br Immunofluorescence analysis of primary mouse
Immunofluorescence analysis of primary mouse PACs revealed that these 85186-71-6 expressed amylase but not CK19 (Fig. 4E and F). To assess the eﬀects of TGFα secreted by PCCs on the plasticity of PACs with or without KrasG12D mutation, we performed an in vitro ADM assay. KrasG12D PAC clusters exhibited greater changes in ductal morpholo-gical than KrasWT clusters. Although changes in ductal morphology
were observed in KrasWT PAC clusters following treatment with TGFα, the eﬀects of this ligand were more pronounced for the KrasG12D clus-
ters (Fig. 4G). Although, KrasWT PAC clusters that were not treated with TGFα did not form a ductal structure, either TGFα treatment or KrasG12D mutation resulted in the formation of large ductal structures that were similar to ADM (Fig. 4H).
3.4. ADM-dependent changes in acinar morphology contribute to pancreatic cancer cell invasion and desmoplasia
To investigate the eﬀect of ADM on the local invasion of PCCs in vivo, we orthotopically transplanted KPC-derived PCCs into KC mice, a
model in which ADM is commonly observed [9,10]. ADM-like ducts were frequently seen in the invasive front of tumors in KrasG12D/+mice,
as determined by histopathologic analysis. However, the histological appearance of the tumor core did not diﬀer between KrasWT and KrasG12D/+ mice (Fig. 5A, S2A). Immunohistochemical analysis re-vealed that ADM-like ducts in KrasG12D/+ mice exhibited acinar-to- ductal changes, with increased expression of CK19 and decreased ex-
pression of amylase (Fig. 5A). Tumor volume was significantly en-hanced in the KrasG12D/+ mice (Fig. 5B and C), which commonly ex-
hibited ADM within the invasive front. Liver metastasis and peritoneal dissemination were not detected in either KrasWT or KrasG12D/+ mice (Table 2). We then examined the extent of desmoplasia in the invasive front of tumors. The desmoplastic area, identified by positive staining
for αSMA, Sirius red and Masson trichrome, was significantly increased around the ADM-like ducts in KrasG12D/+ mice (Fig. 5D and E). Similar observations were made for the other KPC-derived PCCs (Figs. S2B, S2C, S2D). These findings suggest that the morphological changes in acini resulting from ADM-like lesions contribute to desmoplasia and the local invasion of pancreatic cancer in vivo.
Fig. 3. KPC mice demonstrate extensive cancer cell invasion into the local pancreatic parenchyma. (A) Representative photos and a photomicrograph of KPC mouse tumors. (B) Immunohistochemistry analysis of regions of cancer-associated acinar atrophy reveals CK19-positive duct-like structures with αSMA-positive desmo-plasia. Scale bars, 100 μm. (C) Clinical findings for KPC mice summarizing the presence or absence of metastasis, peritoneal dissemination, and ascites. (D) Primary tumor volume was not associated with metastasis and/or dissemination. (E) Overall survival was not associated with metastasis and/or dissemination.
Fig. 4. Autocrine and paracrine eﬀects of TGFα on the tumor microenvironment. (A) TGFα mRNA expression in PCCs and CAFs as determined by qRT-PCR. (B) EGFR mRNA expression in PCCs and CAFs as determined by qRT-PCR. (C) Representative microphotographs of H&E staining of migrating PCCs and CAFs treated with or without TGFα (5 or 10 ng/ml). (D) Representative microphotographs of H&E staining of invading PCCs and CAFs treated with or without TGFα (5 or 10 ng/ml). (E) Representative bright-field images of PACs cultured on type I collagen-coated dishes. Images were acquired after 0, 4, and 7 days in culture. (F) Immunofluorescence analysis confirmed that PACs expressed amylase but were negative for the PCC-marker CK19. (G) Primary acinar cells were isolated from KrasWT or KrasG12D mouse pancreas and cultured as 3D explants in collagen in the presence or absence of TGFα to determine the eﬀects on duct formation. Acinar-to-ductal changes were associated with TGFα treatment and Kras mutation. (H) Quantification of ductal area. Data shown represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 μm.
(caption on next page)
Fig. 5. Pancreatic acinar conversion to ADM-like lesions within the invasive front induces the local invasion of pancreatic cancer cells and desmoplasia in murine orthotopic transplantation models. (A) Immunohistochemical analysis of ADM-like lesions within the invasive front of KrasG12D mice showing increased CK19 expression and decreased amylase expression associated with acinar-to-ductal change. Red-framed images highlight examples of ADM-like lesions. (B) Two weeks after orthotopic transplantation of KPC-derived PCCs, tumor volume in KrasG12D mice was significantly larger than that in KrasWT mice. (C) Representative pho-tographs of the abdomen 2 weeks after implantation; yellow circles show pancreatic tumors. (D) Representative microphotographs of orthotopic tumor sections showing CK19, αSMA, Sirius red and Masson trichrome staining. (E) CK19-positive ADM-like lesions are surrounded by areas of desmoplasia, as determined by αSMA, Sirius red and Masson trichrome staining. Data shown represent the mean ± SD. **P < 0.01, ***P < 0.001. Scale bars for images in the left-hand column of A = 500 μm. All other scale bars = 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)