Liver cancer in a dish: modelling hepatocellular carcinoma using patient-derived tumor organoids

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second most frequent cause of cancer-related mortality worldwide. HCC predominantly arises in cirrhotic livers as a consequence of underlying chronic diseases including viral hepatitis, alcoholic liver disease and non-alcoholic steatohepatitis. Treatment options for advanced HCC are limited. Sorafenib (Nexavar®) has been the only approved drug for the management of advanced HCC for the past ten years. Recently, additional multikinase inhibitors entered the clinic, however, without significantly improving overall survival as compared to sorafenib. Major advancements are expected to be achieved with the introduction of immune checkpoint inhibitors such as nivolumab (Opdivo®), but biomarkers to identify patients who may benefit from the treatment are currently missing. Moreover, several additional drugs have failed to meet clinical end points in large phase III trials, indicating a need for new drug discovery for HCC. A major obstacle for the development of new therapies is the lack of suitable preclinical animal models or cell culture systems that allow a faithful translation of basic research findings into the clinical setting. This thesis describes the generation of organoids derived from needle biopsies of HCCs. The use of tumor biopsies instead of surgically resected HCC specimens is important because it allows to generate organoids from all tumor stages, whereas surgical resection of HCCs is limited to a minority of patients with small, early stage tumors. These tumors are typically not treated with systemic therapies, and material derived from them might have limited value for developing new treatments for advanced HCCs. Because of the very limited amount of tissue that can be obtained with a needle biopsy, generation of HCC organoids was technically challenging. A key to our success was the immediate sample processing. The biobank of tumor organoids described in this study encompasses different etiologies and, most importantly, all clinical tumor stages. Our study design also allowed to compare the organoids with the originating tumor biopsies. We found that HCC organoids preserve the morphological characteristics and tumor marker expression of their originating tumors. Moreover, a comprehensive analysis of the genetic landscape in both, primary tumors and corresponding organoids, revealed a high concordance of the molecular alterations and the genetic heterogeneity, confirming that the organoids are a genuine representation of the originating tumors. In addition, tumor organoids can be successfully transplanted and propagated in immunodeficient mice to generate xenografts. Finally, in a proof of concept study, we show that tumor organoids can be used to test sensitivities to clinically-relevant drugs and provide a promising novel tool for developing tailored therapies

IL-20 subfamily cytokines impair the oesophageal epithelial barrier by diminishing filaggrin in eosinophilic oesophagitis.

OBJECTIVE Disruption of the epithelial barrier plays an essential role in developing eosinophilic oesophagitis (EoE), a disease defined by type 2 helper T cell (Th2)-mediated food-associated and aeroallergen-associated chronic inflammation. Although an increased expression of interleukin (IL)-20 subfamily members, IL-19, IL-20 and IL-24, in Th2-mediated diseases has been reported, their function in EoE remains unknown. DESIGN Combining transcriptomic, proteomic and functional analyses, we studied the importance of the IL-20 subfamily for EoE using patient-derived oesophageal three-dimensional models and an EoE mouse model. RESULTS Patients with active EoE have increased expression of IL-20 subfamily cytokines in the oesophagus and serum. In patient-derived oesophageal organoids stimulated with IL-20 cytokines, RNA sequencing and mass spectrometry revealed a downregulation of genes and proteins forming the cornified envelope, including filaggrins. On the contrary, abrogation of IL-20 subfamily signalling in Il20R2 -/- animals resulted in attenuated experimental EoE reflected by reduced eosinophil infiltration, lower Th2 cytokine expression and preserved expression of filaggrins in the oesophagus. Mechanistically, these observations were mediated by the mitogen-activated protein kinase (MAPK); extracellular-signal regulated kinases (ERK)1/2) pathway. Its blockade prevented epithelial barrier impairment in patient-derived air-liquid interface cultures stimulated with IL-20 cytokines and attenuated experimental EoE in mice. CONCLUSION Our findings reveal a previously unknown regulatory role of the IL-20 subfamily for oesophageal barrier function in the context of EoE. We propose that aberrant IL-20 subfamily signalling disturbs the oesophageal epithelial barrier integrity and promotes EoE development. Our study suggests that specific targeting of the IL-20 subfamily signalling pathway may present a novel strategy for the treatment of EoE

Proteogenomic characterization of hepatocellular carcinoma

We performed a proteogenomic analysis of hepatocellular carcinomas (HCCs) across clinical stages and etiologies. We identified pathways differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. These pathways are involved in the organization of cellular components, cell cycle control, signaling pathways, transcriptional and translational control and metabolism. Analyses of CNA-mRNA and mRNA-protein correlations identified candidate driver genes involved in epithelial-to-mesenchymal transition, the Wnt-β- catenin pathway, transcriptional control, cholesterol biosynthesis and sphingolipid metabolism. The activity of targetable kinases aurora kinase A and CDKs was upregulated. We found that CTNNB1 mutations are associated with altered phosphorylation of proteins involved in actin filament organization, whereas TP53 mutations are associated with elevated CDK1/2/5 activity and altered phosphorylation of proteins involved in lipid and mRNA metabolism. Integrative clustering identified HCC subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our analysis provides insights into the molecular processes underlying HCCs

Integrative proteogenomic characterization of hepatocellular carcinoma across etiologies and stages.

Proteogenomic analyses of hepatocellular carcinomas (HCC) have focused on early-stage, HBV-associated HCCs. Here we present an integrated proteogenomic analysis of HCCs across clinical stages and etiologies. Pathways related to cell cycle, transcriptional and translational control, signaling transduction, and metabolism are dysregulated and differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. We describe candidate copy number-driven driver genes involved in epithelial-to-mesenchymal transition, the Wnt-β-catenin, AKT/mTOR and Notch pathways, cell cycle and DNA damage regulation. The targetable aurora kinase A and CDKs are upregulated. CTNNB1 and TP53 mutations are associated with altered protein phosphorylation related to actin filament organization and lipid metabolism, respectively. Integrative proteogenomic clusters show that HCC constitutes heterogeneous subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our study provides a comprehensive overview of the proteomic and phophoproteomic landscapes of HCCs, revealing the major pathways altered in the (phospho)proteome

Organoid Models of Human Liver Cancers Derived from Tumor Needle Biopsies

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second most frequent cause of cancer-related mortality worldwide. The multikinase inhibitor sorafenib is the only treatment option for advanced HCC. Due to tumor heterogeneity, its efficacy greatly varies between patients and is limited due to adverse effects and drug resistance. Current in vitro models fail to recapitulate key features of HCCs. We report the generation of long-term organoid cultures from tumor needle biopsies of HCC patients with various etiologies and tumor stages. HCC organoids retain the morphology as well as the expression pattern of HCC tumor markers and preserve the genetic heterogeneity of the originating tumors. In a proof-of-principle study, we show that liver cancer organoids can be used to test sensitivity to sorafenib. In conclusion, organoid models can be derived from needle biopsies of liver cancers and provide a tool for developing tailored therapies

LATS1 but not LATS2 represses autophagy by a kinase-independent scaffold function

Autophagy perturbation represents an emerging therapeutic strategy in cancer. Although LATS1 and LATS2 kinases, core components of the mammalian Hippo pathway, have been shown to exert tumor suppressive activities, here we report a pro-survival role of LATS1 but not LATS2 in hepatocellular carcinoma (HCC) cells. Specifically, LATS1 restricts lethal autophagy in HCC cells induced by sorafenib, the standard of care for advanced HCC patients. Notably, autophagy regulation by LATS1 is independent of its kinase activity. Instead, LATS1 stabilizes the autophagy core-machinery component Beclin-1 by promoting K27-linked ubiquitination at lysine residues K32 and K263 on Beclin-1. Consequently, ubiquitination of Beclin-1 negatively regulates autophagy by promoting inactive dimer formation of Beclin-1. Our study highlights a functional diversity between LATS1 and LATS2, and uncovers a scaffolding role of LATS1 in mediating a cross-talk between the Hippo signaling pathway and autophagy

Human-correlated genetic HCC models identify combination therapy for precision medicine [Pre-print]

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a leading cause of cancer related mortality worldwide. HCC occurs typically from a background of chronic liver disease, caused by a spectrum of predisposing conditions. Tumour development is driven by the expansion of clones that accumulated progressive driver mutations, with hepatocytes the most likely cell of origin. However, the landscape of driver mutations in HCC is independent of the underlying aetiologies. Despite an increasing range of systemic treatment options for advanced HCC outcomes remain heterogeneous and typically poor. Emerging data suggest that drug efficacies depend on disease aetiology and genetic alterations. Exploring subtypes in preclinical models with human relevance will therefore be essential to advance precision medicine in HCC. We generated over twenty-five new genetically-driven in vivo and in vitro HCC models. Our models represent multiple features of human HCC, including clonal origin, histopathological appearance, and metastasis to distant organs. We integrated transcriptomic data from the mouse models with human HCC data and identified four common human-mouse subtype clusters. The subtype clusters had distinct transcriptomic characteristics that aligned with histopathology. In a proof-of-principle analysis, we verified response to standard of care treatment and used a linked in vitro-in vivo pipeline to identify a promising therapeutic candidate, cladribine, that has not been linked to HCC treatment before. Cladribine acts in a highly effective subtype-specific manner in combination with standard of care therapy

Mechano-modulatory synthetic niches for liver organoid derivation

The recent demonstration that primary cells from the liver can be expanded in vitro as organoids holds enormous promise for regenerative medicine and disease modelling. The use of three-dimensional (3D) cultures based on ill-defined and potentially immunogenic matrices, however, hampers the translation of liver organoid technology into real-life applications. We here use chemically defined hydrogels for the efficient derivation of both mouse and human hepatic organoids. Organoid growth is found to be highly stiffness-sensitive, a mechanism independent of acto-myosin contractility and requiring instead activation of the Src family of kinases (SFKs) and yes-associated protein 1 (YAP). Aberrant matrix stiffness, on the other hand, results in compromised proliferative capacity. Finally, we demonstrate the establishment of biopsy-derived human liver organoids without the use of animal components at any step of the process. Our approach thus opens up exciting perspectives for the establishment of protocols for liver organoid-based regenerative medicine. 3D liver organoids hold great promise for regenerative medicine but the use of ill-defined matrices limits their potential. Here, the authors generate human and mouse liver organoids using a chemically defined matrix, and reveal a link between matrix stiffness and organoid growth that does not require acto-myosin contraction

Fam60A plays a role for production stabilities of recombinant CHO cell lines

Recombinant CHO (Chinese hamster ovary) cell lines producing therapeutic proteins often lose their production capability during long-term cultivation. To ensure that CHO production cell lines can be up-scaled to high volume bioreactors labor intensive stability studies of several months have to be performed to deselect clones that are losing productivity over time. The ability to predict whether clones will produce recombinant proteins at constant high levels e.g. through determination of biomarkers such as expression of specific genes, plasmid integration sites or epigenetic patterns, or even to improve CHO host cell lines to increase the probability of the generation of stable clones would be highly beneficial. Previously, we reported that the lack of a telomeric region of chromosome 8 correlates with increased productivities and higher production stabilities of monoclonal antibody expressing CHO cell lines (Ritter et al. 2015). Herein, we describe that the knock-out of the gene Fam60A, which is one of the genes located within the telomeric region of chromosome 8, in CHO-K1a cells leads to the isolation of significantly more clones with higher protein production stabilities of monoclonal antibodies during long-term cultivation

Degradation of recombinant proteins by CHO host cell proteases is prevented by Matriptase-1 knock-out

An increasing number of non-antibody format proteins are entering the clinical development. However, one of the major hurdles for the production of non-antibody glycoproteins is host cell-related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics and genetic knockdowns we have identified, out of the more than 700 known proteases in rodents, Matriptase-1 as the major protease involved in degradation of recombinant proteins expressed in CHO-K1 cells. Subsequently Matriptase-1 was deleted in CHO-K1 cells using “Transcription Activator-Like Effector Nucleases” (TALENs) as well as zinc-finger nucleases (ZFNs). This resulted in a superior CHO-K1 matriptase knockout (KO) cell line with strongly reduced or no proteolytic degradation activity towards a panel of recombinantly-expressed proteins. The matriptase KO cell line was evaluated in spike-in experiments, and showed little or no degradation of proteins incubated in conditioned medium derived from the KO cells. This effect was confirmed when the same proteins were recombinantly expressed in the KO cell line. In summary, the combination of novel cell line engineering tools, next generation sequencing screening methods and the recently published Chinese hamster genome has enabled the development of this novel matriptase KO CHO cell line capable of improving expression yields of intact therapeutic proteins.