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Reduced Angiogenesis and Tumor Progression in Gelatinase A-Deficient Mice1

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Reduced Angiogenesis and Tumor Progression in Gelatinase A-Deficient Mice1 (CANCER RESEARCH.«. 11)48-1(151. March 1. 1998] Reduced Angiogenesis and Tumor Progression in Gelatinase A-deficient Mice1 Takeshi höh,2Masatoshi Tanioka, Hiroshi Yoshida, Takayuki Yoshioka, Hirofumi Nishimoto, and Shigeyoshi Itohara Shionogi Institute for Medical Science. Shionogi & Co., Lid. IT. /.. M. T., H. N.¡and Discovery Research Laboratories II, Shionogi & Co., Ltd. [H. Y., T. Y.J, Fukushima-ku, Osaka 553. and Institute for Virus Research. Kyoto University. Syogo-in. Sakyo-ku, Kyoto 606-01 ¡S././. Japan ABSTRACT normalities and are fertile, thus offering a useful system for assessing the specific role of gelatinase A in tumor progression. We show here Matrix proteolysis is thought to play a crucial role in several stages of that the rates of angiogenesis and experimental tumor growth and tumor progression, including angiogenesis, and the invasion and metas metastasis are markedly reduced in these gelatinase A-deficient mice. tasis of tumor cells. We investigated the specific role of gelatinase A This is the first direct evidence that host-derived gelatinase A plays a (matrix metalloproteinase 2) on these events using gelatinase A-deficient mice. In these mice, tumor-induced angiogenesis was suppressed accord specific role in angiogenesis and tumor progression in vivo. ing to dorsal air sac assay. When B16-BL6 melanoma cells or Lewis lung carcinoma cells were implanted intradermally, the tumor volumes at 3 MATERIALS AND METHODS weeks after implantation in the gelatinase A-deficient mice decreased by 39% for B16-BL6 melanoma and by 24% for Lewis lung carcinoma Animals. The generation of gelatinase A-deficient mice was described (P < 0.03 for each tumor). The number of lung colonies of i.v. injections previously (11). To minimize genetic variances between experimental groups, fell by 54% for B16-BL6 melanoma and 77% for Lewis lung carcinoma we backcrossed the heterozygous mutant mice five times to C57BL/6J mice before using them. The homozygous mutant and control wild-type mice were (P < 0.014 and P < 0.0015, respectively). These results indicated that host-derived gelatinase A plays an important role in angiogenesis and littermates from the crosses between hétérozygotes.Thegenotypes of the mice tumor progression, suggesting the usefulness of gelatinase A inhibitors for were determined by Southern blot analysis (11). Tumor Cells. B16-BL6 melanoma cells (12), provided kindly by Dr. I. J. anticancer chemotherapy. Fidler (M. D. Anderson Cancer Center, Houston, TX), were maintained as monolayer cultures in DMEM containing 10% fetal bovine serum. Lewis lung INTRODUCTION carcinoma (supplied from the National Cancer Institute, NIH, Bethesda, MD) was maintained by serial intradermal implantation in C57BL/6J mice. MMPs1 constitute a family of zinc-requiring matrix-degrading pro- Dorsal Air Sac Method and Image Analysis. The dorsal air sac method teinases, which include the collagenases, gelatinases, stromelysins, was carried out as described elsewhere (13). B16-BL6 melanoma cells were and MT-MMPs. These enzymes are synthesized as proenzymes and washed three times with HBSS and were suspended in HBSS at a concentra must undergo proteolytic cleavage of a NH2-terminal domain to tion of 1 X IO7 cells/ml. A Millipore chamber (diameter, 10 mm; thickness, 2 become catalytically active (1). The catalytic activities are further mm; filter pore size, 0.22 j*m; Millipore Co.) was filled with 0.2 ml of either modulated by interacting with many endogenous inhibitors (2, 3). cell suspension or HBSS and implanted s.c. into the dorsal side of the mice. At 5 days after the implantation, the mice were anesthetized and fixed in the prone Generally, a given cell expresses multiple MMPs. Although these position. A wide, rectangular incision was made in the skin on the dorsal side, enzymes are believed to have crucial roles in various physiological and the skin was carefully ablated. To locate the chamber-contacting region, a and pathological conditions, such as embryonic development and ring (Millipore) of the same shape as the chamber was placed onto the s.c. tissue repair in adults, their complexities as mentioned above have tissues adjacent to the chamber region, and the area was photographed. For made it difficult to definitively evaluate the roles of individual en histológica! analyses, two samples were randomly taken from each skin area, zymes in vivo. However, recent progress on gene-knockout mice embedded in paraffin wax, and sliced into 3 /¿m-thickvertical sections, which offers a way to overcome this problem. were then stained with H&E. From each H&E-stained section, microphoto- Gelatinase A (MMP-2) and gelatinase B (MMP-9), which hydro- graphs of three randomly selected fields were taken at a final magnification of lyze type IV collagen localized in the basement membrane, have been X116. suggested to play a role in tumor progression and metastasis (4, 5). Vascular structures were recognized as luminal or slitlike structures that occasionally contained blood cells within them and were delineated by flat The idea first arose from the finding that many malignant tumor cells tened endothelial cells. Vessels beneath the museali cutáneas were traced secrete large amounts of type IV collagen-degrading enzymes (4). manually without awareness of the genotype. The traced vascular images were Furthermore, studies have suggested that either gelatinase A or gela entered into a computer, and image analyses for the area and number of vessels tinase B or both expressed by endothelial cells play a crucial role in beneath the musculi cutaneus were performed using image analysis software angiogenesis, which is considered to be an important stage in tumor (NIH Image, Version 1.61 PPC). The vascular area and number of vessels were progression (6-8). Recent studies also suggested that host-derived expressed as ;inr/mm width and number/mm width, respectively. A total of gelatinase A binds to the cell-surface proteins MT-MMP (9) and six fields per animal (two samples x three fields) were analyzed, and the mean integrin avß,(10) of tumor or endothelial cells and may modulate the value for the six analyses was used. cell entity. However, there is no clear evidence to support the hypo Intradermal Tumor Growth Assay. B16-BL6 melanoma cells were thetical roles of gelatinases in tumor progression. washed three times with HBSS and were suspended in HBSS. Lewis lung Recently, we produced gelatinase A-deficient mice by gene target carcinoma cells that had been maintained in C57BL/6J mice were treated with 1% collagenase for 10 min at room temperature, washed with HBSS three ing (11). These mice develop normally without any anatomical ab- times, and suspended in HBSS. Mice were inoculated intradermally with B16-BL6 melanoma cells or Lewis lung carcinoma cells (2 X IO5 cells/mouse Received 9/9/97; accepted 12/31/97. for either of the tumor cells). The primary tumor volume was measured by The costs of publication of this article were defrayed in part by the payment of page using a slide caliper, applying the following formula: Volume = 0.5 X charges. This article must therefore be hereby marked advertisement in accordance with (Width)2 X Length. 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was partly supported by a Grant-in-Aid for Scientific Research B from the Lung Colonization Assay. Mice were injected with 1 x IO5 cells/mouse Ministry of Education. Science. Sports and Culture of Japan (to S. 1.). (B16-BL6 melanoma cells) or 3 X 10* cells/mouse (Lewis lung carcinoma - To whom requests for reprints should be addressed, at Shionogi Research Laboratory. cells) in the lateral tail vein. Fourteen days (B16-BL6 melanoma) or 16 days Shionogi & Co., Ltd., 12-4, Sagisu 5-chome. Fukushima-ku, Osaka 553, Japan. Phone: (81) 6-458-5861; Fax: (81) 6-458-0987; E-mail: [email protected]. (Lewis lung carcinoma) later, the mice were killed, and the lungs were ' The abbreviations used are: MMP. matrix metalloproteinase; MT-MMP. membrane- removed and fixed with Bouin's fluid to count the numbers of visible tumor type MMP. colonies. 1048 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1998 American Association for Cancer Research. GELATINASE A-DEFICIENT MICE HBSS Day 3 Day 5 \ Fig. 1. Reduced angiogenesis in gelatinase A-deficient mice. a-f. representative views from the inner side of dorsal skins in wild-type (a-c) and mutant (d-f) mice, a and d, dorsal skin of the mice 5 days after implantation of a chamber filled with HBSS for negative control. b, c, e, and/ dorsal skin of mice 3 (b and e) or 5 (c and» days after implantation of a B16-BL6 melanoma cell-filled chamber. Bar. 1 mm. Gelatin Zymography. Gelatin zymography was carried out as described experiments in which the chamber was filled with HBSS, vessel elsewhere (11). Briefly, the tumor tissues were homogenized in 50 mM Tris- development in mutant mice appeared to proceed normally, suggest HC1 (pH 7.5) and centrifuged at 15,000 rprn for 5 min. The protein concen ing that gelatinase A had little influence or that another enzyme tration of the supernatant was determined by a protein assay (Bio-Rad), and 20 ¡igof supernatant proteins were applied to nonreduced SDS-PAGE using a compensated for the role of gelatinase A in normal vessel develop ment (Fig. 1, a and d). By 3 days after implantation with the tumor- 7.5% gel containing 0.1% gelatin. After electrophoresis, the gel was soaked in 50 mM Tris-HCl (pH 7.5) containing 2.5% Triton X-100 at room temperature filled chamber, the blood vessels had expanded, and newly formed with gentle shaking for 2 h and then incubated overnight in 50 mM Tris-HCl blood vessels had branched out from large blood vessels in wild-type (pH 7.5) containing 10 mM CaCU at 37°C.The gels were then stained with mice (Fig.
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