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Animal Models

Challenges and Opportunities to Determine Optimal Experimental Models of Pancreatitis and Pancreatic Cancer

Saloman, Jami L. PhD*; Albers, Kathryn M. PhD*; Cruz-Monserrate, Zobeida PhD; Davis, Brian M. PhD*; Edderkaoui, Mouad PhD; Eibl, Guido MD§; Epouhe, Ariel Y. BS*; Gedeon, Jeremy Y. BS*; Gorelick, Fred S. MD∥¶#; Grippo, Paul J. PhD**; Groblewski, Guy E. PhD††; Husain, Sohail Z. MD‡‡; Lai, Keane K.Y. MD§§∥∥¶¶; Pandol, Stephen J. MD; Uc, Aliye MD##; Wen, Li MD, PhD***; Whitcomb, David C. MD, PhD†††

doi: 10.1097/MPA.0000000000001335
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At the 2018 PancreasFest meeting, experts participating in basic research met to discuss the plethora of available animal models for studying exocrine pancreatic disease. In particular, the discussion focused on the challenges currently facing the field and potential solutions. That meeting culminated in this review, which describes the advantages and limitations of both common and infrequently used models of exocrine pancreatic disease, namely, pancreatitis and exocrine pancreatic cancer. The objective is to provide a comprehensive description of the available models but also to provide investigators with guidance in the application of these models to investigate both environmental and genetic contributions to exocrine pancreatic disease. The content covers both nongenic and genetically engineered models across multiple species (large and small). Recommendations for choosing the appropriate model as well as how to conduct and present results are provided.

From the *Department of Neurobiology, Pittsburgh Center for Pain Research, University of Pittsburgh, Pittsburgh, PA;

Division of Gastroenterology, Hepatology, and Nutrition, Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, OH;

Basic and Translational Pancreas Research, Cedars-Sinai Medical Center;

§Department of Surgery, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, CA;

Department of Internal Medicine, Section of Digestive Diseases and

Department of Cell Biology, Yale University School of Medicine, New Haven, CT;

#Veterans Affairs Connecticut Healthcare, West Haven, CT;

**Department of Medicine, Division of Gastroenterology and Hepatology, UI Cancer Center, University of Illinois at Chicago, Chicago, IL;

††Department of Nutritional Sciences, University of Wisconsin, Madison, WI;

‡‡Department of Pediatrics, Stanford University, Palo Alto;

§§Department of Pathology (National Medical Center),

∥∥Department of Molecular Medicine (Beckman Research Institute) and

¶¶Comprehensive Cancer Center, City of Hope, Duarte, CA;

##Stead Family Department of Pediatrics, Stead Family Children's Hospital, University of Iowa, Iowa City, IA;

***Department of Pediatrics, Children's Hospital of Pittsburgh, Pittsburgh, PA; and

†††Department of Medicine, University of Pittsburgh, Pittsburgh, PA.

Received for publication January 14, 2019; accepted April 25, 2019.

Address correspondence to: Jami L. Saloman, PhD, Department of Neurobiology, Pittsburgh Center for Pain Research, University of Pittsburgh, 200 Lothrop St, BSWTR E1457, Pittsburgh, PA 15213 (e-mail: jls354@pitt.edu).

L.W. is currently with the Department of Gastroenterology, Shanghai General Hospital and Shanghai Key Laboratory of Pancreatic Disease, Institute of Pancreatic Disease, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

This study was supported by Hirshberg Foundation for Pancreatic Cancer Research and National Institutes of Health (NIH) K01DK120737 (J.L.S.); NIH R01CA177857 (B.M.D.); NIH K01AA019996 (M.E.); NIH P01CA163200 (G.E.); Veterans Administration Merit Award, NIH P01DK098108, and R01DK054021 (F.S.G.); NIH K08AA025112 (K.K.Y.L.); The National Pancreas Foundation and R01CA223204 (Z.C.M.); NIH U01DK108314 (S.J.P.); NIH U01DK108334 and R01DK118752 (A.U.); and NIH U01DK108306 (D.C.W.).

A.U. is a member of the American Board of Pediatrics, Subboard of Pediatric Gastroenterology; associate editor of Pancreatology; and a consultant for Cystic Fibrosis Foundation. The rest of the authors declare no conflict of interest.

Pancreatitis and pancreatic cancer are highly variable diseases with a range of etiologies and disease courses. The use of animal models in the scientific exploration of these diseases is critical. Animal models are vital tools for understanding pathophysiology, and they are a key step in the drug development pipeline as well as disease biomarker discovery. In general, it has been difficult to translate findings from animal studies into meaningful changes in clinical care. During the 2018 PancreasFest conference, a working group met to discuss the challenges with animal models. The goal of this report is to emphasize issues that should be considered when performing animal studies of exocrine pancreatic disease.

Choosing the most appropriate model for a study is contingent upon the specific question being asked, what aspects of the disease are relevant, and the time and resources one has available (Fig. 1). This report introduces nongenic and genetic engineered models of pancreatitis and exocrine pancreatic cancer and identifies the key features of each model. In particular, it emphasizes the multiple factors contributing to intermodel and intramodel variability and their direct effects on the manifestation of disease.

FIGURE 1

FIGURE 1

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NONGENIC MODELS

Pancreatitis

Pancreatitis is an umbrella diagnosis that includes subclassifications such as acute, recurrent, and chronic. It can be further characterized by the presence or absence of necrosis. Multiple nongenetic factors that increase the risk of developing pancreatitis or of more severe disease have been identified; some can be recapitulated in animals. For example, exposure to a specialized (high fat) diet, alcohol feeding, endoscopic retrograde cholangio-pancreatography (ERCP), or duct obstruction can induce pancreatitis in animals in a manner similar to clinical pancreatitis in humans (Table 1). Models have been developed based on the known biological processes contributing to pancreatitis. For instance, acute pancreatitis (AP) involves premature activation of trypsinogen within the pancreas. Administration of supraphysiologic concentrations of the cholecystokinin analog cerulein is a popular method of inducing experimental pancreatitis. Such a hyperstimulation model in rodents is generally used to induce mild to moderate injury and reflects the hyperstimulation pancreatitis observed in humans after exposure to certain scorpion bites and insecticides with cholinesterase activities. There are also several chemicals that, when infused into the pancreatic duct, may mimic the effects of bile reflux and its possible role in inducing biliary pancreatitis (Table 1). The technical challenges associated with bile acid and post-ERCP induced AP have likely prevented their widespread adoption. Another group of animal models that are less commonly used are those that mimic uncommon forms of pancreatitis such as autoimmune pancreatitis. These models use foreign serum, adoptive cell transfer, or infectious pathogens to induce disease. There are potential advantages to using nongenic models of pancreatitis, including their lower expense, and in many cases, the same method of induction can be achieved through multiple routes of administration of an agent and across both small and large animal species. Finally, the initiation of disease can often be highly synchronized across a cohort of animals.

TABLE 1

TABLE 1

Similar induction methods can sometimes be modified to phenocopy both acute and chronic types of pancreatitis. For example, the dosage, frequency, and route of administration of cerulein are regularly manipulated to experimentally induce both AP and chronic pancreatitis (CP). Cerulein was first used to induce AP in rats using intravenous infusion, but it was later discovered that 6 to 8 hourly intraperitoneal injections also provoke mild AP, whereas 12 hourly injections provoke more severe AP.1–4 By repeating daily dosing with cerulein, one can replicate recurring episodes to develop a model of recurrent AP. However, many investigators use this paradigm, repeated dosing of cerulein, to generate CP models. The dosing regimen is not standardized, and the variability of the repeated cerulein paradigms was recently outlined by Klauss et al.15 For example, biweekly sessions of 6 to 8 hourly injections for 6 to 10 weeks result in histological changes consistent with CP, but dramatic differences in fibrosis are seen in different mouse strains. Unlike clinical CP, when cerulein dosing is terminated, pancreata recover within 3 to 6 weeks.16 Other groups have accelerated the repeated cerulein model and show that administering 6 hourly injections on 3 days per week for 3 to 4 weeks also recapitulates CP.17 It is unclear at this time whether there are meaningful differences between the variable dosing paradigms for CP. It is also unknown whether a longer dosing schedule would generate irreversible pancreatic changes. Many laboratories currently choose the dosing paradigm based on what is most convenient for laboratory personnel; however, they have shown their chosen regimen works for their individual studies.

One step that must be taken to improve the comparability of results across studies and models is to standardize endpoints. The diagnosis and severity of pancreatitis rely heavily on pathologist-specific interpretations of histology: interstitial edema, acinar cell death (necrosis or vacuolization), parenchymal loss, hemorrhage, fat necrosis, inflammatory cell infiltrate, and fat and fibrotic tissue replacement.5 However, there are quantitative measures available for exocrine function, edema, immune responses, and endocrine function that should be used alongside histological measures.6–10,18 For example, serum enzyme levels (eg, amylase or lipase) or intrapancreatic trypsinogen activation are standard exocrine-related endpoints that should be included in all studies, particularly those focused on AP or early phases of CP. Furthermore, edema can be evaluated through objective comparisons of organ weight. Inflammation can be measured using a standard myeloperoxidase assay as well as pancreas-specific or systemic measures of cytokine expression. For severe or advanced disease models, measures of endocrine function such as glucose levels or glucose tolerance tests could also be informative. Although preliminary studies of stains for fibrosis are important, quantitative measures of collagen expression (collagen subtypes, hydroxproline levels) should be performed when fibrosis is an endpoint. With all AP and CP models, the time course of pancreatitis can differ among species and within strains of the same species, as well as by age or by sex in some strains of rodents. Equally important is the time point at which a particular endpoint is measured. The peak and resolution of an endpoint are likely parameter specific, and more thorough investigations are ongoing. For example, in the acute cerulein model initial zymogen activation within the acinar cell begins between 15 minutes and 1 to 2 hours after the first cerulein injection, but inflammatory cells mediate this at later time points.91–94 Thus, it is particularly important to consider previous literature and conduct preliminary studies to decide on the most appropriate time points to assess your chosen parameters (eg, 1 hour, 6 hours, or 24 hours after the first or last cerulein injection).95,96

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Pancreatic Cancer

Multiple chemicals have been used to model tumorigenesis (Table 2). If these carcinogens are introduced systemically, tumors develop across multiple organ systems. For instance, administration through oral gavage will incite lesions throughout the gastrointestinal tract, whereas intraductal instillation will restrict tumor development to the pancreas.97–99,115 More recently, investigators have largely moved away from chemically induced tumor models; instead, a variety of transplantation models have gained significant favor. There are several sources for the transplants that will be discussed below. The site of the transplant in the host animal can also vary, but the most popular locations are subcutaneous or directly into the pancreas. Investigators opt for subcutaneous placement because it is easy to observe tumor growth in the absence of advanced imaging technologies as well as to perform behavioral assays. For investigators interested in metastases, tumor cells can also be implanted into the target of interest such as liver or lung. Orthotopic transplantation of pancreatic tumor cells can be used to model primary tumor growth in a synchronized manner such that an entire cohort will have similar progression of disease.

TABLE 2

TABLE 2

The source of pancreatic tumor cells that are transplanted is critical. There are a multitude of human pancreatic tumor cell lines available.116–118 To perform xenografts (interspecies transplantation), nude or SCID mice must be used, so the foreign cells will not be rejected. More recently, the development of the patient-derived xenograft (PDX) provided a great hope toward the development of personalized therapies because drugs could be directly tested on each individual patient's tumor. The PDX model of pancreatic ductal adenocarcinoma (PDAC) has facilitated translational (from mouse to man) studies on pancreatic cancer. Manegold et al119 recently reported on serial subcutaneous implantations of PDX PDAC tissue into immunocompromised NOD/SCID/IL2rγ−/− (NSG) mice to study the role of CBP/β-catenin-induced pancreatic cancer cell stemness in human PDAC carcinogenesis. The investigators found that the first subcutaneous implantation of human PDAC into mice that were treated with a specific small molecule CBP/β-catenin antagonist, ICG-001, did not inhibit the growth of the xenograft. However, the ICG-001-treated tumor (which grew during the first implantation) failed to grow during the second implantation. The investigators concluded that CBP/β-catenin antagonism of pancreatic cancer cell stemness was able to prevent propagation of human PDAC. Indeed and as previously reviewed,120 such PDX models provide a couple of key advantages because the PDAC xenografts are comprised of cells which (1) largely retain the appropriate genetic profile even after initial selection in the mouse, (2) display a high degree of heterogeneity characteristic of PDAC at least initially, and (3) are derived from humans.

Xenograft models do have several limitations; the key one being that transplant recipients must have a compromised immune system.121 A relatively more physiological approach is the syngeneic or allograft model in which the donor cells and recipient are of the same species. Like xenografts, either primary tumor cells or cell lines can be used for allografts. Using a syngeneic approach allows a better assessment of how the intact immune system contributes to tumor growth. Previously, the Panc02 cell line was the primary cell line readily available for such syngeneic mouse models. However, the major limitation of the Panc02 cell line is the finding by Logsdon et al120 that they do not harbor mutations in Kras or p53, which represent the most common, classical mutations in PDAC, hence severely diminishing the translational relevance of such models. More recently, Kras mutant cell lines, 6606PDA and 6606I, isolated from PDAC and liver metastasis, respectively, from KrasG12D (KC) mouse,122 as well as Kras mutant and p53 mutant cell lines UN-KPC-960 and UN-KPC-961 derived from KrasG12D;Trp53R172H;Pdx1-Cre (KPC) mice,123 have been developed and have been used in syngeneic models. Given these recently developed mouse cell lines, which harbor relevant PDAC mutations as well as methods for harvesting primary tumor cells,124 the syngeneic mouse model should facilitate more in-depth assessment of how the immune system contributes to the genesis of PDAC. Overall, transplantation models are useful because they are relatively easy and quick to set up. However, most transplant models are based on the implantation of differentiated tumor cells, which could be viewed as a drawback of this approach. In patients, there is a natural progression from normal to neoplastic cells. Transplantation models lack the opportunity to study how bodily systems function together to drive the transformation of normal cells.

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Environmental Modulators

Although nongenic models have weaknesses, there are advantages to using them for the study of exocrine pancreatic diseases. Nongenic models provide an opportunity to study the impact of environmental modulators including alcohol and tobacco, major risk factors for both pancreatitis and pancreatic cancer.125 Combining exposure to these factor(s) with the nongenic models is a useful way to examine the role of the environment in the development and progression of pancreatic disease. The most common method to introduce alcohol exposure is through the Lieber-Decarli liquid diet that allows for easy modifications of the amount of ethanol.32 Ethanol (EtOH) administration alone does not cause a pancreatitis phenotype; however, combined with chemical (eg, dibutyltin dichloride [DBTC], oleic acid) or diet models, it can accelerate disease and increase severity.38,126–130 Furthermore, EtOH in combination with its nonoxidative metabolites, fatty acid ethyl esters or a subclinical stimulus dose of cerulein or lipopolysaccharide is also sufficient to produce pancreatitis.131–134 Ethanol use has also been associated with development of adenocarcinoma in the dimethylbenzanthracene (DMBA) model.107 Ethanol increases multiplicity but not incidence in the rat azaserine model and the hamster N-nitrosobis (2-oxopropyl)amine (BOP) model.100 Interestingly, dietary fat enhances carcinogenesis in both the azaserine and BOP models. Cigarette smoke has also been shown to exacerbate nongenic models of pancreatitis and pancreatic cancer.108,135,136 By combining EtOH, smoke, and diet with nongenic models of exocrine pancreatic disease, investigators have been able to confirm the degree that these environmental exposures affect the risk for development or increased severity of disease.

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GENETIC MODELS

To study pancreatitis and pancreatic cancer, investigators have developed a plethora of genetically engineered animal models. Three key aspects for developing a genetically engineered model include choosing a method to produce the animal, choosing the genetic alteration, and choosing the gene promoter that will drive expression of that genetic alteration. In addition to the CRISPR-Cas9 technology, several classical methodologies to generate genetically engineered models are well described.137 There are several gene promoters that are popular for use in genetically engineered models of exocrine pancreatic disease because they are enriched in the pancreas and, when combined with a conditional technology (eg, Cre-Loxp), are thought to restrict recombination specifically to the pancreas (Table 3). However, it is important to realize that the method and the gene targeted have implications for differing interpretations of results. Specifically, using technologies that are not inducible means that the animal will express the genetic change in all cell types that express the gene at any time throughout development, including extra pancreatic tissues. In instances where the mutation of interest is a known germ line mutation, global recombination approaches may be optimal because they mimic hereditary disease. However, this could be considered a negative when the goal is to achieve pancreas or even cell-type specific mutations. PDX1 and p48/pft1a, the 2 most widely used gene promoters in exocrine pancreatic disease, are expressed in multiple cell types within the pancreas and can result in multiple foci of disease.138,139 PDX1 is also expressed in duodenum and antrum of the stomach.140–142 Moreover, both PDX1 and p48 are expressed in various divisions of the nervous systems. Pft1a/p48 is a key regulator in the differentiation of spinal cord dorsal horn neurons, determining GABAergic versus glutamatergic cell fate.143 PDX1 is expressed in a subset of sensory and proprioceptive neurons and regulates neuronal calcium homeostasis.144 Neuronal PDX1 is also involved in hypothalamic control of glucose metabolism.145,146 Thus, extra pancreatic expression and/or function of pancreas specific genes could contribute to disease phenotype and should be considered when interpreting data.

TABLE 3

TABLE 3

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Pancreatitis

There are several genetically engineered mouse models (GEMMs) that spontaneously exhibit pancreatitis-related phenotypes (Table 4). These models can be useful for questions regarding the course of idiopathic pancreatitis as well as testing pharmacological interventions. Although the majority of genetically engineered pancreatitis models are in the mouse, nonmurine models are also available. The Wistar Bonn/Kobori (WBN/KOB) rat exhibits degeneration of pancreatic parenchyma, widely distributed fibrosis, and infiltration of lymphocytes.179 Overexpressing Sonic hedgehog or Indian hedgehog at the Ptf1a domain in zebrafish results in morphological changes in developing pancreas.180 With age, these zebrafish show progressive pancreatic fibrosis intermingled with proliferating ductular structures and destruction of acinar structures. Our current understanding of hereditary pancreatitis has been significantly improved through the development of GEMMs. Animal models expressing mutations in trypsinogen or SPINK genes exhibit spontaneous pancreatitis or a predisposition to develop more severe disease after experimentally induced pancreatitis.149–151,168 Like the combinatorial approach introduced earlier (nongenic method of induction plus exposure to environmental factors), a similar strategy is often used to study the role of a specific gene or signaling pathway in the development or severity of pancreatitis. In this scenario, a nongenic method of inducing pancreatitis is applied to GEMMs that do not exhibit a spontaneous disease phenotype. Investigators have successfully used this approach to implicate numerous signaling pathways in the development and severity of pancreatitis, including the complement system,181–185 cytokine signaling,147,186–188 immunoglobulins,189,190 and, of course, protease (eg, trypsin) pathways.91,92,191,192 Based on the prior evidence implicating trypsin pathways, Geisz and Sahin-Tóth168 recently created a gain of function trypsinogen mutant that effectively mimics hereditary pancreatitis.

TABLE 4

TABLE 4

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Pancreatic Cancer

The field of basic pancreatic cancer research has exploded with the development of GEMMs for pancreatic cancer. Mutations inducing a gain of function in the GTPase Kras occur in nearly all human pancreatic intraepithelial neoplasia (PanIN) and PDAC.193,194 Several basic GEMMs, described in Table 5, take advantage of oncogenic Kras expression combined with pancreas specific promoters.137,193,194,206–210 Although multiple mutations in Kras have been detected in PDAC patients, the most prevalent is G12D.211–213 Thus, KrasG12D has become the backbone of PDAC GEMMs. The pathophysiology, histology, molecular, and clinical aspects of GEMMs that parallel the disease course of human pancreatic cancers are detailed elsewhere.194,209 Briefly, GEMMs exhibit a slower disease course compared with transplant models. Lesions (eg, PanIN or mucinous) develop followed by tumor formation and, in some cases, metastases.

TABLE 5

TABLE 5

Mutations in additional oncogenes such as TP53, CDKN2A, and SMAD4, occur in more than 50% of human PDAC cases.138,194,207–210 The contribution of these genes to the development and progression of disease can be assessed with the complex Kras-based GEMMS described in Table 6.214,215,226–228 Often one of the most obvious results of introducing additional pathological gene mutations is an acceleration of disease progression. The most popular complex Kras-based GEMM is the KPC (KrasP53Cre) model. KPC was originally coined to describe a GEMM in which PDX1-cre drives KrasG12D and p53R172H mutations.197,229 Over time, however, it has been more loosely applied to GEMMs that use either PDX1-cre or p48/Pft1a-cre as well as those deleting a p53 allele (p53fl/+).198,220,230,231 For mice to be viable, the Kras mutation must be heterozygous in these models. However, it is possible to make viable mice that express different zygosities for other gene alterations. The zygosity of any additional genetic alterations incorporated into a model can directly affect the time course of disease progression. For instance, mice expressing a single mutant p53 allele (KrasG12D;p53(fl/+)) develop tumors around 16 weeks, whereas dual mutant p53 alleles (KrasG12D;p53(fl/fl)) develop lethal tumors by 8 weeks.226 We encourage authors to follow this example and provide explicit descriptions of their models in every publication to avoid confusion in the field.

TABLE 6

TABLE 6

A key challenge with making GEMMs is the complexity of the genetics. As one moves from the basic to more complex models, there is a reduction in both cost-effectiveness and efficiency in breeding and using these models (eg, only 1 in 8 or 1 in 16 mice could have the genotype of interest). Many investigators have also opted to incorporate reporters into their models to allow for tracking of pancreatic-lineage cells as they transform, disseminate, and metastasize.198,230–233 Although a creative improvement on this already valuable technology, the resulting genetic complexity presents an increased risk for unknown or unexpected adverse effects. Bearing in mind the caveats of complex genetic models, GEMMs are a powerful tool to aid in our investigation of PDAC.

Although the majority of current pancreatic cancer models are Kras based, there are non-Kras–based models. The first transgenic mouse models of pancreatic neoplasia were generated by expressing the full-length or truncated oncogenic simian virus 40 T antigen (SV40) under the rat elastase-1 promotor.234,235 These mice exhibit acinar dysplasia that progresses to neoplasia in adulthood. Survival is approximately 4 to 6 months. More recently, non-Kras–based models have been developed to study the contribution of specific tumor suppressor genes to the development of PDAC. Mutations in pten are commonly found in several tumor types. Stanger et al236 developed Pdx1-CreER™;Ptenlox/lox in which pten is deleted from the pancreas and cre activity is confirmed through the Z/AP reporter. Beginning at 3 weeks of age, mice exhibit an age-dependent phenotype with multifocal architectural changes. Acini are progressively replaced by proliferative mucin-expressing ductal structures, centroacinar cells proliferate, and ductal adenocarcinoma develops at 11 weeks. To parse out the contribution of the tumor suppressor genes Ink4a/Arf (p16Ink4a/p19Arf) and TP53, Bardeesy et al237 examined the incidence, latency, and histological phenotype of PDAC after either hemizygous or homozygous loss of p16 and p19 or p16 and p53. These authors demonstrated that PDAC can develop in a non-Kras–based GEMM and also that specific tumor suppressor genotypes directly influence the phenotypes of resulting tumors.

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Environmental Modulators

To explore the interactions between genetics and the environment, factors such as alcohol, diet, and tobacco can be paired with genetic models of disease. For instance, long-term ethanol feeding in the Mist1-creERT2;XBP1+/− model of spontaneous pancreatitis causes increased endoplasmic reticulum stress, thereby enhancing acinar cell pathology.158 Similarly, exposure to tobacco induces flattening of ductal epithelial cells and significantly increases atrophy in the rEla1;sshIL-1β model of spontaneous pancreatitis.147 Ethanol and smoking have also been introduced into genetic models of pancreatic cancer. Exposing KC (Pdx1-Cre; K-Ras+/LSLG12D) mice to the Lieber-DeCarli alcohol diet in combination with cerulein results in synergistic and additive effects on PanIN formation.198,238 Exposing KC mice to tobacco smoke also stimulates PanIN development, fibrosis, activation of stellate cells and M2 macrophages, as well as increased expression of both stem cell and epithelial-mesenchymal transition markers.239,240 Smoking was found to activate histone deacetylases and regulate cytokines such as interleukin (IL) 6 and IL-4, which promote cancer development at the early stage.239 Smoking also activates stem cell features of pancreatic cells, through activation of PAF1 expression in KC mice.240 The same procancer effects were observed in Kras+/LSLG12Vgeo;Elas-tTA/tetO-Cre (Ela-KRAS) mice.241 The effect of alcohol alone on promoting PDAC is less obvious than smoking in animal models. However, the combination of alcohol with pancreatitis further promotes the progression of the disease in KC mice.238 Analogous to the association between pancreatitis and diet, a high-fat diet and obesity are associated with increased risk for pancreatic cancer. Several groups have taken of advantage of GEMMs to study the biological mechanisms linking obesity to pancreatic cancer. A high-fat lard-based diet in the Ela-CreERT;K-Ras+/LSLG12D GEMM promotes immune infiltration in addition to PanIN and PDAC development through regulating inflammation in a COX2 and lipocalin-2 dependent manner.242,243 Similarly, a high-fat, high-calorie, corn oil–based diet given to EL-Kras and KC mice is associated with accrual of additional genetic mutations, as well as more extensive inflammation, fibrosis, and neoplasia.244,245 The incidence of cancer was particularly higher in males, which is thought to be associated with a sex-dependent localization of adipose (visceral vs subcutaneous). Interestingly, a high-calorie diet based on fish oil (menhaden) significantly delays PanIN progression.246 Using these early stage models, including KC mice, is sometimes criticized because PDAC has not yet developed and never develops in most of these mice. However, understanding the changes in the cancer precursor cells is equally important to understanding changes in cancer cells because it will help develop a preventive strategy. Moreover, the data published in KC mice were confirmed in the Pdx1-Cre;LSL-KrasG12D;Trp53R172H/+ (KPC) mice, confirming that smoking and high-fat diet upregulate tumor growth and metastasis.241,247 It is very important to use the best model to study the effect of environmental factors on PDAC promotion; it is equally important to choose the best time to start or stop treatments, as the disease progresses with different kinetics in each model.

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INDUCIBLE GENETIC MODELS

Despite the identical genetic background in models that use inbred strains, disease development and progression are variable across animals. Some investigators minimize this issue by using inducible technologies to improve synchronization of experiments and avoid complications of expressing mutations throughout development of the animal. Many of the models described in Tables 4 to 6 take advantage of these inducible technologies. The most popular choice is to use tamoxifen-dependent cre recombinase systems (eg, CreER, CreERT, CreERT2) in which transgene expression is not induced until tamoxifen treatment. There are also models that take advantage of the tetracycline transactivator (tTA) method in which expression is triggered by the removal of doxycycline from drinking water or food.173,174,200,241

Viruses provide an alternative inducible method that allows more control over the localization of recombinant gene expression. For instance, adeno-associated virus serotype 6 (AAV6) encoding Ela-iCre infused into the pancreatic duct of calcineurin B1 (CnB1)fl/fl mice results in acinar cell specific loss of CnB1 gene expression.79 In newborn pigs, ductal expression of genes can be achieved by injecting AAV9 vectors into the celiac artery, accessed via umbilical catheterization.248 In Swiss Webster mice, intraductal infusion of lentivirus has been used to drive expression of shRNAp53, KrasG12D, and luciferase.249 These mice develop pathology similar to human PDAC without requiring alterations of embryonic development. They develop PanINs with increasing severity followed by tumor formation 28 weeks postvirus injection. Mice also have elevated levels of cancer markers and, in some cases, liver and lung metastases. Inducible methods have advanced the field of GEMMs by improving temporal and spatial control over these models.

Viruses also have the advantage of enabling the development of genetically engineered animal models in larger species. Viral models are becoming a popular route for developing large animal models because of their importance in drug development and improved resemblance to humans. Previously, large animal (eg, dog, nonhuman primate) studies have been restricted to case reports of spontaneous disease. One type of pancreatic disease that has benefited from a viral approach is cystic fibrosis (CF) associated pancreatitis. Mutations in the CFTR gene lead to dysregulation of fluid transport in multiple organs, primarily lung and pancreas. Low flow of secretions leads to duct obstruction, acini destruction, severe inflammation, fibrosis, and fat replacement. With the advent of recent advances in genetics including viral-mediated gene targeting and somatic cell nuclear transfer, global manipulation of the CFTR gene in larger species has become possible. Two pig models, global CFTR-null and CFTR-Δ508, were generated using homologous recombination and somatic cell nuclear transfer.250,251 Fetal and neonatal CF pigs have pancreatic lesions seen typically in humans with CF including progressive acinar cells loss, ductal plugging and fibrosis.252,253 Although islets are morphologically normal, CF pigs demonstrate abnormal glycemic responses and decreased insulin secretion at birth.254 A similar approach has been executed to generate CFTR-null ferrets.255 CF ferrets exhibit a milder exocrine pancreatic phenotype compared with CF pigs. Newborn CF ferrets have only minor histological changes in the pancreas including dilated acini and ductules with inspissated, eosinophilic zymogen secretions, but no acinar atrophy and otherwise preserved pancreatic architecture.256 The pancreatic disease progresses rapidly in CF ferrets after birth (>1 month) with loss of acini, inflammation, fibrosis, islet destruction/remodeling, and hyperglycemia.257,258 Another model that is worthwhile to mention is the CFTR-null zebrafish model.259 Loss of CFTR leads to exocrine pancreatic destruction in zebrafish larva. The CFTR models are based on expression of global alterations, but viral-mediated approaches can also be used to make tissue specific manipulations. The Oncopig cancer model is a novel model in which pigs have combined KrasG12D and TP53R167H mutations under control of a cre-inducible vector.260 Introduction of virus containing Ad-Cre directly into the pancreatic duct drives locally invasive disease that has the hallmarks of human PDAC including a dense fibroblastic stroma and acinar-to-ductal metaplasia.261 The development of large species models creates a unique opportunity in which not only pharmacological therapies but also novel surgical approaches can be tested.

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PAIN

Pain is a prominent feature associated with both pancreatitis and pancreatic cancer. The role of the nervous system, neuroplasticity, and neurogenic inflammation in exocrine pancreatic disease has been reviewed before.262–265 Chronic pain, such as that associated with pancreatic disease, is often a result of sensitization of peripheral neurons. Several studies have used molecular tools to examine the expression of molecules involved in neurogenic inflammation and peripheral sensitization including growth factors, neuropeptides, and TRP channels.11,22,266–271 A few laboratories have also performed functional studies (eg, calcium imaging, electrophysiology) to directly assess sensory neuron excitability and sensitization.11,40,267,272–277 Most studies, however, have focused on pain-associated behaviors (Table 7). There are 2 types of pain that can be assessed, experimentally evoked or ongoing pain. Experimentally evoked mechanical pain involves applying Von Frey monofilaments of increasing force to determine withdrawal thresholds. Application of radiant heat or placing the animal on a hot plate can be used to assess latency to respond to a noxious temperature. These are thought to be modeling referred somatic pain. Finally, direct electrical stimulation of the pancreas evokes an abdominal muscle contraction called the visceromotor reflex (VMR). This technique can be used to determine the threshold to evoke a VMR or changes in the size of the VMR in controls versus animals with pancreatic disease. If the thresholds or latencies to evoke a response using any of these tests are reduced, this is interpreted as the animal experiencing pain. There are no strong models for directly measuring ongoing pain. In humans, however, ongoing pancreatic pain leads to hunching posture as well as a reduction in quality of life and spontaneous activity. Several investigators have taken advantage of this and used a variety of measures thought to indicate ongoing pain: reduced rearing, grooming, wheel running, and ambulation as well as increased hunching, vocalization, and catalepsy. Unfortunately, pancreatic pain–related studies have been limited to rats and mice whose neural innervation, ductal structure, location, and gross anatomy are quite different from humans. Future studies need to address pain in a wider variety of animal models, including larger species, to improve our understanding of pain within the context of exocrine pancreatic disease.

TABLE 7

TABLE 7

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CHOOSING A MODEL

One must consider the strengths and weaknesses implicit to each model as well as available resources and expectations regarding experiments and findings. Just as important, however, is which animal system to choose. Nongenic models can be applied to a greater variety of species, but there can be differential responses across species. Indeed, it is important to choose the most appropriate method (+/− additional environmental factors) to induce pancreatitis and/or pancreatic cancer. For instance, the nitrosamine BOP preferentially drives pancreatic tumorigenesis in rodents, but not dogs (Table 2). There are also species differences associated with the nongenic models of pancreatitis. For example, cerulein administration to rats results in more interstitial edema and intracellular vacuolization, whereas mice develop more acinar cell necrosis, a difference that may be explained by species-dependent roles of apoptosis and autophagy.295,296 Mechanical models of pancreatitis have been applied across the widest range of species, from mice to nonhuman primates, and a diversity of responses has been reported.297 In most species, ductal ligation and bile acid infusion evoke mild disease; however, pigs and opossums exhibit severe disease. The variability in mechanical models is likely a consequence of the anatomical and functional differences across species.298–300 Grossly, mouse and rabbit, for example, have diffuse pancreata scattered within the mesentery, whereas dog, hamster, and pig have compact pancreata more closely resembling the retroperitoneal solid pancreas found in humans. In addition, the ductal structure varies widely across species. Rats have a single outflow channel that may explain why rats develop fibrosis more rapidly than dogs after ductal ligation. Mice, on the other hand, have multiple ducts. This can provide an internal control within each animal if desired, but it also makes it technically challenging to execute a complete obstruction. When discussing data, it is important to consider how the anatomy of the animal may contribute to observed phenotypes.

Within a species, there are also important considerations when choosing a model (eg, age, sex, strain, body fat). For instance, mouse strain directly impacts the severity of pancreatitis and systemic inflammation after intraductal infusion of taurocholate.48 Serum enzymes and morphological damage are increased compared with controls in nine different strains; however, NOD/SHILT and AKR/J mice had enzyme activity significantly higher than the other strains. Furthermore, only half of the strains exhibited elevated IL-6, a marker of inflammation. Even within substrains, differences in the severity of disease can be observed. C57BL/6 J mice, for example, are more susceptible to cerulein-induced pancreatitis than their C57BL/6NHsd counterparts with regard to atrophy, morphological changes, and fibrosis.301 Strain differences have also been reported in GEMMs. A study examining the effects of high-fat diets on PDAC in the Ela-KrasG12D GEMM reported that the incidence, frequency, and size of pancreatic neoplasia were significantly increased in mutant mice with an F1 background (hybrid strain containing FVB/N and C57bl/6 genetics) as compared to the pure FVB strain.302 Differences are not restricted to strain but also appear with respect to sex. Females, for instance, exhibit a much greater sensitivity to the choline-deficient ethionine-supplemented (CDE) diet because CDE induces hemorrhagic necrosis in an estrogen-dependent manner.35,303 Another consideration is body fat status. The coadministration of IL-12 and IL-18, cytokines elevated in AP patients, drives edematous AP in wild type or lean mice.30 However, the same doses induce necrotizing pancreatitis in both diet-induced obese mice and a GEMM of obesity called ob/ob mice.30,31 Finally, age understandably plays a role in the development of disease in noninducible genetically engineered models. However, age has also been implicated in nongenetic models. For example, in the cerulein paradigm for AP, a loss of uncoupling protein 2 aggravated the severity of disease in older but not young mice.304

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CONCLUSIONS

The goal of animal models is to reproduce human disease including etiology, histopathology, pathophysiology, and therapeutic responsiveness. All of the animal models presented have both strengths and weaknesses with regard to disease phenotype. There are also considerations with respect to time and cost. The rationale for which model is chosen for a study should be clearly explained in publications. With the complex nature and variability of these models, it is essential to be overly transparent and provide explicit details regarding the design of the model used in a particular study, including which features of disease it successfully recapitulates. Several of the models available provide an opportunity to examine the synergy between environmental and genetic contributors to disease, which would expand our understanding of the pathogenesis and progression of exocrine pancreatic diseases. However, neuroplasticity and pain are key features of both pancreatitis and pancreatic cancer. If models are going to be truly translational moving forward, they should also recapitulate alterations in the nervous system. At this time, how the nervous system is affected is unknown for most of the available models. To improve translation of basic pancreas research to clinically relevant therapies, there must be methods in place to ensure thorough interpretation of data, comparison across studies, and validation of findings. Toward that end, all future studies should incorporate objective quantitative measures to allow for these direct comparisons. Furthermore, in the event that potential therapeutics are identified, we strongly recommend simultaneous testing and/or validation by an independent laboratory at another institution. Indeed, such cross-validation should be incorporated into studies during the design phase, as potential collaborators are easily identified through the numerous pancreas associations and consortiums. Although we have made much progress, continued refinement of currently available models along with development of newer models will be important for bridging the gap between basic science and the clinic.

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          Keywords:

          pancreatitis; pancreatic cancer; animal models

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