Pancreatic duct glands are distinct ductal compartments that react to chronic injury and mediate Shh-induced metaplasia

Abstract

Background & aims: Pancreatic intraepithelial neoplasia (PanIN) are pancreatic cancer precursor lesions of unclear origin and significance. PanIN aberrantly express sonic hedgehog (Shh), an initiator of pancreatic cancer, and gastrointestinal mucins. A majority of PanIN are thought to arise from ducts. We identified a novel ductal compartment that is gathered in gland-like outpouches (pancreatic duct glands [PDG]) of major ducts and characterized its role in injury and metaplasia.

Methods: The ductal system was analyzed in normal pancreata and chronic pancreatitis in humans and mice. Anatomy was assessed by serial hematoxylin and eosin sections and scanning electron microscopy of corrosion casts. Expression of mucins and developmental genes and proliferation were assessed by immunohistochemistry or real-time quantitative polymerase chain reaction. Effects of Shh on ductal cells were investigated by exposure to Shh in vitro and transgenic misexpression in vivo.

Results: Three-dimensional analysis revealed blind-ending outpouches of ducts in murine and human pancreata. These PDG are morphologically and molecularly distinct from normal ducts; even in normal pancreata they display PanIN and metaplastic features, such as expression of Shh and gastric mucins. They express other developmental genes, such as Pdx-1 and Hes-1. In injury, Shh is up-regulated along with gastric mucins. Expansion of the PDG compartment results in a mucinous metaplasia. Shh promotes this transformation in vitro and in vivo.

Conclusions: PDG are distinct gland-like mucinous compartments with a distinct molecular signature. In response to injury, PDG undergo an Shh-mediated mucinous gastrointestinal metaplasia with PanIN-like features. PDG may provide a link between Shh, mucinous metaplasia, and neoplasia.

Figure 1

Morphologically distinct ductal compartment. Normal mouse (A) and human (B) pancreas. Cells with abundant supranuclear cytoplasm and basally located nuclei (arrowheads) are located in outpouches/small branches (arrowheads) within the ductal wall mesenchyme. This compartment is morphologically distinct from the epithelial lining of the adjacent main lumen (asterisk) and peripheral ducts of comparable size (lower panels). (C) Serial sections reveal that the novel compartment has unique molecular features, such as expression of Muc6 (brown).

Figure 2

Corrosion casts reveal that ducts have blind-ending outpouches: pancreatic duct glands. (A, B) Overviews show complete corrosion casts of the ductal system of (A) mouse and (B) human. (C, D) Light microscopy demonstrates hierarchical ramification of the ductal system rather than multiple small branches of larger ducts. (E) SEM reveals that pancreatic ducts have small gland-like outpouches, often located near branching points (arrowheads). They are predominantly found in large ducts (upper panels) but can be observed in small ducts (mid panels). These glands can appear as single outpouches or exhibit a complex arrangement of several sac-like dilations (lower panels).

Figure 3

PDG undergo hyperplasia in response to chronic injury. Overviews of the proximal ductal systems of a control animal (A) and an animal with chronic injury and metaplasia (B). (A) The normal pancreas has few, small PDG (arrows) with a regular, single-layer lining of mucinous appearance (insets) in a thin ductal mesenchyme. (B) In chronic injury PDG significantly increase in both number and size (arrows). PDG exhibit hyperplasia, a complex morphology (asterisk) and are lined by a less regular epithelium with several layers and papillations (insets). Changes are more pronounced in proximal PDG. The ductal mesenchyme is thickened.

Figure 3

PDG undergo hyperplasia in response to chronic injury. Overviews of the proximal ductal systems of a control animal (A) and an animal with chronic injury and metaplasia (B). (A) The normal pancreas has few, small PDG (arrows) with a regular, single-layer lining of mucinous appearance (insets) in a thin ductal mesenchyme. (B) In chronic injury PDG significantly increase in both number and size (arrows). PDG exhibit hyperplasia, a complex morphology (asterisk) and are lined by a less regular epithelium with several layers and papillations (insets). Changes are more pronounced in proximal PDG. The ductal mesenchyme is thickened.

Figure 4

Mouse pancreas: PDG retain GI mucins and are a source of GI mucinous metaplasia. (A) Normal peripheral pancreatic ducts have a cuboidal to low columnar epithelium with low expression of PAS⁺ mucins (magenta) and Muc-1, but are Alcian blue ⁻. (B) PDG contain a distinct epithelium with PAS⁺ and Alcian blue⁺ (turquoise) mucins. PDG, but not the lining of the main lumen (below), express mucins normally found in the deep gastric mucosa (low panels) (GI tract in E). (C, D) In chronic injury, PDG hypertrophy, along with increased mucin expression and morphologic atypia. Gastric mucins are upregulated (lower two rows). Muc5ac is frequently, Muc2 rarely expressed. This results in a metaplastic phenotype resembling the pyloric region (E), rather than normal pancreatic ducts (A).

Figure 5

PDG are a preferential site of expression of developmental factors and of proliferation. (A) In the normal state, Shh is expressed at a low level specifically in PDG (arrowheads), but not in the luminar epithelium. The receptor Ptch2 is co-expressed with Shh in PDG and additionally in some cells of the ductal surface epithelium (arrowheads); Ptch1 is expressed in the mesenchyme around PDG. Pdx-1 is expressed predominantly in PDG (arrowheads) but can be identified in the ductal epithelium. Hes-1 is expressed predominantly in PDG within large ducts (arrowheads) but can be identified throughout the ductal epithelium. (B) In response to chronic injury, all these markers appear upregulated. Shh remains specific to mucinous cells within PDG. Ptch2 is expressed in PDG but expanded in surface epithelium (arrowheads); Ptch1 is generalized in the ductal mesenchyme. Expression of Pdx-1 and Hes-1 is expanded or generalized in the ductal epithelium. (C) In severe metaplasia and atypia these changes are more pronounced. Shh remains specific for the mucinous epithelium. Pdx-1 and Hes-1 are expanded to the surface epithelium, but remain strongly expressed in the deeper epithelium. (D) In response to injury, proliferation (BrdU incorporation) in large ducts is predominantly located to PDG (arrows). In chronic injury and metaplasia, expression of the proliferation marker Ki-67 is identified predominantly in PDG and the apex of mucosal folds. MD: Main duct epithelial lining.

Figure 5

PDG are a preferential site of expression of developmental factors and of proliferation. (A) In the normal state, Shh is expressed at a low level specifically in PDG (arrowheads), but not in the luminar epithelium. The receptor Ptch2 is co-expressed with Shh in PDG and additionally in some cells of the ductal surface epithelium (arrowheads); Ptch1 is expressed in the mesenchyme around PDG. Pdx-1 is expressed predominantly in PDG (arrowheads) but can be identified in the ductal epithelium. Hes-1 is expressed predominantly in PDG within large ducts (arrowheads) but can be identified throughout the ductal epithelium. (B) In response to chronic injury, all these markers appear upregulated. Shh remains specific to mucinous cells within PDG. Ptch2 is expressed in PDG but expanded in surface epithelium (arrowheads); Ptch1 is generalized in the ductal mesenchyme. Expression of Pdx-1 and Hes-1 is expanded or generalized in the ductal epithelium. (C) In severe metaplasia and atypia these changes are more pronounced. Shh remains specific for the mucinous epithelium. Pdx-1 and Hes-1 are expanded to the surface epithelium, but remain strongly expressed in the deeper epithelium. (D) In response to injury, proliferation (BrdU incorporation) in large ducts is predominantly located to PDG (arrows). In chronic injury and metaplasia, expression of the proliferation marker Ki-67 is identified predominantly in PDG and the apex of mucosal folds. MD: Main duct epithelial lining.

Figure 6

The PDG compartment exists in peripheral ducts. In chronic injury Shh, Alcian blue and Muc6 identify the PDG-compartment in the mesenchyme of interlobular (A) and small intralobular (B) ducts.

Figure 7

Shh misdirects the pancreatic ductal epithelium toward a GI mucinous metaplasia. (A) Shh exposure results in enhanced mucin expression (PAS: magenta, arrowheads) in HPDE cells. Real-time quantitative RT-PCR reveals that HPDE cells are Hh target cells expressing the receptor PTCH2. In response to Shh exposure the pathway is activated (PTCH2 upregulation) along with upregulation of gastric mucins (MUC5ac, MUC6). (B) Transgenic misexpression of Shh in Pdx-Shh mice results in a GI phenotype of pancreatic ducts with expression of PAS⁺ (magenta) and Alcian blue⁺ mucins, including Muc5ac. Scale bars indicate 100μm.

Figure 7

Shh misdirects the pancreatic ductal epithelium toward a GI mucinous metaplasia. (A) Shh exposure results in enhanced mucin expression (PAS: magenta, arrowheads) in HPDE cells. Real-time quantitative RT-PCR reveals that HPDE cells are Hh target cells expressing the receptor PTCH2. In response to Shh exposure the pathway is activated (PTCH2 upregulation) along with upregulation of gastric mucins (MUC5ac, MUC6). (B) Transgenic misexpression of Shh in Pdx-Shh mice results in a GI phenotype of pancreatic ducts with expression of PAS⁺ (magenta) and Alcian blue⁺ mucins, including Muc5ac. Scale bars indicate 100μm.

Figure 8

SHH is expressed in human PDG and upregulated in metaplasia. (A) PDG of the main duct. Control: SHH is expressed at a low level in PDG (arrowheads). PAS staining (magenta) identifies co-expression of mucins. CP: H&E reveals hypertrophic outpouches/branches exhibiting papillary formations (arrowheads) and pseudo-stratified nuclei (arrow), features of PanIN. SHH is upregulated, identified by brown supranuclear granular staining. PAS reveals enhanced mucin expression. (B) Control: Peripheral ducts have cuboidal epithelium without Shh expression and with minimal expression of mucins. CP: Atypical peripheral ducts with PanIN features (arrow, arrowheads) express SHH (arrowheads) along with increased mucin expression. Scale bars indicate 100μm. (C) Expression of HH pathway genes in CP shown as -fold expression over normal pancreata. Shh and the effectors Gli-1 and Gli-2 are significantly upregulated. Receptor genes PTCH1 and SMO are upregulated to a lesser extent. Comparison of expression levels in main duct tissue (black bars) vs. peripheral parenchyma (white bars) reveals that HH pathway activation is mainly localized to the proximal ductal system. Error bars indicate S.E.M. *, P < 0.05.

Figure 8

SHH is expressed in human PDG and upregulated in metaplasia. (A) PDG of the main duct. Control: SHH is expressed at a low level in PDG (arrowheads). PAS staining (magenta) identifies co-expression of mucins. CP: H&E reveals hypertrophic outpouches/branches exhibiting papillary formations (arrowheads) and pseudo-stratified nuclei (arrow), features of PanIN. SHH is upregulated, identified by brown supranuclear granular staining. PAS reveals enhanced mucin expression. (B) Control: Peripheral ducts have cuboidal epithelium without Shh expression and with minimal expression of mucins. CP: Atypical peripheral ducts with PanIN features (arrow, arrowheads) express SHH (arrowheads) along with increased mucin expression. Scale bars indicate 100μm. (C) Expression of HH pathway genes in CP shown as -fold expression over normal pancreata. Shh and the effectors Gli-1 and Gli-2 are significantly upregulated. Receptor genes PTCH1 and SMO are upregulated to a lesser extent. Comparison of expression levels in main duct tissue (black bars) vs. peripheral parenchyma (white bars) reveals that HH pathway activation is mainly localized to the proximal ductal system. Error bars indicate S.E.M. *, P < 0.05.

Figure 8

SHH is expressed in human PDG and upregulated in metaplasia. (A) PDG of the main duct. Control: SHH is expressed at a low level in PDG (arrowheads). PAS staining (magenta) identifies co-expression of mucins. CP: H&E reveals hypertrophic outpouches/branches exhibiting papillary formations (arrowheads) and pseudo-stratified nuclei (arrow), features of PanIN. SHH is upregulated, identified by brown supranuclear granular staining. PAS reveals enhanced mucin expression. (B) Control: Peripheral ducts have cuboidal epithelium without Shh expression and with minimal expression of mucins. CP: Atypical peripheral ducts with PanIN features (arrow, arrowheads) express SHH (arrowheads) along with increased mucin expression. Scale bars indicate 100μm. (C) Expression of HH pathway genes in CP shown as -fold expression over normal pancreata. Shh and the effectors Gli-1 and Gli-2 are significantly upregulated. Receptor genes PTCH1 and SMO are upregulated to a lesser extent. Comparison of expression levels in main duct tissue (black bars) vs. peripheral parenchyma (white bars) reveals that HH pathway activation is mainly localized to the proximal ductal system. Error bars indicate S.E.M. *, P < 0.05.