Alteration of strain background and a high omega-6 fat diet induces earlier onset of pancreatic neoplasia in EL-Kras transgenic mice

Abstract

Diets containing omega-6(ω-6) fat have been associated with increased tumor development in carcinogen-induced pancreatic cancer models. However, the effects of ω-6 fatty acids and background strain on the development of genetically-induced pancreatic neoplasia is unknown. We assessed the effects of a diet rich in ω-6 fat on the development of pancreatic neoplasia in elastase (EL)-Kras^G12D (EL-Kras) mice in two different backgrounds. EL-Kras FVB mice were crossed to C57BL/6 (B6) mice to produce EL-Kras FVB6 F1 (or EL-Kras F1) and EL-Kras B6 congenic mice. Age-matched EL-Kras mice from each strain were compared to one another on a standard chow. Two cohorts of EL-Kras FVB and EL-Kras F1 mice were fed a 23% corn oil diet and compared to age-matched mice fed a standard chow. Pancreata were scored for incidence, frequency, and size of neoplastic lesions, and stained for the presence of mast cells to evaluate changes in the inflammatory milieu secondary to a high fat diet. EL-Kras F1 mice had increased incidence, frequency, and size of pancreatic neoplasia compared to EL-Kras FVB mice. The frequency and size of neoplastic lesions and the weight and pancreatic mast cell densities in EL-Kras F1 mice were increased in mice fed a high ω-6 fatty acid diet compared to mice fed a standard chow. We herein introduce the EL-Kras B6 mouse model which presents with increased frequency of pancreatic neoplasia compared to EL-Kras F1 mice. The phenotype in EL-Kras F1 and FVB mice is promoted by a diet rich in ω-6 fatty acid.

Introduction

Pancreatic adenocarcinoma is the fourth leading cause of cancer-related mortality in the U.S.¹ An estimated 34,290 deaths resulted from pancreatic cancer in 2008 alone.¹ Given the dismal mortality associated with pancreatic cancer, much attention has been given to the prevention of this deadly disease. Several risk factors have been identified with the aim of minimizing these factors for the purpose of prevention.

Obesity is one such risk factor, which has been shown to increase the risk for pancreatic cancer in numerous prospective cohort studies. A body mass index (BMI) ≥30 was found to increase relative risk (RR) for pancreatic adenocarcinoma, with RR values ranging between 1.2 to 2.76.^2-6 However, the exact mechanisms that relate obesity to pancreatic cancer continue to be investigated.

Fatty acid metabolism is an area of research that may help explain the tie between obesity and pancreatic cancer. Our lab previously found that omega-3 fatty acids have a chemopreventive role against the development of pancreatic neoplasia in a murine model.⁷ In contrast, previous studies have shown that diets rich in ω-6 fatty acids stimulated pancreatic tumor growth, in vitro and in vivo.^{8, 9} However, these studies have had conflicting explanations for mechanism. In vitro evidence argued for increased proliferation rates due to ω-6 fatty acids while in vivo studies found no difference in proliferation rates. Furthermore, nearly all related animal studies utilized carcinogen-induced invasive pancreatic adenocarcinoma models. Perhaps more relevant to the effects of a high ω-6 diet is the study of preinvasive disease, namely pancreatic intraepithelial neoplasia (PanIN), intrapapillary mucinous neoplasia (IPMN), and mucinous cystic neoplasia (MCN). Utilizing an established murine model of pancreatic preinvasive disease in three different backgrounds (FVB, F1, and B6), we evaluated the effects that background strain and a diet rich in ω-6 fatty acids (see Table 1) has on the development of neoplastic lesions

Table 1. Diet Composition.

Ingredients and calculated energy levels for the high omega-6 and standard mouse diets.

Ingredient	Standard Diet (g/kg)	High Omega-6 Diet (g/kg)
Corn Oil	0	230
Total Omega-6 Fatty Acids	27.9	139.6
Fish Oil	0	0
Total Omega-3 Fatty Acids	2.9	1.95
Omega-3:Omega-6 Fatty Acid Ratio	1:9.6	1:118
Calculated Values
Protein	19%	18%
Fat	5.6%	23%
Fiber	4.5%a	5%
Ash	6.5%	2.8%
Moisture	10-12%	<5%
Carbohydrate	40.3%	46.2%
Protein Energy	0.76 kcal/g	0.72 kcal/g
Fat Energy	0.51 kcal/g	2.07 kcal/g
Carbohydrate Energy	1.61 kcal/g	1.85 kcal/g
Total Energy	2.90 kcal/g	4.64 kcal/g

^avalue represents crude fiber while actual fiber may be closer to 14–16%

Material and Methods

Production of transgenic study groups

EL-Kras transgenic mice were generated by microinjection of FVB/N fertilized mouse embryos and subsequent implantation into pseudopregnant females as described in a prior publication.¹⁰ PCR genotyping of EL-Kras mice demonstrated that all males were positive for the transgene while all females were negative. EL-Kras mice were subjected to tail biopsy and PCR evaluation of genomic DNA from mouse tail digests (see below). EL-Kras male mice were bred to C57BL/6 (B6) female mice to generate EL-Kras FVB6 F1 (EL-Kras F1) and EL-Kras B6 mice. In order to evaluate the differences in the pancreatic neoplastic phenotype between EL-Kras mice in these three strains (FVB, F1, and B6), a group of 12 EL-Kras FVB mice (8-10 months old) were compared to 12 age-matched EL-Kras F1 mice for incidence (number of mice with lesions per number of mice with respective genotype), frequency (number of lesions per random section/per mouse) and average neoplastic lesion size. The same phenotypic comparisons were performed on 14 EL-Kras FVB mice (12-14 month old) and 14 age matched EL-Kras F1 mice. Similar comparisons were done between 15 EL-Kras F1 mice and 7 EL-Kras B6 mice all at 8-12 months of age. (A broader time window was used for this initial evaluation in order to capture more mice per group.) Table 2 outlines the number of mice per cohort.

Table 2. Experimental Groups.

Number of mice per cohort, with background and high omega-6 diet (Fat) respectively designated.

	FVB				F1					B6
Age	8 – 10mo	8 – 10mo Fat	12 – 14mo	12 – 14mo Fat	8 – 10mo	8 – 10mo Fat	8 – 12mo	12-14mo	12 – 14mo Fat	8 – 12mo
Total	12	6	14	6	12	6	15	14	9	7

Tail biopsy and PCR genotyping

A segment of mouse tail was digested with proteinase K (Fermentas) solution in GNT Buffer (50mM KCl, 1.5mM MgCl₂, 10mM Tris-HCl, 0.01% gelatin, 0.45% NP40, and 0.45% Tween-20 pH=8.5). The digested, heat-inactivated supernatant was used as a DNA template in the following 25 ul PCR reactions: 1 uM of each primer (EL-Kras 1f primer 5′-GAGTGCCGGCCTTGTTCTGTCTTTG -3′ and 2r primer 5′- CTACGCCACAAGCTCC AACTACCAC -3′) in a solution with 1X PCR Buffer, 1.5mM MgCl₂, 1 uM dNTPs, and 0.2 – 0.4 U Taq polymerase (Promega) was used in a standard PCR reaction. The 25 μl samples were incubated at 94°C for 3-5 minutes followed by 35 cycles of 94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute followed by 5 minutes at 72°C and an indefinite soak at 4-10°C.

Dietary Studies & Histology

Groups of mice were administered ad libitum either a standard chow (∼5% fat) or a diet with 23% corn oil (ω-6). The high ω-6 fatty acid diet was prepared by Bio-Serv (Frenchtown, NJ) and 5kg quantities were sealed in a bag and placed in a box for shipment. To minimize oxidation of fatty acids, the following approaches were employed. These boxes were stored at -20°C in the dark for up to 5 months. Individual bags of diet that were opened were used for only 1 month. Diet on the cages was replaced every 2-3 days. After each use, the diets were kept tightly wrapped and stored at 4°C in the dark. All diets were used up to but not past the 6-month expiration. The specific ingredients and omega-3:omega-6 fatty acid ratios for these diets are provided in Table 1. Two time points (8-10 months and 12-14 months) were selected based on previous findings that most EL-Kras F1 mice at 8-10 months of age will present with neoplastic lesions compared with few if any in EL-Kras FVB mice. At 12-14 months of age, we expected half of the EL-Kras FVB and all of the EL-Kras F1 mice to present with neoplasia. The age of mice on the diet studies were also in these ranges. Six EL-Kras FVB mice were fed the 23% corn oil diet and were sacrificed at 8-10 months of age while six mice were sacrificed at 12 - 14 months of age. The 8-10 month-old cohort of mice was compared to twelve EL-Kras FVB age-matched mice fed a standard chow. EL-Kras FVB mice were crossed one generation into a B6 background. Nine EL-Kras F1 mice were fed a 23% corn oil diet, sacrificed at 12-14 months of age, and compared to fourteen age-matched control EL-Kras F1 mice fed standard chow. All mice were weighed at the time of cull. Following euthanasia, mouse pancreatic tissue was fixed in 10% buffered formalin overnight. Tissue blocks were sectioned at 4-6 um and placed on positively charged slides. Following removal of paraffin by xylenes and subsequent hydration, the tissue was stained with H&E and then dehydrated to xylenes and mounted with a cover slip. H&E stained tissue was scored for the presence of ductal carcinoma in situ (pancreatic neoplasia). Several parameters were noted including incidence (number of mice with phenotype over number of mice with genotype), frequency (number of lesions per random section), and size (area) of neoplastic lesions. The size of these lesions was determined by using a retical with a 10mm × 10mm grid (placed within the eyepiece of the microscope) and counting the number of 1mm² grids that correspond to a specific lesion. The number of 1mm² grids is divided by the magnification of the objective lens to provide the actual area of the lesion.

Immunohistochemistry

After deparafinization, the slides were heated in the microwave in Dako antigen retrieval solution [(S1699), Dako (Carpinteria, CA)] and then washed in Dako wash buffer (S3006). Sections were blocked with 0.5% BSA in PBS for 30 minutes at room temperature followed by Dako peroxidase block for 30 minutes. Primary anti-PCNA (Proliferating Cell Nuclear Antigen) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was placed on sections at a concentration of 1:1000 at room temperature for 1 hour. Sections were then treated with Dako secondary antibody (anti-mouse IgG) HRP-linked (Dako Envision Kit K4008) for 30 minutes at room temperature and developed with DAB reagent for 30 sec. The ApopTag Red in situ apoptosis detection kit from Chemicon International (catalog no. S7185) was used according to the manufacturer's instruction, measuring terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). For mast cell labeling, slides were stained with Naphthol-AS-D chloroacetate (CAE) as previously described.¹¹ Counterstaining was performed with Gills-2 hematoxylin. Immunohistochemistry for PCNA, TUNEL, and CAE was independently evaluated manually by two investigators (P.G. and M.S.), verified by ImageJ software (National Institute of Health, Bethesda MD.), and calculated as an average of the percentages of positive nuclei per total number of nuclei per lesion.

Results

Genetic background influences development of pancreatic neoplasia

EL-Kras mice were originally generated in the FVB/N background.¹⁰ These lesions were characterized as ductal carcinoma in situ with features similar to human PanINs, IPMN, and MCN. We hypothesized that changing the genetic background may affect the neoplastic phenotype as several studies have demonstrated that genetic background may affect the phenotype of transgenic mice.^{12, 13} We demonstrated that the incidence of pancreatic neoplasia in EL-Kras FVB mice at 8-10 months of age was zero percent compared to 92% in age-matched EL-Kras F1 mice (12 mice per group)(figure 1 panel a). Furthermore, there was a 43% incidence of neoplasia in 12-14 month old EL-Kras FVB mice compared to a 100% incidence of neoplasia in age-matched EL-Kras F1 mice (14 mice per group)(figure 1 panel a). In the 12-14 month-old groups, there was a statistically significant increase in the frequency of neoplasia in EL-Kras F1 mice compared to EL-Kras FVB mice (6.4±1.6 vs. 0.6±0.2 lesions per mouse, respectively; p<0.005, figure 1 panel b). Moreover, the EL-Kras F1 mice demonstrated significantly larger neoplastic lesions compared to EL-Kras FVB mice at 12-14 months (1569±463 vs. 453±37 μ², respectively; p<0.05, figure 1 panel c).

Figure 1.

Twelve 8-10 month old and fourteen 12-14 month old EL-Kras FVB mice were compared to an equal number of age-matched EL-Kras F1 mice and histologically compared for a. incidence of pancreatic neoplasia (number of mice with lesions per number of mice with genotype) b. frequency of pancreatic neoplasia (number of lesions in a random section/per mouse) and c. size of pancreatic neoplastic lesions (area in μ²). Compared to 12 – 14mo FVB mice, age-matched F1 mice had increased frequency (FVB, 0.57 ± 0.20 versus F1, 4.43 ± 0.70; **, p<0.005) and size (FVB, 453.3 ± 37.56 versus F1, 1,559 ± 463.2; *, p<0.05) of neoplastic lesions. Means shown are ± SEM. To determine statistical significance, t tests were used.

We generated EL-Kras B6 mice to evaluate if this would further increase the severity of neoplastic lesions. Seven EL-Kras B6 mice fed a standard chow were culled at 8-12 months of age. EL-Kras B6 mice had a statistically insignificant increase in their neoplastic incidence, a statistically significant increase in their frequency, and a trend towards significance in the increased size of neoplastic lesions when compared to EL-Kras F1 mice of the same age (figure 2 panel a – c). There was a difference in weight, with EL-Kras B6 mice weighing less than their F1 controls (31.33 ± 0.8819 versus 40.23 ± 1.645, respectively, figure 2 panel d). To summarize, EL-Kras F1 mice demonstrated an earlier onset, increased frequency, and increased size of pancreatic neoplastic lesions than EL-Kras FVB mice, while EL-Kras B6 mice had increased frequency compared to EL-Kras F1 mice. Of note, these neoplastic lesions were morphologically similar with no progression to invasive cancer among the three background strains. These neoplastic lesions are cystic papillary neoplasms with varying degrees of cellular atypia and either a large, empty lumen or one filled with chords of cells that are occasionally mucinous and thus reminiscent of IPMN. There are infrequent lesions that have some similarity to PanINs and appear contiguous with the ductal system. The term neoplasia will be employed to define this heterogeneous population of lesions, many of which are IPMN-like, a few that are PanIN-like, and an even a smaller population that are MCN-like. None of the lesions progress to cancer (unless EL-Kras mice are crossed to TSG knockout mice), and the natural course of these lesions is unknown. These lesions are consistent between background strains (FVB, FVB6 F1, and B6) as well as the FVB and F1 strains on a diet rich in ω-6 fatty acid. The only phenotypic differences among these groups is the enhanced lipoatrophy (particularly noticeable in FVB mice administered this high ω-6 fatty acid diet), the increased frequency of islets, and the presence of fatty liver in mice administered the high ω-6 fatty acid diet.

Figure 2.

EL-Kras mice were further crossed into a B6 background to evaluate for any additional changes in phenotype. Fifteen 8 - 12 month old EL-Kras F1 mice (F1) were compared to seven age-matched EL-Kras B6 mice (B6) fed a standard chow. Phenotype was evaluated for a. incidence, b. frequency, c. size of pancreatic neoplastic lesions, and d. weight of mice. Lesion incidence was 95.85% in EL-Kras F1 mice, while it was 100% in EL-Kras B6 mice. EL-Kras B6 mice had a statistically significant increase in lesion frequency (F1 mice, 3.267 ± 0.6 lesions/mouse versus B6 mice, 6.60 ± 0.60 lesions/mouse; *, p<0.05) and had a reduced weight (F1 mice, 38.90 ± 1.04g versus B6 mice, 31.71 ± 0.99g; ***, p<0.0005). There was a trend towards significance in increased lesion size (F1 mice, 889 ± 189.6μ² versus B6 mice, 3,150 ± 1,058μ²; p=0.09). Means shown are ± SEM. To determine statistical significance, t tests were used.

A high ω-6 fat diet promotes pancreatic neoplasia in EL-Kras FVB mice

Twelve EL-Kras FVB mice were fed the 23% corn oil diet with six being sacrificed at 8-10 months of age and another six at 12-14 months. None of the mice on the standard diet developed lesions at 8-10 months but some ductal metaplasia was observed (figure 3 panel a). In comparison to age-matched EL-Kras FVB mice fed a standard diet, there was a significant increase in pancreatic neoplasia at the 8-10 month time point (representative lesion in figure 3 panel b and depicted in bar graph format in figure 3 panel d). At the 12-14 month time point, there was a large amount of lipoatrophy in ω-6 fed mice (very little parenchyma) and thus lesions could not be scored (figure 3 panel c). At 8-10 and 12-14 months, there was no statistically significant difference in weights of mice fed the ω-6 diet versus the standard chow (data for 8-10 month group shown, Figure 3 panel e).

Figure 3.

Twelve EL-Kras FVB mice were fed the 23% corn oil diet and six were sacrificed at 8-10 months of age while six mice were sacrificed at 12-14 months of age. The 8-10 month old cohort of mice was compared to twelve EL-Kras FVB age-matched control mice fed a standard chow (∼5% fat). a. Representative section of acinar-to-ductal metaplasia in an 8-10 month old EL-Kras FVB mouse on standard chow. b. Representative neoplastic lesion in an 8-10 month old EL-Kras FVB mouse on a high omega-6 fat diet. c. Representative area of lipoatrophy in a 12-14 month old EL-Kras FVB mouse fed a high omega-6 fat diet. d. Six 8-10 month old EL-Kras FVB mice fed a high omega-6 fat diet were compared to fourteen age-matched EL-Kras FVB mice for incidence of pancreatic neoplasia (number of mice with lesions per number of mice with genotype). e. The weights of 8-10 month old EL-Kras FVB mice fed a high omega-6 diet were not statistically different than age-matched EL-Kras FVB mice fed a standard chow. Means shown are ± SEM. To determine statistical significance, t tests were used.

A high ω-6 fat diet increases the frequency of pancreatic neoplasia in EL-Kras F1 mice

Since none of the EL-Kras FVB mice on a standard diet developed lesions at 8-10 months and the 12-14 month old EL-Kras FVB mice on the high ω-6 diet had severe lipoatrophy, we were unable to further compare the neoplastic phenotype in these cohorts. Therefore, the EL-Kras F1 mice, which have an earlier onset and higher frequency of neoplastic lesions, were fed either a standard diet or high ω-6 fatty acid diet and assessed for differences in the phenotype of neoplasia. Nine EL-Kras F1 mice were fed a high ω-6 fatty acid diet and sacrificed at 12-14 months while fourteen age-matched EL-Kras F1 control mice were fed a standard diet. In both the ω-6 diet and standard diet group, there was a 100% incidence of pancreatic neoplasia (figure 4 panel e). On histological examination, neoplastic lesions in the ω-6 fed mice appeared larger than lesions observed in mice fed the standard chow (representative lesions shown in figure 4 panels a - d). Indeed, the ω-6 group had significantly larger lesions with a mean lesion size of 2,570 ± 407.6 μm versus the standard group's mean lesion size of 1,476 ± 240.8 μm (p<0.05, figure 4 panel g). Furthermore, there was a statistically significant increase in the frequency of neoplasia in the ω-6 fed mice compared to standard diet mice (14.50 ± 3.541 lesions per mouse vs. 6.357 ± 1.595 lesions per mouse, respectively; p<0.05, figure 4 panel f). In addition, there was also a significant increase in body weight in mice fed the diet rich in ω-6 fatty acid compared to those fed a standard chow (p<0.0001, figure 4 panel h).

Figure 4.

Nine EL-Kras F1 mice were fed a 23% corn oil diet, sacrificed at 12 - 14 months of age, and compared to age-matched control EL-Kras F1 mice fed standard chow (∼5% fat). a. Representative neoplastic lesion from an EL-Kras F1 mouse fed a standard chow b. Representative neoplastic lesion from an EL-Kras F1 mouse fed a high omega-6 diet; *, preneoplastic lesion. c - d. higher powered magnification of insets in panels a and b, respectively. EL-Kras F1 mice fed a high omega-6 fat diet demonstrated a similar e. incidence of pancreatic neoplasia, a significantly greater f. frequency of pancreatic neoplasia (standard, 7.83 ± 1.74 lesions/mouse versus omega-6, 17.11 ± 3.64 lesions/mouse;*, p<0.05), a significantly increased g. size of neoplastic lesion (standard, 1,529 ± 309.8μ² versus omega-6, 2,706 ± 603.2μ² *, p<0.05), and a greater average h. weight (standard, 40.23 ± 1.65g versus omega-6, 61.00 ± 2.88;***, p<0.0001) compared to standard diet fed mice. Means shown are ± SEM. To determine statistical significance, t tests were used.

A high ω-6 diet does not alter neoplasia proliferation and apoptosis

Because there was an increased frequency and size of lesions in EL-Kras F1 mice fed a high ω-6 diet compared to standard diet mice, we assessed the proliferative index within lesions by immunohistochemistry for PCNA. The accuracy of PCNA staining was confirmed using Ki-67 staining on matching sections of tissue (data not shown). Mice fed the high ω-6 diet demonstrated no statistically significant difference in PCNA staining compared to age-matched mice fed a standard diet, 24.1% vs. 28.9%, respectively; (p=0.71, figure 5 panels a - c). TUNEL staining was also performed which was confirmed qualitatively using caspase-3 staining (data not shown). The ω-6 group also exhibited no difference in TUNEL staining for apoptosis (p=0.961, figure 5 panels d - f).

Figure 5.

The proliferative index within lesions of 12 -14mo EL-Kras F1 mice fed either a high omega-6 or standard diet was assessed by immunohistochemistry for PCNA and TUNEL. Representative PCNA immunohistochemical staining in neoplastic lesions from an EL-Kras F1 mouse on a. a standard chow and b. a high omega-6 fatty acid diet. The high omega-6 diet mice demonstrated c. no statistically significant difference in PCNA staining compared to age-matched standard diet fed mice, 24.10 ± 9.56% vs. 28.96 ± 7.77%, respectively (p=0.71). Representative TUNEL staining of pancreatic neoplastic lesions from EL-Kras F1 mice on d. a standard chow and e. a high omega-6 fatty acid diet. f. There was no statistically significant difference in apoptosis between the standard (1.68 ± 0.37%) and omega-6 groups (1.66 ± 0.22%, p=0.961). Means shown are ± SEM. To determine statistical significance, t tests were used.

A high ω-6 diet increases mast cell infiltration of pancreatic neoplasia

In order to evaluate the effects of a high ω-6 diet on the inflammatory milieu of the pancreas, we performed counts of cells positive for chloracetate esterase (CAE), a marker for mast cells. A diet high in omega-6 significantly increased the density of mast cells in the pancreas (p<0.05, figure 6 panels a - e). In both groups, mast cells were localized specifically in the stroma surrounding pancreatic neoplastic lesions (figure 6 panels a - d).

Figure 6.

Mice fed a high omega-6 diet had a significant increase in stromal mast cell infiltration surrounding pancreatic neoplastic lesions. Representative chloracetate esterase staining (CAE, a marker for mast cells) in an EL-Kras F1 mouse on a. a standard chow (50×) and b. a high-omega 6 diet (50×). c - d. High-powered magnification view (200×) of insets in panels a and b, respectively. e. CAE-positive mast cells were significantly increased in the stroma surrounding pancreatic neoplastic lesions in omega-6 mice (standard, 4.68 ± 0.78 mast cells/high powered field(hpf) versus omega-6, 7.82 ± 1.02 mast cells/hpf;*, p<0.05). Means shown are ± SEM. To determine statistical significance, t tests were used.

Discussion

In a prior study, we reported the development of pancreatic neoplasia in 18 of 40 12-month-old male EL-Kras mice on an FVB/N background.¹⁰ There has emerged clear evidence that the background strain of mouse models of cancer can have significant effects on phenotype. Goulet et al dramatically demonstrated the effects strain can have on inflammation by genetically deleting the 5-lipoxygenase (5-LO) enzyme in the 129, B6, and DBA mouse strains.¹² Although the three strains shared the same enzymatic deletion, the contribution 5-LO byproducts made in several models of inflammatory response differed significantly.¹² Furthermore, inflammatory response differed between tissue types among the three strains.¹² Halberg et al illustrated the effects of genetic background on an intestinal polyposis model that also developed pancreatic adenocarcinoma (primarily, though not exclusively, acinar in histotype).¹³ Mice of 129 and (B6 × 129) F1 backgrounds were mutated for Apc and p53(Apc^Min/+ p53^-/-). These mice subsequently developed massive pancreatic adenocarcinomas. In stark contrast, Apc^Min/+ p53^-/- mice on a B6 background developed only microscopic pancreatic adenomas. Even with the same Apc and p53 mutation, homozygosity for a recessive B6 allele markedly attenuated pancreatic tumorigenesis. Given that both inflammation and pancreatic carcinogenesis differed significantly across mouse strains, we hypothesized that the EL-Kras phenotype would differ between the FVB and B6 strains.

For the purpose of engineering a model that developed pancreatic neoplasia with reduced latency and a higher incidence and frequency, we crossed the EL-Kras mice one generation and twelve generations into B6. Nearly all of the resulting 6-month-old EL-Kras F1 mice developed pancreatic neoplasia at a higher frequency, though these cystic papillary neoplastic lesions were morphologically similar to those in EL-Kras FVB mouse pancreas. Both strains of EL-Kras mice, neither of which develop invasive disease, were utilized to study the role that high fat diets play in the development of pancreatic neoplasia. The generation of two models of neoplasia with different severity of neoplasia serves as a valuable tool to evaluate additional genetic and epigenetic phenomena (including diet) on the development and progression of pancreatic neoplasia. The F1 and B6 models demonstrated more severe neoplasia which is useful in evaluating strategies that may mitigate the phenotype while the less severe FVB model serves as a valuable tool to examine a stimulus that may exacerbate the neoplastic phenotype. In this study, we employed FVB and F1 models to assess the effect of a diet rich in ω-6 fatty acids on the development of pancreatic neoplasia.

Several studies have shown that dietary intake of ω-3 fatty acids suppresses progression of breast, prostate and colon cancers.^14-17 Most recently, our lab found that a high ω-3 fat diet suppresses pancreatic neoplasia by inhibition of cellular proliferation through induction of cell cycle arrest and apoptosis.⁷ In contrast, there is increasing evidence that dietary intake of ω-6 fatty acids promotes the development of breast, prostate, colon, and pancreatic cancers.^{14, 15, 18-22} Diets high in essential fatty acids were found to significantly increase tumor burden in an azaserine-induced rat model of pancreatic cancer.⁸ An increased rate of liver metastases was also identified in BOP-induced pancreatic cancer in hamsters fed a high ω-6 fatty acid diet ^{23, 24}. To date, all of the in vivo studies regarding the effect of different PUFA diets during pancreatic carcinogenesis have utilized carcinogen-induced models that develop invasive pancreatic cancer. However, the effects of high levels of fatty acids in the diet are likely to be more relevant at preinvasive stages. To our knowledge, this is the first study to evaluate the effects of dietary ω-6 fatty acid on transgenic mice predisposed to the development of pancreatic neoplasia.

Our first aim was to quantify the effects that a high ω-6 diet has on the incidence of neoplasia. We therefore utilized the EL-Kras FVB mouse, whose relatively limited incidence of neoplasia made it the ideal model to evaluate if a high ω-6 fatty acid diet would increase development of neoplasia. Indeed, 8-10-month old EL-Kras FVB mice that were fed a high ω-6 fatty acid diet demonstrated a 50% incidence of neoplasia compared to a complete lack of neoplasia in age-matched mice on a standard diet. EL-Kras F1 mice were then utilized to further characterize the lesions that developed from high ω-6 fatty acid versus standard chows. EL-Kras F1 mice administered a high ω-6 fatty acid diet demonstrated an increase in the frequency and size of neoplastic lesions in the pancreas. Interestingly, mice fed high ω-6 fatty acid diets also weighed more than mice fed standard chow (p<0.0001), but only in the F1 background. This concurs with previous human data that linked increased risk for pancreatic cancer with obesity.^2-6

It has been postulated that ω-6 fatty acids contribute to the inflammatory milieu by acting as an arachidonic acid substrate for the enzyme 5-lipoxygenase (5-LO), which plays a significant role in inflammation.²⁵ In particular, 5-LO produces metabolites important for mast cell chemotaxis and expansion.²⁶ More attention has been recently given to the role of mast cells in pancreatic cancer.²⁷ Mast cells were found to be an essential component of myc oncogene-induced pancreatic islet cell tumor development.²⁸ Our own lab recently showed that in vitro, mast cells promote pancreatic cancer growth and invasiveness. Furthermore, we found that increased mast cell infiltration was associated with a poorer prognosis in patients with pancreatic adenocarcinoma.²⁹ Concurring with these findings, we found increased mast cell infiltration in EL-Kras F1 mice fed a diet high in ω-6 fatty acids. Similar to our previous human data, mast cells specifically infiltrated the stroma surrounding the preinvasive lesions. A diet high in ω-6 fatty acids contributes to a higher degree of mast cell infiltration at the site of preinvasive pancreatic disease. Indeed, this may serve as the impetus for earlier onset and increases in frequency and size of neoplastic lesions in EL-Kras F1 on the high ω-6 fatty acid diet compared to those on standard chow since there were no significant changes in the proliferative or apoptotic indices.

Since all EL-Kras F1 mice on either diet develop neoplasia by 6 months of age, it was difficult to assess incidence changes in cohorts of mice provided high fat diets for at least 6 months. We contend that the tendency of these mice towards larger and greater number of lesions is likely due to an earlier onset rather than a higher proliferation rate. Thus, a high ω-6 fatty acid diet likely results in an earlier transformation process with the subsequent lesions proliferating at the same rate once they have developed. Similar findings were observed in the azaserine-induced pancreatic cancer rat model⁸, as well as prostate ³⁰, mammary ³¹, skin ³², and colon²², where development of tumor was enhanced (with a shorter latency) in mice administered a high ω-6 fat diet without any indication of a higher proliferation index.

Although EL-Kras mice administered this high ω-6 fatty acid diet for nearly one year do not present with invasive disease, most of the parameters evaluated in this study (incidence, frequency, and size of neoplastic lesions, and presence of infiltrating mast cells) indicate a more aggressive preinvasive disease in the pancreas. Considering that most dietary effects are long-term, one may speculate that providing a diet rich in ω-6 PUFA over many years may ultimately contribute to creating an environment that is more conducive to the development of neoplastic lesions in the pancreas, potentially contributing to the generation of an invasive phenotype. Other factors to consider are obesity, changes in the ω-3:ω-6 fatty acid ratio, and saturated versus unsaturated dietary fatty acids. Although difficult to prove in a human population, epidemiological studies with this in mind should be considered in addition to longer diet studies where mice with mutant Kras-driven neoplasia are administered these types of diets for more than one year. We are currently pursuing these types of investigations.

Since the standard chow had lower calorie content per gram than the diet rich in ω-6 fatty acids, phenotypic differences may be influenced by total calorie intake. This is a difficult variable to control even with isocaloric diets since each mouse must consume the same amount of isocaloric diet in order to alleviate any possible contributions from caloric intake. EL-Kras FVB mice fed a high ω-6 fatty acid diet had no significant difference in body weight compared to mice fed a control chow, although there was considerable loss of parenchyma that was replaced with fat (lipoatrophy). Pancreatic lipoatrophy is associated with hyperglycemia, hyperinsulinemia, and insulin resistance³³, which may affect development of neoplasia and/or parenchymal loss and is a project for future investigation. In EL-Kras F1 mice, a diet rich in ω-6 fatty acids led to a significant increase in body weight not observed in EL-Kras FVB mice. This implies that a potential metabolic alteration between FVB and FVB6 F1 mouse strains may be induced by combined genetic and epigenetic components as observed in islet amyloid formation in hIAPP transgenic mice.³⁴ Yet, the differences between pancreatic neoplasia development being reduced in mice fed ω–3 fatty acids⁷ but enhanced in mice fed ω–6 fatty acids (this study) demonstrates that the type of high fat diet makes a profound difference on the phenotype. This implies that caloric intake is not the predominant impetus for these phenotypic alterations, although some level of contribution cannot be ruled out.

In summary, we characterized the EL-Kras mouse model in three background strains demonstrating a similar phenotype among FVB, FVB6 F1, and congenic B6 strains, though the kinetics of neoplastic lesion development was more pronounced in the B6 strain than the FVB strain. Also, we utilized an EL-Kras transgenic mouse model in two different genetic backgrounds to evaluate the effect of a high omega-6 fat diet on the development of pancreatic neoplasia. We demonstrated that a diet high in ω-6 fatty acid resulted in an increased incidence, frequency, and size, but no detectable change in the proliferation or apoptotic rates of neoplastic lesions compared to mice fed the standard diet. While further work is needed to elucidate the mechanism(s) of how omega-6 fatty acids affect neoplasia development, this study provides convincing evidence that a high ω-6 fatty acid diet can promote pancreatic neoplasia in vivo.

Supplementary Material

Supp Figure S1. Supplemental Figure 1.

PCNA and TUNEL confirmation. a. Ki-67 staining of a neoplastic lesion at (50×) and b. high-powered (200×) magnification of inset in panel a. c. Graph demonstrating correlation between PCNA and Ki-67 values. d. View of a neoplastic lesion, stained with caspase-3, illustrating qualitative correlation to TUNEL staining.

Abbreviations

ω-6

omega-6

EL

elastase

F1

FVB6 F1

C57BL/6

B6

PCNA

proliferating cell nuclear antigen

BMI

body mass index

RR

relative risk

PanIN

pancreatic intraepithelial neoplasia

IPMN

intrapapillary mucinous neoplasia

MCN

mucinous cystic neoplasia

5-LO

5-lipoxygenase