Experimental Models for Preclinical Research in Hepatocellular Carcinoma

Pedro Molina-Sánchez; Amaia Lujambio

doi:10.1007/978-3-030-21540-8_16

NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Hoshida Y, editor. Hepatocellular Carcinoma: Translational Precision Medicine Approaches [Internet]. Cham (CH): Humana Press; 2019. doi: 10.1007/978-3-030-21540-8_16

Hepatocellular Carcinoma: Translational Precision Medicine Approaches [Internet].

Show details

Contents

< PrevNext >

Chapter 16Experimental Models for Preclinical Research in Hepatocellular Carcinoma

Pedro Molina-Sánchez and Amaia Lujambio.

Author Information and Affiliations

Published online: August 6, 2019.

Hepatocellular carcinoma (HCC), the most frequent form of liver cancer, is among the top fatal malignancies worldwide. The therapeutic options for patients with advanced HCC are limited and poorly efficient. Therefore, it is critical that we identify and develop novel curative strategies in order to address this major health issue. For this, it is essential to gain mechanistic understanding of HCC pathogenesis as clues to therapeutic intervention. Over the last decades, a wide range of HCC models have been developed, aiming to achieve the goal. In particular, animal models and in vitro systems that mimic the major characteristics of the human HCC have emerged to enable functional assessment of candidate therapeutic targets in liver cancer. However, each model recapitulates limited aspects of the whole pathogenesis of the human disease. Nevertheless, combined analysis of multiple different models can collectively provide clinically relevant insights into the complex disease mechanisms. Here we summarize the major approaches of experimental modeling in HCC and their strengths and limitations.

Keywords:

Hepatocellular carcinoma (HCC), Mouse models, Cell lines, Organoids, Genetically engineered mouse models (GEMMs), Chemically induced models, Transplantation models

Introduction

It is estimated that more than ¾ million people in the world are diagnosed with liver cancer every year [1], 85% of them presenting its most common form, hepatocellular carcinoma (HCC) [2]. HCC is described as a poor prognosis malignancy due to its difficult detection in early stages, when curative therapies are available, along with its strong metastatic capacity [3] and high frequency of recurrence [4]. Some of the major risk factors associated with HCC include viral hepatitis, alcohol abuse, obesity, and diabetes [5], which have all been on the raise in recent years. This increased incidence of risk factors has resulted in a dramatic rise in occurrence of HCC cases in the last years [6]. Unfortunately, unlike other tumor malignancies, HCC currently presents a limited number of available therapeutic approaches [2]. Therefore, it is essential to develop a comprehensive research plan in order to harness new diagnostic methods and efficient treatments to attack this disease. To this aim, numerous models have been established in the last decades, enabling scientists to study every biological aspect of human HCC (including malignant transformation, tumor progression and dissemination, and tumor microenvironment) and to evaluate tumor responses to novel treatments (Fig. 16.1). So far, due to their intrinsic properties, murine and human tumor-based models have been the most successful instruments for this purpose. Nevertheless, there is no model able to reproduce the entire nature and complexity of human disease, making model selection a critical step to successfully achieve the desired research objectives. In this chapter we compile some of the most widely used preclinical HCC models, outlining the characteristics that make them suitable to understand the different aspects of liver cancer.

Fig. 16.1

Experimental models of hepatocellular carcinoma

In Vitro Models

HCC Cell Lines

Human and murine cell lines are routinely used in biomedical research laboratories for a better understanding of cancer biology. Their faithfulness is a matter of discussion since it is accepted that cultured cells present important limitations when recapitulating the complexity of the original tumors. For example, cell lines are usually obtained from a limited tumor portion in a very specific time in tumor evolution, fail to reproduce the implications of growing in three dimensions (3D), and are isolated from its natural environment, which includes the host’s immune response [7]. However, cell lines present certain unrivaled advantages for cancer research as they represent a homogenous population (providing consistent data), are effortlessly maintained/expanded, and can be manipulated easily [7]. There are dozens of cell lines established from HCCs, both of human and animal origins, that are currently available for research purposes. This wide variety of HCC cell lines constitutes a very valuable tool for researchers, but a large cell line catalogue can also complicate the selection of the appropriate line or lines for each research purpose. Initiatives such as the Cancer Cell Line Encyclopedia (CCLE), Cellosaurus (ExPASy-SIB), and other platforms (e.g., NCI-60) have emerged with the objective of collecting as much information as possible (gene expression profiles, genomic and chromosomal alterations, mutational landscape, etc.) to provide complete cellular databases to guide investigators.

The most commonly employed cancer cell line in liver research is HepG2. Established from a Caucasian adolescent, this cell line has been traditionally considered as a “pure” HCC cell line since it is not infected with hepatitis virus and presents intact characteristics associated to human neoplastic lesions, such as increased α-fetoprotein (AFP), α2-macroglobulin, and transferrin expression [8]. Nevertheless, the use of HepG2 cells has become a matter of controversy since some studies suggest that HepG2 cell line could have been originated from a more epithelial hepatoblastoma-like tumor [9]. Another very frequently used HCC cell line is Huh7. This cell line was obtained from a well-differentiated HCC tumor from a middle-aged Japanese patient [10], and it has been described to be an appropriate model to study the implications of hepatitis C virus (HCV) infection, in particular related to cancer initiation [11–13]. It is worth noting that Huh7 cell line presents a point mutation at codon 220 of tumor suppressor TP53 which, contrary to most p53 mutations observed in HCC cell lines, results in increased levels of the protein [14]. This characteristic may be of interest when studying the functional consequences of different p53 mutations in liver carcinogenesis. Other cell lines such as HepaRG, BEL-7402, Hep3B, SKHep1, or SMMC-7721 have been traditionally used in HCC studies. However, it has been recently reported that some of them (including SMMC-7721, BEL-7402, or SKHep1) may be contaminated with cells of diverse origins, and it is recommended that these cell lines are not used for HCC studies [15].

Most human HCC cell lines can be obtained from different cell banks, such as the Japanese Collection of Research Bioresources (JCRB), the American Type Culture Collection (ATCC), or the Cell Bank of Chinese Academy of Science. Major applications of HCC cell lines include the study of cell proliferation control, immortality acquisition, and metastatic progression [16]. Furthermore, HCC cell lines are broadly used as tools for target discovery or in drug screens [17] and are essential in the development of in vivo xenograft models.

HCC Organoids

The idea to create 3D cell cultures and/or organoids was born many years ago in an effort to address some of the limitations of traditional cell cultures. But deficient technologies and lack of understanding of stem cell biology delayed their full development until recently. One of the most accepted descriptions for “organoid” was given by Fatehullah et al., being defined as “in vitro 3D cellular clusters derived exclusively from primary tissue, embryonic stem cells, or induced pluripotent stem cells, capable of self-renewal and self-organization, and exhibiting similar organ functionality as the tissue of origin” [18]. Due to their characteristics, organoids have been added to the repertoire of cancer research tools as they theoretically combine the benefits of both in vitro and in vivo models. For example, organoids can be propagated for a long time and amplified from a small tissue sample, can be adapted to high-throughput research approaches, and can keep cellular complexity and 3D structure. In the last few years, a wide variety of human and murine organoids have been established from different organs [19]. In the context of human liver, Tabeke et al. were pioneers by generating 3D aggregates of human pluripotent stem cell-derived hepatocytes in combination with endothelial and mesenchymal cells in a Matrigel matrix [20]. At the same time, Meritxell Huch and colleagues have optimized several protocols to grow adult human and mouse liver organoids [21–23]. These liver organoids exhibit equivalent genetic and histological characteristics to the tissue of origin and are amenable for genetic manipulation [21], which will enable performing functional experiments. Moreover, HCC patient-derived organoids have also been generated [24]. Exome sequencing analyses exhibit low mutation rates during HCC organoid expansion, and gene expression profiles show high correlation with the corresponding human HCCs [24]. In addition, it has been also demonstrated the ability of HCC organoids to grow in vivo when injected into mice, even displaying the ability to induce metastasis [24]. Although this technology is quite recent, the initial results suggest that HCC organoids could be very promising for liver cancer research. Thus, organoids could be used in drug screens and toxicology studies, enable precision medicine when directly derived from HCC patients, and be applied to transplantation strategies. Nonetheless, organoids still require several improvements as they are more expensive and time-consuming than regular cancer cell lines, are difficult to generate, and lack essential components of in vivo systems such as blood vessels or the immune compartment [18].

In Vivo Models

Transplantation-Based Models: Xenografts and Allografts

By definition, xenografting consists in the transplantation of a living entity (cell, tissue, organ, or system) from one species to another. In the HCC field, this method generally involves the transplantation of human HCC samples (tumor-derived cells or tumor tissue) into mice, either in the liver (orthotopic xenograft) or under the skin (ectopic xenograft) [25]. These transplantation-based models emerged to take into consideration the role of tissue microenvironment in cancer progression, an aspect that is absent in the majority of in vitro methods. A fundamental issue to be considered when establishing xenografts is that the recipient must accept the “foreign” tissue so that it can survive and grow in the host organism. This implies the use of immune-deficient mice. Some of the most common mouse strains used for xenografting are nu/nu nude mice (deficient of T cells), severe combined immune-deficient (SCID) mice (lacking T and B cells), athymic nude mice (presenting Foxn1 deletion and consequently abnormal thymus and defective T cells), nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice, and the recombination-activating gene 2 (Rag2) knockout mice (unable to produce mature B and T lymphocytes) [26–28]. Xenograft models are largely designed to test new drugs or combination therapies, which may be administrated through oral gavage, intraperitoneal (IP), intravenous (IV), or intratumoral injection, the latter mimicking the clinically used transcatheter arterial chemoembolization (TACE).

The extent and growth pattern of the xenografts are affected by different parameters such as the size of the transplanted tumor tissue or the nature and number of the cells injected, requiring from 1 week to even more than 5 months to develop [29]. The most common and easiest way to establish HCC xenografts is through the injection of human tumor cell lines subcutaneously, generally placed on the flanks or on the back of the mouse. This is a suited strategy when studying cellular response to drugs as it provides a rapid growth model that can be easily tracked. However, cell line-derived xenografts lack the typical tumor cell diversity or heterogeneity, which can potentially lead to inaccurate experimental conclusions. Some representative examples of this method have been reviewed elsewhere [30]. The subcutaneous transplantation of human tumors into mice (known as patient-derived xenografts or PDXs) can overcome some of these limitations and provides a more reliable model of HCC. However, the establishment of PDXs is challenging as the engraftment success strongly depends on every tumor model. Both ectopic approaches, however, present an unnatural environment for tumor growth. In the orthotopic xenograft models, in contrast, tumor cells are implanted directly in the liver, which provides a more physiological context. The orthotopic model is more technically challenging than the ectopic as it requires surgical intervention, but it also facilitates tumor cell dissemination, enabling the study of metastatic progression [31].

One of the major disadvantages of the xenograft models is the impossibility to faithfully study the role of the immune system in tumor development, which limits the understanding of the contribution of immune cells to HCC origin, progression, and drug response. Similarly, xenografts are also inadequate to test drugs that activate the immune system. This is a major issue since immunotherapy is emerging as a key therapy for HCC treatment [32, 33]. Allograft models, in which tumor cells from one species are transplanted into animals from the same species, are a widely used alternative as they preserve an intact immune system. Allografts can be combined with genome engineering tools to more precisely dissect HCC. While allografts allow studying the interactions between immune cells and HCC cells, there are species-specific differences that are important to keep in mind when reaching conclusions that may not completely apply to humans. Some relevant examples of liver cancer allografts and other transplantation-based models can be found elsewhere [25].

Chemically Induced Models

Undoubtedly, xenobiotic detoxification is one of the major functions of the liver. Humans are exposed during their lifetimes to a very broad spectrum of molecules in different doses and periods, many of them affecting liver homeostasis and inducing disease [34, 35]. The identification of hepatotoxic compounds and the full understanding of the underlying mechanisms of carcinogenesis is an ongoing public health goal. As a consequence, decades of liver investigation have already brought into light a considerable number of compounds with very defined roles in tumorigenesis and cancer progression [36]. Some of those compounds have been frequently tested in animals (mostly mice and rats) in order to reproduce human disease. These molecules are classified as genotoxics (inducing DNA damage) and promoters (accelerating tumor progression after malignant transformation). Chemically induced models are very valuable tools to understand human HCC as they reproduce the typical damage and healing episodes observed in the human setting. However, each hepatotoxin produces particular liver lesions according to its nature, its mechanisms of action, and other extrinsic factors such as administration route, animal strain, gender, age, dose, and treatment schedule. Some of the most relevant models of HCC based on the administration of chemical compounds are described below.

Diethylnitrosamine (DEN)

DEN or diethylnitrosamine (C₄H₁₀N₂O) is a member of the N-nitroso group of compounds and an extensively described carcinogenic molecule. The first time that DEN was described as a tumorigenic agent was precisely in a study to evaluate the oncogenic effect of this compound in the liver using experimental rats [37], although other members of this same family were already considered as potential carcinogens. Since then, DEN administration has been commonly used to induce tumorigenesis in rodents. There are two primary mechanisms underlying DEN tumorigenesis: its capacity to induce DNA adduct formation [38], which promotes genomic point mutations and consequently carcinogenesis (when affecting driver genes), and the stimulation of the cytochrome P450, increasing the production of reactive oxygen species (ROS) in the liver [39, 40]. The effect of DEN in mice is strongly affected by the animal strain (and therefore, the underlying genetic background). SWR, C57BL/6, or BALB/c mouse strains are considered more resistant to tumor formation by DEN than SM/J, FVB, CE/J, P/J, LP, or AKR/J strains, while CBA and C3H strains are highly sensitive to DEN-based liver carcinogenesis [41, 42]. The dose (ranging from 1 mg/kg to 100 mg/kg) and the number of administrations (single administration or multiple administrations over time), together with the gender, the weight, and the age of the animals, are important parameters that dramatically affect the final outcome of DEN treatment [41]. IP injection is the favorite route of administration since it enables a better control and accuracy of the dosage, although we can find studies with very diverse administration methods, such as oral gavage, drinking water, inhalation, and even intragastrical or intratracheal instillation. Regarding gender discrepancies, long-term administration of DEN induces HCC in 100% of male mice but only in approximately a third of females [43, 44], recapitulating the gender discrepancies observed in humans (men’s incidence is 2.5–3 times higher than women’s). DEN can therefore be used to better understand the role of gender in HCC development and suggests that the gender disparities observed in humans are not only attributed to disparate lifestyles but also to the opposite impact that estrogens and androgens have in hepatocarcinogenesis [43, 44]. In terms of genetic damage, recent studies based on whole exome sequencing analysis have provided a detailed mutational characterization of the DEN-induced murine tumors, showing that DEN treatment induces a mutational imprint affecting Hras, Braf, Egfr, and Apc but a higher burden of mutations compared to human HCCs [45]. A detailed review on DEN models in liver cancer is presented elsewhere [42].

DEN-induced liver tumor models present some significant limitations to be considered. DEN-based models are poorly reproducible, consequently requiring high numbers of animals [29]. In addition, metastases are not observed after DEN treatment, restricting its application to the study of primary tumors. Finally, it is worth noting that tumor formation can be relatively slow (taking up to 100 weeks) through most routes of administration and treatment regimens [29]. Regarding this last issue, there are a few chemical agents that do not induce malignant transformation of hepatocytes but accelerate tumorigenesis after DEN-mediated tumor initiation. In this respect, phenobarbital is a widely used partner of DEN. Phenobarbital is a barbiturate commonly used for treating epilepsy that shows an interesting HCC-promoting effect in rodents. The mechanisms by which phenobarbital enhances DEN tumorigenic activity are not totally understood, but they have been attributed to the induction of cytochrome P450 expression [46] and its role in promoting DNA hypermethylation of tumor suppressor genes (TSGs) [47]. The administration of phenobarbital not only results in more aggressive tumors in a significantly shorter time (from 12 to 40 weeks) but also stimulates the induction of metastasis enabling the study of this critical aspect of cancer progression.

Aflatoxin Exposure

Aflatoxins are mycotoxins produced by some members of the Aspergillus gender of fungi (mostly by Aspergillus flavus and Aspergillus parasiticus [48]). These fungi are commonly found as contaminants of diverse types of nuts and cereals (corn, wheat, rice, and other oil plants) when kept in a humid and warm environment and represent a major HCC risk factor in countries with non-strict food control regulations [49]. Aflatoxin B1 is the most carcinogenic molecule within this family. When captured by liver cells, cytochrome P450 transforms it in its exo-eposide form, which is primarily responsible for DNA adduct formation [50]. Aflatoxin B1 mostly induces guanine to thymine transversion in genomic DNA and consequently increases the mutational load in hepatocytes, eventually resulting in malignant transformation [51]. Aflatoxin products have been intimately related with mutations in tumor suppressor p53 (G:C to T:A transversion in 249ser codon) [52], which can be a potential mechanism of tumorigenesis. Nevertheless, single administration of 6 mg/kg of aflatoxin B1 into 1-week-old mice led to liver cancer development within 1 year, presenting features of human tumors but showing no compromising mutations in p53, suggesting that other mechanisms are involved, at least in mice [53]. HCC induction by aflatoxin treatment is very strain-dependent in mice, highlighting that different genetic variants affecting detoxification genes can significantly affect aflatoxin-mediated transformation and, consequently, liver cancer susceptibility [53]. Another interesting fact is that aflatoxin B and hepatitis B and C virus (HBV and HCV) infection could have a synergistic effect in HCC development [54, 55]. One potential explanation is that DNA mutations could appear as a result of ROS production in the chronic inflammatory context produced by the virus [55]. Accordingly, the model may be appropriate not only to identify the pathological mechanisms linked to aflatoxin exposure in humans but also to study the role of hepatitis infection on liver tumorigenesis.

Carbon Tetrachloride (CCl₄)

CCl₄ (carbon tetrachloride) is the most employed hepatotoxic molecule for modeling human liver disease due to its capacity to induce liver damage. In rodents, its administration through IP injection, inhaled, or in drinking water, leads to the production of trichloromethyl radicals after cytochrome P450-mediated transformation, which results in ROS production and inflammatory response induced by hepatic stellate cells (HSCs) and particularly by liver macrophages (Kupffer cells) [56]. CCl₄ does not induce direct mutagenesis in the hepatocytes but contributes to fibrosis after several rounds of injury and healing. Consequently, repeated CCl₄ treatment provokes massive hepatic fibrosis that eventually ends up in tumorigenesis. While this model is very interesting since it recapitulates the common steps that lead to HCC in humans, it can be rather slow, taking up to several months for tumor development depending on the mouse strain [57]. Combined treatment of CCl₄ with other hepatotoxic molecules can however solve this problem. This is the case of the two-staged DEN-CCl₄ model, based on a single IP injection of DEN (1 mg/kg) at 2 weeks of age and followed by repeated doses of CCl₄ (0.2 ml/kg, IP) [58]. In this model, the initial DEN administration produces genotoxic effects that are enhanced by CCl₄-induced fibrosis, increasing the incidence of tumor development in mice [58]. The molecular and genetic alterations detected when following this protocol are similar to those observed in the human disease, which allows the study of the genetic alterations associated with severe liver fibrosis.

Thioacetamide (TAA)

Thioacetamide (TAA) was identified as a hepatotoxin in the mid-twentieth century, shortly after its introduction as a fungicide [59]. Similar to CCl₄, this organosulfur compound has been used routinely to induce fibrosis and cirrhosis in rodents (mostly in rats) due to its capacity to produce liver injury. TAA, while is not a direct genotoxic, can be administered on its own or in combination with other molecules (such as DEN) to induce HCC in mice. Despite being used for a long time, the mechanisms by which TAA leads to tumor formation are still unclear. One possibility is that TAA is transformed into its oxidized forms (TAA-S-oxide and TAA-S,S-oxide) by the FAD-containing and the cytochrome P450-depending monooxygenases, since it has not been described that TAA by itself produces toxic effects in hepatocytes. These secondary forms may be responsible for glutathione depletion and oxidative stress in the cells, causing liver damage. It has also been attributed to TAA the capacity to join covalently to essential cellular components such as lipids and proteins, impairing cellular homeostasis [60]. As in many chemically induced models, the chronic administration of this molecule in rodents causes repetitive cycles of injury and healing in the liver, eventually leading to malignant transformation. TAA has been administered to different rodent strains through very diverse routes, and in different doses, which determines liver injury grade. In most of the cases though, liver histology reported common human-like fibrosis appearance [61]. The most often administration routes selected are IP injection (in repeated administrations) or through drinking water (at a concentration of 0.02–0.05%), which can promote HCC initiation after at least 20 weeks of exposure [62]. Nevertheless, despite constant TAA exposure frequently resulting in malignant transformation in the liver, this model is barely used to study HCC nowadays.

Choline-Deficient Diet (CDD)

Choline is an essential nutrient metabolized by the liver. Its function in cell biology is heterogeneous since it participates in cell membrane signaling pathways as well as in lipid transportation or in the synthesis of neurotransmitters [63]. Low-choline diets have been shown to induce liver damage, starting with fat accumulation and ending with cirrhosis and liver cancer in the more severe cases [64]. The molecular mechanism for choline-induced liver injury is not totally understood, but it is possibly related to defects in lipoprotein production (mostly impaired hepatic very-low-density lipoprotein secretion), anomalous phospholipid metabolism, and/or mitochondrial dysfunction [64]. Choline-deficient diets (CDDs) have been traditionally used as models of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NAFLD and NASH, respectively). However, if CDD is kept for long periods of time, oxidative stress, genomic instability, and mutagenesis become frequent phenomena, leading to HCC development. Nevertheless, this process is long and CDD can take up to 1 year to induce HCC in rodents [65]. Feeding with CDD and similar diets (CDD + methionine-free diet, CDD + L-amino acid-defined + high-fat diet [66]) has also been done in combination with other hepatotoxic stimuli (such as CCl₄ or DEN) in order to accelerate tumor formation [67, 68]. Interestingly, very similar tumor gene expression profiles have been observed in high-fat diet + CDD mouse models and better prognosis human HCCs [69], suggesting that this model could mimic less aggressive HCCs. Highly heterogeneous tumor susceptibility is perhaps the greatest limitation of this model.

Peroxisome Proliferators

Ciprofibrate, fenofibrate, methyl clofenapate, and clofibrate are examples of peroxisome proliferators that can promote liver tumorigenesis in rodents. These molecules are ligands of the peroxisome proliferator-activated receptor family (PPAR), which are nuclear receptors that control lipid metabolism in the cell. Their binding to the corresponding receptor stimulates diverse molecular pathways related to oncogenesis, such as cell proliferation (p53, p27, p18, or p21), apoptosis (caspases), metastasis (metalloproteinases and cadherins), and ROS production [70, 71]. The tumors generated after oral administration of these molecules show well-defined HCC histology, with trabecular pattern. However, it seems that the genetic makeup of these tumors is very different to human neoplasms, which implies clear limitations when results are translated into patients. More information about these models can be found elsewhere [29].

Genetically Engineered Mouse Models (GEMMs)

The previously described models are based on the accumulation of genetic alterations through the action of different chemical compounds, which leads to a complex and heterogeneous genetic landscape. Genome editing technologies, however, enable to target specific genes in different model systems, such as mice, thus providing the tools to systematically study the role of different genes and molecular pathways in cancer, including HCC. As a general rule, GEMMs must fulfill several features to be considered good models for HCC research. Essentially GEMMs should recapitulate as many human features as possible. For that, it is optimal to generate models that recapitulate the most frequent genetic alterations occurring in human disease, something that it is not always possible due to the differences between the human and mouse genomes. It is also preferred to restrict these mutations to a reduced number of hepatocytes, similar to what happens when liver cancer develops in humans. Finally, it would also be ideal to introduce the same mutational load as in human tumors; however, this is technically more challenging since most models only reproduce one or two mutations rather than tens of mutations.

GEMMs can model HCC in many different ways. The most traditional and extended methods involve the in vivo inactivation of TSGs and/or the activation of oncogenes. This can be achieved through diverse genetic engineering strategies. The generation of transgenic mice is the most straightforward option. The transgenic approach is based on the modification of the embryonic genome by transduction of recombinant elements, allowing the expression of oncogenes or dominant-negative TSGs [72]. These inserted genetic elements are in general constitutively and ubiquitously expressed but can also be designed to limit its expression to specific cell types. Conditional expression of transgenes can be achieved through the utilization of tissue-specific promoters, such as the albumin promoter in the context of liver cancer. Transgenic elements can induce non-desirable effects when expressed during embryogenesis or mouse development. To manage the timing of transgene expression and avoid these negative effects, inducible systems based on tetracycline (tet) administration can be used. The tet-inducible system allows the activation or repression of exogenous genes when they are under the tet operon regulating elements (Tet-on and Tet-off systems) [73]. Transgenic models, however, show some limitations. For example, transgene insertion occurs randomly, which could affect the integrity of the landing genes. In addition, the number of copies integrated can lead to an unwanted transgene expression pattern, deeply affecting the final phenotype.

Another interesting strategy is the development of endogenous GEMMs consisting in the targeted loss or gain of function of cancer-related genes. The targeted alteration of selected genes can be achieved by manipulating one or both alleles, in a constitutive or tissue-specific manner, through the employment of Cre-loxP and Cre-loxP-stop recombination mechanisms [74]. Genetic modification can also be induced in adult mouse livers using specific gene transfer procedures, including viral infection or hydrodynamic tail vein delivery. Either way, these models represent invaluable tools for studying human HCC, despite the intrinsic insurmountable differences existing between human and mouse biology. The following is a description of some frequent models recapitulating essential aspects of HCC biology.

Mouse Models of Human Hepatitis

Hepatitis caused by viral infection is the most important risk factor for subsequent HCC development [75]. The host range of hepatitis viruses is mostly limited to humans, which restricts the use of mice as model organisms. As an alternative, GEMMs harboring different components of HBV and HCV have been developed in order to study the effect of these viruses on HCC.

Hepatitis B Virus (HBV) Model

HBV is a DNA virus mainly present in tropical Africa, southeast of Asia, and some areas of China [76]. It is well known that chronically infected HBV patients are more prone to develop HCC due to several factors. First, viral infection can promote hepatocyte dysfunction and the activation of an inflammatory response that causes liver damage, which in turn can stimulate tumor transformation. In addition to this, some of the HBV gene products can be oncogenic per se. But most importantly, HBV DNA gets frequently integrated into the host’s genome. HBV integration promotes the rearrangement of adjacent genomic areas, which can affect essential cellular regulatory sequences or key cancer-related genes [77]. Transgenic mice expressing different components of the HBV (surface protein, HBx genes, or even HBV genome) have been established to better understand HBV’s underlying biology.

Transgenic mice expressing the surface protein of this virus were the first to be developed and studied. These models have demonstrated that the expression of just this protein is sufficient to induce tumorigenesis in the mouse livers, mimicking some of the human features of the disease and reflecting the different incidence observed in men and women [78]. Expression of mutations in the surface antigens can further increase tumor transformation through mechanisms involving ER stress [79]. Similarly, HBx protein, with effects on almost all basic cellular processes in hepatocytes such as cell division, activation of diverse signaling pathways, mitochondrial function, gene expression, or DNA stabilization, among others, can induce tumoral transformation both in vitro [80] and in vivo [81]. As an example, transgenic mice expressing HBx undergo spontaneous tumor formation 1–1,5 years after birth, presenting human histopathological features (such as trabecular structure, aberrant nuclei, and cirrhotic appearance) and preserving sex incidence discrepancies. Interestingly, no malignant transformation has been observed in models harboring full HBV genome, the precore, or core proteins, highlighting the need of further research to fully understand the role of HBV in HCC, at least in the murine context.

Hepatitis C Virus (HCV) Model

Western countries still present considerable HCV infection rates, being a major risk factor for HCC in these regions. The processes underlying malignant transformation caused by HCV are still unknown, but contrary to HBV, HCV (a positive-strand RNA virus) shows no integration into the human genome. This suggests indirect carcinogenic mechanisms, probably involving the succession of liver injury and healing events after infection together with a persistent inflammatory response, thus resulting in hepatocellular mutagenesis and cancer development over the years [82]. While the accumulation of mutations as a result of liver damage and inflammation is the most accepted explanation for HCV-induced HCC, it is important to note that HCV proteins also seem to have oncogenic effects. In this regard, it has been reported that HCC occurs after 2 years in at least 15% of mice when viral proteins are expressed [83], but this effect is influenced by the mouse strain. The hepatic expression of core genes, alone [84] or in combination with E1 and E2 structural proteins [85], can also lead to HCC formation although the specific role of the HCV core in liver carcinogenesis is a matter of discussion. This malignant transformation, however, does not occur when envelope (env) or nonstructural genes are expressed [86]. Other models in the context of HCV are the transgenic full-length HCV polyprotein (FL-N) and the HCV structural protein (S-N) mice, which also develop HCC [83] in a steatotic and non-inflammatory background [83].

Mouse Models of Cancer-Related Genes

HCCs arise as a result of the accumulation of mutations in hepatocytes. Unlike other tumor types, the number of genetic alterations and the nature of the pathways regulated by the genes involved are highly diverse, making HCC an extremely heterogeneous disease that is challenging to model. In this respect, whole exome sequencing studies in HCC have shown the presence of at least 40 somatic mutations in coding regions per tumor, affecting both driver and passenger genes [87]. Among the most frequently mutated genes in HCC, we can find some very-well-known players in human cancer such as TERT (whose mutation in the promoter is estimated to affect ≈50% of the patients), MYC (amplified in ≈20% of the patients), TP53 (mutated in ≈30% of the patients), or CTNNB1 (mutated in ≈30% of the patients) [87, 88]. However, there is also a diverse range of mutated genes related to different biological processes such as cell proliferation (CDKN2A) or chromatin remodeling (ARID1A, ARID2, or members of the KMT2 family) that, regardless of their low frequency of mutation in HCC (less than 10%), can still have a significant role in liver tumorigenesis and contribute to the inter-patient heterogeneity [89]. GEMMs can be used for the systematic identification of novel cancer driver genes and the characterization of the pathways involved in HCC malignant transformation and tumor progression. Some of the most interesting and widely used genetic mouse models of HCC are summarized below.

c-MYC

c-MYC (also known as MYC), a member of the MYC family of transcription factors (which includes c-, l-, and n-MYC), is one of the most frequently deregulated genes in human cancers [90]. It is therefore not surprising that high expression levels of MYC are a very common feature of human HCC, and MYC amplification can potentially be indicative of disease progression [91]. In combination with MAX protein and through the recruitment of histone acetyltransferases, MYC controls the expression of a vast number of genes. When overexpressed (e.g., as a result of gene amplification), MYC leads to the deregulation of closely related pathways (including apoptosis, cell growth, and cell differentiation), thereby resulting in carcinogenesis [92]. In fact, many murine models support this observation. Liver-specific expression of Myc (driven by albumin promoter) can lead to tumor formation in C57BL/6 J mice after 1,5–2 years [93]. High proliferation rates and p53 dysfunction were observed in this model and contributed to a high mutational load and cancer susceptibility. Similarly, conditional expression of Myc, controlled by the liver activator protein (LAP), also promotes tumor formation in mice and demonstrated oncogene addiction to Myc [94]. Similar approaches have also been tested in more cancer-prone settings, with comparable results. Some notable examples are liver-specific Myc overexpression models in combination with E2F1 or TGF-alpha transgenic expression, which exhibited accelerated HCC formation after 8–9 months [93, 95]. The histological analysis of the transgenic Myc-driven tumors shows characteristics that can be observed in human samples, including trabecular structure, well- and poorly differentiated regions, and morphological and cellular diversity.

β-Catenin (CTNNB1 Gene)

β-Catenin is a member of the cadherin protein complex and is a key element of the intracellular signaling transduction of the Wnt signaling pathway. Therefore, mutations changing β-catenin functionality promote pro-malignant phenomena such as cell proliferation, apoptosis, cell adhesion imbalance, and altered cell motility [96, 97]. Like MYC, β-catenin is frequently deregulated in an important number of cancer types [90], including HCC [87, 88]. CTNNB1 gain of function mutations have been described in approximately a quarter of HCC patients [87, 88] and are considered an early event in liver tumorigenesis [98], being traditionally correlated with accelerated tumor progression [99], metastatic transformation [100], and poor prognosis [99]. In this respect, mutations in Wnt/β-catenin pathway can be used for patient stratification, as they correlated with specific gene expression profiles and tumor characteristics [101]. Genetic mouse models have shown that activation of β-catenin is not sufficient to induce HCC, suggesting that additional mutations in the genome are needed for malignant transformation. However, β-catenin activation induces aberrant growth in the liver and hepatomegaly as seen after liver-specific induction of a β-catenin activated form [102]. As an alternative, combination of overexpression of stable forms of β-catenin and other carcinogenic stimuli can induce HCC formation. For example, expression of activated β-catenin together with mutant H-ras overexpression leads to tumor formation in 6 months [103], while activated β-catenin upregulation challenged with DEN administration also induces tumors after 6 months [104].

Cell Cycle Control Genes

Uncontrolled cell growth is a hallmark of cancer [105], and accordingly, alterations in genes regulating cell division are frequent in the vast majority of malignancies, including HCC [87, 88]. To specifically study the role of cell cycle modulators in HCC, several interesting models have been developed. One major example is the tumor suppressor p53, which plays essential roles in DNA repair, cell growth arrest, and apoptosis induction [106]. Mutations and/or deletions in p53 have been observed in around 30% of all human HCC tumors, a percentage that is higher in patients exposed to aflatoxin B or HCV infection [87, 88, 107]. p53’s essential role in liver carcinogenesis is beyond doubt given the high frequency of mutation rates also observed in chemically induced HCC mouse models [108]. However, gene mutations in only p53 are not sufficient to induce liver cancer as it has been seen in different models, but confers aggressiveness features. For example, p53 loss of function in combination with overexpression of the oncogenic polyoma virus middle T antigen (PyMT) confers metastatic capacity to the liver tumors [109]. Similarly, mice expressing just a copy of wild-type p53 undergo tumor formation only after liver damage concomitant with telomerase deregulation [110]. Another example of murine HCC models caused by p53 loss of function includes the conditional p53 and Ink4a/Arf mutant mice injected intrahepatically with polyoma virus middle T antigen (PyMT) [111].

Another key example of liver tumorigenesis induced by deregulation of cell proliferation involves the expression of SV40 virus. The large TAg (T antigen) of SV40 is a well-known oncoprotein which has shown a transforming effect in different contexts by repressing tumor suppressors p53 and retinoblastoma (Rb) and promoting uncontrolled cell proliferation of hepatocytes. Liver-specific induction of SV40-TAg leads to tumor formation in mice after approximately 5 months [112]. Finally, hepatocyte-specific deletion of Rb family members, Rb, p107, and p130, leads to human-like liver tumors after 3–4 months [113] and support the idea of Rb pathway activation as a potential therapeutic option for HCC.

PTEN

The phosphatase and tensin homolog (PTEN) gene is broadly considered as a tumor suppressor [114]. PTEN protein is a phosphatase that exerts multiple functions. On one hand, PTEN inhibits phosphatidylinositol (3,4,5)-trisphosphate – PIP₃ (its major substrate) – which is an activator of the Akt pathway that promotes apoptosis inhibition and cell proliferation [115]. In addition, PTEN loss leads to liver fat accumulation, which is associated with HCC development [116]. On the other hand, PTEN is also involved in chromosomal stability, DNA repair, and cell invasiveness [117, 118]. Despite PTEN mutations not being a common event in HCC cells [87, 88], low levels of PTEN protein are seen in 40–50% of HCC patients and correlate with nonalcoholic fatty liver, increased tumor grade, and advanced tumor stage [119]. In mice, total loss of PTEN function leads to fetal lethality during embryogenesis [120], and while the presence of one functional copy makes them viable, heterozygous mice develop tumors in different organs during their lifetime [121, 122]. Liver-specific PTEN loss, by using albumin-driven Cre recombinase and Pten^loxp/loxp mice, enables the induction of hepatomegaly and changes in fat metabolism in the liver, resulting in steatohepatitis [116]. This model not only reproduces human NAFLD features but also induces the formation of HCC-like tumors in ≈1,5 years, reproducing the gender bias in incidence seen in patients [116].

Telomerase

Telomeres are highly repetitive regions of noncoding DNA located at the end of the chromosomes, being responsible for chromosome stability and preventing loss of genetic information in every cell division [123]. Telomere integrity is maintained by telomerase, TERT, a reverse transcriptase that is activated in very limited number of cell types (mostly in the germline in humans) [124], and TERC, the RNA template for telomere synthesis [123]. As telomeres shorten, the risk of losing important genetic information during cell division increases, inducing senescence or apoptosis in the cell. On the other hand, TERT activation can lead to cell immortalization, which is a key step in malignant transformation. This indicates that TERT can have different roles in tumor initiation and progression.

In the context of liver cancer TERT promoter mutation is the most frequent genetic alteration, affecting around 60% of the patients [87, 88, 98]. In agreement with this, most HCC patients show augmented telomerase activity increased [125, 126]. The role of telomerase dysfunction in liver tumorigenesis is supported by different animal models. One of the most popular is the mTERC-null mice (lacking the telomerase RNA component) [127]. This mouse model, in different oncogenic scenarios, has shown a reduced incidence and growth of HCC in correlation with decreased DNA damage and apoptosis [128, 129], but also associated an augmented number of early malignant lesions in the liver [129], thus confirming the antagonistic effect of telomerase in tumor initiation and progression.

Mouse Models of Inflammation

The importance of inflammation in tumorigenesis is beyond doubt nowadays since it participates in almost every step of cancer development [130]. HCC is the archetypical example of inflammation-driven cancer. Beyond some of the already mentioned viral hepatitis and chemical models (in which there is a significant inflammatory component during tumor formation), there are several GEMMs specifically designed to study the influence of inflammation in HCC. This is the case of those targeting members of the NF-κB pathway. Mice harboring liver-specific deletion of the inhibitor of the nuclear factor Kβ kinase (IKKβ) (in hepatocytes and Kupffer cells), an activator of NF-κB, have shown decrease tumor formation after DEN treatment, demonstrating a promoting role for NF-κB in HCC development [131]. However, when IKKβ loss is restricted to hepatocytes, carcinogenesis is augmented in this same model [131]. Similarly, IKKγ loss in hepatocytes induces tumor formation spontaneously [132], suggesting a dual role for NF-κB in tumorigenesis depending on cellular context. These are just examples of the complex role of the inflammatory pathways in cancer [133]. Additional GEMMs in the context of inflammation-associated liver cancer are the Mdr2 knockout mouse [134], the liver-specific TGF-β transgenic mouse [135], and the IL-6 knockout mouse, [44] among others [136].

Non-germline GEMMs

The previously mentioned GEMMs have been extremely helpful in the last years to unveil key concepts of liver cancer biology. However, their generation is very expensive and time-consuming (several months may be necessary from the construction of the targeting vectors to the generation of the first heterozygous mouse) and presents important limitations (some genes are required in embryogenesis). Liver-specific gene delivery methods have been developed in recent years to overcome these disadvantages. Adeno-associated virus (AAV) vectors with liver-tropic capsid are interesting tools, enabling exogenous gene expression in mice [137, 138], and can be used to overexpress oncogenes or other cancer-related genes. Yet, in spite of being a very promising tool, as they are able to induce efficient and time-lasting transduction, its use is limited by several reasons. On one hand, AAV particles usually remain episomal, which leads to gene expression loss in dividing cells. In addition, AAVs present restricted DNA packaging capacity, which restricts the size or the number of genes to deliver. A few years ago, a novel genetic therapy tool called “hydrodynamic gene delivery” was developed as an attractive alternative. This method [139, 140] allows the transduction of genetic elements directly into the murine hepatocytes in vivo by simply injecting naked DNA. In more detail, the procedure involves the injection of exogenous DNA plasmids, resuspended in a very high volume of saline solution, through the tail vein of the mouse. The administration of a massive bolus (corresponding to 1/10 of the mouse body weight) is executed without interruption and rapidly (in no more than 10 seconds), inducing heart congestion and pushing the solution into the liver through retrograde flow. As a consequence, the capillary vessels of the liver undergo high-pressure forces, enabling the permeabilization of the endothelial cells and the transfection of the surrounding hepatocytes with the injected DNA [141]. This method is highly liver-specific reaching transfection levels of up to 40% of the total hepatocytes, barely affecting other organs or cell types [139].

Hydrodynamic gene delivery presents considerable advantages. First, the time and cost of liver-specific GEMM generation is drastically reduced since it only requires the cloning of specific DNA vectors. Related to this, since DNA vector cloning is relatively easy, hydrodynamic gene delivery enables the study of virtually any gene. Similar to conventional GEMMs, the most extended hydrodynamic gene delivery strategies involve the overexpression of oncogenes and/or loss of function of TSGs. For oncogene overexpression, it is important to notice that time-lasting overexpression is required for tumor formation and it can only be achieved through transgene integration into the genome of the hepatocytes. Otherwise, gene overexpression would only be transient, lasting no more than a few days before plasmid degradation. The use of DNA plasmids expressing Sleeping Beauty transposon systems is a successful way to ensure plasmid integration [142]. To induce loss of function of TSGs, RNA interference [143] or the recent CRISPR technology [144] can be used. Many HCC models have already been generated using this approach, introducing alterations in p53, β-catenin, MET, or AKT as representative examples [145]. A detailed list can be found elsewhere [145]. Lastly, it is worth noting that, similarly to many GEMMs, most HCC models generated by hydrodynamic gene delivery have been produced in a non-inflammatory background, which is a limiting condition when it comes to closely reproducing human HCC. This suggests the possibility to combine these models with hepatotoxic agent administration in order to better reproduce the conditions that drive human carcinogenesis.

Conclusions and Future Directions

Preclinical models of HCC enable a better understanding of major key processes in human liver carcinogenesis. However, it is also evident that there is no single model that can truly encompass all the genetic and cellular aspects of the human disease. It is therefore essential to deeply understand all currently available models in order to select the most appropriate tools depending on our research interest. Broadly speaking, chemically induced models are suitable to reproduce the liver damage that triggers tumorigenesis in humans. On the other hand, GEMMs allow a more systematic interrogation of the role of specific genes and pathways in liver tumor formation. Transplantation-based models are broadly used tools to study metastasis and test new drug treatments. Finally, in vitro techniques are appropriate to understand the tumor-associated molecular mechanisms and to perform high-throughput experiments (for drug testing or genetic screens). In general, the use of multiple models to address one biological question is optimal as this strategy can overcome the limitations of each individual model. In addition, new challenges in the HCC field, including the study of cancer immunotherapies and precision medicine, warrant the optimization of some of these preclinical models. Our efforts should now focus on improving the existing models to address these new challenges.

References

1.: Dhanasekaran R, Limaye A, Cabrera R. Hepatocellular carcinoma: current trends in worldwide epidemiology, risk factors, diagnosis, and therapeutics. Hepat Med. 2012;4:19–37. https://doi.org/10.2147/HMER.S16316. [PMC free article: PMC3846594] [PubMed: 24367230] [CrossRef]
2.: Llovet JM, Zucman-Rossi J, Pikarsky E, Sangro B, Schwartz M, Sherman M, et al. Hepatocellular carcinoma. Nat Rev Dis Primers. 2016;2:16018. https://doi.org/10.1038/nrdp.2016.18. [PubMed: 27158749] [CrossRef]
3.: Tang ZY, Ye SL, Liu YK, Qin LX, Sun HC, Ye QH, et al. A decade’s studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol. 2004;130(4):187–96. https://doi.org/10.1007/s00432-003-0511-1. [PubMed: 14685850] [CrossRef]
4.: Portolani N, Coniglio A, Ghidoni S, Giovanelli M, Benetti A, Tiberio GA, et al. Early and late recurrence after liver resection for hepatocellular carcinoma: prognostic and therapeutic implications. Ann Surg. 2006;243(2):229–35. https://doi.org/10.1097/01.sla.0000197706.21803.a1. [PMC free article: PMC1448919] [PubMed: 16432356] [CrossRef]
5.: Dimitroulis D, Damaskos C, Valsami S, Davakis S, Garmpis N, Spartalis E, et al. From diagnosis to treatment of hepatocellular carcinoma: an epidemic problem for both developed and developing world. World J Gastroenterol. 2017;23(29):5282–94. https://doi.org/10.3748/wjg.v23.i29.5282. [PMC free article: PMC5550777] [PubMed: 28839428] [CrossRef]
6.: Mittal S, El-Serag HB. Epidemiology of hepatocellular carcinoma: consider the population. J Clin Gastroenterol. 2013;47(Suppl):S2–6. https://doi.org/10.1097/MCG.0b013e3182872f29. [PMC free article: PMC3683119] [PubMed: 23632345] [CrossRef]
7.: Gillet JP, Varma S, Gottesman MM. The clinical relevance of cancer cell lines. J Natl Cancer Inst. 2013;105(7):452–8. https://doi.org/10.1093/jnci/djt007. [PMC free article: PMC3691946] [PubMed: 23434901] [CrossRef]
8.: Collection AATC. Hep G2 [HEPG2] (ATCC® HB-8065™). ATCC. 2016. https://www.atcc.org/products/all/HB-8065.aspx#characteristics. 2018.
9.: Lopez-Terrada D, Cheung SW, Finegold MJ, Knowles BB. Hep G2 is a hepatoblastoma-derived cell line. Hum Pathol. 2009;40(10):1512–5. https://doi.org/10.1016/j.humpath.2009.07.003. [PubMed: 19751877] [CrossRef]
10.: Bank JC. JCRB0403 – Huh-7. National Institutes of Biomedical Innovation, Health and Nutrition – Japan. 2015. http://cellbank.nibiohn.go.jp/~cellbank/en/search_res_det.cgi?ID=385.2018.
11.: Sainz B Jr, TenCate V, Uprichard SL. Three-dimensional Huh7 cell culture system for the study of hepatitis C virus infection. Virol J. 2009;6:103. https://doi.org/10.1186/1743-422X-6-103. [PMC free article: PMC2719612] [PubMed: 19604376] [CrossRef]
12.: Fang C, Yi Z, Liu F, Lan S, Wang J, Lu H, et al. Proteome analysis of human liver carcinoma Huh7 cells harboring hepatitis C virus subgenomic replicon. Proteomics. 2006;6(2):519–27. https://doi.org/10.1002/pmic.200500233. [PubMed: 16317778] [CrossRef]
13.: Ali N, Allam H, May R, Sureban SM, Bronze MS, Bader T, et al. Hepatitis C virus-induced cancer stem cell-like signatures in cell culture and murine tumor xenografts. J Virol. 2011;85(23):12292–303. https://doi.org/10.1128/JVI.05920-11. [PMC free article: PMC3209402] [PubMed: 21937640] [CrossRef]
14.: Bressac B, Galvin KM, Liang TJ, Isselbacher KJ, Wands JR, Ozturk M. Abnormal structure and expression of p53 gene in human hepatocellular carcinoma. Proc Natl Acad Sci U S A. 1990;87(5):1973–7. [PMC free article: PMC53607] [PubMed: 2155427] [CrossRef]
15.: Rebouissou S, Zucman-Rossi J, Moreau R, Qiu Z, Hui L. Note of caution: contaminations of hepatocellular cell lines. J Hepatol. 2017;67(5):896–7. https://doi.org/10.1016/j.jhep.2017.08.002. [PubMed: 28807831] [CrossRef]
16.: Ramboer E, Vanhaecke T, Rogiers V, Vinken M. Immortalized human hepatic cell lines for in vitro testing and research purposes. Methods Mol Biol. 2015;1250:53–76. https://doi.org/10.1007/978-1-4939-2074-7_4. [PMC free article: PMC4579543] [PubMed: 26272134] [CrossRef]
17.: Arellanes-Robledo J, Hernández C, Camacho J, Pérez-Carreón JI, editors. In vitro models of HCC, Chapter 42. In: Liver pathophysiology: Academic Press; 2017. https://www.elsevier.com/books/liver-pathophysiology/muriel/978-0-12-804274-8.
18.: Fatehullah A, Tan SH, Barker N. Organoids as an in vitro model of human development and disease. Nat Cell Biol. 2016;18(3):246–54. https://doi.org/10.1038/ncb3312. [PubMed: 26911908] [CrossRef]
19.: Clevers H. Modeling development and disease with organoids. Cell. 2016;165(7):1586–97. https://doi.org/10.1016/j.cell.2016.05.082. [PubMed: 27315476] [CrossRef]
20.: 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. Nature. 2013;499(7459):481–4. https://doi.org/10.1038/nature12271. [PubMed: 23823721] [CrossRef]
21.: Huch M, Gehart H, van Boxtel R, Hamer K, Blokzijl F, Verstegen MM, et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell. 2015;160(1–2):299–312. https://doi.org/10.1016/j.cell.2014.11.050. [PMC free article: PMC4313365] [PubMed: 25533785] [CrossRef]
22.: Broutier L, Andersson-Rolf A, Hindley CJ, Boj SF, Clevers H, Koo BK, et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc. 2016;11(9):1724–43. https://doi.org/10.1038/nprot.2016.097. [PubMed: 27560176] [CrossRef]
23.: Huch M, Dorrell C, Boj SF, van Es JH, Li VS, van de Wetering M, et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature. 2013;494(7436):247–50. https://doi.org/10.1038/nature11826. [PMC free article: PMC3634804] [PubMed: 23354049] [CrossRef]
24.: Broutier L, Mastrogiovanni G, Verstegen MM, Francies HE, Gavarro LM, Bradshaw CR, et al. Human primary liver cancer-derived organoid cultures for disease modeling and drug screening. Nat Med. 2017;23(12):1424–35. https://doi.org/10.1038/nm.4438. [PMC free article: PMC5722201] [PubMed: 29131160] [CrossRef]
25.: He L, Tian DA, Li PY, He XX. Mouse models of liver cancer: progress and recommendations. Oncotarget. 2015;6(27):23306–22. https://doi.org/10.18632/oncotarget.4202. [PMC free article: PMC4695120] [PubMed: 26259234] [CrossRef]
26.: Price JE. Xenograft models in immunodeficient animals: I. Nude mice: spontaneous and experimental metastasis models. Methods Mol Med. 2001;58:205–13. https://doi.org/10.1385/1-59259-137-X:205. [PubMed: 21340860] [CrossRef]
27.: Morton CL, Houghton PJ. Establishment of human tumor xenografts in immunodeficient mice. Nat Protoc. 2007;2(2):247–50. https://doi.org/10.1038/nprot.2007.25. [PubMed: 17406581] [CrossRef]
28.: Richmond A, Su Y. Mouse xenograft models vs GEM models for human cancer therapeutics. Dis Model Mech. 2008;1(2–3):78–82. https://doi.org/10.1242/dmm.000976. [PMC free article: PMC2562196] [PubMed: 19048064] [CrossRef]
29.: Heindryckx F, Colle I, Van Vlierberghe H. Experimental mouse models for hepatocellular carcinoma research. Int J Exp Pathol. 2009;90(4):367–86. https://doi.org/10.1111/j.1365-2613.2009.00656.x. [PMC free article: PMC2741148] [PubMed: 19659896] [CrossRef]
30.: Santos NP, Colaco AA, Oliveira PA. Animal models as a tool in hepatocellular carcinoma research: a review. Tumour Biol. 2017;39(3):1010428317695923. https://doi.org/10.1177/1010428317695923. [PubMed: 28347231] [CrossRef]
31.: Killion JJ, Radinsky R, Fidler IJ. Orthotopic models are necessary to predict therapy of transplantable tumors in mice. Cancer Metastasis Rev. 1998;17(3):279–84. [PubMed: 10352881] [CrossRef]
32.: El-Khoueiry AB, Sangro B, Yau T, Crocenzi TS, Kudo M, Hsu C, et al. Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet. 2017;389(10088):2492–502. https://doi.org/10.1016/S0140-6736(17)31046-2. [PMC free article: PMC7539326] [PubMed: 28434648] [CrossRef]
33.: Zhu AX, Finn RS, Edeline J, Cattan S, Ogasawara S, Palmer D, et al. Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial. Lancet Oncol. 2018; https://doi.org/10.1016/S1470-2045(18)30351-6. [PubMed: 29875066] [CrossRef]
34.: Zimmerman HJ, Lewis JH. Chemical- and toxin-induced hepatotoxicity. Gastroenterol Clin N Am. 1995;24(4):1027–45. [PubMed: 8749910]
35.: Lee WM. Drug-induced hepatotoxicity. N Engl J Med. 2003;349(5):474–85. https://doi.org/10.1056/NEJMra021844. [PubMed: 12890847] [CrossRef]
36.: Zhang YJ. Interactions of chemical carcinogens and genetic variation in hepatocellular carcinoma. World J Hepatol. 2010;2(3):94–102. https://doi.org/10.4254/wjh.v2.i3.94. [PMC free article: PMC2999273] [PubMed: 21160980] [CrossRef]
37.: Heath JC. The production of malignant tumours by cobalt in the rat. Br J Cancer. 1956;10(4):668–73. [PMC free article: PMC2073851] [PubMed: 13426378] [CrossRef]
38.: Verna L, Whysner J, Williams GM. N-nitrosodiethylamine mechanistic data and risk assessment: bioactivation, DNA-adduct formation, mutagenicity, and tumor initiation. Pharmacol Ther. 1996;71(1–2):57–81. [PubMed: 8910949] [CrossRef]
39.: Qi Y, Chen X, Chan CY, Li D, Yuan C, Yu F, et al. Two-dimensional differential gel electrophoresis/analysis of diethylnitrosamine induced rat hepatocellular carcinoma. Int J Cancer. 2008;122(12):2682–8. https://doi.org/10.1002/ijc.23464. [PubMed: 18351647] [CrossRef]
40.: Kolaja KL, Klaunig JE. Vitamin E modulation of hepatic focal lesion growth in mice. Toxicol Appl Pharmacol. 1997;143(2):380–7. [PubMed: 9144454] [CrossRef]
41.: Tolba R, Kraus T, Liedtke C, Schwarz M, Weiskirchen R. Diethylnitrosamine (DEN)-induced carcinogenic liver injury in mice. Lab Anim. 2015;49(1 Suppl):59–69. https://doi.org/10.1177/0023677215570086. [PubMed: 25835739] [CrossRef]
42.: Maronpot RR. Biological basis of differential susceptibility to hepatocarcinogenesis among mouse strains. J Toxicol Pathol. 2009;22(1):11–33. https://doi.org/10.1293/tox.22.11. [PMC free article: PMC3246016] [PubMed: 22271974] [CrossRef]
43.: Nakatani T, Roy G, Fujimoto N, Asahara T, Ito A. Sex hormone dependency of diethylnitrosamine-induced liver tumors in mice and chemoprevention by leuprorelin. Jpn J Cancer Res. 2001;92(3):249–56. [PMC free article: PMC5926710] [PubMed: 11267934] [CrossRef]
44.: Naugler WE, Sakurai T, Kim S, Maeda S, Kim K, Elsharkawy AM, et al. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science. 2007;317(5834):121–4. https://doi.org/10.1126/science.1140485. [PubMed: 17615358] [CrossRef]
45.: Connor F, Rayner TF, Aitken SJ, Feig C, Lukk M, Santoyo-Lopez J, et al. Mutational landscape of a chemically-induced mouse model of liver cancer. J Hepatol. 2018; https://doi.org/10.1016/j.jhep.2018.06.009. [PMC free article: PMC6142872] [PubMed: 29958939] [CrossRef]
46.: Waxman DJ, Azaroff L. Phenobarbital induction of cytochrome P-450 gene expression. Biochem J. 1992;281(Pt 3):577–92. [PMC free article: PMC1130728] [PubMed: 1536639] [CrossRef]
47.: Watson RE, Goodman JI. Effects of phenobarbital on DNA methylation in GC-rich regions of hepatic DNA from mice that exhibit different levels of susceptibility to liver tumorigenesis. Toxicol Sci. 2002;68(1):51–8. [PubMed: 12075110] [CrossRef]
48.: Gourama H, Bullerman LB. Aspergillus flavus and Aspergillus parasiticus: Aflatoxigenic fungi of concern in foods and feeds: a review. J Food Prot. 1995;58(12):1395–404. [PubMed: 31159052] [CrossRef]
49.: Liu Y, Wu F. Global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment. Environ Health Perspect. 2010;118(6):818–24. https://doi.org/10.1289/ehp.0901388. [PMC free article: PMC2898859] [PubMed: 20172840] [CrossRef]
50.: Guengerich FP, Johnson WW, Shimada T, Ueng YF, Yamazaki H, Langouet S. Activation and detoxication of aflatoxin B1. Mutat Res. 1998;402(1–2):121–8. [PubMed: 9675258] [CrossRef]
51.: Hamid AS, Tesfamariam IG, Zhang Y, Zhang ZG. Aflatoxin B1-induced hepatocellular carcinoma in developing countries: geographical distribution, mechanism of action and prevention. Oncol Lett. 2013;5(4):1087–92. https://doi.org/10.3892/ol.2013.1169. [PMC free article: PMC3629261] [PubMed: 23599745] [CrossRef]
52.: Mace K, Aguilar F, Wang JS, Vautravers P, Gomez-Lechon M, Gonzalez FJ, et al. Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell lines. Carcinogenesis. 1997;18(7):1291–7. [PubMed: 9230270] [CrossRef]
53.: McGlynn KA, Hunter K, LeVoyer T, Roush J, Wise P, Michielli RA, et al. Susceptibility to aflatoxin B1-related primary hepatocellular carcinoma in mice and humans. Cancer Res. 2003;63(15):4594–601. [PubMed: 12907637]
54.: Chu YJ, Yang HI, Wu HC, Lee MH, Liu J, Wang LY, et al. Aflatoxin B1 exposure increases the risk of hepatocellular carcinoma associated with hepatitis C virus infection or alcohol consumption. Eur J Cancer. 2018;94:37–46. https://doi.org/10.1016/j.ejca.2018.02.010. [PMC free article: PMC5895495] [PubMed: 29533866] [CrossRef]
55.: Kew MC. Synergistic interaction between aflatoxin B1 and hepatitis B virus in hepatocarcinogenesis. Liver Int. 2003;23(6):405–9. [PubMed: 14986813] [CrossRef]
56.: Boll M, Weber LW, Becker E, Stampfl A. Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites. Z Naturforsch C. 2001;56(7–8):649–59. [PubMed: 11531102] [CrossRef]
57.: Bhathal PS, Rose NR, Mackay IR, Whittingham S. Strain differences in mice in carbon tetrachloride-induced liver injury. Br J Exp Pathol. 1983;64(5):524–33. [PMC free article: PMC2040819] [PubMed: 6639871]
58.: Dapito DH, Mencin A, Gwak GY, Pradere JP, Jang MK, Mederacke I, et al. Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4. Cancer Cell. 2012;21(4):504–16. https://doi.org/10.1016/j.ccr.2012.02.007. [PMC free article: PMC3332000] [PubMed: 22516259] [CrossRef]
59.: Fitzhugh OG, Nelson AA. Liver tumors in rats fed thiourea or thioacetamide. Science. 1948;108(2814):626–8. https://doi.org/10.1126/science.108.2814.626. [PubMed: 17783352] [CrossRef]
60.: Koen YM, Sarma D, Hajovsky H, Galeva NA, Williams TD, Staudinger JL, et al. Protein targets of thioacetamide metabolites in rat hepatocytes. Chem Res Toxicol. 2013;26(4):564–74. https://doi.org/10.1021/tx400001x. [PMC free article: PMC3710294] [PubMed: 23465048] [CrossRef]
61.: Martinez AK, Maroni L, Marzioni M, Ahmed ST, Milad M, Ray D, et al. Mouse models of liver fibrosis mimic human liver fibrosis of different etiologies. Curr Pathobiol Rep. 2014;2(4):143–53. https://doi.org/10.1007/s40139-014-0050-2. [PMC free article: PMC4226463] [PubMed: 25396098] [CrossRef]
62.: Li X, Benjamin IS, Alexander B. Reproducible production of thioacetamide-induced macronodular cirrhosis in the rat with no mortality. J Hepatol. 2002;36(4):488–93. [PubMed: 11943419] [CrossRef]
63.: Zeisel SH, da Costa KA. Choline: an essential nutrient for public health. Nutr Rev. 2009;67(11):615–23. https://doi.org/10.1111/j.1753-4887.2009.00246.x. [PMC free article: PMC2782876] [PubMed: 19906248] [CrossRef]
64.: Corbin KD, Zeisel SH. Choline metabolism provides novel insights into nonalcoholic fatty liver disease and its progression. Curr Opin Gastroenterol. 2012;28(2):159–65. https://doi.org/10.1097/MOG.0b013e32834e7b4b. [PMC free article: PMC3601486] [PubMed: 22134222] [CrossRef]
65.: Chandar N, Lombardi B. Liver cell proliferation and incidence of hepatocellular carcinomas in rats fed consecutively a choline-devoid and a choline-supplemented diet. Carcinogenesis. 1988;9(2):259–63. [PubMed: 3338109] [CrossRef]
66.: Ikawa-Yoshida A, Matsuo S, Kato A, Ohmori Y, Higashida A, Kaneko E, et al. Hepatocellular carcinoma in a mouse model fed a choline-deficient, L-amino acid-defined, high-fat diet. Int J Exp Pathol. 2017;98(4):221–33. https://doi.org/10.1111/iep.12240. [PMC free article: PMC5639266] [PubMed: 28895242] [CrossRef]
67.: Kishida N, Matsuda S, Itano O, Shinoda M, Kitago M, Yagi H, et al. Development of a novel mouse model of hepatocellular carcinoma with nonalcoholic steatohepatitis using a high-fat, choline-deficient diet and intraperitoneal injection of diethylnitrosamine. BMC Gastroenterol. 2016;16(1):61. https://doi.org/10.1186/s12876-016-0477-5. [PMC free article: PMC4906823] [PubMed: 27296438] [CrossRef]
68.: Tsuchida T, Lee YA, Fujiwara N, Ybanez M, Allen B, Martins S, et al. A simple diet- and chemical-induced murine NASH model with rapid progression of steatohepatitis, fibrosis and liver cancer. J Hepatol. 2018;69(2):385–95. https://doi.org/10.1016/j.jhep.2018.03.011. [PMC free article: PMC6054570] [PubMed: 29572095] [CrossRef]
69.: Hill-Baskin AE, Markiewski MM, Buchner DA, Shao H, DeSantis D, Hsiao G, et al. Diet-induced hepatocellular carcinoma in genetically predisposed mice. Hum Mol Genet. 2009;18(16):2975–88. https://doi.org/10.1093/hmg/ddp236. [PMC free article: PMC2714725] [PubMed: 19454484] [CrossRef]
70.: Borbath I, Horsmans Y. The role of PPARgamma in hepatocellular carcinoma. PPAR Res. 2008;2008:209520. https://doi.org/10.1155/2008/209520. [PMC free article: PMC2396389] [PubMed: 18509497] [CrossRef]
71.: Hsu HT, Chi CW. Emerging role of the peroxisome proliferator-activated receptor-gamma in hepatocellular carcinoma. J Hepatocell Carcinoma. 2014;1:127–35. https://doi.org/10.2147/JHC.S48512. [PMC free article: PMC4918273] [PubMed: 27508182] [CrossRef]
72.: Kersten K, de Visser KE, van Miltenburg MH, Jonkers J. Genetically engineered mouse models in oncology research and cancer medicine. EMBO Mol Med. 2017;9(2):137–53. https://doi.org/10.15252/emmm.201606857. [PMC free article: PMC5286388] [PubMed: 28028012] [CrossRef]
73.: Lewandoski M. Conditional control of gene expression in the mouse. Nat Rev Genet. 2001;2(10):743–55. https://doi.org/10.1038/35093537. [PubMed: 11584291] [CrossRef]
74.: Bouabe H, Okkenhaug K. Gene targeting in mice: a review. Methods Mol Biol. 2013;1064:315–36. https://doi.org/10.1007/978-1-62703-601-6_23. [PMC free article: PMC4524968] [PubMed: 23996268] [CrossRef]
75.: El-Serag HB. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology. 2012;142(6):1264–73 e1. https://doi.org/10.1053/j.gastro.2011.12.061. [PMC free article: PMC3338949] [PubMed: 22537432] [CrossRef]
76.: Ott JJ, Stevens GA, Groeger J, Wiersma ST. Global epidemiology of hepatitis B virus infection: new estimates of age-specific HBsAg seroprevalence and endemicity. Vaccine. 2012;30(12):2212–9. https://doi.org/10.1016/j.vaccine.2011.12.116. [PubMed: 22273662] [CrossRef]
77.: Levrero M, Zucman-Rossi J. Mechanisms of HBV-induced hepatocellular carcinoma. J Hepatol. 2016;64(1 Suppl):S84–S101. https://doi.org/10.1016/j.jhep.2016.02.021. [PubMed: 27084040] [CrossRef]
78.: Wang Y, Cui F, Lv Y, Li C, Xu X, Deng C, et al. HBsAg and HBx knocked into the p21 locus causes hepatocellular carcinoma in mice. Hepatology. 2004;39(2):318–24. https://doi.org/10.1002/hep.20076. [PubMed: 14767984] [CrossRef]
79.: Wang HC, Huang W, Lai MD, Su IJ. Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis. Cancer Sci. 2006;97(8):683–8. https://doi.org/10.1111/j.1349-7006.2006.00235.x. [PubMed: 16863502] [CrossRef]
80.: Seifer M, Hohne M, Schaefer S, Gerlich WH. In vitro tumorigenicity of hepatitis B virus DNA and HBx protein. J Hepatol. 1991;13(Suppl 4):S61–5. [PubMed: 1822516] [CrossRef]
81.: Koike K. Hepatitis B virus HBx gene and hepatocarcinogenesis. Intervirology. 1995;38(3–4):134–42. https://doi.org/10.1159/000150424. [PubMed: 8682608] [CrossRef]
82.: Zampino R, Marrone A, Restivo L, Guerrera B, Sellitto A, Rinaldi L, et al. Chronic HCV infection and inflammation: clinical impact on hepatic and extra-hepatic manifestations. World J Hepatol. 2013;5(10):528–40. https://doi.org/10.4254/wjh.v5.i10.528. [PMC free article: PMC3812455] [PubMed: 24179612] [CrossRef]
83.: Lerat H, Honda M, Beard MR, Loesch K, Sun J, Yang Y, et al. Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus. Gastroenterology. 2002;122(2):352–65. [PubMed: 11832450] [CrossRef]
84.: Koike K. Molecular basis of hepatitis C virus-associated hepatocarcinogenesis: lessons from animal model studies. Clin Gastroenterol Hepatol. 2005;3(10 Suppl 2):S132–5. [PubMed: 16234061] [CrossRef]
85.: Kamegaya Y, Hiasa Y, Zukerberg L, Fowler N, Blackard JT, Lin W, et al. Hepatitis C virus acts as a tumor accelerator by blocking apoptosis in a mouse model of hepatocarcinogenesis. Hepatology. 2005;41(3):660–7. https://doi.org/10.1002/hep.20621. [PubMed: 15723444] [CrossRef]
86.: Koike K, Moriya K, Matsuura Y. Animal models for hepatitis C and related liver disease. Hepatol Res. 2010;40(1):69–82. https://doi.org/10.1111/j.1872-034X.2009.00593.x. [PubMed: 20156300] [CrossRef]
87.: Schulze K, Imbeaud S, Letouze E, Alexandrov LB, Calderaro J, Rebouissou S, et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat Genet. 2015;47(5):505–11. https://doi.org/10.1038/ng.3252. [PMC free article: PMC4587544] [PubMed: 25822088] [CrossRef]
88.: Cancer Genome Atlas Research Network. Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell. 2017;169(7):1327–41 e23. https://doi.org/10.1016/j.cell.2017.05.046. [PMC free article: PMC5680778] [PubMed: 28622513] [CrossRef]
89.: Schulze K, Nault JC, Villanueva A. Genetic profiling of hepatocellular carcinoma using next-generation sequencing. J Hepatol. 2016;65(5):1031–42. https://doi.org/10.1016/j.jhep.2016.05.035. [PubMed: 27262756] [CrossRef]
90.: Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J, et al. The landscape of somatic copy-number alteration across human cancers. Nature. 2010;463(7283):899–905. https://doi.org/10.1038/nature08822. [PMC free article: PMC2826709] [PubMed: 20164920] [CrossRef]
91.: Kawate S, Fukusato T, Ohwada S, Watanuki A, Morishita Y. Amplification of c-myc in hepatocellular carcinoma: correlation with clinicopathologic features, proliferative activity and p53 overexpression. Oncology. 1999;57(2):157–63. https://doi.org/10.1159/000012024. [PubMed: 10461064] [CrossRef]
92.: Dang CV. MYC on the path to cancer. Cell. 2012;149(1):22–35. https://doi.org/10.1016/j.cell.2012.03.003. [PMC free article: PMC3345192] [PubMed: 22464321] [CrossRef]
93.: Santoni-Rugiu E, Nagy P, Jensen MR, Factor VM, Thorgeirsson SS. Evolution of neoplastic development in the liver of transgenic mice co-expressing c-myc and transforming growth factor-alpha. Am J Pathol. 1996;149(2):407–28. [PMC free article: PMC1865312] [PubMed: 8701981]
94.: Shachaf CM, Kopelman AM, Arvanitis C, Karlsson A, Beer S, Mandl S, et al. MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer. Nature. 2004;431(7012):1112–7. https://doi.org/10.1038/nature03043. [PubMed: 15475948] [CrossRef]
95.: Calvisi DF, Conner EA, Ladu S, Lemmer ER, Factor VM, Thorgeirsson SS. Activation of the canonical Wnt/beta-catenin pathway confers growth advantages in c-Myc/E2F1 transgenic mouse model of liver cancer. J Hepatol. 2005;42(6):842–9. https://doi.org/10.1016/j.jhep.2005.01.029. [PubMed: 15885355] [CrossRef]
96.: Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36(11):1461–73. https://doi.org/10.1038/onc.2016.304. [PMC free article: PMC5357762] [PubMed: 27617575] [CrossRef]
97.: Khalaf AM, Fuentes D, Morshid AI, Burke MR, Kaseb AO, Hassan M, et al. Role of Wnt/beta-catenin signaling in hepatocellular carcinoma, pathogenesis, and clinical significance. J Hepatocell Carcinoma. 2018;5:61–73. https://doi.org/10.2147/JHC.S156701. [PMC free article: PMC6027703] [PubMed: 29984212] [CrossRef]
98.: Nault JC, Mallet M, Pilati C, Calderaro J, Bioulac-Sage P, Laurent C, et al. High frequency of telomerase reverse-transcriptase promoter somatic mutations in hepatocellular carcinoma and preneoplastic lesions. Nat Commun. 2013;4:2218. https://doi.org/10.1038/ncomms3218. [PMC free article: PMC3731665] [PubMed: 23887712] [CrossRef]
99.: Inagawa S, Itabashi M, Adachi S, Kawamoto T, Hori M, Shimazaki J, et al. Expression and prognostic roles of beta-catenin in hepatocellular carcinoma: correlation with tumor progression and postoperative survival. Clin Cancer Res. 2002;8(2):450–6. [PubMed: 11839663]
100.: Lai TY, Su CC, Kuo WW, Yeh YL, Kuo WH, Tsai FJ, et al. β-catenin plays a key role in metastasis of human hepatocellular carcinoma. Oncol Rep. 2011;26(2):415–22. https://doi.org/10.3892/or.2011.1323. [PubMed: 21617877] [CrossRef]
101.: Hoshida Y, Nijman SM, Kobayashi M, Chan JA, Brunet JP, Chiang DY, et al. Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma. Cancer Res. 2009;69(18):7385–92. https://doi.org/10.1158/0008-5472.CAN-09-1089. [PMC free article: PMC3549578] [PubMed: 19723656] [CrossRef]
102.: Harada N, Miyoshi H, Murai N, Oshima H, Tamai Y, Oshima M, et al. Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin. Cancer Res. 2002;62(7):1971–7. [PubMed: 11929813]
103.: Harada N, Oshima H, Katoh M, Tamai Y, Oshima M, Taketo MM. Hepatocarcinogenesis in mice with beta-catenin and Ha-ras gene mutations. Cancer Res. 2004;64(1):48–54. [PubMed: 14729607] [CrossRef]
104.: Nejak-Bowen KN, Thompson MD, Singh S, Bowen WC Jr, Dar MJ, Khillan J, et al. Accelerated liver regeneration and hepatocarcinogenesis in mice overexpressing serine-45 mutant beta-catenin. Hepatology. 2010;51(5):1603–13. https://doi.org/10.1002/hep.23538. [PMC free article: PMC2908905] [PubMed: 20432254] [CrossRef]
105.: Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74. https://doi.org/10.1016/j.cell.2011.02.013. [PubMed: 21376230] [CrossRef]
106.: Kastenhuber ER, Lowe SW. Putting p53 in context. Cell. 2017;170(6):1062–78. https://doi.org/10.1016/j.cell.2017.08.028. [PMC free article: PMC5743327] [PubMed: 28886379] [CrossRef]
107.: Hussain SP, Schwank J, Staib F, Wang XW, Harris CC. TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer. Oncogene. 2007;26(15):2166–76. https://doi.org/10.1038/sj.onc.1210279. [PubMed: 17401425] [CrossRef]
108.: Chawanthayatham S, Valentine CC 3rd, Fedeles BI, Fox EJ, Loeb LA, Levine SS, et al. Mutational spectra of aflatoxin B1 in vivo establish biomarkers of exposure for human hepatocellular carcinoma. Proc Natl Acad Sci U S A. 2017;114(15):E3101–E9. https://doi.org/10.1073/pnas.1700759114. [PMC free article: PMC5393230] [PubMed: 28351974] [CrossRef]
109.: Lewis BC, Klimstra DS, Socci ND, Xu S, Koutcher JA, Varmus HE. The absence of p53 promotes metastasis in a novel somatic mouse model for hepatocellular carcinoma. Mol Cell Biol. 2005;25(4):1228–37. https://doi.org/10.1128/MCB.25.4.1228-1237.2005. [PMC free article: PMC548003] [PubMed: 15684377] [CrossRef]
110.: Farazi PA, Glickman J, Horner J, Depinho RA. Cooperative interactions of p53 mutation, telomere dysfunction, and chronic liver damage in hepatocellular carcinoma progression. Cancer Res. 2006;66(9):4766–73. https://doi.org/10.1158/0008-5472.CAN-05-4608. [PubMed: 16651430] [CrossRef]
111.: Chen YW, Klimstra DS, Mongeau ME, Tatem JL, Boyartchuk V, Lewis BC. Loss of p53 and Ink4a/Arf cooperate in a cell autonomous fashion to induce metastasis of hepatocellular carcinoma cells. Cancer Res. 2007;67(16):7589–96. https://doi.org/10.1158/0008-5472.CAN-07-0381. [PMC free article: PMC2396788] [PubMed: 17699762] [CrossRef]
112.: Cullen JM, Sandgren EP, Brinster RL, Maronpot RR. Histologic characterization of hepatic carcinogenesis in transgenic mice expressing SV40 T-antigens. Vet Pathol. 1993;30(2):111–8. https://doi.org/10.1177/030098589303000203. [PubMed: 8385835] [CrossRef]
113.: Viatour P, Ehmer U, Saddic LA, Dorrell C, Andersen JB, Lin C, et al. Notch signaling inhibits hepatocellular carcinoma following inactivation of the RB pathway. J Exp Med. 2011;208(10):1963–76. https://doi.org/10.1084/jem.20110198. [PMC free article: PMC3182062] [PubMed: 21875955] [CrossRef]
114.: Hopkins BD, Parsons RE. Molecular pathways: intercellular PTEN and the potential of PTEN restoration therapy. Clin Cancer Res. 2014;20(21):5379–83. https://doi.org/10.1158/1078-0432.CCR-13-2661. [PMC free article: PMC4362520] [PubMed: 25361917] [CrossRef]
115.: Sun H, Lesche R, Li DM, Liliental J, Zhang H, Gao J, et al. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc Natl Acad Sci U S A. 1999;96(11):6199–204. [PMC free article: PMC26859] [PubMed: 10339565] [CrossRef]
116.: Horie Y, Suzuki A, Kataoka E, Sasaki T, Hamada K, Sasaki J, et al. Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas. J Clin Invest. 2004;113(12):1774–83. https://doi.org/10.1172/JCI20513. [PMC free article: PMC420505] [PubMed: 15199412] [CrossRef]
117.: Shen WH, Balajee AS, Wang J, Wu H, Eng C, Pandolfi PP, et al. Essential role for nuclear PTEN in maintaining chromosomal integrity. Cell. 2007;128(1):157–70. https://doi.org/10.1016/j.cell.2006.11.042. [PubMed: 17218262] [CrossRef]
118.: Kotelevets L, van Hengel J, Bruyneel E, Mareel M, van Roy F, Chastre E. Implication of the MAGI-1b/PTEN signalosome in stabilization of adherens junctions and suppression of invasiveness. FASEB J. 2005;19(1):115–7. https://doi.org/10.1096/fj.04-1942fje. [PubMed: 15629897] [CrossRef]
119.: Hu TH, Huang CC, Lin PR, Chang HW, Ger LP, Lin YW, et al. Expression and prognostic role of tumor suppressor gene PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer. 2003;97(8):1929–40. https://doi.org/10.1002/cncr.11266. [PubMed: 12673720] [CrossRef]
120.: Di Cristofano A, Pesce B, Cordon-Cardo C, Pandolfi PP. Pten is essential for embryonic development and tumour suppression. Nat Genet. 1998;19(4):348–55. https://doi.org/10.1038/1235. [PubMed: 9697695] [CrossRef]
121.: Stambolic V, Tsao MS, Macpherson D, Suzuki A, Chapman WB, Mak TW. High incidence of breast and endometrial neoplasia resembling human Cowden syndrome in pten+/− mice. Cancer Res. 2000;60(13):3605–11. [PubMed: 10910075]
122.: Podsypanina K, Ellenson LH, Nemes A, Gu J, Tamura M, Yamada KM, et al. Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc Natl Acad Sci U S A. 1999;96(4):1563–8. [PMC free article: PMC15517] [PubMed: 9990064] [CrossRef]
123.: Blackburn EH. Structure and function of telomeres. Nature. 1991;350(6319):569–73. https://doi.org/10.1038/350569a0. [PubMed: 1708110] [CrossRef]
124.: Cong YS, Wright WE, Shay JW. Human telomerase and its regulation. Microbiol Mol Biol Rev. 2002;66(3):407–25, table of contents. [PMC free article: PMC120798] [PubMed: 12208997] [CrossRef]
125.: Park YM, Choi JY, Byun BH, Cho CH, Kim HS, Kim BS. Telomerase is strongly activated in hepatocellular carcinoma but not in chronic hepatitis and cirrhosis. Exp Mol Med. 1998;30(1):35–40. https://doi.org/10.1038/emm.1998.5. [PubMed: 9873820] [CrossRef]
126.: Nagao K, Tomimatsu M, Endo H, Hisatomi H, Hikiji K. Telomerase reverse transcriptase mRNA expression and telomerase activity in hepatocellular carcinoma. J Gastroenterol. 1999;34(1):83–7. [PubMed: 10204615] [CrossRef]
127.: Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM, DePinho RA, et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell. 1997;91(1):25–34. [PubMed: 9335332] [CrossRef]
128.: Lechel A, Holstege H, Begus Y, Schienke A, Kamino K, Lehmann U, et al. Telomerase deletion limits progression of p53-mutant hepatocellular carcinoma with short telomeres in chronic liver disease. Gastroenterology. 2007;132(4):1465–75. https://doi.org/10.1053/j.gastro.2007.01.045. [PubMed: 17433324] [CrossRef]
129.: Farazi PA, Glickman J, Jiang S, Yu A, Rudolph KL, DePinho RA. Differential impact of telomere dysfunction on initiation and progression of hepatocellular carcinoma. Cancer Res. 2003;63(16):5021–7. [PubMed: 12941829]
130.: Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell. 2010;140(6):883–99. https://doi.org/10.1016/j.cell.2010.01.025. [PMC free article: PMC2866629] [PubMed: 20303878] [CrossRef]
131.: Maeda S, Kamata H, Luo JL, Leffert H, Karin M. IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell. 2005;121(7):977–90. https://doi.org/10.1016/j.cell.2005.04.014. [PubMed: 15989949] [CrossRef]
132.: Luedde T, Beraza N, Kotsikoris V, van Loo G, Nenci A, De Vos R, et al. Deletion of NEMO/IKKgamma in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma. Cancer Cell. 2007;11(2):119–32. https://doi.org/10.1016/j.ccr.2006.12.016. [PubMed: 17292824] [CrossRef]
133.: He G, Karin M. NF-kappaB and STAT3 – key players in liver inflammation and cancer. Cell Res. 2011;21(1):159–68. https://doi.org/10.1038/cr.2010.183. [PMC free article: PMC3193410] [PubMed: 21187858] [CrossRef]
134.: Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, et al. NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature. 2004;431(7007):461–6. https://doi.org/10.1038/nature02924. [PubMed: 15329734] [CrossRef]
135.: Sanderson N, Factor V, Nagy P, Kopp J, Kondaiah P, Wakefield L, et al. Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions. Proc Natl Acad Sci U S A. 1995;92(7):2572–6. [PMC free article: PMC42260] [PubMed: 7708687] [CrossRef]
136.: Newell P, Villanueva A, Friedman SL, Koike K, Llovet JM. Experimental models of hepatocellular carcinoma. J Hepatol. 2008;48(5):858–79. https://doi.org/10.1016/j.jhep.2008.01.008. [PMC free article: PMC2990959] [PubMed: 18314222] [CrossRef]
137.: Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A. 2002;99(18):11854–9. https://doi.org/10.1073/pnas.182412299. [PMC free article: PMC129358] [PubMed: 12192090] [CrossRef]
138.: Nakai H, Fuess S, Storm TA, Muramatsu S, Nara Y, Kay MA. Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice. J Virol. 2005;79(1):214–24. https://doi.org/10.1128/JVI.79.1.214-224.2005. [PMC free article: PMC538708] [PubMed: 15596817] [CrossRef]
139.: Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 1999;6(7):1258–66. https://doi.org/10.1038/sj.gt.3300947. [PubMed: 10455434] [CrossRef]
140.: Zhang G, Budker V, Wolff JA. High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum Gene Ther. 1999;10(10):1735–7. https://doi.org/10.1089/10430349950017734. [PubMed: 10428218] [CrossRef]
141.: Kamimura K, Yokoo T, Abe H, Kobayashi Y, Ogawa K, Shinagawa Y, et al. Image-guided hydrodynamic gene delivery: current status and future directions. Pharmaceutics. 2015;7(3):213–23. https://doi.org/10.3390/pharmaceutics7030213. [PMC free article: PMC4588196] [PubMed: 26308044] [CrossRef]
142.: Aronovich EL, McIvor RS, Hackett PB. The Sleeping Beauty transposon system: a non-viral vector for gene therapy. Hum Mol Genet. 2011;20(R1):R14–20. https://doi.org/10.1093/hmg/ddr140. [PMC free article: PMC3095056] [PubMed: 21459777] [CrossRef]
143.: Wuestefeld T, Pesic M, Rudalska R, Dauch D, Longerich T, Kang TW, et al. A Direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration. Cell. 2013;153(2):389–401. https://doi.org/10.1016/j.cell.2013.03.026. [PubMed: 23582328] [CrossRef]
144.: Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi NS, et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature. 2014;514(7522):380–4. https://doi.org/10.1038/nature13589. [PMC free article: PMC4199937] [PubMed: 25119044] [CrossRef]
145.: Chen X, Calvisi DF. Hydrodynamic transfection for generation of novel mouse models for liver cancer research. Am J Pathol. 2014;184(4):912–23. https://doi.org/10.1016/j.ajpath.2013.12.002. [PMC free article: PMC3969989] [PubMed: 24480331] [CrossRef]

Bookshelf ID: NBK553756PMID: 32078271DOI: 10.1007/978-3-030-21540-8_16

Contents

< PrevNext >

PubReader
Print View
Cite this Page
Molina-Sánchez P, Lujambio A. Experimental Models for Preclinical Research in Hepatocellular Carcinoma. 2019 Aug 6. In: Hoshida Y, editor. Hepatocellular Carcinoma: Translational Precision Medicine Approaches [Internet]. Cham (CH): Humana Press; 2019. Chapter 16. doi: 10.1007/978-3-030-21540-8_16
PDF version of this page (489K)

Recent Activity

Clear Turn Off Turn On

Experimental Models for Preclinical Research in Hepatocellular Carcinoma - Hepat...
Experimental Models for Preclinical Research in Hepatocellular Carcinoma - Hepatocellular Carcinoma

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

Bookshelf

Hepatocellular Carcinoma: Translational Precision Medicine Approaches [Internet].

Chapter 16Experimental Models for Preclinical Research in Hepatocellular Carcinoma

Authors

Affiliations

Keywords:

Introduction

Fig. 16.1

In Vitro Models

HCC Cell Lines

HCC Organoids

In Vivo Models

Transplantation-Based Models: Xenografts and Allografts

Chemically Induced Models

Diethylnitrosamine (DEN)

Aflatoxin Exposure

Carbon Tetrachloride (CCl₄)

Thioacetamide (TAA)

Choline-Deficient Diet (CDD)

Peroxisome Proliferators

Genetically Engineered Mouse Models (GEMMs)

Mouse Models of Human Hepatitis

Hepatitis B Virus (HBV) Model

Hepatitis C Virus (HCV) Model

Mouse Models of Cancer-Related Genes

c-MYC

β-Catenin (CTNNB1 Gene)

Cell Cycle Control Genes

PTEN

Telomerase

Mouse Models of Inflammation

Non-germline GEMMs

Conclusions and Future Directions

References

Views

In this Page

Similar articles in PubMed

Recent Activity

Bookshelf

Hepatocellular Carcinoma: Translational Precision Medicine Approaches [Internet].

Chapter 16Experimental Models for Preclinical Research in Hepatocellular Carcinoma

Authors

Affiliations

Keywords:

Introduction

Fig. 16.1

In Vitro Models

HCC Cell Lines

HCC Organoids

In Vivo Models

Transplantation-Based Models: Xenografts and Allografts

Chemically Induced Models

Diethylnitrosamine (DEN)

Aflatoxin Exposure

Carbon Tetrachloride (CCl4)

Thioacetamide (TAA)

Choline-Deficient Diet (CDD)

Peroxisome Proliferators

Genetically Engineered Mouse Models (GEMMs)

Mouse Models of Human Hepatitis

Hepatitis B Virus (HBV) Model

Hepatitis C Virus (HCV) Model

Mouse Models of Cancer-Related Genes

c-MYC

β-Catenin (CTNNB1 Gene)

Cell Cycle Control Genes

PTEN

Telomerase

Mouse Models of Inflammation

Non-germline GEMMs

Conclusions and Future Directions

References

Views

In this Page

Similar articles in PubMed

Recent Activity

Carbon Tetrachloride (CCl₄)