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  • br ADM is classified into three phenotypical subtypes that a

    2020-08-28


    3.5. ADM is classified into three phenotypical subtypes that are determined by the tissue microenvironment
    To further characterize the ADM phenotype associated with dif-ferent pathological conditions, we performed gene Ezatiostat analyses of tissues associated with three distinct forms of ADM by RNA se-quencing (RNA-seq). Laser-capture microdissection (LCM) was used to capture tissues containing regions of cancer-associated ADM (CA-ADM) from the invasive front of pancreatic cancers, from pancreas with chronic pancreatitis-associated ADM (CP-ADM), and from pancreas with sporadic-ADM (SP-ADM) (Fig. 6A). Total RNA was isolated from the captured tissues, and the expression of 770 genes from 13 cancer-associated canonical pathways were subsequently analyzed using the NanoString nCounter gene expression system [27]. The heatmap ana-lysis revealed that CA-ADM, CP-ADM, and SP-ADM exhibit distinct phenotypical gene expression profiles (Fig. 6B). Ezatiostat Venn diagram analysis identified both differentially and co-expressed genes (Fig. 6C). In our analysis of the RNA-seq data, we found functional annotation clusters of genes uniquely up or down-regulated in each of the three types of ADM, when compared with normal acini (Fig. 6D). For each ADM subtype, representative genes with a fold change > 2 or < 0.5 were identified (Fig. S3B). In particular, growth factor, cytokine, and secretory factor-related genes were up-regulated in CA-ADM. Inflammatory response related genes were up-regulated in CP-ADM. Differentiation-related genes were up-regulated in all three subtypes of ADM. Apoptosis-re-lated genes were up-regulated in both CA-ADM and CP-ADM. However, SP-ADM did not exhibit significant changes that were unique from the others forms of ADM (Fig. 6D). Additionally, GSEA analysis revealed that, when compared with normal acinar tissues, CA-ADM exhibited an up-regulation of pancreatic cancer-related genes, CP-ADM exhibited an up-regulation of inflammatory response genes, and SP-ADM revealed an up-regulation of genes associated with carcinogenesis (Fig. S3A). These results suggest the possibility that the mechanisms driving ADM differ according to the given tissue microenvironment.
    4. Discussion
    The pancreas is an organ in which inflammation, fibrosis and atrophy are easily induced because most of the pancreatic parenchyma is composed of acini that produce digestive enzymes [31]. However, the significance of the changes in acinar morphology within the sur-rounding microenvironment of the tumor during the development and progression of pancreatic cancer are unknown. In this study, we have found that the border between the tumor and the acini is largely di-vided into two histopathologically distinct regions, the invasive front and the non-invasive front. In the invasive front, cancer cells and/or CAFs demonstrate extensive invasion of the pancreatic parenchyma that is accompanied by desmoplastic changes and acinar atrophy. In the 
    non-invasive front, cancer cells and areas of desmoplasia are en-capsulated by fibrosis. We also found extensive ADM-like lesions in areas of CAA observed within the invasive front of pancreatic tumors in both humans and KPC mice. In the orthotopic transplantation model, we also observed extensive ADM-like lesions and desmoplastic changes within the invasive front of tumors in the pancreas of mice. Further-
    more, tumor volumes were also significantly larger, possibly because of the extensive ADM found in KrasG12D/+ mice. These results suggest that
    ADM-like lesions within CAA regions of the invasive front enhance tumor invasion into the local pancreatic parenchyma.
    Pancreatic acinar cells show plasticity termed ADM [12–14]. In previous reports, ADM has been described in humans and mice, espe-cially regarding carcinogenesis [17,18], [32,33]. In our study, ADM-like lesions were observed in the invasive front, especially where acinar atrophy and invasion into the pancreatic parenchyma were extensive. In addition, a strong desmoplastic reaction was observed around these ADM-like lesions. When we induced temporary acute pancreatitis by administration of caerulein in KPC mice that had already formed palpable invasive cancer, tumor progression was accelerated (data not shown). This is possibly a consequence of the Kras mutation, which promotes ADM within the pancreas around the established tumor. Changes in the physical structural of the surrounding tumor micro-environment, including collagen matrix remodeling within regions of desmoplasia, are known to contribute to the process of cancer invasion [20,34]. These data suggest that ADM-like lesions with acinar atrophy and associated areas of desmoplasia within the invasive front of the tumor contribute to the formation of a microenvironment that favors local invasion in pancreatic cancer.