ARTICLE |
Correspondence to: Myriam Polette, Unité INSERM 314, 45, rue Cognacq-Jay, 51 100 Reims, France.
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Summary |
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Tumor cell-derived collagenase stimulatory factor (TCSF) stimulates in vitro the biosynthesis of various matrix metalloproteinases involved in tumor invasion, such as interstitial collagenase, gelatinase A, and stromelysin 1. The expression of TCSF mRNAs was studied in vivo, using in situ hybridization and Northern blotting analysis, in seven normal tissues and in 22 squamous cell carcinomas of the lung, and in seven benign proliferations and in 22 ductal carcinomas of the mammary gland. By in situ hybridization, TCSF mRNAs were detected in 40 of 44 carcinomas, in pre-invasive and invasive cancer cells of both lung and breast cancers. TCSF mRNAs and gelatinase A mRNAs were both visualized in the same areas in serial sections in breast cancers, and were expressed by different cells, tumor cells, and fibroblasts. The histological results were confirmed by Northern blot analysis, which showed a higher expression of TCSF mRNAs in cancers than in benign and normal tissues. These observations support the hypothesis that TCSF is an important factor in lung and breast tumor progression. (J Histochem Cytochem 45:703-709, 1997)
Key Words: TCSF, metalloproteinases, tumor invasion
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Introduction |
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Tumor invasion is a multistep process that involves the degradation of basement membrane and interstitial matrix components by proteolytic enzymes. Many data actually support an important role for the matrix metalloproteinases (MMPs) in this proteolytic event. High levels of MMPs have been described in many cancer cell lines that display high invasive capacity (
The specific detection of MMPs in peritumoral fibroblasts has led to the hypothesis that tumor cells might induce the synthesis of these enzymes implicated in cancer dissemination. In agreement with such an idea, several investigators have demonstrated cooperation between tumor cells and fibroblasts in vitro in the regulation of several MMPs, such as interstitial collagenase (
On the basis of limited information concerning TCSF localization in cancer tissue, the role of TCSF in tumor progression remains unclear. In the present study, to clarify the cell origin of TCSF and to study its role in cancer invasion, we performed in situ hybridization and Northern blot analysis on human lung and breast carcinomas as well as on normal tissues.
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Materials and Methods |
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Source of Tissue
The tissue was obtained from 22 lungs resected for squamous cell carcinomas of Stages I (10 cases), II (eight cases), and III (four cases) according to the TMN classification, from seven normal lung samples, from 22 ductal breast cancers of Grade 1 (four cases), Grade 2 (14 cases), and Grade 3 (four cases) according to the Scarf and Bloom classification, and from seven benign breast proliferations (two fibrocystic disease and five fibroadenoma).
Tissue Preparation
Part of the samples were frozen in liquid nitrogen for Northern blot analysis and the remainder were fixed in formalin and embedded in paraffin for in situ hybridization.
In Situ Hybridization Localization
Tissue sections (5 µm) were deparaffinized, rehydrated, and treated with 0.2 M HCl for 20 min at room temperature, followed by 15 min in 1 µg/ml proteinase K (Sigma Chemical; St Louis, MO) in Tris-EDTA-NaCl, 37C, to remove basic proteins. The sections were washed in 2 x SSC (sodium saline citrate), acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min, and hybridized overnight with 35S-labeled (50C) anti-sense RNA transcripts. TCSF cDNA (1700 BP) and gelatinase A (1500 BP) (a gift from G. Murphy; Cambridge, UK) were subcloned into pBluescript II SK+/- plasmid and pSP64, respectively, and used to prepare 35S-labeled RNA probes. Hybridizations were followed by RNAse treatment (20 µg/ml, 1 h, 37C) to remove unhybridized probe and two stringent washes (50% formamide-2 x SSC, 2 hr at 60C) before autoradiography using D 19 emulsion (Kodak; Rochester, NY). Slides were exposed for 15 days before development. The controls were performed under the same conditions, using 35S-labeled sense RNA probes. All slides were counterstained with HPS (hematoxylin-phloxin-safran), mounted, and examined under a Zeiss Axiophot microscope.
In Situ Hybridization Quantitation by Image Cytometry
Quantitation of the number of hybridization grains/µm2 was performed with the help of an automated image analyzer, the DISCOVERY system (Becton-Dickinson; Mountain View, CA). After thresholding, the number of grains are counted automatically on at least six fields at high magnification (x 500). At this magnification, one field measures 12,688 µm2. We performed these measurements on six different samples (three lung and three breast carcinomas) in which we found normal, in situ, and invasive areas on the same tissue section. Statistical analyses of TCSF mRNA expression levels were compared using the non-parametric Mann-Whitney U-test. Data were expressed as mean of dots/µm2 ± SEM. p values equal to or less than 0.05 were considered significant.
Northern Blot Analysis
Extraction of total RNA from tissues was performed by RNAzol treatment (Biogenesis; Bournemouth, UK). Ten µg of each RNA was analyzed by electrophoresis in 1% agarose gels containing 10% formaldehyde and transferred onto nylon membranes (Hybond-N; Amersham, Poole, UK). The membrane was hybridized with the cDNA probe encoding TCSF (1700 BP) labeled with 32P using random priming synthesis (5 x 108 cpm/µg) (Dupont Nemoursde ; Bruxelles, Belgium). The filters were exposed for 1 day. Membranes were rehybridized to a ubiquitous 36B4 gene probe, which served as a control. Signal intensities were recorded using a CD 60 Desaga (Heidelberg, Germany) laser-scanning densi-tometer and TCSF levels (in arbitrary units) were standardized with their corresponding 36B4 levels to obtain values independent of RNA quantities deposed onto gels. Statistical analyses of TCSF expression levels were compared using the non-parametric Mann-Whitney U-test. Data were expressed as mean ± SEM. Differences or similarities between two populations were considered significant when confidence intervals were <95% (p<0.05).
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Results |
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Lung Lesions
By Northern blotting, TCSF transcripts were detected in 18 of 22 carcinomas. Quantitative analysis showed significantly higher (p<0.05) TCSF mRNA expression in lung carcinomas than in peritumoral lung tissues (Figure 1 and Figure 2A). However, no significant differences between the TCSF mRNA levels were found in accordance with the TNM stage (Figure 2A).
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With in situ hybridization, pre-invasive and invasive cancer cells were labeled in 18 of 22 tumors examined (the same positive samples as those found by Northern blot analysis). Stromal cells surrounding labeled invasive cancer cells, were always negative (Figure 3A). Normal (Figure 3C) or squamous metaplastic epithelium and bronchial glands did not express any TCSF transcripts. Moreover, in the normal or emphysematous adjacent lung, pulmonary alveolar macrophages identified by the CD68 monoclonal antibody (Dako; Carpinteria, CA) on serial sections (not shown) were particularly rich in TCSF mRNAs (Figure 3D). In the three cases analyzed by image cytometry, TCSF mRNAs were significantly expressed in tumor cell nests of both pre-invasive (1.68 ± 0.22 /µm2) and invasive areas (2.14 ± 0.35 /µm2) compared to the extracellular control compartment (0.19 ± 0.02 /µm2) and normal tissue (0.23 ± 0.04 /µm2) (p<0.05).
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Breast Lesions
By Northern blotting, TCSF mRNAs were detected in the 22 breast carcinomas. Quantitative analysis showed significantly higher (p<0.05) expression of TCSF mRNAs in breast carcinomas than in benign breast lesions (Figure 1 and Figure 2B). No significant differences between the TCSF mRNA levels were found in accordance with the Scarf and Bloom staging (Figure 2B).
With in situ hybridization, benign proliferations and normal mammary areas mixed with cancer cells or adjacent to cancer areas did not show any hybridization grains (Figure 3E), whereas the TCSF mRNAs were detected in cancer cells in pre-invasive and invasive areas (Figure 3F) of all 22 tumors examined. Stromal cells did not contain any hybridization grains. In the three cases studied by quantification, the density of markers in both pre-invasive (2.14 ± 0.48/µm2) and invasive (2.95 ± 0.92/µm2) areas was significantly higher than in the extracellular control compartment (0.23 ± 0.12/µm2) and the normal areas (0.28 ± 0.10/µm2) (p<0.05). On serial sections, TCSF mRNAs were localized in cancer cells, whereas fibroblasts close to tumor clusters expressed mRNAs encoding gelatinase A (Figure 3F and Figure 3H) in the same areas.
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Discussion |
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In this study we clearly showed the presence of mRNA encoding TCSF in epithelial tumor cells of lung and breast carcinomas. In agreement with our observations, previous immunohistochemical studies detected TCSF in cancer cells in breast (
Even though the precise function of TCSF is not known, some recent in vitro studies have shown that TCSF is able to stimulate the production of several MMPs by fibroblasts. Recent experimental data have demonstrated that TCSF stimulates the production of interstitial collagenase, stromelysin 1 and gelatinase A but not stromelysin 3 in fibroblasts (
It therefore appears that some MMPs, as well as TCSF, are expressed selectively in both pre-invasive and invasive carcinoma but by different cell types, peritumoral fibroblasts and tumor cells, respectively. Relating our in vivo data to the observation that TCSF enhances the production of some particular MMPs in fibroblasts in vitro, it can be postulated that the TCSF produced by tumor cells in vivo stimulates the expression of some MMPs by peritumoral fibroblasts. However, in vivo, interstitial collagenase and stromelysin 1, which are induced by TCSF in fibroblasts in vitro, are infrequently observed in stromal cells in breast carcinomas (
In conclusion, our observations on lung and breast cancers strongly support the hypothesis that TCSF is an important factor in tumor progression. More precisely, TCSF produced by tumor cells could play a role in the degradation of extracellular matrix associated with tumor invasion by stimulating the synthesis of some MMPs by peritumoral fibroblasts.
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Acknowledgments |
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We gratefully thank Dr Gillian Murphy for the generous gift of gelatinase A probe.
This material is based on work supported by the US Army Medical Research administration under award no. DAMD 17-95-5017, the ARC no. 1096, and the Lyons Club of Soissons.
Received for publication June 4, 1996; accepted October 23, 1996.
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