CD8low) group comprised one significantly enriched taxon. Patients with higher Clostridia abundance (enriched in the CD8hi group) experienced longer overall survival than those with lower abundance. The enrichment of Clostridia in the CD8hi group corresponded with lower CCL2 expression and downregulation of phosphatidylinositol 3-kinase activity, which might decrease myeloid-derived suppressor cell recruitment to the tumor milieu, thus increasing CD8+ lymphocyte infiltration in BTCs. CONCLUSIONS: The tumor microbiome is related to CD8+ T lymphocyte infiltration in patients with resected BTCs. The relationship between tumor Clostridia and high infiltration of CD8+ T lymphocytes might reflect decreased recruitment of myeloid-derived suppressor cells via the PI3K-CCL2-CCR2 axis.

Profiling of tumour-associated microbiota in human hepatocellular carcinoma.

Liver cancer is the fourth leading cause of cancer-related death. Hepatocellular carcinoma (HCC) is a primary liver cancer that results from chronic hepatitis caused by multiple predisposing factors such as viral infection, alcohol consumption, and non-alcoholic fatty liver disease. Accumulating studies have indicated that dysfunction of the gut epithelial barrier and hepatic translocation of gut microbes may be implicated in the pathogenesis of HCC. However, the translocated bacteria in HCC patients remains unclear. Here, we characterised tumour-associated microbiota in patients with liver cancer and focused on HCC. We observed that the number of amplicon sequence variants in tumour-associated microbiota was significantly higher compared with that in non-tumour regions of the liver. The tumour-associated microbiota consisted of Bacteroidetes, Firmicutes, and Proteobacteria as the dominant phyla. We identified an unclassified genus that belonged to the Bacteroides, Romboutsia, uncultured bacterium of Lachnospiraceae as a signature taxon for primary liver cancer. Additionally, we identified Ruminococcus gnavus as a signature taxon for HCC patients infected with hepatitis B and/or hepatitis C viruses. This study suggests that tumour microbiota may contribute to the pathology of HCC.

Characterization of biliary microbiota dysbiosis in extrahepatic cholangiocarcinoma.

Extrahepatic cholangiocarcinoma (CCA) accounts for 3% of digestive cancers. The role of biliary microbiota as an environment-related modulator has been scarcely investigated in CCA, and the putative impact of associated diseases has not been yet assessed. We characterized the biliary microbiota in CCA patients in order to identify a specific CCA-related dysbiosis. The biliary effluents were collected through an endoscopic retrograde pancreatic cholangiography (ERCP) examination involving 28 CCA and 47 patients with gallstones, herein considered as controls. The biliary effluents were submitted to bacterial DNA extraction and 16S rRNA sequencing, using Illumina technology. Overall, 32% of CCA and 22% of controls displayed another associated disease, such as diabetes, pancreatitis, inflammatory bowel disease, or primary sclerosing cholangitis. Such associated diseases were considered in the comparisons that were made. Principal coordinate analysis (PCoA) detected a significant disparity of biliary microbiota composition between CCA patients and controls without an associated disease. Amongst the most abundant phyla, Proteobacteria did not significantly differ between CCA patients and controls, whereas Firmicutes levels were lower and Bacteroidetes higher in CCAs' biliary microbiota than in the controls' microbiota. The most abundant genera were Enterococcus, Streptococcus, Bacteroides, Klebsiella, and Pyramidobacter in CCA's biliary microbiota. Additionally, levels of Bacteroides, Geobacillus, Meiothermus, and Anoxybacillus genera were significantly higher in CCA patients' biliary microbiota, without an associated disease, in comparison with controls. A specific CCA-related dysbiosis was identified as compared to controls independently from associated diseases. This suggests that a microorganism community may be involved in CCA pathogenesis.

Profiling of Bile Microbiome Identifies District Microbial Population between Choledocholithiasis and Cholangiocarcinoma Patients.

OBJECTIVE: Choledocholithiasis (CDL), a potential risk for cholangiocarcinoma (CCA) development, is often a consequence of bacterial infection. Thus, the microbial population that contributes to CDL might also be involved in CCA development. We compared the microbiome in bile fluid of CDL patients and CCA patients. METHODS: Bile samples were collected from CDL (n = 30) and CCA (n =30) patients. Microbial profiling was performed individually by the sequencing of V3-V4 regions of the 16S rRNA gene. RESULTS: Enterobacter, Pseudomonas, and Stenotrophomonas species were much more abundant in bile samples from CCA compared to CDL (p.

Association of Chronic Opisthorchis Infestation and Microbiota Alteration on Tumorigenesis in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a common hepatobiliary cancer in East and Southeast Asia. The data of microbiota contribution in CCA are still unclear. Current available reports have demonstrated that an Opisthorchis viverrini (OV) infection leads to dysbiosis in the bile duct. An increase in the commensal bacteria Helicobacter spp. in OV-infected CCA patients is associated with bile duct inflammation, severity of bile duct fibrosis, and cholangiocyte proliferation. In addition, secondary bile acids, major microbial metabolites, can mediate cholangiocyte inflammation and proliferation in the liver. A range of samples from CCA patients (stool, bile, and tumor) showed different degrees of dysbiosis. The evidence from these samples suggests that OV infection is associated with alterations in microbiota and could potentially have a role in CCA. In this comprehensive review, reports from in vitro, in vivo, and clinical studies that demonstrate possible links between OV infection, microbiota, and CCA pathogenesis are summarized and discussed. Understanding these associations may pave ways for novel potential adjunct intervention in gut microbiota in CCA patients.

Characterization of Gut Microbiota, Bile Acid Metabolism, and Cytokines in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC), a type of bile duct cancer, has a high mortality rate. Gut microbiota, bile acid (BA) metabolism, and cytokines have not been characterized in patients with ICC, and better noninvasive diagnostic approaches for ICC are essential to be established. Therefore, in this study we aimed to improve our understanding of changes in gut microbiota, BA metabolism, and cytokines in patients with ICC. We found that the α-diversities and ß-diversities of ICC were highest and that the abundances of four genera (Lactobacillus, Actinomyces, Peptostreptococcaceae, and Alloscardovia) were increased in patients with ICC compared with those in patients with hepatocellular carcinoma or liver cirrhosis and in healthy individuals. The glycoursodeoxycholic acid and tauroursodeoxycholic acid (TUDCA) plasma-stool ratios were obviously increased in patients with ICC. Furthermore, the genera Lactobacillus and Alloscardovia that were positively correlated with TUDCA plasma-stool ratios were combined to discriminate ICC from the other three diseases. Vascular invasion (VI) frequently led to a poor prognosis in patients with ICC. Compared with patients with ICC without VI, patients with VI had a greater abundance of the family Ruminococcaceae, increased levels of plasma interleukin (IL)-4 and six conjugated BAs, and decreased levels of plasma IL-6 and chenodeoxycholic acid. A positive correlation between plasma taurocholic acid and IL-4 was observed in patients with ICC. Plasma TUDCA was negatively correlated with the abundance of the genus Pseudoramibacter and the survival time of patients with ICC, but had no effect on tumor size, as determined in two murine tumor models. Conclusion: In this study, we identified some biomarkers, including gut microbiota, BAs and inflammatory cytokines, for the diagnosis of ICC and prediction of VI in patients with ICC.

A Preliminary Study of Biliary Microbiota in Patients with Bile Duct Stones or Distal Cholangiocarcinoma.

BACKGROUND AND OBJECTIVE: The distal cholangiocarcinoma (dCCA) is associated with many factors: genes, environment, infection, etc. The current changes in biliary flora are thought to be involved in the formation of many gastrointestinal tract (GIT) diseases, like colon adenocarcinoma. Therefore we want to investigate whether the dCCA has a certain correlation with biliary microecology, and to detect specific strains. METHODS: A total of 68 adults were enrolled, of whom 8 with dCCA, 16 with recurrent choledocholithiasis, and 44 with the onset of common bile duct stones. Endoscopic Retrograde Cholangiopancretography (ERCP) was utilized to collect bile samples for DNA extraction and 16S rRNA gene sequencing, followed by analysis of bile microbiota composition. RESULTS: First, Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria are the most dominant phyla in the bile of patients with dCCA and the onset of common bile duct stoes. Secondly, compared with the onset of common bile duct stones patients, we got a significant increase in the phylum Gemmatimonadetes, Nitrospirae, Chloroflexi, Latescibacteria, and Planctomycetes in dCCA patients. Finally, at the genus level, we obtained sequencing results of 252 bacterial genera from patients with dCCA, recurrent choledocholithiasis, and the new onset of common bile duct stones, revealing heterogeneity among individuals. CONCLUSION: To the best of our knowledge, this is the first study of the dysbiosis of bile flora in patients with dCCA. This micro-ecological disorder may be a decisive factor in the formation of dCCA. At the same time, for the first time, this study provides a test chart of biliary microbial populations that may be associated with recurrent choledocholithiasis. The compositional changes of the core microbial group of the biliary tract have potentially important biological and medical significance for the microbiological biliary disorders of dCCA.

Association between small intestinal bacterial overgrowth and toll-like receptor 4 in patients with pancreatic carcinoma and cholangiocarcinoma.

BACKGROUND/AIMS: Multiple factors have been linked to pathogenesis of pancreatic cancer and cholangiocarcinoma. Until now, few studies have investigated the role of small intestinal bacterial overgrowth (SIBO) and toll-like receptor 4 (TLR-4) signaling in these diseases. This study aimed to examine the relationship between the prevalence of SIBO and the TLR-4 expression in patients with pancreatic carcinoma and cholangiocarcinoma. MATERIALS AND METHODS: A total of 90 human subjects suffering from pancreatic carcinoma (n=30), cholangiocarcinoma (n=30), and healthy controls (n=30) were enrolled in the study. A glucose hydrogen breath test (GHBT) was used to evaluate SIBO. The TLR4 protein expression was measured by immunohistochemistry (IHC). RESULTS: The positive rate of SIBO was 63.3% in the pancreatic cancer group and 46.7% in patients with cholangiocarcinoma, which was significantly greater than 13.3% in the healthy control group (p<0.05). An IHC analysis revealed that the TLR-4 protein expression in the SIBO-positive pancreatic carcinoma patients was significantly higher than that in the SIBO-negative patients (p<0.05), and the same result was in the cholangiocarcinoma subjects. In addition, a correlation analysis identified the positive relationship between the prevalence of SIBO and the TLR-4 protein expression in pancreatic carcinoma (r=0.489), and the same result was in the cholangiocarcinoma subjects. CONCLUSION: Our findings indicate a high prevalence of SIBO in pancreatic carcinoma and cholangiocarcinoma, and SIBO displays a positive correlation with the TLR-4 expression, suggesting that SIBO could be a risk factor for the pathogenesis of pancreatic carcinoma and cholangiocarcinoma, in which the TLR4 signaling may be involved.

Opisthorchiasis-Induced Cholangiocarcinoma: How Innate Immunity May Cause Cancer.

Innate, inflammatory responses towards persistent Opisthorchis viverrini (OV) infection are likely to contribute to the development of cholangiocarcinoma (CCA), a liver cancer that is rare in the West but prevalent in Greater Mekong Subregion countries in Southeast Asia. Infection results in the infiltration of innate immune cells into the bile ducts and subsequent activation of inflammatory immune responses that fail to clear OV but instead may damage local tissues within the bile ducts. Not all patients infected with OV develop CCA, and so tumourigenesis may be dependent on multiple factors including the magnitude of the inflammatory response that is activated in infected individuals. The purpose of this review is to summarize how innate immune responses may promote tumourigenesis following OV infection and if such responses can be used to predict CCA onset in OV-infected individuals. It also hypothesizes on the role that Helicobacterspp., which are associated with liver fluke infections, may play in activation of the innate the immune system to promote tissue damage and persistent inflammation leading to CCA.

Imaging Features on Contrast-Enhanced Ultrasound and Clinical Characteristics of Hepatitis B Virus-Related Combined Hepatocellular-Cholangiocarcinoma: Comparison with Hepatitis B Virus-Related Hepatocellular Carcinoma.

The objective of this study was to compare the clinical characteristics and imaging features on contrast-enhanced ultrasound (CEUS) of hepatitis B virus (HBV)-related combined hepatocellular-cholangiocarcinoma (CHC) and hepatocellular carcinoma (HCC). Thirty-one pathologically proven CHCs were included and 31 HCCs were randomly selected as controls. Elevated carbohydrate antigen (CA) 19-9 alone and simultaneous elevation of α-fetoprotein and CA19-9 were more frequent in CHC than in HCC patients (p = 0.004 and 0.029, respectively). On CEUS, homogeneous, heterogeneous and peripheral irregular rim-like enhancement was illustrated in 8 (25.8%), 12 (38.7%) and 11 (35.5%) CHCs and in 6 (19.4%), 23 (74.1%) and 2 (6.5%) HCCs, respectively (p = 0.007). Multivariate logistic regression analysis revealed CA19-9 elevation (p = 0.011, odds ratio [OR] = 6.545) and peripheral irregular rim-like enhancement on CEUS (p = 0.017, OR = 7.718) were independent variables. A receiver operating characteristic curve was plotted and the area under the curve was 0.740. CHC should be watched for in HBV-infected patients with liver tumor manifesting peripheral irregular rim-like enhancement on CEUS, accompanied by CA19-9 elevation.

Co-infections with liver fluke and Helicobacter species: A paradigm change in pathogenesis of opisthorchiasis and cholangiocarcinoma?

Infection with the fish-borne liver fluke Opisthorchis viverrini is classified by the International Agency for Research on Cancer as a Group 1 carcinogen: definitely carcinogenic in humans. Cofactors likely contribute to bile duct cancer (cholangiocarcinoma) caused by this infection. Here we review recent findings that address the role of liver fluke associated H. pylori in hepatobiliary disease and malignancy. We hypothesize that co-infection by O. viverrini and the bacillus Helicobacter pylori is central of liver fluke infection associated cholangiocarcinoma.

Parasitic and infectious diseases of the biliary tract in migrants and international travelers.

INTRODUCTION: In recent years, global and regional crises have led to extraordinary worldwide migration, accompanied by an increase in long-distance travel from Western countries. Both are linked to a rising incidence of rare parasitic and infectious diseases in first world countries, including in the biliary tract. Areas covered: A selective literature research in PubMed was performed to review the most important parasitic and infectious biliary diseases, which are caused by a wide variety of pathogens and may be latent over long periods, with chronic courses leading to cholangitis, hepatic failure or development of cholangiocarcinoma. Parasites such as Ascaris, Fasciola and Clonorchis/Opisthorchis are particularly important and may trigger biliary diseases or predisposition for bacterial superinfections. Viral or protozoal cholangitis is mainly a problem of impaired immunity. Expert commentary: Currently, these entities are still rare in migrants and long-distance travelers. However, a significant increase in Western countries has to be expected. Incidences are most likely underestimated because of protracted clinical latency. Diagnosis depends on the relevant pathogens, the host's immune status and the extent or distribution of biliary obstruction. Modern tomographic methods, ERCP and specific microbiological/parasitological/virological tests are of crucial diagnostic importance. Antimicrobial/antiparasitic/antiviral therapy along with ERCP and interventional sonography/radiology provide effective treatment options.

Helicobacter Species are Possible Risk Factors of Cholangiocarcinoma.

Several infectious agents are considered to be causes of cancer in human, mainly hepatitis B and C viruses, high-risk human pailloma viruses, Helicobacter pylori, Clonorchis sinensis, and Opisthorchis viverrini. Here we described the evident research and the association between Helicobacter spp. and biliary tract cancer particularly cholangiocarcinoma (CCA). Global epidemiological studies have suggested that Helicobacter spp. are possible risk factors for biliary tract diseases. Molecular studies support a linkage of Helicobacter spp. with CCA development. H. pylori, H. bilis, and H. hepaticus, are found in CCA, but the most common species are H. pylori and H. bilis. The type of CCA are associated with Helicobacter spp. include extrahepatic CCA, and common bile duct cancer. Up to the present, however, the results from different regions, materials and methods, sub-sites of cancer, and controls have not been consistent, thus introducing heterogeneity. Therefore, a comparison between co-Helicobacter spp.-CCA in the countries with low and high incident of CCA is required to settle the question. Furthermore, clarifying variation in the role of Helicobacter species in this CCA, including pathogenesis of CCA through enhanced biliary cell inflammation and proliferation, is necessary.

Microbiota studies in the bile duct strongly suggest a role for Helicobacter pylori in extrahepatic cholangiocarcinoma.

Biliary tract cancer or extrahepatic cholangiocarcinoma (ECCA) represents the sixth commonest cause of cancer in the gastrointestinal tract in western countries. We aimed to characterize the microbiota and its predicted associated functions in the biliary tract of ECCA and benign biliary pathology (BBP). Samples were taken from 100 patients with ECCA and 100 patients with BBP by endoscopic cholangio-pancreatography for DNA extraction. Ten patients with ECCA and ten with BBP were selected for microbiota studies using the V4-16S rRNA gene and sequenced in Illumina platform. Microbiota analyses included sample-to-sample distance metrics, ordination/clustering and prediction of functions. Presence of Nesterenkonia sp. and Helicobacter pylori cagA and vacA genes were tested in the 100 ECCA and 100 BBP samples. Phylum Proteobacteria dominated all samples (60.4% average). Ordination multicomponent analyses showed significant microbiota separation between ECCA and BBP (p 0.010). Analyses of 4002 operational taxonomic units with presence variation in at least one category probed a separation of ECCA from BBP. Among these, Nesterenkonia decreased, whereas Methylophilaceae, Fusobacterium, Prevotella, Actinomyces, Novosphingobium and H. pylori increased in ECCA. Predicted associated functions showed increased abundance of H. pylori virulence genes in ECCA. cagA and vacA genes were confirmed by PCR in ECCA and BBP samples. This is the first microbiota report in ECCA and BBP to show significant changes in microbial composition. Bacterial species unusual for human flora were found: Methylophilaceae and Nesterenkonia are reported in hypersaline soils, and Mesorhizobium is a nitrogen-fixing bacterium. Enrichment of virulence genes confirms previous studies suggesting that H. pylori might be associated with ECCA.

A microRNA profile associated with Opisthorchis viverrini-induced cholangiocarcinoma in tissue and plasma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive tumor of the bile duct, and a significant public health problem in East Asia, where it is associated with infection by the parasite Opisthorchis viverrini. ICC is often detected at an advanced stage and with a poor prognosis, making a biomarker for early detection a priority. METHODS: We have comprehensively profiled miRNA expression levels in ICC tumor tissue using small RNA-Seq and validated these profiles using quantitative PCR on matched plasma samples. RESULTS: Distinct miRNA profiles were associated with increasing histological differentiation of ICC tumor tissue. We also observed that histologically normal tissue adjacent to ICC tumor displayed miRNA expression profiles more similar to tumor than liver tissue from healthy donors. In plasma samples, an eight-miRNA signature associated with ICC, regardless of the degree of histological differentiation of its matched tissue, forming the basis of a circulating miRNA-based biomarker for ICC. CONCLUSIONS: The association of unique miRNA profiles with different ICC subtypes suggests the involvement of specific miRNAs during ICC tumor progression. In plasma, an eight-miRNA signature associated with ICC could form the foundation of an accessible (plasma-based) miRNA-based biomarker for the early detection of ICC.

Infection with Helicobacter bilis but not Helicobacter hepaticus was Associated with Extrahepatic Cholangiocarcinoma.

BACKGROUND AND AIMS: The biliary tract cancer or cholangiocarcinoma (CCA) represents the sixth leading cause of gastrointestinal tumors in the Western world, and mortality varies across the world, with regions such as Chile, Thailand, Japan, and northeastern India presenting the highest rates. CCA may develop in the bile duct, gallbladder, or ampulla of Vater; and risk factors include obesity, parity, genetic background, geographical and environmental factors. Inflammation induced by bacterial infections might play a role in the pathogenesis of CCA. In this work, we investigated whether there is an association between extrahepatic cholangiocarcinoma (ECCA) and infection with S. typhi, H. hepaticus, or H. bilis in a Mexican population. METHODS: A total of 194 patients were included and divided into 91 patients with benign biliary pathology (controls) and 103 with ECCA (cases). Tumor samples were taken during endoscopic retrograde cholangiopancreatography by biliary brushing, followed by DNA extraction and PCR testing for infections. RESULTS: We found that 44/103 cases were positive for H. bilis, compared with 19/91 controls (p = 0.002; OR 2.83, 95% CI 1.49-5.32), and when analyzed by sub-site, H. bilis infection was significantly more associated with cancer in the common bile duct (p = 0.0005; OR 3.56, 95% CI 1.77-7.17). In contrast, H. hepaticus infection was not different between cases (17/103) and controls (13/91) (p = 0.82; OR 1.19, 95% CI 0.54-2.60). None of the samples were positive for S. typhi infection. CONCLUSION: In conclusion, infection with H. bilis but neither H. Hepaticus nor S. typhi was significantly associated with ECCA, particularly with tumors located in the common bile duct.