? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
Oncogenic Kras is required for both
the initiation and maintenance
of pancreatic cancer in mice
Meredith A. Collins,1 Filip Bednar,2 Yaqing Zhang,2 Jean-Christophe Brisset,3
Stefanie Galbán,4 Craig J. Galbán,3 Sabita Rakshit,2 Karen S. Flannagan,2
N. Volkan Adsay,5 and Marina Pasca di Magliano1,2,6,7
1Program in Cellular and Molecular Biology, 2Department of Surgery, 3Department of Radiology, and 4Department of Radiation Oncology,
University of Michigan, Ann Arbor, Michigan, USA. 5Department of Pathology, Emory University, Atlanta, Georgia, USA.
6Department of Cell and Developmental Biology and 7Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan, USA.
Pancreatic ductal adenocarcinoma (PDA), the most common form
of pancreatic cancer, has among the worst prognoses of all human
malignancies. Annually, the number of victims of the disease
approaches the number of new diagnoses, and the average survival
from diagnosis is less than 6 months (1, 2). Surgery is the best option
for the minority of patients who present with localized disease at the
time of diagnosis (about 20% of the total), but even those patients
often experience local or metastatic recurrence (3, 4). Therefore,
there is a dire need for new therapeutic approaches that are likely to
be based on a better understanding of the biology of this disease.
The KRAS oncogene is frequently mutated in human malignan-
cies such as colon, lung, and ovarian cancer. In pancreatic cancer,
mutations in KRAS are found in more than 90% of patient sam-
ples (5, 6). The most frequent mutation is the constitutively active
KRASG12D allele (herein referred to as Kras*) (for review, see refs. 7,
8). Interestingly, KRAS mutations are frequently detected in the
most common precursor lesion to pancreatic cancer, pancreatic
intraepithelial neoplasia (PanIN), indicating a potential role early
in the disease (9). Mouse studies have provided compelling evidence
that oncogenic Kras* is required for the formation of PanINs (10,
11). However, how Kras* contributes to PanIN progression and
PDA maintenance has not been addressed due to the lack of a suit-
able in vivo model. The role of oncogenic Kras* in tumor mainte-
nance has been addressed in lung adenocarcinoma, where Kras* is
required for tumor cell survival, even in advanced stages of the dis-
ease, and in the presence of additional genetic alterations such as
loss of tumor suppressor genes (12). In addition, a subset of pancre-
atic cancer cell lines require Kras* activity for growth and survival
(13, 14). However, a mouse model for study of Kras* dependency in
pancreatic cancer has so far not been developed.
Here, we describe two new mouse models of pancreatic tumori-
genesis defined by tissue-specific, temporally regulated, and revers-
ible expression of Kras*, with or without inactivation of one allele of
the tumor suppressor gene p53, that we have named inducible Kras*
(iKras*) and iKras*-p53+/–, respectively. We use these new models to
address the role of Kras* at several key stages during pancreatic
carcinogenesis: PanIN initiation, established PanIN maintenance,
and the development and maintenance of invasive PDA.
The iKras* mouse model resembles the well-established KC (p48-Cre;LSL-
KrasG12D) model and mimics the progression of the human disease. We used
three genetically modified mouse strains to generate triple trans-
genic p48-Cre;R26-rtTa-IRES-EGFP;TetO-KrasG12D mice, referred
to as iKras* mice. The p48-Cre, or Ptf1a-Cre, allele (15) drives Cre
expression mostly in a pancreas-specific manner, thus recombin-
ing a stop cassette preceding the reverse tetracycline transactivator
(rtTa) IRES-EGFP cassette in the Rosa26 (R26) locus (16). Thus,
rtTa and EGFP are expressed in the pancreatic epithelium during
embryogenesis and into adulthood, even in cell types that down-
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J Clin Invest. 2012;122(2):639–653. doi:10.1172/JCI59227.
640?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
regulate p48 expression in the adult, such as islet and ductal cells
(see Cre lineage tracing and EGFP expression in Supplemental
Figure 1, A and B; supplemental material available online with
this article; doi:10.1172/JCI59227DS1). Once expressed, rtTa is
inactive unless doxycycline (doxy) is administered in the animals’
drinking water (Figure 1A). Activation of rtTa leads to mutant
Kras* expression from the TetO-KrasG12D (12) allele, and its activa-
tion can be reversed by doxy withdrawal (Figure 1A), leading to a
system that allows for organ-specific, temporally regulated, and
reversible expression of Kras*.
The iKras* mouse model of pancreatic tumorigenesis. (A) Genetic makeup of the iKras* model: p48-Cre;R26-rtTa-IRES-EGFP;TetO-KrasG12D.
(B) Experimental design. Kras* expression was induced with doxy for 72 hours before 2 consecutive days of intraperitoneal cerulein injections
to induce pancreatitis and neoplasia. n = 3–5 mice per time point. (C) H&E staining of wild-type murine pancreas. Scale bar: 50: μm. (D) H&E
staining of iKras* murine pancreas 3 weeks after doxy induction of Kras*. Scale bar: 50 μm. (E) H&E staining of iKras* murine pancreas 3 and
5 weeks after induction of Kras* and cerulein injections. Scale bars: 50 μm. (F) H&E staining of iKras* of iKras* murine pancreas 2 days, 1 week,
3 weeks, and 5 weeks after induction of Kras* and cerulein injections. Scale bar: 20 μm. (G) Gomori trichrome staining for interstitial collagen
2 days, 1 week, 3 weeks, and 5 weeks after induction of Kras* and cerulein injections. Scale bar: 20 μm. (H) PAS staining for mucin accumulation
2 days, 1 week, 3 weeks, and 5 weeks after induction of Kras* and cerulein injections. Scale bar: 20 μm.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
We first conducted a series of experiments to compare our iKras*
mouse with the well-established p48-Cre;LSL-KrasG12D model (herein
referred to as KC) (10, 11, 17). The expression of Kras* is differen-
tially regulated in the two models, since the oncogene is expressed
from the endogenous Kras locus in KC mice and from an artificial
transgene in iKras* mice. Moreover, in the KC model Kras* is acti-
vated during embryogenesis. In iKras* mice, we chose to express
Kras* in adult mice (4–6 weeks of age), and tissues were harvested
after 72 hours, 1 week, 3 weeks, 5 weeks, 18 weeks, and 23 weeks
(Supplemental Figure 1C and data not shown). Doxy administra-
tion in control animals did not result in any detectable pancreatic
phenotype (Supplemental Figure 1D; compare with wild-type in
Figure 1C). The iKras* pancreata appeared completely normal up to
1 week following Kras* induction. However, at 3 weeks we observed
rare areas of acinar-ductal metaplasia (ADM) and low-grade PanINs
in 1 of 3 mice (Figure 1D and Supplemental Figure 1D). At 5 weeks
of age, 2 of 3 mice had areas of ADM and low-grade PanIN forma-
tion (data not shown). Unequivocal PanINs, surrounded by areas
of fibrosis and embedded in the acinar parenchyma, were observed
after 18 weeks on doxy (Supplemental Figure 1D), and by 23 weeks,
large areas of the pancreatic parenchyma were substituted with
PanIN lesions of different grade, with frank adenocarcinoma being
observed in 1 of 2 animals (Supplemental Figure 1D).
Previous reports have shown that induction of chronic or acute
pancreatitis acts synergistically with oncogenic Kras* in driving
carcinogenesis (18–20). Therefore, in a second set of experiments,
we induced acute pancreatitis in adult mice by injecting them with
the cholecystokinin analog cerulein. We used age-matched wild-
type KC and iKras* mice with or without doxy. In the absence of
doxy (Kras* OFF), cerulein treatment in iKras* mice led to pan-
creatitis-specific changes such as ADM and infiltration of inflam-
matory cells; however, the damage completely resolved within
3 weeks (Supplemental Figure 2, A and B) as in wild-type animals
(Supplemental Figure 2C). By contrast, recovery from pancre-
atitis was impaired in KC mice as well as in iKras* mice treated
with doxy starting 72 hours prior to the induction of pancreati-
tis and maintained with doxy for the duration of the experiment
(Kras* ON; Figure 1B). At 2 days and at 1 week after induction of
pancreatitis, both iKras* and KC mice pancreata presented with
ADM (Figure 1F and Supplemental Figure 2D) with progressive
accumulation of fibrotic stroma (Figure 1G), but no evidence of
the intracellular mucin accumulation that characterizes PanIN
lesions (Figure 1H and Supplemental Figure 2D). At 3 weeks after
pancreatitis induction, in both iKras* and KC mice, the whole
pancreatic parenchyma was replaced by ductal structures (Figure
1, E and F, and Supplemental Figure 2D) surrounded by collagen-
rich stroma (Figure 1G). The epithelial cells showed intracellular
mucin accumulation (Figure 1H and Supplemental Figure 2D)
and strong positive staining of membrane claudin-18, a marker
that specifically differentiates PanIN and PDA from reactive ducts
in human and mouse (Supplemental Figure 2E and refs. 21, 22);
thus, we identify them as PanINs and refer to them as such here-
in. Our classification of these morphologic lesions as PanINs is
consistent with previous publications using the cerulein model
(19, 20) and activation of oncogenic Kras* in adult animals (23).
As expected, the MAPK/ERK pathway was active in PanINs, as
determined by strong nuclear and cytoplasmic phospho-ERK1/2
staining (Supplemental Figure 2F), thus demonstrating that
Kras* was biologically active in the epithelial cells. In both mod-
els, abundant proliferating cells were present both in the epithe-
lial compartment and in the stroma (Supplemental Figure 2G).
The results of this first set of experiments show that the iKras*
mouse model closely recapitulates the kinetics of PanIN forma-
tion and progression of the well-established KC model, both with
and without induction of pancreatitis.
In order to determine the effect of expressing oncogenic Kras*
for a longer time period, we harvested pancreata of iKras* mice
5 weeks after induction of pancreatitis and found that the paren-
chyma was replaced by low- and high-grade PanIN lesions (Figure
1, E–H) interspersed with areas of carcinoma in situ (PanIN3). In a
series of aging experiments, the iKras* mice were kept on doxy for
up to 17 weeks after induction of pancreatitis without developing
invasive disease. This finding was consistent with those observed
in a cohort of KC mice for the same period of time and support the
notion that oncogenic Kras* inefficiently drives PDA (10, 11).
Oncogenic Kras is required for PanIN maintenance. We took advantage
of the reversibility of Kras* expression in the iKras* model to address
whether Kras* activity is continuously required during pancreatic
carcinogenesis. For this purpose, we kept iKras* mice on doxy for
23 weeks, until the pancreatic parenchyma was largely composed
of PanIN lesions, fibroinflammatory stroma, and interspersed acini
(Supplemental Figure 1E). Then the mice were placed on doxy-free
water, and tissues were harvested after 2 weeks. The pancreata in
those animals appeared fibrotic, atrophic, and largely populated
by acini with some scattered, focal areas of ADM (Supplemental
Figure 1, E and F), with little evidence of inflammation or presence
of PanIN lesions. Thus, these animals showed reversion of PanINs,
indicating that Kras* is indeed required for PanIN maintenance.
In order to study PanIN regression in more detail, we elected
to use acute pancreatitis induction with cerulein, as previously
described, to trigger consistent, tissue-wide PanIN formation, thus
eliminating variability in PanIN onset among individual mice.
Kras* expression was induced by doxy administration in adult mice
(3–5 weeks old). The mice were continuously kept on doxy (Kras*
ON) starting 72 hours before induction of acute pancreatitis and
for 3 weeks following the cerulein treatment. Then we inactivated
oncogenic Kras* expression by returning the animals to normal
water (Kras* OFF) and harvested their pancreata at subsequent
time points as indicated in the scheme in Figure 2A. Transgene
regulation was confirmed by quantitative RT-PCR (qRT-PCR)
analysis for Kras* (Figure 2B). We also directly measured the levels
of Ras-GTP, the active form of the protein, by Ras-GTP pull-down
assays. We used pancreata extracted from wild-type mice and KC
mice 3 weeks after pancreatitis induction and iKras* mice after
pancreatitis induction with Kras* ON for 3 weeks and Kras* OFF
for 2 days, 3 days, and 2 weeks. Figure 2C shows a representative
assay: Ras-GTP levels were significantly increased in the pancre-
atic epithelial compartment of KC and iKras* mice compared
with wild-type mice. Ras-GTP levels were normalized to total Ras
expression (Figure 2D) and to expression of E-cadherin (Figure
2E), a pan-epithelial marker that allowed us to correct for the
possible confounding presence of differing amounts of tumor
stroma in the individual samples. The Ras-GTP levels in KC and
iKras* mice were comparable (KC/WT ratio, 6.6 ± 2.1; iKras*/WT,
5.9 ± 1.6). In iKras* mice, Ras-GTP was rapidly downregulated
upon doxy withdrawal (iKras* OFF 3 days/WT, 1.2 ± 0.8; iKras*
OFF 2 weeks/WT, 0.7 ± 0.8). Phospho-ERK1/2 levels were also
comparable in KC and iKras* mice, but were rapidly downregu-
lated in the latter following Kras* inactivation (Figure 2, C and G).
Our results are consistent with the previously published analy-
642?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
Kras* inactivation in mucinous ADM/early PanINs. (A) Experimental design. Kras* expression was kept ON for 3 weeks following acute pancre-
atitis; then, Kras* was turned OFF; tissues were harvested at the indicated time points (arrows). n = 3–5 mice/time point. (B) Kras* expression
by qRT-PCR. Each point is an individual mouse. Data represent mean ± SEM. (C) Representative Western blot showing Ras protein activity
(Ras-GTP) measured by Raf1-RBD pull-down assay; as well as blots showing levels of phospho-ERK1/2, total ERK1/2, and E-cadherin. (D) Ras
protein activity normalized to total Ras. (E) Ras protein activity normalized to the epithelial marker E-cadherin. (F) Histology of the pancreas at the
indicated time points. Scale bar: 50 μm. (G) Activation of the MAP/ERK kinase pathway measured by phospho-ERK1/2 immunohistochemistry.
Scale bar: 20 μm. (H) Quantification of the change in pancreas size. Data represent mean ± SEM. (I) Quantification of lesions at the indicated
time points. Data represent mean ± SEM.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
sis of the TetO-KrasG12D transgene in the lung (12), where mRNA
expression levels from the transgene were comparable to those of
the endogenous locus, and likely explain the similar phenotype in
the KC and iKras* models.
Analysis of the tissues harvested 2 and 3 days after doxy remov-
al revealed widespread replacement of PanINs with ADM inter-
spersed with pancreatic acini (Figure 2F), and PAS staining was
progressively reduced (Supplemental Figure 3A). Strikingly, by
2 weeks after doxy removal, the pancreas was of normal size (Fig-
ure 2H) and appearance and was largely populated by acinar clus-
ters (Figure 2, F and I). We proceeded to determine whether the
tissue had recovered its normal characteristics also in terms of
gene expression and activity. Analysis of phospho-ERK1/2 levels,
as a readout of the MAPK/ERK pathway, revealed it to be rapidly
inactivated in the epithelial cells upon doxy withdrawal (Figure
2G). Intriguingly, we observed a surge in MAPK/ERK activity in
the stroma during the remodeling process. The expression of other
PanIN markers, such as the ductal markers CK19 and mucin 1
(Muc1), was also rapidly downregulated upon Kras* inactivation
(Supplemental Figure 3, B and C).
In order to determine whether the requirement for Kras* activ-
ity changes over time, we performed another series of experi-
ments in which iKras* mice were kept on doxy for 5 weeks fol-
lowing induction of pancreatitis. At this time point, when the
pancreas was largely composed of PanIN lesions surrounded by
active stroma, we returned the mice to regular water, thus inac-
tivating Kras* (Figure 3A). We collected tissues at several time
points following removal of doxy (see scheme in Figure 3A). Ras-
GTP expression decreased upon doxy withdrawal (Figure 3B),
although the downregulation was slower than observed when
doxy was removed at the 3-week time point (Figure 2C). In fact,
these tissues did not appear to be undergoing significant changes
at 2 and 3 days following Kras* inactivation, based on both his-
tology and expression of PanIN markers (Figure 3C). The dynam-
ics of tissue remodeling appeared dramatically different than in
the 3-week set: PAS-positive PanIN lesions persisted 2 and 3 days
after Kras* inactivation (Supplemental Figure 3D). By 2 weeks
after doxy withdrawal (Kras* OFF), only a small remnant of the
pancreas was present (Figure 3E). We observed partial acinar cell
recovery, as well as occasional pancreatic lipomatosis (a common
reaction to epithelial cell death in the pancreas) (24) and areas of
ADM (Figure 3, C and F). In addition, phospho-ERK1/2 and clau-
din-18 levels were rapidly downregulated in most epithelial cells
(Figure 3, D and G), although a surge of phospho-ERK1/2 in the
stroma was transiently observed. Aberrant expression of CK19
in the basolateral membrane of epithelial cells and intracellular
accumulation of Muc1 were frequently observed with Kras*
ON; upon Kras* inactivation, both CK19 and Muc1 gradually
returned to their normal subcellular localization, with apical
accumulation in ductal cells (Supplemental Figure 3, E and F).
The expression pattern and level of most other markers were
largely comparable to those in normal pancreas (Supplemental
Figure 4, A–F). In addition, we observed β-catenin accumulation
in PanIN lesions and expression of EGFR family members, con-
sistent with previous work in humans and mice (25–29). All of
these changes were reversed upon Kras* inactivation (Supple-
mental Figure 5, A–C). Taken together, our data indicated that
while PanIN lesions could not persist once Kras* was inactivated,
the repair process was not complete and left a small, fibrotic pan-
creas with fewer acini than expected.
In order to determine whether complete pancreatic repair was
delayed, and could be achieved over time, we harvested additional
pancreata 5 weeks following Kras* inactivation. At this time point,
we did not observe evidence of persisting PanINs; however, the
pancreas at histological analysis appeared fibrotic (Figure 3C) and
had not increased in size back to control levels (Figure 3E).
Mechanism of PanIN regression and epithelial tissue repair. Due to the
rapid and dramatic recovery of the pancreas upon inactivation of
Kras* following its expression for 3 weeks, we evaluated whether
this recovery might be due to death of the cells forming PanIN
lesions, followed by active proliferation of residual acinar cells that
had not undergone recombination within the tissues, a mecha-
nism that has been described following pancreatic damage in the
absence of Kras* (30, 31). However, staining for cleaved caspase-3
did not show any significant changes in the number of apoptotic
cells over time upon Kras* inactivation (Figure 4A). Moreover, pro-
liferation analysis using the Ki67 marker showed rare positive cells
immediately following Kras* inactivation, and active proliferation
of acinar cells only at later time points (Figure 4B). Proliferation
of the newly formed acinar cells is likely to play a role in the later
phases of tissue repair, thus explaining the increase in pancreas
size between 3 days and 2 weeks after Kras* inactivation (Figure
2H). We also found that EGFP (linked to rtTa expression in the
R26-rtTa transgene; see Figure 1A) was expressed in the epithelial
compartment both before and after Kras* inactivation (Supple-
mental Figure 6A), indicating that the newly formed acini derived
from cells that had expressed rtTa and EGFP, and thus Kras*.
Kras* has been hypothesized to prevent ductal-to-acinar re-dif-
ferentiation following pancreatitis-induced ADM, thus acting as
a barrier to tissue repair and maintaining the epithelial cells in
a differentiation state that is possibly more prone to neoplastic
transformation (8, 19). Therefore, we investigated the differentia-
tion status of pancreatic cells during Kras* expression and upon
Kras* inactivation. PanIN lesions express the ductal marker CK19
(Figure 4C) and do not express the acinar cell marker amylase.
By contrast, at 2 and 3 days after doxy withdrawal, we frequently
observed cells with mixed acinar-ductal differentiation, co-express-
ing the ductal and PanIN marker CK19 and the acinar marker
amylase (Figure 4, D and E). By 2 weeks after Kras* inactivation,
these intermediate cell types were substituted by acini expressing
amylase, while CK19 was confined to the ducts (Figure 4F). This
may indicate that the PanIN regression occurs by reprogramming
of PanIN cells into acinar cells and explain the complete recovery
of the pancreas, notwithstanding its limited regenerative poten-
tial (32). Quantification of the cell types present at different time
points further disproved the possibility that residual acinar cells
had repopulated the pancreas: upon Kras* inactivation, the num-
ber of acinar cells increased more than 20-fold in 48 hours, an
increase that could not be explained by cell division alone in any
mammalian cell (Figure 4G).
In order to gain additional insight into the repair process, we
addressed the expression of pancreatic progenitor markers in the
PanINs and during the regression process. PanIN lesions expressed
a subset of genes that are associated with pancreatic progenitors
and with maintenance of an undifferentiated status (33–35) and
reactivated in pancreatic cancer (10, 36), such as Sox9, Pdx1, and
Hes1, a Notch signaling component and target gene (Supplemental
Figure 6, B–D). The expression of those markers was maintained
during the initial recovery stages, in structures with mixed acinar
and ductal differentiation, and was repressed once full recovery
644? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
had been achieved (Supplemental Figure 6, B–D). Taken together,
our data support the hypothesis that Kras* is altering the differ-
entiation status of pancreatic epithelial cells, derailing the repair
process following pancreatitis.
We next addressed the mechanism of the recovery process in mice
in which Kras* had been kept on for 5 weeks following pancreati-
tis. In sharp contrast to the observations at the 3-week time point,
here we observed a dramatic increase of apoptotic cells (cleaved
caspase-3 positive) upon Kras* inactivation (Figure 5A). This
observation is consistent with the smaller size of the pancreas in
these animals (Figure 3E). We then assessed the proliferation index
by Ki67 immunostaining. In the presence of oncogenic Kras*, both
the epithelial cells and the surrounding stroma were Ki67 posi-
tive (Figure 5, D and E). Following Kras* inactivation, the overall
Kras* inactivation in established PanINs. (A) Experimental design: Kras* expression was kept ON for 5 weeks following acute pancreatitis; then,
Kras* was turned OFF; tissues were harvested at the indicated time points (arrows). n = 3–5 mice/time point. (B) Representative Western blot
showing Ras protein activity (Ras-GTP) measured by Raf1-RBD pull-down assay. (C) Histology of the pancreas at the indicated time points.
Scale bar: 50 μm. (D) MAP/ERK pathway activation shown by phospho-ERK1/2 immunohistochemistry. Scale bar: 20 μm. (E) Quantifica-
tion of the change in pancreas size. Data represent mean ± SEM. (F) Quantification of pancreatic lesions. Data represent mean ± SEM. (G)
Immunohistochemistry for the PanIN marker claudin-18. Scale bar: 20 μm.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
proliferative index was unchanged; however, the proliferation was
confined to the acinar compartment. In contrast, proliferation in
the stroma was rapidly downregulated. Using EGFP-based lineage
tracing, we were able to determine that the vast majority of the
epithelial cells in the tissue after Kras* inactivation derive from
cells that had previously expressed the Kras* transgene (Figure 5B)
rather than repopulation of the pancreas from acinar cells that had
not undergone Cre recombination and thus never expressed the
Kras* transgene. Thus, some limited re-differentiation of ductal
to acinar cells might play a role in the repair process, but this was
not as prominently observed as at the earlier time points (Figure
5C; compare with Figure 4, C–F). Finally, the expression of Sox9
and Pdx1 was downregulated during the repair process (Supple-
mental Figure 4, A and B); however, this did not occur as rapidly as
seen after the earlier, 3-week time point. The limited proliferative
capability of adult pancreatic cells might account for the incom-
plete repair process. Nevertheless, the mice at this stage appeared
healthy and showed no sign of pancreatic insufficiency.
Interactions between the epithelial cells and their microenvironment are
regulated by oncogenic Kras. In early PanIN lesions, inactivation of
Kras* is accompanied by reversal of enhanced proliferation in the
stroma (Figure 4B) and complete remodeling of this compartment
within 2 weeks (Supplemental Figure 7A). Although the remodel-
ing of the stroma was not complete in tissues where Kras* had
been inactivated at the 5-week time point, rapid downregulation
of proliferation in the stroma was evident (Figure 5, D and E). We
Mechanism of tissue recovery from early PanINs. Kras* expression was maintained ON for 3 weeks following pancreatitis, then turned OFF for
2 days, 3 days, and 2 weeks. n = 3–5 mice/time point. (A) Apoptosis as indicated by cleaved caspase-3 immunohistochemistry. Scale bar: 20 μm.
(B) Co-immunofluorescence of PanIN lesions and tissue proliferation during tissue repair: Ki67 (green), CK19 (red), and DAPI (blue). Scale bar:
20 μm. (C–F) CK19 (green), amylase (red), and DAPI (blue) co-immunofluorescence analysis of PanIN transdifferentiation in iKras* pancreas
after (C) Kras* ON 3 weeks and (D) Kras* OFF 2 days, (E) 3 days, and (F) 2 weeks. Scale bar: 20 μm (G) Quantification of CK19- and amylase-
positive cells at the indicated time points. Data represent mean ± SEM.
646? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
therefore investigated whether Kras* activity is required to main-
tain the extensive fibroinflammatory stroma that is a hallmark of
pancreatic cancer and has been proposed to mediate its resistance
to treatment (37). The pancreatic cancer stroma is largely com-
posed of vimentin- and SMA-positive cells of mesenchymal origin.
SMA is a marker of activated fibroblasts and is not expressed in
quiescent pancreatic stellate cells, the resident mesenchymal cell
population in the pancreas. As expected, the stroma surround-
ing PanIN lesions in the iKras* pancreata was SMA and vimentin
positive with Kras* ON (Figure 6A and Supplemental Figure 7B).
Interestingly, while vimentin expression remained unaltered fol-
lowing Kras* inactivation, SMA was rapidly downregulated. Loss
of SMA expression preceded remodeling of the stroma, both in
the 3-week and in the 5-week tissues. In the 5-week tissues, the
fibrotic areas that persisted were SMA negative and non-prolifera-
tive, features consistent with scar tissue. Thus, our data indicate
that Kras* activity in the epithelium is required to maintain the
surrounding active stroma.
One of the key signaling pathways mediating epithelial-mesen-
chymal interactions in pancreatic cancer is Hedgehog signaling.
The Hedgehog signaling pathway is deregulated during the onset
of pancreatic cancer (38–40), since, unlike normal pancreatic epi-
thelium, PanIN and pancreatic cancer cells secrete the Hedgehog
ligands Shh and Ihh. The Hedgehog ligands act in a paracrine
manner to activate signaling in the stroma (41), and activation of
the pathway has been linked to stroma expansion and induction
of its tumor-promoting ability (37, 42).
In iKras* tissues, Shh expression was induced upon Kras* activa-
tion, and its expression was dependent on continuous Kras activity
(Figure 6, B and C). We also quantified, by qRT-PCR, the expres-
Extensive tissue remodeling in established PanINs following Kras* inacti-
vation. Kras* expression was maintained ON for 5 weeks following pancre-
atitis, then turned OFF for 2 days, 3 days, and 2 weeks. n = 3–5 mice/time
point. (A) Cell death shown by cleaved caspase-3 immunohistochemistry.
Scale bar: 20 μm. (B) Immunohistochemistry for the lineage tracer EGFP.
Scale bar: 20 μm. (C) CK19 (green), amylase (red), and DAPI (blue)
co-immunofluorescence. Scale bar: 20 μm. (D) Tissue proliferation shown
by Ki67 immunohistochemistry. Scale bar: 20 μm. (E) Quantification of
cellular proliferation (Ki67) in each tissue compartment for the indicated
time points. Data represent mean ± SEM.
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sion of Shh, Ptch1, and Gli1 — Hedgehog pathway components and
targets of the pathway — and Gli2, which together with Gli1 medi-
ates the transcriptional output of the Hedgehog pathway. Shh and
Gli2 expression was strictly Kras* dependent, while Ptch1 did not
significantly change, and Gli1 persisted after Kras* inactivation
(Figure 6C). In order to determine which cell compartment had
active Hedgehog signaling, we generated iKras*;Gli1LacZ/+ mice, in
which one copy of the Gli1 gene was replaced by LacZ, and ana-
lyzed their pancreata 3 weeks after inducing pancreatitis and
Kras* expression (Figure 6D), when PanINs were prevalent (Figure
6E). In these animals, the fibroblasts surrounding the epithelial
lesions were LacZ positive (Figure 6F). LacZ expression overlapped
Oncogenic Kras* regulates inter-
actions between epithelial cells
and their microenvironment. Kras*
expression was maintained ON
for 3 weeks following pancreatitis,
then turned OFF for 2 days, 3 days,
and 2 weeks. (A) SMA and (B) Shh
ligand immunohistochemistry. Scale
bar: 20 μm. (C) qRT-PCR analysis
of Hedgehog signaling components
Shh, Ptch1, Gli1, and Gli2. Each
point represents 1 mouse. Data
represent mean ± SEM. (D) Experi-
mental design. In iKras*;Gli1LacZ/+
experimental mice, Kras* expres-
sion was kept ON for 3 weeks fol-
lowing pancreatitis, then turned OFF
for 3 days. n = 2 mice/time point. (E)
Histology. Scale bar: 50 μm. (F)
β-Galactosidase staining for Gli1/
LacZ expression. Scale bar: 20 μm.
(G) CK19 (purple), amylase (red),
Gli1/LacZ (green), and DAPI (blue)
co-immunofluorescence. Scale bar:
20 μm. (H) SMA (purple), Gli1/LacZ
(green), and DAPI (blue) co-immuno-
fluorescence. Scale bar: 20 μm.
648?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
with SMA-positive cells, but not with cells expressing the epithelial
markers CK19 or amylase (Figure 6, G and H). We also collected
tissues in iKras*;Gli1LacZ/+ mice 3 days after Kras* inactivation, at
a stage when SMA expression is downregulated, and found that
Gli1 was still highly expressed in the stroma surrounding the aci-
nar-ductal clusters (Figure 6, F–H). Thus, Gli1 expression persists
following fibroblast inactivation, indicating that although Hedge-
hog signaling is one of the pathways that mediate the paracrine
interactions between the epithelial cells and the stroma, it is not
the only component of a likely complex regulatory mechanism.
In addition to fibroblasts, the pancreatic cancer stroma is rich in
inflammatory cells. This is not surprising, as several inflammatory
cytokines are upregulated in PanIN lesions. We therefore investi-
gated the expression of mediators of inflammatory pathways that
have been shown to be important for pancreatic cancer tumorigen-
esis. Cox2 is overexpressed in PDA, and forced overexpression in
mice has been shown to be sufficient to induce pancreatic dysplasia
(43, 44). More recently, IL-6 and its downstream effector phospho-
Stat3 have been shown to be important not only during the ini-
tial stages of pancreatic cancer development, but also in advanced
disease (20, 45, 46). As previously observed in other mouse mod-
els of PDA (10) and in human tumors, Cox2, IL-6, and phospho-
Stat3 were expressed in the PanIN lesions of iKras* mice, and their
expression was reduced upon Kras* inactivation (Supplemental
Figure 4E and Supplemental Figure 7, C–E). In addition, MMP7,
a matrix metalloproteinase that is expressed in human (47) and
mouse pancreatic cancer (10), was expressed in the PanIN lesions of
the iKras* mice (Supplemental Figure 4F and Supplemental Figure
7F) but downregulated upon Kras* inactivation.
Taken together, these data show that fibroblast activation,
inflammatory cell infiltration, and production of enzymes that
might remodel the extracellular matrix are regulated by Kras*
both during the initiation of pancreatic carcinogenesis and once
PanIN lesions are established, thus highlighting the essential role
of oncogenic Kras is the maintenance of the stroma.
Role of oncogenic Kras in PDA. As previously stated, PanIN lesions
in iKras* animals do not progress to invasive PDA, at least in the
time frame considered. In the KC model, development of PDA has
been shown to require not only Kras* mutation, but also inactiva-
tion of at least one tumor suppressor gene (11, 48, 49). The iKras*
mouse was crossed with p53-null mice (50) in order to obtain
iKras*-p53+/– mice, in which one allele of p53 is inactivated while
the other is present in its wild-type form. Following the experi-
mental design described above, PanIN formation was induced in
iKras*-p53+/– mice by activation of Kras* expression with doxy,
followed by induction of acute pancreatitis. In a first cohort of
animals, the tissue was harvested after 5 weeks (Figure 7A). The
pancreata of iKras*-p53+/– mice presented with PanINs (Figure 7B),
dilated ducts with the presence of intracellular mucins, and exten-
sive fibroinflammatory stroma (data not shown). Ras pathway
activation in the tissues was verified by phospho-ERK1/2 staining
(Figure 7C), and both epithelium and stroma were found to be
highly proliferative (Figure 7D). In a subset of the animals in which
Kras* had been expressed for 5 weeks, doxy was removed to inacti-
vate Kras* and the pancreata were harvested 2 weeks later. At dis-
section, the pancreata appeared as a small, translucent remnant of
tissue, without the characteristic fibrotic appearance characteristic
of pancreata bearing PanIN lesions. Histological analysis verified
the absence of PanIN lesions within 2 weeks after Kras* inactiva-
tion (Figure 7B). However, the pancreata of iKras*-p53+/– mice did
not return to their normal morphology and histology following
Kras* inactivation. Rather, the tissue was characterized by normal
acini interspersed with dilated ducts and ADM and surrounded by
fibrosis and occasional lipomatosis (Figure 7B), but with a mini-
mal inflammatory infiltrate. Phospho-ERK1/2 was inactivated in
the epithelium, but still detectable in the stroma following Kras*
inactivation (Figure 7C). The proliferation index was dramatically
reduced following removal of doxy (Figure 7D), with only acinar
cells staining positive for Ki67 and no staining in ductal structures
or in the fibrotic tissue.
Since no bona fide PDA was observed in the 5-week cohort, a
second cohort of animals was reserved for an aging experiment
(Figure 7F). Between 8 and 18 weeks after Kras* activation, the
animals in this iKras*-p53+/– cohort (n = 9) died or needed to be
euthanized due to weight loss and a deteriorating clinical condi-
tion, in accordance with the animal protocol guidelines, while all
iKras* controls (n = 35) included in this experiment reached this
age without clinical sign of disease (Figure 7K). The difference in
survival between the two cohorts was determined to be highly sig-
nificant, with a P value of 0.0008 in a log-rank test. At necropsy,
the pancreata of these animals demonstrated invasive adenocar-
cinoma, in some cases accompanied by hemorrhagic ascites, with
admixed poorly differentiated and well-differentiated areas, and
duodenal invasion (Figure 7G). The tumors presented with rare
PAS positivity and extensive collagen deposition (data not shown),
strong phospho-ERK1/2 positivity, and proliferation in both the
epithelial and stromal compartments (Figure 7, H and I).
In some of the sick animals, Kras* was inactivated by interrup-
tion of doxy administration. The animals in this cohort that were
removed from doxy returned to good health; in contrast, the animals
that were kept on doxy succumbed to PDA (Figure 7K). At dissection
(2 weeks following Kras* inactivation), the pancreata appeared indis-
tinguishable from those of the 5-week cohort described above: they
were atrophic, with acini interspersed by residual fibrosis (Figure
7G), and showed no or little PAS staining (data not shown). MAPK
activation, measured as phospho-ERK1/2 level, was only rarely
observed in epithelial or stromal cells (Figure 7H), and proliferation
in the stroma was almost completely abrogated, while some of the
acini remained Ki67 positive (Figure 7I). In order to confirm the
tumor regression within the same animal over time, we performed
MRI on iKras*-p53+/– animals (n = 4) before (when showing clini-
cal signs of disease) and after Kras* inactivation. In the presence of
active Kras*, we observed a tumor mass in the head of the pancreas
or, occasionally, in the pancreas tail. Upon Kras* inactivation, the
pancreatic mass regressed, leaving a small pancreatic remnant (Fig-
ure 7L, mass in the head of the pancreas and regression; and Supple-
mental Figure 8A, mass in the tail of the pancreas and regression).
Together, our data indicate that Kras* is required for cancer
maintenance, suggesting that Kras* and/or its downstream effec-
tors are potential therapeutic targets in this disease. In order to
determine whether Kras*-independent cells had persisted that
could lead to cancer recurrence, iKras*-p53+/– mice that had been
on doxy for 10 weeks were taken off doxy and observed over time.
Mice that were kept off doxy (Kras* OFF) for 23 weeks were
healthy, with no evidence of relapse. We harvested the tissues and
confirmed that there was no residual or recurring disease; a few
mucinous ducts present in the pancreas were PAS negative (Figure
7G, inset) and were identified as ectopic Brunner glands.
Analysis of tumors from iKras*-p53+/– mice showed loss of expres-
sion of the wild-type allele of p53 (Supplemental Figure 8, B and C).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
iKras*-p53+/– model and the effect of Kras* inactivation. Experimental design. Kras* expression was maintained ON for 5 weeks (A) or until the
mice developed frank PDA (F) before being turned OFF for 2 weeks or 23 weeks. (B and G) Histology of the pancreata at indicated time points.
Scale bar: 100 μm (top row) and 20 μm (bottom row). Inset: PAS staining. Scale bar: 20 μm. (C and H) Phospho-ERK1/2 immunohistochemistry.
Scale bar: 20 μm. (D and I) Ki67 immunohistochemistry. Scale bar: 20 μm. (E and J) Analysis of genomic instability in iKras*-p53+/– mice by
γ-H2AX immunohistochemistry. Scale bar: 20 μm. (K) Kaplan-Meier survival curve. Log-rank statistical analysis yielded a P value of 0.0008.
(L) In vivo imaging of tumor regression in one iKras*-p53+/– animal using MRI. Total animals imaged, n = 4. T, tumor, outlined in yellow;
S, stomach; Sp, spleen; K, kidney; Int, intestine.
650? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
Nuclear accumulation of a phosphorylated form of histone 2A
(γ-H2AX) reflects oncogenic stress that can be associated with
genomic instability (51) and is a common feature of human and
mouse PDA (52). Analysis of tissues from iKras*-p53+/– mice revealed
nuclear expression of γ-H2AX, both in the epithelial cells and in
the stroma (Figure 7, E and J). Upon Kras* inactivation, γ-H2AX
expression was strongly reduced and, when present, confined to
the cytoplasm, most likely in dying cells, where the integrity of the
nuclear membrane was compromised (Figure 7, E and J). In order to
determine whether oncogenic stress was associated with genomic
instability in our samples, we performed DNA fingerprinting (53,
54) to detect genetic alterations in the tumors compared with
matched genomic DNA from the same mouse. Our results indicate
the presence of genomic instability in the tumors (Supplemental
Figure 8D). Interestingly, some of the tumor-specific bands became
undetectable upon Kras* inactivation, indicating that the cells car-
rying those genetic alterations did not persist. Taken together, our
data indicate that Kras* activity is still required for tumor main-
tenance in pancreatic cells that have accumulated genetic damage
and have lost expression of tumor suppressor genes.
A new approach to modeling Kras inhibition in pancreatic cancer. Mouse
models are widely used to study the events that lead to cancer for-
mation and have represented a powerful tool for new discoveries
(17, 55). Since most cancer patients are diagnosed with advanced
disease, models that mimic Kras* inhibition in the advanced stag-
es of cancer are particularly relevant to the human condition. In
particular, a key point is to determine which oncogenes are impor-
tant during the initiation and progression phases of cancer ver-
sus cancer maintenance. The term “oncogene addiction” has been
used to describe the need for cancer cells to maintain the activity of
oncogenes even after additional genetic and epigenetic events have
occurred (56). The concept has been validated in an elegant model
of pancreatic islet tumors driven by myc overexpression, where
oncogene inactivation leads to tumor regression (57). Oncogenic
addiction for Kras* has been studied in lung adenocarcinoma (12),
where tumors remain Kras* dependent even in the presence of
other genetic alterations. However, whether those findings could
be extended to PDA, a tumor type characterized by activation of
several signaling pathways (5) and by the extensive accumulation
of desmoplastic stroma, had so far not been addressed. Expression
of mutant Kras* from its endogenous locus using the LoxP-STOP-
LoxP-KrasG12D allele (58) results in stepwise PanIN formation that
closely mimics the human disease (10, 11, 59). Additional models
have used Cre-mediated removal of a stop cassette to activate Kras*
expression specifically in the pancreas but not from the endog-
enous locus (60). A limitation of those models is that oncogenic
Kras*, once activated, is constitutively expressed. Different sub-
types of pancreatic cancer have recently been defined in human
patients (14), with potentially differential dependence on onco-
genic Kras*, thus highlighting the need for a suitable model of
reversible Kras* expression in the pancreas. Previous attempts at
generating a reversible form of Kras* expression in the pancreas
using the tTa/TetO system have been limited by the lack of a suit-
able driver for the tTa transcription factor.
Here we used an approach that allows conditional, Cre-mediated
activation of rtTa expression and were thus able to use pancreas-
specific Cre lines to induce its expression. We opted for the broadly
expressed p48-Cre line, since previous studies in mouse models
have shown that PanINs can arise from acini (18, 23), ducts (22),
and even islets during conditions of tissue damage (61). Since Cre
recombination is irreversible, our approach leads to expression of
rtTa in most pancreatic epithelial cells. The approach allows us
to express Kras* in the pancreatic epithelium in a temporally and
spatially regulated manner and, more importantly, in a reversible
manner. It has been recently successfully used in a model of basal
cell carcinoma (62) and could easily be adapted to study other
oncogenes in other organs, and should be of broad interest to sci-
entists interested in tumor maintenance. The iKras* mouse devel-
ops PanINs, and, when crossed with mice with p53 loss of func-
tion, develops PDA that resembles the human disease and mimics
the previously published LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre
model of pancreatic cancer (48).
Oncogenic Kras prevents tissue repair following acute pancreatitis. Acute
pancreatitis can be induced in mice by injection with the cholecys-
tokinin analog cerulein (19) and is characterized, at the tissue level,
by infiltration of inflammatory cells and edema, as well as by ADM,
defined as the replacement of acinar cells with duct-like structures.
Wild-type animals rapidly recover from acute pancreatitis; the
Proposed model for the role of oncogenic Kras in the initiation and maintenance of PanINs and PDA. Initial oncogenic Kras activation leads to
pancreatic dysplasia. When Kras is inactivated at the early time points, the pancreatic tissue reverts back to its original state. However, when
dysplasia is advanced, or if frank PDA is present, turning off Kras will induce apoptosis in the dysplastic epithelium, and the remodeling of the
pancreatic parenchyma is incomplete even after an extended period of time.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
pancreatic parenchyma returns to its normal architecture, and the
inflammatory cell infiltration and edema subside within 1–3 weeks
after treatment. By contrast, it has been observed that in Kras*
mutant animals, recovery is completely prevented (19), and acute
pancreatitis is rapidly followed by more severe ADM surrounded by
fibrosis and, by 3 weeks after the treatment, by extensive mucinous
ADM/early PanINs. It has been hypothesized that Kras* directly
prevents tissue repair, but so far this has not been demonstrated
experimentally (8, 19). In addition, the de-differentiation of acinar
cells to duct-like cells has been hypothesized to be a necessary step
for PanIN formation, at least for PanINs of acinar origin (8, 19). We
have taken advantage of the reversibility of Kras* expression in our
model to investigate the role of this oncogene during the earliest
stages of pancreatic carcinogenesis. Our results show that oncogen-
ic Kras* prevented the repair process by maintaining the ductal dif-
ferentiation of acinar cells; this process was initially fully reversible
upon Kras* inactivation. An additional finding was that expression
of Kras* in the epithelium was responsible for formation and main-
tenance of the fibrotic stroma that accompanies PanIN formation
and is prevalent in PDA. The expansion of a pro-tumor stroma is
one of the “hallmarks of cancer” (63), and in pancreatic cancer, the
extensive desmoplastic stroma has been shown to contribute to this
tumor’s chemoresistance (37).
Kras is required at all stages of pancreatic carcinogenesis. In a last set of
experiments, we have analyzed iKras*-p53+/– mice, in which activa-
tion of oncogenic Kras* is accompanied by loss of one allele of the
p53 tumor suppressor (and by loss of expression from the other
allele). The purpose of this set of experiments was to address the
important question of the role of this oncogene in tumor mainte-
nance, a question of great biologic and therapeutic importance.
The role of Kras in advanced pancreatic cancer has in the past been
addressed in established pancreatic cancer cell lines. Interestingly,
two independent groups have found that pancreatic cancer cell
lines can be subdivided into “Kras*-dependent” and “Kras*-inde-
pendent” subsets (13, 14). Pancreatic cancer, however, is charac-
terized by extensive stroma, which has been shown to alter the
cellular response to treatment (37). Moreover, the established cell
lines do not allow us to study the biology of the precursor lesions
of pancreatic cancer. Kras* inactivation in iKras*-p53+/– mice with
PanINs or adenocarcinoma results in tumor regression, thus
indicating that the tumor cells have become “addicted” to Kras*
expression and activity. Due to the limited regenerative capability
of the adult pancreas, complete repair is not achieved upon Kras*
inactivation; however, even when we let the animals age for several
months, we did not observe any tumor recurrence.
In summary, we have used our iKras* mice to study the effect of
Kras* inhibition at different stages of pancreatic carcinogenesis.
In addition, we have used a model of pancreatitis-induced PanIN
formation to synchronize the appearance of the lesions, thus facili-
tating quantification and analysis of the data. In our model, acti-
vation of Kras* followed by induction of acute pancreatitis leads
to pancreas-wide PanIN formation within 3 weeks and high-grade
PanINs (including carcinoma in situ) by 5 weeks. In the presence
of one loss-of-function allele of p53, the mice showed tissue-wide
PanIN3 by 5 weeks and invasive adenocarcinoma by 8 to 18 weeks.
Our findings are summarized in Figure 8: inactivation of Kras* at
the early PanIN stage led to rapid and complete tissue recovery. At
the mechanistic level, the recovery was accompanied by re-differ-
entiation of PanIN cells into acinar cells and by remodeling of the
stroma. However, once high-grade PanINs had formed, Kras* inac-
tivation was linked to massive cell death, indicating that those cells
had become addicted to the continuous expression of oncogenic
Kras*. Finally, Kras* inhibition also led to regression of invasive
adenocarcinoma. However, in both cases partial recovery of pan-
creatic acini was accompanied by persistence of metaplastic areas
surrounded by fibrotic scar tissue. These findings differ from what
was observed in lung adenocarcinoma upon Kras* inactivation
(12). In that case, Kras* inactivation led to full regression, simi-
lar to what we observed when Kras* was inactivated in low-grade
PanIN lesions (Figure 2). Taken together, our results indicate that
targeting Kras* is likely to have a profound effect on pancreatic
cancer. These data support the potential utility of targeting Kras*
or its downstream signaling pathways as a therapeutic approach
in patients with pancreatic cancer.
Mice. Animals were housed in specific pathogen–free facilities of the Uni-
versity of Michigan Comprehensive Cancer Center. p48Cre (Ptf1a-Cre)
mice (15) (provided by Christopher V. Wright, Vanderbilt University,
Nashville, Tennessee, USA) were intercrossed with TRE-KrasG12D (The
Jackson Laboratory, stock #004735) (12) and R26-rtTa (The Jackson Labo-
ratory, stock #005670) (16) mice to create iKras* triple mutants: p48-Cre;
R26-rtTa-IRES-EGFP;TetO-KrasG12D. iKras* mice were also crossed
with p53-null mice to create iKras*-p53+/– quadruple mutants: p48-Cre;
TRE-KrasG12D;R26-rtTa;p53+/– (The Jackson Laboratory, stock #002101) or
with Gli1LacZ/LacZ (The Jackson Laboratory, stock #008211) (64) mice to gen-
erate iKras*;Gli1LacZ/+ mice. Combinations of single or double mutant lit-
termates were used as controls. LSL-KrasG12D mice were provided by David
Tuveson (Cambridge Research Institute, Cambridge, United Kingdom)
and were bred with p48-Cre mice to generate KC double transgenics. A
Kaplan-Meier survival curve was created to represent animals that had to
be euthanized, according to the animal protocol, or died during the time
of the experiments. Statistical significance was established with a log-rank
test, carried out using GraphPad Prism version 5.00 for Windows (Graph-
Pad Software). A P value less than 0.05 was considered significant, and a
P value less than 0.01 was considered highly significant.
Doxy treatment. Doxy was administered in the drinking water at a concen-
tration of 0.2 g/l in a solution of 5% sucrose and replaced every 3–4 days.
Induction of pancreatitis. Mice were subjected to two series of 8 hourly
intraperitoneal injections of cerulein (Sigma-Aldrich) at a concentration
of 75 μg/kg over a 48-hour period, as previously described (19). Littermate
controls were injected in parallel with the experimental animals.
Immunohistochemistry and immunofluorescence. Pancreatic tissues from
both experimental and control mice were fixed overnight in 10% neutral
buffered formalin, embedded in paraffin, and sectioned. Embedding and
sectioning was performed by the University of Michigan Cancer Cen-
ter Histopathology Core. H&E, PAS, Gomori trichrome, and immuno-
histochemistry stainings were performed as previously described (65). For
a list of the antibodies used, see Supplemental Table 1. β-Galactosidase
staining was performed on tissues fixed in 4% PFA and embedded in OCT
for cryosectioning. Tissues were equilibrated in rinse buffer (100 mM sodi-
um phosphate pH 7.3, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02%
NP-40 w/v) for 10 minutes, then incubated overnight in X-gal stain solution
(rinse buffer plus 1 mg/ml X-gal, 0.5 mM potassium ferricyanide, 0.5 mM
potassium ferrocyanide). Samples were post-fixed in 10% neutral buffered
formalin and counterstained with FastRed (Vector Laboratories). Images
were taken with an Olympus BX-51 microscope, Olympus DP71 digital
camera, and DP Controller software. For immunofluorescence, second-
ary antibodies labeled with FITC, Texas red, and Alexa Fluor (Invitrogen)
were used. Cell nuclei were counterstained with DAPI (Invitrogen). The
652?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 122 Number 2 February 2012
immunofluorescence images were acquired using an Olympus IX-71 con-
focal microscope and FluoView FV500/IX software.
qRT-PCR. Tissue for RNA extraction was prepared through overnight
incubation in RNAlater-ICE (Ambion) at –20°C, then isolated using
RNeasy Protect (QIAGEN) according to the manufacturer’s instructions.
Reverse transcription reactions were conducted using a High-Capacity
cDNA Reverse Transcription Kit (Applied Biosystems). Samples for qRT-
PCR were prepared with 1× SYBR Green PCR Master Mix (Applied Biosys-
tems) and various primers (sequences in Supplemental Table 2). All prim-
ers were optimized for amplification under reaction conditions as follows:
95°C 10 minutes, followed by 40 cycles of 95°C 15 seconds and 60°C
1 minute. Melt curve analysis was performed for all samples after comple-
tion of the amplification protocol. Gapdh was used as the housekeeping
gene expression control.
Histopathological analysis. Histopathological analysis was performed as
previously described. A minimum of 50 total acinar or ductal cluster were
counted from at least 3 independent animals for each group (66). Five ran-
domly selected, non-overlapping high-power images (×20 objective) were
taken for each slide. Each cluster was classified as acinar, PanIN1A, -1B, -2,
or -3, or PDA based on the classification consensus (67).
Quantification of pancreas size. Images of control and iKras* H&E sections
of pancreatic tissue were taken with a Leica MZFLIII dissection microscope
and Olympus DP72 camera. Pancreatic tissue area was determined with
Image Pro Plus v4 software (MediaCybernetics). Averages of the area of the
iKras* pancreata per time point were normalized to control averages to
determine fold change.
Proliferation analysis. Three randomly selected, non-overlapping high-
power images (×20 objective) were taken from Ki67-stained slides from 2–3
independent animals for each group. Nuclei positive for Ki67 were counted
as actively proliferating cells. Epithelial and stromal compartments for each
image were counted separately, and data were expressed as percentage of
total counted nuclei for each compartment. Errors bars represent SEM.
Active Ras pull-down assay. Pull-down of active Ras was performed using
an Active Ras pull-down kit (Pierce). Protein bands were visualized on
Kodak Biomax XAR film for 15 seconds to 3 minutes. Activity levels were
normalized to total Ras as well as E-cadherin levels using ImageJ software
Western blot analysis. Tissues were homogenized in RIPA buffer (Sigma-
Aldrich, R0278) and protease inhibitor (Sigma-Aldrich, P8340). Equal
amounts of protein were electrophoresed in 12% SDS-PAGE gels, trans-
ferred to PVDF membrane (Bio-Rad). Membranes were blocked with milk,
and primary antibody incubations were performed at room temperature
for 2 hours (phospho-ERK1/2, ERK1/2, and E-cadherin 1:1,000 dilution).
Secondary antibody HRP-conjugated anti-rabbit (1:5,000) was used and
detected with Supersignal West Pico substrate (Thermo Scientific). Protein
bands were visualized on Kodak Biomax XAR film.
MRI. Mice were anesthetized with 1%–2% isoflurane/air, and body tem-
perature was maintained by blowing warm air through the bore of the
magnet using an Air-Therm (World Precision Instruments). MRI scanning
was performed using a 7T Agilent Direct Drive system with a quadrature
rat head volume coil (M2M). Mice were placed supine in the coil, taped
below the thoracic cavity on the bed to reduce respiratory motion. A sub-
cutaneous catheter was placed into skin of the neck for delivery of con-
trast agent. Two T1-weighted images were acquired before and 10 minutes
after a subcutaneous bolus injection of Gd-DTPA (gadopentetate dime-
glumine, Bayer HealthCare) at a dose of 0.7 mmol/kg using a spin-echo
sequence, with fat saturation and the following parameters: repetition
time (TR)/echo time (TE) = 757/15 ms, field of view (FOV) = 25 × 25 mm2,
matrix size = 128 × 128, slice thickness = 1 mm, number of slices = 25,
no gap, interleaved, and 4 dummy scans. T2-weighted images were
acquired using a fast spin echo multi-slice sequence with TR/TE: 4,000/
30 ms, 8 echo trains, 4 averages, 2 dummy scans, and the same slice pack-
age as the T1-weighted sequence. Using in-house software, the tumor
boundary was manually defined on each slice and then integrated across
slices to measure the volume.
Random amplified polymorphic DNA analysis (RAPD). Genomic DNA
was isolated from tumor and normal tissues from each animal using a
QIAGEN DNeasy Blood and Tissue Kit per the manufacturer’s instruc-
tions. Inter-simple sequence repeat (inter-SSR) PCR was performed as
described previously (68) using (CA)8RY, (CA)8RG, and (AAC)6Y primers.
PCR products were separated with 2.5% agarose gels and visualized with
SYBR Safe DNA gel stain (Invitrogen).
Study approval. All animal protocols were approved by the University of
Michigan University Committee on Use and Care of Animals (UCUCA).
We would like to thank Diane Simeone, Anj Dlugosz, Jörg Zeller,
Ben Allen, and Mats Ljungman (University of Michigan, Ann Arbor)
for scientific discussion and critical reading of the manuscript. We
thank Jimmy Hogan, Ace Josifoki, and Beth Skendrovich for help
with immunostaining and histology. The p48-Cre (Ptf1a-Cre) mouse
was a gift from Chris Wright (Vanderbilt University). The CK19
antibody (Troma III) was obtained from the Iowa Developmental
Hybridoma Bank. The Hes1 antibody was a gift from Ben Stanger
(University of Pennsylvania, Philadelphia, Pennsylvania, USA). Work
in the Pasca di Magliano laboratory is supported by the University
of Michigan Biological Scholar Program, NCI 1R01CA151588-01,
GI SPORE P50 CA 13810, a Pancreatic Cancer Action Network
(PanCan)/American Association for Cancer Research (AACR) Career
Development grant, and the Michigan Gastrointestinal Peptide
Research Center (P30 DK 034933-25). M.A. Collins is supported by
a University of Michigan Program in Cellular and Molecular Biology
training grant (NIH T32 GM07315) and by a University of Michi-
gan Center for Organogenesis training grant (5-T32-HD007515).
F. Bednar is supported by the American College of Surgeons Resi-
dent Research Scholarship and by NIH T32 HD007505.
Received for publication June 9, 2011, and accepted in revised form
November 16, 2011.
Address correspondence to: Marina Pasca di Magliano, Depart-
ment of Surgery and Cell and Developmental Biology, 1500 E Med-
ical Center Drive, Ann Arbor, Michigan 48109-5936, USA. Phone:
734.615.7424; Fax: 734.647.9654; E-mail: firstname.lastname@example.org.
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