Acute Pancreatitis and Pancreatic Cancer Figure 2. Caerulein treatment leads to major PanIN lesion progression 2 months following caerulein treatment. (A–F) H&E staining of 4 months old N/K pancreata: (A–B) untreated pancreas displaying rare low-grade lesions (*), (B) inset from (A) shows a single PanIN-1A lesion; (C–F) caerulein-treated pancreata show extensive dysplasia with a mix of low- (*) and high-grade (arrowhead) lesions (D, inset of C) and (E) as well as ADM foci (marked by dotted line)(F). (G) Morphometric analyses show dramatic increases in the number of animals displaying high grade PanIN lesions and areas of microinvasion in caerulein-treated pancreata. (H) Significant increases in proliferation are observed in caerulein treated pancreata (* P,0.05, ** P,0.01). Scale bar: 50 mm. doi:10.1371/journal.pone.0027725.g002 from treated and untreated N/K mice. While most sections from untreated N/K pancreata exhibited low-grade lesions only and less than 5% of the section presented ADM, most caerulein-treated pancreata showed significant increases in PanIN-2 and -3 (P,0.0002) as well as extensive ADM covering 15 to 95% of the pancreas (Fig. 2G). These findings are similar to those we observed in a parallel cohort of P/K subjected at 2 month-old to caerulein- induced AP (Fig.S2); P/K mice were sacrificed 6 months post treatment (at age 8 months) which explains the higher rates of high-grade lesions and PDAC/microinvasion both in untreated and treated mice. As observed in N/K mice, all the caerulein- treated P/K mice show extensive ADM covering 25–95% of the sections (P,0.001) and dramatic progression to high-grade lesions and PDAC (Fig.S2). In contrast, in all control animals (wild type or carrying a single mutation, Nestin-Cre, Pdx1-Cre or LSL- KrasG12D only) treated with caerulein, the pancreata appeared to be normal with occasionally residual traces of inflammation (not shown). Low-grade lesions in N/K pancreata showed a proliferation index of 13%63% similar to what is observed in P/K mice [4]. The high-grade lesions observed after caerulein treatment displayed as expected a high proliferation index (55%63%), but interestingly low-grade lesions also showed a higher proliferation index of 31%64% (Fig. 2H, S3A). Thus, the general increase in proliferation observed in the pancreas immediately following AP episodes [21] was maintained 2 months post injury but only in the neoplastic lesions in the presence of an activated Kras allele. As observed in human PDAC samples, PanINs expressed high level of CK19, underscoring their epithelial nature, displayed mucus accumulation as shown by Muc5a expression (Fig.S3B–C), and were surrounded by collagen-rich stroma (Fig. S3F). The lesions showed a reactivation of the early pancreatic progenitor marker Pdx1 as well as the Notch signaling marker Hes1, both known to be reactivated in early stages of PDAC (Fig.S3D–E). ADM was demonstrated by the co-expression of acinar and ductal markers in the same cells (Fig.S3G–I) as observed by others [24]. These data confirm that caerulein-induced AP does not affect the nature of the lesions at the molecular level but accelerates their progression to high grade. It was shown recently that the inflammatory mediator Stat3 and the interleukin IL6 were essential contributors to PanIN progression and PDAC development at least in the early stages of the disease and were essential components for the acute response to caerulein-induced AP [16,25]. Immunohistochemical analysis in N/K pancreata showed that phospho-Stat3 was still highly expressed 2 months post AP (Fig. 3) while IL6 was not detected (not shown). Phospho-Stat3 was shown previously to be highly activated in PanINs immediately following AP episodes [16,25]. However, two months later phospho-Stat3 did not appear to be expressed in the lesions themselves but was abundantly detected in the inflammatory cells, in ADM foci and in groups of apparently normal acinar cells (Fig. 3A–D). Similarly, in untreated N/K mice, phospho-Stat3 was rarely detected and only in cells surrounding the PanINs (not shown). Oncogenic Kras activation in the Nestin cell lineage leads to PDAC development In half the mice subjected to AP, areas of microinvasion can be observed in the pancreata as shown by the loss of the basement membrane in some PanIN-3 (Fig. 4A–B) and CK19 expression in cells within the adjacent stroma (Fig. 4C). From a small group of 4 N/K animals treated with caerulein at later stages (4 month-old), 3 survived until age 6 months and of these, 2 displayed tumors (diameter ,5 mm) visible at the gross anatomical level. Only one tumor was available for histological analysis, displaying the morphological characteristic of PDAC, and revealing local invasion of the fat and fibrous tissues (Fig. 4D). Despite the fact that the nuclei were enlarged and crowded, a glandular PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27725