Protein Kinase C Iota Regulates Pancreatic Acinar-to-Ductal Metaplasia

Pancreatic acinar-to-ductal metaplasia (ADM) is associated with an increased risk of pancreatic cancer and is considered a precursor of pancreatic ductal adenocarcinoma. Transgenic expression of transforming growth factor alpha (TGF-α) or K-rasG12D in mouse pancreatic epithelium induces ADM in vivo. Protein kinase C iota (PKCι) is highly expressed in human pancreatic cancer and is required for the transformed growth and tumorigenesis of pancreatic cancer cells. In this study, PKCι expression was assessed in a mouse model of K-rasG12D-induced pancreatic ADM and pancreatic cancer. The ability of K-rasG12D to induce pancreatic ADM in explant culture, and the requirement for PKCι, was investigated. PKCι is elevated in human and mouse pancreatic ADM and intraepithelial neoplastic lesions in vivo. We demonstrate that K-rasG12D is sufficient to induce pancreatic ADM in explant culture, exhibiting many of the same morphologic and biochemical alterations observed in TGF-α-induced ADM, including a dependence on Notch activation. PKCι is highly expressed in both TGF-α- and K-rasG12D-induced pancreatic ADM and inhibition of PKCι significantly reduces TGF-α- and K-rasG12D-mediated ADM. Inhibition of PKCι suppresses K-rasG12D–induced MMP-7 expression and Notch activation, and exogenous MMP-7 restores K-rasG12D–mediated ADM in PKCι-depleted cells, implicating a K-rasG12D-PKCι-MMP-7 signaling axis that likely induces ADM through Notch activation. Our results indicate that PKCι is an early marker of pancreatic neoplasia and suggest that PKCι is a potential downstream target of K-rasG12D in pancreatic ductal metaplasia in vivo.


Introduction
Oncogenic KRAS mutations are found in .90% of pancreatic ductal adenocarcinomas (PDACs). [1] Mutational activation of KRAS is thought to occur early in PDAC development, as KRAS mutations are observed in ,30% of PDAC precursor lesions, pancreatic intraepithelial neoplasia (PanIN). [1] A mouse model for conditional expression of an activated Kras (Kras G12D ) allele in the pancreas from its physiological promoter has been utilized to investigate the role of oncogenic K-ras in initiation and progression of PDAC. [2,3,4] Expression of oncogenic K-ras induces formation of preneoplastic lesions in mice that are histologically similar to human PanINs (mouse PanINs, mPanINs). [2,4] K-ras G12D -induced mPanINs become increasingly dysplastic, with a small percent progressing to invasive and metastatic adenocarcinomas, strongly suggesting that acquisition of an oncogenic Kras mutation can be an initiating event in pancreatic cancer. [2,4] Acinar-to-ductal metaplasia (ADM), the replacement of acinar cells with metaplastic ductal cells, is thought to be a source of neoplasia in the initiation of human PDAC. [4,5,6] Dysplastic features often arise in areas of ductal metaplasia, and metaplastic ductal cells exhibit many properties of embryonic progenitor cells, including Nestin expression. [4,7,8] The K-ras G12D -initiated mouse model of PDAC exhibits morphological, molecular and biochemical features indicative of ADM as early as 4 weeks of age, prior to the development of mPanINs. [2,4] Aberrant activation of EGFR signaling in mouse pancreas also induces ADM and subsequent formation of PDAC. [7,9,10] EGFR-mediated ADM has been further characterized in an explant model. [11,12] TGFa induces primary mouse pancreatic acinar cells to transition through a de-differentiated, Nestin-positive intermediate to form metaplastic ductal structures. [7,11,12] Additional studies revealed that Notch signaling is both necessary and sufficient for acinar cell de-differentiation, Nestin expression and ADM in explant culture. [2,12] MMP-7, which is also upregulated in human and mouse PanINs and PDAC, promotes activation of Notch signaling and ADM. [13,14] MMP-7 is required for ADM in explant culture, and expression of a constitutively active Notch construct reconstitutes ADM in MMP-7-depleted acinar cells, indicating that MMP-7-dependent Notch activity is required for ADM. [14] These studies demonstrate the utility of the pancreatic acinar cell explant model for characterization of ADM, and strengthen the link between pancreatic metaplasia, neoplasia and initiation of PDAC.
We have identified PKCi as an important effector in oncogenic K-ras-induced transformation of lung and intestinal epithelial cells. [15,16] We have also demonstrated that PKCi expression is elevated in a large percent of primary pancreatic adenocarcinomas, and high PKCi expression predicts poor patient survival. [17] In the current study, we demonstrate that PKCi is elevated in pancreatic metaplasia associated with human PDAC tumors and in K-ras G12D -mediated pancreatic metaplasia in vivo. To further characterize the molecular mechanism of K-ras G12D -mediated pancreatic ADM we employed a well-characterized mouse pancreatic acinar cell explant model. In this context, we evaluated the role of PKCi in K-ras G12D -mediated pancreatic ADM. Expression of oncogenic K-ras, the most frequently mutated oncogene in PDAC, is sufficient to induce pancreatic ADM in explant culture. PKCi expression is elevated in K-ras G12D -and TGFa-induced ADM. Inhibition of PKCi significantly reduces both K-ras G12D -and TGFa-induced ADM and also significantly reduces K-ras G12D -mediated Nestin expression, Notch activation and MMP-7 expression. Exogenous MMP-7 partially but significantly reconstitutes K-ras G12D -mediated ADM in PKCidepleted cells, suggesting that PKCi mediates initiation of ADM, at least in part, by regulating MMP-7 expression. Our results demonstrate that K-ras G12D -mediated ADM in explant culture is regulated by PKCi.

PKCi is induced in oncogenic K-ras-mediated ADM in vivo
PKCi expression is elevated in the vast majority of primary PDAC, and high PKCi expression predicts poor patient survival. [17] PKCi is also elevated in PanINs and pancreatic metaplastic ducts associated with human PDAC ( Figure 1A). In normal mouse pancreas, PKCi is detected in interlobular ductal cells, but not in acinar cells ( Figure 1B). PKCi expression was also detected in mPanINs ( Figure 1C) from P48-Cre;LSL-Kras mice. PKCi expression tended to increase, with a redistribution from apical to cytoplasmic localization, in more progressed mPanIN lesions and in adenocarcinoma ( Figure S1). Interestingly, PKCi was also expressed in the metaplastic ductal cells, but not in the morphologically normal acinar cells of K-ras G12D -induced ADM ( Figure 1D). K-ras G12D -induced pancreatic ADM exhibits some of the same properties of mPanINs, including increased proliferation and Notch signaling, [2,4,11,12,14] suggesting ADM is a precursor to mPanINs and therefore relevant to the initiation of PDAC. [4] The increased PKCi expression observed in Kras G12D -induced ADM prompted us to investigate a possible role for PKCi in K-ras G12D -induced ADM using an explant culture amenable to evaluation of the molecular mechanisms involved in the specific transdifferentiation of pancreatic acinar cells to metaplastic duct-like cells.
PKCi regulates TGF-a-mediated ADM As described, mouse pancreatic acinar cells plated in collagen matrix undergo TGF-a-induced ADM, characterized by morphological conversion from clusters of zymogen-containing acinar cells to cystic structures with a ductal morphology ( Figure S2A). [11,14] This morphological transformation is associated with a loss of acinar differentiation, as assessed by amylase expression and a concomitant increase in ductal differentiation, characterized by expression of cytokeratin 19 (CK-19) ( Figure S2B). [11] PKCi expression is undetectable in isolated acinar cells, but is significantly increased as cells undergo TGF-a-induced ADM (Figure 2A), consistent with PKCi playing a role in the transdifferentiation of pancreatic acinar cells to metaplastic ducts.
To investigate the role of PKCi in TGF-a-mediated ADM, we utilized pancreatic acinar cells isolated from Prkci f/f mice. [18] Prkci f/f acinar cells were transduced with control adeno-virus (adeno-null) or adeno-virus expressing Cre-recombinase (adeno-Cre) to induce genetic recombination and deletion of the loxPflanked Prkci allele ( Figure S3A). Adeno-null-treated Prkci f/f acinar cells underwent ADM in response to TGF-a, while adeno-Cre-treated Prkci f/f acinar cells were largely refractory to TGF-a-induced ADM ( Figure 2B). Adeno-Cre treatment did not inhibit TGF-a-mediated ADM in R26R acinar cells ( Figure  S3B and C). Consistent with a specific requirement for PKCi, addition of the molecularly-targeted inhibitor of PKCi signaling, aurothiomalate, [19,20,21] to the explant culture significantly reduced TGF-a-induced ADM ( Figure 2C). These data demonstrate at least a partial requirement for PKCi for TGFa-induced ADM.

K-ras G12D induces ADM in explant culture
The earliest morphological alteration observed in the pancreata of P48-Cre;LSL-Kras mice is the formation of metaplastic structures containing both acinar-and duct-like cells. [4] Molecular analysis of these metaplastic structures suggests that K-ras G12D induces ADM. [4] To evaluate the role of PKCi in K-ras G12D -induced ADM, we first characterized the ability of K-ras G12D to induce ADM in explant culture. Pancreatic acinar cells were isolated from LSL-Kras mice and incubated with adeno-Cre-GFP to induce genomic recombination ( Figure S4A) and expression of Kras G12D . K-ras G12D was sufficient to induce ADM in explant culture in the absence of exogenous TGF-a, as determined by transition from acinar to ductal morphology ( Figure 3A) with a single layer of cells surrounding a clear lumen, indicative of a mature ductal structure ( Figure S4B). Likewise, a loss of expression of acinar cell markers and a gain of expression of ductal cell markers was also observed in K-ras G12D -induced ADM ( Figure 3B and Figure S4C) confirming transition from acinar to ductal gene expression profile.
While K-ras G12D induced ADM in explant culture in the absence of exogenous TGF-a, TGF-a mRNA was elevated in Kras G12D -mediated ADM ( Figure 3C). K-ras G12D -induced ADM was partially, but significantly reduced by Erlotinib, an EGFR inhibitor ( Figure 3D). Furthermore, inhibition of Rac1 blocks Kras G12D -mediated ADM ( Figure 3D), consistent with a recent report that Rac1 activity regulates ADM. [22] K-ras G12D -induced ADM was also accompanied by a significant increase in PKCi expression ( Figure 3E) in CK-19-positive duct cells ( Figure  S4D). Taken together, these results demonstrate that K-ras G12D induces metaplastic duct formation in explant culture, as in mouse pancreas in vivo, [4] and that PKCi expression is induced in Kras G12D -mediated metaplastic ducts in vitro and in vivo.

PKCi regulates K-ras G12D -induced ADM
We next tested the hypothesis that PKCi plays a role in Kras G12D -induced ADM in explant culture, using acinar cells from LSL-Kras;Prkci f/f mice which allow simultaneous Cre-mediated activation of expression of K-ras G12D and genetic knockout of PKCi. [16] K-ras G12D induced ADM in LSL-Kras acinar cells, but not LSL-Kras;Prkci f/f acinar cells ( Figure 4A, B). Expression of PKCi and CK-19 remained low in adeno-Cre-GFP-treated LSL-Kras;Prkci f/f explant cultures, compared to adeno-Cre-GFP-treated LSL-Kras explant cultures (compare Figure S5A to Figure S4D). GFP expression confirmed highly efficient viral infection of both LSL-Kras and LSL-Kras;Prkci f/f acinar cells ( Figure S5B) and PCR analysis demonstrated adeno-Cre-mediated recombination of both the LSL-Kras and Prkci f/f floxed alleles in the LSL-Kras;Prkci f/f acinar cells ( Figure S5C). Furthermore, addition of aurothiomalate to the explant culture also significantly reduced K-ras G12D -mediated ADM ( Figure 4C), without a significant effect on cell viability (data not shown). aurothiomalate did not prevent K-ras G12D -induced PKCi expression ( Figure S5D and E), however, PKCi was detected primarily in the cytoplasm of aurothiomalate-blocked acinar-like cells, in contrast to the more basolateral localization of PKCi in K-ras G12D -induced metaplastic ducts ( Figure S5D). Therefore, genetic and pharmacological inhibition of PKCi significantly reduce K-ras G12D -mediated ADM, strongly supporting a role for PKCi activity in K-ras G12D -mediated ADM.  [11,12,14] We asked whether K-ras G12D -induced ADM also proceeds through a Nestin-positive intermediate. Nestin expression was undetectable in K-ras G12D -expressing explant cultures on day 1, but increased significantly by day 3 ( Figure 5), similar to the kinetics of Nestin expression in TGFa-induced ADM. [11,12,14] PKCi ablation blocked K-ras G12D -induced Nestin expression on day 3 ( Figure 5), implicating PKCi in the initial de-differentiation step of K-ras G12D -induced ADM.
Notch signaling is activated in K-ras G12D -mediated ADM in vivo, [4] and is both required and sufficient to induce pancreatic ADM in explant culture. [12] We therefore evaluated whether Notch was activated by K-ras G12D in explant culture ( Figure 6). Gamma-secretase-dependent cleavage of the Notch receptor is required for activation of Notch signaling. [23] Using an antibody specific for gamma-secretase cleaved (activated) Notch1, we detected little to no activated Notch1 in K-ras G12D -expressing acinar cell explant culture on day 1, but by day 3 the amount of activated Notch was significantly increased ( Figure 6A). Kras G12D -induced Notch1 activation was inhibited in PKCideficient cells ( Figure 6A). Likewise, expression of Hes1, a Notch transcriptional target, was induced in K-ras G12D -expressing explant culture, but the increased Hes1 expression was blocked by loss of PKCi expression ( Figure 6B), implicating PKCi in the regulation of Notch1 activation. Finally, K-ras G12D -induced ADM was significantly reduced by a gamma-secretase inhibitor (L-685,458) [24] (Figure 6C), suggesting that K-ras G12D -induced ADM may require Notch activity.

MMP-7 overcomes PKCi deficiency to recover ADM
Our data strongly suggest that PKCi regulates acinar-to-ductal transdifferentiation prior to Notch activation. Sawey et al. demonstrated that MMP-7 is both necessary and sufficient for Notch activation in ADM in explant culture. [14] MMP-7 expression is elevated in K-ras G12D -induced mPanINs in vivo, suggesting a role for MMP-7 in K-ras G12D -initiated neoplasia. [2] Consistent with these findings, we found that K-ras G12D -induced ADM was accompanied by a significant increase in MMP-7 expression, whereas PKCi-null explants showed no induction of MMP-7 ( Figure 7A). Genetic knockout of PKCi expression in Kras G12D -expressing explant culture significantly reduced the Kras G12D -induced increase in MMP-7 mRNA expression ( Figure  S6A), suggesting that PKCi may regulate MMP-7 transcription. To test whether restoration of MMP-7 rescues K-ras G12D -induced ADM in PKCi-deficient acinar cells, we added recombinant MMP-7 to the explant culture. Indeed, MMP-7 significantly enhanced ADM in PKCi-deficient cells ( Figure 7B, C). PKCi expression remains low in MMP-7-induced ducts (compare Figure S6B to Figure S5A), suggesting that addition of exogenous MMP-7 by-passes PKCi in promoting ADM, and providing support for the hypothesis that PKCi regulates ADM, at least in part, by controlling MMP-7 expression. [14] The lack of complete reconstitution of ADM by MMP-7 in PKCi deficient acinar cells may be due to the reduced diffusion of MMP-7 in collagen matrix, but may also indicate the requirement of additional factors downstream of PKCi. Our results demonstrate that K-ras G12D -induced ADM utilizes signaling pathways implicated in TGF-a-induced ADM in explant culture and K-ras G12Dinduced pancreatic carcinogenesis in vivo. [2,4,25] Importantly, we make the novel observation that PKCi regulates K-ras G12D -and TGF-a-mediated pancreatic ADM in explant culture.

Discussion
PKCi is highly overexpressed in human pancreatic cancer and expression of PKCi-targeted RNAi significantly reduces PDAC cell transformed growth and tumorigenicity in vivo. [17] These data suggest that PKCi plays a required role in human pancreatic cancer. We have previously defined a required role for PKCi in oncogenic K-ras-mediated initiation of preneoplastic lesions of the lung and intestinal epithelium. [15,16] In this study, we investigated the role of PKCi in oncogenic K-ras signaling and initiation of pancreatic metaplasia using a well-characterized pancreatic explant culture model.
Increasing evidence suggest that PanINs can develop from acinar cells and that ADM may be a critical intermediate in the development of PanINs. [4,26] PKCi expression is significantly higher in K-ras G12D -mediated ductal metaplasia than in morphologically normal regions of mouse pancreatic acinar cells, and remains elevated in mPanINs and adenocarcinoma. To directly investigate the role of PKCi in K-ras G12D -mediated ADM, we utilized an acinar cell explant model of ADM [11] in which TGFa induces acinar cell de-differentiation to Nestin-positive, precursor-like intermediates that subsequently convert to cytokeratin-expressing metaplastic ducts. [12,14] Indeed, several studies have concluded that the rate limiting step in K-ras G12D -mediated mPanIN formation appears to be de-differentiation of mature pancreatic exocrine cells. For example, creating an expanded, dedifferentiated cell population through genetic knockout of Mist1 (an acinar cell-restricted transcription factor) or pancreatic injury, enhanced the rate of formation of K-ras G12D -mediated mPanINs. [27,28] Likewise, targeting K-ras G12D only to Nestin-expressing progenitor cells yielded similar levels of mPanINs as targeting the entire exocrine cell population, [3] suggesting that this dedifferentiated, progenitor-like population of cells may be the target for K-ras G12D -mediated initiation of PDAC.  . K-ras G12D induces ADM in explant culture. Pancreatic acinar cells were isolated from LSL-Kras mice, incubated with adeno-Cre-GFP virus, and embedded in collagen (without exogenous TGF-a). A) Representative bright field and fluorescent images were captured on days 1, 3 and 7. GFP fluorescence indicates infection by adeno-Cre-GFP virus. Scale bar, 200 mm. B) ADM was confirmed by co-immunofluorescence of amylase (red) and CK-19 (green) in K-ras G12D -induced ductal cells on day 7. C) mRNA was isolated from day 1 and 6 explant cultures of Ad-Cre virus-treated LSL-Kras acinar cells and analyzed by qPCR for TGF-a expression. Data is presented relative to 18S abundance (610 5 ) and is representative of two independent experiments. D) Pancreatic acinar cells were isolated from LSL-Kras mice, incubated with Ad-Cre and embedded in collagen 6 1 mM or 10 mM Erlotinib, or 10 mM NSC23766 for 5 days. Quantitative analysis of metaplastic duct formation is plotted for each treatment. Bars = mean 6 SD. *P,0.05 (Student T-test). Plots are representative of two independent experiments. E) PKCi (red) was undetectable in LSL-Kras explant culture on day 1, but was elevated in K-ras G12D -induced ductal cells on day 7. Scale bar, 25 mm. doi:10.1371/journal.pone.0030509.g003 In this study, we demonstrate that K-ras G12D induces ADM in explant culture in a manner similar to TGF-a-induced ADM, including progression through a Nestin-positive intermediate and a dependence on PKCi. Inhibition of PKCi significantly reduced K-ras G12D -induced Nestin expression, suggesting a role for PKCi in K-ras G12D -mediated de-differentiation of mature acinar cells. K-ras G12D -induced ADM does not require exogenous TGF-a, however, activation of K-ras G12D induced TGF-a mRNA expression and inhibition of EGFR decreased K-ras G12D -induced ADM in explant culture. Since EGFR expression and activation is induced in K-ras G12D -induced ADM in vivo [4] our data suggests that K-ras G12D may induce ADM, at least in part by up-regulation of autocrine EGFR signaling. This hypothesis is supported by the observation that EGFR signaling synergizes with K-ras G12D to promote progression of mPanINs in the LSL-Kras mouse model of pancreatic cancer. [29] The Notch signaling pathway, which blocks pancreatic acinar cell differentiation and maintains cells in a non-differentiated, proliferative state, is required for normal pancreatic development. [30] Notch signaling is aberrantly reactivated in PanINs and PDAC, as well as K-ras G12D -initiated mPanINs. [12,31] These observations suggest a required role for Notch signaling in Kras G12D -mediated initiation of PDAC. Notch signaling is activated by TGF-a in mouse pancreas in vivo and in explant culture, and Notch signaling is required and sufficient for TGF-a-induced ADM in explant culture. [12,14] K-ras G12D also induces Notch activation in acinar cell explant culture, and K-ras G12D -mediated ADM is significantly reduced by a gamma-secretase inhibitor, suggesting that K-ras G12D -mediated ADM may require Notch activation. Inhibition of gamma-secretase activity, which blocks activation of Notch signaling, inhibits progression of K-rasmediated mPanINs in vivo and reduces the transformed growth of pancreatic cancer cells. [25,32] Likewise, expression of a constitutively-active Notch promoted formation and progression of K-ras-mediated mPanINs, suggesting a tumor-promotive role for Notch signaling in the mouse model of PDAC. [26] Conversely, genetic knockout of Notch1 expression promoted formation and progression of K-ras-mediated mPanINs, suggesting that under some conditions, or at certain stages of cancer development, Notch signaling may suppress pancreatic cancer. [33] In this context, it will be interesting to determine whether PKCi regulates Notch activation in mPanINs and PDAC, since PKCi remains elevated as mPanINs become increasingly dysplastic.
Inhibition of PKCi significantly reduced K-ras G12D -mediated MMP-7 expression, Notch activation and ADM in explant culture. Addition of exogenous MMP-7 to the explant culture partially, but significantly, recovered the inhibitory effect of PKCi deficiency. These results implicate MMP-7 as a likely downstream effector of PKCi in K-ras G12D -mediated ADM, and a possible mechanism by which PKCi regulates Notch1 activation, since MMP-7 can cleave and activate Notch1 in metaplastic acinar cells. [14] PKCi is required for mutant Apc-induced intestinal adenoma formation. [34] Tumorigenesis in the Apc min/+ mouse model also requires MMP-7 and Notch activation. [35,36] MMP-7 has been identified as a target gene of Rac1 in colorectal carcinoma cells, [37] suggesting regulation of Rac1 activity as a possible mechanism by which PKCi may regulate MMP-7 expression and initiation of pancreatic and colon cancer. In addition, PKCi regulates expression of another MMP, MMP-10, in lung cancer cells. [38] Both PKCi and MMP-10 are required for lung cancer cell transformed growth, [38,39] suggesting that regulation of expression of MMPs may be a general mechanism by which PKCi controls initiation and maintenance of the transformed phenotype in cancer.
In this study, we use both genetic and pharmacological means to demonstrate that PKCi regulates TGF-a-and K-ras G12D -induced ADM in explant culture. Our results indicate that PKCi is an early marker of pancreatic neoplasia. Our results further suggest that Kras G12D -mediated ADM utilizes a PKCi-MMP-7 signaling pathway, and that, similar to lung and colon cancer, [15,16] PKCi may play a promotive role in the initiation of PDAC. Tri-transgenic P48-Cre;LSL-Kras;Prkci f/f mice would be useful to test the hypothesis that PKCi is required for K-ras G12D -mediated ADM and mPanIN formation in vivo. However, these tri-transgenic mice are currently unavailable due to difficulties in breeding. Overcoming these breeding difficulties, whose cause is currently unknown, will be important for future studies to test the prediction of our in vitro results, namely, that PKCi plays a role in K-ras G12D -mediated pancreatic metaplasia and carcinogenesis in vivo.

Ethics Statement
Biospecimens were obtained from the Mayo Clinic SPORE in Pancreatic Cancer Tissue Core under an approved Mayo Clinic Institutional Review Board protocol (08-001607). All animal experiments performed were approved by the Mayo Clinic Institutional Animal Care and Use Committee (Mayo Clinic Institutional Animal Care and Use Committee protocols A6508, A48510).

Reagents
A list of antibodies used in this study and their sources can be found in Table S1. Other reagents utilized: recombinant human infection, MOI) overnight at 37uC, with gentle rocking every 15 minutes for the first hour. Thereafter, the cells were embedded in collagen matrix and grown for up to 7 days in explant culture. For detection of b-gal activity, collagen explants were washed, fixed and stained in X-gal overnight at 37uC. [11] Transduction efficiency calculation is described in Supporting Materials and Methods S1.

Immunofluorescence
Pancreatic explant cultures were fixed and labeled with fluorescent antibodies as described. [11,14] Fluorescent images were captured on a Zeiss LSM-510 Meta confocal microscope and bright field and GFP images were captured on an Olympus IX71/ IX51 inverted microscope.

Statistical analysis
Unless otherwise noted, two-way Analysis of Variance (AN-OVA) was used to evaluate the statistical significance of the difference between groups, and a P value,.05 was considered statistically significant.  Table S2 for PCR primer sequences. B) Representative bright field images of primary acinar cells from WT mice incubated with Ad-Cre and embedded in collagen 6 TGF-a for 7 days. Scale bar, 200 mm. C) Pancreatic acinar cells were isolated from R26R mice, incubated with Ad-null or Ad-Cre and embedded in collagen 6 TGF-a for 7 days. b-galactosidase staining indicates Ad-Cre-mediated recombination of the RO-SA26R allele. Scale bar, 50 mm.  of Ad-Cre-treated LSL-Kras mouse pancreatic acinar cells. See Table S2 for PCR primer sequences. B) Representative image of H&E stained, formalin-fixed, paraffin-embedded day 7 explant culture of Ad-Cre-treated LSL-Kras cells. Note the single layer of duct-like cells that surround the luminal structure is more easily distinguished in fixed and sectioned explant culture. C) Coimmunofluorescence of chymotrypsin (green) and carbonic anhydrase II (red) in Ad-Cre-treated LSL-Kras on day 1 and 7. DAPI (blue) staining is shown. D) Co-immunofluorescence of PKCi (red) and CK-19 (green) in Ad-Cre-treated LSL-Kras on day 7. DAPI (blue) staining is shown. Scale bar, 50 mm.  Table S2 for PCR primer sequences. D) Detection of PKCi (red) in Ad-Cre-treated LSL-Kras acinar cells in explant culture (day 7). Untreated (left panel) or+aurothiomalate (ATM; right panel). PKCi expression is elevated in ATM-treated cells, relative to non-K-ras G12D -expressing acinar cells (panel A), but cell-type-specific differences in PKCi subcellular distribution makes determination of relative PKCi expression in K-ras G12Dinduced cells 6 ATM (panel D), difficult. DAPI (blue) staining is shown. Scale bar, 25 mm. E) mRNA was isolated from day 1 and 6 explant cultures of Ad-Cre virus-treated LSL-Kras acinar cells +/2 ATM and analyzed by qPCR for PKCi expression. Data is presented relative to 18 S abundance and presented relative to PKCi mRNA expression on day 1. Data presented is representative of two independent experiments. (TIF) Figure S6 Characterization of the relationship between PKCi and MMP-7 in K-ras G12D -mediated ADM. A) mRNA was isolated from day 1 and 6 explant cultures of Ad-Cre virustreated LSL-Kras and LSL-Kras;Prkci f/f acinar cells and analyzed by qPCR for MMP-7 expression. Data is presented relative to 18 S abundance (610 5 ) and is representative of two independent experiments. B) Immunofluorescence of PKCi (red) in Ad-Cretreated LSL-Kras;Prkci f/f cells plated with 200 ng/ml active recombinant MMP-7 (rMMP-7) in explant culture (day 6). DAPI (blue) staining is shown. Scale bar, 50 mm. (TIF)