Differential Expression of Laminin Isoforms in Ovarian Epithelial Carcinomas Suggesting Different Origin and Providing Tools for Differential Diagnosis
Department of Pathology (MM,HA-H), Collagen Research Unit, Biocenter Oulu, Department of Medical Biochemistry and Molecular Biology (JL,TP), and Department of Biochemistry (SS), University of Oulu, Oulu, Finland; Department of Pathology, HUSLAB, Haartman Institute and Department of Obstetrics and Gynecology, Research Laboratory, Biomedicum, Helsinki University Central Hospital, Finland (RB); Institute of Biomedicine/Anatomy, University of Helsinki, Helsinki, Finland (NP,IV); and Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Maioka-cho, Totsuka-ku, Yokohama, Japan (KM)
Correspondence to: Marko Määttä, MD, PhD, Department of Ophthalmology, University of Helsinki, PO Box 220, 00029 HUS Helsinki, Finland. E-mail: mmaatta{at}mailcity.com
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Summary |
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(J Histochem Cytochem 53:12931300, 2005)
Key Words: basement membrane carcinoma collagen invasion laminin Lutheran ovary
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Introduction |
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It has been considered that the ovarian surface epithelial (OSE) cell layer plays a central role in vitro and in vivo as a precursor for development of ovarian carcinomas (Auersperg et al. 2001; Feeley and Wells 2001
). It is not fully clear which factors take part in the tumor development clinically seen as a four main histological tumor typesnamely serous, mucinous, endometrioid, and clear cell adenocarcinomas. OSE presents many typical epithelial features, such as cytokeratin filaments, mucin, desmosomes, and apical microvilli, but also simultaneously mesenchymal properties including vimentin filaments and N-cadherin (Auersperg et al. 2002
). Based on multipotent features, OSE has been suggested to represent an immature precursor and therefore would be susceptible to mutations (Feeley and Wells 2001
; Auersperg et al. 2002
). It has also been suggested that, during tumor development, these features allow OSE to express properties related to fallopian tube epithelium (serous tumors), endocervix or colonic epithelium (mucinous tumors), endometrium (endometrioid tumors), or urogenital tract (clear cell tumors) (Van Niekerk et al. 1993
, Auersperg et al. 2002
). However, it has also suggested that OSE may not fully correspond to all premalignant lesions of the ovary and that the secondary Müllerian system would also have contribution (Dubeau 1999
).
In the beginning of the neoplastic process, surface epithelium or inclusion cyst becomes stratified and adenomas are formed (Resta et al. 1993; Scully 1995
). At this stage, BM is usually still continuous (Visser et al. 1995
). BMs form natural barriers for tumor cell invasion, and, after malignant change, ovarian epithelial BMs often show variable disruptions or even total loss (Kuwashima et al. 1995
; Skubitz et al. 1996
; Capo-Chichi et al. 2002
).
Specific receptors, such as integrins and the dystroglycan glycoprotein complex, mediate cell adhesion to the BM macromolecules (Belkin and Stepp 2000). Previously, it has been shown that
6ß4 integrin, which normally mediates adhesion of basal epithelial cells to the underlying BM, is decreased or simultaneously lost with Ln and type IV collagen in tumor nests of invasive ovarian carcinomas (Skubitz et al. 1996
). Lutheran blood group glycoprotein (Lu), a member of immunoglobulin superfamily, is a recently described receptor for Ln
5 chain-containing heterotrimers, Ln-10 (
5ß1
1) and Ln-11 (
5ß2
1) (Parsons et al. 1995
; Udani et al. 1998
). Lu binds to LG3 module of G-domain of the long arm of Ln
5 chain (Parsons et al. 2001
; Kikkawa et al. 2002
), is widely distributed in normal tissues, and localizes to the basal aspect of the cells. However, a solely nonpolarized distribution has previously been reported in ovarian tumors (Gabin-Chesa et al. 1994
; Campbell et al. 1994
).
In this study, we wanted to clarify the immunohistochemical profile of BM proteins in different types of ovarian adenocarcinomas and to relate the results to the distribution of these components in the normal OSE. The results show that different ovarian carcinomas differ from each other, which may serve as diagnostic help in distinguishing serous, mucinous, and endometrioid adenocarcinomas from each other.
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Materials and Methods |
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Antibodies and Immunohistochemistry
The antibodies employed for this study are presented in Table 1. Frozen samples were fixed in cold acetone for 10 min, and all the steps in the immunoreactivity procedures were followed by washes with PBS. Sections were incubated with fetal calf serum (1:5; Hyclone Laboratories, Inc., Logan, UT) in PBS for 20 min; with primary antibody or, for negative controls, with PBS for 1 hr. Two methods were used: avidin-biotin-peroxidase and alkaline-phosphatase-anti-alkaline-phosphatase. In avidin-biotin method biotinylated secondary antibody (Dako; Glostrup, Denmark) was used for 30 min; avidin-peroxidase complex was used for 30 min. The color was developed with diaminobenzidine tetrahydrochloride-H2O2 (Sigma; St Louis, MO). In the alkaline-phosphatase-anti-alkaline-phosphatase method, the reaction was visualized with a substrate solution containing naphthol-AS-BI-phosphate, levamisole, and new fuchsin (all from Sigma). Finally, all sections were counterstained with hematoxylin.
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Results |
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Serous Carcinomas
Chains of Lns-10 5 (Figure 2A), ß1 (Figure 2B), and
1 (Figure 2C); chains of Ln-5
3 (Figure 2D), ß3, and
2 (Figure 2E); and collagen types IV and XVIII (Figure 2F) were found in BMs of practically all tumors (15/1818/18). Their immunoreactivities were more constant and linear in grade I tumors than in grade II-III tumors; all except one grade I tumor showed a moderate or high BM linearity according to the scoring, but none of the grade III tumors did. A distinct immunoreactivity in the stroma was occasionally found for Ln ß1, ß2, and
2 chains and collagen types IV and XVIII. Interestingly, immunoreactivities for Ln ß1 and ß2 chains appeared to be reciprocally expressed in the specimens. Ln chains
2 (4/18 cases),
4 (Figure 2G) (5/18 cases),
1 (Figure 2H) (8/18 cases), and collagen type VII (Figure 2I) (7/18 cases) were found only in some of the tumors. In some of the tumors, Ln
2 chain was usually found only as a short, faint strip around tumor cell islands and was also expressed in the stroma. Similarly, Ln
4 chain immunoreactivity was detected, together with tumor BMs, exclusively and diffusely in the tumor stroma per se and endothelial BMs (Figure 2G). Immunoreactivities for Ln
1 chain (Figure 2H) and type VII collagen (Figure 2I) were solely confined to epithelial BM. Type VII collagen immunoreactivity was mainly found in grade I tumors, because only two of the tumors represented grade III tumors.
Mucinous Carcinomas
Ln chains 3,
5, ß1 (Figure 3A), ß3 (Figure 3B),
1, and
2 (Figure 3C) and collagen types IV (Figure 2D) and XVIII were found in BMs of most or all mucinous tumors. Contrary to serous tumors, immunoreactivity for Ln ß2 chain (Figure 3E) was detected in BMs of two tumors. Ln
4 chain (Figure 3F) showed a constant immunoreactivity in tumor epithelial BMs (8/8 cases) and was also abundantly found in the tumor stroma per se and in vascular endothelium BMs. Immunoreactivity for Ln
1 (2/8 cases) and
2 (0/8 cases) chains and collagen type VII (2/8 cases) could not be detected in most mucinous carcinomas.
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Lutheran
Most of the cases 33/36 (92%) showed immunoreactivity for Lu, being found either in a polarized manner localizing to the basal aspect of the tumor cells (Figure 5A) or in a more diffuse manner on cell surfaces (Figure 5B). These two patterns of immunoreactivities were usually simultaneously present. A solely polarized immunoreactivity was found in 7 tumors and a mainly diffuse cell surface distribution was detected in 20 tumors. The central parts of tumor islands lacked Lu immunoreactivity. Well-differentiated serous carcinomas tended to show the most intense overall Lu expression, followed by endometrioid carcinomas, but mucinous tumors showed only an occasional immunoreactivity. Three tumors lacked Lu-immunoreactivity when Ln 5 chain was present, but no Lu lacking cases when Ln
5 chain was not present. Lu immunoreactivity was constantly present in the vascular endothelium and on stromal fibroblasts.
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Discussion |
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The present study shows immunoreactivity for several different Ln -chains in BMs of ovarian carcinomas, suggesting the presence of more than one Ln heterotrimer in the tumor BMs. We have previously shown that Ln isoforms found in tumors usually reflect the BM composition of their normal tissue of origin (Määttä et al. 2001
; Rissanen et al. 2003
); when comparing the results obtained from tumors and OSE, this is likely true also in the case of ovary (Table 2). However, there were some differences; for instance, Ln
2 chain and collagen type VII were not detected in BM of OSE, whereas they were present in variable amounts in BM of ovarian carcinomas. This may represent a true neoexpression, as has been reported for some other tumor types (Lohi et al. 1998
; Catusse et al. 2000
), or to be related to the origin of the tumor (as previously discussed). Collagen type VII normally localizes to the anchoring fibrils together with Ln-5, but in ovarian carcinomas, such a colocalization was not observed and it was mainly confined to the well-differentiated serous carcinomas. Lack of type VII collagen in tumor BMs has earlier been reported by Tani et al. (1996)
and Catusse et al. (2000)
. This might reduce adhesion of the carcinoma cells to the BMs and hence enhance the ability of Ln-5 to promote cell migration (Katayama and Sekiguchi 2004
).
Chains of laminin-10 and collagens type IV and XVIII were found in BMs of most tumors in all three ovarian carcinomas. Chains of Ln-5 were detected in BMs of serous and mucinous tumors, but only inconsistently in endometrioid tumors. We have previously reported that chains of Ln-10 can be found in most carcinomas (Määttä et al. 2001). In serous carcinomas, the presence of a pronounced Ln ß2 chain immunoreactivity instead of Ln ß1 chain suggested the presence of Ln-11 in these tumors.
Our results show that there are differences in the laminin composition of BMs among the three ovarian tumor types. The BMs of mucinous carcinomas were characterized by the constant presence of Ln 4 chain, suggesting the presence of mostly Ln-8 (
4ß1
1), because only occasional immunoreactivity for Ln ß2 chain was found. Ln
4 chain was also found in the tumor stroma and vascular BMs, in line with the previous studies (Miner et al. 1997
; Petäjäniemi et al. 2002
). Inflammatory cells may also be a significant source for this chain (Geberhiwot et al. 2001
). Interestingly, in vitro Ln
4 chain is poorly adhesive, but effectively stimulates cell migration (Fujiwara et al. 2001
), a feature that may affect the metastatic potential of carcinomas of the ovary. In this respect, it is of interest that Ljubimova et al. (2001)
recently showed that
4 chain is upregulated in human gliomas and antisense inhibition of Ln
4 chain resulted in effective invasion block by glioma cells (Khazenzon et al. 2003
).
We also found that Ln 1 chain was constantly present in BMs of endometrioid carcinomas in contrast to findings on serous and mucinous carcinomas. Endometrioid tumors are characterized by endometriosis in significant numbers of cases, and deep endometriotic deposits induce metaplastic Müllerian transformation of adjacent mesenchymal cells (Van Niekerk et al. 1993
; Leyendecker et al. 1998
). We found that normal OSE contains Ln
1 chain, which suggests that endometrioid adenocarcinoma might be derived from OSE. However, it is also known that endometrial surface epithelium and glandular BM contain Ln
1 chain (Virtanen et al. 2000
), which would therefore also support the implantation theory (for review, see Leyendecker et al. 1998
). Additionally, it has been noted that endometrioid carcinomas of the ovary and endometrium are often positive for vimentin and carcinoembryonic antigen, contrary to endometrioid carcinomas of endocervix, which are usually negative (Dabbs et al. 1996
). Together, these differences may help clinically to detect the origin of the tumor.
We decided to include Lu glycoprotein in the study because it is known to effectively bind to Ln 5 chaincontaining heterotrimers, which appeared to be ubiquitously present in normal and neoplastic ovary. BM of normal OSE expressed Ln
5 chain and showed a basally confined immunoreactivity for Lu. In carcinomas, however, Lu showed either a basally confined immunoreactivity or a more diffuse cell surface distribution. There were also three cases that totally lacked Lu immunoreactivity, but presented immunoreactivity for Ln
5 chain. Together our results implicate that during tumorigenesis Lu loses its normal polarized expression in significant amounts of ovarian carcinomas, which indirectly suggests that it also simultaneously may lose its function as a receptor for Ln
5 chaincontaining heterotrimers. When comparing our results with those previously reported by Gabin-Chesa et al. (1994)
, the overall expression was similar (87% vs our 92%), but we found Lu to be mostly localized to basal epithelial cells, whereas they reported that ovarian carcinomas show mainly uniform staining pattern over the tumor area. Many ECM receptor alterations have been observed in ovarian carcinomas, of which some are reported to have decreased expression, whereas others are upregulated in tumor tissues (Skubitz et al. 1996
; Strobel and Cannistra 1999
; Goldberg et al. 2001
; Skubitz 2002
). Nonpolarized Lu expression, together with changes in other ECM receptors, may modulate tumor cell adhesion to BMs and influence on migration of tumor cells.
In conclusion, the present results show that ovarian carcinomas present BMs around tumor cell nests consisting mostly of Ln-5 and Ln-10 and collagen types IV and XVIII. Mucinous tumors were characterized by immunoreactivity for Ln 4 chain, whereas BMs of endometrioid tumors were found to have, on the other hand, distinct immunoreactivity for Ln
1 chain. These differences suggest that the latter two tumor types have distinct features that may have a different histogenetic origin or differentiation pattern compared with serous tumors. These features may serve a diagnostic tool to distinguish these carcinoma types from each other.
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Acknowledgments |
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The antibody to the Ln ß2 chain developed by Dr. Dale Hunter and Dr. Joshua Sanes was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, Iowa.
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Footnotes |
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Literature Cited |
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Auersperg N, Ota T, Mitchell GW (2002) Early events in ovarian epithelial carcinogenesis: progress and problems in experimental approaches. Int J Gynecol Cancer 12:691703[CrossRef][Medline]
Auersperg N, Wong AS, Choi KC, Kang SK, Leung PC (2001) Ovarian surface epithelium: biology, endocrinology, and pathology. Endocrine Rev 22:255288
Barsky SH, Siegal GP, Jannotta F, Liotta LA (1983) Loss of basement membrane components by invasive tumors but not by their benign counterparts. Lab Invest 49:140147[Medline]
Belkin AM, Stepp MA (2000) Integrins as receptors for laminins. Microsc Res Tech 51:280301[CrossRef][Medline]
Campbell IG, Foulkes WD, Senger G, Trowsdale J, Garin-Chesa P, Rettig WJ (1994) Molecular cloning of the B-CAM cell surface glycoprotein of epithelial cancers: a novel member of the immunoglobulin superfamily. Cancer Res 54:57615765[Abstract]
Capo-Chichi CD, Smith ER, Yang DH, Roland IH, Vanderveer L, Cohen C, Hamilton TC, et al. (2002) Dynamic alterations of the extracellular environment of ovarian surface epithelial cells in premalignant transformation, tumorigenicity, and metastasis. Cancer 95:18021815[CrossRef][Medline]
Catusse C, Polette M, Coraux C, Burlet H, Birembaut P (2000) Modified basement membrane composition during bronchopulmonary tumor progression. J Histochem Cytochem 48:663669
Colognato H, Yurchenco PD (2000) Form and function: the laminin family of heterotrimers. Dev Dyn 218:213234[CrossRef][Medline]
Dabbs DJ, Sturtz K, Zaino RJ (1996) The immunohistochemical discrimination of endometrioid adenocarcinomas. Hum Pathol 27:172177[CrossRef][Medline]
Dubeau L (1999) The cell of origin of ovarian epithelial tumors and the ovarian surface epithelium dogma: does the emperor have no clothes? Gynecol Oncol 72:437442[CrossRef][Medline]
Erickson AC, Couchman JR (2000) Still more complexity in mammalian basement membranes. J Histochem Cytochem 48:12911306
Feeley KM, Wells M (2001) Precursor lesions of ovarian epithelial malignancy. Histopathology 38:8795[CrossRef][Medline]
Fujiwara H, Kikkawa Y, Sanzen N, Sekiguchi K (2001) Purification and characterization of human laminin-8. Laminin-8 stimulates cell adhesion and migration through 3ß1 and
6ß1 integrins. J Biol Chem 276:1755017558
Gabin-Chesa P, Sanz-Moncasi M-P, Campbell IG, Rettig J (1994) Non-polarized expression of basal cell adhesion molecule B-CAM in epithelial ovarian cancers. Int J Oncol 5:12611266
Geberhiwot T, Assefa D, Kortesmaa J, Ingerpuu S, Pedraza C, Wondimu Z, Charo J, et al. (2001) Laminin-8 (4ß1
1) is synthesized by lymphoid cells, promotes lymphocyte migration and co-stimulates T cell proliferation. J Cell Sci 114:423433
Goldberg I, Davidson B, Reich R, Gotlieb WH, Ben-Baruch G, Bryne M, Berner A, et al. (2001) v integrin expression is a novel marker of poor prognosis in advanced-stage ovarian carcinoma. Clin Cancer Res 7:40734079
Katayama M, Sekiguchi K (2004) Laminin-5 in epithelial tumour invasion. J Mol Histol 35:277286[Medline]
Khazenzon NM, Ljubimov AV, Lakhter AJ, Fujita M, Fujiwara H, Sekiguchi K, Sorokin LM, et al. (2003) Antisense inhibition of laminin-8 expression reduces invasion of human gliomas in vitro. Mol Cancer Ther 2:985994
Kikkawa Y, Moulson CL, Virtanen I, Miner JH (2002) Identification of the binding site for the Lutheran blood group glycoprotein on laminin alpha 5 through expression of chimeric laminin chains in vivo. J Biol Chem 277:4486444869
Kuwashima Y, Uehara T, Kurosumi M, Kishi K, Shiromizu K, Matsuzawa M, Takayama S (1995) Basement membrane status in undifferentiated carcinomas of the ovary. Immunohistochemical distribution of type IV collagen and laminin. Eur J Gynaecol Oncol 16:181186[Medline]
Leyendecker G, Kunz G, Noe M, Herbertz M, Mall G (1998) Endometriosis: a dysfunction and disease of the archimetra. Hum Reprod Update 4:752762
Ljubimova JY, Lakhter AJ, Loksh A, Yong WH, Riedinger MS, Miner JH, Sorokin LM, et al. (2001) Overexpression of alpha4 chain-containing laminins in human glial tumors identified by gene microarray analysis. Cancer Res 61:56015610
Lohi J, Leivo I, Owaribe K, Burgeson RE, Franssila K, Virtanen I (1998) Neoexpression of the epithelial adhesion complex antigens in thyroid tumours is associated with proliferation and squamous differentiation markers. J Pathol 184:191196[CrossRef][Medline]
Määttä M, Virtanen I, Burgeson RE, Autio-Harmainen H (2001) Comparative analysis of the distribution of laminin chains in the basement membranes in some malignant epithelial tumors: the 1 chain of laminin shows a selected expression pattern in human carcinomas. J Histochem Cytochem 49:711726
Miner JH, Patton BL, Lentz SI, Gilbert DJ, Snider WD, Jenkins NA, Copeland NG, et al. (1997) The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha15, identification of heterotrimeric laminins 811, and cloning of a novel alpha3 isoform. J Cell Biol 137:685701
Miner JH, Yurchenco PD (2004) Laminin functions in tissue morphogenesis. Annu Rev Cell Dev Biol 20:255284[CrossRef][Medline]
Parsons SF, Lee G, Spring FA, Willig TN, Peters LL, Gimm JA, Tanner MJ, et al. (2001) Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind alpha5 chain-containing human laminin with high affinity. Blood 97:312320
Parsons SF, Mallinson G, Holmes CH, Houlihan JM, Simpson KL, Mawby WJ, Spurr NK, et al. (1995) The Lutheran blood group glycoprotein, another member of the immunoglobulin superfamily, is widely expressed in human tissues and is developmentally regulated in human liver. Proc Natl Acad Sci USA 92:54965500
Patarroyo M, Tryggvason K, Virtanen I (2002) Laminin isoforms in tumor invasion, angiogenesis and metastasis. Semin Cancer Biol 12:197207[CrossRef][Medline]
Petäjäniemi N, Korhonen M, Kortesmaa J, Tryggvason K, Sekiguchi K, Fujiwara H, Sorokin L, et al. (2002) Localization of laminin alfa-4-chain in developing and adult human tissues. J Histochem Cytochem 50:11131130
Resta L, Russo S, Colucci GA, Prat J (1993) Morphologic precursors of ovarian epithelial tumors. Obstet Gynecol 82:181186[Abstract]
Rissanen J, Korhonen M, Lehto VP, Virtanen I (2003) Laminin alpha1 chain in human renal cell carcinomas and integrin-mediated adhesion of renal cell carcinoma cells to human laminin isoforms. J Pathol 200:157167[CrossRef][Medline]
Rousselle P, Lunstrum GP, Keene DR, Burgeson RE (1991) Kalinin: an epithelium specific basement membrane adhesion molecule that is a component of anchoring filaments. J Cell Biol 114:567576[Abstract]
Scully RE (1995) Pathology of ovarian cancer precursors. J Cell Biochem Suppl 23:208218[Medline]
Skubitz AP (2002) Adhesion molecules. Cancer Treat Res 107:305329[Medline]
Skubitz AP, Bast RC Jr, Wayner EA, Letourneau PC, Wilke MS (1996) Expression of alpha 6 and beta 4 integrins in serous ovarian carcinoma correlates with expression of the basement membrane protein laminin. Am J Pathol 148:14451461[Abstract]
Stenback F, Wasenius VM (1985) Basement membrane structures in tumors of the ovary. Eur J Obstet Gynecol Reprod Biol 20:357371[CrossRef][Medline]
Strobel T, Cannistra SA (1999) ß1-integrins partly mediate binding of ovarian cancer cells to peritoneal mesothelium in vitro. Gynecol Oncol 73:362367[CrossRef][Medline]
Tani T, Karttunen T, Kiviluoto T, Kivilaakso E, Burgeson RE, Sipponen P, Virtanen I (1996) 6ß4 integrin and newly deposited laminin-1 and laminin-5 form the adhesion mechanism of gastric carcinoma. Continuous expression of laminins but not that of collagen VII is preserved in invasive parts of the carcinomas: implications for acquisition of the invading phenotype. Am J Pathol 149:781793[Abstract]
Udani M, Zen Q, Cottman M, Leonard N, Jefferson S, Daymont C, Truskey G, et al. (1998) Basal cell adhesion molecule/lutheran protein. The receptor critical for sickle cell adhesion to laminin. J Clin Invest 101:25502558
Van Niekerk CC, Ramaekers FC, Hanselaar AG, Aldeweireldt J, Poels LG (1993) Changes in expression of differentiation markers between normal ovarian cells and derived tumors. Am J Pathol 142:157177[Abstract]
Virtanen I, Gullberg D, Rissanen J, Kivilaakso E, Kiviluoto T, Laitinen LA, Lehto V-P, et al. (2000) Laminin -1 chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues. Exp Cell Res 257:298309[CrossRef][Medline]
Visser R, Arends JW, Bosman FT (1995) Basement membrane patterns in borderline tumors of the ovary. An immunohistochemical study with antibodies to laminin and type IV collagen. Int J Gynecol Cancer 5:286290[CrossRef][Medline]
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