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The disease relevance of human hepatocellular xenograft models: Molecular characterization and review of the literature
Cancer Letters 286 (2009) 121–128
Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Mini-review
The disease relevance of human hepatocellular xenograft models: Molecular characterization and review of the literature K. Kashofer a, M.M. Tschernatsch a, H.J. Mischinger b, F. Iberer c, K. Zatloukal a,* a b c
Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria Department of Surgery, Medical University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria Department of Transplantation Medicine, Medical University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria
a r t i c l e
i n f o
Article history: Received 14 July 2008 Accepted 4 November 2008
Keywords: Hepatocellular carcinoma HCC Xenografts Gene expression profiles
a b s t r a c t In recent years a number of new therapeutics has been developed that were not general toxins and inhibitors of cell division like classical chemotherapeutics, but were designed to target a specific pathway. A prerequisite for this development was the comprehensive characterization of molecular alterations occurring in human hepatocellular carcinoma (HCC). However, while much knowledge of the molecular pathogenesis of human HCC has been gained, the model systems used to test the functional relevance of these alterations and applied for preclinical evaluation of drug candidates are still poorly characterized. In this paper, we reviewed the literature about several commonly used HCC cell lines and xenotransplantation models and present our own data on the molecular characterization of these. Results obtained demonstrate that it is important to have a sound knowledge of the specific molecular constitution of the experimental model and to carefully evaluate the functional status of the pathway of interest. For this reason, we make the gene expression profiles publicly available to help researchers making an informed decision about which model to use. Ó 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinomas (HCCs) are a heterogeneous tumor entity with multiple etiologies and risk factors which may result in different pathways being deregulated in each tumor [1]. Although many molecular aberrations have recently been identified in HCC, the molecular pathways involved in hepatocarcinogenesis are still the target of intense research. In this paper, we present a review of the literature and our own data on the molecular characterization of several murine xenograft models focused on similarities to human HCC. In the last several years an improved understanding of the mechanisms of hepatocarcinogenesis has provided the opportunity to develop and study new therapeutic * Corresponding author. Tel.: +43 316 385 4404; fax: +43 316 384 329. E-mail addresses: [email protected], [email protected] (K. Zatloukal). 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.11.011
agents for advanced HCC. Many carcinogenic pathways like angiogenesis, altered signal transduction and aberrant cell cycle control are involved in HCC development [2,3]. In HCC, cancer cells are surrounded by stromal tissue containing extracellular matrix proteins (ECM), growth factors and proteolytic enzymes such as matrix metalloproteinases (MMPs) due to the underlying cirrhosis. It has been reported that these molecules play a key role in regulating the biological behavior and clinical outcome of HCC [4]. The expression of several EGF family members, specifically EGF, TGF-a and heparin binding epidermal growth, factor as well as EGFR have been described in several HCC cell lines and tissues [5,6]. Studies have led to a theory of an autocrine, paracrine and endocrine effect of TGF-a and EGFR/HER-1 on the proliferation of human HCC [7]. Despite that, treatment with the EGFR tyrosine kinase inhibitor erlotinib or monoclonal antibodies against EGFR have shown only modest or no benefit for the patient [8,9]. HCCs are tumors with increased levels of vascular endothelial
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growth factor (VEGF) and microvessel density [10]. VEGF represents an important therapeutic target in several malignancies [11] and there have been reports of modest clinical benefit in HCC patients if VEGF signaling is inhibited by an anti-VEGF antibody (bevacizumab) in combination with standard therapy [12]. However, the most promising compound which has made a big impact recently is sorafenib [13]. It is a novel signal transduction inhibitor that targets the Raf/mitogen-activated protein kinase-extracellular signal-related kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway and the VEGF receptor VEGFR-2 and PDFG receptor PDGFR-b. It has also shown promising clinical activity in HCC in a large phase II study with 137 patients [14]. More recently, a large multi-center international double-blind placebo controlled trial (SHARP) was conducted. In SHARP, researchers randomized 602 patients with Child–Pugh A cirrhosis and HCC to receive either placebo or sorafenib. It could be shown that sorafenib-treated patients survived 46.3 weeks as compared to 34.4 weeks in the placebo group. The study was stopped in February 2007 because results favoring sorafenib were found in the second planned interim analysis in October 2006. Based on this study, sorafenib seems to be the first systemic-targeted therapeutic agent for stabilization of HCC progression. The high specificity of drugs aimed against molecularly defined targets makes it increasingly important to have a good understanding of the molecular basis of the development of hepatocellular carcinoma. In recent years, much insight into molecular pathways and their deregulation in HCC has been gained. This development should present possibilities to develop small molecule compounds which are targeted against key regulator pathways in HCC. However, the murine models used in preclinical assessment of these highly specific drugs are few in number and overall poorly understood. Clinical relevance of murine transplantation models can only be obtained if careful attention is paid to the experimental conditions. The cell lines used must be free of pathogens and the mice have to be kept under specific pathogen-free conditions (SPF), as contamination of either the grafted cells or the mice leads to natural killer cell (NK) activation and thus to changes in the tumor take rate and growth [15]. The National Cancer Institute (NCI) started a large screening study to select agents for evaluation as clinical candidates in 1955. Initially, three transplanted rodent tumor cell lines (Sarcoma 180, Carcinoma 755 and Leukaemia 1210) were used. The spectrum of models was later broadened and in 1975 the murine P388 leukaemia model was added. In the early eighties, human tumor xenografts were added to the panels [16]. In this study, 1085 compounds were screened in five murine and three xenograft models. It was confirmed that screening against a fast growing murine leukaemia (P388) was useful as an initial cost-effective prescreen which can be followed by more stringent human xenograft models. In 1995 another rodent tumor model, the hollow fiber assay, in which polyvinylidine fluoride fibers loaded with tumor cells were implanted subcutaneously and intraperitoneally [17],
was added to the NCI screen. Johnson et al., performed a retrospective comparison of the results from the testing of compounds in the in-vitro NCI screen, the hollow fiber assay and the standard xenograft models with respect to their ability to predict the activity of a compound in each other model and in phase II clinical trials [18]. In this study, no close correlation could be found between activity of a compound in a xenograft model derived from a particular tumor type and activity of the compound against the same human tumor type in patients. However, if a compound displayed activity in at least one-third of the tested xenograft models there was correlation with activity in at least some Phase II trials. This study highlights the problem that xenografts only represent a tumor from a single patient, while human cancer is known to be very heterogeneous. The group of Snorri Thorgeirsson performed a comparison of the expression profiles of several murine hepatocellular cancers with human HCC [19]. In this study, Lee et al., used a list of orthologous genes that were present both in the murine and the human microarrays and changed significantly in both systems (n = 1650). After clustering it could be shown that the group of murine tumors that arose in Myc Tgf-a transgenic mice or diethyl nitrosamine-induced mice which harbour extensive chromosomal damage are most similar to a group of human HCCs with poor survival prognosis. A second group of murine tumors from Myc, E2F1 and Myc + E2F1 transgenic mice were most similar to HCCs with better prognosis while a third subgroup of Acox1 / and ciprofibrate-induced HCCs did not correlate with many human HCCs. This demonstrates that the different mouse models mimic different subclasses of human HCC which may to a large extent be due to a similarity in the underlying biology of the disease. This also demonstrates the need to identify the mouse models that most closely resemble human HCC in general or a subclass of human HCC under investigation. The transplantation of human primary HCC or established HCC tumor cell lines into immunodeficient mice (the human-into-murine-xenograft model) is frequently used to generate human tumors in a model system for investigation in the laboratory. This model has been used in basic research to test the activity of novel anti-cancer compounds and also as a general model of hepatocarcinogenesis. Hence, we have performed a detailed analysis of the gene expression profiles of several readily available HCC xenograft models. We chose three different carcinoma cell lines derived from HCC which are widely used and well published, i.e., Hep3B, HuH-7 and SK-Hep. The HuH-7 cell line was established from hepatoma tissue from a 57-year-old male Japanese patient with well-differentiated hepatocellular carcinoma. HuH-7 can be grown in serumfree RPMI 1640 supplemented with 3 10 8 M Na2SeO3 and maintains liver-specific functions like plasma protein production and expression of G6Pase and FDPase. When injected subcutaneously into nude mice these cells create tumors which histologically resemble the original hepatoma tissue of the patient [20]. HepG2 and Hep3B cell lines were established from hepatocellular carcinoma obtained during extended lobectomies of a 15-year-old caucasian male (HepG2) and a 8-year-old black male (Hep3B) in
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1975 and 1976, respectively. Both cell lines produce human albumin and a-fetoprotein (AFP). Hep3B cells additionally produce HBsAg. The amount of HBsAg is minute during exponential growth and increases in the stationary phase mimicking the expression of AFP. No viral particles were observed in both apparently healthy and necrotising cells or in the cellular debris [21]. SK-Hep was derived from the ascitic fluid of a patient with carcinoma of the liver [22]. This cell line is often referenced as a HCC cell line [23,24] despite the fact that several reports show that it does not have properties of hepatocytes and instead displays mainly features characteristic of endothelial origin [25]. The earliest animal tumor models were murine leukaemic cell lines (L1210 and P388) which were intraperitoneally injected into mice and gave rise to tumor ascites. These models were successful in identifying active compounds against leukaemias and some lymphomas, and in general mostly fast growing tumor entities [26]. However, they were not suitable for the identification of therapeutics against solid tumors [27]. Tymectomy of newborn mice allowed transplantation of human cells into mice but the feasibility of human-into-mouse xenografts was greatly enhanced after discovery of the nude mouse, a mouse which lacks hair and thymus development and thus adaptive immune response [28,29]. The nude mouse was soon found to be a suitable host for human tumor xenografts [22,30,31]. The gene responsible for this phenotype is FoxN1, a member of the winged-helix or forkhead family of transcription factors [32,33]. Another important mouse strain used in tumor xenografts is the SCID mouse which harbours a mutation of the Prkdc protein kinase leading to defects in VDJ recombination and DNA repair [34,35]. Both strains still have functional NK cells which impairs uptake of xenografts. Removal of the NK compartment can be achieved by anti-asialo antibody [36] or by backcrossing onto the NOD/Lt strain [37] leading to improved xenograft take rate and growth.
Table 1 Summary of microarray experiments Tissue/cell line
Method
Hepatocellular carcinoma Cirrhotic liver Non-neoplastic liver Hep3B Hep3B Hep3B HuH-7 HuH-7 HuH-7 SK-Hep SK-Hep Total
No. of microarray datasets 14 7 5
Tissue culture Subcutaneous graft Orthotopic graft
2 2
Tissue culture Subcutaneous graft Orthotopic graft
2 3
Tissue culture Subcutaneous graft
2 2
3
1
43
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2. Material and methods 2.1. Human samples Frozen human HCC, cirrhosis and non-neoplastic liver samples were obtained from the Biobank at the Medical University of Graz. All use of human material was approved by the Ethical Committee at the Medical University of Graz. H&E staining of cryosections were inspected by a pathologist (K. Zatloukal) and only samples containing the desired tissue type and tumor cell content were used for analysis. 2.2. Animal models Five- to six-week-old nu/nu mice were obtained from Harlan Winkelmann Germany. Hep3B (HB-8064) and SKHep (HTB-52) cell lines were obtained from the American Type Culture Collection (ATCC) and HuH-7 (01042712) was obtained from the European Collection of Cell Cultures (ECACC). All cell lines were cultured under standard cell culture conditions to sub-confluency. For tissue culture samples cells were trypsinized, washed and pelleted. To obtain subcutaneous xenografts 5 mio cells of each cell line were suspended in 100 ll PBS and injected subcutaneously laterally into 3 mice each. For orthotopic xenografts we opened the peritoneum and injected 5 mio cells suspended in PBS into the pole of the spleen. The spleen pole was then ligated and the peritoneum and the skin were closed with sutures. We could not obtain orthotopic grafts from SKHep cells and only one orthotopic graft for HuH-7 cells. All animal experimentation was approved by the Austrian Ministry for Science and Research. After 4 (SK-Hep) to 8 (Hep3B and HuH-7) weeks the tumors were harvested and processed by fixation in 4% buffered formalin or snap-frozen and stored in liquid nitrogen. 2.3. Microarrays RNA was extracted from cell pellets, xenografts and human tissue with TriZol Reagent (Invitrogen). All microarrays were obtained from Applied Biosystems (Human Genome Survey Microarray V2.0), had the same lot number, and were processed according to manufacturer’s instructions. 2.4. Data analysis Gene expression data was subsequently analyzed with Genespring software (Agilent Technologies). First, we selected genes which are deregulated in human HCC but not in cirrhosis when normalized to non-neoplastic liver. To do this, we performed ANOVA (p = 0.05) comparing HCC and non-neoplastic livers (NNL) and subtracting from this list the genes similarly deregulated between cirrhosis and NNL. In total, 2635 genes were specifically deregulated in our set of HCC tumor samples. We then performed hierarchical clustering based on Pearson correlation coefficients to group genes on the basis of similarity in the pattern over all tissues and tissues on the basis of similarity in the pattern over all genes.
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2.5. Immunostaining Tissues were fixed in formalin, paraffin embedded and 1.5-lm sections were cut. Slides were deparaffinized and boiled in citrate buffer at pH 6 for 30 min. After blockade
of endogenous peroxidases by immersion in 3% hydrogen peroxide in methanol, primary antibody for GLUL (BD Biosciences, Cat. No. 10702024) was applied in 1:200 dilution and subsequently visualized with DAKO Envision system.
Fig. 1. Analysis of microarray data. Gene expression profiles were generated with ABI Human Genome Survey V2.0 Microarrays, data were normalized to non-neoplastic liver and genes deregulated in human HCC but not in cirrhosis were selected by ANOVA (p = 0.05). (a) Hierarchical clustering of the 2635 most deregulated genes showing xenografts from each separate cell line forming a cluster together. (ortho, orthotopic; sc, subcutaneous and tc, tissue culture). (b) PCA of this gene list demonstrates the degree of representation of the gene expression pattern of HCC by the models. The Hep3B xenografts and cells are closely clustered together and nearest to HCC indicating the best representation of human HCC deregulated genes by these models.
K. Kashofer et al. / Cancer Letters 286 (2009) 121–128 3. Results For our experiments, we chose the nude/nude mouse (FoxN1 / ) as it has been extensively used in the past and is a good compromise with respect to immunodeficiency, meaning it readily takes human xenografts but still has some resistance mechanisms against infections, so it is not too difficult to maintain a colony in a standard SPF facility. The experimental details of the xenografts are described in Section 2. In addition to the experimental models we also selected 10 human hepatocellular carcinomas, 5 samples with liver cirrhosis and 5 samples from normal non-neoplastic livers to be processed on the same microarray platform as the xenograft tumor samples to maximize the comparability of the results. The details of our collection of samples (n = 43) are given in Table 1. We performed the same microarray analysis with the ABI Whole Genome Survey V2.0 on all the samples and used Genespring software to analyze the resulting gene expression values. As can be seen in Fig. 1a, all animal models cluster together according to the cell line used with the HCC samples clustering close to the xenografts. This indicates that the choice of the cell line used in the transplant is more important than the site of implantation. We then performed principal component analysis (PCA) on the sample groups shown in Fig. 1b. The PCA plots sample groups on two axes according to the similarity of their expression of two gene groups (components) when compared to each other. Cirrhosis and NNL samples were excluded from the PCA as the genes evaluated by PCA were specifically deregulated in HCC. Fig. 1b clearly shows that all xenograft tumors derived from Hep3B cells clustered closer to human HCC than the other samples. It can thus be speculated that from the three models analyzed in our dataset Hep3B
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xenografts show the best overall representation of genes deregulated in HCC. It is also interesting that the gene expression values of the three different types of tissue, cells from tissue culture, subcutaneous xenografts and orthotopic xenografts did not show a great variation in grafts from the Hep3B cell line. With the HuH-7 cell line and also in the SK-Hep model the differences of the gene expression values in the principal components seem to be much greater than with Hep3B. We conclude that the identity of the cell line is not only important for the overall representation of genes deregulated in HCC, but does also influence the choice of the model best suited for an experiment. However, one has to keep in mind that the PCA is strongly dependent on the parameters used for derivation of the principal components, possibly leading to different conclusions if the gene set of interest is restricted by other experimental parameters. In the next step, we selected 57 genes from the aforementioned list which were additionally deregulated between HCC and the xenograft samples thus highlighting differences between the human primary tumor samples and the cell line xenografts. Upon close inspection of the heatmap generated with this set of genes several features of the xenograft tumors could be determined. One cluster of genes highly upregulated in HCC while being silent in normal liver tissue and cirrhosis contains glutamine synthetase (GLUL). GLUL upregulation in HCC is associated with bcatenin-activating mutations [38], however b-catenin does not seem to be the only activator of GLUL in HCC [39]. As is evident in the heatmap in Fig. 2, the overexpression of GLUL in HCC was not reproduced in any of the animal models used in this study. We performed immunohistochemistry to prove this hypothesis. Fig. 3 shows immunostaining for GLUL on tumor tissues from HCC (Fig. 3a–c) which were clearly positive for GLUL expression. Fig. 3d–f shows immunostaining on xenograft samples. Obvi-
Fig. 2. Differences in xenograft models: 53 genes which are deregulated in HCC but not in cirrhosis, and also strongly differentially deregulated between the xenograft models were selected by ANOVA (p = 0.05). The heatmap represents genes which are upregulated (red) or downregulated (green). Three distinct clusters of genes can be recognized showing genes which are not represented by either model (1) not represented by the SK-Hep cell line (2) or not represented by the HuH-7 cell line (3).
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Fig. 3. Expression of GLUL in various tumor tissues: sections of formaldehyde-fixed, paraffin-embedded tumor samples were stained with glutamine synthase antibody (BD Biosciences). Three examples of human HCC (a–c), xenografts (d, Hep3B; e, HuH-7 and f, SK-Hep), murine tumors (g, MDR2 / ; h, DDC and i, DEN). Note the strong cytoplasmic reaction in the examples of human primary HCC, whereas all HCC xenografts were negative. ously the xenograft samples were negative for GLUL expression in concordance with the microarray data. Murine genetic or toxicological models however seem to also show the GLUL overexpression as can be seen for the MDR2 knockout model [40] (Fig. 3g), the DDC model (Fig. 3h) and the DEN model [41] (Fig. 3i). A second cluster of genes in the heatmap in Fig. 2 separates the SK-Hep-1 cell line from all the other xenografts and also from HCC. This cluster consisting of eight genes includes a-fetoprotein (AFP) the lack of expression of which further corroborates the notion that SK-Hep is not of hepatocellular origin. The third cluster of genes remarkable in Fig. 2 is a cluster consisting of PAGE3 and two genes of the MAGE family, which are also known as cancer-testis genes. This group of genes represents members of a category of tumor-specific antigens which share several distinct features. They are expressed predominantly in tumors of different origins and in testis but not in other normal tissues, and the genes encoding the CT antigens locate to the X chromosome [42]. The overexpression of these genes is not represented by the HuH-7 cell line and its xenografts suggesting that HuH-7 might be a poor choice for studies focusing on immunotherapy. Using the KEGG database [43–45] we have further evaluated the differences in gene expression of specific pathways in HCC and the xenotransplantation models. The activation of the Ras/Raf/MEK/ERK pathway in HCC is clearly visible in the ErbB signaling pathway depicted in Supplemental Fig. 1. The genes of the RAS/Raf/MEK/ERK pathway were strongly upregulated in human HCC and cirrhosis of different etiologies when compared to normal liver. This deregulation was mirrored in some but not all of the transplantation models. For instance, epiregulin (EPR), a potent vascular smooth muscle-derived mitogen [46], was upregulated in Hep3B and SK-Hep models, while it was not deregulated in HCC and downregulated in all HuH-7 models. Amphiregulin (AR), a major autocrine growth factor for cultured cells, was highly upregulated in the Hep3B cell line both in vitro and in vivo but only in vitro in HuH-7 cells. In human samples, AR was not deregulated and repressed in the in vivo models of HuH-7 and SK-Hep. The growth factor receptor ErbB-3 also showed a diverse expression pattern in the HCC models. It was not detectable in SK-Hep cells, but upregulated in human HCC and cirrhosis, and also in the HuH-7 and Hep3B cell lines. Expression of ErbB-3 also var-
ied depending on the model system used, being highest in tissue culture cells and lowest in the orthotopic xenograft. The transcription factor cmyc, the target of the map-kinase cascade, is highly upregulated in SKHep and Hep3B in all conditions and downregulated in the in vivo models of HuH-7 while ERK, the kinase phosphorylating c-myc, is highly expressed in all models as well as HCC and cirrhosis. We think that the analysis of the ErbB pathway regulation in different models can be useful in defining which of the models is most suitable to answer a given question. We propose that gene expression data should be consulted in the design phase of projects involving in vivo HCC models to ensure the validity of data generated in the model of choice.
4. Discussion In this paper, we demonstrated strengths and weaknesses of the in-vivo HCC xenograft models. The xenograft models which are widely used in laboratories clearly have limitations in their predictive power for the human diseases. However, we think that if these differences between the available models and primary human tumors are properly considered, it should be possible to select a model which is reliable for the particular field of interest. In general, the xenograft model in the nude mouse is a very suitable model for the evaluation of questions relating to the molecular pathology of HCC, and can also be used for preclinical evaluation of drug candidates. Although the orthotopic Hep3B xenograft model was most similar to primary human HCC, a general recommendation for the best model cannot be made since models have to be evaluated on the individual pathway level. Interestingly, the overall gene expression pattern was characteristic for the various cell
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lines investigated and revealed only minor changes under the different in vitro and in vivo experimental conditions suggesting a robust molecular phenotype. The gene expression dataset presented in this paper has been generated to aid researchers in making an informed decision which model is most appropriate for the needs of a planned experiment. All microarray data presented here are publicly available on our webpage (http://www. meduni-graz.at/pathology-graz/hcc (user = hcc_user, pass = xeno)).
[13]
[14]
[15]
[16]
Acknowledgements [17]
We thank Helmut Denk for helpful discussions and critical reviewing of the manuscript and we are grateful for the technical assistance of Daniela Pabst and Iris Kufferath. We also thank Christian Gülly for supervision of the microarray analysis which was performed at the Center for Medical Research, Medical University of Graz. We are grateful to Marcus Otte and ORIDIS Biomed for stimulating discussions. This work was financially supported by the EU-FP6 Project PONT (#505781) and the Austrian Genome Program GEN-AU. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.canlet.2008.11.011.
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Mini-review
The disease relevance of human hepatocellular xenograft models: Molecular characterization and review of the literature K. Kashofer a, M.M. Tschernatsch a, H.J. Mischinger b, F. Iberer c, K. Zatloukal a,* a b c
Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria Department of Surgery, Medical University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria Department of Transplantation Medicine, Medical University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria
a r t i c l e
i n f o
Article history: Received 14 July 2008 Accepted 4 November 2008
Keywords: Hepatocellular carcinoma HCC Xenografts Gene expression profiles
a b s t r a c t In recent years a number of new therapeutics has been developed that were not general toxins and inhibitors of cell division like classical chemotherapeutics, but were designed to target a specific pathway. A prerequisite for this development was the comprehensive characterization of molecular alterations occurring in human hepatocellular carcinoma (HCC). However, while much knowledge of the molecular pathogenesis of human HCC has been gained, the model systems used to test the functional relevance of these alterations and applied for preclinical evaluation of drug candidates are still poorly characterized. In this paper, we reviewed the literature about several commonly used HCC cell lines and xenotransplantation models and present our own data on the molecular characterization of these. Results obtained demonstrate that it is important to have a sound knowledge of the specific molecular constitution of the experimental model and to carefully evaluate the functional status of the pathway of interest. For this reason, we make the gene expression profiles publicly available to help researchers making an informed decision about which model to use. Ó 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinomas (HCCs) are a heterogeneous tumor entity with multiple etiologies and risk factors which may result in different pathways being deregulated in each tumor [1]. Although many molecular aberrations have recently been identified in HCC, the molecular pathways involved in hepatocarcinogenesis are still the target of intense research. In this paper, we present a review of the literature and our own data on the molecular characterization of several murine xenograft models focused on similarities to human HCC. In the last several years an improved understanding of the mechanisms of hepatocarcinogenesis has provided the opportunity to develop and study new therapeutic * Corresponding author. Tel.: +43 316 385 4404; fax: +43 316 384 329. E-mail addresses: [email protected], [email protected] (K. Zatloukal). 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.11.011
agents for advanced HCC. Many carcinogenic pathways like angiogenesis, altered signal transduction and aberrant cell cycle control are involved in HCC development [2,3]. In HCC, cancer cells are surrounded by stromal tissue containing extracellular matrix proteins (ECM), growth factors and proteolytic enzymes such as matrix metalloproteinases (MMPs) due to the underlying cirrhosis. It has been reported that these molecules play a key role in regulating the biological behavior and clinical outcome of HCC [4]. The expression of several EGF family members, specifically EGF, TGF-a and heparin binding epidermal growth, factor as well as EGFR have been described in several HCC cell lines and tissues [5,6]. Studies have led to a theory of an autocrine, paracrine and endocrine effect of TGF-a and EGFR/HER-1 on the proliferation of human HCC [7]. Despite that, treatment with the EGFR tyrosine kinase inhibitor erlotinib or monoclonal antibodies against EGFR have shown only modest or no benefit for the patient [8,9]. HCCs are tumors with increased levels of vascular endothelial
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growth factor (VEGF) and microvessel density [10]. VEGF represents an important therapeutic target in several malignancies [11] and there have been reports of modest clinical benefit in HCC patients if VEGF signaling is inhibited by an anti-VEGF antibody (bevacizumab) in combination with standard therapy [12]. However, the most promising compound which has made a big impact recently is sorafenib [13]. It is a novel signal transduction inhibitor that targets the Raf/mitogen-activated protein kinase-extracellular signal-related kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway and the VEGF receptor VEGFR-2 and PDFG receptor PDGFR-b. It has also shown promising clinical activity in HCC in a large phase II study with 137 patients [14]. More recently, a large multi-center international double-blind placebo controlled trial (SHARP) was conducted. In SHARP, researchers randomized 602 patients with Child–Pugh A cirrhosis and HCC to receive either placebo or sorafenib. It could be shown that sorafenib-treated patients survived 46.3 weeks as compared to 34.4 weeks in the placebo group. The study was stopped in February 2007 because results favoring sorafenib were found in the second planned interim analysis in October 2006. Based on this study, sorafenib seems to be the first systemic-targeted therapeutic agent for stabilization of HCC progression. The high specificity of drugs aimed against molecularly defined targets makes it increasingly important to have a good understanding of the molecular basis of the development of hepatocellular carcinoma. In recent years, much insight into molecular pathways and their deregulation in HCC has been gained. This development should present possibilities to develop small molecule compounds which are targeted against key regulator pathways in HCC. However, the murine models used in preclinical assessment of these highly specific drugs are few in number and overall poorly understood. Clinical relevance of murine transplantation models can only be obtained if careful attention is paid to the experimental conditions. The cell lines used must be free of pathogens and the mice have to be kept under specific pathogen-free conditions (SPF), as contamination of either the grafted cells or the mice leads to natural killer cell (NK) activation and thus to changes in the tumor take rate and growth [15]. The National Cancer Institute (NCI) started a large screening study to select agents for evaluation as clinical candidates in 1955. Initially, three transplanted rodent tumor cell lines (Sarcoma 180, Carcinoma 755 and Leukaemia 1210) were used. The spectrum of models was later broadened and in 1975 the murine P388 leukaemia model was added. In the early eighties, human tumor xenografts were added to the panels [16]. In this study, 1085 compounds were screened in five murine and three xenograft models. It was confirmed that screening against a fast growing murine leukaemia (P388) was useful as an initial cost-effective prescreen which can be followed by more stringent human xenograft models. In 1995 another rodent tumor model, the hollow fiber assay, in which polyvinylidine fluoride fibers loaded with tumor cells were implanted subcutaneously and intraperitoneally [17],
was added to the NCI screen. Johnson et al., performed a retrospective comparison of the results from the testing of compounds in the in-vitro NCI screen, the hollow fiber assay and the standard xenograft models with respect to their ability to predict the activity of a compound in each other model and in phase II clinical trials [18]. In this study, no close correlation could be found between activity of a compound in a xenograft model derived from a particular tumor type and activity of the compound against the same human tumor type in patients. However, if a compound displayed activity in at least one-third of the tested xenograft models there was correlation with activity in at least some Phase II trials. This study highlights the problem that xenografts only represent a tumor from a single patient, while human cancer is known to be very heterogeneous. The group of Snorri Thorgeirsson performed a comparison of the expression profiles of several murine hepatocellular cancers with human HCC [19]. In this study, Lee et al., used a list of orthologous genes that were present both in the murine and the human microarrays and changed significantly in both systems (n = 1650). After clustering it could be shown that the group of murine tumors that arose in Myc Tgf-a transgenic mice or diethyl nitrosamine-induced mice which harbour extensive chromosomal damage are most similar to a group of human HCCs with poor survival prognosis. A second group of murine tumors from Myc, E2F1 and Myc + E2F1 transgenic mice were most similar to HCCs with better prognosis while a third subgroup of Acox1 / and ciprofibrate-induced HCCs did not correlate with many human HCCs. This demonstrates that the different mouse models mimic different subclasses of human HCC which may to a large extent be due to a similarity in the underlying biology of the disease. This also demonstrates the need to identify the mouse models that most closely resemble human HCC in general or a subclass of human HCC under investigation. The transplantation of human primary HCC or established HCC tumor cell lines into immunodeficient mice (the human-into-murine-xenograft model) is frequently used to generate human tumors in a model system for investigation in the laboratory. This model has been used in basic research to test the activity of novel anti-cancer compounds and also as a general model of hepatocarcinogenesis. Hence, we have performed a detailed analysis of the gene expression profiles of several readily available HCC xenograft models. We chose three different carcinoma cell lines derived from HCC which are widely used and well published, i.e., Hep3B, HuH-7 and SK-Hep. The HuH-7 cell line was established from hepatoma tissue from a 57-year-old male Japanese patient with well-differentiated hepatocellular carcinoma. HuH-7 can be grown in serumfree RPMI 1640 supplemented with 3 10 8 M Na2SeO3 and maintains liver-specific functions like plasma protein production and expression of G6Pase and FDPase. When injected subcutaneously into nude mice these cells create tumors which histologically resemble the original hepatoma tissue of the patient [20]. HepG2 and Hep3B cell lines were established from hepatocellular carcinoma obtained during extended lobectomies of a 15-year-old caucasian male (HepG2) and a 8-year-old black male (Hep3B) in
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1975 and 1976, respectively. Both cell lines produce human albumin and a-fetoprotein (AFP). Hep3B cells additionally produce HBsAg. The amount of HBsAg is minute during exponential growth and increases in the stationary phase mimicking the expression of AFP. No viral particles were observed in both apparently healthy and necrotising cells or in the cellular debris [21]. SK-Hep was derived from the ascitic fluid of a patient with carcinoma of the liver [22]. This cell line is often referenced as a HCC cell line [23,24] despite the fact that several reports show that it does not have properties of hepatocytes and instead displays mainly features characteristic of endothelial origin [25]. The earliest animal tumor models were murine leukaemic cell lines (L1210 and P388) which were intraperitoneally injected into mice and gave rise to tumor ascites. These models were successful in identifying active compounds against leukaemias and some lymphomas, and in general mostly fast growing tumor entities [26]. However, they were not suitable for the identification of therapeutics against solid tumors [27]. Tymectomy of newborn mice allowed transplantation of human cells into mice but the feasibility of human-into-mouse xenografts was greatly enhanced after discovery of the nude mouse, a mouse which lacks hair and thymus development and thus adaptive immune response [28,29]. The nude mouse was soon found to be a suitable host for human tumor xenografts [22,30,31]. The gene responsible for this phenotype is FoxN1, a member of the winged-helix or forkhead family of transcription factors [32,33]. Another important mouse strain used in tumor xenografts is the SCID mouse which harbours a mutation of the Prkdc protein kinase leading to defects in VDJ recombination and DNA repair [34,35]. Both strains still have functional NK cells which impairs uptake of xenografts. Removal of the NK compartment can be achieved by anti-asialo antibody [36] or by backcrossing onto the NOD/Lt strain [37] leading to improved xenograft take rate and growth.
Table 1 Summary of microarray experiments Tissue/cell line
Method
Hepatocellular carcinoma Cirrhotic liver Non-neoplastic liver Hep3B Hep3B Hep3B HuH-7 HuH-7 HuH-7 SK-Hep SK-Hep Total
No. of microarray datasets 14 7 5
Tissue culture Subcutaneous graft Orthotopic graft
2 2
Tissue culture Subcutaneous graft Orthotopic graft
2 3
Tissue culture Subcutaneous graft
2 2
3
1
43
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2. Material and methods 2.1. Human samples Frozen human HCC, cirrhosis and non-neoplastic liver samples were obtained from the Biobank at the Medical University of Graz. All use of human material was approved by the Ethical Committee at the Medical University of Graz. H&E staining of cryosections were inspected by a pathologist (K. Zatloukal) and only samples containing the desired tissue type and tumor cell content were used for analysis. 2.2. Animal models Five- to six-week-old nu/nu mice were obtained from Harlan Winkelmann Germany. Hep3B (HB-8064) and SKHep (HTB-52) cell lines were obtained from the American Type Culture Collection (ATCC) and HuH-7 (01042712) was obtained from the European Collection of Cell Cultures (ECACC). All cell lines were cultured under standard cell culture conditions to sub-confluency. For tissue culture samples cells were trypsinized, washed and pelleted. To obtain subcutaneous xenografts 5 mio cells of each cell line were suspended in 100 ll PBS and injected subcutaneously laterally into 3 mice each. For orthotopic xenografts we opened the peritoneum and injected 5 mio cells suspended in PBS into the pole of the spleen. The spleen pole was then ligated and the peritoneum and the skin were closed with sutures. We could not obtain orthotopic grafts from SKHep cells and only one orthotopic graft for HuH-7 cells. All animal experimentation was approved by the Austrian Ministry for Science and Research. After 4 (SK-Hep) to 8 (Hep3B and HuH-7) weeks the tumors were harvested and processed by fixation in 4% buffered formalin or snap-frozen and stored in liquid nitrogen. 2.3. Microarrays RNA was extracted from cell pellets, xenografts and human tissue with TriZol Reagent (Invitrogen). All microarrays were obtained from Applied Biosystems (Human Genome Survey Microarray V2.0), had the same lot number, and were processed according to manufacturer’s instructions. 2.4. Data analysis Gene expression data was subsequently analyzed with Genespring software (Agilent Technologies). First, we selected genes which are deregulated in human HCC but not in cirrhosis when normalized to non-neoplastic liver. To do this, we performed ANOVA (p = 0.05) comparing HCC and non-neoplastic livers (NNL) and subtracting from this list the genes similarly deregulated between cirrhosis and NNL. In total, 2635 genes were specifically deregulated in our set of HCC tumor samples. We then performed hierarchical clustering based on Pearson correlation coefficients to group genes on the basis of similarity in the pattern over all tissues and tissues on the basis of similarity in the pattern over all genes.
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2.5. Immunostaining Tissues were fixed in formalin, paraffin embedded and 1.5-lm sections were cut. Slides were deparaffinized and boiled in citrate buffer at pH 6 for 30 min. After blockade
of endogenous peroxidases by immersion in 3% hydrogen peroxide in methanol, primary antibody for GLUL (BD Biosciences, Cat. No. 10702024) was applied in 1:200 dilution and subsequently visualized with DAKO Envision system.
Fig. 1. Analysis of microarray data. Gene expression profiles were generated with ABI Human Genome Survey V2.0 Microarrays, data were normalized to non-neoplastic liver and genes deregulated in human HCC but not in cirrhosis were selected by ANOVA (p = 0.05). (a) Hierarchical clustering of the 2635 most deregulated genes showing xenografts from each separate cell line forming a cluster together. (ortho, orthotopic; sc, subcutaneous and tc, tissue culture). (b) PCA of this gene list demonstrates the degree of representation of the gene expression pattern of HCC by the models. The Hep3B xenografts and cells are closely clustered together and nearest to HCC indicating the best representation of human HCC deregulated genes by these models.
K. Kashofer et al. / Cancer Letters 286 (2009) 121–128 3. Results For our experiments, we chose the nude/nude mouse (FoxN1 / ) as it has been extensively used in the past and is a good compromise with respect to immunodeficiency, meaning it readily takes human xenografts but still has some resistance mechanisms against infections, so it is not too difficult to maintain a colony in a standard SPF facility. The experimental details of the xenografts are described in Section 2. In addition to the experimental models we also selected 10 human hepatocellular carcinomas, 5 samples with liver cirrhosis and 5 samples from normal non-neoplastic livers to be processed on the same microarray platform as the xenograft tumor samples to maximize the comparability of the results. The details of our collection of samples (n = 43) are given in Table 1. We performed the same microarray analysis with the ABI Whole Genome Survey V2.0 on all the samples and used Genespring software to analyze the resulting gene expression values. As can be seen in Fig. 1a, all animal models cluster together according to the cell line used with the HCC samples clustering close to the xenografts. This indicates that the choice of the cell line used in the transplant is more important than the site of implantation. We then performed principal component analysis (PCA) on the sample groups shown in Fig. 1b. The PCA plots sample groups on two axes according to the similarity of their expression of two gene groups (components) when compared to each other. Cirrhosis and NNL samples were excluded from the PCA as the genes evaluated by PCA were specifically deregulated in HCC. Fig. 1b clearly shows that all xenograft tumors derived from Hep3B cells clustered closer to human HCC than the other samples. It can thus be speculated that from the three models analyzed in our dataset Hep3B
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xenografts show the best overall representation of genes deregulated in HCC. It is also interesting that the gene expression values of the three different types of tissue, cells from tissue culture, subcutaneous xenografts and orthotopic xenografts did not show a great variation in grafts from the Hep3B cell line. With the HuH-7 cell line and also in the SK-Hep model the differences of the gene expression values in the principal components seem to be much greater than with Hep3B. We conclude that the identity of the cell line is not only important for the overall representation of genes deregulated in HCC, but does also influence the choice of the model best suited for an experiment. However, one has to keep in mind that the PCA is strongly dependent on the parameters used for derivation of the principal components, possibly leading to different conclusions if the gene set of interest is restricted by other experimental parameters. In the next step, we selected 57 genes from the aforementioned list which were additionally deregulated between HCC and the xenograft samples thus highlighting differences between the human primary tumor samples and the cell line xenografts. Upon close inspection of the heatmap generated with this set of genes several features of the xenograft tumors could be determined. One cluster of genes highly upregulated in HCC while being silent in normal liver tissue and cirrhosis contains glutamine synthetase (GLUL). GLUL upregulation in HCC is associated with bcatenin-activating mutations [38], however b-catenin does not seem to be the only activator of GLUL in HCC [39]. As is evident in the heatmap in Fig. 2, the overexpression of GLUL in HCC was not reproduced in any of the animal models used in this study. We performed immunohistochemistry to prove this hypothesis. Fig. 3 shows immunostaining for GLUL on tumor tissues from HCC (Fig. 3a–c) which were clearly positive for GLUL expression. Fig. 3d–f shows immunostaining on xenograft samples. Obvi-
Fig. 2. Differences in xenograft models: 53 genes which are deregulated in HCC but not in cirrhosis, and also strongly differentially deregulated between the xenograft models were selected by ANOVA (p = 0.05). The heatmap represents genes which are upregulated (red) or downregulated (green). Three distinct clusters of genes can be recognized showing genes which are not represented by either model (1) not represented by the SK-Hep cell line (2) or not represented by the HuH-7 cell line (3).
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Fig. 3. Expression of GLUL in various tumor tissues: sections of formaldehyde-fixed, paraffin-embedded tumor samples were stained with glutamine synthase antibody (BD Biosciences). Three examples of human HCC (a–c), xenografts (d, Hep3B; e, HuH-7 and f, SK-Hep), murine tumors (g, MDR2 / ; h, DDC and i, DEN). Note the strong cytoplasmic reaction in the examples of human primary HCC, whereas all HCC xenografts were negative. ously the xenograft samples were negative for GLUL expression in concordance with the microarray data. Murine genetic or toxicological models however seem to also show the GLUL overexpression as can be seen for the MDR2 knockout model [40] (Fig. 3g), the DDC model (Fig. 3h) and the DEN model [41] (Fig. 3i). A second cluster of genes in the heatmap in Fig. 2 separates the SK-Hep-1 cell line from all the other xenografts and also from HCC. This cluster consisting of eight genes includes a-fetoprotein (AFP) the lack of expression of which further corroborates the notion that SK-Hep is not of hepatocellular origin. The third cluster of genes remarkable in Fig. 2 is a cluster consisting of PAGE3 and two genes of the MAGE family, which are also known as cancer-testis genes. This group of genes represents members of a category of tumor-specific antigens which share several distinct features. They are expressed predominantly in tumors of different origins and in testis but not in other normal tissues, and the genes encoding the CT antigens locate to the X chromosome [42]. The overexpression of these genes is not represented by the HuH-7 cell line and its xenografts suggesting that HuH-7 might be a poor choice for studies focusing on immunotherapy. Using the KEGG database [43–45] we have further evaluated the differences in gene expression of specific pathways in HCC and the xenotransplantation models. The activation of the Ras/Raf/MEK/ERK pathway in HCC is clearly visible in the ErbB signaling pathway depicted in Supplemental Fig. 1. The genes of the RAS/Raf/MEK/ERK pathway were strongly upregulated in human HCC and cirrhosis of different etiologies when compared to normal liver. This deregulation was mirrored in some but not all of the transplantation models. For instance, epiregulin (EPR), a potent vascular smooth muscle-derived mitogen [46], was upregulated in Hep3B and SK-Hep models, while it was not deregulated in HCC and downregulated in all HuH-7 models. Amphiregulin (AR), a major autocrine growth factor for cultured cells, was highly upregulated in the Hep3B cell line both in vitro and in vivo but only in vitro in HuH-7 cells. In human samples, AR was not deregulated and repressed in the in vivo models of HuH-7 and SK-Hep. The growth factor receptor ErbB-3 also showed a diverse expression pattern in the HCC models. It was not detectable in SK-Hep cells, but upregulated in human HCC and cirrhosis, and also in the HuH-7 and Hep3B cell lines. Expression of ErbB-3 also var-
ied depending on the model system used, being highest in tissue culture cells and lowest in the orthotopic xenograft. The transcription factor cmyc, the target of the map-kinase cascade, is highly upregulated in SKHep and Hep3B in all conditions and downregulated in the in vivo models of HuH-7 while ERK, the kinase phosphorylating c-myc, is highly expressed in all models as well as HCC and cirrhosis. We think that the analysis of the ErbB pathway regulation in different models can be useful in defining which of the models is most suitable to answer a given question. We propose that gene expression data should be consulted in the design phase of projects involving in vivo HCC models to ensure the validity of data generated in the model of choice.
4. Discussion In this paper, we demonstrated strengths and weaknesses of the in-vivo HCC xenograft models. The xenograft models which are widely used in laboratories clearly have limitations in their predictive power for the human diseases. However, we think that if these differences between the available models and primary human tumors are properly considered, it should be possible to select a model which is reliable for the particular field of interest. In general, the xenograft model in the nude mouse is a very suitable model for the evaluation of questions relating to the molecular pathology of HCC, and can also be used for preclinical evaluation of drug candidates. Although the orthotopic Hep3B xenograft model was most similar to primary human HCC, a general recommendation for the best model cannot be made since models have to be evaluated on the individual pathway level. Interestingly, the overall gene expression pattern was characteristic for the various cell
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lines investigated and revealed only minor changes under the different in vitro and in vivo experimental conditions suggesting a robust molecular phenotype. The gene expression dataset presented in this paper has been generated to aid researchers in making an informed decision which model is most appropriate for the needs of a planned experiment. All microarray data presented here are publicly available on our webpage (http://www. meduni-graz.at/pathology-graz/hcc (user = hcc_user, pass = xeno)).
[13]
[14]
[15]
[16]
Acknowledgements [17]
We thank Helmut Denk for helpful discussions and critical reviewing of the manuscript and we are grateful for the technical assistance of Daniela Pabst and Iris Kufferath. We also thank Christian Gülly for supervision of the microarray analysis which was performed at the Center for Medical Research, Medical University of Graz. We are grateful to Marcus Otte and ORIDIS Biomed for stimulating discussions. This work was financially supported by the EU-FP6 Project PONT (#505781) and the Austrian Genome Program GEN-AU. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.canlet.2008.11.011.
[18]
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[20]
[21]
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