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Novel patient-derived preclinical models of liver cancer

  • Erin Bresnahan
    Affiliations
    Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA

    Liver Cancer Program, Division of Liver Diseases, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA

    The Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, USA
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  • Pierluigi Ramadori
    Affiliations
    Division of Chronic Inflammation and Cancer, German Cancer Research Center Heidelberg (DKFZ), Heidelberg, Germany
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  • Mathias Heikenwalder
    Correspondence
    Corresponding authors. Address: Division of Chronic Inflammation and Cancer, German Cancer Research Center Heidelberg (DKFZ), Heidelberg, Germany (M. Heikenwalder), or Department of Medical Oncology and Pneumology, University Hospital Tuebingen, 72076 Tuebingen, Germany (L. Zender), or Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA (A. Lujambio).
    Affiliations
    Division of Chronic Inflammation and Cancer, German Cancer Research Center Heidelberg (DKFZ), Heidelberg, Germany
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  • Lars Zender
    Correspondence
    Corresponding authors. Address: Division of Chronic Inflammation and Cancer, German Cancer Research Center Heidelberg (DKFZ), Heidelberg, Germany (M. Heikenwalder), or Department of Medical Oncology and Pneumology, University Hospital Tuebingen, 72076 Tuebingen, Germany (L. Zender), or Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA (A. Lujambio).
    Affiliations
    Department of Medical Oncology and Pneumology, University Hospital Tuebingen, 72076 Tuebingen, Germany

    German Cancer Research Consortium (DKTK), German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany

    iFIT Cluster of Excellence EXC 2180, Image Guided and Functionally Instructed Tumour Therapies, University of Tuebingen, Tuebingen, Germany
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  • Amaia Lujambio
    Correspondence
    Corresponding authors. Address: Division of Chronic Inflammation and Cancer, German Cancer Research Center Heidelberg (DKFZ), Heidelberg, Germany (M. Heikenwalder), or Department of Medical Oncology and Pneumology, University Hospital Tuebingen, 72076 Tuebingen, Germany (L. Zender), or Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA (A. Lujambio).
    Affiliations
    Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, USA

    Liver Cancer Program, Division of Liver Diseases, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA

    The Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, USA

    Graduate School of Biomedical Sciences at Icahn School of Medicine at Mount Sinai, New York, USA
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      Summary

      Preclinical models of cancer based on the use of human cancer cell lines and mouse models have enabled discoveries that have been successfully translated into patients. And yet the majority of clinical trials fail, emphasising the urgent need to improve preclinical research to better interrogate the potential efficacy of each therapy and the patient population most likely to benefit. This is particularly important for liver malignancies, which lack highly efficient treatments and account for hundreds of thousands of deaths around the globe. Given the intricate network of genetic and environmental factors that contribute to liver cancer development and progression, the identification of new druggable targets will mainly depend on establishing preclinical models that mirror the complexity of features observed in patients. The development of new 3D cell culture systems, originating from cells/tissues isolated from patients, might create new opportunities for the generation of more specific and personalised therapies. However, these systems are unable to recapitulate the tumour microenvironment and interactions with the immune system, both proven to be critical influences on therapeutic outcomes. Patient-derived xenografts, in particular with humanised mouse models, more faithfully mimic the physiology of human liver cancer but are costly and time-consuming, which can be prohibitive for personalising therapies in the setting of an aggressive malignancy. In this review, we discuss the latest advances in the development of more accurate preclinical models to better understand liver cancer biology and identify paradigm-changing therapies, stressing the importance of a bi-directional communicative flow between clinicians and researchers to establish reliable model systems and determine how best to apply them to expanding our current knowledge.

      Keywords

      Introduction

      Liver cancer is the sixth most frequent type of cancer and the fourth-leading cause of cancer-related death globally.
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      However, developing biomarkers and stratification systems to predict which patients will respond to immune checkpoint blockade is critical, given that only a minority of patients respond and these drugs are associated with considerable toxicity and cost. The limited success of current therapies in patients with liver cancer could potentially be explained by a lack of stratification according to the genetic makeup of their tumours.
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      highlighting the functional relevance of TERT in HCC. Other genes that are frequently mutated in patients with HCC include the cell cycle regulator TP53 (around 30% of cases), the WNT pathway regulator CTNNB1 (around 30% of cases), and chromatin remodelers ARID1A and ARID2 (around 10% and 5% of cases, respectively).
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      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      It is estimated that a potentially actionable mutation is identifiable in around 10–28% of HCCs; however, the most frequent driver mutations in HCC are currently not targetable.
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      In addition, HCC is characterised by a particularly high inter-patient heterogeneity,
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      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
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      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      which makes biomarker-guided treatments critical.
      Preclinical models that can recapitulate the genetic heterogeneity, immune interactions and specific microenvironment of HCC will be crucial for the development of personalised treatments.
      The development of effective personalised treatments for HCC has traditionally been hampered by the scarcity of preclinical models that recapitulate the genetic heterogeneity of human tumours while preserving critical features of in vivo tumour growth, such as interactions with the immune system and the tumour microenvironment. So far, most HCC preclinical models have been based on the use of genetically engineered mouse models (GEMMs), diet or chemotoxin-induced murine HCC models, human tumour-derived 2D cell lines in culture, or xenografts in immunodeficient mice. Two-dimensional cancer cell line culture has been the main preclinical model system in cancer biology but lacks critical features of in vivo tumour growth, including the interactions with the immune system and the tumour microenvironment.
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      Cell line-based platforms to evaluate the therapeutic efficacy of candidate anticancer agents.
      A number of methods can be used to generate liver tumours in mice, including genetic engineering, chemotoxic agents, intrahepatic or intrasplenic injection of tumour cells, and xenograft approaches.
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      GEMMs, which harbour activated oncogenes or inactivated tumour suppressor genes that lead to liver tumour formation, are useful for interrogating the effects of specific genetic alterations on tumour biology and therapeutic responsiveness.
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      Mouse models of liver cancer.
      However, the ability to isolate individual mutations and analyse their effects on tumorigenesis comes at the price of oversimplifying the genetic complexity of human tumours and eliminating the ability to see important differences present in a tumour with a number of other mutated genes. Since HCC most frequently develops on the background of a diseased liver, there are also models that recapitulate the insults that promote liver cancer, including viral hepatitis infection, NAFLD, fibrosis, alcohol-induced liver disease, and cholestasis.
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      Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research.
      Modelling of HCC development in an environment of chronic inflammation and liver disease can also be accomplished using GEMMs generated to express specific fragments of HBV or HCV, or to induce chronic liver damage which in turn leads to tumour formation.
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      Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research.
      Chemically induced liver tumour models are based on the use of chemicals that directly promote mutations in hepatocytes or induce liver damage that favours the proliferation of preneoplastic cells.
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      Experimental mouse models for hepatocellular carcinoma research.
      For example, diethylnitrosamine (DEN) and 9,10-dimethyl-1,2-benzanthracene (DMBA) are carcinogens that can induce a variety of mutations, including in the oncogene Ras, when given to mice neonatally.
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      and liver damage-induced models,
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      which indicated that DEN-driven models are the least similar to human tumours.
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      While GEMMs and diet-induced mouse models definitely help us answer mechanistic questions and provide information on the biological processes underlying cancer development and treatment response, they may lack the genetic complexity of human tumours. Patient-derived model systems may be better able to account for the complexity of human tumours and result in more directly applicable results that can inform personalised treatment decisions. In this review, we discuss the latest developments in patient-derived models of liver cancer, with a special focus on models of HCC. In particular, we will examine recent advances in 2D cancer cell culture, the generation of patient-derived organoids (PDOs) and xenografts (PDXs), and novel strategies to integrate the immune system into these models and mimic the genetic and immunological complexity of human HCC tumours.

      Patient-derived cell lines

      For many years, most preclinical research in HCC focused on the use of 2D cancer cell line cultures and xenografted mice.
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      Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5.
      In vitro HCC cancer cell lines are relatively inexpensive, allow for seamless high-throughput drug testing, and most importantly, retain genomic and transcriptomic landscapes similar to primary HCCs.
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      Hepatocellular carcinoma cell lines retain the genomic and transcriptomic landscapes of primary human cancers.
      Whole genome analyses of 4 newly established HCC cell lines showed that mutations, copy number alterations, and HBV integration were preserved during cell line establishment.
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      Hepatocellular carcinoma cell lines retain the genomic and transcriptomic landscapes of primary human cancers.
      However, there is the caveat that although these cell lines harbour driver mutations similar to primary HCCs, they do not retain the same profile of genetic alterations as the specific patient tumour from which they were derived, and they are able to acquire new mutations in vitro as well.
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      An in-depth analysis of the genetic, RNA, and protein profiles of 34 liver cancer cell lines was recently performed and revealed unique drug sensitivities based on the genetic makeup of the tumour cells.
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      Analysis of liver cancer cell lines identifies agents with likely efficacy against hepatocellular carcinoma and markers of response.
      These profiles were aligned with those of primary human HCCs to delineate groups of patients with characteristics that predicted differential responses to drug treatments in vitro. In addition, a similar effort by Qiu and colleagues recently generated a Liver Cancer Model Repository (LIMORE) with 50 newly established patient-derived liver cancer cell lines in addition to 31 previously available lines.
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      Through modification of previous cell line establishment protocols, the authors were able to achieve around a 50% success rate for generating lines from primary liver tumours. The authors report that with this new database, 85% of cancer-functioning genes in HCC were found to be represented by at least 1 of the models, and 51% of cancer-functioning genes had at least 3 models in which they were represented.
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      A pharmacogenomic landscape in human liver cancers.
      The utility of the LIMORE 2D cell culture models was also shown, through identification of specific genetic markers of drug sensitivity or resistance, as well as synergistic drug combinations that were validated in patients.
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      A pharmacogenomic landscape in human liver cancers.
      This newly expanded repertoire of 2D cell lines offers the opportunity to conduct drug screens and other studies within a context that more accurately represents the diversity and genetic complexity of human liver cancer.
      Despite its ease of use and utility for finding drug candidates and response profiles applicable to patients, 2D cell culture has a number of disadvantages: most notably the loss of intratumoural heterogeneity, inadequate preservation of the tumour microenvironment, and the inability to sufficiently study interactions between tumours and the immune system.
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      Remodelling and improvements in organoid technology to study liver carcinogenesis in a dish.
      Given the early successes of immunotherapies in advanced liver cancer, models that enable interrogation of the immune-tumour interface are critical for dissecting mechanisms of response and resistance, and for predicting patient response to immuno-active drugs.

      Patient-derived organoids

      Initial attempts were made in the late 1990s to overcome these limitations of 2D cell culture and more realistically mimic in vivo tumour growth in cell culture systems.
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      For example, a 3D culture set up was developed based on the use of a bioreactor, beads coated with extracellular matrix, and liver cells (including hepatocytes, cholangiocytes, hepatic stellate cells, and sinusoidal endothelial cells), which was able to sustain fully functional hepatocytes.
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      In the mid-2000s, human precision-cut liver slices containing both tumoural and non-tumoural liver tissue were maintained ex vivo for periods of 3–5 days.
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      but their use is restricted to short periods of time.

       Organoids derived from adult liver cells

      3D organoids that mimic the in vivo architecture of organs have been developed both from stem cells and primary human hepatocytes.
      Recently, 3D organoids, defined by Clevers as being “characterized by a self-organizing 3D structure which mimics the original in vivo architecture of organs or tumours and can be derived from different sources”, have been successfully established from liver tissue.
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      Cancer modeling meets human organoid technology.
      Thus far, organoids can be derived from organ-specific adult stem cells, from pluripotent stem cells (PSCs), from embryonic stem cells or induced pluripotent stem cells (iPSCs), and from tumours (called tumouroids).
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      Cancer modeling meets human organoid technology.
      The first stem cell-derived organoids were established in 2009 from intestinal stem cells
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      • Stange D.E.
      • et al.
      Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.
      while the first tumour organoids were described in 2012, also from an intestinal source.
      • Sato T.
      • Stange D.E.
      • Ferrante M.
      • Vries R.G.
      • Van Es J.H.
      • Van den Brink S.
      • et al.
      Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.
      Huch and colleagues established the first organoids from murine
      • Huch M.
      • Dorrell C.
      • Boj S.F.
      • van Es J.H.
      • Li V.S.
      • van de Wetering M.
      • et al.
      In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.
      and human liver stem cells
      • Huch M.
      • Gehart H.
      • van Boxtel R.
      • Hamer K.
      • Blokzijl F.
      • Verstegen M.M.
      • et al.
      Long-term culture of genome-stable bipotent stem cells from adult human liver.
      in 2013 and 2015, respectively, through the isolation and expansion of epithelial progenitor cells derived from adult ductal cells. These progenitor cells retain the ability to differentiate into either of the 2 epithelial cell types of the liver: cholangiocytes (also known as ductal cells) or hepatocytes. Interestingly, despite their massive proliferative capacity in vivo, hepatocytes have proven difficult to grow in vitro, and the development of bipotent progenitor cells was only successfully achieved in these initial studies through derivation from cholangiocytes.
      • Michalopoulos G.K.
      Liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas.
      • Mitaka T.
      The current status of primary hepatocyte culture.
      The bipotent progenitor cells can be expanded for over 6 months and can differentiate into a biliary or hepatic fate depending on the composition of the culture media.
      • Huch M.
      • Gehart H.
      • van Boxtel R.
      • Hamer K.
      • Blokzijl F.
      • Verstegen M.M.
      • et al.
      Long-term culture of genome-stable bipotent stem cells from adult human liver.
      Organoids established from liver biopsies of patients with α-1-antitrypsin-deficiency and Alagille syndrome, genetic syndromes that manifest as liver pathologies, were able to mirror the corresponding human pathology.
      • Huch M.
      • Gehart H.
      • van Boxtel R.
      • Hamer K.
      • Blokzijl F.
      • Verstegen M.M.
      • et al.
      Long-term culture of genome-stable bipotent stem cells from adult human liver.
      In addition, liver organoids can be genetically modified for functional studies or therapeutic purposes.
      • Broutier L.
      • Andersson-Rolf A.
      • Hindley C.J.
      • Boj S.F.
      • Clevers H.
      • Koo B.K.
      • et al.
      Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.
      More recently, long-term 3D organoid culture systems have been established from mouse and human primary hepatocytes. Two studies in parallel have grown hepatocyte-derived liver organoids using very similar culture protocols,
      • Hu H.
      • Gehart H.
      • Artegiani B.
      • LÖpez-Iglesias F.
      • Dekkers F.
      • Basak O.
      • et al.
      Long-term expansion of functional mouse and human hepatocytes as 3D organoids.
      • Peng W.C.
      • Logan C.Y.
      • Fish M.
      • Anbarchian T.
      • Aguisanda F.
      • Alvarez-Varela A.
      • et al.
      Inflammatory cytokine TNFalpha promotes the long-term expansion of primary hepatocytes in 3D culture.
      in which organoids were established from single hepatocytes and grown for multiple months, while retaining key in vivo morphological, functional, and gene expression features. In the study by Peng and colleagues, TNFα, an injury-induced inflammatory cytokine, was critical for promoting the expansion of hepatocytes, suggesting that tissue repair signals could be harnessed to promote the expansion of otherwise hard-to-culture cell types.
      • Peng W.C.
      • Logan C.Y.
      • Fish M.
      • Anbarchian T.
      • Aguisanda F.
      • Alvarez-Varela A.
      • et al.
      Inflammatory cytokine TNFalpha promotes the long-term expansion of primary hepatocytes in 3D culture.
      Further improvements of culture and transplantation conditions (possibly by combining the 2 protocols) may enable important clinical applications.
      Although they are not technically derived from patients with liver cancer, normal liver organoids developed from adult primary liver cells
      • Huch M.
      • Gehart H.
      • van Boxtel R.
      • Hamer K.
      • Blokzijl F.
      • Verstegen M.M.
      • et al.
      Long-term culture of genome-stable bipotent stem cells from adult human liver.
      • Hu H.
      • Gehart H.
      • Artegiani B.
      • LÖpez-Iglesias F.
      • Dekkers F.
      • Basak O.
      • et al.
      Long-term expansion of functional mouse and human hepatocytes as 3D organoids.
      or PSCs
      • Takebe T.
      • Sekine K.
      • Enomura M.
      • Koike H.
      • Kimura M.
      • Ogaeri T.
      • et al.
      Vascularized and functional human liver from an iPSC-derived organ bud transplant.
      could be modified by gene editing technologies to recapitulate specific mutations present in patients with liver cancer. As a proof of principle, murine CCA organoids were established and later modified by RNA interference and clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology to produce tractable CCA organoids that recapitulate the multiple steps involved in liver tumorigenesis, eliminating the need for repeated establishment of liver organoids.
      • Saborowski A.
      • Wolff K.
      • Spielberg S.
      • Beer B.
      • Hartleben B.
      • Erlangga Z.
      • et al.
      Murine liver organoids as a genetically flexible system to study liver cancer in vivo and in vitro.
      A similar strategy has also been employed with human cholangiocyte organoids to study the role of the tumour suppressor BAP1 in CCA.
      • Artegiani B.
      • van Voorthuijsen L.
      • Lindeboom R.G.H.
      • Seinstra D.
      • Heo I.
      • Tapia P.
      • et al.
      Probing the tumor suppressor function of BAP1 in CRISPR-engineered human liver organoids.
      BAP1 deletion by CRISPR/Cas9 technology led to increased motility but not full tumoural transformation. Additional loss of tumour suppressors, including TP53, PTEN, SMAD4, and NF1, led to full malignant transformation.
      • Artegiani B.
      • van Voorthuijsen L.
      • Lindeboom R.G.H.
      • Seinstra D.
      • Heo I.
      • Tapia P.
      • et al.
      Probing the tumor suppressor function of BAP1 in CRISPR-engineered human liver organoids.
      Organoid technology combined with CRISPR/Cas9, while potentially not recapitulating the complexity of human tumours, provides an experimental platform for mechanistic studies of cancer gene function in a human context.

       Organoids from pluripotent stem cells

      Apart from primary epithelial cells and stem cells, PSCs serve as a tool for the establishment of tissue-specific organoids.
      • Tharehalli U.
      • Svinarenko M.
      • Lechel A.
      Remodelling and improvements in organoid technology to study liver carcinogenesis in a dish.
      In the liver, Takebe and colleagues used iPSCs to generate liver organoids.
      • Takebe T.
      • Sekine K.
      • Enomura M.
      • Koike H.
      • Kimura M.
      • Ogaeri T.
      • et al.
      Vascularized and functional human liver from an iPSC-derived organ bud transplant.
      Human iPSCs were first differentiated into endodermal spheres and then cultured in matrigel and mixed with stromal and endothelial cells, which formed functional vasculature. In vivo transplantation experiments demonstrated that endothelial cells are essential to establish vascular structures. This novel organoid platform could potentially be harnessed for liver transplantation or the treatment of genetic diseases after genetic manipulation in vitro.
      • Takebe T.
      • Sekine K.
      • Enomura M.
      • Koike H.
      • Kimura M.
      • Ogaeri T.
      • et al.
      Vascularized and functional human liver from an iPSC-derived organ bud transplant.
      Liver organoids can now be generated from a variety of cells, using different cell mixtures and protocols, including: primary human hepatocytes modified to undergo proliferation, mixed with liver sinusoidal endothelial cells and mesenchymal stem cells;
      • Ramachandran S.D.
      • Schirmer K.
      • Munst B.
      • Heinz S.
      • Ghafoory S.
      • Wolfl S.
      • et al.
      In vitro generation of functional liver organoid-like structures using adult human cells.
      iPSC-derived liver organoids on perfusable micropillar chips;
      • Wang Y.
      • Wang H.
      • Deng P.
      • Chen W.
      • Guo Y.
      • Tao T.
      • et al.
      In situ differentiation and generation of functional liver organoids from human iPSCs in a 3D perfusable chip system.
      primary hepatocytes mixed with Kupffer cells, quiescent stellate cells, and liver sinusoidal endothelial cells;
      • Sendi H.
      • Mead I.
      • Wan M.
      • Mehrab-Mohseni M.
      • Koch K.
      • Atala A.
      • et al.
      miR-122 inhibition in a human liver organoid model leads to liver inflammation, necrosis, steatofibrosis and dysregulated insulin signaling.
      or primary hepatocytes, stellate cells, and Kupffer cells.
      • Ouchi R.
      • Togo S.
      • Kimura M.
      • Shinozawa T.
      • Koido M.
      • Koike H.
      • et al.
      Modeling steatohepatitis in humans with pluripotent stem cell-derived organoids.
      iPSCs can also be used to generate hepatobiliary organoids by inducing endodermal and mesodermal differentiation in a modified media.
      • Wu F.
      • Wu D.
      • Ren Y.
      • Huang Y.
      • Feng B.
      • Zhao N.
      • et al.
      Generation of hepatobiliary organoids from human induced pluripotent stem cells.
      Some reports suggest that genetic and epigenetic aberrations may occur during the derivation and reprogramming processes,
      • Liang G.
      • Zhang Y.
      Genetic and epigenetic variations in iPSCs: potential causes and implications for application.
      • Pera M.F.
      Stem cells: the dark side of induced pluripotency.
      • Lund R.J.
      • Narva E.
      • Lahesmaa R.
      Genetic and epigenetic stability of human pluripotent stem cells.
      including the development of chromosomal abnormalities
      • Laurent L.C.
      • Ulitsky I.
      • Slavin I.
      • Tran H.
      • Schork A.
      • Morey R.
      • et al.
      Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture.
      , “de novo” copy number variations,
      • Hussein S.M.
      • Batada N.N.
      • Vuoristo S.
      • Ching R.W.
      • Autio R.
      • Narva E.
      • et al.
      Copy number variation and selection during reprogramming to pluripotency.
      and point mutations in protein-coding regions.
      • Gore A.
      • Li Z.
      • Fung H.L.
      • Young J.E.
      • Agarwal S.
      • Antosiewicz-Bourget J.
      • et al.
      Somatic coding mutations in human induced pluripotent stem cells.
      Such changes may complicate their use in regenerative medicine;
      • Bayart E.
      • Cohen-Haguenauer O.
      Technological overview of iPS induction from human adult somatic cells.
      however, novel advances in gene editing technologies and the establishment of iPSCs may circumvent these problems.

       Organoids from liver tumours (tumouroids)

      The first efforts to establish organoid-like structures were based on the growth of tumour spheroids. For example, Takai et al. demonstrated that HCC cells in porous alginate scaffolds can generate organoid-like spheroids that mimic numerous features of glandular epithelium in vivo, such as acinar morphogenesis and apical expression patterns of EpCAM, a hepatic stem/progenitor cell marker highly expressed in a subset of HCCs with stemness features.
      • Takai A.
      • Fako V.
      • Dang H.
      • Forgues M.
      • Yu Z.
      • Budhu A.
      • et al.
      Three-dimensional organotypic culture models of human hepatocellular carcinoma.
      Interestingly, EpCAM+ HCC cells cultured as spheroids showed tumourigenic and metastatic potential in vivo that was absent in conventionally cultured 2D cells.
      • Takai A.
      • Fako V.
      • Dang H.
      • Forgues M.
      • Yu Z.
      • Budhu A.
      • et al.
      Three-dimensional organotypic culture models of human hepatocellular carcinoma.
      The laboratory of Dr. Huch recently modified the protocol for growing liver organoids to accommodate the growth of liver tumouroids.
      • Broutier L.
      • Mastrogiovanni G.
      • Verstegen M.M.
      • Francies H.E.
      • Gavarro L.M.
      • Bradshaw C.R.
      • et al.
      Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.
      The protocol for tissue digestion was also adapted to facilitate selection of tumour cells in preference to non-tumoural cells, and basement membrane extract was used to support the stability of tumour cells. In addition, 2 distinct culture media were developed: one enabling the growth of both non-tumour and tumour cells, and the other lacking key growth factors to prevent contamination from non-tumour cells.
      • Broutier L.
      • Mastrogiovanni G.
      • Verstegen M.M.
      • Francies H.E.
      • Gavarro L.M.
      • Bradshaw C.R.
      • et al.
      Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.
      The latter protocol was able to generate tumouroids from HCC, CCA, and mixed HCC/CCA tumour samples.
      • Broutier L.
      • Mastrogiovanni G.
      • Verstegen M.M.
      • Francies H.E.
      • Gavarro L.M.
      • Bradshaw C.R.
      • et al.
      Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.
      In a subsequent study by an independent group, HCC and CCA tumouroids were established from patient-derived needle biopsies with around a 30% success rate, and could be maintained in culture for up to 32 weeks
      • Nuciforo S.
      • Fofana I.
      • Matter M.S.
      • Blumer T.
      • Calabrese D.
      • Boldanova T.
      • et al.
      Organoid models of human liver cancers derived from tumor needle biopsies.
      Through the use of the needle biopsy sample acquisition protocol, non-tumoural liver tissue was also collected and used to generate patient-derived liver organoids for normalisation of data generated from the corresponding patient’s tumouroid.
      • Wu F.
      • Wu D.
      • Ren Y.
      • Huang Y.
      • Feng B.
      • Zhao N.
      • et al.
      Generation of hepatobiliary organoids from human induced pluripotent stem cells.
      More recently, organoid technology has been applied to the generation of tumouroids which can be established from patient-derived needle biopsies. These tumouroids recapitulate the key features of the original tumour biopsy.
      Most liver tumouroids established to date closely resemble the original tumour biopsy in terms of growth pattern, differentiation grade, expression of HCC-specific markers, genomic alterations, and ability to form tumours in xenograft models.
      • Broutier L.
      • Mastrogiovanni G.
      • Verstegen M.M.
      • Francies H.E.
      • Gavarro L.M.
      • Bradshaw C.R.
      • et al.
      Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.
      • Nuciforo S.
      • Fofana I.
      • Matter M.S.
      • Blumer T.
      • Calabrese D.
      • Boldanova T.
      • et al.
      Organoid models of human liver cancers derived from tumor needle biopsies.
      This represents a difference from 2D cancer cell lines, which tend to lose their original patient-specific genetic signature and acquire additional mutations, possibly because of the need to adapt to a non-physiologic environment which lacks signalling from surrounding stromal tissue.
      • Iorio F.
      • Knijnenburg T.A.
      • Vis D.J.
      • Bignell G.R.
      • Menden M.P.
      • Schubert M.
      • et al.
      A landscape of pharmacogenomic interactions in cancer.
      • Drexler H.G.
      • Fombonne S.
      • Matsuo Y.
      • Hu Z.B.
      • Hamaguchi H.
      • Uphoff C.C.
      p53 alterations in human leukemia-lymphoma cell lines: in vitroartifact or prerequisite for cell immortalization?.
      In both studies, the efficiency of liver tumouroid establishment was similar but relatively low (around 20–30%), and was influenced by the proliferation rate and differentiation of primary tumours.
      • Broutier L.
      • Mastrogiovanni G.
      • Verstegen M.M.
      • Francies H.E.
      • Gavarro L.M.
      • Bradshaw C.R.
      • et al.
      Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.
      • Nuciforo S.
      • Fofana I.
      • Matter M.S.
      • Blumer T.
      • Calabrese D.
      • Boldanova T.
      • et al.
      Organoid models of human liver cancers derived from tumor needle biopsies.
      The ability to establish liver tumouroids from needle biopsies overcomes a major limitation, as surgical resection is not a treatment option for the majority of patients with HCC and liver biopsy is not required for diagnosis in advanced cases with classic imaging findings.
      • Nuciforo S.
      • Fofana I.
      • Matter M.S.
      • Blumer T.
      • Calabrese D.
      • Boldanova T.
      • et al.
      Organoid models of human liver cancers derived from tumor needle biopsies.
      This could potentially facilitate the establishment of liver tumouroid biobanks, similar to what has been done for colorectal tumours.
      • van de Wetering M.
      • Francies H.E.
      • Francis J.M.
      • Bounova G.
      • Iorio F.
      • Pronk A.
      • et al.
      Prospective derivation of a living organoid biobank of colorectal cancer patients.
      Liver cancer tumouroids have also been established from murine tumours, including both HCC
      • Cao W.
      • Liu J.
      • Wang L.
      • Li M.
      • Verstegen M.M.A.
      • Yin Y.
      • et al.
      Modeling liver cancer and therapy responsiveness using organoids derived from primary mouse liver tumors.
      and CCA,
      • Saborowski A.
      • Wolff K.
      • Spielberg S.
      • Beer B.
      • Hartleben B.
      • Erlangga Z.
      • et al.
      Murine liver organoids as a genetically flexible system to study liver cancer in vivo and in vitro.
      enabling generation of tumouroids that have specific genetic manipulations and thereby expanding the potential applications of organoids for scientific study.

      Patient-derived xenografts

      Although 3D culture has enabled pioneering advances in cancer biology,
      • Sachs N.
      • de Ligt J.
      • Kopper O.
      • Gogola E.
      • Bounova G.
      • Weeber F.
      • et al.
      A living biobank of breast cancer organoids captures disease heterogeneity.
      • Vlachogiannis G.
      • Hedayat S.
      • Vatsiou A.
      • Jamin Y.
      • Fernandez-Mateos J.
      • Khan K.
      • et al.
      Patient-derived organoids model treatment response of metastatic gastrointestinal cancers.
      it fails to recapitulate certain critical features of a growing tumour in vivo, most notably preservation of the tumour microenvironment and dynamic interaction between the growing tumour and the immune system.
      • Shamir E.R.
      • Ewald A.J.
      Three-dimensional organotypic culture: experimental models of mammalian biology and disease.
      Injection of liver cancer cells or liver tumours subcutaneously or into the liver more accurately recapitulates the genetic complexity of human tumours and models the interactions of the tumour with its surrounding tissue in vivo.
      • Brown Z.J.
      • Heinrich B.
      • Greten T.F.
      Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research.
      Subcutaneous injection of cells/tumours (heterotopic models) is the easiest in vivo method and enables more accurate measurement of tumour growth and response to therapies. Orthotopic models, established via injection of tumour cells into the tissue of interest, are more challenging to establish as they require surgery for implantation of HCC into the liver or spleen.
      • Brown Z.J.
      • Heinrich B.
      • Greten T.F.
      Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research.
      However, orthotopic models more realistically recapitulate a native tumour environment, including vascularisation and interactions with the stroma and immune microenvironment of the relevant tissue, and are better able to model tumour metastasis.
      • Hernandez-Gea V.
      • Toffanin S.
      • Friedman S.L.
      • Llovet J.M.
      Role of the microenvironment in the pathogenesis and treatment of hepatocellular carcinoma.

       Patient-derived xenograft models in immunodeficient mice

      Murine xenograft models are established by heterotopically or orthotopically implanting human cancer cell lines, human tumours (PDXs), or potentially PDOs, into mice. To avoid rejection of human tumours by the murine immune system, implantation is performed into immunodeficient mice, of which a number of strains are available (Table 1).
      • Jung J.
      Human tumor xenograft models for preclinical assessment of anticancer drug development.
      When tumours reach a reasonable size, they can be cryopreserved, studied, or propagated again;
      • Choi Y.
      • Lee S.
      • Kim K.
      • Kim S.H.
      • Chung Y.J.
      • Lee C.
      Studying cancer immunotherapy using patient-derived xenografts (PDXs) in humanized mice.
      or, therapies can be tested, as PDX models can closely recapitulate clinical responses to treatment.
      • Krumbach R.
      • Schuler J.
      • Hofmann M.
      • Giesemann T.
      • Fiebig H.H.
      • Beckers T.
      Primary resistance to cetuximab in a panel of patient-derived tumour xenograft models: activation of MET as one mechanism for drug resistance.
      For example, in a pilot study, using drug screening in PDX to determine real-life personalised cancer treatments, 14 PDX models of solid tumours of various origin (not including liver cancer) were developed; treating the xenografted mice with a large panel of drugs identified targeted treatments for 12 of the 14 corresponding patients, which led to durable partial responses in the majority of cases.
      • Hidalgo M.
      • Bruckheimer E.
      • Rajeshkumar N.V.
      • Garrido-Laguna I.
      • De Oliveira E.
      • Rubio-Viqueira B.
      • et al.
      A pilot clinical study of treatment guided by personalized tumorgrafts in patients with advanced cancer.
      In a subsequent larger effort, around 1,000 PDX models of a spectrum of solid tumours that included 85 liver tumours were generated and subjected to high-throughput drug screens, revealing novel mechanisms of resistance to treatments that had been used in patients.
      • Gao H.
      • Korn J.M.
      • Ferretti S.
      • Monahan J.E.
      • Wang Y.
      • Singh M.
      • et al.
      High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response.
      Table 1Mouse strains commonly used for PDX.
      StrainDescriptionNotes
      nu/nuAthymic nude mice; deficient in T cells.
      • Morton C.L.
      • Houghton P.J.
      Establishment of human tumor xenografts in immunodeficient mice.
      Rag2−/−Recombination activating gene 2 knockout; deficient in T and B cells.
      • Morton C.L.
      • Houghton P.J.
      Establishment of human tumor xenografts in immunodeficient mice.
      scid/scidSeverely compromised immunodeficient; deficient in T and B cells.
      • Morton C.L.
      • Houghton P.J.
      Establishment of human tumor xenografts in immunodeficient mice.
      Retain NK cell activity.
      NOD-scidNon-obese diabetic, severely compromised immunodeficient; deficient in T and B cells, retain low level of NK cell activity.Retain NK cell activity, but at lower level than scid/scid; much higher HSC engraftment rate compared with scid/scid.
      • Shultz L.D.
      • Lyons B.L.
      • Burzenski L.M.
      • Gott B.
      • Chen X.
      • Chaleff S.
      • et al.
      Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.
      NOD/Ltsz-scid Il2rg−/− (NSG)Non-obese diabetic, severely compromised immunodeficient, Il2rg knockout; deficient in mature lymphocytes and NK cells.Survival >16 months, resistant to thymic lymphoma development; higher human HSC engraftment rates compared with NOD-scid, with multilineage differentiation.
      • Shultz L.D.
      • Lyons B.L.
      • Burzenski L.M.
      • Gott B.
      • Chen X.
      • Chaleff S.
      • et al.
      Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.
      NOD/LtSz-scid Il2rg−/−-SGM3 (NSG-SGM3, or NSGS)Express human IL-3, GM-CSF/CSF2, and stem cell factor [KITLG] to facilitate engraftment of human HSCs for humanisation
      • Wunderlich M.
      • Chou F.S.
      • Link K.A.
      • Mizukawa B.
      • Perry R.L.
      • Carroll M.
      • et al.
      AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3.
      Enhanced engraftment of HSCs compared with NSG mice.
      • Shultz L.D.
      • Lyons B.L.
      • Burzenski L.M.
      • Gott B.
      • Chen X.
      • Chaleff S.
      • et al.
      Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.
      NOD/LtSz-scid Il2rg−/− KitW41/W41 (NBSGW)NSG mouse with mutant c-kit allele.
      • McIntosh B.E.
      • Brown M.E.
      • Duffin B.M.
      • Maufort J.P.
      • Vereide D.T.
      • Slukvin I.I.
      • et al.
      Nonirradiated NOD, B6.SCID Il2rgamma-/- Kit(W41/W41) (NBSGW) mice support multilineage engraftment of human hematopoietic cells.
      Can achieve HSC engraftment with enhanced humanisation, without the need for irradiation.
      • McIntosh B.E.
      • Brown M.E.
      • Duffin B.M.
      • Maufort J.P.
      • Vereide D.T.
      • Slukvin I.I.
      • et al.
      Nonirradiated NOD, B6.SCID Il2rgamma-/- Kit(W41/W41) (NBSGW) mice support multilineage engraftment of human hematopoietic cells.
      SRG-15Rag2−/− Il2rg−/− mice with human IL15 and human SIRPA knock-in.Enable growth of NK cells, intraepithelial lymphocytes, and innate lymphoid cell subsets
      • Herndler-Brandstetter D.
      • Shan L.
      • Yao Y.
      • Stecher C.
      • Plajer V.
      • Lietzenmayer M.
      • et al.
      Humanized mouse model supports development, function, and tissue residency of human natural killer cells.
      HSCs, haematopoietic stem cells; NK, natural killer; NOD, non-obese diabetic; PDX, patient-derived xenografts; scid, severely compromised immunodeficient.
      The first HCC PDX was established in 1996;
      • Sun F.X.
      • Tang Z.Y.
      • Lui K.D.
      • Ye S.L.
      • Xue Q.
      • Gao D.M.
      • et al.
      Establishment of a metastatic model of human hepatocellular carcinoma in nude mice via orthotopic implantation of histologically intact tissues.
      however, the development of additional HCC PDXs has been slow and cumbersome, with low efficiency rates.
      • Xin H.
      • Wang K.
      • Hu G.
      • Xie F.
      • Ouyang K.
      • Tang X.
      • et al.
      Establishment and characterization of 7 novel hepatocellular carcinoma cell lines from patient-derived tumor xenografts.
      • Yan M.
      • Li H.
      • Zhao F.
      • Zhang L.
      • Ge C.
      • Yao M.
      • et al.
      Establishment of NOD/SCID mouse models of human hepatocellular carcinoma via subcutaneous transplantation of histologically intact tumor tissue.
      More recently, an impressive cohort of 65 liver cancer PDX models was generated.
      • Gu Q.
      • Zhang B.
      • Sun H.
      • Xu Q.
      • Tan Y.
      • Wang G.
      • et al.
      Genomic characterization of a large panel of patient-derived hepatocellular carcinoma xenograft tumor models for preclinical development.
      In this cohort, lenvatinib, a multikinase inhibitor that targets FGFR1, showed better therapeutic efficacy than sorafenib in models with higher levels of FGFR1.
      • Gu Q.
      • Zhang B.
      • Sun H.
      • Xu Q.
      • Tan Y.
      • Wang G.
      • et al.
      Genomic characterization of a large panel of patient-derived hepatocellular carcinoma xenograft tumor models for preclinical development.
      The clinical relevance of this finding is underscored by the recent approval of lenvatinib as a first-line treatment for patients with advanced HCC following a clinical trial showing non-inferiority to sorafenib.
      • Kudo M.
      • Finn R.S.
      • Qin S.
      • Han K.H.
      • Ikeda K.
      • Piscaglia F.
      • et al.
      Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial.
      Future studies in patients may validate FGFR1 expression levels as a predictor of response to lenvatinib, which would demonstrate feasibility of biomarker-guided selection of patients for treatments based on data garnered from PDX modelling. The generation of additional HCC PDX models will aid in the identification of novel drug targets and the corresponding patient populations most likely to benefit. Recently, a comprehensive dataset named PDXliver was assembled with 116 HCC PDXs: 51 newly generated, and the remainder from the established literature.
      • He S.
      • Hu B.
      • Li C.
      • Lin P.
      • Tang W.G.
      • Sun Y.F.
      • et al.
      PDXliver: a database of liver cancer patient derived xenograft mouse models.
      The database contains information about clinical features, expression profiles, and genetic alterations, and the models adequately represent the diversity seen in patients with HCC.
      • Schulze K.
      • Imbeaud S.
      • Letouze E.
      • Alexandrov L.B.
      • Calderaro J.
      • Rebouissou S.
      • et al.
      Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets.
      • Cancer Genome Atlas Research Network
      • Electronic address wbe
      • Cancer Genome Atlas Research N.
      Comprehensive and integrative genomic characterization of hepatocellular carcinoma.
      Moreover, drug response data is also included, which may lead to the nomination of biomarkers for specific therapies.
      • He S.
      • Hu B.
      • Li C.
      • Lin P.
      • Tang W.G.
      • Sun Y.F.
      • et al.
      PDXliver: a database of liver cancer patient derived xenograft mouse models.
      A comprehensive dataset of patient-derived HCC xenografts has been assembled, providing information that could be applied to the development of biomarkers for specific therapies.
      Just as it was established that needle biopsy samples of livers could be developed into liver tumouroids,
      • Nuciforo S.
      • Fofana I.
      • Matter M.S.
      • Blumer T.
      • Calabrese D.
      • Boldanova T.
      • et al.
      Organoid models of human liver cancers derived from tumor needle biopsies.
      it has recently been demonstrated that liver cancer PDXs can also be generated from needle biopsies.
      • Blumer T.
      • Fofana I.
      • Matter M.S.
      • Wang X.
      • Montazeri H.
      • Calabrese D.
      • et al.
      Hepatocellular carcinoma xenografts established from needle biopsies preserve the characteristics of the originating tumors.
      In this particular study, 11 out of 54 human HCC needle biopsies were successfully engrafted into immunodeficient mice. Interestingly, the histological, transcriptomic, and genomic characteristics of the original HCC biopsies were maintained over subsequent re-transplantations.
      • Blumer T.
      • Fofana I.
      • Matter M.S.
      • Wang X.
      • Montazeri H.
      • Calabrese D.
      • et al.
      Hepatocellular carcinoma xenografts established from needle biopsies preserve the characteristics of the originating tumors.
      The engraftment efficiency was only 20%, which may be influenced by the amount of available tumoural tissue or the engraftment protocol. PDX models of CCA are still underdeveloped but also closely recapitulate the primary tumours.
      • Vaeteewoottacharn K.
      • Pairojkul C.
      • Kariya R.
      • Muisuk K.
      • Imtawil K.
      • Chamgramol Y.
      • et al.
      Establishment of highly transplantable cholangiocarcinoma cell lines from a patient-derived xenograft mouse.
      Until recently, only a few PDX models of paediatric liver cancer had been developed.
      • Hata Y.
      • Uchino J.
      • Sato K.
      • Sasaki F.
      • Une Y.
      • Naito H.
      • et al.
      Establishment of an experimental model of human hepatoblastoma.
      • Desdouets C.
      • Fabre M.
      • Gauthier F.
      • Brechot C.
      • Sobczak-Thepot J.
      Proliferation and differentiation of a human hepatoblastoma transplanted in the Nude mouse.
      • Fuchs J.
      • Schmidt D.
      • Pietsch T.
      • Miller K.
      • von Schweinitz D.
      Successful transplantation of human hepatoblastoma into immunodeficient mice.
      More recently, 2 independent studies have established comprehensive cohorts of paediatric liver tumour PDXs.
      • Bissig-Choisat B.
      • Kettlun-Leyton C.
      • Legras X.D.
      • Zorman B.
      • Barzi M.
      • Chen L.L.
      • et al.
      Novel patient-derived xenograft and cell line models for therapeutic testing of pediatric liver cancer.
      • Nicolle D.
      • Fabre M.
      • Simon-Coma M.
      • Gorse A.
      • Kappler R.
      • Nonell L.
      • et al.
      Patient-derived mouse xenografts from pediatric liver cancer predict tumor recurrence and advise clinical management.
      One of the studies generated 6 out of 15 (40% success rate) PDXs from HB, hepatocellular malignant neoplasm, and childhood HCC.
      • Bissig-Choisat B.
      • Kettlun-Leyton C.
      • Legras X.D.
      • Zorman B.
      • Barzi M.
      • Chen L.L.
      • et al.
      Novel patient-derived xenograft and cell line models for therapeutic testing of pediatric liver cancer.
      These paediatric liver cancer PDXs recapitulated the histology, genetic alterations, and biology of the original primary tumours. In addition, the generation of cell lines from 2 of the PDXs enabled the identification of suitable therapies for the corresponding PDXs,
      • Bissig-Choisat B.
      • Kettlun-Leyton C.
      • Legras X.D.
      • Zorman B.
      • Barzi M.
      • Chen L.L.
      • et al.
      Novel patient-derived xenograft and cell line models for therapeutic testing of pediatric liver cancer.
      highlighting the potential of this novel pipeline to guide the selection of personalised therapies. The second study was able to establish PDX models from 20 HBs, 1 HCC, 2 malignant rhabdoid tumours, and 1 transitional liver cell tumour.
      • Nicolle D.
      • Fabre M.
      • Simon-Coma M.
      • Gorse A.
      • Kappler R.
      • Nonell L.
      • et al.
      Patient-derived mouse xenografts from pediatric liver cancer predict tumor recurrence and advise clinical management.
      These novel models also mimicked the histology and genetic alterations, and the ability to engraft tumours correlated with poor prognosis.
      • Nicolle D.
      • Fabre M.
      • Simon-Coma M.
      • Gorse A.
      • Kappler R.
      • Nonell L.
      • et al.
      Patient-derived mouse xenografts from pediatric liver cancer predict tumor recurrence and advise clinical management.
      Like the other paediatric study, drug responses were unique in different PDX models and could potentially predict patient treatment outcomes.
      • Nicolle D.
      • Fabre M.
      • Simon-Coma M.
      • Gorse A.
      • Kappler R.
      • Nonell L.
      • et al.
      Patient-derived mouse xenografts from pediatric liver cancer predict tumor recurrence and advise clinical management.
      A PDX model of fibrolamellar carcinoma has already been established
      • Oikawa T.
      • Wauthier E.
      • Dinh T.A.
      • Selitsky S.R.
      • Reyna-Neyra A.
      • Carpino G.
      • et al.
      Model of fibrolamellar hepatocellular carcinomas reveals striking enrichment in cancer stem cells.
      and there are several others in preparation.
      • Kastenhuber E.R.
      • Craig J.
      • Ramsey J.
      • Sullivan K.M.
      • Sage J.
      • de Oliveira S.
      • et al.
      Road map for fibrolamellar carcinoma: progress and goals of a diversified approach.

       Patient-derived xenograft models in humanised mice

      One limitation of these PDX models is that they require immunocompromised mice to avoid rejection by the murine immune system and are therefore not suitable for the interrogation of immunotherapeutic approaches.
      • Jung J.
      Human tumor xenograft models for preclinical assessment of anticancer drug development.
      Immunocompromised mice cannot mimic full immune responses, lymphangiogenesis, or critical chemotactic signalling from the tumour microenvironment.
      • Shultz L.D.
      • Ishikawa F.
      • Greiner D.L.
      Humanized mice in translational biomedical research.
      To study cancer immunotherapy, it is critical to reproduce the complexity of the human immune system. One strategy for accomplishing this is to use humanised mouse models which have been modified to include human immune cells.
      • Brown Z.J.
      • Heinrich B.
      • Greten T.F.
      Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research.
      A frequently used method for creating humanised mice is to transfer human haematopoietic stem cells (HSCs) and precursor cells isolated from foetal cord blood to the marrow of sub-lethally irradiated mice, to promote the development of a functional immune system.
      • Zhou Q.
      • Facciponte J.
      • Jin M.
      • Shen Q.
      • Lin Q.
      Humanized NOD-SCID IL2rg-/- mice as a preclinical model for cancer research and its potential use for individualized cancer therapies.
      • Shultz L.D.
      • Goodwin N.
      • Ishikawa F.
      • Hosur V.
      • Lyons B.L.
      • Greiner D.L.
      Human cancer growth and therapy in immunodeficient mouse models.
      Novel immunodeficient mice have been developed for the purpose of generating humanised mice (Table 1). Humanisation of both human liver and human immune cells in mice has also been achieved, through crossing of immunodeficient mice (NOD;Rag2−/−;Il2rg−/−) with fumarylacetoacetate hydrolase deficient (Fah−/−) mice and co-transplantation with HSCs and adult human hepatocytes.
      • Wilson E.M.
      • Bial J.
      • Tarlow B.
      • Bial G.
      • Jensen B.
      • Greiner D.L.
      • et al.
      Extensive double humanization of both liver and hematopoiesis in FRGN mice.
      Fah deficiency facilitates the successful engraftment of hepatocytes, which are fully functional.
      Humanised liver PDX models are generated by implanting human liver tumour fragments into these humanised mice, either orthotopically or heterotopically.
      • Choi Y.
      • Lee S.
      • Kim K.
      • Kim S.H.
      • Chung Y.J.
      • Lee C.
      Studying cancer immunotherapy using patient-derived xenografts (PDXs) in humanized mice.
      In an initial study, human HCC PDXs were generated in NOD-scid-Il2rg−/− mice, and the animals were repopulated with adoptive chimeric antigen receptor (CAR) T cells.
      • Jiang Z.
      • Jiang X.
      • Chen S.
      • Lai Y.
      • Wei X.
      • Li B.
      • et al.
      Anti-GPC3-CAR T cells suppress the growth of tumor cells in patient-derived xenografts of hepatocellular carcinoma.
      The 3 PDXs mirrored the primary tumours at the transcriptional, morphological, and immunological levels,
      • Jiang Z.
      • Jiang X.
      • Chen S.
      • Lai Y.
      • Wei X.
      • Li B.
      • et al.
      Anti-GPC3-CAR T cells suppress the growth of tumor cells in patient-derived xenografts of hepatocellular carcinoma.
      and CAR T cells directed against glypican 3, a well-established HCC tumour-associated antigen,
      • Prieto J.
      • Melero I.
      • Sangro B.
      Immunological landscape and immunotherapy of hepatocellular carcinoma.
      were able to suppress tumour growth with a response that was directly proportional to the levels of glypican 3 expression.
      • Jiang Z.
      • Jiang X.
      • Chen S.
      • Lai Y.
      • Wei X.
      • Li B.
      • et al.
      Anti-GPC3-CAR T cells suppress the growth of tumor cells in patient-derived xenografts of hepatocellular carcinoma.
      In a more recent study, human HCCs were established in NSG (NOD-scid gamma) mice with human leukocyte antigen-matched human immune systems, which were affected by the tumours and showed an immune exhaustion phenotype.
      • Zhao Y.
      • Shuen T.W.H.
      • Toh T.B.
      • Chan X.Y.
      • Liu M.
      • Tan S.Y.
      • et al.
      Development of a new patient-derived xenograft humanised mouse model to study human-specific tumour microenvironment and immunotherapy.
      Two out of the 4 models were responsive to immunotherapies but only in the humanised mice, not in the NSG mice.
      • Zhao Y.
      • Shuen T.W.H.
      • Toh T.B.
      • Chan X.Y.
      • Liu M.
      • Tan S.Y.
      • et al.
      Development of a new patient-derived xenograft humanised mouse model to study human-specific tumour microenvironment and immunotherapy.
      Ideally, the humanised mice should be made with the immune system from which the PDX will be generated. Unfortunately, HSCs from cancer patients are not optimal at repopulating mice.
      • Choi Y.
      • Lee S.
      • Kim K.
      • Kim S.H.
      • Chung Y.J.
      • Lee C.
      Studying cancer immunotherapy using patient-derived xenografts (PDXs) in humanized mice.
      Nevertheless, this work represents an important step towards the development of more fully personalised and humanised mouse models for liver cancer research.
      • Zhao Y.
      • Shuen T.W.H.
      • Toh T.B.
      • Chan X.Y.
      • Liu M.
      • Tan S.Y.
      • et al.
      Development of a new patient-derived xenograft humanised mouse model to study human-specific tumour microenvironment and immunotherapy.
      The use of patient-derived models of liver cancer holds great promise for the development of personalised treatment approaches that will improve outcomes in patients.

      Future directions

      Patient-derived models of liver cancer show great promise for personalised medicine and the improvement of outcomes in HCC. The recent expansion of available 2D HCC cell lines will allow for a more thorough investigation of diverse tumour types and their drug responses. Additionally, building on what has been accomplished in 2D models, the creation of biobanks with PDOs established from a large number of patients with liver cancer could be a new application, of the rapidly developing 3D culture system technology, that better represents the high inter-patient heterogeneity seen in human HCC and the natural tumour growth environment.
      • van de Wetering M.
      • Francies H.E.
      • Francis J.M.
      • Bounova G.
      • Iorio F.
      • Pronk A.
      • et al.
      Prospective derivation of a living organoid biobank of colorectal cancer patients.
      • Sachs N.
      • de Ligt J.
      • Kopper O.
      • Gogola E.
      • Bounova G.
      • Weeber F.
      • et al.
      A living biobank of breast cancer organoids captures disease heterogeneity.
      • Vlachogiannis G.
      • Hedayat S.
      • Vatsiou A.
      • Jamin Y.
      • Fernandez-Mateos J.
      • Khan K.
      • et al.
      Patient-derived organoids model treatment response of metastatic gastrointestinal cancers.
      • Yan H.H.N.
      • Siu H.C.
      • Law S.
      • Ho S.L.
      • Yue S.S.K.
      • Tsui W.Y.
      • et al.
      A comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening.
      The newly established tumouroids could be genetically profiled and then used to test different treatments and identify genotype-specific strategies for personalised treatment of patients with liver cancer.
      • Tharehalli U.
      • Svinarenko M.
      • Lechel A.
      Remodelling and improvements in organoid technology to study liver carcinogenesis in a dish.
      Since patient-derived liver tumouroids can be established in a relatively fast manner compared to patient-derived xenografts, treatment selection could potentially be applied to the corresponding patients from whom the grafts were derived. Banked liver tumouroids could also be used to research novel therapies, interrogate underlying biological processes, and delineate mechanisms of resistance and susceptibility to different drug regimens.
      • Tharehalli U.
      • Svinarenko M.
      • Lechel A.
      Remodelling and improvements in organoid technology to study liver carcinogenesis in a dish.
      It is important to keep in mind that liver cancer, in particular HCC, tends to develop in the context of underlying liver damage. Therefore, strategies that mimic viral hepatitis, fatty liver disease, fibrosis, NAFLD, non-alcoholic steatohepatitis (NASH), and cholestasis will need to be incorporated into model systems.
      • Yuan L.
      • Liu X.
      • Zhang L.
      • Li X.
      • Zhang Y.
      • Wu K.
      • et al.
      A Chimeric humanized mouse model by engrafting the human induced pluripotent stem cell-derived hepatocyte-like cell for the chronic hepatitis B virus infection.
      • Bility M.T.
      • Cheng L.
      • Zhang Z.
      • Luan Y.
      • Li F.
      • Chi L.
      • et al.
      Hepatitis B virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and M2-like macrophages.
      • Benten D.
      • Kluwe J.
      • Wirth J.W.
      • Thiele N.D.
      • Follenzi A.
      • Bhargava K.K.
      • et al.
      A humanized mouse model of liver fibrosis following expansion of transplanted hepatic stellate cells.
      It is estimated that the incidence of HCC in NASH-diagnosed patients, with or without cirrhosis, ranges from 2.4% to 12.8%.
      • Anstee Q.M.
      • Reeves H.L.
      • Kotsiliti E.
      • Govaere O.
      • Heikenwalder M.
      From NASH to HCC: current concepts and future challenges.
      Beyond the possibility of modifying nutritional and lifestyle habits, there is no approved therapeutic approach to tackle the increasing incidence of this disease. Given its multifactorial aetiology and the complex inter-organ networks involved in its progression, NASH-induced HCC still represents a challenging pathological context to model in vitro or in vivo.
      NASH, and more generally NAFLD, encompass a wide range of conditions characterised by the common feature of steatosis; the alteration of cellular metabolism caused by excessive fatty acid uptake or systemic metabolic disorders leads to increased accumulation of lipids, inducing cellular stress and sensitising hepatocytes to cell death.
      • Anstee Q.M.
      • Reeves H.L.
      • Kotsiliti E.
      • Govaere O.
      • Heikenwalder M.
      From NASH to HCC: current concepts and future challenges.
      • Ringelhan M.
      • Pfister D.
      • O’Connor T.
      • Pikarsky E.
      • Heikenwalder M.
      The immunology of hepatocellular carcinoma.
      Recent evidence indicates that the evolution and burden of the disease and the progression to HCC seem to be critically influenced by a plethora of signals generated from various organs (e.g. adipose tissue, intestine) and by several cell populations, including the innate and adaptive immune systems.
      • Anstee Q.M.
      • Reeves H.L.
      • Kotsiliti E.
      • Govaere O.
      • Heikenwalder M.
      From NASH to HCC: current concepts and future challenges.
      • Ringelhan M.
      • Pfister D.
      • O’Connor T.
      • Pikarsky E.
      • Heikenwalder M.
      The immunology of hepatocellular carcinoma.
      In recent years, many efforts have been made by the scientific community to develop experimental models of NASH-induced HCC able to recapitulate as faithfully as possible the human clinical situation. For example, the choline-deficient high-fat diet (CD-HFD) and western diet are recognised as some of the most relevant nutritional models reproducing features typical of the human NASH condition (hepatocyte cell death, ballooning, Mallory body formation, myeloid and lymphoid inflammatory infiltrate, and fibrosis) in mice and resulting in a long-term tumour incidence comparable to that observed in humans.
      • Anstee Q.M.
      • Reeves H.L.
      • Kotsiliti E.
      • Govaere O.
      • Heikenwalder M.
      From NASH to HCC: current concepts and future challenges.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      It is now generally accepted that tumorigenesis observed in mice treated with CD-HFD or western diet feeding is strongly influenced by the immune system,
      • Ringelhan M.
      • Pfister D.
      • O’Connor T.
      • Pikarsky E.
      • Heikenwalder M.
      The immunology of hepatocellular carcinoma.
      and it was shown that the cooperative activity of natural killer T and CD8+ T cells is a central event in CD-HFD-induced liver damage and the NASH-to-HCC transition.
      • Wolf Monika J.
      • Adili A.
      • Piotrowitz K.
      • Abdullah Z.
      • Boege Y.
      • Stemmer K.
      • et al.
      Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes.
      It was also recently demonstrated in the CD-HFD nutritional model that the interaction between Kupffer cells and platelets plays a central role in the recruitment of lymphoid cells to the steatotic liver, and critically affects the progression to cancer.
      • Malehmir M.
      • Pfister D.
      • Gallage S.
      • Szydlowska M.
      • Inverso D.
      • Kotsiliti E.
      • et al.
      Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer.
      The importance of the immune system in the progression of these NASH models to HCC underscores the need for in vivo models that incorporate both steatohepatitis and a functional immune system. Humanised PDX models are ideal for accurate modelling of immune interactions, and development of a system that combines this ability with a background of steatosis would be ideal for studying a tumour microenvironment representative of liver cancer in the setting of NASH.
      Regarding strategies to model NASH in vitro, the effects of lipid stimulation at a cellular level have been widely investigated in classical 2D cell culture models but present the obvious limitation of being a simplified system lacking the cellular complexity of an in vivo model.
      • Müller F.A.
      • Sturla S.J.
      Human in vitro models of nonalcoholic fatty liver disease.
      To overcome this limitation, novel hepatic 3D cellular systems have been generated to allow for spatio-temporal exploration of the inter-cellular dynamics resulting from metabolic alterations.
      • Kozyra M.
      • Johansson I.
      • Nordling Å.
      • Ullah S.
      • Lauschke V.M.
      • Ingelman-Sundberg M.
      Human hepatic 3D spheroids as a model for steatosis and insulin resistance.
      The establishment of liver organoid cultures on specific matrigels and under specific cell culture conditions was shown to be a feasible system for use with different species-specific fresh liver biopsies.
      • Kruitwagen H.S.
      • Oosterhoff L.A.
      • Vernooij I.G.W.H.
      • Schrall I.M.
      • van Wolferen M.E.
      • Bannink F.
      • et al.
      Long-term adult feline liver organoid cultures for disease modeling of hepatic steatosis.
      Moreover, it turned out to be a versatile model of hepatic steatosis, displaying proper lipid accumulation as characterised by phenotypic and genotypic changes in response to fatty acid challenge. Furthermore, the generation of 3D spheroids composed of multicellular co-cultures of human non-parenchymal cells and hepatocytes enabled an in vitro analysis of the fibrotic process in response to lipid accumulation.
      • Pingitore P.
      • Sasidharan K.
      • Ekstrand M.
      • Prill S.
      • Lindén D.
      • Romeo S.
      Human multilineage 3D spheroids as a model of liver steatosis and fibrosis.
      More recently, Takebés group set up a promising model of liver organoids generated via differentiation of human iPSCs that represented the cellular heterogeneity typical of the in vivo-derived tissue, thus showing better potential to faithfully recapitulate the inflammatory response and fibrosis occurring upon fatty acid treatment.
      • Ouchi R.
      • Togo S.
      • Kimura M.
      • Shinozawa T.
      • Koido M.
      • Koike H.
      • et al.
      Modeling steatohepatitis in humans with pluripotent stem cell-derived organoids.
      Recreating an environment that mimics steatotic and cirrhotic livers (both in vitro and in vivo) in which patient-derived tumours can be studied would likely reveal important relationships inherent to the tumour microenvironment that could influence tumour progression, treatment response, and prognosis. Modification of organoid, tumouroid, and PDX models to incorporate features of diseased livers is one potential direction of development to enhance our patient-derived models so that they more realistically mimic the environment in which human liver cancer develops and interacts with the immune system and therapeutic agents.
      One of the main limitations of these novel technologies is that the efficiency of establishment is low. The successful growth of tumouroids or PDXs could also be biased by the genetic makeup of the tumours or other features in addition to tumour cell proliferation. The genetic profiling of the original tumours from both successful and unsuccessful tumouroids and PDXs will be critical to better understand this process. Furthermore, while tumouroids maintain 3D architecture, the tumour microenvironment is lost. One possible barrier could be that the tumouroid/PDX establishment protocol may select for specific cells, meaning that these models do not represent intratumour heterogeneity. A preliminary study that established 27 liver tumouroids and performed drug testing concluded that organoids do maintain tumour heterogeneity, at least at the drug response level.
      • Li L.
      • Knutsdottir H.
      • Hui K.
      • Weiss M.J.
      • He J.
      • Philosophe B.
      • et al.
      Human primary liver cancer organoids reveal intratumor and interpatient drug response heterogeneity.
      Nevertheless, additional studies will shed light on the suitability of liver tumouroids to capture intratumour heterogeneity and to predict patient responses to therapy.
      The ideal context in which to study HCC therapies would balance the degree of similarity to a physiologic human liver and the ability to rapidly screen for drug responsiveness in a time-sensitive and cost-effective manner. To this end, a combination of patient-derived 2D cell lines, organoids/tumouroids, and xenograft models will allow a multifaceted approach to effective and personalised treatment selection (Table 2). One could envision establishing a pipeline in which freshly resected liver tumours or needle biopsies are sequenced and expanded as organoids and PDXs in parallel (Fig. 1). Ideally, several regions of the tumour could be used, to better estimate intratumour heterogeneity and the risk of resistance. While the patient is being treated with the standard therapy, drug sensitivities and genomic profiling could be performed to delineate potential second-line therapies. The optimisation and use of patient-derived liver cancer organoids co-cultured with immune cells from the patient
      • Dijkstra K.K.
      • Cattaneo C.M.
      • Weeber F.
      • Chalabi M.
      • van de Haar J.
      • Fanchi L.F.
      • et al.
      Generation of tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids.
      and humanised PDX models will be needed for such a pipeline, especially considering the potential of immunotherapies for liver cancer treatment. It will be necessary to advance and optimise the current technology further to ensure a reasonable timeline and cost, and it is critical that researchers prove that it confers a significant benefit to patients, since it would involve performing a resection or needle biopsy that are not currently the standard of care in advanced disease. Potentially, with further development of methods for extracting circulating tumour cells, liquid biopsies could be used for those cases in which a resection or needle biopsy is not an option. However, it is unlikely that sufficient cells could be recovered for such an ambitious pipeline. Future studies will expand on the inherent promise of patient-derived cell lines, organoids, and xenografts for liver cancer research and treatment.
      Table 2Relative advantages and disadvantages related to patient-derived 2D cell lines, organoids, and xenografts.
      2D cell linesOrganoidsXenografts
      Establishment efficacy++
      High-throughput screening+++
      Cost efficiency+++
      Recapitulates tumoural heterogeneity++
      Preservation of tumor microenvironment+++
      Preservation of immune system interactions++
      Figure thumbnail gr1
      Fig. 1Potential pipeline involving organoid and humanised PDX establishment from liver tumours, genomic profiling, and drug testing to nominate personalised and biomarker-guided therapies. Immune cells would be retrieved from the blood of the patient. PDX, patient-derived xenografts.

       Abbreviations

      CAR, chimeric antigen receptor; CCA, cholangiocarcinoma; CD-HFD, choline-deficient high-fat diet; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9; FLC, fibrolamellar carcinoma; HB, hepatoblastoma; HCC, hepatocellular carcinoma; HSCs, haematopoietic stem cells; iPSCs, induced PSCs; LIMORE, Liver Cancer Model Repository; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NK, natural killer; NOD, non-obese diabetic; NSG, NOD-scid gamma; PDO, patient-derived organoids; PDX, patient-derived xenografts; PSCs, pluripotent stem cells; scid, severely compromised immunodeficient.

      Financial support

      M.H. was supported by an ERC Consolidator grant (HepatoMetaboPath), the SFBTR 209 and SFBTR179, the Helmholtz-Gemeinschaft , Zukunftsthema “Immunology and Inflammation” (ZT-0027), and the European Union’s Horizon 2020 research and innovation program under grant agreement No 667273. A.L was supported by Damon Runyon-Rachleff Innovation Award (DR52-18), R37 Merit Award (R37CA230636), DoD Career Development Award (CA150178), DoD Translational Team Science Award (CA150272P2) and The Tisch Cancer Institute (P30 CA196521).

      Conflict of interest

      The authors declare no conflict of interest.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      The authors contributed equally to this review.

      Supplementary data

      The following are the Supplementary data to this article:

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