br Fig TGF is expressed in ADM like
Fig. 2. TGFα is expressed in ADM-like lesions associated with CAA and is associated with poor prognosis. (A) Representative micrographs of TGFα staining in normal acini and regions of CAA. (B) ADM-like lesions positive for TGFα expression are found in regions of CAA (n = 46) but not in regions of normal acini (n = 49). (C) Representative micrographs of H&E, amylase, CK19 and TGFα staining in normal acini, pancreatic ductal adenocarcinoma (PDAC) and ADM-like lesion. (D) As with ADM-like lesions (n = 46), pancreatic cancer Caspofungin (n = 106) were positive for TGFα expression. (E) Representative immunohistochemistry images of pancreatic cancer tissue sections demonstrating low and high-intensity TGFα staining. (F) Kaplan–Meier analysis of overall survival according to TGFα expression in patients with pancreatic cancer (n = 106). TGFα expression was associated with shorter patient survival times. Data shown represent the mean ± SD. ***P < 0.001. Scale bars, 100 μm.
associated fibroblasts (CAFs) invade the pancreatic parenchyma. Acinar atrophy is frequently observed in this region while fibrotic encapsula-tion is uncommon. We define such acinar atrophy within the invasive front of pancreatic cancer as cancer-associated acinar atrophy (CAA). Within the non-invasive front, the tumor is encapsulated by fibrotic tissue and there are no cancer cells and CAFs invading the pancreatic parenchyma (Fig. 1A). Close examination of the CAA region within the invasive front revealed duct-like structures, which are similar to ADM (Fig. 1B). It is diﬃcult to distinguish between small intra-acinar term-inal ducts and ADM by morphology alone. Therefore, to confirm the origin of the duct-like structures within the invasive front, we per-formed immunohistochemistry for amylase and CK19 using serial sec-tions of pancreatic tissues. The duct-like lesions stained positive for both amylase and CK19 (Fig. 1B), suggest that duct-like changes in acini such as ADM occur in the invasive front of pancreatic cancer. We define such duct-like changes in acini within the invasive front as cancer-as-sociated ADM-like lesions. We next assessed the number of ADM-like lesions positive for TGFα expression (Fig. 2A). TGFα is a member of the epidermal growth factor (EGF) family of protein ligands, and transgenic mice overexpressing TGFα in the pancreas display multiple duct-like structures derived from diﬀerentiated acinar cells [29,30]. As expected, our analysis revealed that ADM-like lesions were positive for TGFα. Moreover, these lesions were frequently seen in regions of CAA, when compared with regions containing normal acini (Fig. 2B). Interestingly, as with the CAA region, the cancerous region was also positive for TGFα (Fig. 2C and D). We also evaluated TGFα expression in the cancer cells of 106 resected tumor samples. We divided pancreatic cancer patients into two groups according to the intensity of TGFα staining in cancer cells (Fig. 2E) and found that TGFα expression was associated with shorter postoperative survival times (Fig. 2F). Analysis of mRNA ex-pression data from The Cancer Genome Atlas (TCGA) pancreatic cancer database also revealed that high TGFA gene expression was associated with shorter overall survival times (Fig. S1). TGFα expression was significantly associated with pathologic T category (Table 1), sug-gesting that TGFα is associated with the local progression of pancreatic cancer.
3.2. In KPC mice, tumor cell invasion into the local pancreatic parenchyma is extensive
To assess the mechanism driving cancer cell invasion into the local pancreatic parenchyma, we used KPC mice that recapitulate the clinical
Table 1 Relationship between TGFα expression and clinicopathologic factors.
TGFα low staining TGFα high staining P value
and pathological features of human pancreatic cancer (Fig. 3C). In KPC mice, as with human pancreatic cancer, ADM-like ducts with acinar atrophy were seen in the invasive front of the tumor (Fig. 3B). Inter-estingly, the tumors of KPC mice demonstrated extensive local invasion, almost replacing the entirety of the pancreatic parenchyma, even before the formation of metastatic lesions (Fig. 3A). Indeed, there was no significant diﬀerence in primary tumor volume between KPC mice with or without metastasis and/or dissemination (Fig. 3D). There was also no significant diﬀerence in the overall survival rate between these two groups of mice (Fig. 3E).
3.3. Autocrine and paracrine eﬀects of TGFα on pancreatic tumor, stellate and acinar cells
To investigate the autocrine and paracrine eﬀects of TGFα in the tumor microenvironment, we examined the relative expression of this ligand and its cognate receptor, epidermal growth factor receptor (EGFR), in PCCs, PSCs and PACs, and we also determined the eﬀects of TGFα treatment on the migratory and invasive behavior of these cells. TGFα and EGFR mRNA expression levels were measured in eleven PCC lines and three independent PSC primary cultures using qRT-PCR. All PCC lines exhibited higher levels of TGFα expression than the three PSC lines tested (Fig. 4A). Although relative TGFα expression was lower in PSCs, EGFR expression was detected in all cell lines tested (Fig. 4B). Both PCCs and PSCs exhibited a dose-dependent increase in migration and invasion in response to TGFα treatment (Fig. 4C and D).