# BUILDING STRATEGIES FOR PORCINE CANCER MODELS

EDITED BY : Tiago Collares, Fabiana K. Seixas, Laurie Rund and Lawrence B. Schook PUBLISHED IN : Frontiers in Genetics

#### Frontiers Copyright Statement

© Copyright 2007-2018 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA ("Frontiers") or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers.

The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. For the conditions for downloading and copying of e-books from Frontiers' website, please see the Terms for Website Use. If purchasing Frontiers e-books from other websites or sources, the conditions of the website concerned apply.

Images and graphics not forming part of user-contributed materials may not be downloaded or copied without permission.

Individual articles may be downloaded and reproduced in accordance with the principles of the CC-BY licence subject to any copyright or other notices. They may not be re-sold as an e-book.

As author or other contributor you grant a CC-BY licence to others to reproduce your articles, including any graphics and third-party materials supplied by you, in accordance with the Conditions for Website Use and subject to any copyright notices which you include in connection with your articles and materials.

All copyright, and all rights therein, are protected by national and international copyright laws.

The above represents a summary only. For the full conditions see the Conditions for Authors and the Conditions for Website Use. ISSN 1664-8714 ISBN 978-2-88945-650-5 DOI 10.3389/978-2-88945-650-5

#### About Frontiers

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

#### Frontiers Journal Series

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

#### Dedication to Quality

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

#### What are Frontiers Research Topics?

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office<EMAIL_ADDRESS>
# BUILDING STRATEGIES FOR PORCINE CANCER MODELS

Topic Editors:

Tiago Collares, Federal University of Pelotas, Brazil Fabiana K. Seixas, Federal University of Pelotas, Brazil Laurie Rund, University of Illinois at Urbana-Champaign, United States Lawrence B. Schook, University of Illinois at Chicago, United States

Cover image: Volodymyr Burdiak/Shutterstock.com

The eBook *Building Strategies for Porcine Cancer Models* presents a series of articles demonstrating the state-of-the-art developments in pig models for cancer research. Renowned researchers dedicated to the reproduction, genomic and biological engineering of the pig model for biomedicine contribute to this special research area. Although advances in these areas are occurring at surprising speeds, they are still far from realizing all the potential benefits that this biological model could provide to science. The current biomedical models may limit the frontier of knowledge in the cancer research.

Citation: Collares, T., Seixas, F. K., Rund, L., Schook, L. B., eds (2018). Building Strategies for Porcine Cancer Models. Lausanne Frontiers Media. doi: 10.3389/978-2-88945-650-5

# Table of Contents

*04 Editorial: Building Strategies for Porcine Cancer Models*

Tiago Collares, Fabiana K. Seixas, Laurie A. Rund and Lawrence B. Schook

### SECTION 1

#### STRATEGIES BASED ON PORCINE GENOMIC INFORMATION

Lawrence B. Schook, Laurie Rund, Karine R. Begnini, Mariana H. Remião, Fabiana K. Seixas and Tiago Collares

### SECTION 2

#### BREAST CANCER, LEUKEMIAS, CUTANEOUS AND CANCER VACCINE MODELS

Nana H. Overgaard, Thomas M. Frøsig, Simon Welner, Michael Rasmussen, Mette Ilsøe, Maria R. Sørensen, Mads H. Andersen, Søren Buus and Gregers Jungersen

# Editorial: Building Strategies for Porcine Cancer Models

Tiago Collares 1 \*, Fabiana K. Seixas 1 , Laurie A. Rund2 and Lawrence B. Schook 2,3

*1 Laboratory of Cancer Biotechnology, Biotechnology Postgraduate Program, Technology Development Center, Federal University of Pelotas, Pelotas, Brazil, 2 Animal Science, University of Illinois at Urbana-Champaign, Champaign, IL, United States, 3 Department of Radiology, University of Illinois at Chicago, Chicago, IL, United States*

Keywords: oncopig, pig models, genome editing technologies, swine, cancer models

**Editorial on the Research Topic**

**Building Strategies for Porcine Cancer Models**

#### INTRODUCTION

Pigs are proven models for biomedical studies due to their anatomical and physiological similarities with humans. Pig models have increasingly been validated for mimicking human diseases (Prather, 2013). The sequencing of the swine genome has demonstrated genetic similarities with humans (Groenen et al., 2012).

These considerations led Schook et al. (2015) to develop a transgenic swine model of cancer the oncopig cancer model (OCM). In addition, recent transcription profile studies of the oncopig's soft tissue sarcoma cells demonstrated altered TP53 signaling, activation of Wnt signaling, and epigenetic reprogramming—all transcriptional features found in human soft tissue sarcoma tumors (Schachtschneider et al., 2017a).

In addition to sarcomas, other cancers have been developed to date using the OCM platform: hepatocellular carcinoma (HCC) and pancreatic cancer (PA) (Schachtschneider et al., 2017c). Oncopig HCC acquired histopathological characteristics similar to human HCC [arginase expression and alpha-fetoprotein (AFP) secretion] and formed tumors after autologous injection (Schachtschneider et al., 2017b). In addition, human HCC transcriptional characteristics were also detected in porcine HCC (Schachtschneider et al., 2017b). In a review recently published by Segatto et al. pigs were proposed as a complementary platform for the discovery of new therapies against cancer through phenotypic screening of compounds, due to the metabolic, physiological, and genetic similarities of pigs with humans (Segatto et al., 2017).

On the research topic "Building strategies for porcine cancer models," seven papers have been published, with contributions from 40 authors from different institutions around the world, with a focus on molecular and cellular approaches for the development of porcine cancer models.

Watson et al. have reviewed the limitations of rodent models of cancer through a comparison of knockout mouse models to human patients. The authors have highlighted the advantages of using the swine as the biomedical model for cancer research, reviewing special aspects from the swine genome sequence and potential homologies to the human genome. They present the advantages of targeted gene editing using custom endonucleases—specifically TALENs and CRISPRs—and transposon systems, to make novel pig models of cancer with broad preclinical applications.

Schook et al. have discussed genetic modification technologies successfully used to produce porcine biomedical models, in particular the Cre-Lox system as well as the major advances and perspectives of the CRISPR/Cas9 system, highlighting its capacity to induce mutations at a chosen time and space, a characteristic that is especially important when creating genetic models of cancer. Recent advancements in porcine tumor modeling and genome editing will bring porcine models to the forefront of translational cancer research.

Duran-Struuck et al. have reviewed the limitations in using rodents to model human diseases, including the large differences in size, anatomy, physiology, drug metabolism, chromosome

Edited and reviewed by: *Geoff Clark, University of Louisville, United States*

> \*Correspondence: *Tiago Collares<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Genetics, a section of the journal Frontiers in Genetics*

Received: *30 May 2018* Accepted: *24 August 2018* Published: *02 October 2018*

#### Citation:

*Collares T, Seixas FK, Rund LA and Schook LB (2018) Editorial: Building Strategies for Porcine Cancer Models. Front. Genet. 9:377. doi: 10.3389/fgene.2018.00377* structures, and genetics. The authors have highlighted an exciting perspective from their experience with myeloid and lymphoid tumors in major histocompatibility complex characterized miniature swine and future approaches regarding the development of a large animal transplantable tumor model. This work covered the incidence of chronic myeloid leukemias in swine, highlighting the importance of genetic studies of these tumors that can provide a new platform for the development of novel human therapeutics for genetically similar human tumors. Moreover, they discussed the potential of swine models to study post-transplant lymphoproliferative disease (PTLD), since immunosuppressed swine present several characteristics that closely resemble human PTLD. The authors reported their attempt to establish an immortal cell line that could induce PTLD when inoculated into the same inbred line animals.

Gutierrez et al. have presented the potential applications and advantages of using pigs, particularly minipigs, as indispensable large animal models in fundamental and clinical research, including the development of therapeutics for inherited and chronic disorders including cancers. The authors have reviewed examples of naturally occurring conditions in pigs that closely mimic those affecting humans (like malignant spontaneously regressing melanomas, dwarf phenotype, and ventricular septal defect) as well as examples of induced swine models of diseases (for type I diabetes, obesity and metabolic syndromes, and liver cancer models) and established engineered pig models (for cystic fibrosis, heart arrhythmias, xenotransplants, and several types of cancer).

Overgaard et al. have shared original research regarding the use of pigs as a large animal model for cancer vaccine development. Their work demonstrated that the pig model is highly appropriate for addressing the questions related to optimal adjuvant composition and vaccine formulations. The authors investigated whether it is possible for pigs to generate immune responses to cancer antigens RhoC and IDO, using three different adjuvants (CAF09, CASAC, or ISA 51 VG). The results showed that all adjuvants tested were capable of generating

#### REFERENCES

some cytotoxic T lymphocytes (CTL) response to the cancer antigens following peptide immunization.

Clark et al. have demonstrated that BRCA1 inactivation in pig cells promotes transformation and thus serves as a model for human cancers. The authors established an immortalized porcine breast cell line and stably inactivated BRCA1 using miRNA. The cell line developed the characteristics of breast cancer stem cells and exhibited a transformed phenotype. These results validate the concept of using pigs as a model to study BRCA1 defects in breast cancer and establish the first porcine breast tumor cell line.

Bourneuf has reviewed melanoma genetics, discussing some of the most common mutations found in this type of tumor in humans. Examples of melanoma animal models have also been discussed, with emphasis on the porcine MeLiM model. This work suggests that the spontaneous tumor progression and regression occurring in these models could shed light on the interplay between endogenous retroviruses, melanomagenesis, and adaptive immune response.

#### CONCLUSION

Combined, the studies on this research topic have demonstrated that pigs are proven useful models for cancer studies including in (1) the development of genetically engineered pigs by using different technologies like TALENs, CRISPRs, transposons, and the Cre-Lox system; and (2) models for myeloid, lymphoid, breast, and melanoma cancers. Thus, the porcine genome sequence coupled with somatic cell cloning has led to the development of innovative porcine cancer models to support translational cancer research.

### AUTHOR CONTRIBUTIONS

TC and FS are responsible for organizing the materials and writing this editorial. LR and LS are responsible for proofreading this editorial.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Collares, Seixas, Rund and Schook. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Efficacy of the porcine species in biomedical research

*Karina Gutierrez1, Naomi Dicks1, Werner G. Glanzner1, Luis B. Agellon2\* and Vilceu Bordignon1\**

*1 Department of Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC, Canada, 2 School of Dietetics and Human Nutrition, McGill University, Sainte-Anne-de-Bellevue, QC, Canada*

Since domestication, pigs have been used extensively in agriculture and kept as companion animals. More recently they have been used in biomedical research, given they share many physiological and anatomical similarities with humans. Recent technological advances in assisted reproduction, somatic cell cloning, stem cell culture, genome editing, and transgenesis now enable the creation of unique porcine models of human diseases. Here, we highlight the potential applications and advantages of using pigs, particularly minipigs, as indispensable large animal models in fundamental and clinical research, including the development of therapeutics for inherited and chronic disorders, and cancers.

#### *Edited by:*

*Tiago Collares, Federal University of Pelotas, Brazil*

#### *Reviewed by:*

*Thomas John, Ludwig Institute for Cancer Research, Australia Howard Donninger, University of Louisville, USA*

#### *\*Correspondence:*

*Vilceu Bordignon, Department of Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada<EMAIL_ADDRESS>Luis B. Agellon, School of Dietetics and Human Nutrition, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Cancer Genetics, a section of the journal Frontiers in Genetics*

*Received: 30 June 2015 Accepted: 04 September 2015 Published: 16 September 2015*

#### *Citation:*

*Gutierrez K, Dicks N, Glanzner WG, Agellon LB and Bordignon V (2015) Efficacy of the porcine species in biomedical research. Front. Genet. 6:293. doi: 10.3389/fgene.2015.00293* Keywords: Large animal models, biomedical research, swine, pigs, minipigs, clones, transgenics

#### Introduction

The first evidence of swine domestication dates back to approximately 7000–9000 years ago (Jones, 1998; McGlone and Pond, 2003; Köhn, 2011; Larson et al., 2011; **Figure 1A**). China and Europe have been, since domestication, the pig-breeding centers dictating the profile of the pig breeds (Jones, 1998; Amills et al., 2001). The reason for domestication was to provide meat as a source of food protein, which stimulated pig selection and farming (Jones, 1998; Köhn, 2011). Studies have been conducted using genome-wide genotyping and genetic variability to trace the migration, selection, and improvement from ancient wild species to modern swine (Giuffra et al., 2000; Bosse et al., 2014a,b). It is generally accepted that the majority of all modern breeds are derived from the Eurasian wild boar (European and Asian wild boars; Porter, 1993; Bosse et al., 2014b). Although pig selection started just after domestication, it has only been since the mid-20th century that performance has been used as the main tool in the animal selection process (Safranski, 2008). More recently, molecular biology technologies, genome-wide association studies, and next-generation sequencing have been applied to enhance the selection process of domesticated pig breeds (e.g., Duroc, Landrace, Pietrain, Yorkshire, etc.) to further improve traits of high economic value such as feed conversion, meat quality, growth, precocious puberty, and prolificity (Sahana et al., 2013; Tart et al., 2013; Jiang et al., 2014; Sanchez et al., 2014).

The variety of modern pig breeds available today (Buchanan and Stalder, 2011), are a product of human intervention since domestication, but especially during the last century (**Figure 1A**). Besides breeds specialized for food production, smaller sized breeds (miniature- and micro-pigs) with certain characteristics such as obedience, friendly nature, and cognitive ability have also been selected for the purpose of companion animals. In addition, their use in biomedical research has been increasing considerably in the last years (**Figure 1B**).

Compared with other animals used in research (e.g., mice, rats, rabbits, and dogs), domestic farm pigs are much larger (*>*300 kg adult size), therefore, requiring more space and feed, and

making them harder to handle. Mini- or micro-pigs are hence more desirable for research use. The adult sizes vary among breeds, reaching around 20–30 kg for a Panepinto micropig to 100 kg for a Munich minipig (Köhn, 2011). Although many minipig breeds are a product of crossbreeding, some breeds, like the Yucatan pigs, are naturally occurring stocks (Panepinto, 1996; Köhn, 2011). Since the late 1940s, minipigs have been further

articles indexed by PubMed, from 1970 to the present date).

developed specifically for biomedical research purposes (England and Panepinto, 1986; Köhn, 2011).

There are now several minipig breeds available for use in research (Panepinto, 1996). The main breeds developed in the USA are Yucatan, Sinclair (also known as Minnesota or Hormel miniature pig), Hanford, NIH minipig and Panepinto miniature pig. The minipig breeds developed in Europe are Göttingen, Munich, Berlin, Mini-Lewe, Czech-Republic, Vietnamese potbellied and Mini-Sib. In Asia, the breeds include Ohmini, Clawn, Lee Sung, and Chinese minipigs. The Göttingen and Yucatan breeds are the most commonly used minipigs in research, although there is no apparent clear reason for preference. Unlike the Yucatan, a natural breed, the Göttingen minipig was developed specifically for research use. Other breeds are used only by specific research groups, thus limiting their widespread availability in research. Nevertheless, the interest in the use of pigs in biomedical research has been rising over the last 40–45 years (**Figure 1B**).

#### Use of Pigs in Biomedical Research

Biomedical research is broad, spanning studies on underlying disease mechanisms to the evaluation of safety and effectiveness of preventative measures, diagnostic tests, and therapies. Most animal studies in recent times have used the murine species due to their small size, fast reproductive cycles and short lifespan. In addition, the availability of murine embryonic stem cells, fully annotated genome, and facile tools for targeted genetic manipulation have all contributed to the elucidation of gene functions and disease pathophysiology. However, in many cases, mouse models do not adequately represent features of human disorders (Seok et al., 2013). In this regard, animals that better represent human pathophysiology are required. Pigs and humans share many similarities such as size, physiology, anatomy, metabolic profile, and longer lifespan (Panepinto, 1996; Spurlock and Gabler, 2008; Kuzmuk and Schook, 2011; Swindle et al., 2012).  Pigs also share anatomical and physiological similarities with respect to the renal system, making them valuable for pharmacological studies (Dalgaard, 2014; Huppertz et al., 2015). Pigs can also be useful in the study of nutrient absorption and intestinal transport, as well as the pathogenesis of gastrointestinal diseases (Sangild et al., 2014). All these characteristics contribute to the development of superior models of human conditions (Kuzmuk and Schook, 2011).

The choice between outbred or inbred strains can have a significant impact on research outcomes (Festing, 2014). While, outbred strains may be better suited for quantitative trait loci studies, experiments addressing mechanistic aspects would benefit from the use of inbred strains (Chia et al., 2005). Some minipig breeds are already established for specific applications due to their unique characteristics (**Table 1**). Pigs have also been used for testing new therapies, devices, and efficacy and safety of new drugs prior to human trials. For instance, a novel endovascular chemotherapy filter, designed to reduce circulatory drug excess *in vitro*, was successfully tested in pigs (Patel et al., 2014). As well, a new method for pediatric liver transplantation was validated using pigs (Leal et al., 2015). Regarding pharmacokinetic and cytotoxic tests, pigs have been used for testing topical skin formulations (Mitra et al., 2015), and are considered a better choice compared to dogs for the

study of drugs that are metabolized by the aldehyde oxidase (AOX1), *N*-acetyltransferase (NAT1 or NAT2) or cytochrome (CYP2C9-like) enzymes (Dalgaard, 2014).

In general, there is low incidence of naturally occurring pathologies described in pigs. The reason for this is twofold. First, human intervention by way of selective breeding has eliminated genes that increased disease susceptibility. Second, the majority of the domestic farm pigs are slaughtered at a young age (*<* 6 months old), precluding the detection of late onset diseases such as cancer. On the other hand, Vietnamese potbellied minipigs raised as companion animals do reach old ages. Indeed, a retrospective study found a variety of neoplasms with widespread metastases in these pigs of advanced age (∼11 years; Newman and Rohrbach, 2012). The most common malignances found included hepatic and intestinal carcinomas, and uterine and ovarian smooth muscle tumors (Newman and Rohrbach, 2012).

Occurrence of malignant spontaneously regressing melanomas has been described in Sinclair minipigs (Millikan et al., 1974; Oxenhandler et al., 1979). Selective interbreeding, by removing animals with red coat color that do not develop the lesions, increased the frequency of tumor formation in these selected minipigs (Millikan et al., 1974). The tumors appear from birth and culminate in skin depigmentation after tumor regression showing a phenotype similar to human vitiligo (Millikan et al., 1974). Studies conducted in these minipigs have shown decreased telomerase activity during melanoma regression (Pathak et al., 2000), which has also been observed by inhibiting telomerase activity in human melanoma cells (Burchett et al., 2014). Therefore, these minipigs may represent a useful model to study malignant melanomas because the tumors appear spontaneously and then either regress or grow progressively and metastasize similarly to human melanomas (Oxenhandler et al., 1979).

Another example of a naturally occurring condition in pigs is the dwarf phenotype, caused by a single amino acid change in the α1 chain of type X collagen (Nielsen et al., 2000). The *COL10A1* gene, which encodes type X collagen, is expressed in hypertrophic chondrocytes during endochondral ossification. In humans, an amino acid variation in the same position of the type X collagen protein has been shown to be the cause of Schmid metaphyseal chondrodysplasia (SMCD), a mild skeletal disorder associated with dwarfism (Warman et al., 1993). Since mice lacking type X collagen do not develop abnormalities in long bone development (Rosati et al., 1994), pigs represent a better animal model of human SMCD.

Another naturally occurring disease observed in Yucatan minipigs mimics human ventricular septal defect (VSD; Swindle et al., 1990). The VSD in pigs can be observed in fetal stages similar to the congenital anomaly in humans, and can be used for the study of new methods of diagnosis or therapies (Swindle et al., 1990; Amin et al., 2006).

Despite a number of natural occurring pig phenotypes that resemble human diseases, for most of human pathologies it is difficult to find representative animal models in nature. Thus, manipulation of diet, use of drugs and/or surgeries has been necessary to generate appropriate models. For example, minipig models for Type I diabetes were induced

#### TABLE 1 | Characteristics and applications of minipig breeds for the study of human conditions.

∗*The animals died 18 days after birth.*

†*Cloned animals were not yet born at the time of publication.*

via administration of streptozotocin or alloxan to selectively destroy insulin-producing cells (Phillips et al., 1980; Larsen et al., 2002). High-energy diets in young minipigs lead to the development of obesity and metabolic syndromes, with increased visceral fat deposition, glucose intolerance, decreased insulin sensitivity, and higher levels of blood cholesterol and triglycerides, which progress to Type 2 Diabetes mellitus (Xi et al., 2004; Neeb et al., 2010; Koopmans and Schuurman, 2015). Other chemicals have been used to induce cellular dysregulation and damage in pigs including the administration of *N*-nitrosodiethylamine to produce a liver cancer model (Li et al., 2006).

#### Use of Engineered Pigs in Biomedical Research

Genetically modified animals have been instrumental in advancing our understanding of gene function and significance of inappropriate gene expression in metabolic malfunction in mammals. Genome editing holds great promise in generating these models, and has already permitted the rapid development of new pig models of several human diseases (Rogers et al., 2008; Prather et al., 2013; Hai et al., 2014; Dicks, 2015).

The cystic fibrosis (CF) model is an example of genetically engineered pigs created by targeted inactivation of the cystic fibrosis transmembrane conductance regulator (*CFTR*) gene (Rogers et al., 2008). The resulting pigs exhibit clinical features and disease progression consistent with those observed in CF infants. In contrast, inactivation of the *CFTR* gene in mice did not produce the comorbidities typically observed in human CF patients (Snouwaert et al., 1992).

Advanced reproductive technologies, such as somatic cell nuclear transfer (SCNT), can now be routinely applied to large animal species, including minipigs. Minipigs of different breeds have been cloned from different cell types, including genetically modified cells (Estrada et al., 2008; Kurome et al., 2008; Wakai et al., 2008; Zhao et al., 2009). In addition SCNT offers the possibility of creating isogenic and immunocompatible animals from the same cell line. Importantly, models of severe disorders can be generated from engineered cultured cells without the need of breeding sick animals. The sequencing of the pig genome is another key development in the production of genemodified pigs in the post-genomic era (Schook et al., 2015a). Genome editing techniques, including zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered, regularly interspaced, short palindromic repeats (CRISPR) together with CRISPR associated (Cas) nucleases (CRISPR/Cas), now allow the precise manipulation of gene sequences in germ, embryonic and somatic cells (Hauschild et al., 2011; Carlson et al., 2012; Cong et al., 2013; Hai et al., 2014; Whitworth et al., 2014; Dicks, 2015). Among these methods, the CRISPR/Cas9 system is emerging as the method of choice because it permits gene editing to be accomplished in only one step by injecting both the specific guide RNAs and endonuclease into zygotes (Hai et al., 2014; Whitworth et al., 2014).

Another example of human disease that has the potential to be studied in genetically engineered pigs is heart arrhythmias (Park et al., 2015). Mutations in the *SCN5A* gene, which encodes a subunit of the cardiac sodium channel Nav1.5 required for excitability and conduction in the myocardium, were found in patients with Bruguda syndrome (Hedley et al., 2009). *SCN5AE*558*X/*+ engineered Yucatan minipigs with reduced expression of the sodium channel Nav1.5 have been created and these animals exhibit conduction abnormalities and susceptibility to ventricular arrhythmias (Park et al., 2015). There has also been considerable interest in genetically modified pig strains suitable for xenotransplantation. Most research into the development of appropriate xenotransplantation strains focused on addressing hyperacute rejection, which is initiated rapidly and involves preformed natural human antibodies and the complement system (Cooper et al., 2002). This has been possible by targeting cell surface antigens such as α-1,3 galactosyltransferase (Miyagawa et al., 2001; Lai et al., 2002; Phelps et al., 2003; Takahagi et al., 2005) or complement regulatory proteins such as human decay accelerating factor (Murakami et al., 2002). The pigs made deficient of α-1,3 galactosyltransferase have contributed to the reduction of immunogenicity of donor tissue/organs (Phelps et al., 2003). Transgenic pigs expressing antibodies against cytotoxic T-cell lymphocyte antigen receptor, a cell-mediated immune response suppressor, were also developed (Phelps et al., 2009).

A pig model for the human familial adenomatous polyposis was generated by inactivation of the adenomatous polyposis coli (*APC*) gene (Flisikowska et al., 2012). Mice lacking the *APC* gene exhibit non-metastatic neoplasias only in the small intestine (Su et al., 1992). However, the pig model of colon and rectal cancer reproduces the human features of the disease, which includes the development of polyps spread along the whole large bowel in young animals. A candidate gene for the development of breast and ovarian cancer models is the breast cancer-associated gene 1 (*BRCA1*), which has been manipulated in both Yucatan and Göttingen cells, but lines of modified minipigs remain to be produced (Luo et al., 2011, 2012). The *TP53* gene, which encodes the tumor suppressor protein p53 and is the most commonly observed suppressed gene in human tumors, was found to be mutated in Li-Fraumeni patients having increased risk to develop multiple types of cancers (Gonzalez et al., 2009). Suppression of p53 in mesenchymal stem cells derived from pig bone marrow exhibits chemoresistance *in vitro* (Leuchs et al., 2012). Mutation of *TP53* gene in Yucatan minipigs resulted in development of lymphomas and osteogenic tumors (Sieren et al., 2014). More recently, a new engineered pig strain termed "oncopig" was developed, which promises inducible formation of a wide variety of cancers that are potentially novel platforms for research and therapeutics development (Schook et al., 2015b). These examples illustrate the potential of genetically engineered pigs as robust models for the study of human pathologies that are not well represented in small laboratory animal species.

#### Improving the Usefulness of Pigs in Biomedical Research

Rodents have been the choice animal model for basic research, but are not always suitable for translational research due to marked differences in size, lifespan as well as metabolic, anatomical, and physiological discrepancies. On the other hand, the pig is more closely related to humans in terms of these parameters (Swindle et al., 2012) and, therefore, is better suited for recapitulation of human diseases. Indeed, the use of the pig in translational research is increasingly gaining acceptance (**Figure 1C**). Dogs and non-human primates have traditionally been used for this purpose, but rising ethical concerns have reduced their favor and increased demand for alternatives (Swindle et al., 2012). The number of peer-reviewed papers describing the use of pigs as biomedical models has risen eightfold over the past 30 years (**Figure 1B**). Already, the pig has become well established in many areas of research and training. For instance, in the past 20 years the pig has replaced the dog as a model for surgical training and has also gained FDA approval for the testing of surgical implantation devices intended for human use (Swindle et al., 2012; Schook et al., 2015a). Minipig models, which are much smaller in size compared to the domestic farm breeds, offer lower operating costs compared to other large animal models and also reduce the concern of ethical acceptance given the already widespread use of pigs in agriculture (Bollen and Ellegaard, 1997; Swindle et al., 2012).

Pigs offer many exciting applications, including stem cell research, tissue engineering and xenotransplantation.  Porcine induced pluripotent stem cells (iPSCs) have been produced (Esteban et al., 2009) and chimeric pigs were generated using iPSC (West et al., 2010, 2011). This is highly relevant since study of porcine iPSCs have eventual human applications (Esteban et al., 2009), such as cell-based therapies. However, the mechanisms of cellular reprogramming, directed cell differentiation and species-specific cell culture requirements necessitate further investigation (Ezashi et al., 2012). The International Society for Stem Cell Research has indicated in their guidelines for translational use that validation must occur in both small and large animal models (Aigner et al., 2010). Tissue repair is another potential application of engineered pig models. Cartilage tissue grafts have been created using chondrocytes isolated from infant minipigs (Deponti et al., 2014), and mandibular condyle grafts have been generated from Yucatan minipig adipose-derived mesenchymal stem cells (Abukawa et al., 2003). There has also been successful regeneration of bone defects using engineered bone graft tissues in minipig models (Gröger et al., 2003). If custom donor transgenic minipig strains can be created, this could open the doors to other engineered tissue replacements for human uses. For example, the use of blastocyst complementation and pluripotent stem cells has been applied to direct the development of otherwise missing organs in pigs (Matsunari et al., 2013). This has increased the hope that it may one day be possible to create non-immunogenic donor organs in pigs using human iPSCs (Matsunari et al., 2013; Feng et al., 2015). Finally, similarities in the porcine and human immune system have sparked interest in vaccine development and efficacy testing in pigs (Meurens et al., 2012).

The completion of the porcine genome project in 2012 has further facilitated the use of pigs in research. Data from this project has enabled the comparative analysis of genetic sequences and development of the necessary tools to create and validate targeted genetic alterations in the porcine genome (Gun and Kues, 2014; Schook et al., 2015a). In addition, the development of RNASeq technology has facilitated transcriptome analysis, which further improves our ability to identify important targets related to certain phenotypic traits (Ropka-Molik et al., 2014). Other recent achievements in the pig include the use of inducible or conditional systems to control transgene expression (Kues et al., 2006; Klymiuk et al., 2012), and tissue-specific expression of the Cre recombinase (Li et al., 2009; Luo et al., 2014). These advances will ensure the continued development of various pig strains for research, similar to what has already been accomplished in mice.

#### References

#### Summary

It is clear that the use of the pig as a biomedical model is increasingly gaining approval due to physiopathological similarities with humans. However, some obstacles remain to be overcome in order to realize the full potential of the porcine species in developing new diagnostic and therapeutic approaches. Despite the sequencing of the porcine genome, full annotation has yet to be completed. This is essential to facilitate interrogation of the pig genome and investigation of less characterized genes. Efforts to develop a complete porcine proteome map as well as epigenome map are currently underway (Meurens et al., 2012; Schook et al., 2015a). These databases are necessary to understand disease pathogenesis (Meurens et al., 2012; Schook et al., 2015a). Moreover, the availability of both inbred and outbred breeds of minipigs extends the utility of these species as a viable large animal model. Continuing refinements and adaptation of technologies for genome editing, cell/tissue-specific gene targeting strategies, stem cells and somatic cell cloning will further facilitate the creation of specialized pig strains for biomedical research.

#### Acknowledgments

Research in our laboratories is funded by grants from the Natural Sciences and Engineering Research Council of Canada (to VB and to LA). KG is supported by a scholarship from the Science without Borders Program of the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES). WG is supported by a scholarship from the Brazilian National Council for Scientific and Technological Development (CNPq). ND is supported by an Alexander Graham Bell Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada.

of families with p53 germline mutations. *J. Clin. Oncol.* 27, 1250–1256. doi: 10.1200/JCO.2008.16.6959

from two different pig breeds using RNA-Seq method. *Anim. Genet.* 45, 674– 684. doi: 10.1111/age.12184

gene knockout pigs expressing both human decay-accelerating factor and N-acetylglucosaminyltransferase III. *Mol. Reprod. Dev.* 71, 331–338. doi: 10.1002/mrd.20305

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Gutierrez, Dicks, Glanzner, Agellon and Bordignon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Engineered Swine Models of Cancer

Adrienne L. Watson1 \*, Daniel F. Carlson1 , David A. Largaespada1, 2, 3, 4, Perry B. Hackett 1, 3, 5 and Scott C. Fahrenkrug1

*1 Recombinetics, St. Paul, MN, USA, 2 Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA, 3 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA, 4 Pediatrics, University of Minnesota, Minneapolis, MN, USA, 5 Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA*

Over the past decade, the technology to engineer genetically modified swine has seen many advancements, and because their physiology is remarkably similar to that of humans, swine models of cancer may be extremely valuable for preclinical safety studies as well as toxicity testing of pharmaceuticals prior to the start of human clinical trials. Hence, the benefits of using swine as a large animal model in cancer research and the potential applications and future opportunities of utilizing pigs in cancer modeling are immense. In this review, we discuss how pigs have been and can be used as a biomedical models for cancer research, with an emphasis on current technologies. We have focused on applications of precision genetics that can provide models that mimic human cancer predisposition syndromes. In particular, we describe the advantages of targeted gene-editing using custom endonucleases, specifically TALENs and CRISPRs, and transposon systems, to make novel pig models of cancer with broad preclinical applications.

#### Edited by:

*Lawrence Schook, University of Illinois, USA*

#### Reviewed by:

*Geoff Clark, University of Louisville, USA Karine Begnini, Universidade Federal de Pelotas, Brazil*

\*Correspondence:

*Adrienne L. Watson<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Genetics, a section of the journal Frontiers in Genetics*

Received: *15 February 2016* Accepted: *18 April 2016* Published: *09 May 2016*

#### Citation:

*Watson AL, Carlson DF, Largaespada DA, Hackett PB and Fahrenkrug SC (2016) Engineered Swine Models of Cancer. Front.  The National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) data, estimated that in 2012 there were more than 13.7 million people living with cancer in the U.S. (Siegel et al., 2012). Trends suggest that in 2015 there will be over 1.6 million new cancer diagnoses in the U.S. and a staggering 589,000 deaths due to cancer (SEER). Throughout history there have been dramatic improvements in the methods by which we detect, diagnose, and treat cancer, yet the 5-year survival for all types of cancer remains at a dismal 66.5% (N.C.I. Surveillance Research Program; Mukherjee, 2011; Siegel et al., 2012). While the overall trends in cancer mortality in the U.S. have been reduced over the past decades, there are still several types of cancer for which the prognosis is very poor and for which few improvements have been made (**Figure 1**). Indeed, it has been suggested that the apparent increase in 5-year survival rates is due to earlier diagnoses, rather than improvements in treatment, for many types of cancer. The lifetime risk of developing cancer in the U.S. is over 40%, which emphasizes the need to better understand this deadly disease and improve outcomes for patients diagnosed with cancer (Siegel et al., 2012).

Cancer is a genetic disease in which cells acquire or inherit mutations, leading to uncontrolled growth of cells in the blood or solid organs (Hanahan and Weinberg, 2011). Animal models, and specifically mouse models, have played a major role in our understanding of the genetic basis of cancer and the role of specific genes and gene mutations in the development and progression of cancer. Mice led the way for the identification of new therapies to treat cancer owing to advances in constructing specific mutants in the late 20th century. Hundreds of mouse models of cancer have

**15**

been made and studied. However, gaining a complete understanding of cancer, which turns out to be an astonishing number of variant diseases, and translating this knowledge to better treatments and ultimately a cure has been elusive. Clearly, there are limitations to using rodents to model human diseases including large differences in their size, anatomy, physiology, drug metabolism, chromosome structures, and their genetics. Most cancer studies done in mice involve inbred lines of mice in which every locus is homozygous—a condition that inhibits translation of murine studies to humans (Hunter, 2012). To augment studies in the mice, new animal models of cancer are needed. Swine may turn out to be the best alternative models due to their size, physiological, genetic, and biochemical similarities to humans (Prather et al., 2008; Schook et al., 2008; Ganderup et al., 2012; Swindle et al., 2012; Flisikowska et al., 2013; Helke and Swindle, 2013). High-throughput genome sequencing and a collection of precision-genetic tools combined with tools for bioinformatics analysis, and profiling of gene expression/proteomics can be applied in swine. The ability to modify mammalian genomes through transgenesis and targeted nucleases, united with the development of advanced reproductive technologies including cloning, allows researchers to create complex and unique models of cancer in swine that are more applicable to human disease.

### THE LIMITATIONS OF RODENT MODELS OF CANCER

Due to the vast differences between rodents and humans, the ability to model the complex diseases such as cancer is quite limited (Cheng et al., 2014). Humans are 3,000 times larger than mice, live 30–50 times longer, and therefore undergo about 105 more cell divisions in a lifetime (Rangarajan and Weinberg, 2003). Without genetic modification, mice develop cancers of mainly mesenchymal origin, such as sarcomas and lymphomas, whereas humans have a bias toward the development of epithelial cancers with age (Rangarajan and Weinberg, 2003). The small size and short lifespan of mice means that loss of certain tumor suppressor genes is insufficient to result in development of cancer in a highly penetrant manner, particularly when such mutations are heterozygous. Accordingly, investigators have used the Cre-Lox system to homozygously inactivate tumor suppressors in a tissue or cell type-specific manner. While this is often sufficient to drive tumor formation, such a situation does not mimic the disease course in patients in which rare loss of heterozygosity (LOH), a genetic condition in which one copy of a gene (or genetic locus, portion of chromosome, etc.) is lost or deleted due to a mutation or chromosomal event, occurs in a field of heterozygous cells. LOH is a common phenomenon in cancer, resulting in homozygous loss of tumor suppressor genes in a subset of cells in the body, often leading to the development of a tumor or the progression of an existing tumor. Because mouse chromosomes are telocentric, LOH often occurs in mouse models by loss of the entire chromosome carrying the wild type tumor suppressor gene allele in cells heterozygous for a tumor suppressor gene mutation (Luongo and Dove, 1996). However, in human tumors LOH usually occurs via sub-chromosomal deletions covering the wild type tumor suppressor gene locus (Thiagalingam et al., 2001; Petursdottir et al., 2004).

On a cellular level, murine cells have a lower threshold for genetic and/or epigenetic changes that lead to transformation in culture, which demonstrates fundamental differences in the mechanistic properties of cancer development between mice and humans (Holliday, 1996). Arguably, the most profound difference between rodent models and humans is the essentially 100% homozygosity of every locus in inbred mouse lines, which may represent only a single individual in the entire population, making extrapolation back to entire human populations challenging (Kaiser, 2015). Mouse cells are immortalized much more readily than are human cells (Rangarajan and Weinberg, 2003). It has also been suggested that mouse cells respond to oncogenic Ras expression in a different manner than human cells; RAS oncogenes require RAL signaling in human cells, whereas the requirement for this signaling pathway is much reduced in RAS oncogene transformation of mouse cells (Hamad et al., 2002). Laboratory mouse strains have very long telomeres, and readily re-express TERT, in contrast to human cells (Holliday, 1996; Kim Sh et al., 2002). Moreover, mice do not develop the same kinds of genetic instability that human cells do during tumorigenesis, perhaps due to their shorter lifespan that could restrict the number of sequential mutations that accumulate in human tumors (Kim Sh et al., 2002).

Many organ systems vary so greatly between rodents and humans that certain types of cancer cannot be accurately modeled. For example, when one copy of the tumor suppressor gene adenomatous polyposis coli (APC) is inherited in humans, LOH leads to polyps in the large intestine that progress to invasive carcinoma. In contrast, mice that are heterozygous for Apc develop polyps in the small intestine that rarely show disease progression (Karim and Huso, 2013). Such differences in cancer development are due to inherent biological differences between man and rodent and are not limited to the intestinal polyps, but are seen in many mouse models of cancer (**Table 1**). There are fundamental differences in how tumorigenesis occurs in rodents and humans. This is well illustrated by variations in tumor spectrum when certain tumor suppressor genes, known to cause specific cancers in humans, are knocked out in mice (**Table 1**). Specifically, the five deadliest cancers in the U.S. (**Figure 1**) either cannot be modeled in rodents, or have ineffective models for identification of treatments that translate to the clinic.

The size limitation in rodents makes the development of novel imaging modalities and surgical techniques nearly impossible, yet these are key techniques needed to diagnose and treat a wide variety of tumor types in patients. Moreover, the rate of metabolism is much, much higher in mice compared to humans (Rangarajan and Weinberg, 2003). These differences mean that the pathways by which tumor progression occurs can vary dramatically when comparing mouse models to human cancer. As a consequence, the tumors that develop in a mouse model may respond differently to therapy. For the genetic and physiological reasons, including vast differences in drug metabolism and xenobiotic receptors, rodents also poorly model toxicity, sensitivity, and efficacy when used in preclinical drug studies (Swanson et al., 2004). The ability to establish toxicity and drug sensitivity pre-clinically in animal models is immensely

important because less than 8% of cancer drugs translate successfully in Phase I clinical trials from animal models (Mak et al., 2014). While mice have provided numerous insights into the biology of cancer, their historical limitations emphasize the need to develop new models for cancer research, such as swine.

#### ADVANTAGES OF USING SWINE CANCER MODELS

The anatomical, physiological, and genetic similarities between swine and humans are striking, suggesting that disease modeling in this large animal may better represent the development and progression of cancer seen in human patients (Swindle et al., 2012). Swine have been widely used in many areas of biomedical research due to such a high resemblance in organ systems. For these reasons pigs are commonly used in cardiovascular research where models of atherosclerosis, thrombosis, and myocardial infarction have been used to understand these health conditions in patients and to develop therapeutic and medical device interventions (Dixon and Spinale, 2009; Vilahur et al., 2011). The similarity in size and anatomy of the swine cardiovascular system allows design and testing of stents and tissue engineering of blood vessels (Bedoya et al., 2006; Gyongyosi et al., 2006). Further, comprehensive studies of the skin, urinary, integumentary, and digestive systems demonstrate extensive similarities to humans (Swindle et al., 2012). This history suggests that swine may be extremely useful as models of human cancer.

Perhaps of greater importance is the degree of genetic variation in pigs, including those used for disease models. Numerous genetically distinct lines of pigs exist and are available for model development, with various levels of diversity and inbreeding. Cultivation and characterization of these lines provides the opportunity to address both basic science and preclinical research needs. Lines that are low in variation provide a predictable platform for the development of therapeutics and toxicology research. But, like patients, many swine herds are highly outbred, with tremendous genetic and phenotypic heterogeneity that is more reflective of the patient population. This heterogeneity has two major consequences. First, genes that act as "drivers" or are otherwise critical to cellular transformation are more likely to be evident in pigs because they must act in the presence of other genetic variations. Also, therapeutic interventions that show efficacy will have to operate in many genetic environments in pigs, likely more accurately predicting safety and efficacy in patients.

The swine genome (sus scrofa) has been completely sequenced and, as expected, it shares considerable homology to the human genome (Schook et al., 2005; Groenen et al., 2012). Extensive conservation between pigs and people at the protein and primary sequence level, and extensive chromosomal synteny provide opportunities to address the initiation and progression of cancers, including frequently observed indels, inversions, and translocations, an outcome prohibitive in rodents, where synteny is more fragmented (Schook et al., 2005; Groenen et al., 2012). For instance, one study identified conservation between human and pigs of 112 loci, wherein a human amino acid that is implicated in a human disease is the same in swine (Groenen et al., 2012). Further, gene expression profiling and proteomics have been rapidly advancing in swine (Garbe et al., 2010). With the full genomic sequence in swine, the advancement of bioinformatics tools, the ability to modify somatic swine cells with transgenesis and targeted nucleases, and the development of techniques such as somatic cell nuclear transfer (SCNT), we are now able to create genetically engineered swine models of human disease (Prather et al., 2008). The homology between human and swine genes will be a guide for engineering exact human disease alleles into the swine genome. In the past 4 years, a platform for genetically engineering the swine genome using targeted nucleases and homology-dependent repair (HDR) has been developed (Carlson et al., 2012a; Tan et al., 2013). As described below, this technology allows the replication of precise amino acid changes and/or truncating mutations in oncogenes and tumor suppressor genes known to drive initiation, progression, or metastasis in human cancer. The use of targeted nucleases to engineer swine genocopies, or exact mutant alleles that cause cancer in human patients, represent a more accurate animal model than removing entire exons using standard knockout strategies or overexpressing oncogenes using transgenesis, which have been the mainstays in murine models for decades. Further, recent precision genetic technologies can support the development of single-gene modifications and complex, multiple-gene changes as well as chromosomal translocations in a single generation in swine.

In addition to recent advances in making precise genetic modifications to large animal genomes, there has been significant progress in technologies for testing consequences of genetic changes. Imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) can be easily applied to large animals such as pigs, whereas application of analogous clinical protocols is difficult in rodents (Sieren et al., 2014). By applying these imaging modalities to swine models of cancer we can improve detection techniques, better monitor progression, and more accurately measure response to therapy. The size of the pig allows for radiation-directed therapies to be tested and optimized. Surgical resection is the first line of defense and often the standard of care for many cancers. The size of the pig allows for refinement of surgical techniques and studies of local tumor recurrence, both of which are difficult or impossible to perform in rodents. Tumor natural history is an area that is difficult to study in rodents due to their short lifespan, about 1/30th that of humans (Rangarajan and Weinberg, 2003). Swine can live up to 10 years thereby enabling researchers to carefully follow the development of tumors, tumor progression, invasion, and metastasis in the absence of intervention over time. Additionally, the identification of biomarkers may be feasible in these animals due to the facile nature of accessing blood and tissue samples, the abundance of sample material and the ability to perform longitudinal blood sampling over long periods of time. Understanding tumor heterogeneity may be well suited for a large animal, as samples could be collected from many different tumors over time and followed for variations in somatic mutations, gene expression, or differential responses to treatment.

One of the main drawbacks of rodent models of cancer has been their inability to identify safe and effective drugs to treat cancer. Mouse models of cancer have been poor predictors of drug safety, toxicity, and efficacy (Gould et al., 2015). Further, routes of administration in mice are largely limited to intravenous (i.v.), intraperitoneal (i.p.) or oral gavage. Pigs have been widely used in preclinical drug toxicology, and are a standard large animal model for preclinical toxicology prior to human studies (Ganderup et al., 2012). The size and ease in handling pigs allows for drugs to be administered in the same way that patients are given them, including orally, intravenous (i.v.), intraperitoneal (i.p.), inhalation, dermal absorption, subcutaneous, intramuscular, and transmucosal. Longitudinal blood sampling can be performed to assess drug exposure and metabolism over long periods of time, and the amount of blood samples that can be taken from swine, in a short period of time, enhances the ability of pharmacologists to get precise kinetic data following drug exposure. There is significant homology in xenobiotic receptors in swine and human that regulate drug metabolism and pharmacokinetic properties (Myers et al., 2001). The cytochrome P450 (CYP) superfamily of proteins play a critical role in the processing and metabolism of drugs, and again, many studies have shown parallels in the structure and function of these molecules in pigs and humans (Myers et al., 2001). Importantly for pediatric cancer drug studies, juvenile pigs have been shown to have human-similar pharmacokinetic responses to certain drugs that cannot be modeled in other animals (Roth et al., 2013). The use of pigs in preclinical drug testing may identify safer and more effective therapies as well as establish dosing and routes of administration for new drugs prior to human clinical trials. Furthermore, a facile porcine genome engineering platform enables future humanization of drug metabolism in swine models.

#### APPLICATIONS OF PRECISION GENETICS TO MODEL CANCER IN SWINE

There have been three main types of disease models in swine that have been applied toward cancer modeling—spontaneous, induced, and genetically modified. Spontaneous models of pig cancers are rare, because like humans, pigs develop cancer with age, and as an agricultural animal produced mostly for food, most pigs do not survive to the age where cancer would be commonly seen. Sinclair miniature white swine have been a valuable model of malignant melanoma, which was identified as occurring in these animals in 1967 and has since been selected for by breeding (Oxenhandler et al., 1979). Another study identified 92 cases of leukemia in 3.7 million pigs tested, and 58% of those cases were in pigs under 6 months of age (Anderson and Jarrett, 1968). A range of rarer cancers have been described in older pigs (Brown and Johnson, 1970). However, owing to the economic necessities of keeping costs low in animals of agricultural importance, pigs harboring diseases due to rare mutations are euthanized without further study. Induced models of cancer in swine have been developed and are providing valuable insights into triggers of tumorigenesis found in some agricultural environments. In one study, researchers exposed pigs to N-nitrosodiethylanime to induce hepatocellular carcinoma (HCC) that resembles human HCC (Li et al., 2006). Another induced model took advantage of a naturally occurring severe combined immunodeficiency (SCID)-like Yorkshire pig line and transplanted human melanoma and pancreatic carcinoma cells, demonstrating the usefulness of these animals as human tumor xenografts (Basel et al., 2012).

Genetic engineering and gene editing technologies are being developed for cancer modeling in pigs (**Figure 2**, **Table 2**). A transgenic pig carrying the MMTV-v-Ha-ras oncogene was developed by microinjection of DNA into embryos, although no tumors developed in these animals (Yamakawa et al., 1999). A basal cell carcinoma model was created by making a transgenic pig in which Gli2 was expressed under control of a keratinocytespecific promoter (McCalla-Martin et al., 2010). Standard genetargeting methods developed in mice have been applied to pigs and led to the development of a familial adenomatous polyposis (FAP) model in which gene-targeting was used to introduce premature termination codons (PTCs) in APC by electroporation of linearized vector DNA into mesenchymal stem cells and subsequent SCNT to produce animals (Flisikowska et al., 2012). Similarly, gene-targeting was used to introduce a dominant-negative missense mutation, R167H, in the swine tumor suppressor gene TP53 using a recombinant adenoassociated virus (rAAV) in fetal fibroblasts, which were cloned to produce animals that when bred to homozygosity for the R167H mutation developed lymphomas, osteogenic tumors, and renal tumors at varying rates (Sieren et al., 2014). These animals represent a great model for humans with germline mutant TP53 mutations, seen in Li-Fraumeni patients, as they develop hematopoietic malignancies, bone tumors, and adrenal gland tumors in the kidney as well. A breast cancer model was attempted in the pig using rAAV-mediated BRCA1 knockout, but no phenotype was observed due to death of the animals by unknown causes before they reached 3 weeks of age (Luo et al., 2011).

The ability to activate or inactivate genes in a temporal or spatially-specific manner has been a critical aspect of many murine models of cancer, and this technology is now being developed in the pig by use of the Cre-Lox system (Flisikowska et al., 2013; Schook et al., 2016). There have been three swine models that use the Cre-Lox system for inducible cancer gene expression (Leuchs et al., 2012; Li et al., 2015; Schook et al., 2015). A pig was engineered to harbor a latent TP53R167H mutation by the insertion of a transcriptional termination signal between two LoxP sites upstream of the gene, allowing expression of TP53R167H only in the presence of Cre recombinase. Cre can be given in a time- or tissue-specific manner to induce recombination and results in the expression of the dominant negative TP53 allele, and in the absence of Cre Recombinase, the latent form of the gene is a knockout; however, at this time, the effects of this allele in vivo have not been reported (Leuchs et al., 2012). A second inducible model was generated using

Gene-targeted swine are created using large, homologous DNA sequences to integrate exogenous DNA into the swine genome at targeted loci, but this method has low efficiency, often requiring the introduction of some exogenous markers such as drug resistance genes. Gene-edited swine are made using designer nucleases, such as TALENs or CRISPRs, and use a double stranded break at a specific locus to induce efficient homologous recombination to make a targeted alteration in the genome (Yamakawa et al., 1999; McCalla-Martin et al., 2010; Luo et al., 2011; Flisikowska et al., 2012; Leuchs et al., 2012; Sieren et al., 2014; Li et al., 2015).

#### TABLE 2 | Genetically engineered swine models of cancer.

gene-targeting to express a Cre-activated KRAS G12D mutation, although the effect of Cre-induced activation of this allele has yet to be tested in these pigs (Li et al., 2015). Most recently, a transgenic "oncopig" was developed in which a Cre- inducible transgene expressing KRAS G12D and TP53 R167H was engineered, in hopes to model the many types of human cancers that have KRAS and TP53 mutations (Schook et al., 2015). Indeed, upon transgene activation, porcine cells were transformed in culture, formed tumors in immunodeficient mice, and led to tumors of mesenchymal origin when activated by AdCre injection directly into these animals (Schook et al., 2015). In addition to using the Cre-Lox system for conditional gene expression, as described in the models above, this system can also be applied to conditional deletions of short coding sequences and used as a strategy for inducing chromosomal rearrangements (Schook et al., 2016).

#### USING SITE-SPECIFIC NUCLEASES FOR GENE-EDITING TO MODEL CANCER IN PIGS

The use of designer nucleases is the latest technological platform being used to modify the germline of model species. This technology includes zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPRs). All of these methods to engineer precise changes into genomes have been used to make genetically engineered mouse models. In short, site-specific nucleases are designed to bind to user-defined regions of the DNA. ZFNs utilize a zinc finger domain which generally contains 3–6 zinc finger repeats recognizing 9–18 base pairs of DNA (Pabo et al., 2001). TALENs utilize a DNA binding domain contains repeated amino acid sequences, each which harbors a Repeat Variable Diresidue (RVD) (Boch et al., 2009). The RVD sequence gives specific nucleotide recognition, allowing TALENs to bind in a sequence specific manner (Boch et al., 2009). Typically, both ZFNs and TALENs utilize a cleavage domain with a bacterial type IIS restriction endonuclease, FokI, which requires dimerization in order for DNA cleavage to occur, creating even a higher level of specificity for site-specific nucleases (Pabo et al., 2001; Boch et al., 2009). The CRISPR/Cas9 system, derived from the prokaryotic immune system, consists of guide RNA (gRNA) sequences that guide Cas9, an RNA-guided DNA endonuclease, which then cleaves the DNA at these recognition sequences (Jinek et al., 2012; Mali et al., 2013). All three site-specific nuclease systems result in a double stranded break in the DNA, which can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR) when a repair template is provided. NHEJ often results in small insertions and deletions which can be used to disrupt the function of a gene, where HR allows users to engineer defined genetic changes at specific sites within genome (Carlson et al., 2012a,b; Tan et al., 2012, 2013). These genome engineering systems can be applied to primary cells, which, upon modification, can be used for SCNT to generate animals with germline genetic changes (Carlson et al., 2012a; Tan et al., 2012, 2013). Alternatively, these custom nuclease systems can be applied directly to embryos for in vivo modification (Bedell et al., 2012; Tan et al., 2012; Lillico et al., 2013).

The use of custom nucleases to enhance the efficiency of HR in swine has been dramatic from less than 104 using standard HR to rates as high as 25–75% using a recently developed TALEN/homology dependent repair (HDR) platform (Carlson et al., 2012a; Tan et al., 2013). Our group has used CRISPRs and TALENs to engineer several pig models of human disease, including models of infertility and atherosclerosis (Carlson et al., 2012a; Tan et al., 2013). Despite these advantages, a disadvantage to using the various site-specific nucleases, is the potential of undesirable collateral mutations that can accompany those that are desired (Kim et al., 2013; Lin et al., 2014; Mussolino et al., 2014; Frock et al., 2015; Hendel et al., 2015). Because the RNAguided site-specific platforms (e.g., CRISPR-based) may allow U-G base-pairing, their fidelity may be lower than the proteinbased platforms (e.g., TALENs). Consequently, although we have used most of the site-specific nuclease platforms, for fidelity and efficiency, we find TALEN-induced cleavages are the best balance for reliable gene-editing (Carlson et al., 2012a; Tan et al., 2013). TALEN technology allows replication of exact cancer mutations found in patients in pigs. Indeed, we have used TALENs to construct a swine models of the cancer predisposition disease, familial adenomatous polyposis by engineering a premature termination codon in APC (Tan et al., 2013).

TALEN-based genome editing for making swine genocopies of human cancer mutations is demonstrated for colorectal cancer in **Figure 3**. More generally, for a given type of cancer or known cancer driver gene, the method begins with the identification of common mutations within a gene of interest (**Figures 3A,B**; Cerami et al., 2012; Gao et al., 2013). Second, the location of the human mutation must be identified in the swine gene using bioinformatic approaches in which the amino acid sequence of the human and swine genes are aligned and the mutated human amino acid in humans is identified in swine (**Figure 3C**; Flicek et al., 2014). Third, custom nucleases such as TALENs are designed to induce a DSB at the appropriate site and HDR-oligonucleotides are designed to introduce the desired mutation (**Figures 3D,E**; Cermak et al., 2011; Doyle et al., 2012). We can employ a strategy in which the HDRoligo design includes not only the mutation of interest, but a novel restriction length polymorphism (RFLP) that allows facile screening of a large number of cells for the desired mutation, although introduction of a single point mutation in the absence of an RFLP allele can be engineered as well. Fourth, TALENs and HDR-oligos are transfected into primary swine fibroblasts, where they will cut the DNA and induce HDR to introduce the desired mutation (**Figures 3F–H**). Transfected cells can be easily screened by RFLP and sequenced to confirm that the intended mutation is present (Carlson et al., 2012a; Tan et al., 2013). In the last step, these cells are used for SCNT to produce gene-edited swine that have the precise human cancer-causing mutation. It should be noted that this technology has also be applied to gene-editing in pig embryos, in addition to somatic cells, avoiding the need for the somatic cell nuclear transfer step (Lillico et al., 2013; Whitworth et al., 2014; Wei et al., 2015).

resulting in truncation of APC protein (Cerami et al., 2012; Gao et al., 2013). (B) This figure shows the rate of specific mutations across the *APC* gene, with truncating mutations shown in red and missense mutations shown in green (Cerami et al., 2012; Gao et al., 2013). One of the most common mutations in colorectal cancer is an R876X truncation mutation that was seen in 22/727 mutations analyzed (Cerami et al., 2012; Gao et al., 2013). (C) Human and swine *APC* genes were aligned and human exon 16 where R876 is found (shown in blue) aligns with swine amino acid 661 in exon 10 of *APC* (shown in green) (Flicek et al., 2014). (D) The locus surrounding swine *APC* R661 was inputted into software that identified TALENs that would bind and cleave the DNA near this site (TALEN binding sites shown in red; Cermak et al., 2011; Doyle et al., 2012). (E) A 90mer HDR-oligo was designed with homology upstream and downstream of targeted mutation including the TALEN binding sites. A C →T base pair change was introduced to create a premature termination codon. Additionally, four nucleotides were added which would cause a frameshift and a novel RFLP (*Hin*dIII) site (shown in green). (F) TALENs and HDR-oligos are then transfected into primary swine fibroblasts. (G) TALENs cut at the specified location resulting in a double-stranded break (DSB) and the HDR-oligo acts as a template for homologous recombination (HR). (H) HR results in the desired allele with the novel premature STOP colon, RFLP site, and frameshift incorporated into the genome of the swine fibroblasts (Carlson et al., 2012b; Tan et al., 2013).

### UNIQUE OPPORTUNITIES FOR GENE-EDITED PIG CANCER MODELS

The ability to engineer specific human mutations into the swine genome is critical for accurately modeling cancer in the pig. While gene-targeting with either rAAV or other vectors has the ability to generate animals with swine genocopies of human disease alleles, these methods generally require the introduction of exogenous DNA into the swine genome in the form of antibiotic resistance genes such as puromycinor neomycin-resistance since standard gene targeting via HR in swine is very inefficient with rates less than 1 in 104 . In contrast, the method of gene editing by employing TALENs and HDR-oligos of ∼90 nucleotides is highly efficient, with homologous recombination occurring at an average efficiency of about 45% across several TALEN pairs tested (Tan et al., 2013). This allows facile isolation of cellular clones that are heterozygous or homozygous for the engineered allele of interest.

We identified several types of cancer-causing mutations that can be engineered into swine using TALEN-mediated gene editing (**Figure 2**). Premature STOP codons can be introduced, resulting in truncated gene products, a common phenomenon for tumor suppressor genes in cancer (**Figure 3**). Tumor suppressor genes have also been shown to contain point mutations that result in a dominant negative protein product, as is the case for the human TP53 mutation R175H, which functions as a dominant negative in swine (R167H) and can be introduced efficiently using TALEN-mediated HDR (Leuchs et al., 2012; Sieren et al., 2014). Similarly, oncogene activation can be modeled by introducing point mutations such as KRASG12V using TALEN-mediated HDR. TALEN-mediated gene-editing and HDR repair can be used to create large deletions encompassing one or more genes, which are also seen in several types of cancer, such as microdeletions of NF1 seen in Neurofibromatosis Type 1 patients (Pasmant et al., 2010).

In addition to making point mutations using custom nucleasemediated HDR, TALENs can be utilized to increase the efficiency of HDR with much larger constructs such as HDR templates with homology arms of 750 bp or larger that include LoxP sites flanking large exons. By inducing a double-stranded break, HDR occurs at a much higher efficiency than when plasmids or viral vectors are put into cells alone (Shin et al., 2014). Using TALENs to induce large HDR events has many applications in developing swine models of cancer. Many oncogenes and tumor suppressor genes are somatically mutated and therefore they must be expressed or disrupted in specific tissue types, at specific times. Using custom nuclease-mediated HDR, one could introduce LoxP flanked exons of tumor suppressor genes, or introduce latent oncogenes that would need to be activated by Crerecombinase to support tissue/temporal regulation. This method allows for the introduction of transgenes at specific loci in the genome thereby avoiding unwanted insertional mutagenesis effects. In order to introduce multiple genetic changes that occur in cancer, TALEN-mediated, site-specific mutagenesis can be used to introduce simultaneous targeted disruption of a tumor suppressor gene along with one or more transgenes for oncogene expression.

The ability to edit multiple genes at one time is necessary due to multiple genetic alterations in each cancer cell. The efficiency at which the TALEN-mediated gene-editing and small HDRoligo platform works, allows this technology to be applied toward multiplex gene-editing to model more complex genotypes of human tumors. Epidemiological data and mathematical models in colorectal cancer has suggested that it takes about five to seven rate limiting "steps" for transformation to occur (Renan, 1993). More recently, whole exome sequencing analysis in colorectal and breast cancer has shown that tumors have an average of 90 mutant genes, with 11 of these mutations being "cancer-causing" (Sjoblom et al., 2006). There are many types of cancer for which mutations in two or more genes are clearly demonstrated (**Figure 4**; Cerami et al., 2012; Gao et al., 2013). Recapitulating this phenomenon would be ideal when engineering animal models of cancer. Developing these multi-hit models is virtually impossible by standard gene-targeting techniques due to such low efficiencies, and would therefore require either serial cloning or animal breeding to obtain multiple alleles in one animal. Both of these alternatives are expensive and time-consuming in large animals such as swine. In contrast, the efficient custom nucleasestimulated HDR allows the engineering of multiple cancer genes in a single generation.

Multiplex gene editing can also be applied in making models of cancer with associated co-morbidities. Treating patients with cancer becomes quite complex when the patient is suffering from other diseases as well, and this phenomenon of co-morbidity is quite common (**Table 3**; Sogaard et al., 2013). A study of 15,962 patients showed various types of cancers that are more highly associated with comorbidities (Ogle et al., 2000). The overall frequency of any single comorbidity occurring in this population of cancer patients was 68.7% and 32.6% of patients had two or more comorbidities (Ogle et al., 2000). Some of the effects of these comorbidities on cancer patients and their subsequent survival and cancer treatment are shown in **Table 3** (Sogaard et al., 2013). Several studies have demonstrated that patients with comorbidities are less likely to complete chemotherapy, more likely to suffer complications from treatment and/or surgery, and the 5-year mortality hazard ratio for cancer patients with comorbidities ranges from 1.1 up to 5.8 (Sogaard et al., 2013). A multiplex gene editing approach can be taken to model complex disease associations with cancer to understand the impact on survival and treatment approaches. For example, we have used our gene-editing technology to develop swine models of hypercholesterolemia, heart failure, and hypertension and have the ability to generate models of various cancer types in conjunction with these common comorbidities (Carlson et al., 2012a; Tan et al., 2013). Using multiplex gene-editing, it is possible to engineer swine models of cancer in the background of other comorbidity diseases in a single generation.

 The ability to model cancer-causing translocations has been limited to the expression of genefusion transgenes in mice and has yet to be demonstrated in swine. Chromosomal translocations can be induced by double-stranded DNA breaks and TALEN technology allows engineering of exact human translocations at endogenous loci in the swine genome resulting in expression from the native promoter (**Figure 5**). Indeed, cancer translocations have been previously engineered using TALENs and ZFNs in human cells (Piganeau et al., 2013) and could also be applied in porcine cells. Applying TALEN-mediated site-specific mutagenesis to swine opens up a broad field of new research into the mechanism of oncogene activation via genomic rearrangements, the pathogenicity of chromosomal translocations in various cancer types, and investigations into therapies targeting novel gene-fusions and mechanisms of resistance in translocationdriven cancer types.

### PIGS AS MODELS FOR CANCER XENOGRAFTS

An additional application of TALEN-based multiplex gene editing is the ability to simultaneously knock out and add in genes involved in immune system development to facilitate even a broader range of cancer research applications. We and others have developed a severe combined immunodeficiency (SCID) swine model by knocking out genes necessary for both B-cell and T-cell development (**Figure 6**) (Shultz et al., 2007; Suzuki et al., 2012; Watanabe et al., 2013; Huang et al., 2014; Ito et al., 2014). These animals will allow xenograft experiments to proceed in which one could engraft cells or tissues from human tumors into the pig and monitor these xenografts for growth and development properties, as well as for efficacy studies with novel therapies. These animals can also be engrafted with human immune cells by either blastocyst complementation or transplantation, making a "humanized pig." This animal would serve several purposes including to investigate the role of the immune system in response to chemo- and radio-therapies for the treatment of cancer and the role of the human immune system in cancer development and progression if combined with a tumor xenograft (Zitvogel et al., 2011). These animals may also have a major impact on immunological research and treatments including the evaluation of: (1) immune-modulatory drugs (Pardoll, 2012; Ileana et al., 2013), (2) cell-based therapies (Fischbach et al., 2013), (3) adoptive T-cell transfer (June, 2007), (4) autologous immune enhancement therapy (Rosenberg, 1984), (5) genetically engineered T-cells (Restifo et al., 2012), and (6) studies of inflammation and infectious disease in the context of cancer (Cibelli et al., 2013).

An alternative approach to using swine as cancer xenograft models is by a method called in utero cell transplantation (Fisher et al., 2013). This method relies on the ability of a fetus to become tolerized to foreign antigens by exposing an immunologically immature fetus to xenogenic cells (Fisher et al., 2013). This allows the recipient fetus to recognize human cells as "self," when the foreign cells are injected prior to population of the pig thymus by CD3+ lymphocytes (Sinkora et al., 2000). This approach has allowed stable and long-term engraftment of both allogenic and xenogenic cells into immunocompetent host animals (Flake et al., 1986; Zanjani et al., 1992a,b, 1994). Using this method, one could perform in utero injections of a xenograft cell line of interest, tolerizing the host pig, and allowing for engraftment of human cancer cells in the pig post-natally. The xenogenic cells could be established human cancer cell lines, or human cells engineered with specific mutations and/or transgenes to determine their contribution to tumor formation, tumor growth, or drug responsiveness.

#### APPLYING TRANSPOSON SYSTEMS IN PIGS TO STUDY CANCER DEVELOPMENT, PROGRESSION, IMMUNE RESPONSE, AND DRUG RESISTANCE

Transposable elements have been widely used in in forward genetic screens to identify genes involved in cancer, as well as

#### TABLE 3 | Cancer comorbidities (Ogle et al., 2000).

#### TABLE 4 | Common cancer-associated chromosomal translocations (Nambiar et al., 2008).

in reverse genetic studies to produce transgenic animals and determine the contribution a gene or set of genes makes in the development of cancer (Tschida et al., 2014). Transgenesis via transposon systems have produced transgenic mice, rats, fish, frogs, and more recently, pigs (Clark et al., 2007; Carlson et al., 2011; Garrels et al., 2011; Jakobsen et al., 2011). Using cytoplasmic or pronuclear injection, transposon systems can efficiently deliver genes of interest into the porcine genome, allowing for the development of transposon-mediated transgenic porcine cancer models (Carlson et al., 2011; Garrels et al., 2011).

Another application of transposon systems in swine is to utilize the ability of transposon mutagenesis systems to screen for genes involved in cancer. Historically, these studies have been done in mice in which one chromosome contains a concatemer of transposons, and upon expression of transposase, these transposons randomly integrate throughout the genome (Moriarity and Largaespada, 2015). By random chance, certain cells will have the right combination of oncogenes activated and/or tumor suppressor genes inactivated to cause a tumor to form (Moriarity and Largaespada, 2015). These studies depend on the development of many, many tumors, which are then sequenced to determine genes that were activated or inactivated by transposon mutagenesis (Moriarity and Largaespada, 2015). Bioinformatics analysis can predict which genes are potential drivers in the development of cancer, because they will undergo mutagenesis at a rate higher than would be expected by random chance (Moriarity and Largaespada, 2015).

The pig offers unique opportunities for applying transposon mutagenesis screens. For example, due to the large size of the pig, one could engineer a swine model harboring transposon concatemers and expressing a transposase, and look for tumors by imaging using MRI, CT, PET, or ultrasound analysis. By using sophisticated imaging techniques, coupled with the large size of the pig and availability of tissue samples, one could

biopsy tumors, and look at transposon insertion sites over time to determine tumor evolution genes involved in development, progression, and metastasis (DeNicola et al., 2015). Further, pigs that develop tumors could be treated with drugs, and their tumors monitored over time to look for tumor regression followed by development of resistance. Tumors that show initial regression to a certain drug could be sequenced to identify genes involved in drug sensitivity. Similarly, tumors that develop resistance to a drug or therapy could be sequenced to identify genes involved in the development of drug resistance. Lastly, new and innovative applications of transposon mutagenesis screens can be applied to swine models of cancer.  Interestingly, these pigs show a nearly 100% spontaneous regression of cutaneous melanomas (Oxenhandler et al., 1982). This spontaneous development and regression of melanoma model is a perfect opportunity to utilize a transposon mutagenesis system to identify genes involved in the suppression of regression. Lastly, methods for transposonmediated transgene delivery to somatic cells have been developed in the mouse and could be applied in the pig as well (Wiesner et al., 2009).

#### PIG MODELS OF CANCER: UNANSWERED QUESTIONS AND LOOKING FORWARD

Cancer remains the second leading cause of death in the U.S. There is a chronic need to understand the etiology and biology of this collection of diseases as well as identify new treatments. The anatomical, physiological, and genetic variations between mice and humans limit the prospects of meeting the needs of patients by modeling cancer in rodents. However, for any novel animal model to be useful in cancer research, it must be adopted and fully tested in many laboratories under many circumstances. Even though pigs may turn out to be better models to investigate cancer and potential therapeutics, the considerable expense associated with large animals over their extended lifetimes coupled with the perceived need to run experiments under various experimental and controlled conditions may impede the rate of their widespread adoption into mainstream science. That will be determined by the scientific community plus funding and private entities that support cancer research and therapeutic development. As a large animal with striking similarities in anatomical structure, physiological function, and genetic makeup to humans, we expect the pig will become an improved model animal to advance decades of cancer research studies conducted

knockout pig to model SCID has many practical applications for cancer research.

in rodents. Custom endonucleases, such as TALENs, coupled with cloning, enable the engineering of swine genocopies of human cancers mutations, providing a myriad of unique and exciting opportunities in cancer research that may ultimately better model cancer seen in human patients and lead to novel biological insights into the mechanism of cancer and more effective treatments for patients.

#### AUTHOR CONTRIBUTIONS

AW provided intellectual content and drafted the review article, figures and tables. DC, DL, PH, and SF contributed significant ideas and intellectual content including conception and design and provided critical review and editing.

#### REFERENCES

of tumor suppressor genes. Clin. Med. Insights Oncol. 5, 235–257. doi: 10.4137/CMO.S7516

**Conflict of Interest Statement:** All authors are owners and employees of Recombinetics Inc. Additionally, DL is a consultant, co-founder, and equity holder of NeoClone Biotechnology, Inc., a company providing antibodies to it's customers. DL is a consultant, co-founder, and equity holder of Discovery Genomics, Inc., a company pursuing human gene therapy. DL is a consultant, cofounder, and equity holder of B-MoGen Biotechnologies, Inc., a gene delivery and gene editing company. DL has a collaborative research agreements with Novartis and Genentech. Genentech is funding a research project DL's laboratory.

Copyright © 2016 Watson, Carlson, Largaespada, Hackett and Fahrenkrug. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Emerging Technologies to Create Inducible and Genetically Defined Porcine Cancer Models

Lawrence B. Schook1,2 \*, Laurie Rund2 , Karine R. Begnini3 , Mariana H. Remião3 , Fabiana K. Seixas3 and Tiago Collares3 \*

1 University of Illinois Cancer Center, University of Illinois at Chicago, Chicago, IL, USA, 2 Department of Animal Sciences, University of Illinois at Urbana–Champaign, Champaign, IL, USA, 3 Postgraduate Program in Biotechnology, Biotechnology Unit, Technology Development Center, Federal University of Pelotas, Pelotas, Brazil

There is an emerging need for new animal models that address unmet translational cancer research requirements. Transgenic porcine models provide an exceptional opportunity due to their genetic, anatomic, and physiological similarities with humans. Due to recent advances in the sequencing of domestic animal genomes and the development of new organism cloning technologies, it is now very feasible to utilize pigs as a malleable species, with similar anatomic and physiological features with humans, in which to develop cancer models. In this review, we discuss genetic modification technologies successfully used to produce porcine biomedical models, in particular the Cre-loxP System as well as major advances and perspectives the CRISPR/Cas9 System. Recent advancements in porcine tumor modeling and genome editing will bring porcine models to the forefront of translational cancer research.

#### Edited by:

Ji Luo, National Institute of Health, USA

#### Reviewed by:

Christina Stuelten, National Cancer Institute, USA Bryan Raymond George Williams, Monash Institute of Medical Research, Australia

#### \*Correspondence:

Lawrence B. Schook<EMAIL_ADDRESS>Tiago Collares<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cancer Genetics, a section of the journal Frontiers in Genetics

Received: 03 November 2015 Accepted: 13 February 2016 Published: 29 February 2016

#### Citation:

Schook LB, Rund L, Begnini KR, Remião MH, Seixas FK and Collares T (2016) Emerging Technologies to Create Inducible and Genetically Defined Porcine Cancer Models. Front. Genet. 7:28. doi: 10.3389/fgene.2016.00028 Keywords: oncopigs, cancer, biotechnology, transgenesis, genome editing

#### INTRODUCTION

Animal models have played a central role over the centuries in scientific investigations of human disease and treatment strategies. Genetic strategies for the development of cancer models using human mutations in targeted oncogenic pathways demonstrated that porcine fibroblasts could be transformed in vitro and could be tumorigenic with four to six gene alterations (Adam et al., 2007). The authors used retroviral vectors carrying pairs of human and murine oncogenic cDNAs (hTERT and p53DD, cyclin D1 and CDK4R24C, and c-MycT58A, and H-RasG12V) to transform porcine fibroblasts. These altered cells showed a transformed phenotype in culture and formed tumors following autologous transfer. These induced changes demonstrated that the pig/tumorigenic pathway recapitulated those observed in human much more closely than murine cells (Adam et al., 2007). Although this approach was limited because the animals needed to be immuno-suppressed for tumors to grow in vivo, this work was the first to demonstrate that genetically defined tumors could be induced in a large animal (Schook et al., 2015a).

Recent innovations in reproductive, cloning and transgene technologies have enhanced efficacy and efficiency or producing targeted porcine genome modifications. With the successful cloning of animals by somatic cell nuclear transfer (SCNT), it is now possible to produce genetically modified pigs from genetically engineered somatic donor cells using a wide variety of techniques from random genomic insertion of plasmid DNA (Hyun et al., 2003; Watanabe et al., 2005), to genomic integration of transduced retroviral or lentiviral vectors (Lai et al., 2002; Park et al., 2002), and to modern genome editing with molecular methods using endonucleases such as transposases, recombinases, and programmable nucleases (Zhou et al., 2015). Genetic modification technologies successfully used to produce porcine biomedical models, in particular the Cre-loxP System as well as major advances and perspectives the CRISPR/Cas9 System will be presented in this mini review.

#### Cre-loxP SYSTEM

fgene-07-00028 February 26, 2016 Time: 16:55 # 2

The ability to activate latent genes in defined tissues and at defined times is a key factor to enable the development of inducible temporally and spatially regulated cancer models. The activation of an oncogenic mutation(s) in a chosen tissue could mimic the spontaneous somatic events that initiate many human cancers and enable replication of diverse cancer types using the same mutant gene(s) (Flisikowska et al., 2013). Those conditional gene expressions are now well established in mice using sitespecific recombinase (SSR) systems that allow the precise recombination between genomic sites, resulting in deletion or inversion of the intervening sequences (Frese and Tuveson, 2007; Oh-McGinnis et al., 2010). The use of SSR technology in genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites (Wang et al., 2011). Site-specific recombination occurs at a specific sequence or recognition site and involves cleavage and reunion leading to integration, deletion or inversion of a DNA fragment without the gain or loss of nucleotides (Wang et al., 2011). Because of the efficiency of the SSR systems, it can be applied to conditional deletions of relatively short coding sequences or regulatory elements but also to more extensive chromosomal rearrangement strategies (Oh-McGinnis et al., 2010).

Cre-recombinase system is one of the best-studied and most commonly used SSR in mammalian cell cultures. Since its first use for mammalian genome editing in 1988 (Sauer and Henderson, 1988) many adaptations have expanded the utility of the Cre system from flies to mammalian cells beyond mouse to include porcine and humans cell lines (Lanza et al., 2012). Cre-recombinase is derived from the bacteriophage P1 and recognizes a distinct sequence-specific motif termed as recombination target sites (loxP) catalyzing efficient conservative DNA rearrangements (Wirth et al., 2007). The loxP site is a 34 bp palindromic sequence with an 8-bp asymmetric spacer region (Feng et al., 1999; Siegel et al., 2001; Araki et al., 2002; Sauer, 2002; Schnutgen et al., 2003; Garcia-Otin and Guillou, 2006) and acts upon the neighboring DNA sequences. The Cre-loxP system is a bidirectional tyrosine recombinase that enables the recombinase-mediated genetic cross-over between two identical loxP recognition sites promoting intermolecular or intramolecular recombination. Intermolecular recombination is a translocation between two DNA fragments with corresponding loxP sites, while the intramolecular recombination involves removal of genetic material between two loxP sites, with the last one been the preferred function of Cre-recombinase (Feng et al., 1999). Because of the identical nature of the recognition sites, the recombination reaction is fully reversible, although intramolecular recombination (excision) is highly favored over intermolecular reactions (integration) (Wang et al., 2011).

One of the most powerful and widely used applications of the Cre/loxP system is in conditional gene expression (Gu et al., 1994). This strategy allows for tissue and time-specific gene expression when recombination is triggered by Cre-recombinase, and is even more important in cancer models where oncogenic activation in a chosen tissue could mimic the spontaneous somatic events that initiate many human cancers (Schook et al., 2015a). Endogenous engineered mice are usually conditional alleles constructed by the insertion of a transcriptional and translational LoxStopLox 'stop' cassette between the promoter and first coding exon of the oncogenic allele. Providing the expression of an active Cre-recombinase, the stop cassette is excised and the mutant oncogene is subsequently expressed (de Alboran et al., 2001; Jackson et al., 2001). In pigs, this conditional gene expression strategy has been used to promote oncogenic expression in three cancer models (Leuchs et al., 2012; Li et al., 2015; Schook et al., 2015b). Leuchs et al. (2012) have generated gene-targeted pigs with a conditionally activated oncogenic mutant form of p53, which in latent form is a gene knockout. The construction used a porcine BAC vector with CAGGS promoter-mCherry cassette (in reverse orientation) as a fluorescent counter-selectable marker; a short arm of homology corresponding to a region of TP53 intron 1 from a point of exon 2 to a PmlI restriction enzyme site of exon 2; a floxed transcriptional termination cassette (LSL); and a region extending from the PmlI site in intron 1 to a point of exon 11 that includes a G to A substitution in exon 5 changing arginine to histidine in codon 167 (R167H) (Leuchs et al., 2012). In this same model, viable gene-targeted pigs carrying a latent KrasG12D mutant allele that could be activated by Crerecombinase was constructed (Li et al., 2015). The KRAS-neo vector comprised: a short homology arm in KRAS intron 1; a transcriptional stop cassette comprising: a loxP site; adenoviral splice acceptor; promoterless neomycin phosphotransferase resistance gene (neo); three poly-adenylation signals derived from SV40, bovine growth hormone and cytomegalovirus; and a second loxP site inserted into a ClaI site in KRAS intron 1; and a region of porcine KRAS extending from the ClaI site in intron 1 to a SacI site in intron 2, which also included an engineered G to A point mutation within exon 2 that results in a glycine to aspartic acid substitution at codon 12 (G12D) (Li et al., 2015). Both KRAS and TP53 transgenic pigs cells were transduced with 5 µM of Cre protein produced in vitro with the vector pTriEx-HTNC (Addgene plasmid 13763; Leuchs et al., 2012; Li et al., 2015).

Transgenic oncopigs (**Figure 1**) have also been engineered to contain oncogenic KrasG12D and dominant-negative p53R167H downstream of a LoxP-polyA(STOP)-LoxP sequence (LSL) and CAG promoter (Schook et al., 2015b). Site-directed mutagenesis was then used to introduce the oncogenic G12D mutation into the porcine KRAS cDNA and the R167H mutation was chosen for TP53 as its human equivalent (R175H) is commonly found in human cancers as well as the cancer predisposition Li-Fraumeni Syndrome. These two cDNAs were then introduced into a Creinducible vector, followed by the aforementioned LSL sequence,

KRASG12D, an IRES sequence to allow for bicistronic expression, TP53R167H and a poly A sequence. This design allows for coexpression of both KRASG12D and TP53R167H in ostensibly any cells of the pig by transient expression of AdCre (Ad5CMVCreeGFP, AdGFP, Gene Transfer Vector Core; Schook et al., 2015b).

#### CRISPR/Cas9 SYSTEM

The discovering of molecules that recognize specific sequences of DNA was one of the most important advances in gene editing technology allowing site specific genetic modifications to be made. These DNA binding proteins include the zinc fingers and transcriptional activator-like effector (TALE; Wood et al., 2011; Gaj et al., 2013). When they are fused to nucleases, they generate a double-strand break (DSB) in the DNA at the desired genomic loci, triggering the endogenous DNA repair machinery (Gaj et al., 2013; Zhu et al., 2014); if fused to transcription factors or inhibitor molecules, they can bind to promoter regions of target genes, modulating gene expression (Gilbert et al., 2014; Kearns et al., 2014). However, there is a disadvantage of utilizing these proteins that interact with DNA: production of these proteins involves a complicated and more expensive assembly process (Pan et al., 2014).

An easier, cheaper, and yet highly efficient tool for directed genome edition appeared to be more worthwhile and profitable than proteins: the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR associated proteins) system. This system is simpler then zinc fingers and TALEs because the CRISPR/Cas system uses the RNA-DNA interaction for genome loci recognition, which is more specific than protein-DNA (Gasiunas and Siksnys, 2013; Pan et al., 2014).

CRISPR/Cas system has been recently discovered as an adaptive immune system of some bacteria and archaea and protects them against invading viruses and plasmids (Barrangou et al., 2007). The transcription of the repeat-spacer elements from CRISPR locus generates a precursor non-coding CRISPR RNA (pre-crRNA) that later will be cleaved to have short CRISPR

RNAs (crRNA) (Garneau et al., 2010; Jinek et al., 2012). The crRNA will be homologous to the DNA or RNA from foreign sequences, and when the invasion occurs, the crRNA will be directed just by Watson-Crick base pairing (Jinek et al., 2012; Wade, 2015). There are different types of CRISPR systems in different organisms (I–III), and the one that have been most developed as a new tool for genome editing, the CRISPR/Cas9 system, is the type II CRISPR originating from Streptococcus pyogenes SF370 (Jinek et al., 2012; Qi et al., 2013). The type II is different from types I and III, that crRNA hybridize with another RNA molecule, the trans-activating crRNA (tracrRNAs), to direct Cas9 protein to specific DNA sequences (Jinek et al., 2012; Mali et al., 2013; Doudna and Charpentier, 2014). For genome editing, the researchers created a single chimeric guide RNA's (sgRNA), which is a fusion of a precursor crRNA and a transactivating crRNA (tracrRNA) (Jinek et al., 2012; Pan et al., 2014). Beyond the polymerization, the genome sequence from invader has a complementary genome sequence containing a tri-nucleotide protospacer adjacent motif (PAM) that will be required for initial binding of Cas9 protein (Guilinger et al., 2014). Cas9 protein has an endonuclease activity that cleaves on both strands a few nucleotides away from the PAM generating DSB, preventing the invader genome translation (Jinek et al., 2012). This has been used to generate knockin and knockout transgenic animals, as the DSB activates the endogenous DNA repair machinery by nonhomologous joining (Ma et al., 2014; Flemr and Buhler, 2015; Yang, 2015; Zhu et al., 2015).

However, the study of CRISPR/Cas9 identified a new application for Cas9: without its nuclease activity, Cas9 protein, attached to a molecule that modulates gene expression, could bind to the promoter region of some gene of interest, changing the genic expression pattern (Qi et al., 2013). The catalytically dead Cas9 (dCas9), lacking endonuclease activity, contains two mutations in the nuclease domains (D10A and H840A) (Choudhary et al., 2015). Since dCas9 was reported, new studies have been described using it for genome regulation creating different segments to use this tool: CRISPRi, for gene interference, and CRISPRa, for activation of gene translation. When these strategies uses an effector domain attached to dCas9, it can be called CRISPRe. For gene interference (CRISPRi), dCas9 recognizes sgRNA attached to the promoter region of target gene, impairing transcription (Qi et al., 2013). However, this strategy is not efficient for gene repression in eukaryotic cells, so dCas9 can be fused to a transcription repression domain to enhance gene knockdown (Gilbert et al., 2013). The most described strategy for CRISPRi is dCas9 fused to a KRAB (Krüppel- associated box domain of Kox1), a repressive chromatin modifier domain, which have been demonstrating increased gene expression repression in relation to dCas9 alone (Gilbert et al., 2013, 2014). Some authors mention that CRISPRi can be an alternative strategy to RNAi for repressing gene expression in mammalian cells (Gilbert et al., 2013).

Another approach for using dCas9 is fused to transcriptional activator domains, which can be called CRISPRa (Gilbert et al., 2014) or CRISPR-on system (Cheng et al., 2013a), to induce expression of target genes. To achieve that, dCas9 fused to the transcriptional activator is guided by the sgRNA complementary

to the promoter region of the gene. The well-characterized tetramer of herpes simplex virus protein, VP16 (VP64) is one of the most reported transcription activator attached to dCas9 and it has been shown to induce gene expression in eukaryotic cells, including human cells (Gilbert et al., 2013, 2014; Maeder et al., 2013; Perez-Pinera et al., 2013; Kearns et al., 2014). Some studies also report that target genes can be simultaneity artificially activated by just adding complementary sgRNAs of promoters of each one of the interest genes (Cheng et al., 2013b; Maeder et al., 2013). This strategy has been tested in human and mouse transformed cells, as well as in ES cells, in one-cell embryo (Cheng et al., 2013b).

The use of CRISPR/Cas9 strategy to build an animal for model of cancer disease is a recently developed approach. For lung adenocarcinoma, Maddalo et al. (2014) describe a methodology of in vivo chromosomal rearrangement using CRISPR/Cas9 delivered by virus infection. Rearranging chromosomes by fusing EML4 and ALK genes generated a new murine model for lung adenocarcinoma. An in vivo somatic cancer mutation in adult animals was described by Xue et al. (2014), which they developed a different strategy using a hydrodynamic delivery of plasmids with CRISPR components that occasioned to efficient hepatocyte transfection to edit oncogenes and suppressor-tumor genes.

Most frequently, rodents are used to test new strategies for genome editing with CRISPR/Cas9 system to develop cancer and other biomedical models of human disease. However, a new strategy for enrichment of cells with chromosomal deletions made by CRISPR/Cas9 to generate cancer genotype was developed in porcine embryonic fibroblasts (He et al., 2015). For employment in xenotransplants, CRISPR/Cas9 technology has already been applied to inactivate porcine endogenous retroviruses in porcine kidney epithelial cell line (Yang et al., 2015).

Not only modifications in genome sequence can induce cancer phenotype, epigenetic modifications can also be a target to develop animal models for cancer. Falahi et al. (2015) supposes that dCas9 can contribute for epigenome engineering to develop animals for cancer study. Effector domains attached to dCas9 could generate epigenetic mutations known to evolve to different cancer types. Also using dCas9, attached or not to KRAB domain, initial studies in human cells HEK293 and HEK293T, showed repression of TP53 (Lawhorn et al., 2014).

The recent advances generated by CRISPR/Cas9 system in genome editing are extremely important for development of new strategies to generate animal models of cancer. The simplicity, low cost, and low off-target effects put this strategy as one alternative not only for ZFN and TALEN, but also for RNAi technology and Cre-loxP systems.

#### REFERENCES

#### PERSPECTIVES

To unite Cre-loxP and CRISPR/Cas9 system has been a promising approach to develop animal models for cancer. CreloxP affords to conditional gene expression, while CRISPR/Cas9 can be used for target gene insertion and also for gene expression regulation. Some promising works already showed how these technologies can be used together. Using Cre-loxP system for induced expression, Sánchez-Rivera et al. (2014) used a system with CRISPR/Cas9 and Cre recombinase to evaluate new candidates for cancer genome, developing adenocarcinoma by editing tumor-suppressor genes sequences in mice models. A different association of both techniques is a study that a mouse model had Cas9 expressed by Cre dependence, and when expressed in conjunction with sgRNAs for Kras, p53, and LKB1 genes, it generated a change of function of those proteins, taking to macroscopic tumors of adenocarcinoma pathology (Platt et al., 2014). Probably, the next step is to standardize those techniques and employ them for a next-generation models for human cancer (Sanchez-Rivera and Jacks, 2015), and pigs fits for those purpose.

#### AUTHOR CONTRIBUTIONS

TC: acquisition of data, data analysis/interpretation, drafting of the manuscript and figure; FS: acquisition of data, data analysis/interpretation, drafting of the manuscript; KB: acquisition of data, data analysis/interpretation, drafting of the manuscript and table; MR: acquisition of data, data analysis/interpretation, drafting of the manuscript; LR: critical revision of the manuscript, drafting of the manuscript and figure; LS: critical revision of the manuscript. All authors approved the manuscript.

#### FUNDING

TC, FS, and KB were supported by the Brazilian Scholarship Program "Science Without Borders" promoted by the National Counsel of Technological and Scientific Development. LS was supported by USDA (AG 58-5438-2-307), the U.S. National Institutes of Health (was supported by USDA (AG 58-5438- 2-307), the U.S. National Institutes of Health (CA 153132), the Edward William and Jane Marr Gutgsell Foundation, and the Cooperative Research Program for Agriculture Science and Technology Development (No. PJ009103), Rural Development Administration, Republic of Korea.

and differentiation in human pluripotent stem cells. Development 141, 219–223. doi: 10.1242/dev.103341

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer CS and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

Copyright © 2016 Schook, Rund, Begnini, Remião, Seixas and Collares. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# A porcine model system of BRCA1 driven breast cancer

Howard Donninger 1 , Katharine Hobbing2 , M. L. Schmidt 3 , Eric Walters 4 , Laurie Rund5 , Larry Schook 5 and Geoffrey J. Clark 2 \*

*1 Department of Medicine, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA, 2 Department of Pharmacology and Toxicology, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA, 3 Department of Biochemistry, University of Louisville, Louisville, KY, USA, 4 Division of Animal Sciences, National Swine Resource and Research Center, University of Missouri, Columbia, MO, USA, 5 Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA*

#### Edited by:

*Ingrid A. Hedenfalk, Lund University, Sweden*

#### Reviewed by:

*Parvin Mehdipour, Tehran University of Medical Sciences, Iran Shree Ram Singh, National Cancer Institute, USA*

#### \*Correspondence:

*Geoffrey J. Clark, Department of Pharmacology and Toxicology, James Graham Brown Cancer Center, University of Louisville, Room 417, CTR Building, 505 S. Hancock St., Louisville, KY 40202, USA<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Genetics, a section of the journal Frontiers in Genetics*

Received: *30 April 2015* Accepted: *06 August 2015* Published: *25 August 2015*

#### Citation:

*Donninger H, Hobbing K, Schmidt ML, Walters E, Rund L, Schook L and Clark GJ (2015) A porcine model system of BRCA1 driven breast cancer. Front. Genet. 6:269. doi: 10.3389/fgene.2015.00269* BRCA1 is a breast and ovarian tumor suppressor. Hereditary mutations in BRCA1 result in a predisposition to breast cancer, and BRCA1 expression is down-regulated in ∼30% of sporadic cases. The function of BRCA1 remains poorly understood, but it appears to play an important role in DNA repair and the maintenance of genetic stability. Mouse models of BRCA1 deficiency have been developed in an attempt to understand the role of the gene *in vivo*. However, the subtle nature of BRCA1 function and the well-known discrepancies between human and murine breast cancer biology and genetics may limit the utility of mouse systems in defining the function of BRCA1 in cancer and validating the development of novel therapeutics for breast cancer.  To determine if BRCA1 inactivation in pig cells promotes their transformation and may serve as a model for the human disease, we developed an immortalized porcine breast cell line and stably inactivated BRCA1 using miRNA. The cell line developed characteristics of breast cancer stem cells and exhibited a transformed phenotype. These results validate the concept of using pigs as a model to study BRCA1 defects in breast cancer and establish the first porcine breast tumor cell line.

#### Keywords: breast cancer, BRCA1, miRNA, SV40 LT, transformation

#### Introduction

Breast cancer is a leading cause of death in women and is one of the most common cancers in the world today.  The underlying causes of breast cancer development remain very much under investigation, but we now know that the BRCA1 tumor suppressor gene plays an important role in many breast cancers.  Although somatic BRCA1 mutations are rare in sporadic breast cancer, BRCA1 expression is down-regulated in ∼30% of sporadic cases by allele loss or epigenetic mechanisms (Welcsh and King, 2001; Yang et al., 2001).

The function of BRCA1 remains poorly understood. It has a ubiquitin ligase activity and can control the stability/activity of proteins such as Claspin (Sato et al., 2012) and estrogen receptor alpha (Savage and Harkin, 2015). It is also a key player in modulating DNA repair (Zhang and Powell), replication fork stability (Pathania et al., 2011), senescence (Tu et al., 2013), oxidative stress (Marks, 2013), genomic stability (Savage and Harkin, 2015), and checkpoint induced cell cycle arrest (Huen et al., 2010). The complex role of BRCA1 in cellular homeostasis has made elucidating its key functions in cancer difficult.

Mouse models of BRCA1 deficiency have been developed in an attempt to understand the role of the gene in vivo (Ma et al., 2010). Although BRCA1 knockout provokes embryonic lethality in mice, conditional knockout of BRCA1 in breast tissue leads to tumor development after a long latency. The latency period can be strongly reduced by introducing defects in the p53 tumor suppressor to the animal system. These animal models have allowed the validation of therapies designed against BRCA1 defective tumors. However, even therapeutic approaches that were effective resulted in the emergence of resistant tumors (Ma et al., 2010). Further studies to examine approaches to overcome the resistance are limited by the short lifespan of the mice. Moreover, the subtle nature of BRCA1 function and the wellknown discrepancies between human and murine breast biology (Dine and Deng, 2013) and cancer genetics (Kendall et al., 2005) may limit the utility of mouse systems in defining the function of BRCA1 in human cancer.

In contrast to mice, pigs exhibit very similar cancer genetics to humans (Adam et al., 2007). Moreover, their physiology and biochemistry is similar (Swindle et al., 2012) and their lifespan extends for decades. Consequently, a porcine model for breast cancer could prove a powerful tool for validating breast cancer therapies, preventative strategies and the clinical response to the emergence of drug resistance.

In order to validate the use of porcine systems in breast cancer research, we generated an immortalized porcine breast cell line using the SV40 LT oncoprotein (Chen and Hahn, 2003). We then used BRCA1 miRNA to generate a stable matched pair of cell lines that are positive or negative for BRCA1 expression. Characterization of the cells showed that BRCA1 knockdown induced enhanced growth and induced a transformed phenotype on the cells. Moreover, the transformed cells expressed markers characteristic of cancer stem cells. These results establish the first porcine breast cancer cell line and validate the concept of using porcine systems as a model to study BRCA1 defects in breast cancer.

#### Materials and Methods

#### Porcine Cell Lines and Transfections

Primary porcine breast epithelia cells were isolated as described in Prather et al. (1999) using a protocol approved by the IACUC of the University of Missouri-Columbia, Columbia, Missouri. They were transfected with pbabe puro SV40LT (Addgene #13970) using Lipofectamine 2000 (Invitrogen, Carlsbad CA) according to the manufacturer's instructions. Cells were selected in puromycin (Sigma, St Louis, MO) at 1µg/ml. miRNA sequences corresponding to two different regions of porcine BRCA1 were designed using the Block-iT™ RNAi Designer (Invitrogen). Two single-stranded DNA oligonucleotides were designed for each sequence, one encoding the target pre-miRNA (top strand) and the other, its compliment (bottom). Each oligonucleotide also contained five nucleotides (TGCTG) derived from the endogenous miR-155 at the 5′ end and 19 nucleotides derived from miR-155 to form a terminal loop. The sequences of the two different oligo sets are as follows: #1 Top: 5 ′ -TGCTGATTGTTTGCAAACTGCAATCCGTTTTGGCCAC TGACTGACGGATTGCATTGCAAACAAT-3′ , #1 Bottom: 5′ -C CTGATTGTTTGCAATGCAATCCGTCAGTCAGTGGCCAA AACGGATTGCAGTTTGCAAACAATC-3′ ; #2 Top: TGCTG TATTAAAGCACCATGAGGGTCGTTTTGGCCACTGACTG ACGACCCTCAGTGCTTTAATA-3′ ; #2 Bottom: 5′ -CCTGTAT TAAAGCACTGAGGGTCGTCAGTCAGTGGCCAAAACGA CCCTCATGGTGCTTTAATAC-3′ .

The corresponding single-stranded oligos were annealed to generate a double-stranded oligo which was then cloned into the pcDNA™ 6.2-GW/EmGFP-miR vector (Invitrogen). Generation of the double-stranded oligos and cloning into the expression vector were performed using the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit (Invitrogen) as described by the manufacturer. Stable transfectants were generated by transfecting the transformed pig mammary epithelial cells with 2µg of the two different miRNA expression vectors, as well as a negative control consisting of a miRNA to LacZ, using Lipofectamine 2000 according to the manufacturer's instructions and selecting with Blasticidin (4µg/ml).

#### qRT-PCR

qRT-PCR was performed on total RNA isolated from the cells with Trizol using an iCycler Real-Time Detection System (Bio-Rad Laboratories, Inc., Hercules, CA) with the Quantitect SYBR Green RT-PCR Kit (Qiagen, Inc., Valencia, CA) as per the manufacturer's instructions. The fold change for each gene was calculated using the 2−11CT method (Livak and Schmittgen, 2001) with GAPDH as the reference gene. The primers used were BRCA1 For: 5′ -GTCCAAAGCGAGCAAGAGAA -3′ , BRCA1 Rev: 5′ - ACAGAAGCCCCACAGAGGA -3′ ; GAPDH For: 5′ - CGATGCTGGTGCTGAGTATG- 3′ , GAPDH Rev: 5′ - GAAGGGGCAGAGATGATGAC- 3′ .

#### Western Blots

Total cell lysates were prepared by lysing the cells in modified RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1% NP-40) supplemented with 100µg/ml leupeptin, 100µg/ml aprotinin and 1 mM sodium orthovanadate. BRCA1 and ALDH1 antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA., Actin antibodies were from Sigma (St. Louis MO) and EpCAM antibodies were from AbCam. HRP conjugated Trueblot secondary antibodies were purchased from eBioscience (eBioscience Inc. San Diego, CA) and western blots were developed using a Pierce ECL detection system (Thermo Scientific, Rockford IL).

#### Growth Curves

2 × 104 cells/well were plated in six-well plates in normal growth medium and incubated for 6 days. Cell number was determined each day by counting the number of viable cells. Experiments were performed twice in duplicate.

#### Matrigel

Fifty micro liters of Matrigel (BD Biosciences, San Jose, CA) was plated in a 96 well plate and allowed to set. Cells were trypsinized, washed in growth medium and plated at 5000 cells per well in 100µl of growth medium. Hundred micro liters of medium +4% Matrigel was added and the medium changed every 4 days.

#### Soft Agar

Six well plates were prepared with 2 ml bottom agar (16 ml 1.8% molten Difco Bacto agar cooled to 42◦C and mixed with 1.6 ml serum, 1.6 ml 10X PBS and mixed with 30.3 ml DMEM) and allowed to set. Cells were trypsinized, washed, and 3 × 104 cells suspended in 1.5 ml growth medium. Three milli liters of liquid bottom agar was added to the cell dilution and 1.5 ml aliquoted into each well to set.

#### Anoikis

Twelve well plates were treated with polyHEMA (Sigma) and allowed to dry overnight. 1 × 106 cells were plated in each well and the cell viability measured after 48 h by trypan blue exclusion.

 Transfected cells were isolated by selection in puromycin and surviving colonies pooled. As the cells were passaged, the SV40 LT transfected cells lost the senescent morphology apparent in the parental cells (**Figure 1**). They were then serially passaged to determine if they had been immortalized. Transfected cells have been passaged more than 26 times without apparent loss of viability. In contrast, parental cultures lose proliferative capacity by passage 8.

#### Identification of an Effective Porcine BRCA1 miRNA

The Block-iT™ RNAi Designer tool from Invitrogen was used to identify potentially effective miRNA sequences against porcine BRCA1. Two were generated and cloned into the vector pcDNA GW 6.2 EmGFPmiRNA. The vectors were then transiently transfected into the immortalized breast epithelial cells and assayed for the degree of knockdown by RT-PCR. Only one of the miRNAs proved effective (**Figure 2A**). This

miRNA and the empty vector were stably transfected into the immortalized pig breast cells to generate a matched pair +/− for BRCA1. Western analysis confirmed that the miRNA transfected cells had almost completely lost BRCA1 protein expression (**Figure 2B**).

#### Suppression of BRCA1 Enhances Porcine Epithelial Cell Growth

As the cells were passaged, the BRCA1 suppressed cells progressively adopted a noticeably different morphology than the vector control cell line (**Figure 3A**). To characterize the effect of the BRCA1 suppression on the cell cycle, we measured the relative growth of the matched pair of cell lines transfected with vector or miBRCA1. Cells were plated and counted every day for 1 week. The BRCA1 suppressed cells exhibited an enhanced growth rate (**Figure 3B**).

#### Suppression of BRCA1 Alters Differentiation

Non-transformed human breast epithelial cell lines can be induced to differentiate into acini with hollow lumens when plated in 3D in matrigel. This differentiation is thought to mimic the process that occurs during the development of breast ducts. The process is disrupted by suppression of BRCA1 (Furuta et al., 2005). To examine the loss of BRCA1 in porcine cells on this process, we plated the BRCA1+/− matched cell lines in matrigel for 10 days. After 10 days, the immortalized cells transfected with vector alone formed acini, reminiscent of human immortalized breast cells. The BRCA1 knockdown cells mostly grew as disordered masses (**Figure 4**).

#### Suppression of BRCA1 Promotes Transformation

The BRCA1 knockdown appeared to have induced enhanced growth and reduced differentiation (**Figures 3**, **4**). In order

FIGURE 2 | miRNA-mediated BRCA1 knockdown in the immortalized pig mammary epithelial cells.  Forty-eight hours later, BRCA1 mRNA levels were determined by qRT-PCR analysis. (B) The immortalized pig mammary epithelial cells were transfected with BRCA1 miRNA#1 or the miLacZ control and selected with blasticidin to obtain cells that were stably knocked down for BRCA1. Western blot analysis confirmed efficient knockdown. β-actin served as control for equal protein loading. Error bars show standard error, *p* < 0.05 for miRNA#1, mRNA #2 was not significant.

to determine if it was sufficient to induce the tumorigenic phenotype, we plated the cells in soft agar and counted colony formation after 14 days. Anchorage-independent growth is one of the hallmarks of cell transformation and is considered the most accurate and stringent in vitro assay for detecting malignant transformation of cells (Colburn et al., 1978). **Figure 5A** shows that the BRCA1 positive cells failed to form colonies in agar. In contrast, the BRCA1 knockdown cells formed numerous, large colonies, indicative of highly transformed cells.