ORIGINAL ARTICLE

Pancreatic Cancer Induced by In Vivo Electroporation-Enhanced Sleeping Beauty Transposon Gene Delivery System in Mouse June-Shine Park,* Kyung-Min Lim, PhD,Þ Sung Goo Park, PhD,þ Sun Young Jung, BS,§ Hyun-Ji Choi, BS,§ Do Hee Lee, PhD,|| Woo-Jin Kim,¶ Seung-Mo Hong, MD, PhD,# Eun-Sil Yu, MD, PhD,# and Woo-Chan Son, DVM, PhD§

Objective: The aim of this study was to establish a pancreatic tumor model of mouse using the electroporation-enhanced Sleeping Beauty (SB) transposon system. Methods: The SB transposon system was used in conjunction with electroporation to deliver oncogenes, c-Myc and HRAS, and shRNA against p53 into the mouse pancreas to induce tumors. Oncogenes (c-Myc and HRAS) and shRNA against p53 gene were directly injected into the pancreas of the mouse along with in vivo electroporation applied on the injection site. The tumors were identified grossly and confirmed using animal positron emission tomographic imaging. The tumors were then characterized using histological and immunohistochemical techniques. The expression of the targeted genes (c-Myc, HRAS, and p53) was analyzed by a real-time quantitative polymerase chain reaction. Results: Pancreatic tumors were successfully induced. The tumor phenotype was a sarcomatoid carcinoma, which was verified through immunohistochemistry. Some cysts or duct-like structures suggested to be metaplastic acinar cells were visible in the induced tumor. Conclusions: The SB transposon enhanced with electroporation can readily generate pancreatic tumors in the mice, and thus, this model serves as a valuable resource for the mouse models of pancreatic cancer. Key Words: Sleeping Beauty transposon, electroporation, pancreatic tumor, mouse (Pancreas 2014;43: 614Y618)

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ancreatic cancer is the fourth leading cause of cancer death for both men and women.1 Approximately 75% of patients die within a year of diagnosis, and the 5-year survival rate has remained unchanged at 4% to 6% for 4 decades.2,3 In addition, only 20% of the diagnosed cases are surgically operable, and approximately 50% are found to be metastatic.4

From the *Asan Institute for Life Sciences and College of Veterinary Medicine, Jeju National University, Jeju; †College of Pharmacy, Ewha Womans University, Seoul; ‡Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon; §Asan Institute for Life Sciences and Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center; ||Department of Biotechnology, Seoul Women’s University; ¶University of Ulsan College of Medicine; and #Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea. Received for publication April 29, 2013; accepted December 12, 2013. Reprints: Woo-Chan Son, DVM, PhD, Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul, 138-736, Korea (e-smooth muscle actin (1:400) for leiomyosarcoma; pan-cytokeratin (1:3000), cytokeratin 20 (1:200), and cytokeratin 7 (1:4000) for undifferentiated carcinoma; and cyclin-dependent kinase 4 (CDK4, 1:1000) and murine double minute 2 (MDM2, 1:1000) for pleomorphic liposarcoma.

Analysis of Gene Expression by Real-Time Quantitative Polymerase Chain Reaction The expression of the transferred oncogenes, c-Myc and HRAS, and that of p53 were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). Samples were obtained from the pancreas tumor tissue and normal pancreas tissue. The Paradise Whole Transcript RT Reagent System (Arcturus, Calif ) was used for the RNA isolation and reverse transcription of the samples. All PCR reactions were performed in a Lightcycler 2.0 (Roche Applied Science). The primers (all purchased from Roche Diagnostics, Mannheim, Germany) used for the identification of the transcript levels were as follows: for c-Myc forward, TCC TGT ACC TCG TCC GAT TC; for c-Myc reverse, GGA GGA CAG CAG CGA GTC; for HRAS forward, GGA CGA ATA CGA CCC CAC TAT; for HRAS reverse, TGT CCA ACA GGC ACG TCT C; for p53 forward, TAA AGG ATG CCC GTG CTG; for p53 reverse, TCT TGG TCT TCG GGT AGC TG; the probes #12, #38, and #94 were individually from the Universal Probe Library (Roche Applied Science). Mouse

TABLE 1. Immunohistochemical Profile of Tumor Developed in the Pancreas Tissue Marker

Immunohistochemical Antibody

Epithelial tissue marker

Pan-cytokeratin Cytokeratin 7 Cytokeratin 20 Myogenin Desmin Smooth muscle actin CD45 CD163 CD68 Melanoma S100 CDK4 MDM2

Muscular tissue marker

Hematopoietic cell marker

Melanoma marker Adipose tissue marker

Reactivity

Dilution

Type

+ j j j j j j j j j j j j

1:1000 1:1000 1:1000 1:1000 1:4000 1:1000 1:2000 1:2000 1:2000 1:100 1:400 1:1000 1:1000

M P M M M M M M M M M M M

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All antibodies were purchased from Abcam (Cambridge, England). M, monoclonal; P, polyclonal.

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RESULTS Tumor Observation Although the mice were nontransgenic, all recipients subjected to the injection with transposons and SB transposase followed by in vivo electroporation developed single or coupled and nodular neoplasms in the pancreas approximately 3 weeks after injection. Tumor-bearing animals exhibiting bulging abdomen were easily recognized through observation. Decedents were seen on week 4 and, subsequently, the rest of the animals died by week 6.

Gross and Microscopic Findings Well-demarcated, ovoid nodules were found at the pancreas where the plasmid DNA was injected. Tumor masses were only present at injected sites. By contrast, no metastases were evident. The tumors showed high cellularity with both epithelial and mesenchymal components as well as abundant mitotic and apoptotic cells. Tumor cells displayed undifferentiated pleomorphic features. The cells were round to oval with pale basophilic cytoplasms and hyperchromatic nuclei with prominent nucleoli. There were some cysts or duct-like structures found in a single epithelial layer along the lumen, which resembled disorganized acinar cells. These were considered to be metastatic acinar cells. The central region of the tumors showed necrosis. All tumors had the same morphological features (Fig. 1A and B).

Immunohistochemical Findings As summarized in Table 1, only pan-cytokeratin was found to be positively expressed in the tumors, suggesting that the tumors were epithelial in origin. Because other immunohistochemical markers were negative, we diagnosed the tumors as sarcomatoid carcinomas (Fig. 1C).

Animal PET Imaging Coronal, sagittal, and transverse PET computed tomographic imaging showed the presence of neoplasms at the injection sites of the pancreas. The tumors indicated by red-pinkish color were apparently visible (Fig. 2).

Confirmation of Gene Expression by Quantitative RT-PCR Analysis Quantitative RT-PCR showed successful expression of the transferred genes c-Myc and HRAS. Expression of HRAS was increased in the tumor tissue compared with that in the pancreas tissue, and that of c-Myc was increased more than 20-fold in the tumor tissue. In addition, p53 decreased 7-fold in the tumor tissue compared with that in the pancreas tissue (Fig. 3). FIGURE 1. Microscopic image of pancreatic tumor tissue. A, Induced pancreatic tumor (original magnification, 100). Tumors consisted of highly pleomorphic and undifferentiated cells. Some of the tumor cells resembled epithelial cells, and others appeared to be of mesenchymal cell origin. B, Cysts or duct-like structures, which resembled disorganized acinar cells, were seen in a single epithelial layer along the lumen (red arrows) (original magnification, 200). C, The only positive immunoreactivity the tumors exhibited was pan-cytokeratin, suggesting the epithelial origin of the tumor. Positively stained normal bile duct epithelium is indicated by blue arrows (original magnification, 200). Editor’s note: A color image accompanies the online version of this article.

GAPDH gene (forward, GAG CCA AAC GGG TCA TCA; reverse, CAT ATT TCT CGT GGT TCA CAC C), analyzed by the TaqMan probe (TIB MOLBIOL, Berlin, Germany), was used as a ‘‘reference gene’’ to normalize gene expression. For reproducibility, triplicate experiments were performed.

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DISCUSSION Tumorigenesis is understood to involve a stepwise acquisition of mutations over time. This understanding provides a basic framework for the field of cancer genetics, but animal cancer model to precisely identify the gene mutations that contribute to each individual tumor is lacking.13 Therefore, in the current study, 2 oncogenes (c-Myc and HRAS) and shRNA against p53 were directly introduced into the pancreas using the SB transposon system and in vivo electroporation to establish a novel pancreatic cancer model in mouse. All mice subjected to the injection produced pancreatic tumors. Subsequent analysis revealed that the induced tumors were positive only for pan-cytokeratin, although the general microscopic morphology mimics sarcoma, indicating a sarcomatoid carcinoma. The pancreatic tumors produced in this study showed highly malignant features under microscopic analysis. However, we failed to identify any metastases to other organs, contrary * 2014 Lippincott Williams & Wilkins

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Pancreatic Tumor Induction by SB System in Mice

FIGURE 2. PET imaging of induced pancreatic tumors. Neoplasms were evident in coronal (A), sagittal (B), and transverse (C) dimensions of the PET computed tomographic images of the injection sites. Tumors are indicated by red-pinkish color (arrows). Editor’s note: A color image accompanies the online version of this article.

to the previous study that used the SB transposon method for genetic modification.13 Unlike our methods, integration of the SB transposon into the host chromosomal DNA was possible in other sites, declared to be metastases, in previous cases involving blastocyst injection method. Acinar-ductal metaplasia is currently suggested to be one of the precursor lesions of pancreatic tumor.5 As previously reported, the KRAS mutation is found in 90% of pancreatic tumors.5 An earlier study conducted in mice, which used the KRAS mutation, showed the presence of acinar-ductal metaplasia.14 Further research into the role of KRAS and acinarductal metaplasia should be conducted to determine their in fluence on pancreatic cancer. We suggest that subsequent studies with mice killed gradually over time would allow us to carefully monitor the changes in acinar-ductal metaplasia lesions, uncovering the progression of pancreatic cancer more systematically. Tumor animal models are of great value in cancer research for the preclinical testing of new cancer therapeutics and for further understanding of initiating factors and tumor origins.10 The commonly used xenograft models and genetically engineered mice cannot sufficiently mimic human cancer progression15 because they do not exactly reflect genetic backgrounds, different genetic variants/mutations, and subsequent protein expression patterns in human.16Y18 A distinctive feature of the human cancer is its genetic complexity, often involving a number of different mutations.19 The complexity of such genetic alterations is a key aspect of the different types of cancers, each exhibiting distinct histological features, and makes it possible to explain the heterogeneous nature of a given type of neoplasm.19 Our SB-based transposon system can be used for the production of genetically modified mouse models that are tailored to individual experiment because specific gene-induced tumorigenesis can be easily performed. This should allow researchers to evaluate the therapeutic effects of antitumor drugs that suppress oncogenes in vivo more efficiently. Conventionally, tumor mouse models created by SB transposon-mediated genetic modification have used hydrodynamic methods, such as injecting a bolus of SB transposon and transposase into the tail vein and exploiting back-pressure to produce tumors in the liver.11 In this study, we demonstrated that the SB transposon system in conjunction with in vivo electroporation successfully induces pancreatic tumors in mice. Compared with hydrodynamic methods, electroporation is a relatively simple and easy-to-do method that is suitable for overcoming the low integration efficiency of the SB transposon system. Gene transfers have traditionally been carried out by * 2014 Lippincott Williams & Wilkins

using gene therapy agents, such as viral vectors and cationic liposomes.20Y28 Electroporation, with a high transfection efficiency and low cytotoxicity,29 is as useful as those other gene transfer methods.30,31 Various types of electrodes developed for different kinds of situations have enabled the current study.32Y37 These electrodes, which are positioned in the target tissue, administer short and intense electrical pulses that make it possible to produce mutagenesis in the target sites where traditional methods may be difficult.38 With this method, tumors can be produced in the lung, mammary gland, prostate, and pancreas, among others, where cancers have not yet been generated using the SB transposon system.39 Conversely, the SB transposon system compensates for the decrease in the level of gene expression after the injection of DNA using electroporation alone because the majority of the DNA is degraded in cells or cleared from the circulation.40 In conclusion, our results suggest a new tool of developing laboratory tumor models with genetic complexity, involving a number of different mutations that can give rise to various histological subtypes, a feat that is difficult in current mouse tumor model systems.15Y19 Especially, in vivo electroporationenhanced SB transposon method can produce diverse gene mutations embodied in a tumor, which resembles human cancers that involve multiple gene mutations.

FIGURE 3. Analysis of gene expression by RT-qPCR. Expression of the transferred genes c-Myc, HRAS, and p53 was analyzed by RT-qPCR as described in the Materials and Methods section. The graph shows the representative RT-qPCR results (each gene plus A-actin as a control). Data shown in the bar graph represent a mean T SEM obtained from 3 independent experiments. www.pancreasjournal.com

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ACKNOWLEDGMENT The authors thank VGX International, Seoul, Korea, for technical assistance. REFERENCES 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62:10Y29. 2. Hidalgo M. Pancreatic cancer. N Engl J Med. 2010;362:1605Y1617. 3. Vincent A, Herman J, Schulick R, et al. Pancreatic cancer. Lancet. 2011;378:607Y620. 4. Mann KM, Ward JM, Yew CCK, et al. Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma. Proc Natl Acad Sci U S A. 2012;109:5934Y5941. 5. Westphalen CB, Olive KP. Genetically engineered mouse models of pancreatic cancer. Cancer J. 2012;18:502Y510. 6. Sharpless NE, DePinho RA. The mighty mouse: genetically engineered mouse models in cancer drug development. Nat Rev Drug Discov. 2006;5:741Y754. 7. Huijbers IJ, Krimpenfort P, Berns A, et al. Rapid validation of cancer genes in chimeras derived from established genetically engineered mouse models. Bioessays. 2011;33:701Y710. 8. Ivics Z, Hackett PB, Plasterk RH, et al. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell. 1997;91:501Y510. 9. Belur LR, Podetz-Pedersen KM, Sorenson BS, et al. Inhibition of angiogenesis and suppression of colorectal cancer metastatic to the liver using the Sleeping Beauty Transposon System. Mol Cancer. 2011;10:14. 10. Howell VM. Sleeping BeautyVa mouse model for all cancers? Cancer Lett. 2012;317:1Y8. 11. Carlson CM, Frandsen JL, Kirchhof N, et al. Somatic integration of an oncogene-harboring Sleeping Beauty transposon models liver tumor development in the mouse. Proc Natl Acad Sci U S A. 2005;102:17059Y17064. 12. Dupuy AJ, Akagi K, Largaespada DA, et al. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature. 2005;436:221Y226. 13. Dupuy AJ, Rogers LM, Kim J, et al. A modified Sleeping Beauty transposon system that can be used to model a wide variety of human cancers in mice. Cancer Res. 2009;69:8150Y8156. 14. Shi G, Direnzo D, Qu C, et al. Maintenance of acinar cell organization is critical to preventing Kras-induced acinar-ductal metaplasia. Oncogene. 2013;32:1950Y1958. 15. Kim IS, Baek SH. Mouse models for breast cancer metastasis. Biochem Biophys Res Commun. 2010;394:443Y447. 16. Thyagarajan T, Totey S, Danton MJ, et al. Genetically altered mouse models: the good, the bad, and the ugly. Crit Rev Oral Biol Med. 2003;14:154Y174. 17. Rivera J, Tessarollo L. Genetic background and the dilemma of translating mouse studies to humans. Immunity. 2008;28:1Y4. 18. Radiloff DR, Rinella ES, Threadgill DW. Modeling cancer patient populations in mice: complex genetic and environmental factors. Drug Discov Today Dis Models. 2008;4:83Y88. 19. Andrechek ER, Nevins JR. Mouse models of cancers: opportunities to address heterogeneity of human cancer and evaluate therapeutic strategies. J Mol Med (Berl). 2010;88:1095Y1100.

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Pancreatic cancer induced by in vivo electroporation-enhanced sleeping beauty transposon gene delivery system in mouse.

The aim of this study was to establish a pancreatic tumor model of mouse using the electroporation-enhanced Sleeping Beauty (SB) transposon system...
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