Abstract
The in vitro release of matrix-degrading proteinases from breast cancer cells is associated in part with shed membrane vesicles. To determine whether shed vesicles might play a similar role in ovarian cancer cells, we analyzed the shedding phenomenon in vivo and in vitro as well as the enzymatic content of their vesicles. This is the first time that an immunoelectron microscopical analysis revealed membrane vesicles carrying tumor-associated antigen α-Folate Receptor (α-FR), circulating in biological fluids (ascites and serum) of an ovarian carcinoma patient. These vesicles were trapped in a fiber network with characteristic fibrin periodicity. An ovarian cancer cell line (CABA I) established from ascitic fluid cells of this patient, grew in Matrigel and formed tubular structures suggesting invasive capability. Immunofluorescence analysis demonstrated strong cytoplasmic staining of CABA I cells with anti-matrix metalloproteinase-9 (MMP-9) and anti-urokinase-type plasminogen activator (uPA) antibodies. CABA I cells shed membrane vesicles, which were morphologically similar to those identified in vivo, as determined by electron microscopy. Gelatin zymography of vesicles isolated both in vivo and in vitro revealed major gelatinolytic bands of the MMP family, identified as the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2). By casein-plasminogen zymography we observed high-molecular weight (HMW)-uPA and plasmin bands. Incubation of purified vesicles from CABA I cells with Matrigel led to cleavage of Matrigel components. Taken together, our results point to a possible role of shed vesicles, both in vivo and in vitro, in proteolysis that mediates invasion and spread of ovarian epithelial carcinoma cells.
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References
Young TN, Rodriguez GC, Rinehart AR et al. Characterization of gelatinases linked to extracellular matrix invasion in ovarian adenocarcinoma: purification of matrix metalloproteinase 2. Gynecol Oncol 1996; 62: 89–99.
Basbaum CB, Werb Z. Focalized proteolysis: spatial and temporal regulation of extracellular matrix degradation at the cell surface. Curr Opin Cell Biol 1996; 8: 731–8.
Nagase H. Activation mechanisms of matrix metalloproteinases. Biol Chem 1997; 378: 151–60.
Taylor DD, Black PH. Shedding of plasma membrane fragments. Neoplastic and developmental importance. In: Steinberg M (ed) Developmental Biology. New York: Plenum Press 1986; 33–57.
Doljanski F, Kapeller M. Cell surface shedding-The phenomenon and its possible significance. J Theor Biol 1976; 62: 253–70.
Dolo V, Ginestra A, Cassarà D et al. Selective localization of MMP-9, b1 integrin and HLA I molecules on membrane vesicles shed by 8701–BC breast carcinoma cells. Cancer Res 1998; 58: 4468–74.
Taylor DD, Black PH. Inhibition of macrophage Ia antigen expression by shed plasma membrane vesicles from metastatic murine melanoma cells. J Natl Cancer Inst 1995; 74: 859–63.
Tarin D. Epithelial-Mesenchymal Interactions in Carcinogenesis. London: Academic Press 1972; 228–89.
Van Blitterswijk WJ, Dever G, Krol J et al. Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemia GSRL cells. Biochim Biophys Acta 1986; 68: 495–504.
Chiba I, Jin R, Hamada J, Hosokawa M et al. Growth-associated shedding of a tumor antigen (CE7) detected by a monoclonal antibody. Cancer Res 1989; 49: 3972–75.
Dolo V, Ginestra A, Ghersi G, Nagase H, Vittorelli ML. Human breast carcinoma cells cultured in the presence of serum shed membrane vesicles rich in gelatinolytic activities. J Submicrosc Cytol Pathol 1994; 26: 173–80.
Dolo V, Adobati E, Canevari S et al. Membrane vesicles shed into the extracellular medium by human breast carcinoma cells carry tumorassociated surface antigens. Clin Exp Metastasis 1995; 13: 277–86.
Dolo V, Pizzurro P, Ginestra A et al. Inhibitory effects of vesicles shed by human breast carcinoma cells on lymphocyte 3H-thymidine incorporation, are neutralized by anti-TGF-b antibodies. J Submicrosc Cytol Pathol 1995; 2: 535–41.
Miotti S, Canevari S, Ménard S, Mezzanzanica D et al. Characterization of human ovarian carcinoma-associated antigens defined by novel monoclonal antibodies with tumor-restricted specificity. Int J Cancer 1987; 3: 297–303.
Coney LR, Tomassetti A, Carayannopoulos L et al. Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein. Cancer Res 1991; 51: 6125–32.
Cambell IG, Jones TA, Foulkes WD et al. Folate-binding protein is a marker for ovarian cancer. Cancer Res 1991; 51: 5329–38.
Mantovani LT, Miotti S, Ménard S et al. Folate binding protein distribution in normal tissues and biological fluids from ovarian carcinoma patients as detected by the monoclonal antibodies MOv18 and MOv19. Eur J Cancer 1994; 30A: 363–9.
Dolo V, Ginestra A, Violini S, Miotti S et al. Ultrastructural and phenotypic characterization of CABA I, a new human ovarian cancer cell line. Oncol Res 1997; 9: 129–38.
Thompson E, Nakamura S, Shima T et al. Supernatants of acquired immunodeficiency syndrome-related Kaposi's sarcoma cells induce endothelial cell chemotaxis and invasiveness. Cancer Res 1991; 51: 2670–1.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-day binding. Anal Biochem 1976; 72: 248–54.
Albini A, Iwamoto Y, Kleinman HK et al. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 1987; 47: 3239–45.
Albini A, Melchiori A, Garofalo A et al.Matrigel promotes retinoblastoma cell growth in vitro and in vivo. Int J Cancer 1992; 52: 234–40.
Azzam HS, Thompson E. Collagen-induced activation of the Mr 72,000 type IV collagenase in normal and malignant fibroblast cells. Cancer Res 1992; 52: 4540–4.
Festuccia C, Dolo V, Guerra F et al. Plasminogen activator system modulates invasive capability and cell proliferation in prostatic tumor cells. Clin Exp Metastasis 1998; 16(6): 513–28.
Moser TL, Young TN, Rodriguez GC et al. Secretion of extracellular matrix-degrading proteinases is increased in epithelial ovarian carcinoma. Int J Cancer 1994; 56: 552–9.
Dvorak HF, Quay SC, Orestein NS et al. Tumor shedding and coagulation. Science 1981; 212: 923–4.
Buist MR, Molthoff CF, Kenemans P et al. Distribution of OV-TL 3 and MOv18 in normal and malignant ovarian tissue. J Clin Pathol 1995; 48: 631–6.
Toffoli G, Cernigoi C, Russo A et al. Overexpression of folate binding protein in ovarian cancers. Int J Cancer 1997; 74: 193–8.
Ross JF, Chaudhuri PK, Ratnam M. Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines: Physiologic and clinical implications. Cancer 1994; 73: 2432–43.
Kleiner D Jr, Stetler-Stevenson WG. Structural biochemistry and activation of matrix metalloproteinases. Curr Opin Cell Biol 1993; 5: 891–7.
Mignatti P, Rifkin DB. Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 1993; 7: 161–95.
Matrisian LM. Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet 1990; 6: 121–5.
Zucker S, Moll UM, Lysik RM et al. Extraction of type-IV collagenase/ gelatinase from plasma membranes of human cancer cells. Int J Cancer 1990; 45: 1137–42.
Naylor MS, Stamp GW, Davies BD et al. Expression and activity of MMPS and their regulators in ovarian cancer. Int J Cancer 1994; 58: 50–6.
Ginestra A, Monea S, Seghezzi G et al. Urokinase plasminogen activator and gelatinases are associated with membrane vesicles shed by human HT1080 fibrosarcoma cells. J Biol Chem 1997; 272: 17216–221.
Zucker S, Lysik RM, Zarrabi MH et al. Mr 92,000 type IV collagenase is increased in plasma of patients with colon cancer and breast cancer. Cancer Res 1993; 53: 140–6.
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Dolo, V., D'Ascenzo, S., Violini, S. et al. Matrix-degrading proteinases are shed in membrane vesicles by ovarian cancer cells in vivo and in vitro. Clin Exp Metastasis 17, 131–140 (1999). https://doi.org/10.1023/A:1006500406240
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DOI: https://doi.org/10.1023/A:1006500406240