Tissue Plasminogen Activator in Murine Exocrine Pancreas Cancer
2004; Elsevier BV; Volume: 165; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63374-3
ISSN1525-2191
AutoresSusana Aguilar, Josep M. Corominas, Núria Malats, José António Pereira, Marlène Dufresne, Francisco X. Real, Pilar Navarro,
Tópico(s)Cell Adhesion Molecules Research
ResumoTissue plasminogen activator (tPA) is absent from normal human pancreas and is expressed in 95% of human pancreatic adenocarcinomas. We have analyzed the expression of components of the tPA system in murine pancreatic tumors and the role of tPA in neoplastic progression. Transgenic mice expressing T antigen and c-myc under the control of the elastase promoter (Ela1-TAg and Ela1-myc, respectively) were used. tPA was undetectable in normal pancreas, acinar dysplasia, ductal complexes, and in all acinar tumors. By contrast, it was consistently detected in Ela1-myc tumors showing ductal differentiation. Crossing transgenic Ela1-myc with tPA−/− mice had no effect on the proportion of ductal tumors, indicating that tPA is not involved in the acinar-to-ductal transition. Ela1-myc:tPA−/− mice showed an increased survival in comparison to control mice. All ductal tumors, and none of the acinar tumors, overexpressed the tPA receptor annexin A2, suggesting its participation in the effects mediated by tPA. Our findings indicate that murine and human pancreatic ductal tumors share molecular alterations in the tPA system that may play a role in tumor progression. Tissue plasminogen activator (tPA) is absent from normal human pancreas and is expressed in 95% of human pancreatic adenocarcinomas. We have analyzed the expression of components of the tPA system in murine pancreatic tumors and the role of tPA in neoplastic progression. Transgenic mice expressing T antigen and c-myc under the control of the elastase promoter (Ela1-TAg and Ela1-myc, respectively) were used. tPA was undetectable in normal pancreas, acinar dysplasia, ductal complexes, and in all acinar tumors. By contrast, it was consistently detected in Ela1-myc tumors showing ductal differentiation. Crossing transgenic Ela1-myc with tPA−/− mice had no effect on the proportion of ductal tumors, indicating that tPA is not involved in the acinar-to-ductal transition. Ela1-myc:tPA−/− mice showed an increased survival in comparison to control mice. All ductal tumors, and none of the acinar tumors, overexpressed the tPA receptor annexin A2, suggesting its participation in the effects mediated by tPA. Our findings indicate that murine and human pancreatic ductal tumors share molecular alterations in the tPA system that may play a role in tumor progression. Exocrine pancreatic cancer is the fifth leading cause of death from malignant disease in Western society and it is one of the most aggressive human tumors.1American Gastroenterological Association Medical Position Statement Epidemiology, diagnosis, and treatment of pancreatic ductal adenocarcinoma.Gastroenterology. 1999; 117: 1463-1484Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 2Jaffee EM Hruban RH Canto M Kern SE Focus on pancreas cancer.Cancer Cell. 2002; 2: 25-28Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 3Real FX A “catastrophic hypothesis” for pancreas cancer progression.Gastroenterology. 2003; 124: 1958-1964Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 4Bardeesy N Sharpless NE DePinho RA Merlino G The genetics of pancreatic adenocarcinoma: a roadmap for a mouse model.Semin Cancer Biol. 2001; 11: 201-218Crossref PubMed Scopus (29) Google Scholar, 5Evans DB Abbruzzese JL Willet CG De Vita VH Hellman S Rosenberg S Cancer of the pancreas. 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Raven Press, New York1986: 443-458Google Scholar Except for duodenopancreatectomy with radical intention, there is no curative treatment and the 2-year survival of patients with a 2-cm tumor is approximately 20%.1American Gastroenterological Association Medical Position Statement Epidemiology, diagnosis, and treatment of pancreatic ductal adenocarcinoma.Gastroenterology. 1999; 117: 1463-1484Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar The reasons for these biological features are not known. They may be related to the anatomical location of the gland, the genetic alterations involved in tumor development, or other epigenetic factors, including the stromal reaction associated with the tumor.3Real FX A “catastrophic hypothesis” for pancreas cancer progression.Gastroenterology. 2003; 124: 1958-1964Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar New approaches to improve the prevention, diagnosis, and treatment of this disease are necessary to decrease mortality and an improved knowledge of its biology should contribute to these aims.There has been extensive debate as to the cell of origin of ductal adenocarcinomas,4Bardeesy N Sharpless NE DePinho RA Merlino G The genetics of pancreatic adenocarcinoma: a roadmap for a mouse model.Semin Cancer Biol. 2001; 11: 201-218Crossref PubMed Scopus (29) Google Scholar, 8Schmid RM Acinar-to-ductal metaplasia in pancreatic cancer development.J Clin Invest. 2002; 109: 1403-1404Crossref PubMed Scopus (45) Google Scholar, 9Real FX The cell biology of pancreatic cancer: an overview.in: Neoptolemos J Lemoine NR Pancreatic Cancer. 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Blockade of tPA using neutralizing antibodies or chemical inhibitors leads to reduced in vitro tumor invasion.19Paciucci R Tora M Diaz VM Real FX The plasminogen activator system in pancreas cancer: role of t-PA in the invasive potential in vitro.Oncogene. 1998; 16: 625-633Crossref PubMed Scopus (65) Google ScholarThe plasminogen system plays a critical role in intravascular thrombolysis as well as in other biological processes that require cellular migration, such as angiogenesis, inflammatory reactions, tissue remodeling, and tumor progression.20Collen D Lijnen HR Molecular basis of fibrinolysis, as relevant for thrombolytic therapy.Thromb Haemost. 1995; 74: 167-171Crossref PubMed Scopus (93) Google Scholar, 21Dano K Andreasen PA Grondahl-Hansen J Kristensen P Nielsen LS Skriver L Plasminogen activators, tissue degradation, and cancer.Adv Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2291) Google Scholar, 22DeClerck YA Imren S Montgomery AM Mueller BM Reisfeld RA Laug WE Proteases and protease inhibitors in tumor progression.Adv Exp Med Biol. 1997; 425: 89-97Crossref PubMed Scopus (115) Google Scholar, 23Liotta LA Rao CN Barsky SH Tumor invasion and the extracellular matrix.Lab Invest. 1983; 49: 636-649PubMed Google Scholar There are two types of plasminogen activators that catalyze plasmin generation from plasminogen: tissue-type and urokinase-type (uPA). Activation of plasminogen to plasmin results in progressive degradation of fibrin and other extracellular matrix components and may also lead to activation of metalloproteases, latent growth factors, and proteolysis of membrane glycoproteins.21Dano K Andreasen PA Grondahl-Hansen J Kristensen P Nielsen LS Skriver L Plasminogen activators, tissue degradation, and cancer.Adv Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2291) Google Scholar, 22DeClerck YA Imren S Montgomery AM Mueller BM Reisfeld RA Laug WE Proteases and protease inhibitors in tumor progression.Adv Exp Med Biol. 1997; 425: 89-97Crossref PubMed Scopus (115) Google Scholar, 24Carmeliet P Moons L Lijnen R Baes M Lemaitre V Tipping P Drew A Eeckhout Y Shapiro S Lupu F Collen D Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation.Nat Genet. 1997; 17: 439-444Crossref PubMed Scopus (571) Google Scholar, 25Mars WM Zarnegar R Michalopoulos GK Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA.Am J Pathol. 1993; 143: 949-958PubMed Google Scholar, 26Houck KA Leung DW Rowland AM Winer J Ferrara N Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms.J Biol Chem. 1992; 267: 26031-26037Abstract Full Text PDF PubMed Google Scholar, 27Saksela O Rifkin DB Cell-associated plasminogen activation: regulation and physiological functions.Annu Rev Cell Biol. 1988; 4: 93-126Crossref PubMed Scopus (714) Google Scholar All these processes may contribute to tumor development and metastasis. There is extensive evidence supporting the notion that the uPA system, including its receptor and plasminogen activator inhibitor PAI-1, can contribute to tumorigenesis in a variety of tissue types28Ossowski L Aguirre-Ghiso JA Urokinase receptor and integrin partnership: coordination of signaling for cell adhesion, migration and growth.Curr Opin Cell Biol. 2000; 12: 613-620Crossref PubMed Scopus (355) Google Scholar but there is less evidence for such a role regarding tPA and annexin A2 (Anx A2), a putative tPA receptor.29Hajjar KA Jacovina AT Chacko J An endothelial cell receptor for plasminogen/tissue plasminogen activator: I. Identity with annexin II.J Biol Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar, 30Cesarman GM Guevara CA Hajjar KA An endothelial cell receptor for plasminogen/tissue plasminogen activator (t-PA): II. Annexin II-mediated enhancement of t-PA-dependent plasminogen activation.J Biol Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar, 31Kassam G Choi KS Ghuman J Kang HM Fitzpatrick SL Zackson T Zackson S Toba M Shinomiya A Waisman DM The role of annexin II tetramer in the activation of plasminogen.J Biol Chem. 1998; 273: 4790-4799Crossref PubMed Scopus (130) Google Scholar We have proposed that, in the pancreas, the tPA system plays an important role in tumor development and/or progression whereas the uPA system may play a more dominant role in pancreatitis.19Paciucci R Tora M Diaz VM Real FX The plasminogen activator system in pancreas cancer: role of t-PA in the invasive potential in vitro.Oncogene. 1998; 16: 625-633Crossref PubMed Scopus (65) Google Scholar More recent studies have shown that tPA stimulates cell proliferation and angiogenesis in exocrine pancreatic tumors.32Diaz VM Planaguma J Thomson TM Reventos J Paciucci R Tissue plasminogen activator is required for the growth, invasion, and angiogenesis of pancreatic tumor cells.Gastroenterology. 2002; 122: 806-819Abstract Full Text Full Text PDF PubMed Scopus (55) Google ScholarOne strategy to facilitate progress in the identification of the genes/molecules that are crucial in tumor progression, and in the analysis of their role in these processes, is the use of genetically modified mice. Transgenic mice harboring mutated oncogenes and tumor suppressor genes have proven useful to show that tumorigenic pathways in mice and humans are largely conserved.33Balmain A Cancer as a complex genetic trait: tumor susceptibility in humans and mouse models.Cell. 2002; 108: 145-152Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Several mouse models are currently available which recapitulate important aspects of human pancreatic cancer. Most of them target transgenes to acinar cells taking advantage of the well-characterized promoter/enhancers of genes coding for acinar enzymes. Tumors arising in these mice display mainly acinar characteristics. As a consequence, they may not faithfully reproduce the ductal phenotype of human pancreatic cancer.4Bardeesy N Sharpless NE DePinho RA Merlino G The genetics of pancreatic adenocarcinoma: a roadmap for a mouse model.Semin Cancer Biol. 2001; 11: 201-218Crossref PubMed Scopus (29) Google Scholar, 34Wagner M Greten FR Weber CK Koschnick S Mattfeldt T Deppert W Kern H Adler G Schmid RM A murine tumor progression model for pancreatic cancer recapitulating the genetic alterations of the human disease.Genes Dev. 2001; 15: 286-293Crossref PubMed Scopus (177) Google Scholar However, pancreatic epithelial cells display a tremendous plasticity, with metaplastic interconversion between acinar, ductal, and islet lineages playing substantial roles in pathological situations.4Bardeesy N Sharpless NE DePinho RA Merlino G The genetics of pancreatic adenocarcinoma: a roadmap for a mouse model.Semin Cancer Biol. 2001; 11: 201-218Crossref PubMed Scopus (29) Google Scholar, 9Real FX The cell biology of pancreatic cancer: an overview.in: Neoptolemos J Lemoine NR Pancreatic Cancer. Blackwell Science Press, Oxford1995: 3-17Google Scholar Acinar cells from normal exocrine pancreas transdifferentiate in vitro to acquire a phenotype, as well as functional properties, of ductal cells.35Vila MR Lloreta J Real FX Normal human pancreas cultures display functional ductal characteristics.Lab Invest. 1994; 71: 423-431PubMed Google Scholar Similarly, mice overexpressing transforming growth factor-α (TGF-α) under the control of the metallothionein/elastase promoter display acinar-ductal metaplasia, with ductal complexes similar to those observed in the pancreas of patients with chronic pancreatitis, and occasionally develop ductal adenocarcinomas at advanced age.34Wagner M Greten FR Weber CK Koschnick S Mattfeldt T Deppert W Kern H Adler G Schmid RM A murine tumor progression model for pancreatic cancer recapitulating the genetic alterations of the human disease.Genes Dev. 2001; 15: 286-293Crossref PubMed Scopus (177) Google Scholar, 36Sandgren EP Luetteke NC Palmiter RD Brinster RL Lee DC Overexpression of TGF-α in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast.Cell. 1990; 61: 1121-1135Abstract Full Text PDF PubMed Scopus (600) Google Scholar, 37Song SY Gannon M Washington MK Scoggins CR Meszoely IM Goldenring JR Marino CR Sandgren EP Coffey Jr, RJ Wright CV Leach SD Expansion of Pdx1-expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing transforming growth factor-α.Gastroenterology. 1999; 117: 1416-1426Abstract Full Text Full Text PDF PubMed Google ScholarIn an attempt to explore the role of tPA in pancreatic tumorigenesis, we have taken advantage of two well-established transgenic mouse models: Ela1-Tag(1–127) and Ela1-myc. In these mice, transgenes are targeted to acinar cells using the Elastase-1 enhancer/promoter. Ela1-Tag(1–127) mice, here designated as Ela1-TAg, develop multi-focal acinar cell dysplasia with areas of progression to acinar cell carcinomas.38Tevethia MJ Bonneau RH Griffith JW Mylin L A simian virus 40 large T-antigen segment containing amino acids 1 to 127 and expressed under the control of the rat elastase-1 promoter produces pancreatic acinar carcinomas in transgenic mice.J Virol. 1997; 71: 8157-8166PubMed Google Scholar By contrast, Ela1-myc transgenic mice develop acinar cell carcinomas that, in approximately 50% of cases, evolve to display areas of ductal differentiation at late stages of tumor progression. The latter are particularly remarkable because they resemble human ductal adenocarcinomas in their morphology, expression of differentiation markers, and extensive desmoplasia.39Sandgren EP Quaife CJ Paulovich AG Palmiter RD Brinster RL Pancreatic tumor pathogenesis reflects the causative genetic lesion.Proc Natl Acad Sci USA. 1991; 88: 93-97Crossref PubMed Scopus (130) Google ScholarIn this work, we have analyzed the expression of components of the tPA system in normal pancreas and in tumors from these mice and have found that tPA is selectively expressed in late-stage Ela1-myc tumors displaying ductal differentiation. To determine the functional role of tPA in tumor progression, tPA-deficient mice were mated to Ela1-myc mice. Histological analysis of tumors indicated that tPA is not required for the progression of acinar to ductal tumors. By contrast, Ela1-myc:tPA−/− mice displayed a modest but significant increase in survival in comparison to Ela1-myc or Ela1-myc:tPA+/− control mice. This effect was associated with reduced microvessel density in ductal tumors from Ela1-myc:tPA−/− mice, although a contribution of the genetic background to this effect cannot be completely ruled out. In addition, we have found that Anx A2, a tPA receptor that greatly enhances its catalytic activity,30Cesarman GM Guevara CA Hajjar KA An endothelial cell receptor for plasminogen/tissue plasminogen activator (t-PA): II. Annexin II-mediated enhancement of t-PA-dependent plasminogen activation.J Biol Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar, 31Kassam G Choi KS Ghuman J Kang HM Fitzpatrick SL Zackson T Zackson S Toba M Shinomiya A Waisman DM The role of annexin II tetramer in the activation of plasminogen.J Biol Chem. 1998; 273: 4790-4799Crossref PubMed Scopus (130) Google Scholar, 40Hajjar KA Krishnan S Annexin II: a mediator of the plasmin/plasminogen activator system.Trends Cardiovasc Med. 1999; 9: 128-138Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar is also selectively overexpressed in tumors with a ductal phenotype, suggesting the coordinated participation of tPA and Anx A2 in tumor progression. Conservation of this signaling axis in human and mouse tumors supports the usefulness of the latter as models for the identification of novel therapeutic strategies for human pancreatic cancer.Materials and MethodsTransgenic and Knockout MiceFounder pairs of Ela1-TAg (C57Bl/6 genetic background) and Ela1-myc (C57Bl/6 genetic background) transgenic mice were obtained from Drs. M.J. Tevethia (Department of Microbiology, Pennsylvania State University College of Medicine, Hershey, PA) and E. Sandgren (Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI), respectively. Animals were housed and fed as previously described.38Tevethia MJ Bonneau RH Griffith JW Mylin L A simian virus 40 large T-antigen segment containing amino acids 1 to 127 and expressed under the control of the rat elastase-1 promoter produces pancreatic acinar carcinomas in transgenic mice.J Virol. 1997; 71: 8157-8166PubMed Google Scholar, 39Sandgren EP Quaife CJ Paulovich AG Palmiter RD Brinster RL Pancreatic tumor pathogenesis reflects the causative genetic lesion.Proc Natl Acad Sci USA. 1991; 88: 93-97Crossref PubMed Scopus (130) Google Scholar Male transgenic mice were mated with C57Bl/6 females and the offspring screened for the presence of the transgene using PCR. The following primers were used: GCA TCC CAG AAG CCT CCA AAG and GAA TCT TTG CAG CTA ATG GAC C for Ela1-TAg mice and CAC CGC CTA CAT CCT GTC CAT TCA AGC and TTA GGA CAA GGC TGG TGG GCA CTG for Ela1-myc. PCR conditions were as follows: after 5 minutes at 95°C, 40 cycles of denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute were carried out, followed by a final extension at 72°C for 10 minutes. Mouse strains overexpressing TGF-α from the metallothionein (MT-TGF-α) promoter, or the type 2 cholecystokinin receptor (CCK2) from elastase promoter (Ela1-CCK2), have been reported elsewhere.36Sandgren EP Luetteke NC Palmiter RD Brinster RL Lee DC Overexpression of TGF-α in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast.Cell. 1990; 61: 1121-1135Abstract Full Text PDF PubMed Scopus (600) Google Scholar, 41Saillan-Barreau C Clerc P Adato M Escrieut C Vaysse N Fourmy D Dufresne M Transgenic CCK-B/gastrin receptor mediates murine exocrine pancreatic secretion.Gastroenterology. 1998; 115: 988-996Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Expression analyses carried out using these strains were performed on pancreatic tissue from adult mice.Homozygous knockout mice for tPA (tPA−/− in 75% C57Bl/6, 25% 129SV/SL background) were a kind gift of Dr. P. Carmeliet (Center for Transgene Technology and Gene Therapy, Katholieke Universiteit Leuven, Leuven, Belgium). The basic features of their phenotype have been reported.42Carmeliet P Schoonjans L Kieckens L Ream B Degen J Bronson R De Vos R van den Oord JJ Collen D Mulligan RC Physiological consequences of loss of plasminogen activator gene function in mice.Nature. 1994; 368: 419-424Crossref PubMed Scopus (906) Google Scholar Mice were bred and genotyped by PCR according to described procedures (Dr. V. Attenburrow, Center for Transgene Technology and Gene Therapy, Katholieke Universiteit Leuven, Leuven, Belgium).Pancreatic Duct LigationC57Bl/6 and tPA−/− mice (n = 3 for each) were subjected to duct ligation as previously reported.43Scoggins CR Meszoely IM Wada M Means AL Yang L Leach SD p53-dependent acinar cell apoptosis triggers epithelial proliferation in duct-ligated murine pancreas.Am J Physiol. 2000; 279: G827-G836Google Scholar Briefly, mice were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (20 mg/kg). The peritoneal cavity was explored through a midline laparotomy and the stomach, pancreas, and spleen were mobilized. After ligation of the main pancreatic duct, the viscera were replaced in anatomical position, and the incision was closed. Animals were sacrificed 7 days later and the pancreas was processed for histological analysis.Analysis of Tumor Development in Transgenic and Hybrid Transgenic-Knockout MiceEla1-myc or Ela1-TAg mice were mated to tPA−/− mice to generate Ela1-myc (or Ela1-TAg):tPA+/− F1 hybrid progeny. Subsequently, the F1 hybrid progeny was mated to tPA−/− mice to generate Ela1-myc (or Ela1-TAg):tPA−/− or tPA+/− F2 hybrid progeny. Mice were housed and regularly followed according to procedures established and approved by the Institutional Animal Experimentation Committee. For some experiments, Ela1-TAg and Ela1-myc transgenic mice were sacrificed at defined time points (1, 2, 3, 4, and 5 months of age), an autopsy was performed, and the pancreas was resected and processed for histological analysis. Tumors from transgenic-knockout hybrid mice (Ela1-myc:tPA−/− or Ela1-TAg:tPA−/−) were collected at defined periods of time as indicated above. For the survival study, tumors were resected from animals sacrificed when they showed obvious signs of health deterioration (ie, wasting or abdominal distension) or at the time of death. All animal procedures were approved by the Institutional Animal Experimentation Committee.Tissue Samples and Histopathological AnalysisResected tumors were measured and their macroscopic appearance was carefully registered (ie, size, vascularization, consistence, presence of additional macroscopic masses). For histology, samples were fixed in buffered formalin for 24 hours and embedded in paraffin. For immunohistochemical assays using antibodies detecting von Willebrand factor (vWF) and Ki-67, tissues were fixed with fresh 4% paraformaldehyde for 24 hours and embedded in paraffin. Blocks from tumors arising in Ela1-CCK2 mice were obtained as described elsewhere.41Saillan-Barreau C Clerc P Adato M Escrieut C Vaysse N Fourmy D Dufresne M Transgenic CCK-B/gastrin receptor mediates murine exocrine pancreatic secretion.Gastroenterology. 1998; 115: 988-996Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Five-μm sections from all blocks were stained with hematoxylin and eosin (H&E) and scored blindly at ×10 to 20 magnification by a pathologist with extensive experience in pancreatic diseases (J.M.C.).ImmunohistochemistrytPA was detected using a rabbit antiserum raised against murine tPA that was kindly provided by Dr. L. Moons (Center for Transgene Technology and Gene Therapy, Flanders Institute for Biotechnology, Leuven, Belgium) at a 1:300 dilution. Anx A2 was detected using an antiserum obtained in our laboratory by immunizing rabbits with purified recombinant human Anx A2. The antiserum specifically detected a 36-kd molecular species in lysates from Panc-1 cells, in agreement with the reported molecular mass of Anx A2 (data not shown). To identify endothelial cells in tissue sections, rabbit polyclonal antibody against vWF (NeoMarkers, Fremont, CA) was used at 1:80 dilution; proliferating cells were identified using polyclonal rabbit anti-Ki-67 antibody NCL-Ki67p (Novocastra, Newcastle on Tyne, United Kingdom) at a 1:1500 dilution.Immunohistochemical analyses were performed using 5-μm sections of paraffin-embedded tissue blocks. Briefly, antigen retrieval was performed by immersing slides in 10 mmol/L citrate (pH 7.3) at 120°C for 1 minute in an autoclave. A Tech-Mate 500 automated immunostainer (Ventana Medical System, Tucson, AZ) was used. Primary antibodies were added for 30 minutes. As secondary antibody, the Envision+ anti-rabbit reagent was applied (Dako, Glostrup, Denmark). Reactions were developed using diaminobenzidine as chromogenic substrate. Sections were counter-stained with hematoxylin, dehydrated, and mounted. As negative controls, tissues were incubated with non-immune (Dako) or pre-immune rabbit serum. The specificity of the antisera used in immunohistochemical assays is described below. Details on methods for detection of vWF have been published elsewhere.44Montaner S Sodhi A Molinolo A Bugge TH Sawai ET He Y Li Y Ray PE Gutkind JS Endothelial infection with KSHV genes in vivo reveals that vGPCR initiates Kaposi's sarcomagenesis and can promote the tumorigenic potential of viral latent genes.Cancer Cell. 2003; 3: 23-36Abstract Full Text Full Text PDF PubMed Scopus (312) Google ScholarTo obtain semi-quantitative data on the expression of markers of angiogenesis and cell proliferation in Ela1-myc tumors arising in mice with a tPA wild-type or −/− genotype, tissue sections were incubated with antibodies detecting vWF and Ki-67, respectively, as described above. An independent investigator recorded digitalized images of each tumor displaying predominantly acinar or predominantly ductal differentiation. Tumor areas selected for analysis were classified as “ductal”, “mixed with ductal predominance”, “mixed with acinar predominance”, and “acinar” by a pathologist who was blind to the origin of the tumors (J.M.C.). An independent investigator who was also blind to the origin of the tumor images counted the number of vessels (vWF) and the proportion of proliferating cells (Ki-67), either on a computer screen or on a printed microphotograph.Statistical AnalysisTo assess the independence of two categorical variables, the χ2 test was
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