DAPI br Several mechanisms linking ADM
Several mechanisms linking ADM to carcinogenesis have been de-scribed [18,35], but the significance of ADM within the invasive front of the tumor has remained unclear. It has been reported that factors secreted by cancer cells, such as TGFα and TGFβ, are involved in ADM formation [17,36]. Our immunohistochemical analysis revealed that ADM lesions within the invasive front of the tumor were positive for TGFα expression. In addition, when PACs were stimulated with TGFα in an in vitro 3D explant culture model, they formed ADM-like structures. ADM can also be induced following pancreatic inflammation  and in response to macrophage-secreted cytokines . Our present find-ings suggest that ADM can be classified according to three distinct phenotypical profiles that are dependent upon the properties of the local pancreatic microenvironment.
The Klf4 gene, which is known to be essential for the induction of ADM [35,38], was up-regulated in all three subtypes of ADM. In CA-ADM, growth factor, cytokine, and secretory factor-related genes, such as Bmp, Fgf, Il, Tnf and Mmp, were up-regulated. Bone morphogenetic protein belongs to the TGFβ superfamily and has been implicated in the invasion and metastasis of cancer DAPI during epithelial–mesenchymal transition . Fibroblast growth factor and Interleukin are
Table 2 Eﬀects of Kras mutation on ADM-associated changes in acini in the orthotopic pancreatic cancer models employed in this study.
Group Pancreatic tumor Tumor volume (mm3) Liver metastasis Peritoneal dissemination Body weight (g)
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Fig. 6. ADM is associated with distinct phenotypical profiles that are determined by the specific tissue microenvironment. (A) Representative images of LCM. CA-ADM was derived from the invasive front of pancreatic cancer. CP-ADM was derived from chronic pancreatitis surrounded by inflammatory cells. SP-ADM was derived from regions of normal pancreas. Tissues from ADM lesions were captured from two cases of each ADM subtype. (B) Heatmap summarizing the results of the ADM gene expression analysis. Data represent log2 fold changes in mean gene expression for each ADM subtype vs. normal acini. (C) Venn diagrams for diﬀerentially expressed genes (up or down-regulated) associated with each subtype of ADM. (D) Representative annotation chart of changes in gene expression (up or down-regulated) for the three subtypes of ADM. (E) Proposed model for ADM induction associated with diﬀerent microenvironments.
extracellular signaling factors that regulate fibroblasts and in-flammatory cells [40,41]. Tumor necrosis factor (TNF) activates NF-ĸB in acinar cells to induce the expression of numerous genes related to extracellular matrix degradation, ADM, and cancer cell invasion, such as matrix metalloproteinases (MMPs) [16, 34]. These data suggest that CA-ADM exerts eﬀects on the surrounding microenvironment, im-pacting on various cell types including cancer cells, CAFs and in-flammatory cells. In CP-ADM, inflammatory response-related genes, such as Mecom, Il15 and Nfe2l2, were up-regulated, indicating that this subtype of ADM is induced by inflammation associated with pancrea-titis. Interestingly, Nfe2l2, which is an anti-inflammatory gene that is associated with resentence responses to various forms of environmental stress [42,43], was up-regulated in addition to the inflammatory re-sponse genes. This suggests that CP-ADM is a defense mechanism in-duced to protect acinar cells from damage resulting from adverse conditions such as pancreatitis. However, CAF and inflammatory cell infiltration were not apparent features of SP-ADM, which is associated with a relatively normal microenvironment, suggesting that SP-ADM has less of an impact on the surrounding tissue. Further studies are now needed to understand the biological significance of, and molecular mechanisms associated with, the changes in gene expression observed for each specific subtype of ADM.
In conclusion, we have found that ADM lesions are associated with the invasive front of pancreatic cancer. The mechanism of ADM in-duction is dependent upon the characteristics of the local micro-environment. Such environmental factors can include interactions with cancer cells and infiltration by CAFs and/or inflammatory cells. CA-ADM, which exists within a tumor microenvironment, promotes des-moplastic changes and the invasion of cancer cells into the local pan-creatic parenchyma. Our present data suggest that inhibition of ADM formation within the invasive front of the tumor may be a new ther-apeutic strategy to regulate the local invasion of pancreatic cancer.