[CANCER RESEARCH 56. 2891-2895. July I, 1996] Advances in Brief Overexpression of Tissue Inhibitor of Metalloproteinases-2 by Retroviral-mediated Gene Transfer in Vivo Inhibits Tumor Growth and Invasion1 Suzan Imren, Donald B. Kohn, Hiroyuki Shimada, Laurence Blavier, and Yves A. DeCIerck2 Division of Hematology-Oncology and Division of Research Immunology and Bone Marrow Transplantation, Departments of Pediatrics [S. /.. D. B. K., L. B., Y. A. DJ, Biochemistry and Molecular Biology [Y. A. D.]. Molecular Microbiology and Immunology ¡D.B. K.I. and Pathology ¡H.S.I. Children.1; Hospital Los Angeles and University of Southern California, Los Angeles, California 90027 Abstract We have demonstrated previously that overexpression of tissue inhib itor of metalloproteinases-2 (TIMP-2), an inhibitor of matrix-degrading metalloproteinases, not only inhibits the invasive and metastatic behavior of tumor cells but also significantly decreases tumor growth in vivo (Y. A. DeCIerck et al., Cancer Res., 52: 701-708, 1992). This latter effect was found to be dependent on the ability of TIMP-2 to prevent the degradation of the collagen matrix (A. M. Montgomery et al., Cancer Res., 54: 5467- 5473,1994). In this report, we have overexpressed TIMP-2 in tumor tissue by retroviral-mediated gene transfer into tumor cells by co-injecting s.c. in nude mice tumorigenic c-Ha-ras-transfected rat embryo fibroblasts with irradiated packaging cells producing high titer retroviral vectors contain ing the human TIMP-2 cDNA. The growth rate of tumors derived from cells co-injected with the TIMP-2 vector producer cells was significantly slower than the growth rate of tumors derived from cells co-injected with packaging cells producing a retrovirus containing the Escherichia coli ß-galactosidase gene. The transduction efficiency was estimated at 13%, and the production of a functional human TIMP-2 in tumor cells trans duced with the TIMP-2-containing vector was documented. Furthermore, histológica! analysis of tumors derived from tumor cells co-injected with the TIMP-2 vector producer cells revealed the presence of a thick con nective tissue capsule and a lack of local invasion. The data indicate that retroviral-mediated transduction of TIMP-2 cDNA into a limited popu lation of tumor cells in vivo is sufficient to increase the accumulation of connective tissue proteins in tumor tissue, to inhibit growth, and to prevent local invasion. Introduction MMPs3 consist of a family of Zn++ -dependent neutral endopepti- dases that have a broad spectrum of proteolytic activity for most components of the ECM ( 1). In the extracellular space, the activity of these proteases is regulated by a specific class of natural inhibitors, designated TIMPs (2). The balance between MMPs and TIMPs is critical in maintaining the integrity of the ECM, and its regulatory role in organ development, cell growth, and differentiation has been well documented (3). During tumor invasion, the balance between MMPs and TIMPs is often shifted in favor of the proteases (4), resulting in an excessive proteolytic degradation of the ECM. It has been dem onstrated that alteration of this balance in tumor cells by genetic Received 3/25/96; accepted 5/15/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported in part by Grant CA 59318 (to Y. A. D. and D. B. K.) from the NIH. Grant BE84 from the American Cancer Society (to Y. A. D.I. and by a private donation to Childrens Hospital Los Angeles and the Norris Cancer Center (to Y. A. D. and D. B. K.). 2 To whom requests for reprints should be addressed, at Division of Hematology- Oncology, MS #54, Childrens Hospital Los Angeles. 4650 Sunset Boulevard, Los Ange les. CA 90027. Phone: (213) 669-5648; Fax; (213) 664-9455; E-mail: ydeclerck9i-smtpgate@chlais.usc.edu. 3 The abbreviations used are: MMP, matrix metalloproteinase; ECM, extracellular matrix; TIMP. tissue inhibitor of metalloproteinases; LTR. long terminal repeat; CMV, cytomegalovirus; X-Gal. 5-bromo-4-chloro-3-indolyl-S-D-galactopyranoside: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LacZ. ß-galactosidase. manipulation has a significant effect on tumor progression and tumor growth (5-7). We have demonstrated that overexpression of TIMP-2 in tumorigenic and invasive c-Ha-ras-transfected rat embryo fibro blasts (4R) injected s.c. in nude mice not only limits experimental metastasis but also inhibits the growth of the primary tumors in vivo and results in the formation of a peritumoral capsule of connective tissue that prevents local invasion (8). A similar growth-inhibitory effect of TIMP-2 overexpression was observed in human melanoma cells injected s.c. in SCID mice, and this inhibitory effect was found to be dependent on the integrity of the collagen matrix (9). Altogether, these observations indicate that genetic manipulation of the protease- protease inhibitor balance in tumors in favor of the inhibitors may have a significant cytostatic effect in cancer. Over the last 5 years, it has been suggested that genetic manipula tion in tumor cells by gene therapy may provide an alternative to conventional approaches for the treatment of cancer. Cytotoxic genes such as the herpes simplex-thymidine kinase suicide gene (10), tumor suppressor genes (11), and immunostimulatory cytokine genes (12) have been among the first candidates to be tested in animal models, and some of them are currently being tested in a variety of human gene therapy trials for cancer (13). A major limiting factor in cyto- toxic suicide and tumor suppressor genes is that they have to be targeted to virtually every single tumor cell by a combination of direct gene transfer and bystander effect to be able to stop growth of the malignant cells (10). Thus, in this aspect, protease inhibitor genes may represent attractive candidates in a gene therapy approach to cancer because it may not be necessary to deliver and express these genes in every single tumor cell as long as the level of expression in a limited number of transduced cells is sufficient to prevent the excessive breakdown of the ECM. To test this hypothesis, we have overex pressed TIMP-2 in tumor cells in vivo by retroviral-mediated gene transfer, and we demonstrate a significant inhibitory effect on growth and local invasion. Materials and Methods Construction of Retroviral Vectors. A Nco\-Stu\ fragment of the human TIMP-2 cDNA, extending from the ATG codon to 5 bp downstream of the stop codon (8) was inserted into plasmid PIC20H to generate PIC20H.TIMP-2. From this plasmid, a 735-bp Hindlll fragment was further subcloned into retroviral vector plasmid, LNCX, to generate LNCTIMP-2. In this plasmid, the 5' Moloney murine leukemia virus LTR is driving expression of the bacterial neomycin resistance (neoR) gene, and the CMV promoter is driving expression of the TIMP-2 cDNA (Fig. 1A). Generation of Retroviral Vector-producing Packaging Cells. The ret roviral vector LNCTIMP-2 was transfected into the murine GP+E-86 ecotropic packaging cell line (14) by lipofection using A'-[l-(2,3)-dioleoyl- oxyl)propyl]-/V,A',/V-trimethylammoniummethylsulfate transfection reagent (DOTAP, Boehringer-Mannheim, Indianapolis, IN). The transiently produced retroviral particles were collected from the culture medium of these cells after 48 h and were used to infect the murine PA317 amphotropic packaging cell line (15). Transduced cells were then selected in the presence of 0.5 mg/ml of 2891 Research. on November 7, 2015. © 1996 American Association for Cancer cancerres.aacrjournals.org Downloaded from