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115 Plasminogen activation in melanoma: An immuno-histochemical study
SESSION IX. Physiology and Disease Posters
116
115 PLASMINOGEN ACTIVATION HISTOCHEMICAL STUDY
IN
MELANOMA;
AN
IMMUNO-
Cilia Ferrier, Winny van Geloof, Huub Straatman, Goos van Muijen, Stefan Suciu*, Dirk Ruiter Department of Pathology, University Hospital Nijmegen, The Netherlands *EORTC Data Center, Brussels, Belgium The plasminogen activation system (PA-system) is a proteolysis-controlling network which is involved in invasion and metastasis in various types of tumors including melanoma. Earlier studies from our group (Am J Pathol 1994, 144:70-81) investigating mRNA and antigen expression of the various PA-components on frozen sections of all types of melanocytic lesions showed that expression could not be detected in all non-malignant and early malignant stages but appeared frequently in advanced primary melanoma and melanoma metastasis lesions. To study a possible prognostic value of PA--eomponents expression in melanoma, we investigated primary melanomas from one hundred patients with welldocumented follow-up. After a systematic analysis of the sensitivity and specificity of immunohistocbemical stainings for the five PA-components on formalin-fixed and paraffin-embedded tissue, these routinely processed primary melanomas were stained for all PA-components. The lesions were scored semi-quantitatively. By linkage of staining results with follow-up data, we found that PAI-2 tumor cell positivity may be an independent parameter of favorable prognosis, uPA, PAL1 and uPAR tumor cell positivity were also related to survival.
ELISA DETERMINATIONOF THE COMPLEX BETWEEN UROKINASE PLASMINOGEN ACTIVATOR AND ITS TYPE-I INHIBITOR IN TUMOR EXTRACTS Ander~N. Pedersen i, Gunilla H~yer-Hansen ', Nils BrQnner", Gary M. Clark b, Birthe Larsen ', Hans S. Poulsen c, Keid Dano °, Ross W. Stephens" ' Finsen Laboratory, Strandboulevarden 49, 2100 Copenhagen O, DK bDept. of Med.,UTHSC at S. Antonio, 7703 Floyd Curl Drive, TX 78284-7884 ° Dept. of Oncology, Rigsbospitalet, Blegdamsvej 9, 2100 Copenhagen O, DK ELISAs for urokinase plasminogen activator (uPA) and for its type-1 inhibitor (PAl-l) have shown that tumor levels of these molecules are prognostic parameters in several types of cancer. These ELISAs measure the total amount of the given component, including preforms, active, inactive, and complex-bound forms. However, the amount of the active forms of a component may more closely reflect the ongoing level of proteolytic activity and thereby be particularly related to prognosis. Since the complex between uPA and PAI-1 can only be formed from the active forms of the individual components we have developed a specific and sensitive uPA:PAI-I complex ELISA consisting of a sandwich format with two monoclonal capture antibodies against PAl-1 and three biotinylalexi detector antibodies against uPA. The data were collected as kinetic measurements of bound alkaline phosphatase activity. A standard of uPA:PAI-1 complex could be specifically measured in the assay with a detection limit of 8 pg/ml and a linear relationship between signal and complex concentrationup to 4 ng/ml. Neither free uPA nor free PAl-1 were detected by this assay and the addition to the internal standard of free PAI-1 in amounts up to 20 ng/ml or uPA did not reduce the detection of complex by the assay. This ELISA was applied to extracts from twenty individual breast cancers. Each tumor was extracted in two different buffers and the median concentration of uPA:PAI-1 complex in the optimal extraction buffer was 0.8 ng/mg protein, range 0.4 to 2.8 ng/mg protein. Extraction of the tumor tissue at a low pH prevented in vitro formation of complex from free uPA and PAI-1 in the tissue without destabilizing preformed uPA:PAl- 1 complex. During incubation of the assay plate at neutral pH further uPA:PAI-I complex formation from free components in the extracts was blocked by pnitrophenyl guanidinobenzoate. Thus, the present assay selectively quantifies in vivo formed complex in tumor extracts and the results of measurements of breast, lung, and brain tumor tissues will be presented.
117
118
ISOLATION AND CHARACTERIZATION OF GENE EXPRESSION IN NONMETASTASIZING VERSUS METASTASIZING HUMAN BREAST CANCER CELLS.
POOR DIFFERENTIATION IN MALIGNANT SEROUS OVARIAN TUMORS IS ACCOMPANIED BY STROMAL EXPRESSION OF THE UROKINASE PLASMINOGEN ACTIVATOR Chfister Borgfel_~, Bettil Cassltn, Iagegerd Lecander. Department of Obstetrics and Gynecology, University Hospital, Lued, Sweden,
1 IKiilgaard, J.F., IFrandsen, T,L., 2Fuqua, S., 3Clarke, R., IBrtinner, ~ . , N.. Fiusen Laboratory, Copenhagen University Hospital, DK. 2UTHSC, San Antonio, TX. 3Lombardi Cancer Center, DC, USA.
We have isolated a number of subelones from the human MCF-7 breast cancer cell line, with different metastatic potential together with various degrees of endocrine resistance. The subelones were selected for progression in vivo in nude mice, as well as in vitro by selecting MCF-7 cells for growth in the absence of estrogen (E2). The phenotype of the subelones range from the non-metastatic hormone dependent cell line to a multi hormone resistant and metastatic cell line (phenntypes of cell lines shown in table' E2 Metastatic 4-OHTAM" ICI 182.780" MCF-7
Dependent
Sensitive
Sensitive
MCF-7/LCCI
Independent
+
Sensitive
Sensitive
MCF-7/LCC2
Independent
++
Resistant
Sensitive
MCF-7/LCC9 Independent + Resistant Resistant "Anti-estrogen In order to investigate genes involved in metastasizing and endocrine resistance the technique of Differential Display (comparison of eDNA, amplified by PCR, from several cell lines) was used. By this technique it was possible to detect a number of differentially expressed mRNAs. We isolated a eDNA (MCF7/LCC9p2) that was expressed in the metastasizing MCF-7/LCC9 cell line and not in the non-invasive MCF-7 ceil line. This finding has been verified beth on mRNA and Protein level by KT-PCR and Western blotting. By using the sequence information from the eDNA, it was possibly to clone a full length human sequence of the gene by RACE techniques. Search in Genebank revealed a homolog gene cloned from a regenerating rat liver. Analysis of the umino acid composition reveals several hydrophobie domains which indicate that the protein could be membrane bound. This theory is also supported by the fact, that is was only possible to detect the protein by western analysis in the detergent phase. The function of the gene is so far unknown but it could be involved in the mechanism behind invasion or in the resistance against anti-estrogens.
Background: The urokinase plasminogen activator (u-PA) has a pivotal role in tumor stroma degradation, tumor spread and metastasis. The expression ofuPA and its receptor is increased in ovarian tumors. U-PA stimulates signal transduction leading to cell proliferation and migration in certain ceils. Materials and methods: Tissue samples from 26 serous ovarian tumors were formalin fixed or frozen. Immuno histochemistry (IHC) using monoclonal antibodies localized the u-PA antigen (American Diagnostica #3689, and our own 8E7) and human maerophages (CD 68 DAKO). Aspergillus niger (lgG1 DAKO) was used as negative control. This was followed by preformed ABCcomplex, u-PA m-RNA was detected by in situ hybridisation with 35S-laballed cRNA-antisense-prnbe. The corresponding sense probe was used as control. Results: The m-RNA u-PA expression was almost exclusivelypositive in the epithelium of benign, borderline and well differentiated tumors, however stronger in the malignant tumors. The IHC staining for u-PA antigen was also positive in the epithelium and in occasional stromal cells. Intermediately differentiated tumors showed m-RNA u-PA expression in epithelium and stroma. Poorly differentiated tumors and metastasis had strong m-RNA u-PA expression in the stroma, whereas it was weak or absent in the epithelium. The 1HC staining for u-PA antigen was positive in the epithelium and to some extent in the stroma. In consecutive sections the macrophages corresponded with some oftbe m-RNA u-PA expression but could not explain all of it. Conclusions:. Stromal u-PA may partly be related to macrophages, but is also likely to be produced by regular stromal cells, u-PA produced by the stromal cells may bind to receptors either on these cells or on other cells, like the cancer cells as well as capillary endothelial ceils, which would imply potential paracrine stimulation of prnteolysis, migration or proliferation in these ceils.
33
116
115 PLASMINOGEN ACTIVATION HISTOCHEMICAL STUDY
IN
MELANOMA;
AN
IMMUNO-
Cilia Ferrier, Winny van Geloof, Huub Straatman, Goos van Muijen, Stefan Suciu*, Dirk Ruiter Department of Pathology, University Hospital Nijmegen, The Netherlands *EORTC Data Center, Brussels, Belgium The plasminogen activation system (PA-system) is a proteolysis-controlling network which is involved in invasion and metastasis in various types of tumors including melanoma. Earlier studies from our group (Am J Pathol 1994, 144:70-81) investigating mRNA and antigen expression of the various PA-components on frozen sections of all types of melanocytic lesions showed that expression could not be detected in all non-malignant and early malignant stages but appeared frequently in advanced primary melanoma and melanoma metastasis lesions. To study a possible prognostic value of PA--eomponents expression in melanoma, we investigated primary melanomas from one hundred patients with welldocumented follow-up. After a systematic analysis of the sensitivity and specificity of immunohistocbemical stainings for the five PA-components on formalin-fixed and paraffin-embedded tissue, these routinely processed primary melanomas were stained for all PA-components. The lesions were scored semi-quantitatively. By linkage of staining results with follow-up data, we found that PAI-2 tumor cell positivity may be an independent parameter of favorable prognosis, uPA, PAL1 and uPAR tumor cell positivity were also related to survival.
ELISA DETERMINATIONOF THE COMPLEX BETWEEN UROKINASE PLASMINOGEN ACTIVATOR AND ITS TYPE-I INHIBITOR IN TUMOR EXTRACTS Ander~N. Pedersen i, Gunilla H~yer-Hansen ', Nils BrQnner", Gary M. Clark b, Birthe Larsen ', Hans S. Poulsen c, Keid Dano °, Ross W. Stephens" ' Finsen Laboratory, Strandboulevarden 49, 2100 Copenhagen O, DK bDept. of Med.,UTHSC at S. Antonio, 7703 Floyd Curl Drive, TX 78284-7884 ° Dept. of Oncology, Rigsbospitalet, Blegdamsvej 9, 2100 Copenhagen O, DK ELISAs for urokinase plasminogen activator (uPA) and for its type-1 inhibitor (PAl-l) have shown that tumor levels of these molecules are prognostic parameters in several types of cancer. These ELISAs measure the total amount of the given component, including preforms, active, inactive, and complex-bound forms. However, the amount of the active forms of a component may more closely reflect the ongoing level of proteolytic activity and thereby be particularly related to prognosis. Since the complex between uPA and PAI-1 can only be formed from the active forms of the individual components we have developed a specific and sensitive uPA:PAI-I complex ELISA consisting of a sandwich format with two monoclonal capture antibodies against PAl-1 and three biotinylalexi detector antibodies against uPA. The data were collected as kinetic measurements of bound alkaline phosphatase activity. A standard of uPA:PAI-1 complex could be specifically measured in the assay with a detection limit of 8 pg/ml and a linear relationship between signal and complex concentrationup to 4 ng/ml. Neither free uPA nor free PAl-1 were detected by this assay and the addition to the internal standard of free PAI-1 in amounts up to 20 ng/ml or uPA did not reduce the detection of complex by the assay. This ELISA was applied to extracts from twenty individual breast cancers. Each tumor was extracted in two different buffers and the median concentration of uPA:PAI-1 complex in the optimal extraction buffer was 0.8 ng/mg protein, range 0.4 to 2.8 ng/mg protein. Extraction of the tumor tissue at a low pH prevented in vitro formation of complex from free uPA and PAI-1 in the tissue without destabilizing preformed uPA:PAl- 1 complex. During incubation of the assay plate at neutral pH further uPA:PAI-I complex formation from free components in the extracts was blocked by pnitrophenyl guanidinobenzoate. Thus, the present assay selectively quantifies in vivo formed complex in tumor extracts and the results of measurements of breast, lung, and brain tumor tissues will be presented.
117
118
ISOLATION AND CHARACTERIZATION OF GENE EXPRESSION IN NONMETASTASIZING VERSUS METASTASIZING HUMAN BREAST CANCER CELLS.
POOR DIFFERENTIATION IN MALIGNANT SEROUS OVARIAN TUMORS IS ACCOMPANIED BY STROMAL EXPRESSION OF THE UROKINASE PLASMINOGEN ACTIVATOR Chfister Borgfel_~, Bettil Cassltn, Iagegerd Lecander. Department of Obstetrics and Gynecology, University Hospital, Lued, Sweden,
1 IKiilgaard, J.F., IFrandsen, T,L., 2Fuqua, S., 3Clarke, R., IBrtinner, ~ . , N.. Fiusen Laboratory, Copenhagen University Hospital, DK. 2UTHSC, San Antonio, TX. 3Lombardi Cancer Center, DC, USA.
We have isolated a number of subelones from the human MCF-7 breast cancer cell line, with different metastatic potential together with various degrees of endocrine resistance. The subelones were selected for progression in vivo in nude mice, as well as in vitro by selecting MCF-7 cells for growth in the absence of estrogen (E2). The phenotype of the subelones range from the non-metastatic hormone dependent cell line to a multi hormone resistant and metastatic cell line (phenntypes of cell lines shown in table' E2 Metastatic 4-OHTAM" ICI 182.780" MCF-7
Dependent
Sensitive
Sensitive
MCF-7/LCCI
Independent
+
Sensitive
Sensitive
MCF-7/LCC2
Independent
++
Resistant
Sensitive
MCF-7/LCC9 Independent + Resistant Resistant "Anti-estrogen In order to investigate genes involved in metastasizing and endocrine resistance the technique of Differential Display (comparison of eDNA, amplified by PCR, from several cell lines) was used. By this technique it was possible to detect a number of differentially expressed mRNAs. We isolated a eDNA (MCF7/LCC9p2) that was expressed in the metastasizing MCF-7/LCC9 cell line and not in the non-invasive MCF-7 ceil line. This finding has been verified beth on mRNA and Protein level by KT-PCR and Western blotting. By using the sequence information from the eDNA, it was possibly to clone a full length human sequence of the gene by RACE techniques. Search in Genebank revealed a homolog gene cloned from a regenerating rat liver. Analysis of the umino acid composition reveals several hydrophobie domains which indicate that the protein could be membrane bound. This theory is also supported by the fact, that is was only possible to detect the protein by western analysis in the detergent phase. The function of the gene is so far unknown but it could be involved in the mechanism behind invasion or in the resistance against anti-estrogens.
Background: The urokinase plasminogen activator (u-PA) has a pivotal role in tumor stroma degradation, tumor spread and metastasis. The expression ofuPA and its receptor is increased in ovarian tumors. U-PA stimulates signal transduction leading to cell proliferation and migration in certain ceils. Materials and methods: Tissue samples from 26 serous ovarian tumors were formalin fixed or frozen. Immuno histochemistry (IHC) using monoclonal antibodies localized the u-PA antigen (American Diagnostica #3689, and our own 8E7) and human maerophages (CD 68 DAKO). Aspergillus niger (lgG1 DAKO) was used as negative control. This was followed by preformed ABCcomplex, u-PA m-RNA was detected by in situ hybridisation with 35S-laballed cRNA-antisense-prnbe. The corresponding sense probe was used as control. Results: The m-RNA u-PA expression was almost exclusivelypositive in the epithelium of benign, borderline and well differentiated tumors, however stronger in the malignant tumors. The IHC staining for u-PA antigen was also positive in the epithelium and in occasional stromal cells. Intermediately differentiated tumors showed m-RNA u-PA expression in epithelium and stroma. Poorly differentiated tumors and metastasis had strong m-RNA u-PA expression in the stroma, whereas it was weak or absent in the epithelium. The 1HC staining for u-PA antigen was positive in the epithelium and to some extent in the stroma. In consecutive sections the macrophages corresponded with some oftbe m-RNA u-PA expression but could not explain all of it. Conclusions:. Stromal u-PA may partly be related to macrophages, but is also likely to be produced by regular stromal cells, u-PA produced by the stromal cells may bind to receptors either on these cells or on other cells, like the cancer cells as well as capillary endothelial ceils, which would imply potential paracrine stimulation of prnteolysis, migration or proliferation in these ceils.
33