Therapeutic cancer vaccines: advancements, challenges, and prospects.

With the development and regulatory approval of immune checkpoint inhibitors and adoptive cell therapies, cancer immunotherapy has undergone a profound transformation over the past decades. Recently, therapeutic cancer vaccines have shown promise by eliciting de novo T cell responses targeting tumor antigens, including tumor-associated antigens and tumor-specific antigens. The objective was to amplify and diversify the intrinsic repertoire of tumor-specific T cells. However, the complete realization of these capabilities remains an ongoing pursuit. Therefore, we provide an overview of the current landscape of cancer vaccines in this review. The range of antigen selection, antigen delivery systems development the strategic nuances underlying effective antigen presentation have pioneered cancer vaccine design. Furthermore, this review addresses the current status of clinical trials and discusses their strategies, focusing on tumor-specific immunogenicity and anti-tumor efficacy assessment. However, current clinical attempts toward developing cancer vaccines have not yielded breakthrough clinical outcomes due to significant challenges, including tumor immune microenvironment suppression, optimal candidate identification, immune response evaluation, and vaccine manufacturing acceleration. Therefore, the field is poised to overcome hurdles and improve patient outcomes in the future by acknowledging these clinical complexities and persistently striving to surmount inherent constraints.

Cancer-associated fibroblasts nurture LGR5 marked liver tumor-initiating cells and promote their tumor formation, growth, and metastasis.

BACKGROUND & AIMS: In liver cancer, leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) compartment represents an important tumor-initiating cell (TIC) population and served as a potential therapeutic target. Cancer-associated fibroblasts (CAFs) is a critical part of the tumor microenvironment, heavily influenced TIC function and fate. However, deeply investigations have been hindered by the lack of accurate preclinical models to investigate the interaction between CAFs and TIC. Organoids model have achieved major advancements as a precious research model for recapitulating the morphological aspects of organs, and thus also serving as a candidate model to investigate the mutual interaction between different cell types. Consequently, this study aimed to construct a three-dimensional (3D) co-culture organoid model of primary LGR5-expressing tumor stem cells from primary murine liver tumors with CAFs to investigate the impact of CAFs on LGR5 marked TICs in liver cancer. MATERIALS AND METHODS: First, both of the transgenic LGR5-diphtheria toxin receptor (DTR)-GFP knock-in mice and transgenic Rosa26-mT mice developed primary liver tumors by diethylnitrosamine (DEN) administration. Tumor organoids and CAFs were generated from those primary liver cancer separately. Second, LGR5-expressing TICs organoid with CAFs were established ex vivo based on cell-cell contact or trans-well co-culture system, and the mutual influence between those two types of cells was further investigated. Subsequently, immunodeficient mouse-based xenograft model was further adopted to evaluate the influence of CAFs to LGR5 tumor stem cell, tumor formation, and metastasis. RESULTS: The co-culture organoid model composed of murine liver tumor LGR5+ tumor-initiating cells and CAFs in 3D co-culture was successfully established, with the intention to investigate their mutual interaction. The existence of CAFs upon engrafting tumor organoids resulted in dramatic higher number of LGR5+ cells in the neoplasia when compared with engrafting tumor organoids alone. Furthermore, ex vivo culture of isolated LGR5+ cells from tumors of co-engrafted mice formed significantly larger size of organoids than mono-engrafted. Our results also indicated significantly larger size and number of formed organoids, when LGR5+ cells co-cultured with CAF in both cell-cell contact and paracrine signaling in vitro, comparing to LGR5+ cells alone. Furthermore, we found that specific knockout of LGR5 expressing cells suppressed CAF-mediated promotion of tumor formation, growth, and metastasis in the experimental mice model. CONCLUSIONS: Altogether, in a 3D co-culture type of murine liver LGR5+ cells and cancer-associated fibroblasts, we have demonstrated robust effects of CAFs in the promotion of LGR5 marked liver TICs. We also further revealed the influence of tumor microenvironment on stem cell-related therapy, suggesting the possibility of combing CAF-targeted and tumor stem cell targeted therapy in treating liver cancer.

Kidney Organoids Are Capable of Forming Tumors, but Not Teratomas.

Induced pluripotent stem cell (iPSC)-derived kidney organoids are a potential tool for the regeneration of kidney tissue. They represent an early stage of nephrogenesis and have been shown to successfsully vascularize and mature further in vivo. However, there are concerns regarding the long-term safety and stability of iPSC derivatives. Specifically, the potential for tumorigenesis may impede the road to clinical application. To study safety and stability of kidney organoids, we analyzed their potential for malignant transformation in a teratoma assay and following long-term subcutaneous implantation in an immune-deficient mouse model. We did not detect fully functional residual iPSCs in the kidney organoids as analyzed by gene expression analysis, single-cell sequencing and immunohistochemistry. Accordingly, kidney organoids failed to form teratoma. Upon long-term subcutaneous implantation of whole organoids in immunodeficient IL2Ry-/-RAG2-/- mice, we observed tumor formation in 5 out of 103 implanted kidney organoids. These tumors were composed of WT1+CD56+ immature blastemal cells and showed histological resemblance with Wilms tumor. No genetic changes were identified that contributed to the occurrence of tumorigenic cells within the kidney organoids. However, assessment of epigenetic changes revealed a unique cluster of differentially methylated genes that were also present in undifferentiated iPSCs. We discovered that kidney organoids have the capacity to form tumors upon long-term implantation. The presence of epigenetic modifications combined with the lack of environmental cues may have caused an arrest in terminal differentiation. Our results indicate that the safe implementation of kidney organoids should exclude the presence of pro-tumorigenic methylation in kidney organoids.

Recapitulating lipid accumulation and related metabolic dysregulation in human liver-derived organoids.

Fatty liver disease has grown into a major global health burden, attributed to multi-factors including sedentary lifestyle, obesogenic diet and prevalence of metabolic disorders. The lack of robust experimental models is hampering the research and therapeutic development for fatty liver disease. This study aims to develop an organoid-based 3D culture model to recapitulate key features of fatty liver disease focusing on intracellular lipid accumulation and metabolic dysregulation. We used human liver-derived intrahepatic cholangiocyte organoids and hepatocyte differentiated organoids. These organoids were exposed to lactate, pyruvate, and octanoic acid (LPO) for inducing lipid accumulation and mitochondrial impairment. Lipid accumulation resulted in alternations of gene transcription with major effects on metabolic pathways, including triglyceride and glucose level increase, which is consistent with metabolic changes in fatty liver disease patients. Interestingly, lipid accumulation affected mitochondria as shown by morphological transitions, alternations in expression of mitochondrial encoded genes, and reduction of ATP production. Meanwhile, we found treatment with obeticholic acid and metformin can alleviate fat accumulation in organoids. This study demonstrated that LPO exposure can induce lipid accumulation and associated metabolic dysregulation in human liver-derived organoids. This provides an innovative model for studying fatty liver disease and testing potential therapeutics. KEY MESSAGES: Lactate, pyruvate, and octanoic acid induce lipid accumulation in liver organoids. Organoids of human compared to mouse origin are more efficient in lipid accumulation. Lipid accumulation dysregulates metabolic pathway and impairs mitochondrial function. Demonstrating a proof-of-concept for testing medications in organoids.

Estimating Global Prevalence of Metabolic Dysfunction-Associated Fatty Liver Disease in Overweight or Obese Adults.

BACKGROUND & AIMS: Metabolic dysfunction-associated fatty liver disease (MAFLD) is a new terminology updated from non-alcoholic fatty liver disease (NAFLD). In this study, we aim to estimate the global prevalence of MAFLD specifically in overweight and obese adults from the general population by performing a systematic review and meta-analysis through mining the existing epidemiological data on fatty liver disease. METHODS: We searched Medline, Embase, Web of Science, Cochrane and google scholar database from inception to November, 2020. DerSimonian-Laird random-effects model with Logit transformation was performed for data analysis. Sensitivity analysis and meta-regression were used to explore predictors of MAFLD prevalence in pooled statistics with high heterogeneity. RESULTS: We identified 116 relevant studies comprised of 2,667,052 participants in general population with an estimated global MAFLD prevalence as 50.7% (95% CI 46.9-54.4) among overweight/obese adults regardless of diagnostic techniques. Ultrasound was the most commonly used diagnostic technique generating prevalence rate of 51.3% (95% CI, 49.1-53.4). Male (59.0%; 95% CI, 52.0-65.6) had a significantly higher MAFLD prevalence than female (47.5%; 95% CI, 40.7-54.5). Interestingly, MAFLD prevalence rates are comparable based on classical NAFLD and non-NAFLD studies in general population. The pooled estimate prevalence of comorbidities such as type 2 diabetes and metabolic syndrome was 19.7% (95% CI, 12.8-29.0) and 57.5% (95% CI, 49.9-64.8), respectively. CONCLUSIONS: MAFLD has an astonishingly high prevalence rate in overweight and obese adults. This calls for attention and dedicated action from primary care physicians, specialists, health policy makers and the general public alike.

Recapitulating Cholangiopathy-Associated Necroptotic Cell Death In Vitro Using Human Cholangiocyte Organoids.

BACKGROUND & AIMS: Liver and bile duct diseases often are associated with extensive cell death of cholangiocytes. Necroptosis represents a common mode of programmed cell death in cholangiopathy, however, detailed mechanistic knowledge is limited owing to the lack of appropriate in vitro models. To address this void, we investigated whether human intrahepatic cholangiocyte organoids (ICOs) can recapitulate cholangiopathy-associated necroptosis and whether this model can be used for drug screening. METHODS: We evaluated the clinical relevance of necroptosis in end-stage liver diseases and liver transplantation by immunohistochemistry. Cholangiopathy-associated programmed cell death was evoked in ICOs derived from healthy donors or patients with primary sclerosing cholangitis or alcoholic liver diseases by the various stimuli. RESULTS: The expression of key necroptosis mediators, receptor-interacting protein 3 and phosphorylated mixed lineage kinase domain-like, in cholangiocytes during end-stage liver diseases was confirmed. The phosphorylated mixed lineage kinase domain-like expression was etiology-dependent. Gene expression analysis confirmed that primary cholangiocytes are more prone to necroptosis compared with primary hepatocytes. Both apoptosis and necroptosis could be specifically evoked using tumor necrosis factor α and second mitochondrial-derived activator of caspases mimetic, with or without caspase inhibition in healthy and patient-derived ICOs. Necroptosis also was induced by ethanol metabolites or human bile in ICOs from donors and patients. The organoid cultures further uncovered interdonor variable and species-specific drug responses. Dabrafenib was identified as a potent necroptosis inhibitor and showed a protective effect against ethanol metabolite toxicity. CONCLUSIONS: Human ICOs recapitulate cholangiopathy-associated necroptosis and represent a useful in vitro platform for the study of biliary cytotoxicity and preclinical drug evaluation.

Targeting the complex I and III of mitochondrial electron transport chain as a potentially viable option in liver cancer management.

Liver cancer is one of the most common and lethal types of oncological disease in the world, with limited treatment options. New treatment modalities are desperately needed, but their development is hampered by a lack of insight into the underlying molecular mechanisms of disease. It is clear that metabolic reprogramming in mitochondrial function is intimately linked to the liver cancer process, prompting the possibility to explore mitochondrial biochemistry as a potential therapeutic target. Here we report that depletion of mitochondrial DNA, pharmacologic inhibition of mitochondrial electron transport chain (mETC) complex I/complex III, or genetic of mETC complex I restricts cancer cell growth and clonogenicity in various preclinical models of liver cancer, including cell lines, mouse liver organoids, and murine xenografts. The restriction is linked to the production of reactive oxygen species, apoptosis induction and reduced ATP generation. As a result, our findings suggest that the mETC compartment of mitochondria could be a potential therapeutic target in liver cancer.

Lipid droplets and their interactions with other organelles in liver diseases.

Lipid droplets are cellular organelles used for lipid storage with a hydrophobic core of neutral lipids enclosed by a phospholipid monolayer. Besides presenting as giant single organelles in fat tissue, lipid droplets are also widely present as a multitude of small structures in hepatocytes, where they play key roles in health and disease of the liver. In addition to lipid storage, lipid droplets are also directly involved in lipid metabolism, membrane biosynthesis, cell signaling, inflammation, pathogen-host interaction and cancer development. In addition, they interact with other cellular organelles to regulate cellular biology. It is fair to say that the exact functions of lipid droplets in cellular physiology remain largely obscure. Thus prompted, here we aim to analyze the corpus of contemporary biomedical literature to create a framework as to how the role of lipid droplets in hepatocyte physiology and pathophysiology should be understood. The resulting framework should help understanding the interaction of lipid droplets with other organelles in important liver diseases, including fatty liver disease, viral hepatitis and liver cancer and direct further research directions.

Cancer-Associated Fibroblasts Provide a Stromal Niche for Liver Cancer Organoids That Confers Trophic Effects and Therapy Resistance.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play a key role in the cancer process, but the research progress is hampered by the paucity of preclinical models that are essential for mechanistic dissection of cancer cell-CAF interactions. Here, we aimed to establish 3-dimensional (3D) organotypic co-cultures of primary liver tumor-derived organoids with CAFs, and to understand their interactions and the response to treatment. METHODS: Liver tumor organoids and CAFs were cultured from murine and human primary liver tumors. 3D co-culture models of tumor organoids with CAFs and Transwell culture systems were established in vitro. A xenograft model was used to investigate the cell-cell interactions in vivo. Gene expression analysis of CAF markers in our hepatocellular carcinoma cohort and an online liver cancer database indicated the clinical relevance of CAFs. RESULTS: To functionally investigate the interactions of liver cancer cells with CAFs, we successfully established murine and human 3D co-culture models of liver tumor organoids with CAFs. CAFs promoted tumor organoid growth in co-culture with direct cell-cell contact and in a Transwell system via paracrine signaling. Vice versa, cancer cells secrete paracrine factors regulating CAF physiology. Co-transplantation of CAFs with liver tumor organoids of mouse or human origin promoted tumor growth in xenograft models. Moreover, tumor organoids conferred resistance to clinically used anticancer drugs including sorafenib, regorafenib, and 5-fluorouracil in the presence of CAFs, or the conditioned medium of CAFs. CONCLUSIONS: We successfully established murine and human 3D co-culture models and have shown robust effects of CAFs in liver cancer nurturing and treatment resistance.

LGR5 marks targetable tumor-initiating cells in mouse liver cancer.

Cancer stem cells (CSCs) or tumor-initiating cells (TICs) are thought to be the main drivers for disease progression and treatment resistance across various cancer types. Identifying and targeting these rare cancer cells, however, remains challenging with respect to therapeutic benefit. Here, we report the enrichment of LGR5 expressing cells, a well-recognized stem cell marker, in mouse liver tumors, and the upregulation of LGR5 expression in human hepatocellular carcinoma. Isolated LGR5 expressing cells from mouse liver tumors are superior in initiating organoids and forming tumors upon engraftment, featuring candidate TICs. These cells are resistant to conventional treatment including sorafenib and 5-FU. Importantly, LGR5 lineage ablation significantly inhibits organoid initiation and tumor growth. The combination of LGR5 ablation with 5-FU, but not sorafenib, further augments the therapeutic efficacy in vivo. Thus, we have identified the LGR5+ compartment as an important TIC population, representing a viable therapeutic target for combating liver cancer.

The global epidemiology of hepatitis E virus infection: A systematic review and meta-analysis.

BACKGROUND AND AIMS: Hepatitis E virus (HEV), as an emerging zoonotic pathogen, is a leading cause of acute viral hepatitis worldwide, with a high risk of developing chronic infection in immunocompromised patients. However, the global epidemiology of HEV infection has not been comprehensively assessed. This study aims to map the global prevalence and identify the risk factors of HEV infection by performing a systematic review and meta-analysis. METHODS: A systematic searching of articles published in Medline, Embase, Web of science, Cochrane and Google scholar databases till July 2019 was conducted to identify studies with HEV prevalence data. Pooled prevalence among different countries and continents was estimated. HEV IgG seroprevalence of subgroups was compared and risk factors for HEV infection were evaluated using odd ratios (OR). RESULTS: We identified 419 related studies which comprised of 1 519 872 individuals. A total of 1 099 717 participants pooled from 287 studies of general population estimated a global anti-HEV IgG seroprevalence of 12.47% (95% CI 10.42-14.67; I2  = 100%). Notably, the use of ELISA kits from different manufacturers has a substantial impact on the global estimation of anti-HEV IgG seroprevalence. The pooled estimate of anti-HEV IgM seroprevalence based on 98 studies is 1.47% (95% CI 1.14-1.85; I2  = 99%). The overall estimate of HEV viral RNA-positive rate in general population is 0.20% (95% CI 0.15-0.25; I2  = 98%). Consumption of raw meat (P = .0001), exposure to soil (P < .0001), blood transfusion (P = .0138), travelling to endemic areas (P = .0244), contacting with dogs (P = .0416), living in rural areas (P = .0349) and receiving education less than elementary school (P < .0001) were identified as risk factors for anti-HEV IgG positivity. CONCLUSIONS: Globally, approximately 939 million corresponding to 1 in 8 individuals have ever experienced HEV infection. 15-110 million individuals have recent or ongoing HEV infection. Our study highlights the substantial burden of HEV infection and calls for increasing routine screening and preventive measures.

Mitochondrial Fusion Via OPA1 and MFN1 Supports Liver Tumor Cell Metabolism and Growth.

Metabolic reprogramming universally occurs in cancer. Mitochondria act as the hubs of bioenergetics and metabolism. The morphodynamics of mitochondria, comprised of fusion and fission processes, are closely associated with mitochondrial functions and are often dysregulated in cancer. In this study, we aim to investigate the mitochondrial morphodynamics and its functional consequences in human liver cancer. We observed excessive activation of mitochondrial fusion in tumor tissues from hepatocellular carcinoma (HCC) patients and in vitro cultured tumor organoids from cholangiocarcinoma (CCA). The knockdown of the fusion regulator genes, OPA1 (Optic atrophy 1) or MFN1 (Mitofusin 1), inhibited the fusion process in HCC cell lines and CCA tumor organoids. This resulted in inhibition of cell growth in vitro and tumor formation in vivo, after tumor cell engraftment in mice. This inhibitory effect is associated with the induction of cell apoptosis, but not related to cell cycle arrest. Genome-wide transcriptomic profiling revealed that the inhibition of fusion predominately affected cellular metabolic pathways. This was further confirmed by the blocking of mitochondrial fusion which attenuated oxygen consumption and cellular ATP production of tumor cells. In conclusion, increased mitochondrial fusion in liver cancer alters metabolism and fuels tumor cell growth.

Pregnane X receptor activation constrains mucosal NF-κB activity in active inflammatory bowel disease.

BACKGROUND: The Pregnane X Receptor (PXR) is a principal signal transducer in mucosal responses to xenobiotic stress. It is well-recognized that inflammatory bowel disease is accompanied by xenobiotic stress, but the importance of the PXR in limiting inflammatory responses in inflammatory bowel disease remains obscure at best. METHODS: We stimulate a total of 106 colonic biopsies from 19 Crohn's disease patients with active disease, 36 colonic biopsies from 8 control patients, colonic organoids and various cell culture models (either proficient or genetically deficient with respect to PXR) in vitro with the PXR ligand rifampicin or vehicle. Effects on NF-κB activity are assessed by measuring interleukin-8 (IL-8) and interleukin-1ß (IL-1ß) mRNA levels by qPCR and in cell culture models by NF-κB reporter-driven luciferase activity and Western blot for signal transduction elements. RESULTS: We observe a strict inverse correlation between colonic epithelial PXR levels and NF-κB target gene expression in colonic biopsies from Crohn's disease patients. PXR, activated by rifampicin, is rate-limiting for mucosal NF-κB activation in IBD. The correlation between colonic epithelial PXR levels and NF-κB target gene expression was also observed in intestinal organoids system. Furthermore, in preclinical in vitro models of intestinal inflammation, including intestinal organoids, genetic inactivation of PXR unleashes NF-κB-dependent signal transduction whereas conversely NF-κB signaling reduces levels of PXR expression. CONCLUSIONS: Our data indicate that the PXR is a major and clinically relevant antagonist of NF-κB activity in the intestinal epithelial compartment during inflammatory bowel disease.

Smoothened-dependent and -independent pathways in mammalian noncanonical Hedgehog signaling.

Hedgehog proteins are pivotal morphogens acting through a canonical pathway involving first activation of ligand binding to Patched followed by alleviation of Smoothened receptor inhibition, leading to activation of Gli transcription factors. Noncanonical Hedgehog signaling remains poorly characterized but is thought to be mainly dependent on Smoothened. However, Smoothened inhibitors have yielded only partial success in combating Hedgehog signal transduction-dependent cancer, suggesting that noncanonical Smoothened-independent pathways also are clinically relevant. Moreover, several Smoothened-dependent effects (e.g. neurite projection) do not require transcriptional activation, further suggesting biological importance of noncanonical Smoothened-dependent pathways. We comprehensively characterized the cellular kinome in Hedgehog-challenged murine WT and Smoothened-/- fibroblasts as well as Smoothened agonist-stimulated cells. A peptide assay-based kinome analysis (in which cell lysates are used to phosphorylate specific kinase substrates), along with endocytosis, Lucifer Yellow-based, and immunoblotting assays, identified an elaborate signaling network of both Smoothened-dependent and -independent pathways that mediates actin reorganization through Src-like kinases, activates various proinflammatory signaling cascades, and concomitantly stimulates Wnt and Notch signaling while suppressing bone morphogenetic protein (BMP) signaling. The contribution of noncanonical Smoothened-independent signaling to the overall effects of Hedgehog on cellular physiology appears to be much larger than previously envisioned and may explain the transcriptionally independent effects of Hedgehog signaling on cytoskeleton. The observation that Patched-dependent, Smoothened-independent, noncanonical Hedgehog signaling increases Wnt/Notch signaling provides a possible explanation for the failure of Smoothened antagonists in combating Hedgehog-dependent but Smoothened inhibitor-resistant cancer. Our findings suggest that inhibiting Hedgehog-Patched interaction could result in more effective therapies as compared with conventional Smoothened-directed therapies.

Suppression of Hepatocellular Carcinoma by Mycophenolic Acid in Experimental Models and in Patients.

BACKGROUND: Tumor recurrence is a major complication following liver transplantation (LT) as treatment for hepatocellular carcinoma (HCC). Immunosuppression is an important risk factor for HCC recurrence, but conceivably may depend on the type of immunosuppressive medication. Mycophenolic acid (MPA) is a currently widely used immunosuppressant. This study investigated the effects of MPA on HCC. METHODS: Three human HCC cell lines and organoids from mouse primary liver tumor were used as experimental models. MTT, Alamar Blue assay, cell cycle analysis, colony formation, and [3H]-thymidine assays were performed. An LT database was used for retrospective analysis of the effect of mycophenolate mofetil, the prodrug of MPA, on HCC recurrence. RESULTS: With clinically achievable concentrations, MPA effectively inhibited HCC cell proliferation and single-cell colony-forming unit. In short-term experiments, MPA effectively elicited S phase arrest in HCC cell lines. In addition, the initiation and growth of liver tumor organoids were effectively inhibited by MPA. Most importantly, the use of mycophenolate mofetil in patients with HCC-related LT was significantly associated with less tumor recurrence and improved patient survival. CONCLUSIONS: MPA can specifically counteract HCC growth in vitro and tumor recurrence in LT patients. These results warrant prospective clinical trials into the role of MPA-mediated immunosuppression following LT of patients with HCC.

Direct-acting antiviral agents for liver transplant recipients with recurrent genotype 1 hepatitis C virus infection: Systematic review and meta-analysis.

BACKGROUND: Comprehensive evaluation of safety and efficacy of different combinations of direct-acting antivirals (DAAs) in liver transplant recipients with genotype 1 (GT1) hepatitis C virus (HCV) recurrence remains limited. Therefore, we performed this systematic review and meta-analysis in order to evaluate the clinical outcome of DAA treatment in liver transplant patients with HCV GT1 recurrence. METHODS: Studies were included if they contained information of 12 weeks sustained virologic response (SVR12) after DAA treatment completion as well as treatment related complications for liver transplant recipients with GT1 HCV recurrence. RESULTS: We identified 16 studies comprising 885 patients. The overall pooled estimate proportion of SVR12 was 93% (95% confidence interval (CI): 0.89, 0.96), with moderate heterogeneity observed (τ2  = 0.01, P < 0.01, I2 =75%). High tolerability was observed in liver transplant recipients reflected by serious adverse events (sAEs) with pooled estimate proportion of 4% (95% CI: 0.01, 0.07; τ2  = 0.02, P < 0.01, I2  = 81%). For subgroup analysis, a total of five different DAA regimens were applied for treating these patients. Sofosbuvir/Ledipasvir (SOF/LDV) led the highest pooled estimate SVR12 proportion, followed by Paritaprevir/Ritonavir/Ombitasivir/Dasabuvir (PrOD), Daclatasvir (DCV)/Simeprevir (SMV) ± Ribavirin (RBV), and SOF/SMV ± RBV, Asunaprevir (ASV)/DCV. There was a tendency for favoring a higher pooled SVR12 proportion in patients with METAVIR Stage F0-F2 of 97% (95% CI: 0.93, 0.99) compared to 85% (95% CI: 0.79, 0.90) for stage F3-F4 (P < 0.01). There was no significant difference between LT recipients treated with or without RBV (P = 0.23). CONCLUSIONS: Direct-acting antiviral treatment is highly effective and well-tolerated in liver transplant recipients with recurrent GT1 HCV infection.

Modeling liver cancer and therapy responsiveness using organoids derived from primary mouse liver tumors.

The current understanding of cancer biology and development of effective treatments for cancer remain far from satisfactory. This in turn heavily relies on the availability of easy and robust model systems that resemble the architecture/physiology of the tumors in patients to facilitate research. Cancer research in vitro has mainly been based on the use of immortalized 2D cancer cell lines that deviate in many aspects from the original primary tumors. The recent development of the organoid technology allowing generation of organ-buds in 3D culture from adult stem cells has endowed the possibility of establishing stable culture from primary tumors. Although culturing organoids from liver tumors is thought to be difficult, we now convincingly demonstrate the establishment of organoids from mouse primary liver tumors. We have succeeded in culturing 91 lines from 129 liver tissue/tumors. These organoids can be grown in long-term cultures in vitro. About 20% of these organoids form tumors in immunodeficient mice upon (serial) transplantation, confirming their tumorigenic and self-renewal properties. Interestingly, single cells from the tumor organoids have high efficiency of organoid initiation, and a single organoid derived from a cancer cell is able to initiate a tumor in mice, indicating the enrichment of tumor-initiating cells in the tumor organoids. Furthermore, these organoids recapitulate, to some extent, the heterogeneity of liver cancer in patients, with respect to phenotype, cancer cell composition and treatment response. These model systems shall provide enormous opportunities to advance our research on liver cancer (stem cell) biology, drug development and personalized medicine.

Oncogenic STRAP Supports Hepatocellular Carcinoma Growth by Enhancing Wnt/ß-Catenin Signaling.

Aberrant activation of Wnt/ß-catenin signaling plays a key role in the onset and development of hepatocellular carcinomas (HCC), with about half of them acquiring mutations in either CTNNB1 or AXIN1. The serine/threonine kinase receptor-associated protein (STRAP), a scaffold protein, was recently shown to facilitate the aberrant activation of Wnt/ß-catenin signaling in colorectal cancers. However, the function of STRAP in HCC remains completely unknown. Here, increased levels of STRAP were observed in human and mouse HCCs. RNA sequencing of STRAP knockout clones generated by gene editing of Huh6 and Huh7 HCC cells revealed a significant reduction in expression of various metabolic and cell-cycle-related transcripts, in line with their general slower growth observed during culture. Importantly, Wnt/ß-catenin signaling was impaired in all STRAP knockout/down cell lines tested, regardless of the underlying CTNNB1 or AXIN1 mutation. In accordance with ß-catenin's role in (cancer) stem cell maintenance, the expressions of various stem cell markers, such as AXIN2 and LGR5, were reduced and concomitantly differentiation-associated genes were increased. Together, these results show that the increased STRAP protein levels observed in HCC provide growth advantage among others by enhancing Wnt/ß-catenin signaling. These observations also identify STRAP as a new player in regulating ß-catenin signaling in hepatocellular cancers. IMPLICATIONS: Elevated STRAP levels in hepatocellular cancers provide a growth advantage by enhancing Wnt/ß-catenin signaling.

Dichotomal functions of phosphorylated and unphosphorylated STAT1 in hepatocellular carcinoma.

Interferons (IFNs) with antiviral and immune-stimulatory functions have been widely used in prevention and treatment of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 1 (STAT1) is a key element of the IFN signaling, and the function of STAT1 is critically determined by its phosphorylation state. This study aims to understand the functions of phosphorylated (p-) and unphosphorylated (u-) STAT1 in HCC. We found that u-STAT1 is significantly elevated in patient HCC tumor tissues and predominantly expressed in cytoplasm; while p-STAT1 is absent. Loss of u-STAT1 potently arrested cell cycle and inhibited cell growth in HCC cells. Induction of p-STAT1 by IFN-α treatment effectively triggers the expression of interferon-stimulated genes (ISGs), but has moderate effect on HCC cell growth. Interestingly, both u-STAT1 and p-STAT1 are induced by IFN-α, through with distinct time-dependent process. Furthermore, the ISG induction patterns mediated by p-STAT1 and u-STAT1 are also distinct. Importantly, artificial blocking of the induction of u-STAT1, but not p-STAT1, sensitizes HCC cells to treatment of IFNs. Therefore, p-STAT1 and u-STAT1 exert dichotomal functions and coordinately regulate the responsiveness to IFN treatment in HCC. KEY MESSAGES: STAT1 is upregulated and predominantly presented as u-STAT1 in HCC, while p-STAT1 is absent. U-STAT1 sustains but p-STAT1 inhibits HCC growth. The dynamic change of phosphorylation state of STAT1 control the responsiveness to IFN treatment.

Action and clinical significance of CCAAT/enhancer-binding protein delta in hepatocellular carcinoma.

CCAAT/enhancer-binding protein delta (CEBPD) is associated with the regulation of apoptosis and cell proliferation and is a candidate tumor suppressor gene. Here, we investigated its role in hepatocellular carcinoma (HCC). We observe that CEBPD mRNA expression is significantly downregulated in HCC tumors as compared with adjacent tissues. Protein levels of CEBPD are also lower in tumors relative to adjacent tissues. Reduced expression of CEBPD in the tumor correlates with worse clinical outcome. In both Huh7 and HepG2 cells, shRNA-mediated CEBPD knockdown significantly reduces cell proliferation, single cell colony formation and arrests cells in the G0/G1 phase. Subcutaneous xenografting of Huh7 in nude mice show that CEBPD knockdown results in smaller tumors. Gene expression analysis shows that CEBPD modulates interleukin-1 signaling. We conclude that CEBPD expression uncouples cancer compartment expansion and clinical outcome in HCC, potentially by modulating interleukin-1 signaling. Thus, although our results support the notion that CEBPD acts as a tumor suppressor in HCC, its action does not involve impairing compartment expansion per se but more likely acts through improving anticancer immunity.