Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells

Background & Aims: Tumor desmoplasia is one of the representative histopathologic findings in ductal pancreatic adenocarcinoma. The aims of this study were to examine the cellular and molecular mechanisms of fibrogenesis associated with pancreatic adenocarcinomas. Methods: Immunostainings were performed with human pancreatic adenocarcinomas (n = 27) and tumors induced in nude mice (n = 36) by subcutaneously injecting MiaPaCa2, Panc1, and SW850 with and without pancreatic stellate cells. Matrix-producing cells were isolated from pancreatic adenocarcinomas and compared with pancreatic stellate cells isolated from tissue of chronic pancreatitis. Paracrine stimulation of pancreatic stellate cells by carcinoma cells was studied regarding matrix synthesis (collagen and c-fibronectin on protein and messenger RNA level) and cell proliferation (bromodeoxyuridine incorporation). Results: High numbers of desmin and α-smooth muscle actin-positive cells were detected in 26 of 27 pancreatic adenocarcinomas. Intense fibronectin and collagen stainings were associated with these cells. By using cytofilament stainings, gene expression profiling, and morphological examinations, the matrix-producing cells obtained by the outgrowth method from pancreatic adenocarcinomas were identified as pancreatic stellate cells. Supernatants of MiaPaCa2, Panc1, and SW850 cells stimulated proliferation and collagen type I and c-fibronectin synthesis of cultured pancreatic stellate cells. Preincubation of the carcinoma cell supernatants with neutralizing antibodies against fibroblast growth factor 2, transforming growth factor β1, and platelet-derived growth factor significantly reduced the stimulatory effects. Subcutaneous injection of carcinoma cells and pancreatic stellate cells induced fast-growing subcutaneous fibrotic tumors in nude mice. Morphometric analysis of carcinoma cells (cytokeratin stainings) showed a high density of carcinoma cells in these tumors. Conclusions: Pancreatic stellate cells strongly support tumor growth in the nude mouse model. The increased deposition of connective tissue in pancreatic carcinoma is the result of a paracrine stimulation of pancreatic stellate cells by carcinoma cells. Background & Aims: Tumor desmoplasia is one of the representative histopathologic findings in ductal pancreatic adenocarcinoma. The aims of this study were to examine the cellular and molecular mechanisms of fibrogenesis associated with pancreatic adenocarcinomas. Methods: Immunostainings were performed with human pancreatic adenocarcinomas (n = 27) and tumors induced in nude mice (n = 36) by subcutaneously injecting MiaPaCa2, Panc1, and SW850 with and without pancreatic stellate cells. Matrix-producing cells were isolated from pancreatic adenocarcinomas and compared with pancreatic stellate cells isolated from tissue of chronic pancreatitis. Paracrine stimulation of pancreatic stellate cells by carcinoma cells was studied regarding matrix synthesis (collagen and c-fibronectin on protein and messenger RNA level) and cell proliferation (bromodeoxyuridine incorporation). Results: High numbers of desmin and α-smooth muscle actin-positive cells were detected in 26 of 27 pancreatic adenocarcinomas. Intense fibronectin and collagen stainings were associated with these cells. By using cytofilament stainings, gene expression profiling, and morphological examinations, the matrix-producing cells obtained by the outgrowth method from pancreatic adenocarcinomas were identified as pancreatic stellate cells. Supernatants of MiaPaCa2, Panc1, and SW850 cells stimulated proliferation and collagen type I and c-fibronectin synthesis of cultured pancreatic stellate cells. Preincubation of the carcinoma cell supernatants with neutralizing antibodies against fibroblast growth factor 2, transforming growth factor β1, and platelet-derived growth factor significantly reduced the stimulatory effects. Subcutaneous injection of carcinoma cells and pancreatic stellate cells induced fast-growing subcutaneous fibrotic tumors in nude mice. Morphometric analysis of carcinoma cells (cytokeratin stainings) showed a high density of carcinoma cells in these tumors. Conclusions: Pancreatic stellate cells strongly support tumor growth in the nude mouse model. The increased deposition of connective tissue in pancreatic carcinoma is the result of a paracrine stimulation of pancreatic stellate cells by carcinoma cells. Ductal adenocarcinomas of the pancreas are characterized by rapid progression, early metastasis, diagnosis at an advanced stage, and a limited response to chemotherapy and radiotherapy.1Brand R.E. Tempero M.A. Pancreatic cancer (review).Curr Opin Oncol. 1998; 10: 362-366Crossref PubMed Scopus (71) Google Scholar, 2Warshaw A.L. Fernandez-del Castillo C. Pancreatic carcinoma.N Engl J Med. 1992; 326: 455-465Crossref PubMed Scopus (1557) Google Scholar Tumor desmoplasia, a process in which fibrous tissue infiltrates and envelops neoplasms, is one of the representative histopathologic findings in ductal pancreatic adenocarcinomas (PAC).3Cubilla A.L. Fitzgerald P.J. Morphological lesions associated with human primary invasive non-endocrine pancreas cancer.Cancer Res. 1976; 36: 2690-2698PubMed Google Scholar, 4Cubilla A.L. Fitzgerald P.J. Tumors of the exocrine pancreas.in: 2nd ed. Atlas of tumor pathology. 19. Armed Forces Institute of Pathology, Washington, DC1984: 1-40Google Scholar, 5Klöppel G. Lingenthal G. Bülow M.V. Kern H.F. Histological and fine structural features of pancreatic ductal adenocarcinomas in relation to growth and prognosis studies in xenografted tumours and clinco-histopathological correlation in a series of 75 cases.Histopathology (England). 1985; 9: 841-856Crossref PubMed Scopus (150) Google Scholar, 6Kuniyasu H. Abbruzzese J.L. Cleary K.R. Fidler I.L. Induction of ductal and stromal hyperplasia by basic fibroblast growth factor produced by human pancreatic carcinoma.Int J Oncol. 2001; 19: 681-685PubMed Google Scholar, 7Mollenhauer 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 (120) Google Scholar In all cases of pancreatic ductal adenocarcinomas studied by Mollenhauer et al,7Mollenhauer 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 (120) Google Scholar a remarkable increase in interstitial connective tissue (collagen type I and fibronectin) was observed. A similar distribution of collagens was observed in lymph node and liver metastases and in tumors xenografted into nude mice.7Mollenhauer 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 (120) Google Scholar The mean collagen content in pancreatic cancer tissue and tumor-associated chronic pancreatitis is 3-fold higher than in normal pancreas, and there is no difference in the proportion of collagen types I, III, and V among alcoholic chronic pancreatitis, tumor-associated chronic pancreatitis, and pancreatic cancer tissue.8Imamura T. Iguchi H. Manabe T. Ohshio G. Yoshimura T. Wang Z.H. Suwa H. Ishigami S. Imamura M. Quantitative analysis of collagen and collagen subtypes I, III, and V in human pancreatic cancer, tumor-associated chronic pancreatitis, and alcoholic chronic pancreatitis.Pancreas. 1995; 11: 357-364Crossref PubMed Scopus (80) Google Scholar Although pancreatic carcinoma cell (CC) lines are able to produce collagen types I, III, and IV and fibronectin, laminin, vitronectin, and undulin in vitro and in vivo,9Löhr M. Trautmann B. Gottler M. Peters S. Zauner I. Maillet B. Klöppel G. Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.Br J Cancer. 1994; 69: 144-151Crossref PubMed Scopus (91) Google Scholar most reports indicate that the fibrotic extracellular matrix (ECM) associated with PAC is produced by stromal cells.6Kuniyasu H. Abbruzzese J.L. Cleary K.R. Fidler I.L. Induction of ductal and stromal hyperplasia by basic fibroblast growth factor produced by human pancreatic carcinoma.Int J Oncol. 2001; 19: 681-685PubMed Google Scholar, 10Gress T.M. Menke A. Bachem M.G. Müller-Pillasch F. Ellenrieder V. Weidenbach H. Wagner M. Schmid R.M. Adler G. Extracellular matrix and pancreatic diseases.Digestion. 1998; 59 (review): 625-637Crossref PubMed Scopus (55) Google Scholar In situ hybridization studies using human pancreatic cancer tissues localized transcripts coding for collagen types I and III to spindle-shaped cells, whereas transcripts for matrix metalloproteinase 2, matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1, and tissue inhibitor of metalloproteinase 2 were found in both stromal and tumor cells.11Gress T.M. Müller-Pillasch F. Lerch M.M. Friess H. Buchler M. Adler G. Expression and in-situ localization of genes coding for extracellular matrix proteins and extracellular matrix degrading proteases in pancreatic cancer.Int J Cancer. 1995; 62: 407-413Crossref PubMed Scopus (224) Google Scholar In liver, activated myofibroblasts derived from hepatic stellate cells (HSCs) are responsible for an increased peritumor collagen production in hepatocellular carcinomas12Faouzi S. Lepreux S. Bedin C. Dubuisson L. Balabaud C. Bioulac-Sage P. Desmouliere A. Rosenbaum J. Activation of cultured rat hepatic stellate cells by tumoral hepatocytes.Lab Invest. 1999; 79: 485-493PubMed Google Scholar, 13Ooi L.P. Crawford D.H. Gotley D.C. Clouston A.D. Strong R.W. Gobe G.C. Halliday J.W. Bridle K.R. Ramm G.A. Evidence that “myofibroblast-like” cells are the cellular source of capsular collagen in hepatocellular carcinoma.J Hepatol. 1997; 26: 798-807Abstract Full Text PDF PubMed Scopus (78) Google Scholar, 14Ooi L.L. Bay B.H. Ng R.T. Song I.C. Mack P.O. An animal model for the study of hepatic stellate cell and hepatocellular carcinoma interaction.Ann Acad Med Singapore. 1999; 28: 95-98PubMed Google Scholar and in metastases of colorectal tumors.14Ooi L.L. Bay B.H. Ng R.T. Song I.C. Mack P.O. An animal model for the study of hepatic stellate cell and hepatocellular carcinoma interaction.Ann Acad Med Singapore. 1999; 28: 95-98PubMed Google Scholar In addition, in experimental hepatocarcinogenesis, the number of activated HSCs increased in fibrous septa around and within dysplastic and carcinomatous nodules, thus suggesting that HSC activation may result from direct stimulation by factors released from dysplastic hepatocytes.15Johnson S.J. Burr A.W. Toole K. Dack C.L. Mathew J. Burt A.D. Macrophage and hepatic stellate cell responses during experimental hepatocarcinogenesis.J Gastroenterol Hepatol. 1998; 13: 145-151Crossref PubMed Scopus (17) Google Scholar Since the first reports on the identification, isolation, and characterization of pancreatic stellate cells (PSCs)16Apte M.V. Haber P.S. Applegate T.L. Norton I.D. McCaughan G.W. Korsten M.A. Pirola R.C. Wilson J.S. Periacinar stellate-shaped cells in rat pancreas identification, isolation and culture.Gut. 1998; 43: 128-133Crossref PubMed Scopus (741) Google Scholar, 17Bachem M.G. Schneider E. Groß H. Weidenbach H. Schmid R. Menke A. Siech A. Beger H. Grünert 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 (861) Google Scholar numerous in vivo and in vitro studies have provided strong evidence of a central role for PSCs in fibrogenesis associated with acute and chronic pancreatitis (see reviews18Apte M.V. Wilson J.S. Alcohol-induced pancreatic injury.Best Pract Res Clin Gastroenterol. 2003; 17 (review): 593-612Crossref PubMed Scopus (77) Google Scholar, 19Bachem M.G. Schmid-Kotsas A. Gross H.J. Schneider E. Menke A. Weidenbach H. Adler G. Siech M. Beger H. Grünert A. Pancreatic stellate cells and their role in human pancreatic fibrogenesis.in: Büchler M.W. Friess H. Uhl W. Malfertheiner P. Chronic pancreatitis—novel concepts in biology and therapy. Blackwell Science, Oxford, UK2002: 134-147Google Scholar). PSCs share homologies to HSCs, including storage of retinyl palmitate, retinol esterification, expression of the cytofilaments vimentin and desmin, and phenotypic transition to an α-smooth muscle actin (α-SMA)-positive matrix-producing myofibroblast-like cell.16Apte M.V. Haber P.S. Applegate T.L. Norton I.D. McCaughan G.W. Korsten M.A. Pirola R.C. Wilson J.S. Periacinar stellate-shaped cells in rat pancreas identification, isolation and culture.Gut. 1998; 43: 128-133Crossref PubMed Scopus (741) Google Scholar, 17Bachem M.G. Schneider E. Groß H. Weidenbach H. Schmid R. Menke A. Siech A. Beger H. Grünert 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 (861) Google Scholar, 20Apte M.V. Haber P.S. Darby S.J. Rodgers S.C. McCaughan G.W. Korsten M.A. Pirola R.C. Wilson J.S. Pancreatic stellate cells are activated by proinflammatory cytokines implications for pancreatic fibrogenesis.Gut. 1999; 44: 534-541Crossref PubMed Scopus (494) Google Scholar, 21Haber P.S. Keogh G.W. Apte M.V. Moran C.S. Stewart N.L. Crawford D.H. Pirola R.C. McCaughan G.W. Ramm G.A. Wilson J.S. Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis.Am J Pathol. 1999; 155: 1087-1095Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar Transforming growth factor (TGF)-β1 and tumor necrosis factor α stimulate the change in the cell phenotype.22Schneider E. Schmid-Kotsas A. Zhao J. Weidenbach H. Schmid R.M. Menke A. Adler G. Waltenberger J. Grunert A. Bachem M.G. Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells.Am J Physiol Cell Physiol. 2001; 281: C532-C543Crossref PubMed Google Scholar In addition, we have shown that platelet-derived growth factor (PDGF) is the most effective mitogen and that fibroblast growth factor (FGF)-2 and TGF-β1 are fibrogenic mediators that stimulate ECM synthesis of the activated phenotype of cultured rat and human PSCs.17Bachem M.G. Schneider E. Groß H. Weidenbach H. Schmid R. Menke A. Siech A. Beger H. Grünert 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 (861) Google Scholar, 22Schneider E. Schmid-Kotsas A. Zhao J. Weidenbach H. Schmid R.M. Menke A. Adler G. Waltenberger J. Grunert A. Bachem M.G. Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells.Am J Physiol Cell Physiol. 2001; 281: C532-C543Crossref PubMed Google Scholar Supernatants (SN) of activated macrophages23Schmid-Kotsas A. Gross H.J. Menke A. Weidenbach H. Adler G. Siech M. Beger H. Grünert A. Bachem M.G. LPS-activated macrophages stimulate the synthesis of collagen type I and c-fibronectin in cultured pancreatic stellate cells.Am J Pathol. 1999; 155: 1749-1758Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar and platelet lysate24Luttenberger T. Schmid-Kotsas A. Menke A. Siech M. Beger H. Adler G. Grünert A. Bachem M.G. Platelet derived growth factors stimulate proliferation and extracellular matrix synthesis of cultured human pancreatic stellate cells.Lab Invest. 2000; 80: 47-55Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar also stimulated proliferation and matrix synthesis of cultured PSCs. Others have shown that acetaldehyde, which is produced primarily in the liver by ethanol oxidation, and ethanol itself directly stimulate collagen synthesis of cultured PSCs.25Apte M.V. Phillips P.A. Fahmy R.G. Darby S.J. Rodgers S.C. McCaughan G.W. Korsten M.A. Pirola R.C. Naidoo D. Wilson J.S. Does alcohol directly stimulate pancreatic fibrogenesis? Studies with rat pancreatic stellate cells.Gastroenterology. 2000; 118: 780-794Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar In addition to these cell-culture data, it was shown that PSCs are the cell type responsible for connective tissue synthesis in experimental pancreatic fibrosis21Haber P.S. Keogh G.W. Apte M.V. Moran C.S. Stewart N.L. Crawford D.H. Pirola R.C. McCaughan G.W. Ramm G.A. Wilson J.S. Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis.Am J Pathol. 1999; 155: 1087-1095Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 26Neuschwander-Tetri B.A. Bridle K.R. Wells L.D. Marcu M. Ramm G.A. Repetitive acute pancreatic injury in the mouse induces procollagen alpha1(I) expression colocalized to pancreatic stellate cells.Lab Invest. 2000; 80: 143-150Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 27Neuschwander-Tetri B.A. Burton F.R. Presti M.E. Britton R.S. Janney C.G. Garvin P.R. Brunt E.M. Galvin N.J. Poulos J.E. Repetitive self-limited acute pancreatitis induces pancreatic fibrogenesis in the mouse.Dig Dis Sci. 2000; 45: 665-674Crossref PubMed Scopus (98) Google Scholar and in human chronic alcoholic pancreatitis.21Haber P.S. Keogh G.W. Apte M.V. Moran C.S. Stewart N.L. Crawford D.H. Pirola R.C. McCaughan G.W. Ramm G.A. Wilson J.S. Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis.Am J Pathol. 1999; 155: 1087-1095Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 28Casini A. Galli A. Pignalosa P. Frulloni L. Grappone C. Milani S. Pederzoli P. Cavallini G. Surrenti C. Collagen type I synthesized by pancreatic periacinar stellate cells (PSC) co-localizes with lipid peroxidation-derived aldehydes in chronic alcoholic pancreatitis.J Pathol. 2000; 192: 81-89Crossref PubMed Scopus (118) Google Scholar Recently Yen et al29Yen T.W.F. Aardal N.P. Bronner M.P. Thorning D.R. Savard C.E. Lee S.P. Bell 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 (115) Google Scholar described a pronounced increase in the numbers of α-SMA-positive cells in PAC. The authors suggested that these cells might be activated stellate cells producing the connective tissue surrounding and infiltrating PAC. In addition, very recently Apte et al30Apte M.V. Park S. Phillips P.A. Santucci N. Goldstein D. Kumar R.K. Ramm G.A. Buchler M. Friess H. McCarroll J.A. Keogh G. Merrett N. Pirola R. Wilson J.S. Desmoplastic reaction in pancreatic cancer role of pancreatic stellate cells.Pancreas. 2004; 29: 179-187Crossref PubMed Scopus (487) Google Scholar identified activated stellate cells in fibrotic areas of pancreatic cancers by staining for desmin, α-SMA, and glial acidic fibrillary protein. They also suggested that interactions between tumor cells and stromal cells (PSCs) may play an important role in the pathobiology of pancreatic cancer.30Apte M.V. Park S. Phillips P.A. Santucci N. Goldstein D. Kumar R.K. Ramm G.A. Buchler M. Friess H. McCarroll J.A. Keogh G. Merrett N. Pirola R. Wilson J.S. Desmoplastic reaction in pancreatic cancer role of pancreatic stellate cells.Pancreas. 2004; 29: 179-187Crossref PubMed Scopus (487) Google Scholar To date, the role of the ECM in pancreatic cancer progression or restriction has not been defined. A role of the microenvironment playing a decisive role in tumor growth and metastatic spreading has been shown in a variety of experimental systems.31Fidler I.J. Critical factors in the biology of human cancer metastasis twenty-eighth G.H.A. Clowes memorial award lecture.Cancer Res. 1990; 50: 6130-6138PubMed Google Scholar, 32Fridman R. Giaccone G. Kanemoto T. Martin G.R. Gazdar A.F. Mulshine J.L. Reconstituted basement membrane (matrigel) and laminin can enhance the tumorigenicity and the drug resistance of small cell lung cancer cell lines.Proc Natl Acad Sci U S A. 1990; 87: 698-702Crossref Scopus (225) Google Scholar In small-cell lung cancer, ECM proteins protect cancer cells against apoptosis and accelerate cancer growth,33Sethi T. Rintoul R.C. Moore S.M. MacKinnon A.C. Salter D. Choo C. Chilvers E.R. Dransfield I. Donnelly S.C. Strieter R. Haslett C. Extracellular matrix proteins protect small cell lung cancer cells against apoptosis a mechanism for small cell lung cancer growth and drug resistance in vivo.Nat Med. 1999; 5: 662-668Crossref PubMed Scopus (643) Google Scholar thus indicating that, at least in this tumor type, the stroma seems to be beneficial for the tumor. Another recent report showed that an interaction between pancreatic CCs and fibroblasts contributed to the development of chemoresistance.34Muerkoester S. Wegehenkel K. Arlt A. Witt M. Sipos B. Kruse M.L. Sebens T. Kloppel G. Kalthoff H. Folsch U.R. Schafer H. Tumor stroma interactions induce chemoresistance in pancreatic ductal carcinoma cells involving increased secretion and paracrine effects of nitric oxide and interleukin-1beta.Cancer Res. 2004; 64: 1331-1337Crossref PubMed Scopus (242) Google Scholar In addition, Buchholz and coworkers35Buchholz M. Biebl A. Neeße A. Wagner M. Iwamura T. Leder G. Adler G. Gress T.M. SERPINE2 (protease nexin I) promotes extracellular matrix production and local invasion of pancreatic tumors in vivo.Cancer Res. 2003; 63: 4945-4951PubMed Google Scholar hypothesized recently that pancreatic cancer cells might stimulate PSCs to create a reactive microenvironment favoring invasive growth of the tumor. The objective of this study was to examine the cellular and molecular mechanisms of fibrogenesis associated with human PAC. To examine this issue, we (1) performed immunohistology of human PACs, (2) isolated and characterized PSCs from PACs, (3) studied paracrine stimulation of cultured PSCs by CC-SNs, and (4) induced subcutaneous tumors into nude mice by injecting CCs with PSCs. We showed that the increased deposition of connective tissue in pancreatic carcinoma is the result of a paracrine stimulation of PSCs by cancer cells. Furthermore, we conclude from our data that PSCs support tumor growth in the nude mouse model. Reagents were purchased from the following sources: acetone from Merck (Darmstadt, Germany); bisbenzimide from Hoechst (Frankfurt, Germany); fetal calf serum (FCS), amphotericin B, anti-α-SMA, High Pure RNA Extraction Kit, LightCycler FastStart SYBR Green, and LightCycler DNA Master SYBR Green I from Roche (Mannheim, Germany); gelatin type B, ethidium bromide, bovine albumin fraction V, ethanol, l(+)-ascorbic acid, calf thymus DNA, yeast transfer RNA, and RedTaq DNA Polymerase from Sigma (Deisenhofen, Germany); l-glutamine and Superscript from GibcoBRL (Paisley, Scotland); Delfia Eu-labeled streptavidin, Delfia Eu-labeled anti-rabbit immunoglobulin G, and enhancement solution from PerkinElmer-Life Science-Wallac (Turku, Finland); rabbit anti-human collagen type I and biotin-labeled goat anti-human collagen type III from Chemicon International (Temecula, CA); rabbit anti-fibronectin from Dade-Behring (Marburg, Germany); goat polyclonal neutralizing anti-PDGF(AA), anti-PDGF(BB), and anti-FGF-2 and rabbit neutralizing anti-TGF-β1 from R&D Systems Inc. (Minneapolis, MN); anti-human collagen type I from Southern Biotechnology Associates (Birmingham, AL), poly-dA/dU homopolymer from Pharmacia LKB (Freiburg, Germany); Hybond-N membrane, Hybond-C extra membrane, and Random Priming Kit (RPN 1601Y) from Amersham-Buchler (Braunschweig, Germany); anti-bromodeoxyuridine (BrdU), biotinylated swine anti-rabbit, horseradish peroxidase (HRP) swine anti-rabbit, HRP rabbit anti-mouse, fluorescein isothiocyanate (FITC)-conjugated streptavidin, biotinylated rabbit anti-mouse, monoclonal mouse anti-α-SMA, monoclonal mouse anti-desmin, monoclonal mouse anti-vimentin, monoclonal mouse anti-human cytokeratin, and HRP-conjugated streptavidin from DAKO (Glostrup, Denmark); trypsin-ethylenediaminetetraacetic acid, Dulbecco’s modified Eagle medium (DMEM), Ham’s F12 medium, and penicillin from Biochrom (Berlin, Germany); Biotin-Tyramide Signal Amplification (TSA) reagent from PerkinElmer Life Science (Boston, MA); and RNeasy Kit and QIAQuick Gel Extraction Kit from Qiagen (Hilden, Germany). The complementary DNA (cDNA) probes for collagen α1 I, collagen α1 III, laminin, and fibronectin were purchased from the American Type Culture Collection (Manassas, VA), oligonucleotide primers were synthesized by Interactiva (Ulm, Germany), and x-ray film and Ekta-chrome 400 film were obtained from Eastman Kodak (Rochester, NY). Small pancreatic tissue blocks (100–150 mg) were obtained during pancreas surgery from patients with resectable pancreatic carcinomas (n = 6) or from patients with chronic pancreatitis (n = 8). The tissue blocks were cut (0.5–1 mm3) and seeded in 10-cm2 uncoated culture wells (6 per plate; 3–5 pieces per well) in the presence of 10%–20% FCS in a 1:1 (vol/vol) mixture of DMEM with Ham’s F12 medium. l-glutamine (2 mmol/L), penicillin/streptomycin, and amphotericin were freshly added. Tissue blocks were cultured at 37°C in a 5% CO2/air humidified atmosphere. Eighteen hours after seeding, culture medium was changed, and 24 hours later, the small tissue blocks were transferred to new culture plates. The PSCs grew out in high number and purity from the tissue blocks 1 to 3 days later. The small tissue blocks were removed after 2–3 weeks. After reaching confluence, monolayers were trypsinized and passaged 1:3. The purity of the cells was assessed by morphology (most cells were stellate-like, with long cytoplasmatic extensions; some were also spindle shaped) and cytofilament stainings of α-SMA (>95%), vimentin (100%), and desmin (20%–40%). Cell populations between passage 3 and 6 were used to study the effects of CC-SNs on proliferation and matrix synthesis. Cultured MiaPaCa2, Panc1, and SW850 cells in 75-cm2 flasks were washed with a 1:1 (vol/vol) mixture of DMEM with Ham’s F12 medium. Thereafter, 10 mL of fresh DMEM/Ham’s F12 (with antibiotics and without FCS) was added and conditioned for 24 hours. Conditioned media were removed under sterile conditions, centrifuged (800 rpm, 5 minutes, 4°C) to remove cell debris, and stored at −80°C until their addition to cultured PSCs. Aliquots of the conditioned media were preincubated for 1 hour with neutralizing antibodies to PDGF(AA) and PDGF(BB) (each 1 μg/mL), FGF-2 (1 μg/mL), and TGF-β1 (10 μg/mL) before the conditioned media were added to cultured PSCs. PSC proliferation was determined by BrdU incorporation as previously described.24Luttenberger T. Schmid-Kotsas A. Menke A. Siech M. Beger H. Adler G. Grünert A. Bachem M.G. Platelet derived growth factors stimulate proliferation and extracellular matrix synthesis of cultured human pancreatic stellate cells.Lab Invest. 2000; 80: 47-55Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar Furthermore, DNA was measured with fluorometry by using bisbenzimide and calf thymus DNA as a standard.17Bachem M.G. Schneider E. Groß H. Weidenbach H. Schmid R. Menke A. Siech A. Beger H. Grünert 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 (861) Google Scholar DNA measurements (standards, controls, and samples) were performed in triplicate. Fibronectin and collagen type I concentrations in PSC-SNs were measured by time-resolved fluorescence immunoassay as described previously.23Schmid-Kotsas A. Gross H.J. Menke A. Weidenbach H. Adler G. Siech M. Beger H. Grünert A. Bachem M.G. LPS-activated macrophages stimulate the synthesis of collagen type I and c-fibronectin in cultured pancreatic stellate cells.Am J Pathol. 1999; 155: 1749-1758Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 24Luttenberger T. Schmid-Kotsas A. Menke A. Siech M. Beger H. Adler G. Grünert A. Bachem M.G. Platelet derived growth factors stimulate proliferation and extracellular matrix synthesis of cultured human pancreatic stellate cells.Lab Invest. 2000; 80: 47-55Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar Fibronectin and collagen I concentrations were referred to the DNA content in the corresponding culture well. All measurements (standards, controls, and samples) were performed in duplicate. Variations of duplicate measurements were usually between 0.5% and 5% and did not exceed 8%. To show the effects of CCs on cell-associated collagens and fibronectin, cultured PSCs grown on glass coverslips in the presence of 0.1% FCS were incubated with 500 μL/mL MiaPaCa2, Panc1, and SW850 conditioned media. After 48 hours, cultures were acetone-fixed and immunostained for collagen types I and III and fibronectin. Immunofluorescence microscopy was performed as described previously,17Bachem M.G. Schneider E. Groß H. Weidenbach H. Schmid R. Menke A. Siech A. Beger H. Grünert 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 (861) Google Scholar, 22Schneider E. Schmid-Kotsas A. Zhao J. Weidenbach H. Schmid R.M. Menke A. Adler G. Waltenberger J. Grunert A. Bachem M.G. Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells.Am J Physiol Cell Physiol. 2001; 281: C532-C543Crossref PubMed Google Scholar, 23Schmid-Kotsas A. Gross H.J. Menke A. Weidenbach H. Adler G. Siech M. Beger H. Grünert A. Bachem M.G. LPS-activated macrophages stimulate the synthesis of collagen type I and c-fibronectin in cultured pancreatic