Gut Microbiota Modulation:A Viable Strategy to Address Medical Needs in Hepatocellular Carcinoma and Liver Transplantation

Hepatocellular carcinoma(HCC)is the most common malignancy of the liver,posing a significant threat to public health.Although liver transplantation(LT)is an effective treatment for HCC,ischemia–reperfusion(I/R)injury,transplant rejection,and complications after LT can greatly reduce its effectiveness.In recent years,transplant oncology has come into being,a comprehensive discipline formed by the intersection and integration of surgery,oncology,immunology,and other related disciplines.Gut microbiota,an emerging field of research,also plays a crucial role.Through the microbiome–gut–liver axis,the gut microbiota has an impact on the onset and progression of HCC as well as LT.This review summarizes the mechanisms by which the gut microbiota affects HCC and its bidirectional interactions with chronic liver disease that can develop into HCC as well as the diagnostic and prognostic value of the gut microbiota in HCC.In addition,gut microbiota alterations after LT were reviewed,and the relationship between the gut microbiota and liver I/R injury,the efficacy of immunosuppressive drugs used,and complications after LT were discussed.In the era of LT oncology,the role of the gut microbiota in HCC and LT should be emphasized,which can provide new insights into the management of HCC and LT via gut microbiota modulation.

TNFR2 is a potent prognostic biomarker for post-transplant lung metastasis in patients with hepatocellular carcinoma

Objective:Lung metastasis is a common and fatal complication of liver transplantation for hepatocellular carcinoma(HCC).The precise prediction of post-transplant lung metastasis in the early phase is of great value.Methods:The mRNA profiles of primary and paired lung metastatic lesions were analyzed to determine key signaling pathways.We enrolled 241 HCC patients who underwent liver transplantation from three centers.Tissue microarrays were used to evaluate the prognostic capacity of tumor necrosis factor(TNF),tumor necrosis factor receptor 1(TNFR1),and TNFR2,particularly for post-transplant lung metastasis.Results:Comparison of primary and lung metastatic lesions revealed that the TNF-dependent signaling pathway was related to lung metastasis of HCC.The expression of TNF was degraded in comparison to that in para-tumor tissues(P<0.001).The expression of key receptors in the TNF-dependent signaling pathway,TNFR1 and TNFR2,was higher in HCC tissues than in para-tumor tissues(P<0.001).TNF and TNFR1 showed no relationship with patients’outcomes,whereas elevated TNFR2 in tumor tissue was significantly associated with worse overall survival(OS)and increased recurrence risk(5-year OS rate:31.9%vs.62.5%,P<0.001).Notably,elevated TNFR2 levels were also associated with an increased risk of post-transplant lung metastasis(hazard ratio:1.146;P<0.001).Cox regression analysis revealed that TNFR2,Hangzhou criteria,age,and hepatitis B surface antigen were independent risk factors for post-transplant lung metastasis,and a novel nomogram was established accordingly.The nomogram achieved excellent prognostic efficiency(area under time-dependent receiver operating characteristic=0.755,concordance-index=0.779)and was superior to conventional models,such as the Milan criteria.Conclusions:TNFR2 is a potent prognostic biomarker for predicting post-transplant lung metastasis in patients with HCC.A nomogram incorporating TNFR2 deserves to be a helpful prognostic tool in liver transplantation for HCC.

Molecular phenotypes reveal heterogeneous engraftments of patient-derived hepatocellular carcinoma xenografts

Objective:Patient-derived xenograft(PDX)models provide a promising preclinical platform for hepatocellular carcinoma(HCC).However,the molecular features associated with successful engraftment of PDX models have not been revealed.Methods:HCC tumor samples from 76 patients were implanted in immunodeficient mice.The molecular expression was evaluated by immunohistochemistry.Patient and tumor characteristics as well as tumor molecular expressions were compared for PDX engraftment using the Chi-square test.The independent prediction parameters were identified by logistic regression analyses.Results:The engraftment rate for PDX models from patients with HCC was 39.47%(30/76).Tumors from younger patients and patients with elevated preoperative alpha-fetoprotein level had higher engraftment rates.Tumors with poor differentiation and vascular invasion were related to engraftment success.The positive expression of CK19,CD133,glypican-3(GPC3),and Ki67 in tumor samples was associated with engraftment success.Logistic regression analyses indicated that GPC3 and Ki67 were two of the strongest predictors of PDX engraftment.Tumors with GPC3/Ki67 phenotypes showed heterogeneous engraftment rates,with 71.9%in GPC3^(+)/Ki67^(+)tumors,30.8%in GPC3^(-)/Ki67^(+)tumors,15.0%in GPC3^(+)/Ki67^(-)tumors,and 0 in GPC3^(-)/Ki67^(-)tumors.Conclusions:Successful engraftment of HCC PDXs was significantly related to molecular features.Tumors with the GPC3+/Ki67+phenotype were the most likely to successfully establish HCC PDXs.

Genetic engineering drives the breakthrough of pig models in liver disease research

Compared with the widely used rodents,pigs are anatomically,physiologically,and genetically more similar to humans,making them high-quality models for the study of liver diseases.Here,we review the latest research progress on pigs as a model of human liver disease,including methods for establishing them and their advantages in studying cystic fibrosis liver disease,acute liver failure,liver regeneration,non-alcoholic fatty liver disease,liver tumors,and xenotransplantation.We also emphasize the importance of genetic engineering techniques,mainly the CRISPR/Cas9 system,which has greatly enhanced the utility of porcine models as a tool for substantially advancing liver disease research.Genetic engineering is expected to propel the pig as one of the irreplaceable animal models for future biomedical research.

ZIP4 inhibits Ephrin-B1 ubiquitination,activating Wnt5A/JNK/ZEB1 to promote liver cancer metastasis

Hepatocellular carcinoma(HCC),a highly malignant tumor,faces a major challenge with its high post-hepatectomy recurrence impacting patient survival.Mechanisms and preventive strategies remain to be resolved.Zrt-/lrt-like protein 4(ZIP4,also called SLC39A4/solute carrier family 39 member 4)belongs to the zinc transporter ZiP superfamily of which the members play an important role in maintaining zinc steady state,and its high expression in various tumors is associated with the prognosis of cancer patients.

Stratified Analysis of Survival Benefit for ABO-incompatible Deceased-donor Liver Transplantation:Multicenter Propensity Score-matched Study

Background and Aims:Liver transplantation(LT)using ABO-incompatible(ABOi)grafts can extend the donor pool to a certain extent and hence reduce the waiting time for transplantation.However,concerns of the impending prognosis associated with this option,especially for patients with liver failure and higher model for end-stage liver disease(MELD)scores,who tend to be more fragile during the waiting period before LT.Methods:Recipients undergoing LT for acute-onchronic liver failure or acute liver failure were retrospectively enrolled at four institutions.Overall survival was compared and a Cox regression analysis was performed.Propensity score matching was performed for further comparison.Patients were stratified by MELD score and cold ischemia time(CIT)to determine the subgroups with survival benefits.Results:Two hundred ten recipients who underwent ABOi LT and 1,829 who underwent ABO compatible(ABOc)LT were enrolled.The 5-year overall survival rate was significantly inferior in the ABOi group compared with the ABOc group after matching(50.6%vs.75.7%,p<0.05).For patients with MELD scores≤30,using ABOi grafts achieved a comparable overall survival rate as using ABOc grafts(p>0.05).Comparison of the survival rates revealed no statistically significant difference for patients with MELD scores≥40(p>0.05).For patients with MELD scores of 31-39,the overall survival rate was significantly inferior in the ABOi group compared with the ABOc group(p<0.001);however,the rate was increased when the liver graft CIT was<8 h.Conclusions:For recipients with MELD scores≤30,ABOi LT had a prognosis comparable to that of ABOc LT and can be regarded as a feasible option.For recipients with MELD scores≥40,ABOi should be adopted with caution in emergency cases.For recipients with MELD scores of 31-39,the ABOi LT prognosis was worse.However,those patients benefited from receiving ABOi grafts with a CIT of<8 h.

Fibroblast growth factor 21(FGF21) attenuates tacrolimus-induced hepatic lipid accumulation through transcription factor EB(TFEB)-regulated lipophagy

Tacrolimus(TAC),also called FK506,is one of the classical immunosuppressants to prevent allograft rejection after liver transplantation.However,it has been proved to be associated with post-transplant hyperlipemia.The mechanism behind this is unknown,and it is urgent to explore preventive strategies for hyperlipemia after transplantation.Therefore,we established a hyperlipemia mouse model to investigate the mechanism,by injecting TAC intraperitoneally for eight weeks.After TAC treatment,the mice developed hyperlipemia(manifested as elevated triglyceride(TG)and low-density lipoprotein cholesterol(LDL-c),as well as decreased high-density lipoprotein cholesterol(HDL-c)).Accumulation of lipid droplets was observed in the liver.In addition to lipid accumulation,TAC induced inhibition of the autophagy-lysosome pathway(microtubule-associated protein 1light chain 3β(LC3B)II/I and LC3B II/actin ratios,transcription factor EB(TFEB),protein 62(P62),and lysosomal-associated membrane protein 1(LAMP1))and downregulation of fibroblast growth factor 21(FGF21)in vivo.Overexpression of FGF21may reverse TAC-induced TG accumulation.In this mouse model,the recombinant FGF21 protein ameliorated hepatic lipid accumulation and hyperlipemia through repair of the autophagy-lysosome pathway.We conclude that TAC downregulates FGF21and thus exacerbates lipid accumulation by impairing the autophagy-lysosome pathway.Recombinant FGF21 protein treatment could therefore reverse TAC-caused lipid accumulation and hypertriglyceridemia by enhancing autophagy.

A novel nomogram for prognosis stratification in salvage liver transplantation:a national-wide study with propensity score matching analysis in China

Background:Salvage liver transplantation(SLT)has been reported to be an efficient treatment option for patients with recurrent hepatocellular carcinoma(HCC)after liver resection(LR).However,for recipients who underwent liver transplantation(LT)due to recurrent HCC after LR in China,the selection criteria are not well established.Methods:In this study,data from the China Liver Transplant Registry(CLTR)of 4,244 LT performed from January 2015 to December 2019 were examined,including 3,498 primary liver transplantation(PLT)and 746 SLT recipients.Propensity score matching(PSM)analysis was used to minimize between-group imbalances.The overall survival(OS)and disease-free survival(DFS)between PLT and SLT in recipients fulfilling the Milan or Hangzhou criteria were compared based on the multivariate analysis,nomograms were plotted to further classify the SLT group into low-and high-risk groups.Results:In this study,the 1-,3-and 5-year OS and DFS of SLT recipients fulfilling Milan criteria(OS,P=0.01;DFS,P<0.001)or Hangzhou criteria(OS,P=0.03;DFS,P=0.003)were significantly reduced when compared to that of PLT group after PSM analysis.Independent risk factors,including preoperative transarterial chemoembolization(TACE),alpha fetoprotein(AFP)level,tumor maximum size and tumor total diameter were selected to draw a prognostic nomogram.The low-risk SLT recipients(1-year,95.34%;3-year,84.26%;5-year,77.20%)showed a comparable OS with PLT recipients fulfilling Hangzhou criteria (P=0.107). Conclusions: An optimal nomogram model for prognosis stratification and clinical decision guidance of SLT was established. The low-risk SLT recipients based on the nomograms showed comparable survival with those fulfilling Hangzhou criteria in PLT group.

Zinc finger protein 296 promotes hepatocellular carcinoma progression via intervening interaction between macrophages and B cells

Objective:Hepatocellular carcinoma(HCC)is a prevalent malignancy with poor survival.Different cell types in the tumor microenvironment participate in the tumorigenesis and progression of HCC.This study aimed to analyze the immune microenvironment of HCC and its relationship with clinical outcomes.Methods:We analyzed HCC RNA-seq for cell type identification and prognosis by estimating relative subsets of RNA transcripts using CIBERSORTx.The interaction between B cells and macrophages in HCC was analyzed using a Hepa1-6 orthotopic transplantation mouse model and flow cytometry.The effect of Zinc finger protein 296(ZNF296)on the interaction of B cells and macrophages was verified using human HCC tissues analyzed through western blot,quantitative real-time polymerase chain reaction(qPCR),and multiplex immunofluorescence.A comparative analysis of immune cells associated with HCC prognosis was performed using RNA-seq data from The Cancer Genome Atlas(TCGA),bulk multimodal data,and single-cell transcriptomic data from existing HCC single-cell transcriptomic data employing the Single Cell Inferred Site Specific Omics Resource for Tumor Microenvironments(SCISSOR).Results:Liver hepatocellular carcinoma(LIHC)RNA-seq analysis of TCGA showed that high eosinophil infiltration promoted HCC progression.The proportion of B cells correlated with that of macrophages(r=−0.24)and affected the infiltration and programmed death ligand 1(PD-L1)expression of macrophages in HCC.ZNF296 may participate in the interaction between B cells and macrophages to accelerate the HCC progression by regulating PAFAH1B3 and H2AFX.Moreover,ZNF296 expression positively correlated with LAG3(r=0.27)and CTLA4(r=0.31)expression levels.Among the immune cell phenotypes related to survival and death identified by SCISSOR analysis,T cells correlated with an excellent prognosis of HCC.The normal function of liver and dendritic cells was also associated with a good prognosis in HCC.Conclusions:This study analyzed the interaction of the immune microenvironment with HCC prognosis,identifying ZNF296 as a promising diagnostic and therapeutic target for HCC.

Severity of early allograft dysfunction following donation after circulatory death liver transplantation:a multicentre study

Background:Early allograft dysfunction(EAD)is associated with decreased graft and patient survival rates.This study aimed to identify the severity of EAD and develop a predictive model for EAD after donation after circulatory death(DCD)liver transplantation(LT).Furthermore,the influence of operative time on EAD incidence was also evaluated.Methods:In this retrospective,multicentre cohort study,nomograms were established based on a single-centre training cohort(n=321)and validated in a 3-center validation cohort(n=501).Results:The incidence rate of EAD was 46.4%(149/321)in the training cohort and 40.5%(203/501)in the validation cohort.Of the 149 EAD patients in the training cohort,77 patients with either elevated alanine aminotransferase(ALT)or aspartate aminotransferase(AST)were classified as having EAD type A,and the rest of the EAD patients were classified as having EAD type B.Recipients with EAD type B had lower graft and patient survival rates than recipients with EAD type A(P=0.043 and 0.044,respectively).We further developed a nomogram to predict EAD(graft weight,cold ischemia time,donor age,model for end-stage liver disease(MELD)score)and another nomogram to predict EAD type B(graft weight,cold ischemia time,MELD score).The nomograms for the prediction of EAD and EAD type B had good discrimination[concordance index(C-index)=0.712(0.666-0.758),0.707(0.641-0.773)]and calibration[Hosmer-Lemeshow(HL)P=0.384,P=0.425]in the validation cohort.An increased operative time(>6 h)was associated with increased EAD and EAD type B incidence in the high-risk group(P=0.005,P=0.020,respectively).Conclusions:EAD type B was associated with decreased graft and patient survival rates.The novel nomograms effectively predicted the incidence of EAD and EAD type B in DCD LT patients.

Extrahepatic organs in the development of non-alcoholic fatty liver disease in liver transplant patients

Background and Objective:Non-alcoholic fatty liver disease(NAFLD)is highly prevalent in patients who undergo liver transplantation(LT).Whereas there is huge data on NAFLD,little is known about NAFLD in LT.In this review,we aim to explore extrahepatic organs and their potential mechanisms in the development of NAFLD in LT patients and discuss current limitations in preclinical and clinical scenarios with suggestions for future study.Methods:The following keywords,such as NAFLD,NASH,liver transplant,therapy,pathogenesis and biomarkers,were set for literature retrieval.The articles which were published articles in English till 25th June 2020 in PubMed database were included,and there is no limit for the study design type.Key Content and Findings:Following LT,there are significant shifts in the microbiota and farnesoid X receptor may be a potential therapeutic target for NAFLD in LT settings.The roles of probiotics and diet on NALFD remain inconclusive in LT background.Nevertheless,the adipokines and cytokines disorder and local insulin resistance of adipose tissue may contribute to NAFLD process.Bariatric surgeries are promising in controlling de novo and recurrent NAFLD with significant reduction in abdominal adipose tissue,despite the optimal timing is inconclusive in LT cases.Furthermore,circumstantial evidence indicates that miRNA-33a may function as a mediator bridging sarcopenia and NAFLD of post-LT.β-Hydroxy-β-Methyl-Butyrate treatment could improve muscle status in graft recipients and shows protective potential for NAFLD in LT settings.Conclusions:Gut,adipose tissue and muscle are intricately intertwined in promoting NAFLD in LT cases.Further animal studies are needed to deepen our understanding of mechanisms in multi-organ crosstalk.High quality clinical trials are warrant for making guidelines and developing management strategies on NAFLD after LT.

Visual analysis of mesenchymal stem cell research in liver disease based on bibliometrics

The research of mesenchymal stem cells(MSCs)in the field of liver diseases has received more and more attention.This paper introduces the current situation,hot spots,and development trends in this field.Compre-hensive searches were conducted using Web of Science Core Collection from January 1,2000 to December 13,2021 with the following keywords:TS(topic)¼(liver*OR hepatic*OR hepatocyte)AND TS(topic)¼(Mesen-chymal stem cell*).VOSviewer(version 1.6.16)and CiteSpace V are used as bibliometric tools to analyze and visualize the knowledge graph.A total of 4452 papers were included in this study,and the number of research papers on MSCs in the field of liver diseases increased from January 2000 to December 2020.Eighty-four countries and regions have published articles on research in this field,among which China and the United States are the main two countries of publication.Based on the keyword burst detection,we find that the research in this field has shifted from basic research to clinical application,from medical research to interdisciplinary research.Tissue engineering and regenerative medicine are the frontier fields of MSCs research in liver diseases.Multicountry,multi-author cooperation,and multi-disciplinary intersection are the research trends in this field.Exocrine body,obesity,and tissue engineering are the hotspots in this field.

CCL16 inhibits tumor proliferation and metastasis in HCC by impacting CK19 phenotype

Background and aims:Cytokeratin 19-positive(CK19t)hepatocellular carcinoma(HCC)is an aggressive subtype with poor outcomes.The initiation and development of CK19t HCC in the background of liver cirrhosis remains unclear.This study investigated the role of the cirrhosis-related gene C-C motif chemokine ligand 16(CCL16)in the development of CK19t HCC.Methods:Datasets from Gene Expression Omnibus(GEO),The Cancer Genome Atlas(TCGA)and International Cancer Genome Consortium(ICGC)were analyzed to screen and validate the genes associated with CK19t HCC.A total of 102 HCC patients were included for tissue microarray analysis.Gain-of-function experiments were conducted to investigate the biological functions of CCL16.CIBERSORT was used to investigate the correlation of CCL16 and immune infiltration.Results:GEO dataset analysis showed that CK19t HCC had lower expression of CCL16.In both TCGA dataset and our HCC cohort,CCL16 expression was negatively correlated with CK19 expression(P<0.05)and its expression was higher in para-tumor than tumor tissues(P<0.001).Moreover,low CCL16 expression was related to advanced stage and poor overall survival(P<0.05).CCL16 overexpression downregulated CK19 expression and impacted the sphere formation ability of HCC cells.Overexpression of CCL16 inhibited the cell proliferation,migration,and invasion of HCC cell lines.Immune analysis showed HCC with high CCL16 expression had more infiltration of mast cells.HCC patients with both low CCL16 expression and low mast cells had the worst prognosis(P<0.001).Conclusion:Our data indicated that CCL16 downregulated the expression of CK19 and inhibited the malignant phenotype of HCC.