The Myofibroblast Is the Predominant Plasminogen Activator Inhibitor-1-Expressing Cell Type in Human Breast Carcinomas
2003; Elsevier BV; Volume: 163; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63547-x
ISSN1525-2191
AutoresBirgitte Vrou Offersen, Boye Schnack Nielsen, Gunilla Høyer‐Hansen, Fritz Rank, Stephen Hamilton‐Dutoit, Jens Overgaard, Peter A. Andreasen,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe tumor level of plasminogen activator inhibitor-1 (PAI-1) is an informative biochemical marker of a poor prognosis in several cancer types. However, the tumor biological functions of PAI-1 and the identity of PAI-1-expressing cells are controversial. With the aim of immunohistochemically localizing PAI-1 in formalin-fixed, paraffin-embedded invasive ductal breast carcinoma samples, we raised new polyclonal antibodies against PAI-1 from different expression systems. The antibodies were affinity purified by absorption on immobilized preparations of PAI-1 different from those used for immunization. The specificity of the antibodies was ensured by immunoblotting analysis. In immunohistochemistry, the staining pattern obtained with the antibodies showed a good correlation with the PAI-1 mRNA expression pattern. In all 25 cases analyzed, PAI-1 immunoreactivity was predominantly localized in fibroblast-like cells. Double-immunofluorescence analyses showed co-expression of PAI-1 and α-smooth muscle actin in these cells, suggesting that they are myofibroblasts. PAI-1 was also seen in some myoepithelial cells surrounding occasional foci of ductal carcinoma in situ (9 of 25), some endothelial cells (8 of 25), some cancer cells (3 of 25), and some mast cells (6 of 25). In conclusion, we have provided a robust immunohistochemical procedure for detection of PAI-1 and shown that the majority of the PAI-1-expressing cells in invasive ductal breast carcinomas are myofibroblasts. The tumor level of plasminogen activator inhibitor-1 (PAI-1) is an informative biochemical marker of a poor prognosis in several cancer types. However, the tumor biological functions of PAI-1 and the identity of PAI-1-expressing cells are controversial. With the aim of immunohistochemically localizing PAI-1 in formalin-fixed, paraffin-embedded invasive ductal breast carcinoma samples, we raised new polyclonal antibodies against PAI-1 from different expression systems. The antibodies were affinity purified by absorption on immobilized preparations of PAI-1 different from those used for immunization. The specificity of the antibodies was ensured by immunoblotting analysis. In immunohistochemistry, the staining pattern obtained with the antibodies showed a good correlation with the PAI-1 mRNA expression pattern. In all 25 cases analyzed, PAI-1 immunoreactivity was predominantly localized in fibroblast-like cells. Double-immunofluorescence analyses showed co-expression of PAI-1 and α-smooth muscle actin in these cells, suggesting that they are myofibroblasts. PAI-1 was also seen in some myoepithelial cells surrounding occasional foci of ductal carcinoma in situ (9 of 25), some endothelial cells (8 of 25), some cancer cells (3 of 25), and some mast cells (6 of 25). In conclusion, we have provided a robust immunohistochemical procedure for detection of PAI-1 and shown that the majority of the PAI-1-expressing cells in invasive ductal breast carcinomas are myofibroblasts. One of the proteolytic enzyme systems involved in the degradation of extracellular matrix during tumor growth, invasion, and metastasis is the urokinase-type plasminogen activator (uPA) system.1Dano 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 (2467) Google Scholar, 2Mignatti P Rifkin DB Biology and biochemistry of proteinases in tumor invasion.Physiol Rev. 1993; 73: 161-195Crossref PubMed Scopus (1186) Google Scholar, 3Andreasen PA Egelund R Petersen HH The plasminogen activation system in tumor growth, invasion, and metastasis.Cell Mol Life Sci. 2000; 57: 25-40Crossref PubMed Scopus (860) Google Scholar, 4Andreasen PA Kjoller L Christensen L Duffy MJ The urokinase-type plasminogen activator system in cancer metastasis: a review.Int J Cancer. 1997; 72: 1-22Crossref PubMed Scopus (1458) Google Scholar. uPA catalyzes the conversion of the inactive zymogen plasminogen to the active broad-spectrum protease plasmin, which is able to degrade many extracellular proteins, eg, fibrin and laminin.5Bugge TH Kombrinck KW Flick MJ Daugherty CC Danton MJ Degen JL Loss of fibrinogen rescues mice from the pleiotropic effects of plasminogen deficiency.Cell. 1996; 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Studies with animal tumor models have failed to give a consistent picture. 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Nevertheless, using PAI-1 gene-deficient mice in in vivo and ex vivo angiogenesis model systems,22McMahon GA Petitclerc E Stefansson S Smith E Wong MK Westrick RJ Ginsburg D Brooks PC Lawrence DA Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis.J Biol Chem. 2001; 276: 33964-33968Crossref PubMed Scopus (251) Google Scholar, 24Bajou K Noel A Gerard RD Masson V Brunner N Holst-Hansen C Skobe M Fusenig NE Carmeliet P Collen D Foidart JM Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization.Nat Med. 1998; 4: 923-928Crossref PubMed Scopus (625) Google Scholar, 25Bajou K Masson V Gerard RD Schmitt PM Albert V Praus M Lund LR Frandsen TL Brunner N Dano K Fusenig NE Weidle U Carmeliet G Loskutoff D Collen D Carmeliet P Foidart JM Noel A The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. 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Such information may also be valuable for selection of relevant animal tumor models for experimental studies of the role of PAI-1, and may even provide information of additional prognostic value. There are several reports on the localization of PAI-1 in primary breast carcinoma, but a consistent picture cannot be drawn.29Sumiyoshi K Baba S Sakaguchi S Urano T Takada Y Takada A Increase in levels of plasminogen activator and type-1 plasminogen activator inhibitor in human breast cancer: possible roles in tumor progression and metastasis.Thromb Res. 1991; 63: 59-71Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 30Jankun J Merrick HW Goldblatt PJ Expression and localization of elements of the plasminogen activation system in benign breast disease and breast cancers.J Cell Biochem. 1993; 53: 135-144Crossref PubMed Scopus (94) Google Scholar, 31Bianchi E Cohen RL Dai A Thor AT Shuman MA Smith HS Immunohistochemical localization of the plasminogen activator inhibitor-1 in breast cancer.Int J Cancer. 1995; 60: 597-603Crossref PubMed Scopus (60) Google Scholar, 32Christensen L Wiborg Simonsen AC Heegaard CW Moestrup SK Andersen JA Andreasen PA Immunohistochemical localization of urokinase-type plasminogen activator, type-1 plasminogen-activator inhibitor, urokinase receptor and alpha(2)-macroglobulin receptor in human breast carcinomas.Int J Cancer. 1996; 66: 441-452Crossref PubMed Scopus (105) Google Scholar, 33Costantini V Sidoni A Deveglia R Cazzato OA Bellezza G Ferri I Bucciarelli E Nenci GG Combined overexpression of urokinase, urokinase receptor, and plasminogen activator inhibitor-1 is associated with breast cancer progression: an immunohistochemical comparison of normal, benign, and malignant breast tissues.Cancer. 1996; 77: 1079-1088Crossref PubMed Scopus (119) Google Scholar, 34Umeda T Eguchi Y Okino K Kodama M Hattori T Cellular localization of urokinase-type plasminogen activator, its inhibitors, and their mRNAs in breast cancer tissues.J Pathol. 1997; 183: 388-397Crossref PubMed Scopus (46) Google Scholar, 35Ferrier CM de Witte HH Straatman H van Tienoven DH van Geloof WL Rietveld FJ Sweep CG Ruiter DJ van Muijen GN Comparison of immunohistochemistry with immunoassay (ELISA) for the detection of components of the plasminogen activation system in human tumour tissue.Br J Cancer. 1999; 79: 1534-1541Crossref PubMed Scopus (46) Google Scholar, 36Dublin E Hanby A Patel NK Liebman R Barnes D Immunohistochemical expression of uPA, uPAR, and PAI-1 in breast carcinoma: fibroblastic expression has strong associations with tumor pathology.Am J Pathol. 2000; 157: 1219-1227Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 37Zhao H Morimoto T Sasa M Tanaka T Izumi K Immunohistochemical expression of uPA, PAI-1, cathepsin D and apoptotic cells in ductal carcinoma in situ of the breast.Breast Cancer. 2002; 9: 118-126Crossref PubMed Scopus (13) Google Scholar The aim of this study was to develop polyclonal antibodies that could be used on routinely processed paraffin-embedded breast cancer samples. Careful affinity purification steps were used to provide two specific high-affinity antibody preparations that showed identical staining patterns, which were in good agreement with the pattern obtained with a monoclonal antibody against PAI-1 as well as the expression pattern of PAI-1 mRNA obtained by in situ hybridization. Tumor specimens from 25 patients with primary invasive ductal breast carcinoma (7 grade I, 11 grade II, 7 grade III) registered in the Danish Breast Cancer Cooperative Group were obtained from the Department of Pathology, Copenhagen University Hospital, Copenhagen, Denmark. The tumor specimens were fixed in 10% buffered formalin for 24 hours at room temperature and paraffin-embedded. Sections from each of these paraffin blocks were used for histopathological analysis, and confirmed the presence of cancer tissue within the samples. Frozen tissue specimens were obtained from eight invasive ductal carcinomas and were used for protein extraction. The study was approved by the Regional Scientific-Ethical Committee for Aarhus (j.nr. 1991/2106) and the Regional Scientific-Ethical Committee for Copenhagen and Frederiksberg (j.nr. KF 01-456/93). A rabbit polyclonal anti-PAI-1 antibody was generated after repeated immunizations with recombinant human PAI-1 expressed in Pichia pastoris.38Rodenburg KW Kjoller L Petersen HH Andreasen PA Binding of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein and very-low-density lipoprotein receptor involves basic residues in the inhibitor.Biochem J. 1998; 329: 55-63Crossref PubMed Scopus (87) Google Scholar The IgG fraction (AB-1) was purified from the rabbit serum by chromatography on a protein A-Sepharose column. The IgG fraction was then passed six times through a Sepharose column with immobilized recombinant human PAI-1 expressed in Escherichia coli.39Jensen JK Wind T Andreasen PA The vitronectin binding area of plasminogen activator inhibitor-1, mapped by mutagenesis and protection against an inactivating organochemical ligand.FEBS Lett. 2002; 521: 91-94Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar The sixth run-through, which was depleted for most anti-PAI-1 IgG, was used as negative control IgG and named AB-1D. An affinity-purified anti-PAI-1 IgG preparation (AB-1A) was prepared by eluting the column with a buffer of 0.1 mol/L glycin, pH 2.5, and 1 mol/L NaCl. As evaluated by enzyme-linked immunosorbent assay, the reactivity of AB-1A toward PAI-140Munch M Heegaard C Jensen PH Andreasen PA Type-1 inhibitor of plasminogen activators. Distinction between latent, activated and reactive centre-cleaved forms with thermal stability and monoclonal antibodies.FEBS Lett. 1991; 295: 102-106Crossref PubMed Scopus (55) Google Scholar was increased by a factor of 3.3 compared to the starting material, whereas AB-1D showed an ∼500-fold reduction in PAI-1 reactivity. In brief, this enzyme-linked immunosorbent assay was performed as follows: wells were coated with HT-1080 PAI-1 and a dilution series of fractions of anti-PAI-1 IgG and anti-PAI-1-depleted IgG were added. The captured antibodies were detected with horseradish-peroxidase-conjugated swine anti-rabbit antibodies and a peroxidase reaction. Another polyclonal anti-PAI-1 antibody preparation was raised against PAI-1 purified from HT-1080 cells.40Munch M Heegaard C Jensen PH Andreasen PA Type-1 inhibitor of plasminogen activators. Distinction between latent, activated and reactive centre-cleaved forms with thermal stability and monoclonal antibodies.FEBS Lett. 1991; 295: 102-106Crossref PubMed Scopus (55) Google Scholar This IgG preparation will be referred to as AB-2. Affinity purification was done as for AB-1A, but using a Sepharose column with immobilized P. pastoris PAI-1. This affinity-purified anti-PAI-1 antibody preparation (AB-2A) showed a fivefold increased reactivity compared with the starting material. The sixth run-through (AB-2D) had an ∼150-fold reduced PAI-1 reactivity. A mouse monoclonal anti-PAI-1 antibody, clone 380, was purchased from American Diagnostica (Greenwich, CT). A rabbit polyclonal antibody raised against uPA from Serono, Aubonne, Switzerland, was a gift from the late Dr. I. Clemmensen (DakoCytomation, Glostrup, Denmark). IgG was purified from the rabbit serum by the use of protein A-Sepharose. The uPA immunogen appeared pure on Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and we previously used absorption against the immunogen for preparing an antibody to be used as a negative control.32Christensen L Wiborg Simonsen AC Heegaard CW Moestrup SK Andersen JA Andreasen PA Immunohistochemical localization of urokinase-type plasminogen activator, type-1 plasminogen-activator inhibitor, urokinase receptor and alpha(2)-macroglobulin receptor in human breast carcinomas.Int J Cancer. 1996; 66: 441-452Crossref PubMed Scopus (105) Google Scholar To improve the specificity of the negative control preparation, the uPA immunogen was affinity-purified by chromatography on an anti-uPA monoclonal antibody immobilized on Sepharose.41Nielsen LS Hansen JG Skriver L Wilson EL Kaltoft K Zeuthen J Dano K Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody.Biochemistry. 1982; 21: 6410-6415Crossref PubMed Scopus (194) Google Scholar Another Sepharose column was made containing this affinity-purified uPA preparation, and the protein A-purified anti-uPA IgG fraction was passed over the column until the reactivity against uPA had been reduced more than 100-fold. This run-through IgG preparation will be referred to as "polyclonal antibodies depleted of uPA-reactivity." Polyclonal anti-tPA IgG was a gift from the late Dr. I. Clemmensen (DakoCytomation, Denmark). Tumors were taken from −80°C and homogenized immediately in 0.1 mol/L Tris, pH 8.1, 0.5% Triton X-100, 10 mmol/L ethylenediaminetetraacetic acid, and 10 μg/ml aprotinin (10 μl/mg tissue) with a Ultraturrax with a S 25 N8G head (24,000 rpm) at 4°C. The homogenate was centrifuged at 10,000 × g for 10 minutes to remove cell debris and nuclei. The supernatants will be referred to as "tumor extracts." Several pools, each consisting of extracts of 5 to 10 tumors, were analyzed by immunoblotting analysis. Samples of the pools, each corresponding to ∼200 μg of total protein, were subjected to SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride filters (Immobilon-P; Millipore, Bedford, MA).32Christensen L Wiborg Simonsen AC Heegaard CW Moestrup SK Andersen JA Andreasen PA Immunohistochemical localization of urokinase-type plasminogen activator, type-1 plasminogen-activator inhibitor, urokinase receptor and alpha(2)-macroglobulin receptor in human breast carcinomas.Int J Cancer. 1996; 66: 441-452Crossref PubMed Scopus (105) Google Scholar The filters were incubated with affinity-purified polyclonal anti-PAI-1 antibodies AB-1A and AB-2A, polyclonal antibodies depleted of PAI-1 reactivity AB-1D and AB-2D, polyclonal anti-uPA antibodies, polyclonal anti-tPA antibodies, polyclonal antibodies depleted of uPA immunoreactivity, or preimmune IgG. The primary antigen-antibody reactions were visualized with peroxidase-conjugated swine anti-rabbit antibodies (P217, DakoCytomation) and the ECL system (Amersham Pharmacia, Uppsala, Sweden). Paraffin sections (3- to 4-μm-thick sections of formalin-fixed and paraffin-embedded tissue samples) were deparaffinized in xylene and ethanol. For retrieval of PAI-1 antigen, sections were boiled in a microwave oven in 10 mmol/L Tris containing 0.5 mmol/L EGTA (pH 9.0, TEG buffer) for 15 minutes.42Ferrier CM van Geloof WL de Witte HH Kramer MD Ruiter DJ van Muijen GN Epitopes of components of the plasminogen activation system are re-exposed in formalin-fixed paraffin sections by different retrieval techniques.J Histochem Cytochem. 1998; 46: 469-476Crossref PubMed Scopus (30) Google Scholar, 43Floridon C Nielsen O Holund B Sweep F Sunde L Thomsen SG Teisner B Does plasminogen activator inhibitor-1 (PAI-1) control trophoblast invasion? A study of fetal and maternal tissue in intrauterine, tubal and molar pregnancies.Placenta. 2000; 21: 754-762Abstract Full Text PDF PubMed Scopus (48) Google Scholar We could not retrieve PAI-1 antigen by boiling in citrate buffer, pH 6.0, or by proteolytic digestion with 0.05% pronase (S2013, DakoCytomation) in Tris-buffered saline (TBS) buffer, pH 7.2, for 15 minutes at 37°C. The sections were left for 20 minutes at room temperature and then incubated with 2% H2O2 in 99% ethanol for 20 minutes to block endogenous peroxidase activity. Sections were washed in TBS containing 0.5% Triton X-100 (TBS-T) for 30 minutes, and incubated with Protein Block (X909, DakoCytomation) for 10 minutes to reduce unspecific adhesion of the antibodies. The affinity-purified rabbit polyclonal antibodies against PAI-1, as well as the anti-PAI-1-depleted IgG preparations, were diluted in Antibody Diluent (S809, DakoCytomation) and incubated at 0.5 μg/ml (if not otherwise stated) overnight at 4°C in a humidifying chamber, followed by ∼1 hour at room temperature. Primary antibodies were detected with anti-rabbit IgG-horseradish peroxidase-conjugated polymers (EnVision rabbit reagent, K4003; DakoCytomation). Each antibody incubation step was followed by thorough washes in TBS-T. Finally, sections were developed for 15 minutes with either 3-amino-9-ethylcarbazole (Sigma A-5754) or NovaRed (Vector Laboratories, Burlingame, CA), resulting in red staining. Finally Mayer's hematoxylin was used for nuclear counterstaining. The monoclonal antibody against PAI-1 (clone 380) was used in some experiments. Antigen retrieval was in this case performed by boiling in citrate buffer, pH 6.0, for 10 minutes.31Bianchi E Cohen RL Dai A Thor AT Shuman MA Smith HS Immunohistochemical localization of the plasminogen activator inhibitor-1 in breast cancer.Int J Cancer. 1995; 60: 597-603Crossref PubMed Scopus (60) Google Scholar Clone 380 and a monoclonal antibody against trinitrophenyl hapten (TNP44Shulman M Wilde CD Kohler G A better cell line for making hybridomas secreting specific antibodies.Nature. 1978; 276: 269-270Crossref PubMed Scopus (1123) Google Scholar) used as negative control (both antibodies are IgG1 subtype), were both used at 5 μg/ml and incubated overnight at 4°C and detected with Envision mouse reagent (K4003, DakoCytomation), followed by tyramine signal amplification using biotinyl tyramine (NEN, Boston, MA) according to the manufacturer's instructions. All other steps were as for the rabbit polyclonal antibodies specified above. Frozen sections (5-μm cryostat sections) were immediately dried on a heating plate at 60°C for 2 minutes and fixed in neutral-buffered formalin overnight at 4°C. After thorough washes in water, the sections were heat-treated at 99°C for 3 minutes in TEG-buffer using a Micromed T/T microwave processor (Milestones, Sorisol, Italy) and subsequently retempered for 30 minutes at room temperature. Endogenous peroxidase was blocked by 15 minutes of incubation in 1% H2O2. Sections were incubated with 0.5 μg/ml of AB-2A for 2 hours and subsequently with EnVision rabbit reagent for 30 minutes at room temperature. Sections were developed with NovaRed chromogene as specified above for the paraffin sections. Four-μm paraffin sections were hydrated and boiled in TEG buffer as specified above. Primary antibodies aga
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