ARTICLE |
Correspondence to: Myriam Polette, INSERM U 514, IFR 53, Laboratorie Pol Bovin, CHU de Reims, 45 rue Cognacq-Jay, 51100 Reims, France.
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Summary |
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Tumor cells interact with stromal cells via soluble or cell-bound factors stimulating the production of matrix metalloproteinases (MMPs), a group of enzymes largely involved in the extracellular matrix (ECM) remodeling in tumor invasion. Among these factors, extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to stimulate in vitro the fibroblast production of various MMPs such as interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), and gelatinase A (MMP-2). In this study, the EMMPRIN protein was detected by immunohistochemistry prominently in malignant proliferations of the breast and the lung. It was present at the surface of both tumor epithelial and peritumor stromal cells. Because previous studies have reported that stromal cells do not express EMMPRIN mRNAs, it is very likely that EMMPRIN is bound to stromal cells via a specific receptor. Moreover, our observations also demonstrated that the same peritumor stromal cells strongly express MMP-2. Our results show that EMMPRIN is an important factor in tumor progression by causing tumor-associated stromal cells to increase their MMP-2 production, thus facilitating tumor invasion and neoangiogenesis. (J Histochem Cytochem 47: 15751580, 1999)
Key Words: metalloproteinases, tumor invasion, breast cancer, lung cancer
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Introduction |
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Tumor invasion and metastasis are the result of a multistep process that includes basement membrane disruption, stromal infiltration, intravasation and extravasation, and invasion of a target organ by tumor cells. All these processes require the degradation or remodeling of basement membrane components and of extracellular matrix (ECM) macromolecules by proteolytic enzymes. Among these proteinases, matrix metalloproteinases (MMPs) are particularly implicated in the metastastic cascade because of their broad spectrum of substrates (
Tumor cells may interact with stromal cells via soluble or cell-bound factors, stimulating MMP production. The best characterized of these factors, a tumor collagenase stimulatory factor (TCSF), recently renamed extracellular matrix metalloproteinase inducer (EMMPRIN), was originally isolated from the LX-1 human pulmonary carcinoma cell line (
All these data prompted us to examine by immunohistochemistry the distribution of EMMPRIN and MMP-2 with regard to the persistence of intact basement membrane, which could be an obstacle for the MMP induction by cellcell contact in various normal, benign, and malignant proliferations of the breast and lung.
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Materials and Methods |
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Source of Tissue
Tissues were obtained from four normal human bronchi, from 20 human lungs resected for squamous cell carcinoma (14 cases) and adenocarcinoma (six cases), from eight fibroadenomas of the breast, from four intraductal carcinomas of the breast, and from 20 invasive ductal breast carcinomas (four of grade 1, 12 of grade 2, and four of grade 3, according to the Scarf and Bloom classification).
Western Blotting Analyses
Tissue samples were homogenized with a turrax mixer in a lysis buffer (8 g/liter NaCl, 0.01% leupeptin, 1% pefablock, 1% aprotinin). The extracts were centrifuged at 14000 x g for 10 min and the supernatants were kept at -20C until use. Amounts of protein were evaluated with the DC Protein Assay kit (BioRad; Hercules, CA). Ten µg of protein from tissue extracts and 2 µg of recombinant EMMPRIN were 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% milk overnight at 4C before exposure with the G6.2 antibody (10 µg/ml; Chemicon, Temecula, CA) for 1 hr at room temperature (RT). The membranes were incubated with a secondary biotinylated sheep anti-mouse antibody (1:1500; Amersham, Arlington Heights, IL) 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
Fresh samples were frozen in liquid nitrogen, cut at -20C in a ReichertJung 2800 Frigocut cryostat at a thickness of 58 µm, and transferred onto gelatin-coated slides. Sections were incubated overnight at 4C in a moist chamber with the monoclonal antibody against EMMPRIN (G6.2 at a concentration of 100 µg/ml; Chemicon) and then with anti-mouse IgG biotinylated complex (Amersham) at a 1:50 dilution in PBSBSA solution for 60 min. Finally, sections were treated with streptavidinfluorescein isothiocyanate (FITC) (Amersham) at a 1:50 dilution in PBS for 30 min.
Double immunostainings were performed for the simultaneous localization of EMMPRIN/Type IV collagen and EMMPRIN/MMP-2. For these double immunostainings, two sucessive labeling reactions for EMMPRIN/Type IV collagen and EMMPRIN/MMP-2 were done sequentially as follows: (a) detection of EMMPRIN using the monoclonal antibody G6.2 at a concentration of 100 µg/ml in PBSBSA solution; (b) F(ab')2-fragments anti-mouse IgGdigoxigenin (Boehringer; Mannheim, Germany) at a concentration of 4 µg/ml in PBS; (c) anti-digoxigenin fluorescein Fab fragments (Boehringer) at a concentration of 1.3 µg/ml in PBSBSA; (d) detection of Type IV collagen using a rabbit biotinylated polyclonal antibody (Institut Pasteur; Paris, France) diluted 1:1000 in PBSBSA; (e) detection of MMP-2 using the rabbit polyclonal antibody AB809 (Chemicon) then using an anti-rabbit IgG biotinylated complex (Amersham) at a 1:50 dilution in PBSBSA for 60 min; and (f) streptavidinTexas Red conjugate (Amersham) at a 1:50 dilution in PBS.
We tested the absence of crossreactivity either by omitting the incubation step with the primary antibody or by replacing the primary antibody by a nonimmune IgG. The sections were counterstained with Harris' hematoxylin solution for 10 sec, mounted in Citifluor antifading solution, and observed with an Axiophot microscope (Zeiss; Oberkochen, Germany) using epifluorescence for conventional microscopy or under a confocal laser scanning microscope (BioRad MRC 600).
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Results |
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EMMPRIN Detection in Lung and Breast Tissues
In Western blotting analyses, the antibody directed against EMMPRIN recognized two major bands of 57 kD (glycosylated form) and 30 kD (non glycosylated form) when evaluated vs normal or tumor tissue extracts (Figure 1). We observed an identical electrophoretic profile between EMMPRIN extracted from tumors and from normal tissues. The detection of the glycosylated form in normal and tumor tissues proves EMMPRIN functionality in both conditions.
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By immunohistochemistry, EMMPRIN was detected in all normal and tumor tissues of the breast and lung (Table 1). In the normal ducts and lobules and in fibroadenomas of the breast, EMMPRIN was frequently confined to the apical membrane of epithelial cells (Figure 2a). In the same way, in the normal bronchi there was an accumulation of EMMPRIN at the apical pole of the ciliated cells, as confirmed by confocal microscopic examination (Figure 2b). In these normal or benign conditions, EMMPRIN was also detected with weak labeling at the basolateral pole of epithelial cells. In tumor clusters, EMMPRIN was highly expressed in all malignant cells (Figure 2c), with more intense staining on the cells located at the periphery of the well-differentiated nests (Figure 2d). Positive staining was distributed at the outer cell membrane of these cancer cells with a punctiform pattern (Figure 2e). Furthermore, EMMPRIN was also found in some stromal cells in breast and lung carcinomas close to tumor cells (Figure 2g). There was positivity at the cell surface of isolated elongated cells considered to be fibroblasts. Moreover, some endothelial cells displayed spotty labeling on their cell membranes (Figure 2c). Because our previous data have shown no EMMPRIN mRNAs in peritumor stromal cells (
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Co-localization of EMMPRIN and Type IV Collagen
We next investigated whether basement membrane integrity could be an obstacle to the diffusion of EMMPRIN from tumor cells. Using double immunofluorescence labeling, we found that the stromal positivity of EMMPRIN was not necessarily associated with the absence of Type IV collagen around tumor clusters, suggesting that basement membrane integrity is not a limiting factor to EMMPRIN translocation (Figure 2f).
Colocalization of EMMPRIN and MMP-2
Because EMMPRIN has been shown to stimulate fibroblast production of several MMPs, we looked at MMP-2 localization in relation to that of EMMPRIN. As expected, MMP-2 protein was detected in the benign neoplasms and malignant lesions, whereas this enzyme was rarely detected in normal tissues adjacent to tumors. In the carcinomas, the MMP-2 was largely found both in cancer and peritumor stromal cells, whereas in fibroadenomas it was present only in some sparse fibroblasts at a distance from the proliferating ducts (Table 1). In all carcinomas, we observed that the presence of EMMPRIN in/on stromal cells coincided with the detection of MMP-2 in the same cells (Figure 2g and Figure 2h).
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Discussion |
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This immunohistochemical study demonstrates epithelial expression of EMMPRIN in normal tissues and in various benign and malignant proliferations of the breast and the lung. These data are in agreement with previous reports using a different antibody (E11F4) in the mammary gland (
In addition to this distribution of EMMPRIN on cancer cells, we also found this protein in peritumor stromal cells in malignant lesions. Because the biosynthesis of EMMPRIN by fibroblasts has never been reported in previous in vitro and in vivo studies (
Even though the cellular mechanism of action of EMMPRIN is not yet well understood, several hypotheses can be drawn from our results. In general, it has been suggested that the plasma membrane localization of EMMPRIN at the periphery of tumor clusters serves to restrict its bioactivity to cells in close proximity. EMMPRIN attached to the plasma membrane via a transmembrane domain could then interact with a cell surface receptor present on peritumor stromal cells via an extracellular domain (
In conclusion, our results describing intense expression of EMMPRIN in cancer tissue clearly show the implication of EMMPRIN in tumor invasion. The detection of EMMPRIN protein on peritumor stromal cells also supports the hypothesis that this factor could be shed from tumor cells and bound to tumor-associated stromal cells. EMMPRIN would further stimulate the stromal production of MMP-2, facilitating tumor invasion and neoangiogenesis. Taken together, these results improve our understanding of the cooperation between cancer and host cells during the process of invasion and metastatis.
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Acknowledgments |
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Supported by the Lions Club of Soissons.
We thank Chemicon (Temecula, CA) for generously providing the antibody to EMMPRIN.
Received for publication February 3, 1999; accepted August 3, 1999.
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