Human primary liver cancer–derived organoid cultures for disease modeling and drug screening

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.M.H. is a Wellcome Trust Sir Henry Dale Fellow and is jointly funded by the Wellcome Trust and the Royal Society (104151/Z/14/Z). L.B. is supported by an EMBO Postdoctoral Fellowship (EMBO ALTF 794-2014) and Marie-Curie Postdoctoral Fellowship (grant no. 656193_H2020-MSCA-IF-2014). G.M. was supported by a Marie Curie Initial Training Network (Marie Curie ITN WntsApp 608180) and a H2020 LSMF4LIFE grant (ECH2020-668350). This work was funded by an NC3Rs International prize, a Beit Prize, a Cambridge Cancer Center-pump priming award (CRUK-RG83267) and, partially, by a NC3Rs project grant (NC/R001162/1), all of them awarded to M.H. Work at the L.J.W.v.d.L lab was funded by the research program InnoSysTox (project number 114027003), by the Netherlands Organisation for Health Research and Development (ZonMw), and part of the research program financed by the Dutch Digestive Foundation (MLDS-Diagnostics project number D16-26). Work in the M.J.G. lab is funded by the Wellcome Trust (102696), Stand Up To Cancer (SU2C-AACRDT1213) and Cancer Research UK (C44943/A22536)

Design by Nature: Emerging Applications of Native Liver Extracellular Matrix for Cholangiocyte Organoid-Based Regenerative Medicine

Organoid technology holds great promise for regenerative medicine. Recent studies show feasibility for bile duct tissue repair in humans by successfully transplanting cholangiocyte organoids in liver grafts during perfusion. Large-scale expansion of cholangiocytes is essential for extending these regenerative medicine applications. Human cholangiocyte organoids have a high and stable proliferation capacity, making them an attractive source of cholangiocytes. Commercially available basement membrane extract (BME) is used to expand the organoids. BME allows the cells to self-organize into 3D structures and stimulates cell proliferation. However, the use of BME is limiting the clinical applications of the organoids. There is a need for alternative tissue-specific and clinically relevant culture substrates capable of supporting organoid proliferation. Hydrogels prepared from decellularized and solubilized native livers are an attractive alternative for BME. These hydrogels can be used for the culture and expansion of cholangiocyte organoids in a clinically relevant manner. Moreover, the liver-derived hydrogels retain tissue-specific aspects of the extracellular microenviron-ment. They are composed of a complex mixture of bioactive and biodegradable extracellular matrix (ECM) components and can support the growth of various hepatobiliary cells. In this review, we provide an overview of the clinical potential of native liver ECM-based hydrogels for applications with human cholangiocyte organoids. We discuss the current limitations of BME for the clinical applications of organoids and how native ECM hydrogels can potentially overcome these problems in an effort to unlock the full regenerative clinical potential of the organoids

Mitochondrial dysfunction and oxidative stress in liver transplantation and underlying diseases: new insights and therapeutics

Mitochondria are essential organelles for cellular energy and metabolism. Like with any organ, the liver highly depends on the function of these cellular powerhouses. Hepatotoxic insults often lead to an impairment of mitochondrial activity and an increase in oxidative stress, thereby compromising the metabolic and synthetic functions. Mitochondria play a critical role in ATP synthesis and the production or scavenging of free radicals. Mitochondria orchestrate many cellular signaling pathways involved in the regulation of cell death, metabolism, cell division, and progenitor cell differentiation. Mitochondrial dysfunction and oxidative stress are closely associated with ischemia-reperfusion injury during organ transplantation and with different liver diseases, including cholestasis, steatosis, viral hepatitis, and drug-induced liver injury. To develop novel mitochondria-targeting therapies or interventions, a better understanding of mitochondrial dysfunction and oxidative stress in hepatic pathogenesis is very much needed. Therapies targeting mitochondria impairment and oxidative imbalance in liver diseases have been extensively studied in preclinical and clinical research. In this review, we provide an overview of how oxidative stress and mitochondrial dysfunction affect liver diseases and liver transplantation. Furthermore, we summarize recent developments of antioxidant and mitochondria-targeted interventions

Kinome profiling of cholangiocarcinoma organoids reveals potential druggable targets that hold promise for treatment stratification

Background: Cholangiocarcinoma is a rare but lethal cancer of the biliary tract. Its first-line treatment is currently restricted to chemotherapy, which provides limited clinical benefit. Kinase inhibitors targeting oncogenic intracellular signaling have changed the treatment paradigm of cancer over the last decades. However, they are yet to be widely applied in cholangiocarcinoma therapy. Cholangiocarcinoma has marked molecular heterogeneity, which complicates the discovery of new treatments and requires patient stratification. Therefore, we investigated whether a commercial kinome profiling platform could predict druggable targets in cholangiocarcinoma. Methods: Kinase activity in patient-derived cholangiocarcinoma organoids, non-tumorous adjacent tissue-derived and healthy donor-derived intrahepatic cholangiocyte organoids was determined using the PamChip® phosphotyrosine kinase microarray platform. Kinome profiles were compared and correlated with RNA sequencing and (multi-)kinase inhibitor screening of the cholangiocarcinoma organoids. Results: Kinase activity profiles of individual cholangiocarcinoma organoids are different and do not cluster together. However, growth factor signaling (EGFR, PDGFRβ) and downstream effectors (MAPK pathway) are more active in cholangiocarcinoma organoids and could provide potential druggable targets. Screening of 31 kinase inhibitors revealed several promising pan-effective inhibitors and compounds that show patient-specific efficacy. Kinase inhibitor sensitivity correlated to the activity of its target kinases for several inhibitors, signifying them as potential predictors of response. Moreover, we identified correlations between drug response and kinases not directly targeted by those drugs. Conclusions: In conclusion, kinome profiling is a feasible method to identify druggable targets for cholangiocarcinoma. Future studies should confirm the potential of kinase activity profiles as biomarkers for patient stratification and precision medicine

Protocol for inducing branching morphogenesis in human cholangiocyte and cholangiocarcinoma organoids

Summary: Bile ducts are essential for bile transport and consist of complex branching tubular networks. Human patient-derived cholangiocyte develops a cystic rather than branching duct morphology. Here, we present a protocol to establish branching morphogenesis in cholangiocyte and cholangiocarcinoma organoids. We describe steps for the initiation, maintenance, and expansion of intrahepatic cholangiocyte organoids branching morphology. This protocol enables the study of organ-specific and mesenchymal-independent branching morphogenesis and provides an improved model to study biliary function and diseases.For complete details on the use and execution of this protocol, please refer to Roos et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Drug repurposing screen identifies vidofludimus calcium and pyrazofurin as novel chemical entities for the development of hepatitis E interventions

Hepatitis E virus (HEV) infection can cause severe complications and high mortality, particularly in pregnant women, organ transplant recipients, individuals with pre-existing liver disease and immunosuppressed patients. However, there are still unmet needs for treating chronic HEV infections. Herein, we screened a best-in-class drug repurposing library consisting of 262 drugs/compounds. Upon screening, we identified vidofludimus calcium and pyrazofurin as novel anti-HEV entities. Vidofludimus calcium is the next-generation dihydroorotate dehydrogenase (DHODH) inhibitor in the phase 3 pipeline to treat autoimmune diseases or SARS-CoV-2 infection. Pyrazofurin selectively targets uridine monophosphate synthetase (UMPS). Their anti-HEV effects were further investigated in a range of cell culture models and human liver organoids models with wild type HEV strains and ribavirin treatment failure-associated HEV strains. Encouragingly, both drugs exhibited a sizeable therapeutic window against HEV. For instance, the IC50 value of vidofludimus calcium is 4.6–7.6-fold lower than the current therapeutic doses in patients. Mechanistically, their anti-HEV mode of action depends on the blockage of pyrimidine synthesis. Notably, two drugs robustly inhibited ribavirin treatment failure-associated HEV mutants (Y1320H, G1634R). Their combination with IFN-α resulted in synergistic antiviral activity. In conclusion, we identified vidofludimus calcium and pyrazofurin as potent candidates for the treatment of HEV infections. Based on their antiviral potency, and also the favorable safety profile identified in clinical studies, our study supports the initiation of clinical studies to repurpose these drugs for treating chronic hepatitis E.</p

Scalable Production of Size-Controlled Cholangiocyte and Cholangiocarcinoma Organoids within Liver Extracellular Matrix-Containing Microcapsules

Advances in biomaterials, particularly in combination with encapsulation strategies, have provided excellent opportunities to increase reproducibility and standardization for cell culture applications. Herein, hybrid microcapsules are produced in a flow-focusing microfluidic droplet generator combined with enzymatic outside-in crosslinking of dextran-tyramine, enriched with human liver extracellular matrix (ECM). The microcapsules provide a physiologically relevant microenvironment for the culture of intrahepatic cholangiocyte organoids (ICO) and patient-derived cholangiocarcinoma organoids (CCAO). Micro-encapsulation allowed for the scalable and size-standardized production of organoids with sustained proliferation for at least 21 days in vitro. Healthy ICO (n = 5) expressed cholangiocyte markers, including KRT7 and KRT19, similar to standard basement membrane extract cultures. The CCAO microcapsules (n = 3) showed retention of stem cell phenotype and expressed LGR5 and PROM1. Furthermore, ITGB1 was upregulated, indicative of increased cell adhesion to ECM in microcapsules. Encapsulated CCAO were amendable to drug screening assays, showing a dose-response response to the clinically relevant anti-cancer drugs gemcitabine and cisplatin. High-throughput drug testing identified both pan-effective drugs as well as patient-specific resistance patterns. The results described herein show the feasibility of this one-step encapsulation approach to create size-standardized organoids for scalable production. The liver extracellular matrix-containing microcapsules can provide a powerful platform to build mini healthy and tumor tissues for potential future transplantation or personalized medicine applications

Characterization and Comparison of Canine Multipotent Stromal Cells Derived from Liver and Bone Marrow

Liver-derived multipotent stromal cells (L-MSCs) may prove preferable for treatment strategies of liver diseases, in comparison to the widely studied bone marrow-derived MSCs (BM-MSCs). Canines are a large animal model, in which the pathologies of liver diseases are similar to man. This study further promotes the implementation of canine models in MSC-based treatments of liver diseases. L-MSCs were characterized and compared to BM-MSCs from the same individual. Both cell types demonstrated a spindle-shaped fibroblast-like morphology, possessed the same growth potential, and demonstrated similar immunomodulation gene expression of CD274, PTGS-1, and PTGS-2. Marked differences in cell surface markers, CD105 and CD146, distinguished these two cell populations, and L-MSCs retained a liver-specific imprinting, observed by expression of CK18 and CK19. Finally, both populations differentiated toward the osteogenic and adipogenic lineage; however, L-MSCs failed to differentiate into the chondrogenic lineage. In conclusion, characterization of canine L-MSCs and BM-MSCs demonstrated that the two cell type populations are highly comparable. Although it is still unclear which cell source is preferred for clinical application in liver treatment strategies, this study provides a foundation for future controlled studies with MSC therapy in various liver diseases in dogs before their application in man

Rescue of chloride and bicarbonate transport by elexacaftor-ivacaftor-tezacaftor in organoid-derived CF intestinal and cholangiocyte monolayers

Background: In cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion precipitates the accumulation of viscous mucus in the lumen of respiratory and gastrointestinal epithelial tissues. We investigated whether the combination of elexacaftor (ELX), ivacaftor (IVA) and tezacaftor (TEZ), apart from its well-documented effect on chloride transport, also restores Phe508del-CFTR-mediated bicarbonate transport. Methods: Epithelial monolayers were cultured from intestinal and biliary (cholangiocyte) organoids of homozygous Phe508del-CFTR patients and controls. Transcriptome sequencing was performed, and bicarbonate and chloride transport were assessed in the presence or absence of ELX/IVA/TEZ, using the intestinal current measurement technique. Results: ELX/IVA/TEZ markedly enhanced bicarbonate and chloride transport across intestinal epithelium. In biliary epithelium, it failed to enhance CFTR-mediated bicarbonate transport but effectively rescued CFTR-mediated chloride transport, known to be requisite for bicarbonate secretion through the chloride-bicarbonate exchanger AE2 (SLC4A2), which was highly expressed by cholangiocytes. Biliary but not intestinal epithelial cells expressed an alternative anion channel, anoctamin-1/TMEM16A (ANO1), and secreted bicarbonate and chloride upon purinergic receptor stimulation. Conclusions: ELX/IVA/TEZ has the potential to restore both chloride and bicarbonate secretion across CF intestinal and biliary epithelia and may counter luminal hyper-acidification in these tissues