phospho-Stat3 was shown to be directly responsible for the accelerated neoplastic progression in P/K mice following AP [16]. In our model two months post AP, phospho-Stat3 was only rarely detected in PanINs. In contrast, it was expressed mainly in the surrounding inflammatory cells, and in ADM foci as well as in small groups of histologically normal acinar cells. These observations suggest that Stat3 activation could be involved in metaplastic processes as the initiating steps in neoplastic transformation. The fact that extensive areas of ADM are still observed after 2 months also suggests that other events are required for the ADM to progress to PanIN. Increased levels of KrasG12D activity could be an essential component for this progression [31,32]. Recent studies showed that PDAC grows slowly over several decades and metastasize only in ‘‘late’’ stages [33,34]. This suggests that early detection and prevention could be the most valuable approaches to confront this highly lethal cancer. Defining the cell types in which cancer can initiate as well as understanding the mechanisms by which environmental injuries can affect cancer progression are both essential for these approaches. Materials and Methods All animal experiments were approved by the Institutional Animal Care and Use Committee at Dartmouth College, protocol number: 07-09-07. Mouse colony generation The LSL-KrasG12D (01XJ6—B6; 129-Kras2tm4Tyj) mice were generated by D.A. Tuveson and T. Jacks [35] and obtained from MMHCC, NCI. The Nestin-Cre mice (B6.Cg-Tg(Nes-cre)1Kln/J) generated by R. Klein [17] were purchased from the Jackson laboratory. Pdx1-Cre mice were a gift from G. Gu [35]. All genotyping were done by PCR following the conditions of the providers. Two episodes of acute pancreatitis were induced by a series of seven hourly intraperitoneal injections of caerulein given on 2 consecutive days, as described [14]. Caerulein (Sigma, St. Louis MO) was diluted in PBS and injected at a dose of 50 mg/kg of body weight. A second group of compound mutant and control animals received injections of PBS only. All animals were fasted for 12 hours before the experiment. Histology and immunohistochemistry Mice were perfused with PBS then 10% formalin/PBS and the pancreata dissected. For bGalactosidase staining, tissues were transferred directly after perfusion into 30% sucrose at 4uC, and 24 hours later cryoembedded in OCT compound. For histology and most immunostaining, pancreata were fixed overnight and processed for paraffin embedding. Routine Hematoxylin and Eosin (H&E) staining was performed using standard procedures. For immunostaining, sections were deparaffinized, rehydrated and antigens were retrieved if required using a 2100-Retriever and References 1. Warshaw AL, Fernandez-del Castillo C (1992) Pancreatic carcinoma. N Engl J Med 326: 455–465. 2. Hruban RH, Wilentz RE, Maitra A (2005) Identification and analysis of precursors to invasive pancreatic cancer. Methods Mol Med 103: 1–13. 3. Tuveson DA, Hingorani SR (2005) Ductal pancreatic cancer in humans and mice. Cold Spring Harb Symp Quant Biol 70: 65–72. 4. Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, et al. (2003) Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4: 437–450. 5. Aguirre AJ, Bardeesy N, Sinha M, Lopez L, Tuveson DA, et al. (2003) Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma. Genes Dev 17: 3112–3126. Acute Pancreatitis and Pancreatic Cancer antigen unmasking solution (Vector Laboratories). For cytokeratin 19 (CK19) immunostaining, Proteinase K antigen retrieval procedure was used. Immunostaining procedures were as described previously [5]. The antibodies and dilution used were: TromaIII (CK19 antibody developed by Rolf Kemler and obtained from Developmental Studies Hybridoma Bank, 1:10), anti-bGalactosidase (rabbit, Cappel, 1:200), anti-Muc5a (Novo- castra, 1:30), anti-Ki67 (mouse, Novocastra, 1:200), anti-Hes1 (a gift from Dr. T. Sudo, Toray Inc., Kamakura, Japan; 1:400), anti- Cox2 and anti-phospho-Stat3 (Tyr705) (both Cell Signaling 1:50), anti-Pdx1 (Upstate1:5000), anti-aSMA (Abcam, 1:100), anti- Nestin (Chemicon, AB5922 for human, MAB353 for mouse, 1:100), and anti-amylase (Calbiochem, 1:500). Pictures were captured with a QImaging EXI Blue camera and analyzed with ImagePro Plus 7.0. Fluorescence pictures were captured using the Black and White settings and artificially colored for analysis, except for Figure 5 in which pictures were taken using the color settings; H&E and immunochemistry pictures were captured using the Color settings. Photoshop was used to process the pictures. Supporting Information Figure S1 Caerulein treatment leads to acute pancrea- titis. H&E staining of pancreata untreated (A) and treated with caerulein (B, C). (B) 48 hours following caerulein treatment (48 h post C), the pancreas displays extensive ADM, exocrine atrophy and inflammatory infiltration. (C) At 1 week post C, the pancreas has regained its normal morphology but high levels of proliferation are still observed as shown by Ki67 expression (D). (TIF) Figure S2 Morphometric analyses of P/K mice, 6 months post caerulein-induced AP. Dramatic increases in the number of animals displaying extensive ADM, high grade lesions and PDAC/microinvasion areas are observed when compared with untreated animals. (TIF) Figure S3 PanIN characterization in NK mice. (A) Ki67 staining shows a clear increase in proliferation in low and high- grade lesions. High-grade PanINs express CK19 (B), Muc5a (C). Embryonic progenitors markers are reactivated (D, E). (F) Masson’s trichrome blue staining shows the presence of collagen around PanIN lesions. (G) Extensive ADM is observed as shown by coexpression of CK19 (green, H) and amylase (red, I) in the same cells (white arrowheads). Scale bar: 20 mm. (TIF) Author Contributions Conceived and designed the experiments: CC MK. Performed the experiments: CC ALY JRG. Analyzed the data: CC DSL MK. Wrote the paper: CC MK. 6. Carriere C, Seeley ES, Goetze T, Longnecker DS, Korc M (2007) The Nestin progenitor lineage is the compartment of origin for pancreatic intraepithelial neoplasia. Proc Natl Acad Sci U S A 104: 4437–4442. 7. Guerra C, Schuhmacher AJ, Canamero M, Grippo PJ, Verdaguer L, et al. (2007) Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice. Cancer Cell 11: 291–302. 8. Habbe N, Shi G, Meguid RA, Fendrich V, Esni F, et al. (2008) Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice. Proc Natl Acad Sci U S A 105: 18913–18918. 9. De La OJ, Emerson LL, Goodman JL, Froebe SC, Illum BE, et al. (2008) Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia. Proc Natl Acad Sci U S A 105: 18907–18912. PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27725