ARTICLE |
Correspondence to: Myriam Polette, INSERM U514, IFR 53, Unité de Biologíe Cellulaire, Laboratoire Pol Bouin, CHU Maison Blanche, 45 rue CognacgJay, 51092 Reims, France.
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Summary |
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During tumor progression, the extracellular matrix (ECM) and particularly the basement membrane (BM) appear to be dynamic structures that are not only degraded but also deposited around tumor clusters. In this study we examined by immunohistochemistry the localization of three chains of Type IV collagen (1,
3 and
5), Type VII collagen, and laminin 5 at different stages of bronchopulmonary cancers. In normal tissues,
1(IV) chain was detected in all BMs (bronchial, vascular, alveolar, and glandular),
5(IV) chain was present only in vascular BM, and laminin 5 and Type VII collagen were co-localized in bronchial and glandular BMs, whereas
3(IV) immunolabeling was totally absent from normal bronchi. In well-differentiated carcinomas,
3(IV) chain staining was found in some neosynthetized BMs interfacing the tumor cell and the stromal compartment, contrasting with the total absence of labeling in normal tissues.
1(IV) chain showed strong reactivity in all BM. Laminin 5 and Type VII collagen were also detected in neosynthetized BM. In poorly differentiated invasive cancers,
3(IV) chain and Type VII collagen were not found, whereas laminin 5 and
1(IV) chain persisted. The most important modifications in BM composition during tumor progression therefore appear to be the appearance of the
3 (IV) chain in well-differentiated carcinomas and its subsequent disappearance in poorly differentiated carcinomas, together with the loss of type VII collagen.
5(IV) chain distribution was restricted in vascular BM of well- and poorly differentiated carcinomas. These results show that the composition of BM is modified during the progression of bronchopulmonary tumor, emphasizing that the BM represents a dynamic element in tumor progression and has an important role in tumor cell invasiveness. (J Histochem Cytochem 48:663669, 2000)
Key Words: tumor invasion, type IV collagen, type VII collagen, laminin 5, basement membrane
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Introduction |
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BASEMENT MEMBRANES (BMs) are a dense meshwork of extracellular matrix (ECM) molecules such as Type IV collagen, laminins, Type VII collagen, and heparan sulfate proteoglycans. Type IV collagen is a major structural component of BMs, forming a network that allows the binding of other BM components. The specific biological functions of the diverse Type IV collagen isoforms are not yet well understood. The major form of Type IV collagen is formed by two 1(IV) chains and one
2(IV) chain and is expressed in most BMs.
3
6(IV) chains have a more restricted tissue distribution.
3(IV) and
4(IV) are co-localized in glomerular BMs, in some tubular BMs, in kidney BMs, in alveolar BMs, and in the synaptic BM at the neuromuscular junction (
5 and
6(IV) chains are also co-expressed in skin BMs, in some renal tubular BMs, in smooth muscle cells, and in adipocytes (
Many studies have demonstrated that a loss of some BM components due to degradation by proteolytic enzymes and/or a lack of biosynthesis is correlated with tumor progression. For example, the 1 chain of Type IV collagen displays a continuous labeling of the BMs lining the normal tissue, whereas this chain has an irregular distribution around tumor clusters (
1(IV) chain in tissues may be correlated with a noninvasive status. On the other hand, recent data have shown a de novo synthesis and/or deposition of some BM components, suggesting an active role for BM molecules in tumor invasion. Immunohistochemical studies have reported the presence of
3(IV) chain around some well-differentiated tumor clusters in bronchopulmonary cancers, whereas it is never found in normal bronchi (
5 chain of Type IV collagen with hemidesmosomal structures. In gastric, liver, and pancreatic carcinomas, laminins are produced and deposited at the invasive front of the tumor clusters. The expression of LN1 in gastric carcinoma has been proposed to be a risk factor for liver metastasis. LN5 is expressed by invasive malignant cells in human carcinomas and can be considered a marker of invasiveness (
To validate this, we examined the expression pattern and localization of the 1,
3, and
5 chains of Type IV collagen, LN5, and Type VII collagen in different types of bronchopulmonary tumors and normal tissues.
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Materials and Methods |
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Sources of Tissues
Tissues were obtained from surgical resection of lung tumors. We used five normal adult bronchi, three normal lungs at a distance from tumors, and 22 primary lung tumors, among which were 11 adenocarcinomas and 11 squamous cell carcinomas (Table 1). In each sample, TNM (tumor node metastasis) staging was done using the international Staging for Lung Cancer of the Union Internationale Contre le Cancer. Histological typing was according to the classification system of the World Health Organization.
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Primary Antibodies
In this study we used three monoclonal antibodies (MAbs) against the globular non-collagenous domain (NC1) of 1(IV) (MAb1),
3(IV) (MAb3), and
5(IV) (MAb5) (Wieslab; Lund, Sweden), an MAb (BMS 4040) against laminin 5 (Boehringer Ingelheim; Heidelberg, Germany), and a rabbit polyclonal antibody against Type VII collagen (a kind gift from Drs. M. Chen and D.T. Woodley).
Western Blotting Analyses
The specificity of the antibodies was evaluated by immunoblotting analyses with normal and tumor tissues. Tissue samples were homogenized with a turrax mixer in a lysis buffer (3.4 M NaCl, 50 mM Tris, 20 mM EDTA, 500 µM NEM, 100 µM PMSF). The extracts were centrifuged at 14,000 x g for 20 min at 4C. The pellets were incubated overnight with solution containing 2 M urea and 50 mM Tris and then were centrifuged at 18,000 x g for 20 min at 4C. The supernatants were collected and stored at -20C until use. Amounts of protein were evaluated with a DC Protein Assay kit (Biorad; Hercules, CA). Ten µg of protein from tissue extracts was mixed with Laemmli buffer (Biorad) containing 5% ß-mercaptoethanol (v/v), boiled for 5 min, separated on a 12% SDS-PAGE gel, and transferred to a nitrocellulose filter (Hybond-C Extra; Amersham, Aylesbury, UK). Transfer was monitored with Ponceau Red reversible staining. The membrane was blocked with 5% nonfat milk overnight at 4C before exposure with the primary antibodies [anti-1(IV), 1 µg/ml; anti-
3(IV), 1 µg/ml; anti-
5(IV), 1 µg/ml; anti-laminin 5, 2 µg/ml; anti-collagen VII, 2 µg/ml] for 2 hr at room temperature (RT). The membranes were incubated with a secondary biotinylated sheep anti-mouse or donkey anti-rabbit antibody (1:1500; Amersham International) for 1 hr at RT and then with the streptavidinperoxidase complex (1:1500; Amersham) for 30 min. Immunoreactive protein bands were detected with ECL Western blotting reagents (Amersham).
Immunohistochemistry
We performed an immunohistochemical study on different tissues in which localization of these proteins has already been described ( 1(IV) chain (normal bronchi BM),
3(IV) chain (normal pulmonary alveoli),
5(IV) chain (normal prostate BM), laminin 5 (dermalepidermal junction), and Type VII collagen (dermalepidermal junction) (not shown).
Fresh samples were frozen in liquid nitrogen, sectioned at 58 µm on a cryostat, and collected on gelatin-coated slides. At the first step, slides were incubated with a PBS buffer containing 3% blocking reagent bovine serum albumin (BSA) for 30 min at RT to block nonspecific binding. The sections were incubated for 60 min in a moist chamber with the primary antibodies directed against the 1,
3, and
5(IV) chains (at a concentration of 10 µg/ml), against laminin 5 (at a concentration of 10 µg/ml), and against Type VII collagen (at a concentration of 10 µg/ml). The sections were incubated with biotinylated anti-mouse or anti-rabbit Ig complex (Amersham) at a 1:50 dilution in a PBSBSA solution for 60 min and then incubated with streptavidin-fluorescein isothiocyanate (FITC) (Amersham) at a 1:50 dilution in PBSBSA for 30 min.
The slides were counterstained with Harris's hematoxylin for 10 sec, mounted in Citifluor anti-fading solution (Agar; Stamsted, UK), and observed with an Axiophot microscope (Zeiss; Oberkochen, Germany) using epifluorescence.
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Results |
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Immunobloting Analysis
To characterize the monoclonal and polyclonal antibodies used in this study, we performed Western blotting analyses on well-differentiated carcinomas and on bronchial tissues. We showed that the antibodies directed against the collagens and the laminin recognized only one band at the correct molecular weight, at 180 kD for the chains of Type IV collagen, 200 kD corresponding to the 3 chain of laminin 5, and 240 kD for the Type VII collagen chain (Fig 1). The
1(IV) chain was present in both normal and tumor bronchial tissues, whereas the
3(IV) chain was detected only in the tumor sample. The
5(IV) chain was not detected in tumor tissue extract. These results confirm that the antibodies against NC1 domains of
1(IV),
3(IV), and
5(IV) are specific.
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Immunohistochemistry of Normal Bronchi and Lung
All the BMs (epithelial and glandular) of the normal bronchi were strongly stained with MAbs against 1(IV) chain, LN 5, and Type VII collagen, whereas they were totally devoid of labeling for
3(IV) and
5(IV) chains (Table 1) (Fig 2a2d ). LN 5 and
1(IV) and
3(IV) chains were also found in alveolar BMs.
1(IV) and
5(IV) chains were localized both in vascular BMs and in smooth muscle cell BM. None of these molecules was found in the connective tissue underlying bronchial epithelia.
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Immunohistochemistry of Bronchopulmonary Carcinomas
In this study we found the same expression and distribution of BM molecules in squamous cell carcinomas and adenocarcinomas (Table 1). In these carcinomas, 1(IV) and
5(IV) chains were found in vascular BMs.
1(IV) and
3(IV) chains, and LN 5 antibodies also stained residual alveolar BMs at the periphery of tumors. The tumor BM showed reactivities with
1(IV) chain (in 22/22 tumors) and LN 5 (in 19/22 tumors) antibodies. The
1(IV) chain and LN 5 labelings were co-localized at the interface between tumor cells and stroma. In relation to the degree of tumor differentiation, the linear immunolabeling of BMs was either thin and continuous in well-differentiated carcinoma ( Fig 2e and Fig 2g ) and disrupted or absent in poorly differentiated carcinoma ( Fig 2i and Fig 2k ) (Table 1). The
3(IV) chain and Type VII collagen were detected in all well-differentiated carcinomas (Fig 2f and Fig 2h), whereas they were not present in poorly differentiated tumor clusters ( Fig 2j and Fig 2l ; Table 1). They were localized around well-differentiated tumor cell clusters as a thin continuous labeling ( Fig 2f and Fig 2h ).
In contrast to their absence in normal bronchial connective tissues, 1(IV) chain (22/22 cancers) and LN 5 (9/22 tumors) were the only BM molecules present in the stromal compartment with a fiber network all around tumor clusters (Table 1).
Concerning the cell labeling, only LN 5 (5/22) and Type VII collagen (8/22) labelings were detected in the cytoplasm of invasive tumor cells close to the BM staining.
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Discussion |
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In this study we examined the particular distribution of some of these macromolecules in relation to tumor progression. We clearly demonstrated here that the redistribution of 1(IV) chain and LN5 and the loss of Type VII collagen and the
3(IV) chain are correlated with invasiveness of tumor cells.
We showed that 1(IV) chain displayed a thin and regular pattern of distribution around well-differentiated tumor clusters, whereas it appeared disrupted or undetectable around poorly differentiated tumor clusters. Such a distribution could reflect the degradation of BM components which has been extensively described during tumor invasion process (
1(IV) in the stromal compartment.
1(IV) chain in lung tumors. The particular distribution of
1(IV) chain could reflect a part of the stromal reaction. Previous studies have demonstrated that interactions between the tumor cells and the ECM could modulate their invasive capacity. More particularly, it has been shown that the
1(IV) 531543 sequence promotes melanoma cell adhesion and enhances migratory activities through matrigel (
1(IV) chain facilitates adhesion and motility of the M4 melanoma cell line. Therefore, it is possible that after the enzyme-mediated degradation of Type IV collagen BMs,
1(IV) chain sequences in the stroma trigger cell motility and enhance tumor progression.
In the same way, LN5 displayed a thin, regular pattern in well-differentiated tumor clusters and a disrupted pattern in poorly differentiated clusters. Likewise, we observed cytoplasmic positivity in cells at the invasive front. Similar results have also shown the overexpression of LN5 in tumor cells at the invasive front, emphasizing its implication in tumor cell invasion of colon carcinomas ( 2 cleavage by a specific protease (MMP-2) exposes cryptic sites that stimulate cell motility in vitro (
These hypotheses strengthened our observations about Type VII collagen distribution because we showed that, in normal epithelia, Type VII collagen staining closely co-distributed with that of LN5. These findings are in agreement with the presence of many hemidesmosomes in bronchial tissue. The anchoring complexes have been well characterized by 6ß4. In the same way,
3-subunit of LN5 played a crucial role in maintenance and nucleation of hemidesmosome structural integrity. Therefore, a co-localization of LN5 with Type VII collagen would characterize a cohesive noninvasive state. In contrast, in invasive cancers, we observed that Type VII collagen staining did not co-express with LN5. Similar results have reported that the hemidesmosomal integrin
6ß4 and Type VII collagen are not co-localized in bladder cancer compared to normal tissue (
Concerning the 3(IV) chain, it was present in some BMs at the interface between tumor clusters and the stromal compartment in bronchopulmonary cancers. Normal bronchial tissues were totally devoid of
3(IV) chain. This observation suggests that this chain is neosynthesized in tumor tissues. Because the
3(IV) chain was detected around well-differentiated tumor clusters, it is likely that the neobiosynthesis of
3(IV) would be implicated in the preinvasive stage of tumor progression. However, at present little is known about the
3(IV) chain function in malignant processes. In vitro experiments suggest that the Type IV collagen
3 chain downregulates activation of human polynuclear leukocytes and thus decreases their capacity to migrate through the capillary walls (
3(IV) chain NC1 domain inhibits proliferation of melanoma cells. It can therefore be speculated that
3(IV) chain deposition could be a part of the desmoplastic reaction protecting normal tissues from tumor expansion by inhibiting malignant cell proliferation. Thus, the
3(IV) chain deposited around tumor clusters would limit tumor progression in bronchopulmonary cancers.
In conclusion, we showed an extensive remodeling of BM structures in bronchopulmonary carcinomas, with a progressive disruption of 1(IV) chain and the two hemidesmosomal components laminin 5 and Type VII collagen and the appearance of the
3(IV) chain around well-differentiated tumor clusters. Together, these results emphasize that the composition of BMs reflect different stages of tumor progression and may have prognostic significance in bronchopulmonary cancers.
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Acknowledgments |
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Supported by a grant from the ARERS (Association Régionale pour l'Enseignement et la Recherche Scientifique) and by the Lions Club of Soissons.
We thank Drs M. Chen and D.T. Woodley (Northwestern University School of Medicine, Chicago, IL) for the generous gift of polyclonal antibody to human Type VII collagen.
Received for publication July 20, 1999; accepted January 5, 2000.
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