and Dysplastic Changes in
Mouse Pancreas Induced by
Jennifer K.L. Colby*, Russell D. Klein†,2,
Mark J. McArthur†, Claudio J. Conti*,
Kaoru Kiguchi*, Toru Kawamoto*,
Penny K. Riggs*,§, Amy I. Pavone*,
Janet Sawicki¶and Susan M. Fischer*
*University of Texas M.D. Anderson Cancer Center,
Science Park – Research Division, Smithville, TX 78957,
USA;†Department of Human Nutrition, Cancer
Chemoprevention Program, The Ohio State University,
Columbus, OH 43210, USA;‡Michale E. Keeling Center
for Comparative Medicine and Research, Department of
Veterinary Sciences, University of Texas M.D. Anderson
Cancer Center, Bastrop, TX 78602, USA;§Department of
Animal Science, Texas A&M University, College Station,
TX, 77843-2471, USA;¶The Lankenau Institute for Medical
Research, Wynnewood, PA, 19016, USA
Cyclooxygenase-2 (COX-2) overexpression is an established factor linking chronic inflammation with metaplastic and
neoplastic change in various tissues. We generated transgenic mice (BK5.COX-2) in which elevation of COX-2 and
its effectors trigger a metaplasia–dysplasia sequence in exocrine pancreas. Histologic evaluation revealed a chronic
pancreatitis-like state characterized by acinar-to-ductal metaplasia and a well-vascularized fibroinflammatory stroma
that develops by 3 months. By 6 to 8 months, strongly dysplastic features suggestive of pancreatic ductal adeno-
carcinoma emerge in the metaplastic ducts. Increased proliferation, cellular atypia, and loss of normal cell/tissue
organization are typical features in transgenic pancreata. Alterations in biomarkers associated with human inflamma-
tory and neoplastic pancreatic disease were detected using immunohistochemistry. The abnormal pancreatic phe-
notype can be completely prevented by maintaining mice on a diet containing celecoxib, a well-characterized COX-2
inhibitor. Despite the high degree of atypia, only limited evidence of invasion to adjacent tissues was observed, with
no evidence of distant metastases. However, cell lines derived from spontaneous lesions are aggressively tumori-
genic when injected into syngeneic or nude mice. The progressive nature of the metaplastic/dysplastic changes ob-
served in this model make it a valuable tool for examining the transition from chronic inflammation to neoplasia.
Neoplasia (2008) 10, 782–796
Abbreviations: BK5, bovine keratin 5; CFP, cyan fluorescent protein; COX, cyclooxygenase; CP, chronic pancreatitis; EP, PGE2receptor; H&E, hematoxylin and eosin; IHC,
immunohistochemistry; K19, keratin 19; (m)PanIN, (mouse) pancreatic intraepithelial neoplasia; PaSC, pancreatic stellate cell; PDAC, pancreatic ductal adenocarcinoma;
PGE2, prostaglandin E2
Address all correspondence to: Susan M. Fischer, The University of Texas M.D. Anderson Cancer Center, Science Park – Research Division, P.O. Box 389, 1808 Park Rd 1C,
Smithville, TX 78957. E-mail: firstname.lastname@example.org
1This work was supported by National Institutes of Health grants CA105345 and CA122815 (to S.M.F.) and a National Institute of Environmental Health Sciences (NIEHS)
training grant T32 ES07247; and by a National Cancer Institute training grant R25CA57730 (J.K.L.C.) and ES07784 from NIEHS.
2In memoriam of Dr. Russell D. Klein (1962–2006): Russell D. Klein died December 1, 2006 after a long battle with leukemia. He was a promising young scientist, an
exemplary mentor, and a fine human being. His presence will be greatly missed.
Received 26 February 2008; Revised 25 April 2008; Accepted 28 April 2008
Copyright © 2008 Neoplasia Press, Inc. All rights reserved 1522-8002/08/$25.00
Volume 10 Number 8August 2008pp. 782–796
Inflammatory conditions of the pancreas predispose individuals to
developing pancreatic ductal adenocarcinoma (PDAC), exemplified
by individuals with heritable or sporadic forms of chronic pancreatitis
(CP) [1–3]. Among the general population, a number of risk factors
for pancreatitis and pancreatic cancer have been identified, e.g., alco-
hol and tobacco use [4,5], high consumption of nitrosamines ,
and diabetes [7,8], which can lead to chronic inflammatory changes
either in specific target tissues  or in the body as a whole [7,8].
Although many forms of CP have been linked to a higher risk for
PDAC, the relationship between the two is ill-defined .
Metaplasia, the replacement of one mature cell type by another,
frequently occurs in chronic inflammation, and is often considered
a preneoplastic condition (e.g., Barrett metaplasia of the esophagus)
. Acinar-to-ductal metaplastic changes in the pancreas are often
found in CP and in association with the recently defined pancreatic
intraepithelial neoplasias (PanINs) and mouse PanIN (mPanIN) pre-
cursor lesions and PDAC [11–15]. Recent articles have attempted to
clarify the relationship between CP and PDAC [12,14], and one
study has demonstrated that acinar cells clearly contribute to the de-
velopment of PanIN lesions . Metaplastic ductal cells seem to
have diminished sensitivity to apoptotic stimuli and, therefore, have
a selective advantage in the diseased pancreas . Although a great
deal of evidence exists supporting high-grade PanINs as precursor
lesions of PDAC, and this is widely accepted, questions remain with
regard to the validity of ductal complexes as precursors to PDAC
. The concept is supported by evidence from groups studying
both rodent and human forms of the disease [18–21].
Cyclooxygenases (COX-1 and -2) are rate-limiting enzymes in
the production of prostaglandins (PGs), which are short-lived lipid-
signaling molecules involved in a number of biologic functions .
COX-1 expression is generally constitutive, whereas COX-2 is usually
induced by stimuli involved in inflammatory responses. Prostaglandin
E2(PGE2), a primary metabolite of COX-2, has been shown to
promote cell survival, proliferation, and angiogenesis and prohibit
apoptosis, all processes influencing cancer development .
Prostaglandin production is frequently a factor in the develop-
ment and maintenance of chronic inflammation, and COX-2 is up-
regulated in numerous human cancers and precancerous conditions,
including pancreatitis and PDAC [24,25]. Cyclooxygenase-2 expres-
sion has also been found to be an early event in the N-nitrosobis-
(2-oxo-propyl)amine model of pancreatic carcinogenesis in hamster
. Forced COX-2 overexpression is associated with the develop-
ment of cancer in mouse models of mammary  and bladder
 tumorigenesis. In the skin of NMRI mice, COX-2 overexpres-
sion driven by the bovine keratin 5 (BK5) promoter does not lead
to spontaneous tumor development but sensitizes the skin to chemical
carcinogenesis . In this same model, high PGE2levels also lead to
fibrocystic changes and epithelial lesions in the mammary gland and,
to some extent, in the pancreas [30,31].
In this article, we present a mouse model, FVB-Tg(KRT5-Ptgs2)7Sf
mice, hereafter referred to as BK5.COX-2 mice, in which the over-
expression of COX-2 under the control of a BK5 promoter drives
pancreatic acinar-to-ductal metaplasia progressing to severe dysplasia
suggestive of PDAC. Histopathologic analyses reveal similarities to
human CP, as well as pancreatic lesions observed in other genetically
[32,33] or chemically induced [34,35] animal models of pancreatic
disease. BK5.COX-2 pancreatic lesions are characterized by acinar-
to-ductal metaplasia, development of a fibroinflammatory stroma,
increased proliferation of metaplastic ductal cells, nuclear pleomor-
phism, and abnormal cell and tissue architecture. Pancreata frequently
develop multilocular cysts. The BK5.COX-2 pancreatic phenotype is
initiated and primarily driven by high levels of COX-2 activity/PGE2
signaling. The malignant potential of spontaneous BK5.COX-2 le-
sions to progress to invasive adenocarcinoma is suggested by the
presence of strongly dysplastic cell/tissue morphology and by the es-
tablishment of cell lines that form aggressive tumors when injected to
either syngeneic or nude mice. We propose that these mice represent
a valuable new model for the assessment of chemopreventive and
chemotherapeutic agents in pancreatic disease, particularly those asso-
ciated with pronounced inflammatory conditions such as CP.
Materials and Methods
All housing and procedures were carried out in an animal facility
accredited by the American Association for the Assessment and Accredi-
tation of Laboratory Animal Care, in accordance with Institutional Ani-
mal Care and Use Committee guidelines. Mice were maintained on
chow ad libitum, unless specified otherwise. FVB-Tg(KRT5-Ptgs2)7Sf
(BK5.COX-2) mice were generated on the FVB background by pro-
nuclear injection of a bovine keratin 5 (BK5) promoter construct con-
taining the coding region of the mouse COX-2 gene, the rabbit
β-globin intron, and an SV40 poly-A tail. The BK5 promoter directs
expression to several tissue types, namely, basal cells of skin, prostate,
bladder, forestomach, mammary myoepithelium, kidney papilla, and
pancreatic ductal epithelia . Transgenic mice can consistently be
identified by a sparse hair coat but showed no other visible abnormalities.
Two founders (Lines 7 and 9) were generated, both of which had a
pancreatic phenotype. Founder 9 produced no offspring; for all sub-
sequent studies, we used mice from Line 7. To maintain the line,
BK5.COX-2 males were bred to wild type female FVB mice. Hemi-
zygous offspring were used for all subsequent analyses. Prostaglandin
production due to higher COX-2 expression in transgenic embryos
was sufficient to trigger premature parturition; to avoid this, dams
were fed low levels of indomethacin (4 ppm of AIN-76A diet; Re-
search Diets, St. Paul, MN) for the last few days of pregnancy. Litters
born to these females were full term and otherwise healthy; we no-
ticed no long-term effects attributable to indomethacin treatment.
COX-2 Genotyping Polymerase Chain Reaction
Primers used recognize the rabbit β-globin intron within the trans-
gene; sequences are as follows: 5′-TCA-AAG-ACA-CTC-AGG-
TAG-AG-3′ (forward); 5′-CTT-GAG-TTT-GAA-GTG-GTA-AC-3′
(reverse). For a single 50-μl reaction, the following amounts were
added: 37.8 μl of double-distilled H2O, 5 μl of 10× polymerase chain
reaction (PCR) buffer, 1.0 μl of 10 mM dNTPs, 0.5 μl each of for-
ward and reverse primers, 0.2 μl of Taq polymerase, and 5 μl sample
from HotShot DNA extraction. DNA was amplified using the fol-
lowing PCR conditions: an initial denaturation at 95°C (1 minute),
30 cycles of denaturation, annealing, and extension at 95°C (30 sec-
onds), 50°C (30 seconds), and 72°C (30 seconds), respectively, and a
final extension at 72°C (5 minutes), followed by a 4°C hold.
Histologic Evaluation and Immunohistochemistry
Tissues were embedded in paraffin blocks, and 4-μm sections were
cut. Slides were deparaffinized in a xylene substitute (CitraSolv; LLC,
Neoplasia Vol. 10, No. 8, 2008COX-2-induced Pancreatic Dysplasia Colby et al.
Danbury, CT) for 2 × 5 minutes. Tissues were hydrated in a series of
alcohols and water before undergoing antigen retrieval. The antigen
retrieval method used was dependent on the specific antibody. Meth-
ods used include microwaving in 10-mM citrate buffer, protease
treatment, and EDTA treatment. After antigen retrieval, endogenous
peroxidase activity was quenched with hydrogen peroxide (3% for
10 minutes), and sections were blocked with 10% normal serum in
phosphate-buffered saline (PBS) for 30 minutes. Primary antibodies
were applied in the concentrations for the lengths of time stated in
Table 1 (detailed antibody protocols are available on request). Slides
were washed two times for 5 minutes in PBS before application of
the secondary antibody. For most antibodies, slides were incubated
with the secondary antibodies for 30 minutes, and again washed sev-
eral times with PBS. Staining was developed by incubating sections
with diaminobenzidine; sections were counterstained with hematoxy-
lin. Slides were dehydrated in a series of alcohols before coverslipping.
Tissue sections were prepared as previously mentioned for hema-
toxylin and eosin (H&E). Toluidine blue: slides were stained with
a 0.1% solution of toluidine blue for 5 minutes and were briefly
destained before dehydration and coverslipping. Alcian blue and
periodic acid Schiff (PAS): slides were stained using kits (KTABP2.5
for Alcian blue and KTPAS for PAS) from American Master-Tech
Scientific, Inc. (Lodi, CA).
BK5.CFP Mice/Detection of Cyan Fluorescent Protein
To construct pK5.CFP, the plasmid pECFP (Clontech, Mountain
View, CA) was digested with BamHI and AflII. A 1-kb fragment con-
taining the CFP sequence was ligated to (BamHI + AflII)–digested
pIND (Stratagene, La Jolla, CA) to create pIND.ECFP. pIND.ECFP
was digested with BamHI and NheI. The 1-kb fragment was ligated
to (BglII + NheI)–digested pMECA  to create pMECA-ECFP. A
7-kb fragment, released by KpnI digestion of the plasmid p3/4 (gift
from Deutsches Krebsforchungzentrum, Heidelberg, Germany),
was digested with SalI. The resulting 5.2-kb fragment containing
the BK5 promoter sequence was ligated to (KpnI + XhoI)–digested
pMECA-ECFP to create pK5.CFP. pK5.CFP was digested with NheI
and KpnI, and the resulting 6.2-kb transgene fragment, containing
the keratin 5 promoter and the cyan fluorescent protein (CFP), was
purified and microinjected into fertilized B6C3F1 mouse oocytes as
described . To observe CFP fluorescence in the pancreas of trans-
genic mice, pancreata were fixed in 4% paraformaldehyde for 30 min-
utes at room temperature, washed three times in PBS, and mounted
in OCT for frozen sectioning. Frozen sections were observed using a
fluorescent microscope (Axioplan; Zeiss, Jena, Germany) equipped
with a CFP filter set and an Axioplan camera.
We used the following protocol to avoid degradation due to the
high levels of RNAses present in pancreas. The protocol is based on
Table 1. List of Antibodies and Conditions Used.
Primary Antibody ApplicationSourceDilution Incubation Time/TemperatureAntigen Retrieval Method
Caspase 3 (active)
Santa Cruz Biotechnology
30 minutes, RT
30 minutes, RT
30 minutes, RT
30 minutes, RT
2 hours, RT
1 hour, RT
1 hour, RT
1 hour, RT
1 hour, RT
1 hour, RT
1 hour, RT
1 hour, RT
1 hour, RT
1 hour, RT
Calbiochem (EMD Biosci)
St. Louis, MO
San Diego, CA
Ann Arbor, MI
San Francisco, CA
Temecula, CA/Billerica, MA
San Diego, CA
All immunohistochemical protocols require blocking sections in 10% serum for 30 minutes (to block nonspecific sites) and in 3% H2O2for 10 minutes (to quench endogenous peroxidase activity).
IHC indicates immunohistochemistry; ON, overnight; RT, 25°C; WB, Western blot.
*Antigen retrieval method used: 10 mM citrate buffer, pH 6.0, for 10 minutes in microwave and 20 minutes of cool down.
†Antigen retrieval method used: 1 mM EDTA, pH 8, for 10 minutes in microwave and 20 minutes of cool down.
‡Antigen retrieval method used: 0.06% protease (type 24) in Tris buffer for 10 minutes.
§Antigen retrieval method used: enzyme-induced epitope retrieval (EIER) with Digest-all 3 (pepsin; Zymed) for 10 minutes at 37°C. All IHC stains were visualized with diaminobenzidine.
¶CD31 IHC uses a tyramide amplification step; contact authors for detailed protocol.
COX-2-induced Pancreatic DysplasiaColby et al.Neoplasia Vol. 10, No. 8, 2008
a combination of methods [39,40] and Tri-Reagent manufacturer’s
instructions (Molecular Research Center, Inc., Cincinnati, OH). Im-
mediately after euthanizing the mice, tissues were harvested and
homogenized with a polytron in 3 ml of 5 M guanidinium thiocyanate
buffer containing 50 mM Tris–HCl, 1% laurosyl-sarcosine (Sarkosyl),
10 mM EDTA, pH 7.5, and 1% β-mercaptoethanol. After a brief
rest on ice, samples were precipitated 24 to 48 hours at 4°C with
seven times the volume of ice-cold 5 M lithium chloride. Samples were
then centrifuged for 30 minutes at 12,000g (9500 rpm) under 4°C.
The supernatant was removed, and the pellet was resuspended in
3 ml of 5 M guanidinium thiocyanate with 1% β-mercaptoethanol
followed by 0.1 volume of 2 M sodium acetate, pH 4. The samples
were then extracted with 3 to 4 ml of Tri-Reagent following manufac-
turer’s instructions. Samples rested for 5 minutes at room temperature;
0.1 volume of the phase separation reagent 1-bromo-3-chloropropane
was then added. Samples were mixed well and allowed to sit at room
temperature for 15 minutes, then centrifuged at 12,000g (at 4°C).
The aqueous layer was removed to a fresh tube, and all extraction
steps were repeated. After the second extraction, samples were washed
in 2 ml of chloroform, and spun down 15 minutes at 12,000g (at
4°C). The top layer was removed, and RNA was precipitated with
6 ml of isopropanol overnight at −20°C. Samples were centrifuged
for 15 minutes at 12,000g, (at 4°C), and the supernatant was poured
off. The RNA pellet was washed once with 75% ethanol made with
diethylpyrocarbonate-treated water. A third extraction was performed
with Tri-Reagent as previously mentioned, and the aqueous layer was
removed to a new tube and precipitated again in 2.5 times the volume
of 100% ethanol (overnight at −20°C or 2 hours at −80°C). Samples
were spun down at 12,000g for 15 minutes and washed once with 1 ml
of 75% ethanol. (Samples were transferred to microfuge tubes for
the remainder of the procedure.) Samples were centrifuged at 4°C for
15 minutes at maximum speed on a microfuge, washed with 100%
ethanol, and recentrifuged. Ethanol was removed, and the pellet was
allowed to air dry briefly (∼10 minutes) and then resuspended in
diethylpyrocarbonate-treated water. An RNAse inhibitor (SUPERaseIn;
Ambion, Austin, TX) was added (final concentration is 1 U/μl). Sam-
ples were stored at −80°C.
Endogenous COX-2 (a.k.a. Ptgs2) Versus
TaqMan primers and probes were designed to the 5′ and 3′ untrans-
lated regions (UTRs) unique to the mouse endogenous COX-2 gene
(Ptgs2) and the BK5.COX-2 transgene. Primers and probe specific to
exon 7 of Ptgs2 were used as positive controls because they amplify
both the endogenous gene and the transgene from genomic DNA
and cDNA (not shown).
Endogenous COX-2 mRNA (Mouse Ptgs2 5′UTR).
amplifies a 113-bp product from the endogenous gene and does
not amplify the transgene. F-primer: 5′ CAG-TCA-GGA-CTC-
TGC-TCA-CGA-A; R-primer: 5′AGC-AGC-ACA-GCT-CGG-
AAG-A; probe: 5′-VIC-CGC-CAC-CAC-TAC-TG-MGB-NFQ.
to the transgene and does not amplify Ptgs2. F-primer: 5′AAA-GGC-
GTT-CAA-CTG-AGC-TGT-AA; R-primer: 5′GGA-GTG-AAT-
TGC-TAG-CGT-ATC-GA; probe: 5′-6FAM-CCG-GGC-TGC-
This assay amplifies a 72-bp product unique
Total RNA was quantified on a spectrophotometer (ND1000;
NanoDrop Technologies, Wilmington, DE), and equal quantities
of RNA were used for each sample. The quality of total RNA was
verified by analysis on NanoChip with a 2100 Bioanalyzer (Agilent
Technologies, Santa Clara, CA). Reverse transcription (RT) of 500 ng
of total RNA was carried out in 20-μl reactions with the High-
Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA)
according to the manufacturer’s recommendations. Antisense primers
for Ptgs2 and BK5.COX-2 (previously mentioned) were substituted
at a final concentration of 300 nM for the random primers included
in the kit.
Quantitative real-time PCR and RT-PCR (qPCR) were carried out
in 25-μl reactions with TaqMan FAST Universal PCR Master Mix
or SYBR Green Master Mix (Applied Biosystems) and 2-μl template
on an ABI Prism 7900HT Sequence Detection System (Applied Bio-
systems). For qPCR, either 20 ng of genomic DNA or the cDNA
generated from RT of 50-ng total RNA was used for each 25-μl re-
action. Primer sets were tested by amplification with SYBR green and
were selected based on amplification efficiency, dissociation curve
analysis, and the presence of single bands of appropriate size visual-
ized on a polyacrylamide gel. Primers were purchased from Integrated
DNA Technologies (Coralville, IA), probes from Applied Biosystems.
Synthetic oligos containing the amplicon were purchased from
MWG Biotech (High Point, NC). The oligos were diluted serially
in a 10-fold dilution series in 25 ng/μl tRNA (Invitrogen, Carlsbad,
CA) to generate a standard curve based on the manufacturer’s stated
concentration and molecular weight. Absolute standard curves were
generated using oligo standards ranging from 3.4 × 1012copies down
to 0.34 copies per reaction. For the endogenous COX-2 assay, reac-
tion efficiency was approximately 100% (slope = −3.32, R = 0.9998),
with a linear range of 340 to 3.4 × 109copies. For the BK5.COX-2
assay, reaction efficiency was approximately 100% (slope = −3.32,
R = 0.9997), with a linear range of 34 to 3.4 × 108copies. Sample
sizes were as follows: 3-week-old wild type, n = 4; 3-week-old trans-
genic, n = 4; 6-month-old wild type, n = 4, and transgenic older than
15 weeks, n = 11.
PGE2Enzyme-Linked Immunosorbent Assay
PGE2levels were determined as previously described using a PGE2
enzyme-linked immunosorbent assay kit from Cayman Chemical
(Ann Arbor, MI) . Levels of PGE2were determined using the
multiple linear regression program on AssayZap (Biosoft, Cambridge,
UK) and were expressed as picograms per microgram protein. Ex-
periments were performed in triplicate and repeated three times. Sam-
ple sizes for PGE2analysis were as follows: wild type, n = 4; young
transgenic (∼4–6 weeks old), n = 4; and older transgenic (>15 weeks
old), n = 5.
Cell Culture and Injections into Nude (Allogeneic) or FVB
Minced tumor pieces (1–3 mm) were incubated overnight at 37°C
in 200 U/ml collagenase type 1A (Sigma, St. Louis, MO) and sus-
pended in supplemented RPMI 1640 medium (Invitrogen) .
After centrifugation, cells were plated in polystyrene flasks. Cultures
were observed for colonies with epithelial phenotype; fibroblast over-
growth was minimized by differential trypsinization. Once lines with
epithelial morphology were established, medium was changed from
high-serum RPMI to DMEM (with high glucose, GlutaMAX, and
sodium pyruvate; Invitrogen) supplemented with 5% fetal bovine
Neoplasia Vol. 10, No. 8, 2008 COX-2-induced Pancreatic DysplasiaColby et al.
serum, insulin (10 μg/ml), and penicillin/streptomycin (100 μg/ml).
Cells from one of the epithelial lines (JC102) were injected into the
spleens of three 7-week-old nude mice (BALB/cAnCr− nu/nu; Na-
tional Cancer Institute, Frederick, MD) and were allowed to grow
for 7 weeks. Cells were injected intraperitoneally (i.p.; JC101 and
JC102) or subcutaneously (JC101) into the FVB mice; mice were
killed and tissues were collected when the tumors were ∼1 cm in
diameter or after 30 days.
Groups of 8 to 12 mice were fed ad libitum diets of AIN-76A diet
(Research Diets, New Brunswick, NJ) containing celecoxib (LKT
Laboratories, St. Paul, MN) at concentrations of 500 (4.5 months),
1000 (3 months), or 1250 ppm (6–10 months). Age- and sex-matched
controls were fed AIN-76A (Research Diets) ad libitum. Mice were
placed on diets at weaning (3–4 weeks) and killed at the end of
the feeding periods. Kaplan–Meier survival curves were generated
using JMP software (SAS Institute, Cary, NC). For survival analysis,
11 transgenic mice fed 1250 ppm of celecoxib up to 33 weeks were
compared with 30 mice fed control diet for the same period.
Statistical differences were performed using analysis of variance
(ANOVA) followed by Student’s t test (one-sided, unequal variance).
PGE2and endogenous COX-2 expression data were analyzed using
Excel (Microsoft Corporation, Redmond, WA). Log-rank analysis
of Kaplan–Meier survival data was performed using JMP software
Necropsies of newborn and perinatal BK5.COX-2 mice did not
reveal any major phenotypic alterations with the exception of a sparse
hair coat. At the histologic level, the skin showed slight to moderate
inflammation in the dermis and mild hyperplasia of the epidermis.
Older mice occasionally developed moderate hyperplasia of the blad-
der epithelium (data not shown). The first significant pathologic al-
teration in the pancreas that distinguished the transgenic from wild
type mice (Figure 1A) was detected at 3 to 4 weeks and consisted of
cells with pyknotic nuclei, occasional apoptotic figures, very mild
degeneration of the acinar structures coupled with widening of the
interlobular spaces, and the presence of a highly vascularized loose
connective tissue in the interacinar spaces (Figure 1B). Foci of in-
filtrated mononuclear inflammatory cells were observed among the
acini and in the interlobular spaces. Concurrently, acini at discrete
sites throughout the pancreas began to undergo a transition to a duc-
tal phenotype, i.e., ductal metaplasia, as evidenced by an increase
in the size of the lumen and change in cellular appearance, e.g., loss
of zymogen granules and reduction in the amount of cytoplasm.
Amylase staining, a marker of acinar differentiation, is retained in
early stages of the transition but is completely lost in metaplastic
ducts (data not shown). A low but widespread level of necrosis was
observed in H&E–stained sections, although no difference was ob-
served in cytoplasmic HMGB1 staining (not shown). Increased pro-
liferation was observed in transitional foci, as evidenced by Ki-67
immunohistochemistry (IHC; Figure 1C, inset). No difference was
seen between Ki-67 staining of 3-week-old wild types and trans-
genics, because the pancreas was still growing to its adult size (not
shown). Full development of the pancreas was complete by 3 months,
at which time Ki-67 was almost undetectable in wild type mice
(Figure 1D, inset), whereas extensive Ki-67 staining is evident in trans-
genics (Figure 1D). A high degree of proliferation continued well
beyond 3 months in BK5.COX-2 mice (Figure 1E).
By 10 weeks, 87% of the transgenic mice develop inflammatory
lesions with distinct similarities to human CP . Figure 1C shows
a representative photomicrograph with characteristic fibrosis, inflam-
matory cell infiltration, and ductal metaplastic foci. After 10 weeks,
an increase in the extent and severity of the pancreatic lesions was
observed, reflected by a significant increase in the size of the gland
relative to wild type (Figure 1F) and the presence of adhesions to
abdominal organs (liver, intestine, and spleen) primarily because of
the inflammatory process. Microscopically, partial or complete ductal
metaplasia of multiple pancreatic lobes was observed, with a signifi-
cant increase in fibrosis. Lobular architecture of the gland was gen-
erally conserved, with a degree of disruption due to extensive fibrosis
(Figure 2A). At this time point, dysplastic changes were observed in
the ductal epithelium in some of the metaplastic foci, consistent with
in situ premalignant lesions. There was considerable loss of architec-
tural orientation, frequent cellular atypia, increased nuclear to cyto-
plasmic ratio, and nuclear atypia with prominent nucleoli. Grossly,
cystic changes were more noticeable and frequent. Taller mucus-
producing cells can be seen in some of the ductal complexes. Lesions
with histologic resemblance to mPanINs can be seen as well (Figure 2,
B and C). Necrotic debris is also found in the lumens of many ductal
complexes (Figures 2, D–G).
Lesions of older mice (3–6 months) show histopathologic fea-
tures that are considered more typical of overt PDAC. Figure 2D
shows a representative example of a lesion with moderately to well-
differentiated ductal structures, an extensive fibroinflammatory reac-
tion, and loss of lobular structure. Mitotic figures were frequently
observed, and nuclei were atypical, with prominent nucleoli (Fig-
ure 2, D and E). Cellular atypia and lack of cellular organization,
i.e., more haphazard growth of ductal complexes, were also observed
(Figure 2, F and G). As in younger mice, occasional adhesions to
other abdominal organs were seen, generally appearing to be the re-
sult of the inflammatory process, although some of the adhesions are
suggestive of early neoplastic invasion. Figure 2H shows an example
of such a case, in which abnormal pancreatic cells have adhered
and possibly begun to invade through the outer muscularis layers
of the intestine. No distant metastases were confirmed, and no signs
of peritoneal carcinomatosis were seen. By 6 to 8 months, 100% of
the animals had greatly enlarged pancreata and were euthanized as
per animal care protocols. Increased volume of the pancreata was
frequently, but only partially, a result of cystic fluid (serous) accumu-
lation. Occasionally, mice presented with some degree of ascites as
well. At this time point, mean ± SD pancreatic mass was significantly
different between wild type and transgenic groups (wild type, 0.28 ±
0.002 g; transgenic, 2.60 ± 1.51 g; P < .001; data were obtained after
draining cystic fluid). Digestive abnormalities associated with pancre-
atic insufficiency were not observed, and mice did not show signs of
pain. It seemed that at the time of euthanasia, mice still retained suf-
ficient acini to allow normal digestive function.
For controls, 24 wild type mice, from 3 to approximately 50 weeks,
were examined histologically. Some of the animals near 1 year were
observed to have small infiltrations of lymphocytes in the interstitium
(data not shown), which is an incidental lesion commonly seen in
aged FVB mice.
COX-2-induced Pancreatic DysplasiaColby et al. Neoplasia Vol. 10, No. 8, 2008
BK5.COX-2 Cell Lines and Allograft Experiments
To better understand the molecular mechanisms of pancreatic le-
sion development and to confirm their neoplastic nature, we devel-
oped epithelial cell lines from BK5.COX-2 pancreatic lesions, JC101
and 102 (Figure 3, A and B). JC102 cells were injected in the spleens
of three nude mice; tumor growth was assessed after 7 weeks. In two
of the mice, metastatic lesions were observed in the liver (Figure 3C)
and/or in the lung (data not shown). Tumors arising from these cells
show COX-2 positivity (Figure 3D). JC101 and JC102 cell lines
were demonstrated to have the capacity to form aggressive, invasive
tumors at a variety of sites. Figure 3E shows invasion of cells into
skin and muscle adjacent to an i.p. injection site; the inset shows
the well-differentiated to moderately differentiated nature of portions
of this tumor. Figure 3F shows a tumor formed by JC101 cells in-
jected subcutaneously. When injected i.p., tumors grew throughout
the peritoneal cavity, invading organs such as intestine, pancreas and
liver (Figure 3, G–I). These tumors, in general, tended to be less well
differentiated, i.e., showing fewer ductal structures. Cell lines JC101
and 102 have been serially passaged through i.p. injection to wild
type FVB mice; cells retain both in vitro and tumorigenic character-
istics of the original cell lines.
Because BK5.COX-2 mice presented with primarily a pancreatic
phenotype, transgene expression was analyzed in detail in the pan-
creas. In 3-week-old transgenic mice, only normal ducts and low
numbers of acinar cells are strongly positive for COX-2. As expected,
islets in both wild types and transgenics show weak COX-2 expres-
sion (Figure 4, A and B). As the transgenic mice aged, COX-2 ex-
pression increased, mainly in ducts (metaplastic and normal) and in
acinar cells (Figure 4C). Because of this unexpected acinar expres-
sion, and because the construct was prepared without a molecular
tag, it was necessary to verify the location of transgene expression
in the pancreas through additional means. To accomplish this, we
Figure 1. Wild type pancreas and BK5.COX-2 pancreatic lesions. (A) Section of normal wild type FVB pancreas. (B) Early changes in the
pancreas of BK5.COX-2 mice. Note prominent nucleoli, acinar atrophy, loss of zymogen granules, and enlargement of interstitial spaces.
(C) Section of BK5.COX-2 transgenic pancreas showing formation of ductular complexes (arrow) and an increase in deposition of stroma
(S). Inset in (C) is Ki-67 staining of a single acinus in a 3-month-old mouse showing active cellular proliferation of ductlike cells. (D)
Comparison of Ki-67 IHC in wild type (inset, D) and transgenic (D) 3-month-old mice. (E) Ki-67 IHC in a more advanced lesion. (F) Gross
appearance of the pancreas from 6-month-old wild type (left) and BK5.COX-2 (right) mice.
Neoplasia Vol. 10, No. 8, 2008 COX-2-induced Pancreatic DysplasiaColby et al.
used a mouse model in which the BK5 promoter drives the expres-
sion of CFP (BK5.CFP mice) to identify those cells in which the
BK5 promoter is active (and therefore COX-2 transgene-positive) be-
fore onset of pathologic changes. CFP and BK5 expression are co-
incident in this model; CFP fluorescence was observed only in the
ductal epithelium of the BK5.CFP mouse pancreas. No fluorescence
was detected in other pancreatic cell types (Figure 4D).
Endogenous COX-2 Expression
On the basis of acinar cell COX-2 expression, we hypothesized
that the transgene was producing PGs, and possibly other cytokines,
that caused expression of the endogenous COX-2 gene. This was
verified through quantitative RT-PCR of pancreas samples from
WT and transgenic mice (Figure 4E). The endogenous gene is ex-
pressed at slightly elevated levels in the young transgenics (3 weeks)
and is greatly, although variably, increased in pancreata harvested
from older transgenics. Variability is likely due to sample hetero-
geneity, as the ratio of stroma to epithelium in lesions can vary consid-
erably. The level of endogenous COX-2 differs significantly between
transgenics >15 weeks and all other groups (Figure 4E).
To confirm transgene functionality, we analyzed PGE2in pancreata
from normal wild type mice, young transgenics with mild disease,
and more severely affected older transgenics. Levels of PGE2were
elevated in young transgenic mice and significantly increased with
age and lesion severity (Figure 4F). Levels were more variable in ad-
vanced lesions due to heterogeneity present in those samples.
Markers of Normal and Diseased Pancreas
Islet beta cells were positive for insulin in both the wild type and
transgenic mice (Figure 5A). α-Smooth muscle actin (α-SMA), a
common marker of activated pancreatic stellate cells (PaSCs), was
increased in the stroma of BK5.COX-2 pancreata (Figure 5B).
Elastase expression was observed in the acinar cell compartment as
expected but was also seen in early ductal structures (Figure 5C).
Figure 2. Increasing metaplastic and dysplastic progression in the BK5.COX-2 pancreas. (A) Pancreas from a 3-month-old BK5.COX-2
with changes histologically similar to CP including metaplastic ductal cells (“D”), mild nuclear atypia, increased reactive stroma (“S”), and
the presence of numerous small vessels (arrows). Note unaffected acini (“A”) in the field. (B and C) Example of “mPanIN”–like lesion in
BK5.COX-2 pancreas. (D–G) Higher-grade dysplastic changes in BK5.COX-2 pancreas. Higher magnifications of the boxed areas in D and
F are shown in E and G. Acini have been replaced by abnormal ductal structures, consisting of cells with significant atypia. Note cellular
disorganization and presence of necrotic debris in lumens of metaplastic ducts. An example of an adhesive (and possibly invasive) lesion
affecting the intestine is shown in H.
COX-2-induced Pancreatic DysplasiaColby et al. Neoplasia Vol. 10, No. 8, 2008
Ductal complexes lose elastase expression as they proliferate. An iden-
tical staining pattern is observed with amylase IHC (data not shown).
Keratin 19 (K19) is an intermediate filament protein found in pan-
creatic ducts and is frequently used to confirm a true ductal pheno-
type . Keratin 19 staining is widespread in BK5.COX-2 ductal
complexes, indicative of a ductal, as opposed to acinar, phenotype
β-Catenin and E-cadherin have critical roles in adhesion and sig-
naling and are frequently dysregulated and/or expressed in aberrant
patterns in both preneoplastic lesions and neoplasia. In BK5.COX-2
mice, cytoplasmic β-catenin levels increase in comparison to wild type,
which have a typical membrane-bound pattern of β-catenin staining
(Figure 5E). A similar pattern is observed with E-cadherin staining
(Figure 5F). The S100 proteins are a group of calcium-binding pro-
teins having numerous effects on cell behavior; they are often over-
expressed in cancer and may have prognostic value . Using a
pan-S100 antibody, we observed a distinct increase in S100 staining
in BK5.COX-2 transgenic pancreas (Figure 5G). Pancreatic ductal
adenocarcinoma frequently shows a dysregulated production of mucins
. Alcian blue staining, used for the detection of mucins, is much
more prominent in BK5.COX-2 lesions versus controls (Figure 5H).
Positive staining for PAS and mucicarmine, additional indicators of
mucins, were also observed in transgenic lesions (data not shown).
An increase in vessel number in the stroma of transgenic lesions was
observed in H&E–stained sections and was confirmed by immuno-
staining for CD31 (Figure 5I). Vascular endothelial growth factor
(VEGF) is another indicator of an angiogenic response; immunoblot
analysis showed up-regulation of VEGF in transgenic pancreata (not
shown). In the pancreas, matrix metalloproteinase 7 (MMP-7) is
involved in acinar-to-ductal transdifferentiation and is up-regulated
in most human PDAC . Matrix metalloproteinase 9 (MMP-9;
gelatinase B) is involved in the remodeling of extracellular matrix in
normal and disease processes, and stromal expression of MMP-9 is
necessary for the angiogenesis and progression of tumors in a nude
mouse model using orthotopically implanted pancreatic cancer cells
[48,49]. In our model, MMP-9 is elevated in transgenic pancreata
as determined by immunoblot analysis (not shown). Matrix metallo-
proteinase 7 expression was evaluated by IHC; areas of positivity are
seen in both stroma and acinar cells undergoing metaplastic change
Inflammatory Cell Markers
A range of cells typical of inflammatory responses is observed in
BK5.COX-2 lesions. The earliest abnormal pancreata have an influx
of neutrophils, and later macrophages, as verified by Ly6G (Gr-1)
and S100A9 IHC (Figure 6, A and B), respectively. More advanced
lesions generally possessed numerous B and T lymphocytes, often
present in large clusters (Figure 6, C and D). B- and T-cell identities
were verified by CD45R (Figure 6C) and CD3 (Figure 6D) IHC,
respectively. Toluidine blue histochemistry demonstrated the pres-
ence of mast cells (Figure 6E).
Celecoxib Feeding Studies
To demonstrate that pancreatic lesions in BK5.COX-2 mice were
due to high levels of PG synthesized by COX-2, transgenic mice
were fed diets containing celecoxib (500–1250 ppm), a selective
COX-2 inhibitor. Kaplan–Meier analysis revealed significant differ-
ences between groups (Figure 7A). None of the mice fed celecoxib
showed any evidence of pancreatic lesions (Figure 7B). Several mice
on the highest dose (1250 ppm) died because of gastrointestinal
toxicity, but none had pancreatic abnormalities. The lower doses of
celecoxib (500 and 1000 ppm) proved equally effective at prevent-
ing lesions with significantly less toxicity (data not shown). As noted
previously, mice on the control diet were euthanized in compliance
with animal care regulations, generally due to the extensive enlarge-
ment of the pancreas due to cystic fluid accumulation/fibrosis lead-
ing to compression/dysfunction of other organs.
Figure 3. Allograft experiments. H&E–stained sections of JC101
(A) and JC102 (B) cells grown on a chamber slide. H&E–stained
section of the liver metastasis (C) of JC102 cells injected into
the spleen. (D) Immunohistochemistry for COX-2 expression in
metastasis shown in (C). H&E–stained section of tumor arising
near the injection site of i.p.–injected JC102 cells (E). Note inva-
sion to muscle and skin. The inset in E shows a portion of the
same tumor that is well to moderately differentiated. (F) Tumor
arising from subcutaneously injected JC101 cells. (G–I) Tumors
arising from i.p.–injected JC101 or 102 cells. Tumors invading
the small intestine (G), normal pancreas (H), and liver (I) are shown.
Neoplasia Vol. 10, No. 8, 2008 COX-2-induced Pancreatic Dysplasia Colby et al.
Figure 4. COX-2 transgene expression in BK5.COX-2 mice and BK5.CFP immunofluorescence. Cyclooxygenase-2 IHC on 3-week-old
wild type FVB (A) and BK5.COX-2 transgenic mice (B). Cyclooxygenase-2 IHC in a more advanced BK5.COX-2 pancreatic lesion (C).
BK5.CFP immunofluorescence in wild type (insert in D) and transgenic mice (D) demonstrating keratin 5 expression in the ductal com-
partment only. Quantitative RT-PCR results (E) showing increased mRNA for endogenous COX-2. Data are presented such that each bar
represents a single animal. When grouped for analysis (ANOVA), significant differences exist between groups (P = .002); transgenic
tumors (including all transgenics 15 weeks or older) differed significantly from all other groups (P values < .0005 for comparisons with 3-
week-old wild type, 3-week-old transgenic, and 6-month-old wild type groups (mean ± SD for groups: 3-week-old wild type, 3.175 ±
1.56; 3-week-old transgenic, 3.575 ± 0.665; 6-month-old wild type, 1.1 ± 0.688; and older transgenics/tumors, 402.1/287.96). PGE2
levels (F) in wild type FVB, prelesion (young) transgenic mouse pancreas, and premalignant transgenic pancreatic lesions. Differences
between groups are significant; ANOVA for all groups, P = .005; significant differences were also detected between wild type and
young transgenic (P = .001), between wild type and lesion (P = .01), and between young transgenic and transgenic lesion (P =
.015). Mean ± SD for groups: wild type, 229 ± 95.10; young transgenic, 3632.25 ± 708.17; and lesion, 27,466.2 ± 16,408.02.
COX-2-induced Pancreatic Dysplasia Colby et al. Neoplasia Vol. 10, No. 8, 2008
We present data supporting a model (Figure 8) of PGE2-driven neo-
plasia in which transgene-expressing ductal cells serve as the initial
source of PGE2, subsequently causing paracrine and autocrine up-
regulation of endogenous COX-2 in acinar and normal ductal cells,
respectively. Whereas additional inflammatory mediators (e.g., tumor
necrosis factor α, interleukin 1β) are likely to be involved in the pro-
cess, PGE2elevation in transgenic pancreata is key in establishing a
proinflammatory environment capable of initiating and supporting
metaplastic and dysplastic changes. All mice develop pancreatic inflam-
mation and metaplasia characteristic of human CP, subsequently fol-
lowed by development of highly dysplastic lesions with features of
Figure 5. Altered protein expression in BK5.COX-2 mouse pancreata (large images) compared to wild type (insets). (A) Insulin expres-
sion is normal in both wild type and transgenic mice. (B) α-Smooth muscle actin expression is increased in BK5.COX-2 transgenics,
reflecting activation and expansion of stellate cells. (C) Elastase staining is restricted to acini in wild type mice but appears in metaplastic
ductal structures as well in transgenics. (D) Keratin 19, a marker of ductal differentiation, shows extensive staining in transgenic lesions,
whereas only normal ducts stain positive (arrow) in wild type mice. (E) β-Catenin staining is restricted to the cytoplasmic membrane in
wild types but becomes more strongly expressed in the cytoplasm of cells making up transgenic lesions. (F) E-Cadherin shows a pattern
similar to that observed with β-catenin (see E). (G) S100 protein is strongly expressed in transgenic lesions, primarily in epithelial cells.
(H) Alcian blue staining reveals the presence of mucin in the lumens of ductal lesions of transgenics; little staining is seen in wild type
mice. (I) The expanding stroma in BK5.COX-2 pancreata is well vascularized as verified by CD31 staining. (J) Matrix metalloproteinase 7
staining has been associated with acinar–ductal metaplasia and is more highly expressed in BK5.COX-2 pancreatic lesions.
Neoplasia Vol. 10, No. 8, 2008COX-2-induced Pancreatic Dysplasia Colby et al.
frank adenocarcinoma, such as the presence of mucins and nuclear
atypia. Increased numbers of fibroblast-like stromal cells expressing
α-SMA occur in our model, representing activated PaSCs. PaSCs nor-
mally make up only 4% of the normal pancreas and have a retinol stor-
age function in their quiescent state . In inflammatory responses,
they become activated and undergo changes leading to deposition of
high levels of extracellular matrix components and fibrosis observed
in pancreatitis and PDAC . Immunocytes are also found in the
Figure 7. Prostaglandins drive the development of pancreatic inflammation and premalignant lesions in BK5.COX-2 mice. (A) Kaplan–
Meier survival curve of mice fed 1250 ppm of celecoxib for 33 weeks (P < .0001). The incidence of pancreatic lesions was zero in the
celecoxib-fed group (red line); see text for details. (B) Hematoxylin and eosin–stained section of pancreas in a transgenic mouse fed the
Figure 6. Inflammatory cells in BK5.COX-2 lesions. (A) The earliest inflammatory cells observed in the BK5.COX-2 pancreata are Ly6G-
positive neutrophils. Macrophages appear slightly later, here identified by S100A9 IHC (B). (C and D) B and T lymphocytes appear as
lesions progress, often forming large clusters of cells. B cells are identified using a CD45R antibody (C) and T cells with CD3 antibody
(D). Mast cells occur frequently in the stroma of transgenic lesions; here, they are identified by toluidine blue (E).
COX-2-induced Pancreatic DysplasiaColby et al. Neoplasia Vol. 10, No. 8, 2008
lesions, including neutrophils, macrophages, mast cells, and B and T
lymphocytes, further indicating the inflammatory nature of this model.
Acinar-to-ductal metaplasia is a key feature of BK5.COX-2 lesions
and has been described in other genetically modified mouse models,
most notably, the Ela-RASG12D(elastase promoter–driven mutant
Ras ) and Ela-TGFα-hGH (elastase promoter–driven transform-
ing growth factor α) . Metaplasia occurs in response to chronic
tissue injury or stress and is seen in several well-characterized human
neoplastic conditions, e.g., Barrett metaplasia of the esophagus, a pre-
cursor lesion for esophageal cancer caused by chronic gastroesopha-
geal reflux . Morphologic changes triggered by the exposure of
acinar cells to unusual stress (culture conditions, inflammatory med-
iators) seems to confer greater resistance to further injury or death,
and greater regenerative potential. The enhanced proliferative re-
sponse, including expansion of the stromal compartment, is also typi-
cal of injured/healing tissue. In the BK5.COX-2 model, chronic
inflammation and the accompanying regenerative response lead to
metaplastic changes similar to those seen in human CP. Pancreatic
acinar–ductal transdifferentiation has also been convincingly demon-
strated in both tissue culture and in vivo models, and it is thought
that this metaplastic change produces a population of cells with
greater proliferative potential that may act as facultative stem cells
[11,54,55]. In further support of the link between metaplasia and
hyperplasia in the pancreas, a recent study by Zhu et al.  suggests
that active cell division does not take place until acinar cells have
undergone metaplastic change to a ductal cell–like phenotype. In
human pancreas, evidence of a “transitional zone” with co-occurrence
of tubular complexes and low-grade PanIN (1A) suggests the exis-
tence of a progression sequence from tubular complexes to PanIN
formation . In addition, a recent review of the literature favors
the idea that the variety of histologic complexity observed in numer-
ous transgenic mouse models is in fact consistent with the hetero-
geneity seen in human PDAC . A study by Strobel et al. 
used lineage tracing of acinar cells and found that in a cerulein model
of CP, certain subsets of metaplastic ductal lesions seem to arise from
transdifferentiation of acinar cells while others do not. Although
these data are intriguing, it is possible that the mechanisms behind
metaplastic events are model-specific. The cerulein pancreatitis mod-
els have been well studied, but there is concern that they do not re-
flect human CP accurately [58,59]. Not all forms of human CP seem
to share the same pathogenetic mechanisms, and there are important
differences between rodents and humans in terms of molecular sig-
naling on which the cerulein model is based . Although there are
multiple mechanisms by which metaplasia may occur (for a review,
see Slack ), the ductal complexes observed in the BK5.COX-2
pancreas do not seem to be due to replacement of dying acinar cells
by rapid proliferation of the ductal compartment. Although we
cannot strictly rule out the co-occurrence of different metaplastic
processes, we believe that we are observing a significant degree of
acinar-to-ductal transdifferentiation. This view is supported by the
observation that extensive simultaneous death of acinar cells is not
seen and by the expression patterns of amylase and K19. Because
PGE2can confer both survival and growth advantages, it may act
both to prevent apoptosis and to promote proliferation.
The precise molecular pathways/mechanisms impacted in the BK5.
COX-2 model are still incompletely described, but multiple processes
are potentially involved, e.g., macromolecular damage either because of
lipid peroxidation or reactive oxygen species (including alterations in
DNA) or because of chronic activation of growth factor receptors. We
assessed several markers frequently associated with CP and/or neo-
plastic transformation using IHC. Stronger cytoplasmic expressions
of β-catenin and E-cadherin, both of which can be dysregulated in
human pancreatic lesions, were observed and suggest alterations in
cell adhesion properties and perhaps Wnt signaling [61,62]. Matrix
metalloproteinases have been associated with increased invasive be-
havior and poor prognosis in other studies [47,49]. S100 proteins
signal through the multiligand receptor RAGE (receptor for advanced
glycation end products) . When present at lower tissue concen-
trations, RAGE ligands activate repair mechanisms, whereas at elevated
levels, they are associated with chronic tissue injury. It is thought
Figure 8. Working model of pancreatic tumorigenesis in BK5.COX-2 mice. Transgene-produced PGE2from the phenotypically normal
transgene-positive ductal epithelium stimulates expression either directly [through signaling through PGE2receptors (EP)] or indirectly
[through up-regulation of additional inflammatory mediators, e.g., tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), interleukin 6
(IL-6)] of the endogenous COX-2 gene in surrounding acinar cells, causing an even greater increase in tissue PGs, leading to concomitant
inflammation and acinar–ductal metaplasia. In this model, a CP-like state ultimately leads to development of high-grade dysplasia with
many cytologic and histologic features of overt PDAC.
Neoplasia Vol. 10, No. 8, 2008 COX-2-induced Pancreatic DysplasiaColby et al.
that in stressful environments such as chronically inflamed tissue, li-
gands for RAGE may be present as higher-order oligomers, possibly
overwhelming normal resolution mechanisms . Up-regulation of
S100 proteins also suggests changes in calcium signaling, which can
affect many aspects of cell behavior, including cell cycling and differ-
In spite of the presence of molecular markers frequently associated
with more aggressive neoplastic behavior, BK5.COX-2 tumors showed
only occasional invasion and no identifiable metastases. Although
we were unable to unequivocally demonstrate metastases originating
from spontaneous lesions, cell lines derived from primary lesions were
shown to be tumorigenic, suggesting the presence of additional genetic
or epigenetic changes within the cells. Most PDAC cases in humans
show mutations in K-ras ; however, mutation-specific sequencing
failed to show any evidence of signature K-ras mutations in BK5.
COX-2 tumors (not shown). Because COX-2 up-regulation is down-
stream of ras activation, our model may bypass the “need” for K-ras
mutation . Activated ras, however, was detected in BK5.COX-2
cell lines used for injection experiments (data not shown). Ras activa-
tion may reflect a critical change responsible for the more aggressive
behavior of tumors arising from the cell lines, although it is still pos-
sible that additional mutations may have been selected for in the pro-
cess of deriving the lines from spontaneous tumors. Interestingly, a
recent article demonstrated that in adult mice expressing mutant ras
(K-RasG12V), inflammatory changes are necessary for the full develop-
ment of neoplastic changes .
An interesting feature seen in most spontaneously occurring BK5.
COX-2 pancreatic lesions is the presence of cystic changes, a feature
shared with several other mouse models of pancreatic disease [32,66–
68], giving the tumors a gross resemblance to serous cystadenoma/
adenocarcinoma . Although the lesions appear grossly like these
neoplasms, microscopically, BK5.COX-2 lesions show much more
dramatic dysplasia, with histologic characteristics more in line with
ductal adenocarcinoma. Newer imaging techniques have lead to
more frequent discovery of pancreatic cysts in humans, and a number
of recent articles have addressed the significance of cystic lesions in
pancreatitis and PDAC [70–74]. Although observed less frequently in
humans than solid neoplasms, cystic variants of many lesions occur,
some with malignant features . Distinguishing benign cysts from
those with malignant potential is a diagnostic challenge, particularly
against a background of CP [73,75]. Although the full significance
of pancreatic cysts is not known, there are data suggesting an increased
risk for PDAC in persons with pancreatic cystic changes .
In a recent publication , a COX-2 transgenic mouse generated
on the NMRI background displays some of the characteristics of our
model but with critical differences. Similarities exist in terms of the
histopathology of ductal lesions, but a lower percentage of these mice
are affected (15% at 6 months; 30% of 12 months), and latency is
greatly increased (lesions take two to three times longer to manifest
themselves). The differences between this model and our own could
be advantageous in efforts to distinguish important genetic or epi-
genetic cofactors influencing the progression of pancreatic disease.
Identification of early, premaligant changes is critical for the devel-
opment of improved prevention or intervention approaches. There
has been mixed success using COX-2 selective inhibitors such as
celecoxib in preclinical/xenograft models of pancreatic cancer, with
some groups reporting inhibition of angiogenesis or growth factor–
associated signaling [76–78] and others showing no effect .
Unfortunately, the efficacy of celecoxib as an adjuvant to standard
chemotherapeutic protocols has been quite limited [79–82], further
highlighting the need for the earlier detection of preneoplastic lesions
and a stronger focus on prevention. A number of studies have dem-
onstrated causal links between tobacco and/or alcohol use and pan-
creatic inflammation and neoplasia [4,5,8]. Cyclooxygenase-2 is
frequently found to be elevated in these studies and can be consid-
ered an important biomarker in CP and PDAC. As an example, a
recent study by Schuller et al.  employed a radiolabeled drug
to help identify precancerous lesions that significantly overexpressed
COX-2 in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone–treated
hamster tissues (liver, lung, and pancreas). In addition, COX-2 inhi-
bition has been shown to limit the progression of mPanINs in the
KrasG12Dmodel . We believe that the BK5.COX-2 model has
attributes that will make it a valuable model in furthering our under-
standing of how unresolved inflammatory processes can initiate pre-
malignant changes linked to pancreatic cancer and how such changes
can be prevented. Data obtained from this and other related models
should broaden our understanding of the complex relationship be-
tween inflammation and pancreatic cancer [12–14,31].
The authors thank Weidan Peng and Yunhua Bao (Lankenau),
Nancy W. Otto, and Kelly Kochan (Science Park) and the personnel
at the Science Park Animal Research Facility for expert technical as-
sistance. Joi Holcombe (Science Park) provided excellent assistance
 Lowenfels AB, Maisonneuve P, DiMagno EP, Elitsur Y, Gates LK Jr, Perrault J,
and Whitcomb DC (1997). Hereditary pancreatitis and the risk of pancreatic
cancer. International Hereditary Pancreatitis Study Group. J Natl Cancer Inst
 Farrow B, Sugiyama Y, Chen A, Uffort E, Nealon W, and Mark Evers B (2004).
Inflammatory mechanisms contributing to pancreatic cancer development. Ann
Surg 239, 763–769; discussion 769–771.
 Hezel AF, Kimmelman AC, Stanger BZ, Bardeesy N, and Depinho RA (2006). Ge-
netics and biology of pancreatic ductal adenocarcinoma. Genes Dev 20, 1218–1249.
Dimagno EP, Andren-Sandberg A, Domellof L, Frulloni L, et al. (2005). Cigarette
smoking accelerates progression of alcoholic chronic pancreatitis. Gut 54, 510–514.
 Schuller HM (2002). Mechanisms of smoking-related lung and pancreatic adeno-
carcinoma development. Nat Rev Cancer 2, 455–463.
 Nkondjock A, Krewski D, Johnson KC, and Ghadirian P (2005). Dietary pat-
terns and risk of pancreatic cancer. Int J Cancer 114, 817–823.
 Jee SH, Ohrr H, Sull JW, Yun JE, Ji M, and Samet JM (2005). Fasting serum
glucose level and cancer risk in Korean men and women. JAMA 293, 194–202.
 Stolzenberg-Solomon RZ, Graubard BI, Chari S, Limburg P, Taylor PR, Virtamo J,
and Albanes D (2005). Insulin, glucose, insulin resistance, and pancreatic cancer
in male smokers. JAMA 294, 2872–2878.
 Jura N, Archer H, and Bar-Sagi D (2005). Chronic pancreatitis, pancreatic adeno-
carcinoma and the black box in-between. Cell Res 15, 72–77.
 Slack JM (2007). Metaplasia and transdifferentiation: from pure biology to the
clinic. Nat Rev Mol Cell Biol 8, 369–378.
 Means AL, Meszoely IM, Suzuki K, Miyamoto Y, Rustgi AK, Coffey RJ Jr,
Wright CV, Stoffers DA, and Leach SD (2005). Pancreatic epithelial plasticity
mediated by acinar cell transdifferentiation and generation of nestin-positive
intermediates. Development 132, 3767–3776.
 Archer H, Jura N, Keller J, Jacobson M, and Bar-Sagi D (2006). A mouse model
of hereditary pancreatitis generated by transgenic expression of R122H trypsino-
gen. Gastroenterology 131, 1844–1855.
 Zhu L, Shi G, Schmidt CM, Hruban RH, and Konieczny SF (2007). Acinar
cells contribute to the molecular heterogeneity of pancreatic intraepithelial neo-
plasia. Am J Pathol 171, 263–273.
 Guerra C, Schuhmacher AJ, Canamero M, Grippo PJ, Verdaguer L, Perez-
Gallego L, Dubus P, Sandgren EP, and Barbacid M (2007). Chronic pancreatitis
COX-2-induced Pancreatic Dysplasia Colby et al. Neoplasia Vol. 10, No. 8, 2008
is essential for induction of pancreatic ductal adenocarcinoma by K-Ras onco-
genes in adult mice. Cancer Cell 11, 291–302.
 Hruban RH, Adsay NV, Albores-Saavedra J, Anver MR, Biankin AV, Boivin GP,
Furth EE, Furukawa T, Klein A, Klimstra DS, et al. (2006). Pathology of geneti-
cally engineered mouse models of pancreatic exocrine cancer: consensus report
and recommendations. Cancer Res 66, 95–106.
 Schmid RM (2002). Acinar-to-ductal metaplasia in pancreatic cancer develop-
ment. J Clin Invest 109, 1403–1404.
 Hruban RH, Takaori K, Klimstra DS, Adsay NV, Albores-Saavedra J, Biankin
AV, Biankin SA, Compton C, Fukushima N, Furukawa T, et al. (2004). An
illustrated consensus on the classification of pancreatic intraepithelial neoplasia
and intraductal papillary mucinous neoplasms. Am J Surg Pathol 28, 977–987.
 Esposito I, Seiler C, Bergmann F, Kleeff J, Friess H, and Schirmacher P (2007).
Hypothetical progression model of pancreatic cancer with origin in the centroacinar–
acinar compartment. Pancreas 35, 212–217.
 Bockman DE, Guo J, Buchler P, Muller MW, Bergmann F, and Friess H (2003).
Origin and development of the precursor lesions in experimental pancreatic
cancer in rats. Lab Invest 83, 853–859.
 Schmid RM, Kloppel G, Adler G, and Wagner M (1999). Acinar–ductal–
carcinoma sequence in transforming growth factor-alpha transgenic mice. Ann
NY Acad Sci 880, 219–230.
 Greten FR, Wagner M, Weber CK, Zechner U, Adler G, and Schmid RM
(2001). TGF alpha transgenic mice. A model of pancreatic cancer development.
Pancreatology 1, 363–368.
 FitzGerald GA (2003). COX-2 and beyond: approaches to prostaglandin inhi-
bition in human disease. Nat Rev Drug Discov 2, 879–890.
 Dannenberg AJ, Lippman SM, Mann JR, Subbaramaiah K, and DuBois RN
(2005). Cyclooxygenase-2 and epidermal growth factor receptor: pharmacologic
targets for chemoprevention. J Clin Oncol 23, 254–266.
 Yip-Schneider MT, Barnard DS, Billings SD, Cheng L, Heilman DK, Lin A,
Marshall SJ, Crowell PL, Marshall MS, and Sweeney CJ (2000). Cyclooxygenase-
2 expression in human pancreatic adenocarcinomas. Carcinogenesis 21, 139–146.
 Schlosser W, Schlosser S, Ramadani M, Gansauge F, Gansauge S, and Beger HG
(2002). Cyclooxygenase-2 is overexpressed in chronic pancreatitis. Pancreas 25,
 Crowell PL, Schmidt CM, Yip-Schneider MT, Savage JJ, Hertzler DA II, and
Cummings WO (2006). Cyclooxygenase-2 expression in hamster and human
pancreatic neoplasia. Neoplasia 8, 437–445.
 Liu CH, Chang SH, Narko K, Trifan OC, Wu MT, Smith E, Haudenschild C,
Lane TF, and Hla T (2001). Overexpression of cyclooxygenase-2 is sufficient to
induce tumorigenesis in transgenic mice. J Biol Chem 276, 18563–18569.
 Klein RD, Van Pelt CS, Sabichi AL, Dela Cerda J, Fischer SM, Furstenberger G,
and Muller-Decker K (2005). Transitional cell hyperplasia and carcinomas in uri-
nary bladders of transgenic mice with keratin 5 promoter–driven cyclooxygenase-
2 overexpression. Cancer Res 65, 1808–1813.
 Muller-Decker K, Neufang G, Berger I, Neumann M, Marks F, and Furstenberger
G (2002). Transgenic cyclooxygenase-2 overexpression sensitizes mouse skin for
carcinogenesis. Proc Natl Acad Sci USA 99, 12483–12488.
 Muller-Decker K, Berger I, Ackermann K, Ehemann V, Zoubova S, Aulmann S,
Pyerin W, and Furstenberger G (2005). Cystic duct dilatations and proliferative
epithelial lesions in mouse mammary glands upon keratin 5 promoter–driven
overexpression of cyclooxygenase-2. Am J Pathol 166, 575–584.
 Muller-Decker K, Furstenberger G, Annan N, Kucher D, Pohl-Arnold A,
Steinbauer B, Esposito I, Chiblak S, Friess H, Schirmacher P, et al. (2006). Pre-
invasive duct-derived neoplasms in pancreas of keratin 5–promoter cyclooxygenase-
2 transgenic mice. Gastroenterology 130, 2165–2178.
 Sandgren EP, Luetteke NC, Palmiter RD, Brinster RL, and Lee DC (1990).
Overexpression of TGF alpha in transgenic mice: induction of epithelial hyper-
plasia, pancreatic metaplasia, and carcinoma of the breast. Cell 61, 1121–1135.
 Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, Ross
S, Conrads TP, Veenstra TD, Hitt BA, et al. (2003). Preinvasive and invasive
ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4,
 Schmied B, Liu G, Moyer MP, Hernberg IS, Sanger W, Batra S, and Pour PM
(1999). Induction of adenocarcinoma from hamster pancreatic islet cells treated
with N-nitrosobis(2-oxopropyl)amine in vitro. Carcinogenesis 20, 317–324.
 Jimenez RE, Z’Graggen K, Hartwig W, Graeme-Cook F, Warshaw AL, and
Fernandez-del Castillo C (1999). Immunohistochemical characterization of
pancreatic tumors induced by dimethylbenzanthracene in rats. Am J Pathol
 Ramirez A, Bravo A, Jorcano JL, and Vidal M (1994). Sequences 5′ of the
bovine keratin 5 gene direct tissue- and cell-type–specific expression of a lacZ
gene in the adult and during development. Differentiation 58, 53–64.
 Thomson JM and Parrott WA (1998). pMECA: a cloning plasmid with 44
unique restriction sites that allows selection of recombinants based on colony
size. Biotechniques 24922–924, 926, 928.
 Hogan BCF and Lacy E (1986). Manipulating the Mouse Embryo. Cold Spring
Harbor, NY: Cold Spring Harbor Laboratory Press.
 Gomez G, Lee HM, He Q, Englander EW, Uchida T, and Greeley GH Jr
(2001). Acute pancreatitis signals activation of apoptosis-associated and survival
genes in mice. Exp Biol Med (Maywood) 226, 692–700.
 Gomez G, Englander EW, Wang G, and Greeley GH Jr (2004). Increased
expression of hypoxia-inducible factor-1alpha, p48, and the Notch signaling
cascade during acute pancreatitis in mice. Pancreas 28, 58–64.
 Fischer SM, Lo HH, Gordon GB, Seibert K, Kelloff G, Lubet RA, and Conti
CJ (1999). Chemopreventive activity of celecoxib, a specific cyclooxygenase-2
inhibitor, and indomethacin against ultraviolet light–induced skin carcino-
genesis. Mol Carcinog 25, 231–240.
 Jaffee EM, Schutte M, Gossett J, Morsberger LA, Adler AJ, Thomas M, Greten
TF, Hruban RH, Yeo CJ, and Griffin CA (1998). Development and character-
ization of a cytokine-secreting pancreatic adenocarcinoma vaccine from primary
tumors for use in clinical trials. Cancer J Sci Am 4, 194–203.
 Bockman DE (1997). Morphology of the exocrine pancreas related to pancrea-
titis. Microsc Res Tech 37, 509–519.
 Schussler MH, Skoudy A, Ramaekers F, and Real FX (1992). Intermediate fila-
ments as differentiation markers of normal pancreas and pancreas cancer. Am J
Pathol 140, 559–568.
 Emberley ED, Murphy LC, and Watson PH (2004). S100 proteins and their
influence on pro-survival pathways in cancer. Biochem Cell Biol 82, 508–515.
 Moniaux N, Andrianifahanana M, Brand RE, and Batra SK (2004). Multiple
roles of mucins in pancreatic cancer, a lethal and challenging malignancy. Br J
Cancer 91, 1633–1638.
 Crawford HC, Scoggins CR, Washington MK, Matrisian LM, and Leach SD
(2002). Matrix metalloproteinase-7 is expressed by pancreatic cancer precursors
and regulates acinar-to-ductal metaplasia in exocrine pancreas. J Clin Invest 109,
 Vu TH and Werb Z (2000). Matrix metalloproteinases: effectors of develop-
ment and normal physiology. Genes Dev 14, 2123–2133.
 Nakamura T, Kuwai T, Kim JS, Fan D, Kim SJ, and Fidler IJ (2007). Stro-
mal metalloproteinase-9 is essential to angiogenesis and progressive growth
of orthotopic human pancreatic cancer in parabiont nude mice. Neoplasia 9,
 Omary MB, Lugea A, Lowe AW, and Pandol SJ (2007). The pancreatic stellate
cell: a star on the rise in pancreatic diseases. J Clin Invest 117, 50–59.
 Apte MV, Park S, Phillips PA, Santucci N, Goldstein D, Kumar RK, Ramm
GA, Buchler M, Friess H, McCarroll JA, et al. (2004). Desmoplastic reaction
in pancreatic cancer: role of pancreatic stellate cells. Pancreas 29, 179–187.
 Grippo PJ, Nowlin PS, Demeure MJ, Longnecker DS, and Sandgren EP
(2003). Preinvasive pancreatic neoplasia of ductal phenotype induced by acinar
cell targeting of mutant Kras in transgenic mice. Cancer Res 63, 2016–2019.
 Jankowski JA, Harrison RF, Perry I, Balkwill F, and Tselepis C (2000). Barrett’s
metaplasia. Lancet 356, 2079–2085.
 Tokoro T, Tezel E, Nagasaka T, Kaneko T, and Nakao A (2003). Differentiation
of acinar cells into acinoductular cells in regenerating rat pancreas. Pancreatology
 Sphyris N, Logsdon CD, and Harrison DJ (2005). Improved retention of
zymogen granules in cultured murine pancreatic acinar cells and induction of
acinar–ductal transdifferentiation in vitro. Pancreas 30, 148–157.
 Liao JD, Adsay NV, Khannani F, Grignon D, Thakur A, and Sarkar FH (2007).
Histological complexities of pancreatic lesions from transgenic mouse models
are consistent with biological and morphological heterogeneity of human pan-
creatic cancer. Histol Histopathol 22, 661–676.
 Strobel O, Dor Y, Alsina J, Stirman A, Lauwers G, Trainor A, Castillo CF,
Warshaw AL, and Thayer SP (2007). In vivo lineage tracing defines the role of
acinar-to-ductal transdifferentiation in inflammatory ductal metaplasia. Gastro-
enterology 133, 1999–2009.
 Perides G, Tao X, West N, Sharma A, and Steer ML (2005). A mouse model of
ethanol dependent pancreatic fibrosis. Gut 54, 1461–1467.
 Schmid RM and Whitcomb DC (2006). Genetically defined models of chronic
pancreatitis. Gastroenterology 131, 2012–2015.
Neoplasia Vol. 10, No. 8, 2008 COX-2-induced Pancreatic DysplasiaColby et al.
 Saluja AK, Lerch MM, Phillips PA, and Dudeja V (2007). Why does pancreatic Download full-text
overstimulation cause pancreatitis? Annu Rev Physiol 69, 249–269.
 Al-Aynati MM, Radulovich N, Riddell RH, and Tsao MS (2004). Epithelial-
cadherin and beta-catenin expression changes in pancreatic intraepithelial neo-
plasia. Clin Cancer Res 10, 1235–1240.
 Zeng G, Germinaro M, Micsenyi A, Monga NK, Bell A, Sood A, Malhotra V,
Sood N, Midda V, Monga DK, et al. (2006). Aberrant Wnt/beta-catenin sig-
naling in pancreatic adenocarcinoma. Neoplasia 8, 279–289.
 Herold K, Moser B, Chen Y, Zeng S, Yan SF, Ramasamy R, Emond J, Clynes R,
and Schmidt AM (2007). Receptor for advanced glycation end products
(RAGE) in a dash to the rescue: inflammatory signals gone awry in the primal
response to stress. J Leukoc Biol 82, 204–212.
 Lohr M, Kloppel G, Maisonneuve P, Lowenfels AB, and Luttges J (2005). Fre-
quency of K-ras mutations in pancreatic intraductal neoplasias associated with
pancreatic ductal adenocarcinoma and chronic pancreatitis: a meta-analysis.
Neoplasia 7, 17–23.
 Smakman N, Kranenburg O, Vogten JM, Bloemendaal AL, van Diest P, and
Borel Rinkes IH (2005). Cyclooxygenase-2 is a target of KRASD12, which
facilitates the outgrowth of murine C26 colorectal liver metastases. Clin Cancer
Res 11, 41–48.
 Jhappan C, Stahle C, Harkins RN, Fausto N, Smith GH, and Merlino GT
(1990). TGF alpha overexpression in transgenic mice induces liver neoplasia
and abnormal development of the mammary gland and pancreas. Cell 61,
 Lewis BC, Klimstra DS, and Varmus HE (2003). The c-myc and PyMT onco-
genes induce different tumor types in a somatic mouse model for pancreatic
cancer. Genes Dev 17, 3127–3138.
 Cano DA, Sekine S, and Hebrok M (2006). Primary cilia deletion in pancreatic
epithelial cells results in cyst formation and pancreatitis. Gastroenterology 131,
 Shintaku M, Arimoto A, and Sakita N (2005). Serous cystadenocarcinoma of
the pancreas. Pathol Int 55, 436–439.
 Strobel O, Z’Graggen K, Schmitz-Winnenthal FH, Friess H, Kappeler A,
Zimmermann A, Uhl W, and Buchler MW (2003). Risk of malignancy in
serous cystic neoplasms of the pancreas. Digestion 68, 24–33.
 Kosmahl M, Pauser U, Anlauf M, and Kloppel G (2005). Pancreatic ductal
adenocarcinomas with cystic features: neither rare nor uniform. Mod Pathol
 Tada M, Kawabe T, Arizumi M, Togawa O, Matsubara S, Yamamoto N, Nakai
Y, Sasahira N, Hirano K, Tsujino T, et al. (2006). Pancreatic cancer in patients
with pancreatic cystic lesions: a prospective study in 197 patients. Clin Gastro-
enterol Hepatol 4, 1265–1270.
 Gomez D, Rahman SH, Won LF, Verbeke CS, McMahon MJ, and Menon KV
(2006). Characterization of malignant pancreatic cystic lesions in the back-
ground of chronic pancreatitis. JOP 7, 465–472.
 Volkan Adsay N (2007). Cystic lesions of the pancreas. Mod Pathol 20 (Suppl 1),
 Kloppel G (2007). Chronic pancreatitis, pseudotumors and other tumor-like
lesions. Mod Pathol 20 (Suppl 1), S113–S131.
 Raut CP, Nawrocki S, Lashinger LM, Davis DW, Khanbolooki S, Xiong H, Ellis
LM, and McConkey DJ (2004). Celecoxib inhibits angiogenesis by inducing
endothelial cell apoptosis in human pancreatic tumor xenografts. Cancer Biol
Ther 3, 1217–1224.
 Wei D, Wang L, He Y, Xiong HQ, Abbruzzese JL, and Xie K (2004). Celecoxib
inhibits vascular endothelial growth factor expression in and reduces angiogene-
sis and metastasis of human pancreatic cancer via suppression of Sp1 transcrip-
tion factor activity. Cancer Res 64, 2030–2038.
 Ali S, El-Rayes BF, Sarkar FH, and Philip PA (2005). Simultaneous targeting of
the epidermal growth factor receptor and cyclooxygenase-2 pathways for pancre-
atic cancer therapy. Mol Cancer Ther 4, 1943–1951.
 Jimeno A, Amador ML, Kulesza P, Wang X, Rubio-Viqueira B, Zhang X,
Chan A, Wheelhouse J, Kuramochi H, Tanaka K, et al. (2006). Assessment
of celecoxib pharmacodynamics in pancreatic cancer. Mol Cancer Ther 5,
 El-Rayes BF, Zalupski MM, Shields AF, Ferris AM, Vaishampayan U, Heilbrun
LK, Venkatramanamoorthy R, Adsay V, and Philip PA (2005). A phase II study
of celecoxib, gemcitabine, and cisplatin in advanced pancreatic cancer. Invest
New Drugs 23, 583–590.
 Xiong HQ, Plunkett W, Wolff R, Du M, Lenzi R, and Abbruzzese JL (2005). A
pharmacological study of celecoxib and gemcitabine in patients with advanced
pancreatic cancer. Cancer Chemother Pharmacol 55, 559–564.
 Cascinu S, Scartozzi M, Carbonari G, Pierantoni C, Verdecchia L, Mariani C,
Squadroni M, Antognoli S, Silva RR, Giampieri R, et al. (2007). COX-2 and
NF-κB overexpression is common in pancreatic cancer but does not predict for
COX-2 inhibitors activity in combination with gemcitabine and oxaliplatin. Am
J Clin Oncol 30, 526–530.
 Schuller HM, Kabalka G, Smith G, Mereddy A, Akula M, and Cekanova M
(2006). Detection of overexpressed COX-2 in precancerous lesions of hamster
pancreas and lungs by molecular imaging: implications for early diagnosis and
prevention. ChemMedChem 1, 603–610.
 Funahashi H, Satake M, Dawson D, Huynh NA, Reber HA, Hines OJ, and
Eibl G (2007). Delayed progression of pancreatic intraepithelial neoplasia in a
conditional Kras(G12D) mouse model by a selective cyclooxygenase-2 inhibitor.
Cancer Res 67, 7068–7071.
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