Peritoneal Gene Transfection of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand for Tumor Surveillance and Prophylaxis.

Peritoneal metastasis is very common in gastrointestinal, reproductive, and genitourinary tract cancers in late stages or postsurgery, causing poor prognosis, so effective and nontoxic prophylactic strategies against peritoneal metastasis are highly imperative. Herein, we demonstrate the first gene transfection as a nontoxic prophylaxis preventing peritoneal metastasis or operative metastatic dissemination. Lipopolyplexes of TNF-related-apoptosis-inducing-ligand (TRAIL) transfected peritonea and macrophages to express TRAIL for over 15 days. The expressed TRAIL selectively induced tumor cell apoptosis while exempting normal tissue, providing long-term tumor surveillance. Therefore, tumor cells inoculated in the pretransfected peritoneal cavity quickly underwent apoptosis and, thus, barely formed tumor nodules, significantly prolonging the mouse survival time compared with chemotherapy prophylaxis. Furthermore, lipopolyplex transfection showed no sign of toxicity. Therefore, this peritoneal TRAIL-transfection is an effective and safe prophylaxis, preventing peritoneal metastasis.

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-on-chronic 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.

SULT2B1-CS-DOCK2 axis regulates effector T-cell exhaustion in HCC microenvironment.

BACKGROUND AND AIMS: HCC is a malignant disease. Compared with tyrosine kinase inhibitors (the classical therapy), immune checkpoint inhibitors are more effective in the treatment of HCC, despite their limited efficacy. Among these restricted factors, exhaustion of tumor-infiltrated lymphocytes, especially CD8 + T cells, is a core event. We aimed to determine the key factors contributing to CD8 + T-cell infiltration in HCC and investigate the underlying mechanisms. APPROACH AND RESULTS: Using machine learning and multiplex immunohistochemistry analysis, we showed that dedicator of cytokinesis protein 2 (DOCK2) was a potential indicator of infiltrated CD8 + T cells in HCC. Using RNA sequencing, flow cytometry analysis, and mouse HCC models, we demonstrated that DOCK2 inactivation accounted for infiltrated CD8 + T-cell exhaustion in tumors. Using quasi-targeted metabolomics, mass spectrum, and mass cytometry by time of flight analysis, we found that cholesterol sulfate synthesized by sulfotransferase 2B1 in tumor cells suppressed DOCK2 enzymatic activity of T cells. Through virtual screening, molecular docking simulation, and experiments validation, we demonstrated that tolazamide reversed DOCK2 inactivation-mediated CD8 + T-cell exhaustion and enhanced anti-programmed death-ligand 1 antibody+apatinib immunotherapeutic effects on HCC. CONCLUSIONS: This study indicates that DOCK2 controls CD8 + T-cell infiltration in HCC, and cholesterol sulfate synthesized by sulfotransferase 2B1 in tumor cells promotes effector T-cell exhaustion. The findings suggest that the usage of conventional drugs affects immunotherapy efficacy in HCC patients.

Identification and Verification of a Novel MAGI2-AS3/miRNA-374-5p/FOXO1 Network Associated with HBV-Related HCC.

BACKGROUND: Hepatocellular carcinoma (HCC) is a very common neoplasm worldwide, and competitive endogenous RNA (ceRNA) plays an important role in the development of HCC. The purpose of this study is to investigate the molecular mechanisms of ceRNAs in HCC. METHODS: This study detects potential ceRNAs from HCC through whole genome analysis of lncRNA, miRNA and mRNA expression. We then performed high-throughput sequencing of tissues from five hepatitis B related HCC patients to screen ceRNAs and those screened ceRNAs expressions were verified on tissues from an independent group of six patients. Finally, the function of ceRNAs of interest was illustrated in vitro. RESULT: Functional and pathway analysis of The Cancer Genome Atlas revealed ceRNA networks. The high-throughput sequencing identified 985 upregulated and 1612 downregulated lncRNAs and 887 upregulated and 1116 downregulated mRNAs in HCC patients. Differentially expressed genes were parallel to cancer-associated processes, comprising 18 upregulated and 35 downregulated significantly enriched pathways including alcoholism and viral carcinogenesis. Among them, a potential ceRNA network was detected and verified in six HCC patients. CeRNAs of the lncRNA MAGI2-AS3/miR-374-5p/FOXO1 pathway were significantly dysregulated in HCC, and validation in vitro showed that FOXO1 is positively regulated by MAGI2-AS3 through the induction of miR-374a/b-5p in HCC cells. In addition, the overexpression of FOXO1 is associated with proliferation, migration, and invasion of HCC cells and increases apoptosis of HCC cells. MiR-374a/b-5p caused an opposite effect by directly suppressing FOXO1 in HCC cells. CONCLUSION: CeRNA networks were found in HCC and aberrantly expressed ceRNAs of lncRNA MAGI2-AS3/miR-374-5p/FOXO1 plays a crucial role in HCC, assisting in diagnosis and providing a method for treatment.

Prediction of tumor recurrence by distinct immunoprofiles in liver transplant patients based on mass cytometry.

Recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT) is a marker of poor prognosis. However, the reliable biomarkers of post-LT HCC recurrence remain to be identified. In this study, serial peripheral blood samples from the LT recipients with and without HCC recurrence were collected at five time points. Single-cell mass cytomertry (CyTOF) was utilized for the in-depth analysis of peripheral blood monocellular cells (PBMCs). CyTOF analysis showed that at 3 weeks post-LT, the activated immune cell population was increased, while the fraction of immune cells with suppressive functions (myeloid-derived suppressive cells) was reduced. The post-LT immune composition in patients with LT for HCC was enormously different from that in patients with LT for causes other than HCC. Furthermore, at 3 weeks after LT, compared with patients without recurrence, the patients with HCC recurrences were high in two subsets of T cells: CD57+ HLA-DR+ CD8+ and CD28+γδ. The CD57+ HLA-DR+ CD8+ T cells presented high levels of perforin, granzyme B, and Ki-67 and displayed a highly cytotoxic and proliferative phenotype, while the CD28+γδ T cells had reduced levels of IFN-γ and, hence, were less activated compared to CD28- cells. Based on these findings, we concluded that analyzing the PBMCs of LT recipients by CyTOF can predict the post-LT HCC recurrence. The distinct immune features can stratify patients with the risk of HCC recurrence at 3 weeks after LT, which will help clinician in further management plan and improve the prognosis of patients.

Proteomics-based identification of the role of osteosarcoma amplified-9 in hepatocellular carcinoma recurrence.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies; its recurrence is associated with high mortality and poor recurrence-free survival and is affected by multisystem and multilevel pathological changes. To identify the key proteins associated with tumor recurrence and the underlying mechanisms, proteomic profiling of tumor specimens from early recurrence and nonrecurrence patients was performed in this study. Proteomics was applied to identify differentially expressed proteins during the early recurrence of HCC after surgery. Osteosarcoma amplified-9 (OS-9) was discovered, and the correlation between OS-9 expression and the clinicopathological characteristics of patients was analyzed. Invasion and migration were examined in SMMC-7721 cells with and without OS-9 overexpression. Proteomics was performed once again using SMMC-7721 cells with OS-9 overexpression to further analyze the proteins with altered expression. OS-9 was overexpressed in the early recurrence group, and OS-9 overexpression was associated with high serum alpha-fetoprotein levels and poor recurrence-free survival in 196 patients with HCC. The invasion and migration abilities of SMMC-7721 cells were enhanced in the OS-9 overexpression group. Bioinformatic functional enrichment methods, including Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis, revealed that the hypoxia-inducible factor 1 (HIF-1) and tumor necrosis factor (TNF) signaling pathways were activated in the OS-9 overexpression group. The migration and invasion capacities of OS-9 overexpressed HCC cell line were weakened while treated with HIF-1α or TNF-α inhibitors. Conclusion: Our results suggest that the overexpression of OS-9 is related to HCC recurrence, thereby contributing to the migration and invasion capacities of HCC cell line by regulating the HIF-1 and TNF pathways.

Development of a Novel Prognostic Nomogram for High Model for End-Stage Liver Disease Score Recipients Following Deceased Donor Liver Transplantation.

Background: A high model of end-stage liver disease (MELD) score (>30) adversely affects outcomes even if patients receive prompt liver transplantation (LT). Therefore, balanced allocation of donor grafts is indispensable to avoid random combinations of donor and recipient risk factors, which often lead to graft or recipient loss. Predictive models aimed at avoiding donor risk factors in high-MELD score recipients are urgently required to obtain satisfactory outcomes. Method: Data of patients with MELD score >30 who underwent LT at three transplantation institutes between 2015 and 2018 were retrospectively reviewed. Early allograft dysfunction (EAD), length of intensive care unit (ICU) stay, and graft loss were recorded. Corresponding independent risk factors were analyzed using stepwise multivariable regression analysis. A prediction model of graft loss was developed, and discrimination and calibration were measured. Results: After applying the exclusion criteria, 778 patients were enrolled. The incidence of EAD was 34.8% (271/778). Donor graft macrovesicular steatosis, graft-to-recipient weight ratio (GRWR), warm ischemia time (WIT), cold ischemia time (CIT), and ABO blood incompatibility, together with donor serum albumins, were independent predictors of EAD. The incidence of ICU stay over 10 days was 64.7% (503/778). Donor age, recipient's MELD score, Child score, and CIT were independent predictors of ICU stay. The 3-year graft survival rates (GSRs) in the training and validation cohorts were 64.2 and 59.3%, respectively. The independent predictors of graft loss were recipient's Child score, ABO blood type incompatibility, donor serum total bilirubin over 17.1 µmol/L, and cold CIT. A nomogram based on these variables was internally and externally validated and showed good performance (area under the receiver operating characteristic curve = 70.8 and 66.0%, respectively). For a recipient with a high MELD score, the avoidance of ABO blood type incompatibility and CIT ≥6 h would achieve a 3-year GSR of up to 78.4%, whereas the presence of the aforementioned risk factors would decrease the GSR to 35.4%. Conclusion: The long-term prognosis of recipients with MELD scores >30 could be greatly improved by avoiding ABO blood type incompatibility and CIT ≥6 h.

The distinct responsiveness of cytokeratin 19-positive hepatocellular carcinoma to regorafenib.

Cytokeratin 19-positive (CK19+) hepatocellular carcinoma (HCC) is an aggressive subtype characterized by early recurrence and chemotherapy tolerance. However, there is no specific therapeutic option for CK19+ HCC. The correlation between tumor recurrence and expression status of CK19 were studied in 206 patients undergoing liver transplantation for HCC. CK19-/+ HCC cells were isolated to screen effective antitumor drugs. The therapeutic effects of regorafenib were evaluated in patient-derived xenograft (PDX) models from 10 HCC patients. The mechanism of regorafenib on CK19+ HCC was investigated. CK19 positiveness indicated aggressiveness of tumor and higher recurrence risk of HCC after liver transplantation. The isolated CK19+ HCC cells had more aggressive behaviors than CK19- cells. Regorafenib preferentially increased the growth inhibition and apoptosis of CK19+ cells in vitro, whereas sorafenib, apatinib, and 5-fluorouracil did not. In PDX models from CK19-/+ HCC patients, the tumor control rate of regorafenib achieved 80% for CK19+ HCCs, whereas 0% for CK19- HCCs. RNA-sequencing revealed that CK19+ cells had elevated expression of mitochondrial ribosomal proteins, which are essential for mitochondrial function. Further experiments confirmed that regorafenib attenuated the mitochondrial respiratory capacity in CK19+ cells. However, the mitochondrial respiration in CK19- cells were faint and hardly repressed by regorafenib. The mitochondrial respiration was regulated by the phosphorylation of signal transducer and activator of transcription 3 (STAT3), which was inhibited by regorafenib in CK19+ cells. Hence, CK19 could be a potential marker of the therapeutic benefit of regorafenib, which facilitates the individualized therapy for HCC. STAT3/mitochondria axis determines the distinct response of CK19+ cells to regorafenib treatment.

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.

A preoperative model for predicting microvascular invasion and assisting in prognostic stratification in liver transplantation for HCC regarding empirical criteria.

PURPOSE: The prediction of microvascular invasion (MVI) has increasingly been recognized to reflect prognosis involving local invasion and distant metastasis of hepatocellular carcinoma (HCC). The aim of this study was to assess a predictive model using preoperatively accessible clinical parameters and radiographic features developed and validated to predict MVI. This predictive model can distinguish clinical outcomes after liver transplantation (LT) for HCC patients. METHODS: In total, 455 HCC patients who underwent LT between January 1, 2015, and December 31, 2019, were retrospectively enrolled in two centers in China as a training cohort (ZFA center; n = 244) and a test cohort (SLA center; n = 211). Univariate and multivariate backward logistic regression analysis were used to select the significant clinical variables which were incorporated into the predictive nomogram associated with MVI. Receiver operating characteristic (ROC) curves based on clinical parameters were plotted to predict MVI in the training and test sets. RESULTS: Univariate and multivariate backward logistic regression analysis identified four independent preoperative risk factors for MVI: α-fetoprotein (AFP) level (p < 0.001), tumor size ((p < 0.001), peritumoral star node (p = 0.003), and tumor margin (p = 0.016). The predictive nomogram using these predictors achieved an area under curve (AUC) of 0.85 and 0.80 in the training and test sets. Furthermore, MVI could discriminate different clinical outcomes within the Milan criteria (MC) and beyond the MC. CONCLUSIONS: The nomogram based on preoperatively clinical variables demonstrated good performance for predicting MVI. MVI may serve as a supplement to the MC.

Interleukin-2 inducible T-cell kinase: a potential prognostic biomarker and tumor microenvironment remodeling indicator for hepatocellular carcinoma.

BACKGROUND: The heterogeneous tumor microenvironment (TME) contributes to poor prognosis of hepatocellular carcinoma (HCC). However, determining the modulation of TME during HCC progression remains a challenge. METHODS: Herein, the stromal score and immune score of HCC samples from The Cancer Genome Atlas database were calculated using the ESTIMATE algorithm and differentially expressed genes (DEGs) were obtained. Key DEGs were identified based on a protein-protein interaction network and survival analysis. Immunohistochemistry was carried out using primary samples to evaluate key DEGs expression. The CIBERSORT algorithm was applied to evaluate immune components. Gene Set Enrichment Analysis (GSEA) and correlation analysis were carried out to determine the relationship between key DEGs and tumor-infiltrating immune cells (TICs). RESULTS: The stromal score, immune score and estimate score correlated significantly with 1-year recurrence-free survival of patients with HCC. Interleukin-2 inducible T-cell kinase (ITK) was identified as the most prognostic DEG for patients with HCC. GSEA revealed that genes in the high ITK subgroup were enriched in inflammatory-immunological terms. CIBERSORT analysis identified nine TIC subsets that correlated with ITK expression. CONCLUSION: We identified ITK as a novel indicator for early post-surgery tumor recurrence and microenvironment remodeling in HCC, providing a potential therapeutic target to treat HCC.

Hangzhou criteria as downstaging criteria in hepatocellular carcinoma before liver transplantation: A multicenter study from China.

BACKGROUND: The downstaging of hepatocellular carcinoma (HCC) has been confirmed to benefit liver transplantation (LT) patients whose tumors are beyond the transplantation criteria. Milan criteria (MC), a tumor size and number-based assessment, is currently used as the endpoint in these patients. However, many studies believe that tumor biological behavior should be added to the evaluation criteria for downstaging efficacy. Hence, this study aimed to explore the feasibility of Hangzhou criteria (HC), which introduced tumor grading and alpha-fetoprotein in addition to tumor size and number, as an endpoint of downstaging. METHODS: We performed a multicenter and retrospective study of 206 patients accepted locoregional therapy (LRT) as downstaging/bridge treatment prior to LT in three centers of China. RESULTS: Recipients were divided into four groups: failed downstaging to the HC (group A, n = 46), successful downstaging to the HC (group B, n = 30), remained within the HC all the time (group C, n = 113), and tumor progressed (group D, n = 17). The 3-year HCC recurrence probabilities of groups B and C were not significantly different (10.3% vs. 11.6%, P = 0.87). The HCC recurrent rate was significantly higher in group A (52.3%) compared with that in group B/C (P < 0.05). Seven patients (7/76, 9.2%) whose tumor exceeded the the HC were successfully downstaged to the MC, and 39.5% (30/76) to the the HC. In group B, 23 patients remained beyond the MC and their survivals were as well as those of patients within the MC. CONCLUSIONS: Compared to the MC, HC downstaging criteria can give more HCC patients access to LT and furthermore, the outcome of these patients is the same as those matching MC downstaging criteria. Hangzhou downstaging criteria therefore is applicable in clinical practice.

Homocysteine: A novel prognostic biomarker in liver transplantation for alpha-fetoprotein- negative hepatocellular carcinoma.

BACKGROUND: Precise recipient selection optimizes the prognosis of liver transplantation (LT) for hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is the most commonly used biomarker for diagnosis and prognosis of HCC in the clinical context. As a crucial molecule in methionine cycle, homocysteine (Hcy) level has been proved to be related to HCC progression and metastasis. OBJECTIVE: We aimed to explore the prognostic capacity of pre-transplant serum Hcy level in LT for HCC. METHODS: This study retrospectively enrolled 161 HCC patients who had underwent LT from donation after cardiac death (DCD) in the First Affiliated Hospital of Zhejiang University from 2015.01.01 to 2018.09.01. Pre-transplant serum Hcy level was incorporated into statistical analysis together with other clinical parameters and pathological features. RESULTS: From an overall perspective, significant difference was observed in Hcy level between recurrence (n= 61) and non-recurrence group (n= 100) though subsequent analysis showed unsatisfactory predicting performance. In the whole cohort, multivariate analysis showed that lnAFP (p= 0.010) and Milan criteria (MC, p< 0.001) were independent risk factors of HCC recurrence after LT. MA score based on MC and lnAFP performed well in predicting post-LT tumor recurrence with the AUROC at 0.836 (p< 0.001) and 3-year recurrence-free survival rate at 96.8% (p< 0.001) in the low risk group (n= 69). According to the clinical practice, serum concentration lower than 20 µg/L is considered as normal range of AFP. Elevated pre-transplant serum AFP (> 20 µg/L) predicts high HCC recurrence after LT. We further divided the 161 recipients into AFP- group (n= 77, AFP ⩽ 20 µg/L) and AFP+ group (n= 84, AFP > 20 µg/L). MA score was still well presented in the AFP+ group and the AUROC for tumor recurrence was 0.823 (p< 0.001), whereas the predicting accuracy was reduced in AFP- group (AUROC: 0.754, P< 0.001). After subsequent analysis, we found that elevated pre-transplant Hcy level (> 12.75 µmol/L) predicted increased tumor recurrence risk in AFP- group. The 3-year recurrence-free survival rates were 92.0% and 53.7% (p< 0.001) in low Hcy subgroup (n= 40) and high Hcy subgroup (n= 37) respectively. Multivariate analysis showed that Hcy (p= 0.040) and Milan criteria (p= 0.003) were independent risk factors for post-transplant tumor recurrence in AFP- group. Further combination of Hcy level and Milan criteria identified a subgroup of AFP- recipients with acceptable outcomes even though beyond Milan criteria (3-year recurrence-free survival rate: 77.7%, p< 0.001). CONCLUSION: As a classic predictor in HCC prognosis, AFP performed well in our study cohort when combined with Milan criteria. Homocysteine was an effective prognostic biomarker in LT for AFP- hepatocellular carcinoma. In recipients exceeding Milan criteria, acceptable post-transplant outcome could be seen in those with low Hcy and AFP level.

The Matching Status Between Donor and Recipient Hepatitis B Seroepidemiology Makes a Difference in Liver Transplantation for Hepatocellular Carcinoma.

INTRODUCTION: Antibody to hepatitis B core antigen (HBcAb) is known to be related with the prognosis for patients with hepatocellular carcinoma (HCC). This study aims to evaluate the prognostic capacity of HbcAb and other donor/recipient hepatitis B seroepidemiological indexes in transplantation for HCC. METHODS: Based on the national liver transplant registry, we analyzed the prognostic capacity of HBcAb in liver transplantation for patients with HCC of different etiological backgrounds. The hepatitis B virus (HBV)-related HCC cohort was further studied regarding donor/recipient hepatitis B seroepidemiology, and then divided into a training cohort (n = 1,222) and a validation cohort (n = 611) to develop a pretransplant recurrence-risk predicting nomogram. RESULTS: Positive HbcAb in recipients was related to an increased risk of post-transplant tumor recurrence in HBV-related (n = 1,833, P = 0.007), HCV-related (n = 79, P = 0.037), and non-B non-C HCC (n = 313, P = 0.017). In HBV-related HCC (n = 1,833), donor hepatitis B surface antigen (HbsAg) was also associated with post-transplant tumor recurrence (P = 0.020). Multivariate analysis showed that the matching status of recipient HbcAb and donor HbsAg (MSHB) was an independent prognostic factor (P = 0.017). HbcAb-positive recipients matched with HbsAg-positive donors displayed the worst post-transplant outcomes (P < 0.001). In the training cohort (n = 1,222), a risk-predicting nomogram was established based on α-fetoprotein, Milan criteria, and MSHB. The model showed excellent prognostic capacity and safely expanded Milan criteria in both training and validation cohorts (P < 0.001). DISCUSSION: Positive HbcAb in recipients increases the risk of post-transplant tumor recurrence in HCC with different etiological backgrounds. The nomogram based on MSHB is effective in predicting tumor recurrence after transplantation for HBV-related HCC.

Transforming a toxic drug into an efficacious nanomedicine using a lipoprodrug strategy for the treatment of patient-derived melanoma xenografts.

Despite the progress made with the recent clinical use of the anticancer compound cabazitaxel, the efficacy in patients remains unsatisfactory, largely due to the high in vivo toxicity of the agent. Therefore, strategies that achieve favorable outcomes and good safety profiles will greatly expand the repertoire of this potent agent. Here, we propose a combinatorial strategy to reform the cabazitaxel agent and the use of sequential supramolecular nanoassembly with liposomal compositions to assemble a prodrug-formulated liposome, termed lipoprodrug, for safe and effective drug delivery. Reconstructing cabazitaxel with a polyunsaturated fatty acid (i.e., docosahexaenoic acid) via a hydrolyzable ester bond confers the generated prodrug with the ability to be readily integrated into the lipid bilayer of liposomes for systemic administration. The resulting lipoprodrug scaffold showed significantly sustained drug release profiles and improved pharmacokinetics in rats as well as a reduction in systemic toxicity in vivo. Notably, the lipoprodrug outperformed free cabazitaxel in terms of in vivo therapeutic efficacy in multiple separate tumor xenograft-bearing mouse models, one of which was a patient-derived xenograft model. Surprisingly, the lipoprodrug was able to reduce tumor invasiveness and reprogram the tumor immunosuppressive microenvironment by proinflammatory macrophage polarization. Our findings validate this lipoprodrug approach as a simple yet effective strategy for transforming the highly toxic cabazitaxel agent into an efficacious nanomedicine with excellent in vivo tolerability. This approach could also be applied to rescue other drugs or drug candidates that have failed in clinical trials due to poor pharmacokinetic properties or unacceptable toxicity in patients.

The circFASN/miR-33a pathway participates in tacrolimus-induced dysregulation of hepatic triglyceride homeostasis.

Dyslipidemia exhibits a high incidence after liver transplantation, in which tacrolimus, a widely used immunosuppressant, plays a fundamental role. MicroRNAs and related circRNAs represent a class of noncoding RNAs that have been recognized as important regulators of genes associated with lipid metabolism. However, their transcriptional activities and functional mechanisms in tacrolimus-related dyslipidemia remain unclear. In this study, we observed that tacrolimus could induce triglyceride accumulation in hepatocytes by stimulating sterol response element-binding proteins (SREBPs) and miR-33a. Our in silico and experimental analyses identified miR-33a as a direct target of circFASN. Tacrolimus could downregulate circFASN and result in elevated miR-33a in vivo and in vitro. Overexpression of circFASN or silencing of miR-33a decreased the promoting effects of tacrolimus on triglyceride accumulation. Clinically, the incidence of dyslipidemia in liver transplant recipients with elevated serum miR-33a after liver transplantation was higher than that in patients without elevated serum miR-33a (46.3% vs. 18.8% p = 0.012, n = 73). Our results showed that the circFASN/miR-33a regulatory system plays a distinct role in tacrolimus-induced disruption of lipid homeostasis. MiR-33a is likely a risk factor for tacrolimus-related dyslipidemia, providing a potential therapeutic target to combat tacrolimus-induced dyslipidemia after liver transplantation.

Sirolimus-based immunosuppression improves outcomes in liver transplantation recipients with hepatocellular carcinoma beyond the Hangzhou criteria.

BACKGROUND: The administration of calcineurin inhibitors (CNIs) posttransplant has been implicated as an independent risk factor for the recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT). The new immunosuppressive agent sirolimus (SRL) acts as a primary immunosuppressant or antitumor agent. In this study we investigated the effect of sirolimus-based immunosuppression compared to CNIs (non-SRL) on the outcomes of LT candidates with HCC. METHODS: We retrospectively analyzed 204 HCC patients who underwent LT in our hospital between January 2, 2014 and December 10, 2017. The median of the follow-up duration of patients was 24.5 months. The patients were divided into a sirolimus (SRL) group (76 patients) and a non-sirolimus (non-SRL) group (128 patients). Patients exceeding the LT criteria were analyzed as subgroups. Disease-free survival (DFS) and overall survival (OS) after tumor recurrence were compared using the Kaplan-Meier method. Univariate and multivariate Cox analyses were used to compare OS between the SRL and non-SRL groups. RESULTS: The SRL group achieved better OS compared to the non-SRL group, while there was no significant difference in DFS. Subgroup (Milan criteria-based or Hangzhou criteria-based) analyses revealed that patients exceeding, rather than meeting, the Milan or Hangzhou criteria benefited from SRL (exceeding the Milan criteria: P=0.002; exceeding the Hangzhou criteria: P<0.001). There was no significant difference in OS between the SRL group and the non-SRL group that met the Milan or Hangzhou criteria. CONCLUSIONS: SRL can improve survival outcomes in LT patients with HCC exceeding the Hangzhou criteria.

Novel fast-acting pyrazole/pyridine-functionalized N-heterocyclic carbene silver complexes assembled with nanoparticles show enhanced safety and efficacy as anticancer therapeutics.

In this study, we designed and synthesized four novel multi-nuclear silver complexes (1-4) coordinated with pyrazole- or pyridine-functionalized N-heterocyclic carbene (NHC) ligands. The crystal structures of the silver-NHC complexes were confirmed by X-ray diffraction analysis. In vitro assays showed that the silver-NHC complexes effectively killed a broad range of cancer cells after short-term drug exposure, serving as fast-acting cytotoxic agents. Of note, in cisplatin-resistant A549 cancer cells, the silver complexes were not cross-resistant with the clinically used cisplatin agent. Detailed mechanistic studies revealed that complex 2 triggered caspase-independent cell necrosis associated with intracellular reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) depletion. By exploiting a facile nano-assembly process, silver-NHC complexes 1, 2 and 4 were successfully integrated into the hydrophobic cores of amphiphilic matrices (DSPE-PEG2K), enabling systemic injection. The silver complex-loaded nanotherapeutics (1-NPs, 2-NPs, and 4-NPs) showed high safety margins with reduced systemic drug toxicities relative to cisplatin in animals. Furthermore, in a xenograft model of human colorectal cancer, the administration of the nanotherapeutics resulted in a marked inhibition of tumor progression.

CC motif chemokine ligand 16 inhibits the progression of liver cirrhosis via inactivating hepatic stellate cells.

BACKGROUND: Liver cirrhosis results from many forms of chronic damage, characterized by accumulation of extracellular matrix. The present study aimed to explore a potential non-invasive biomarker and its mechanism in the progression of liver cirrhosis. METHODS: Gene Expression Omnibus (GEO) dataset (GSE15654, n = 216) was analyzed to screen genes associated with progression of liver cirrhosis. A total of 181 plasma samples, including healthy control (HC, n = 20), chronic hepatitis B (CHB, n = 77) and HBV-related liver cirrhosis (LC, n = 84), were enrolled for validation. In vitro and in vivo experiments were employed for the mechanistic investigation. RESULTS: GEO dataset analysis showed that relatively low mRNA-expression of CC motif chemokine ligand 16 (CCL16) was associated with elevated Child-Pugh score (P = 0.034) and worse prognosis (P = 0.025). Plasma CCL16 level decreased in a stepwise pattern, with a median concentration of 10.29, 6.57 and 4.47 ng/mL in the HC, CHB and LC groups, respectively (P<0.001). Low plasma CCL16 was significantly related to hepatic dysfunction both in the CHB and LC groups (P<0.05). Combination of CCL16 and ALT showed improved distinguishing capability for LC compared to either alone. In vitro, CCL16 expression was downregulated by lipopolysaccharide and hypoxia. Overexpression of CCL16 from human normal liver cell line (LO2) reduced the extracellular matrix associated proteins (Col1 and Col4) in human hepatic stellate cell line (LX-2). In vivo, the pathological feature of cirrhosis was alleviated by the hepatocyte-specific expression of CCL16. CONCLUSIONS: CCL16 could be a feasible plasma marker to predict the occurrence and progression of liver cirrhosis. CCL16 might impact liver cirrhosis through inactivating hepatic stellate cells.

USP22 promotes hypoxia-induced hepatocellular carcinoma stemness by a HIF1α/USP22 positive feedback loop upon TP53 inactivation.

OBJECTIVE: We aimed to elucidate the mutual regulation mechanism of ubiquitin-specific protease 22 (USP22) and hypoxia inducible factor-1α (HIF1α), and the mechanism they promote the stemness of hepatocellular carcinoma (HCC) cells under hypoxic conditions. DESIGN: Cell counting, migration, self-renewal ability, chemoresistance and expression of stemness genes were established to detect the stemness of HCC cells. Immunoprecipitation, ubiquitination assay and chromatin immunoprecipitation assay were used to elucidate the mutual regulation mechanism of USP22 and HIF1α. HCC patient samples and The Cancer Genome Atlas data were used to demonstrate the clinical significance. In vivo USP22-targeting experiment was performed in mice bearing HCC. RESULTS: USP22 promotes hypoxia-induced HCC stemness and glycolysis by deubiquitinating and stabilising HIF1α. As direct target genes of HIF1α, USP22 and TP53 can be transcriptionally upregulated by HIF1α under hypoxic conditions. In TP53 wild-type HCC cells, HIF1α induced TP53-mediated inhibition of HIF1α-induced USP22 upregulation. In TP53-mutant HCC cells, USP22 and HIF1α formed a positive feedback loop and promote the stemness of HCC. HCC patients with a loss-of-function mutation at TP53 and high USP22 and/or HIF1α expression tend to have a worse prognosis. The USP22-targeting lipopolyplexes caused high tumour inhibition and high sorafenib sensitivity in mice bearing HCC. CONCLUSION: USP22 promotes hypoxia-induced HCC stemness by a HIF1α/USP22 positive feedback loop on TP53 inactivation. USP22 is a promising target for the HCC therapy.