Periostin Creates a Tumor-Supportive Microenvironment in the Pancreas by Sustaining Fibrogenic Stellate Cell Activity
2007; Elsevier BV; Volume: 132; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2007.01.031
ISSN1528-0012
AutoresMert Erkan, Jörg Kleeff, Andre Gorbachevski, Carolin Reiser, Tomas Mitkus, Iréne Esposito, Thomas Giese, Markus W. Büchler, Nathalia A. Giese, Helmut Frieß,
Tópico(s)Pancreatic and Hepatic Oncology Research
ResumoBackground & Aims: Pancreatic cancer creates desmoplasia by stimulating stellate cells (PSCs), thereby influencing tumor aggressiveness. The aim of this study was to analyze the impact of the PSC-specific matrix protein periostin on tumor responses to radiochemotherapy. Methods: PSCs and cancer cells in primary and metastatic lesions of patients treated with or without neoadjuvant radiochemotherapy were evaluated by immunohistochemistry. Periostin messenger-RNA levels determined by quantitative reverse-transcription polymerase chain reaction were correlated to patient survival. Interactions between PSCs and cancer cells and the effects of periostin in modulating cellular responses under conditions of hypoxia, starvation, and radiochemotherapy were assessed by immunoblotting and by growth, clonogenicity, and invasion assays. Results: Periostin messenger-RNA levels were elevated 42-fold in cancer, and patients with increased expression had a tendency toward shorter survival (19 vs 12 months; P = .14). Stromal cells were the only source of periostin in the pancreas and in metastatic sites. Cancer cell supernatants stimulated periostin secretion from PSCs. Recombinant periostin increased α–smooth muscle actin, periostin, collagen-1, fibronectin, and transforming growth factor-β1 expression while decreasing PSC invasiveness. These effects were reversed by silencing periostin expression and secretion by small interfering RNA transfection. In cancer cells, periostin stimulated growth and conferred resistance to starvation and hypoxia. In addition, the periostin downstream target collagen-1 significantly increased chemoresistance. Conclusions: Once stimulated by cancer cells, PSCs remain active via an autocrine periostin loop even under radiotherapy and produce excessive extracellular matrix proteins, creating a tumor-supportive microenvironment. Increased periostin expression may therefore reflect a more aggressive tumor phenotype. Background & Aims: Pancreatic cancer creates desmoplasia by stimulating stellate cells (PSCs), thereby influencing tumor aggressiveness. The aim of this study was to analyze the impact of the PSC-specific matrix protein periostin on tumor responses to radiochemotherapy. Methods: PSCs and cancer cells in primary and metastatic lesions of patients treated with or without neoadjuvant radiochemotherapy were evaluated by immunohistochemistry. Periostin messenger-RNA levels determined by quantitative reverse-transcription polymerase chain reaction were correlated to patient survival. Interactions between PSCs and cancer cells and the effects of periostin in modulating cellular responses under conditions of hypoxia, starvation, and radiochemotherapy were assessed by immunoblotting and by growth, clonogenicity, and invasion assays. Results: Periostin messenger-RNA levels were elevated 42-fold in cancer, and patients with increased expression had a tendency toward shorter survival (19 vs 12 months; P = .14). Stromal cells were the only source of periostin in the pancreas and in metastatic sites. Cancer cell supernatants stimulated periostin secretion from PSCs. Recombinant periostin increased α–smooth muscle actin, periostin, collagen-1, fibronectin, and transforming growth factor-β1 expression while decreasing PSC invasiveness. These effects were reversed by silencing periostin expression and secretion by small interfering RNA transfection. In cancer cells, periostin stimulated growth and conferred resistance to starvation and hypoxia. In addition, the periostin downstream target collagen-1 significantly increased chemoresistance. Conclusions: Once stimulated by cancer cells, PSCs remain active via an autocrine periostin loop even under radiotherapy and produce excessive extracellular matrix proteins, creating a tumor-supportive microenvironment. Increased periostin expression may therefore reflect a more aggressive tumor phenotype. Excessive desmoplasia is a typical feature of pancreatic ductal adenocarcinoma (PDAC) but is rarely observed in other tumors of the pancreas.1Mollenhauer J. Roether I. Kern H.F. Distribution of extracellular matrix proteins in pancreatic ductal adenocarcinoma and its influence on tumor cell proliferation in vitro.Pancreas. 1987; 2: 14-24Crossref PubMed Scopus (119) Google Scholar, 2Seymour A.B. Hruban R.H. Redston M. Caldas C. Powell S.M. Kinzler K.W. Yeo C.J. Kern S.E. Allelotype of pancreatic adenocarcinoma.Cancer Res. 1994; 54: 2761-2764PubMed Google Scholar, 3Cubilla A.L. Fitzgerald P.J. Morphological lesions associated with human primary invasive nonendocrine pancreas cancer.Cancer Res. 1976; 36: 2690-2698PubMed Google Scholar, 4Yen T.W. Aardal N.P. Bronner M.P. Thorning D.R. Savard C.E. Lee S.P. Bell Jr, R.H. Myofibroblasts are responsible for the desmoplastic reaction surrounding human pancreatic carcinomas.Surgery. 2002; 131: 129-134Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar Although pancreatic cancer cells (PCCs) can produce several components of the extracellular matrix (ECM), they predominantly stimulate pancreatic stellate cells (PSCs) to synthesize the excessive fibrotic tissue that infiltrates and envelops the normal parenchyma, as well as PCCs themselves.5Lohr M. Trautmann B. Gottler M. Peters S. Zauner I. Maillet B. Kloppel G. Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.Br J Cancer. 1994; 69: 144-151Crossref PubMed Scopus (90) Google Scholar, 6Bachem M.G. Schunemann M. Ramadani M. Siech M. Beger H. Buck A. Zhou S. Schmid-Kotsas A. Adler G. Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells.Gastroenterology. 2005; 128: 907-921Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar Although this fibrotic matrix was initially regarded as a host barrier against tumor invasion, it has become evident that it can modulate and even initiate tumorigenesis.7Lunevicius R. Nakanishi H. Ito S. Kozaki K. Kato T. Tatematsu M. Yasui K. Clinicopathological significance of fibrotic capsule formation around liver metastasis from colorectal cancer.J Cancer Res Clin Oncol. 2001; 127: 193-199Crossref PubMed Scopus (76) Google Scholar, 8Tlsty T.D. Stromal cells can contribute oncogenic signals.Semin Cancer Biol. 2001; 11: 97-104Crossref PubMed Scopus (241) Google Scholar, 9Bissell M.J. Radisky D. Putting tumours in context.Nat Rev Cancer. 2001; 1: 46-54Crossref PubMed Scopus (1711) Google Scholar, 10Paszek M.J. Zahir N. Johnson K.R. Lakins J.N. Rozenberg G.I. Gefen A. Reinhart-King C.A. Margulies S.S. Dembo M. Boettiger D. Hammer D.A. Weaver V.M. Tensional homeostasis and the malignant phenotype.Cancer Cell. 2005; 8: 241-254Abstract Full Text Full Text PDF PubMed Scopus (2855) Google Scholar, 11Hanahan D. Weinberg R.A. The hallmarks of cancer.Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (21926) Google Scholar In fact, the ECM influences growth, differentiation, survival, and motility of cells both by providing a physical scaffold and by acting as a reservoir for soluble mitogens,9Bissell M.J. Radisky D. Putting tumours in context.Nat Rev Cancer. 2001; 1: 46-54Crossref PubMed Scopus (1711) Google Scholar, 11Hanahan D. Weinberg R.A. The hallmarks of cancer.Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (21926) Google Scholar, 12Ingber D.E. Madri J.A. Jamieson J.D. Role of basal lamina in neoplastic disorganization of tissue architecture.Proc Natl Acad Sci U S A. 1981; 78: 3901-3905Crossref PubMed Scopus (171) Google Scholar and cancer cells are believed to exploit this tumor-supportive microenvironment.The replacement of the normal parenchyma by excessive desmoplastic tissue rich in collagen and fibronectin ostensibly plays a role in the aggressive behavior of PDAC. Normally, the collagen IV–rich basement membrane, which separates epithelial cells from the interstitial matrix, exerts inhibitory effects on both cancer cells and stellate cells.12Ingber D.E. Madri J.A. Jamieson J.D. Role of basal lamina in neoplastic disorganization of tissue architecture.Proc Natl Acad Sci U S A. 1981; 78: 3901-3905Crossref PubMed Scopus (171) Google Scholar, 13Sohara N. Znoyko I. Levy M.T. Trojanowska M. Reuben A. Reversal of activation of human myofibroblast-like cells by culture on a basement membrane-like substrate.J Hepatol. 2002; 37: 214-221Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 14Lee C.S. Montebello J. Georgiou T. Rode J. Distribution of type IV collagen in pancreatic adenocarcinoma and chronic pancreatitis.Int J Exp Pathol. 1994; 75: 79-83PubMed Google Scholar In various tumors, including PDAC, breaching of the basement membrane is a critical step in tumor progression. This step brings malignant cells into direct contact with ECM proteins such as collagen 1 (COL1), supporting their growth and contributing to their chemoresistance.1Mollenhauer J. Roether I. Kern H.F. Distribution of extracellular matrix proteins in pancreatic ductal adenocarcinoma and its influence on tumor cell proliferation in vitro.Pancreas. 1987; 2: 14-24Crossref PubMed Scopus (119) Google Scholar, 12Ingber D.E. Madri J.A. Jamieson J.D. Role of basal lamina in neoplastic disorganization of tissue architecture.Proc Natl Acad Sci U S A. 1981; 78: 3901-3905Crossref PubMed Scopus (171) Google Scholar, 14Lee C.S. Montebello J. Georgiou T. Rode J. Distribution of type IV collagen in pancreatic adenocarcinoma and chronic pancreatitis.Int J Exp Pathol. 1994; 75: 79-83PubMed Google Scholar, 15Armstrong T. Packham G. Murphy L.B. Bateman A.C. Conti J.A. Fine D.R. Johnson C.D. Benyon R.C. Iredale J.P. Type I collagen promotes the malignant phenotype of pancreatic ductal adenocarcinoma.Clin Cancer Res. 2004; 10: 7427-7437Crossref PubMed Scopus (230) Google Scholar In liver cirrhosis, COL1, once synthesized by activated hepatic stellate cells, creates a positive feedback loop that sustains their activity and recruits them to regions of inflammation and necrosis, causing a vicious cycle that ultimately leads to the progressive loss of functional parenchyma.16Olaso E. Ikeda K. Eng F.J. Xu L. Wang L.H. Lin H.C. Friedman S.L. DDR2 receptor promotes MMP-2-mediated proliferation and invasion by hepatic stellate cells.J Clin Invest. 2001; 108: 1369-1378Crossref PubMed Scopus (245) Google Scholar Furthermore, an indirect effect of fibrosis is tissue hypoxia, a phenomenon commonly observed in PDAC and several other solid tumors that creates genetic instability, selects more aggressive tumor phenotypes, and increases chemoradioresistance.17Koong A.C. Mehta V.K. Le Q.T. Fisher G.A. Terris D.J. Brown J.M. Bastidas A.J. Vierra M. Pancreatic tumors show high levels of hypoxia.Int J Radiat Oncol Biol Phys. 2000; 48: 919-922Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 18Hockel M. Schlenger K. Hockel S. Vaupel P. Hypoxic cervical cancers with low apoptotic index are highly aggressive.Cancer Res. 1999; 59: 4525-4528PubMed Google Scholar, 19Harris A.L. Hypoxia—a key regulatory factor in tumour growth.Nat Rev Cancer. 2002; 2: 38-47Crossref PubMed Scopus (4206) Google Scholar, 20Couvelard A. O’Toole D. Leek R. Turley H. Sauvanet A. Degott C. Ruszniewski P. Belghiti J. Harris A.L. Gatter K. Pezzella F. Expression of hypoxia-inducible factors is correlated with the presence of a fibrotic focus and angiogenesis in pancreatic ductal adenocarcinomas.Histopathology. 2005; 46: 668-676Crossref PubMed Scopus (104) Google ScholarPeriostin (POSTN), originally isolated as an osteoblast-specific factor, is a disulfide-linked 90-kilodalton secretory protein that functions as a cell adhesion molecule for preosteoblasts and is believed to be involved in osteoblast recruitment, attachment, and spreading.21Horiuchi K. Amizuka N. Takeshita S. Takamatsu H. Katsuura M. Ozawa H. Toyama Y. Bonewald L.F. Kudo A. Identification and characterization of a novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor beta.J Bone Miner Res. 1999; 14: 1239-1249Crossref PubMed Scopus (788) Google Scholar Most of the physiologic functions of POSTN take place at the epithelial-mesenchymal interfaces, where POSTN is strongly induced by transforming growth factor (TGF)-β1 and BMP-2 signaling.21Horiuchi K. Amizuka N. Takeshita S. Takamatsu H. Katsuura M. Ozawa H. Toyama Y. Bonewald L.F. Kudo A. Identification and characterization of a novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor beta.J Bone Miner Res. 1999; 14: 1239-1249Crossref PubMed Scopus (788) Google Scholar, 22Ji X. Chen D. Xu C. Harris S.E. Mundy G.R. Yoneda T. Patterns of gene expression associated with BMP-2-induced osteoblast and adipocyte differentiation of mesenchymal progenitor cell 3T3-F442A.J Bone Miner Metab. 2000; 18: 132-139Crossref PubMed Scopus (120) Google Scholar Pertinently, POSTN-null mice exhibit dwarfism and teeth, bone, and valvular defects.23Kim C.J. Yoshioka N. Tambe Y. Kushima R. Okada Y. Inoue H. Periostin is down-regulated in high grade human bladder cancers and suppresses in vitro cell invasiveness and in vivo metastasis of cancer cells.Int J Cancer. 2005; 117: 51-58Crossref PubMed Scopus (83) Google Scholar POSTN has recently been identified by transcriptome analysis as highly specific for PSC.24Buchholz M. Kestler H.A. Holzmann K. Ellenrieder V. Schneiderhan W. Siech M. Adler G. Bachem M.G. Gress T.M. Transcriptome analysis of human hepatic and pancreatic stellate cells: organ-specific variations of a common transcriptional phenotype.J Mol Med. 2005; 83: 795-805Crossref PubMed Scopus (92) Google ScholarIn colon and ovarian cancers, POSTN exerts prometastatic effects through vitronectin receptors αvβ3 and αvβ5 by increasing cell motility and survival both in vivo and in vitro.25–27 Forced expression of POSTN promotes tumor angiogenesis by up-regulation of vascular endothelial growth factor receptor 2 expression in human breast cancer through an αvβ3-mediated signaling pathway.25Bao S. Ouyang G. Bai X. Huang Z. Ma C. Liu M. Shao R. Anderson R.M. Rich J.N. Wang X.F. Periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway.Cancer Cell. 2004; 5: 329-339Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar In contrast to these promalignant properties, POSTN acts as a tumor suppressor in a variety of human cancer cell lines and in human lung cancer tissue by inhibiting anchorage-independent growth.28Yoshioka N. Fuji S. Shimakage M. Kodama K. Hakura A. Yutsudo M. Inoue H. Nojima H. Suppression of anchorage-independent growth of human cancer cell lines by the TRIF52/periostin/OSF-2 gene.Exp Cell Res. 2002; 279: 91-99Crossref PubMed Scopus (64) Google Scholar Moreover, POSTN is down-regulated in high-grade human bladder cancers and suppresses in vitro invasiveness along with in vivo metastasis of cancer cells.23Kim C.J. Yoshioka N. Tambe Y. Kushima R. Okada Y. Inoue H. Periostin is down-regulated in high grade human bladder cancers and suppresses in vitro cell invasiveness and in vivo metastasis of cancer cells.Int J Cancer. 2005; 117: 51-58Crossref PubMed Scopus (83) Google ScholarMultiple studies have shown that PDAC expresses several integrins, including vitronectin receptors αvβ3 and αvβ5.29Lohr M. Trautmann B. Gottler M. Peters S. Zauner I. Maier A. Kloppel G. Liebe S. Kreuser E.D. Expression and function of receptors for extracellular matrix proteins in human ductal adenocarcinomas of the pancreas.Pancreas. 1996; 12: 248-259Crossref PubMed Scopus (72) Google Scholar, 30Duxbury M.S. Ito H. Ashley S.W. Whang E.E. c-Src-dependent cross-talk between CEACAM6 and alphavbeta3 integrin enhances pancreatic adenocarcinoma cell adhesion to extracellular matrix components.Biochem Biophys Res Commun. 2004; 317: 133-141Crossref PubMed Scopus (39) Google Scholar We and others have previously identified POSTN as a differentially expressed gene by microarray profiling in several cancers, including PDAC.31Friess H. Ding J. Kleeff J. Fenkell L. Rosinski J.A. Guweidhi A. Reidhaar-Olson J.F. Korc M. Hammer J. Buchler M.W. Microarray-based identification of differentially expressed growth- and metastasis-associated genes in pancreatic cancer.Cell Mol Life Sci. 2003; 60: 1180-1199PubMed Google Scholar, 32Amatschek S. Koenig U. Auer H. Steinlein P. Pacher M. Gruenfelder A. Dekan G. Vogl S. Kubista E. Heider K.H. Stratowa C. Schreiber M. Sommergruber W. Tissue-wide expression profiling using cDNA subtraction and microarrays to identify tumor-specific genes.Cancer Res. 2004; 64: 844-856Crossref PubMed Scopus (202) Google Scholar The aim of this study was to analyze the role of POSTN in PSC-PCC interactions and to assess the impact of ECM proteins on pancreatic cancer invasiveness and radiochemoresistance.Materials and MethodsReagentsRPMI 1640, trypsin-EDTA, and penicillin-streptomycin were purchased from Invitrogen (Karlsruhe, Germany); fetal calf serum (FCS) from PAN Biotech (Aidenbach, Germany); and anti–collagen type 1 (sc-8783), anti–γ-tubulin (sc-7396), anti–extracellular signal–regulated kinase (ERK)-2 (sc-154), and donkey anti-goat (sc-2056) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-human smooth muscle actin (SMA) (M0851), immunoglobulin (Ig) G2a (X0943), and IgG1 negative controls, Envision antibody diluent, and liquid DAB+ substrate were purchased from Dako Cytomation (Hamburg, Germany). Anti-POSTN antibody (RD181045050) and recombinant human POSTN (rPOSTN) protein (RD172045100) were purchased from Biovendor (Heidelberg, Germany). Anti-Akt (9272), phospho-Akt (Ser473) (9271), and phospho-p44/42 mitogen-activated protein kinase (MAPK) (9101) were purchased from Cell Signaling Technologies (Danvers, MA). FluoroLink Cy2 (PA42004), FluoroLink Cy3 (PA43002), anti-rabbit (NA934V), and anti-mouse (NA931V) IgG horseradish peroxidase–linked antibodies as well as enhanced chemiluminescence immunoblotting detection reagents were purchased from Amersham Biosciences (Buckinghamshire, England). Mini EDTA-Free Protease Inhibitor was purchased from Roche Molecular Biochemicals (Basel, Switzerland), bicinchoninic acid protein assay from Pierce Chemical Co (Rockford, IL), RNAlater solution and POSTN small interfering RNA (siRNA) from Ambion (Huntingdon, England), and gemcitabine from Lilly (Fegersheim, France). Negative control siRNA and HiPerFect transfection reagents were purchased from Qiagen (Hilden, Germany). Recombinant human BMP-2 (355-BM), recombinant human fibroblast growth factor (FGF) acidic (232-FA), FGF basic (234-FSE), recombinant human platelet-derived growth factor (PDGF)-AA (221-AA), PDGF-BB (220-BB), Pan-specific TGF-β1 neutralizing antibody (AB100NA), control IgG (AB105C), and MMP-2 Quantikine enzyme-linked immunosorbent assay kit (DTM200) were purchased from R&D Systems (Wiesbaden, Germany). Recombinant human TGF-β1 (GF111) was purchased from Chemicon (Temecula, CA). Anti-human fibronectin (F3648) antibody and all other reagents were from Sigma-Aldrich Chemical Co (Taufkirchen, Germany).Pancreatic Tissues and Patient DataTissue collection and preservation were performed as described previously.33Erkan M. Kleeff J. Esposito I. Giese T. Ketterer K. Buchler M.W. Giese N.A. Friess H. Loss of BNIP3 expression is a late event in pancreatic cancer contributing to chemoresistance and worsened prognosis.Oncogene. 2005; 24: 4421-4432Crossref PubMed Scopus (181) Google Scholar Data on patient demographics, tumor stage, applied treatments, and survival were analyzed retrospectively from a prospectively registered database.Real-Time Light Cycler Quantitative Polymerase Chain ReactionAll reagents and equipment for messenger RNA (mRNA)/complementary DNA preparation were purchased from Roche Applied Science Diagnostics (Mannheim, Germany). mRNA extractions and preparations were performed as described previously.33Erkan M. Kleeff J. Esposito I. Giese T. Ketterer K. Buchler M.W. Giese N.A. Friess H. Loss of BNIP3 expression is a late event in pancreatic cancer contributing to chemoresistance and worsened prognosis.Oncogene. 2005; 24: 4421-4432Crossref PubMed Scopus (181) Google Scholar All primers were obtained from Search-LC (Heidelberg, Germany). Results are expressed as number of copies per microliter of input complementary DNA normalized to housekeeping genes.Human PCC LinesPanc-1, SU86.86, and T3M4 cell lines were either purchased from American Type Culture Collection (Rockville, MD) or received as a kind gift of Dr R. S. Metzgar (Durham, NC). Cells were routinely grown in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin) at 37°C, saturated with 5% co2 in a humid atmosphere.Human PSC Isolation and CultureHuman PSC isolation and culture were performed as described by Bachem et al.34Bachem M.G. Schneider E. Gross H. Weidenbach H. Schmid R.M. Menke A. Siech M. Beger H. Grunert A. Adler G. Identification, culture, and characterization of pancreatic stellate cells in rats and humans.Gastroenterology. 1998; 115: 421-432Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar Pancreatic tissue was obtained during surgery from patients with chronic pancreatitis. For the isolation of PSCs, the outgrowth method was used. Cell populations between passage 3 and 6 were used. A 1:1 (vol/vol) mixture of low glucose (1000 mg/L) Dulbecco’s modified Eagle medium with Ham’s F12 medium supplemented with 10% FCS, l-glutamine (2 mmol/L), penicillin/streptomycin, and amphotericin was the standard medium (SM 10%).Immunofluorescence AnalysisPSCs were seeded on Teflon-coated slides (ER-208B-AD-CE24; Erie Scientific Co, Portsmouth, NH) at a density of 2500/well in SM 10%. Twenty-four hours later, the medium was changed to either SM 1% or SM 10%. Forty-eight hours later, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with primary antibodies for 1 hour at 37°C Cy2, Cy3, or fluorescein isothiocyanate–labeled secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI) were used appropriately. Antibody dilutions are shown in Table 1. For immunofluorescence microphotography, exposure time was 2000 milliseconds (250 milliseconds for DAPI).Table 1Antibody Dilutions for Immunoblotting, Immunohistochemistry, and Immunofluorescence AnalysesImmunoblottingImmunohistochemistryImmunofluorescencePrimarySecondaryPrimarySecondaryPrimarySecondaryPOSTN1:50001:25001:4000RtU1:2001:1000COL11:20,0001:10,0001:2501:10001:1001:250Fibronectin1:25,0001:12,5001:1500RtU1:4001:1000α-SMA1:10,0001:50001:400RtU1:1001:500γ-tubulin1:50001:5000————pAkt1:10001:1000————Total Akt1:10001:1000————p-MAPK (p44/42)1:10001:1000————ERK-21:30001:3000————RtU, ready to use. Open table in a new tab Immunohistochemistry AnalysisImmunohistochemistry analyses were performed as described previously.33Erkan M. Kleeff J. Esposito I. Giese T. Ketterer K. Buchler M.W. Giese N.A. Friess H. Loss of BNIP3 expression is a late event in pancreatic cancer contributing to chemoresistance and worsened prognosis.Oncogene. 2005; 24: 4421-4432Crossref PubMed Scopus (181) Google Scholar To localize POSTN expression, 10 normal, 15 PDAC, 5 neoadjuvant radiochemotherapy (NeoRCT)-treated PDAC, 5 liver, and 5 lymph node metastases of PDAC samples were analyzed by immunohistochemistry. To assess whether POSTN, α-SMA, COL1, and fibronectin colocalized, 5 representative blocks from each group (excluding normal) were cut consecutively and stained. A Zeiss Axiocam 3.1 system (Jena, Germany) was used for the evaluation. Antibody dilutions are shown in Table 1.Immunoblotting AnalysisFor the immunoblot analyses, cells were grown on 6-well plates and processed as described previously.33Erkan M. Kleeff J. Esposito I. Giese T. Ketterer K. Buchler M.W. Giese N.A. Friess H. Loss of BNIP3 expression is a late event in pancreatic cancer contributing to chemoresistance and worsened prognosis.Oncogene. 2005; 24: 4421-4432Crossref PubMed Scopus (181) Google Scholar Primary antibodies were applied overnight at 4°C; secondary antibodies were added for 1 hour at room temperature. To detect secreted proteins in the supernatants (SNs), PSCs were grown until 100% confluency in 6-well plates and thereafter kept in serum-free SM for 96 hours. After collection of the SN, cells were counted by trypan blue to exclude the effect of increased cell number (±10%) on the protein level of SN. Fifteen micrograms of protein were size fractioned in the presence of sample buffer without boiling. Antibodies were diluted as specified in Table 1. To quantify the protein expression of POSTN in tissues, protein lysates of 7 normal pancreatic and 15 PDAC tissues were mixed. Each sample contributes an equal amount (mg/mL) of protein to the final mixture. To demonstrate the interplay between PSC activity, POSTN, COL1, and fibronectin synthesis, each membrane was stripped and reblotted consecutively with the specific antibodies. γ-Tubulin was used to verify equal loading.Production of Conditioned Media and Treatment of PSCsTwo types of conditioned media were produced. In the first set of experiments, SM 0% was kept on Panc-1, SU86.86, and T3M4 cell lines for 24 hours (PC-SN). This conditioned medium was then applied to 100% confluent PSCs for 96 hours. After collection of the SN, total mRNA and protein were extracted as described previously.33Erkan M. Kleeff J. Esposito I. Giese T. Ketterer K. Buchler M.W. Giese N.A. Friess H. Loss of BNIP3 expression is a late event in pancreatic cancer contributing to chemoresistance and worsened prognosis.Oncogene. 2005; 24: 4421-4432Crossref PubMed Scopus (181) Google Scholar In the second set, SM 10% was applied to 10-Gy irradiated, 50% confluent PSCs and kept for 7 days (RT-SN). After collection of SN, cell numbers were evaluated by trypan blue. This conditioned medium was used to stimulate PCC lines. All conditioned media were centrifuged for 10 minutes at 1000 rpm and frozen at −80°C until use.Growth Factor Stimulation of PSCsSister clones of PSCs were grown in 6-well plates to 100% confluence. Cells were starved with SM 0% for 24 hours. Fresh SM 0% containing growth factors was then added onto cells for another 96 hours at the following concentrations: FGF-A and FGF-B, 50 ng/mL; PDGF-aa and PDGF-bb, 50 ng/mL; TGF-β1, 5 ng/mL; and BMP-2, 200 ng/mL. Phosphate-buffered saline (PBS) was used as control. The experiments were repeated 3 times.Silencing of POSTN by siRNASeventy percent confluent PSCs were transfected with 20 nmol/L of POSTN siRNA: sense (5′ > 3′) GCAACGUGAAUGUUGAAUUtt, antisense AAUUCAACAUUCACGUUGCtc, or negative control sense r(UUCUCCGAACGU GUCACGU)dTdT, antisense r(ACGUGACACGUUCGGAGAA)dTdT using HiPerFect transfection reagent according to the manufacturer’s recommendations. After the SN was collected, protein and RNA were extracted at 96 hours posttransfection.Serum Deprivation and Resistance to HypoxiaTo assess the effect of POSTN on PCC/PSC growth under serum deprivation and hypoxia, 20,000 cells were seeded in 24-well plates in the presence of either rPOSTN 100 ng/mL or an equal amount of bovine serum albumin dissolved in PBS as control. For assessment of growth and colony formation under serum deprivation (1% FCS), cells were incubated for 5 and 7 days under standard conditions. For hypoxia, sister clones of PCC lines and PSCs were incubated under normoxic and hypoxic conditions (89.25% N2 + 10% CO2 + 0.75% 02) for 5 and 7 days at 37°C supplemented with 10% FCS. Colony formation and growth under hypoxia were visualized using crystal violet staining. After the wells were photographed, the dye was solubilized with methanol and OD was measured at 570 nm by an enzyme-linked immunosorbent assay reader (Opsys MR, Chantilly, VA). All experiments were performed in triplicate and repeated twice.Growth Assays and Response to ChemotherapyCells were seeded in triplicate in normal and collagen-coated (Biocoat, Collagen-I; BD Biosciences, Heidelberg, Germany) 96-well plates at densities of 4000/well for SU86.86 and PSCs and 6000/well for Panc-1 and T3M4. While the cells were still in suspension, either rPOSTN (0, 100 ng/mL, and 1 μg/mL) or equal amounts of bovine serum albumin dissolved in PBS were added to cells. In another set of experiments, either RT-SN or control-SN obtained from PSCs was added to PCCs. For assessment of growth, cells were kept under standard conditions for 48 hours. For the assessment of chemotherapeutic resistance, 5-fluorouracil or gemcitabine was added 12 hours after seeding at gradient concentrations; 0.01% dimethyl sulfoxide and PBS were used as controls, respectively. After 48 hours of incubation, the 3-(4,5-dimethyltriazol-2-yl)-2,5-dephenyltetrazolium bromide (MTT) test was performed to assess cell viability, as described previously.33Erkan M. Kleeff J. Esposito I. Giese T. Ketterer K. Buchler M.W. Giese N.A. Friess H. Loss of BNIP3 expression is a late event in pancreatic cancer contributing to chemoresistance and worsened prognosis.Oncogene. 2005; 24: 4421-4432Crossref PubMed Scopus (181) Google Scholar Growth assays were repeated twice and reported as percent change compared with control. The median effective doses of chemotherapeutic drugs on cancer cells grown on normal and collagen-coated plates were calculated from 5 independent experiments normalized to matching day-0 observations. Because PSCs were practically unresponsive to either drug, it was not possible to calculate median effective doses. Their responses are represented as percent change compared with control.Invasion AssayBD Biocoat Matrigel invasion chambers with 8-μm pore size (BD Biosciences) were used according to the manufacturer’s instructio
Referência(s)