[CANCER RESEARCH 60, 6757– 6762, December 1, 2000] Modulation of Tumor Angiogenesis by Stem Cell Factor 1 Wei Zhang, 2 George Stoica, Serban I. Tasca, Katherine A. Kelly, and Cynthia J. Meininger 3 Cardiovascular Research Institute and Department of Medical Physiology, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114 [W. Z., K. A. K., C. J. M.], and Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas 77843-4467 [G. S., S. I. T.] ABSTRACT Mast cells accumulate within solid tumors and can release many an- giogenic factors, suggesting that they may modulate vascularization of tumors. Stem cell factor (SCF) stimulates mast cell migration, prolifera- tion, and degranulation and therefore may influence mast cell behavior within tumors. We investigated the contribution of SCF to tumor angio- genesis by manipulating its level in mammary tumors. Sense or antisense cDNA fragments of rat SCF were ligated into an episomal expression vector. Ethylnitrosourea-induced rat mammary tumor cell lines were transfected with vector containing either control (no insert, C-P), sense (S-P), or antisense (AS-P) SCF DNA. The functional nature of the trans- fectants was confirmed by measuring SCF in cell lysates and conditioned media. Immunohistochemical analysis of the tumors induced in Berlin- Druckrey rats by these transfected cells demonstrated that mast cell number and microvascular density were significantly higher in S-P tu- mors and significantly lower in AS-P tumors, compared with C-P tumors. The expression of von Willebrand factor, an endothelial cell marker, showed a similar pattern. AS-P tumors were significantly smaller than either C-P or S-P tumors. These data suggest that SCF modulates tumor growth and angiogenesis via the involvement of mast cells. INTRODUCTION Angiogenesis, the growth of new blood vessels from a preexisting microvascular bed (1), is of crucial importance for the growth, main- tenance, and metastasis of solid tumors (2– 4). Therefore, a more complete understanding of the factors contributing to tumor angio- genesis is of paramount importance. Tumor angiogenesis depends on the interaction of different tumor components, e.g., tumor parenchy- mal cells, endothelial cells, infiltrating cells from the bloodstream, and perhaps mast cells. There is much evidence to suggest a link between mast cells and angiogenesis. For example, mast cells are distributed along blood vessels (5), giving them a perfect position to modulate vessel growth. Mast cell degranulation alone is sufficient to induce neovascularization in rat mesentery (6) and in the chick chorioallan- toic membrane (7). Mast cells also accumulate within and around solid tumors (8). When tumor cells are injected into a chick embryo, there is a 40-fold increase in mast cell density around the tumor implantation site compared with normal tissue (9). Injection of mast cell suspensions into animals leads to acceleration of tumor growth (10), whereas decreasing the number of tissue mast cells leads to depression of tumor growth (11). Inhibiting mast cell degranulation with disodium cromoglycate also significantly depresses tumor growth (10, 12). In capillary hemangiomas, which are common benign vascular tumors that inflict young children, the mast cell concentration in the tumor is at least 5-fold higher than in normal tissue (13). When the hemangioma starts to shrink as the child gets older, a decrease in mast cell number precedes tumor shrinkage (13). On the basis of this circumstantial evidence, it has been suggested that mast cells in tumors modulate the neovascularization process. However, the role of mast cells in tumor angiogenesis has not been studied thoroughly. Tumor-associated mast cells are often found to have degranulated and to have released their chemical mediators, especially in the late stages of tumor proliferation (14). Many components of mast cells are angiogenic or can modulate the angiogenesis process (15, 16). These components include basic fibroblast growth factor, vascular endothe- lial growth factor, heparin, heparinase, histamine, tumor necrosis factor-, and various proteases. Therefore, mast cell degranulation may modulate angiogenesis. The activities of mast cells are largely controlled by SCF, 4 a mast cell growth factor. For example, SCF is a chemoattractant for mast cells (17) and repeated injection of SCF into the skin of mice results in the appearance of large numbers of mast cells at the injection site (18, 19). SCF can drive the proliferation of mast cells as well as promote mast cell maturation in vitro (20, 21). Finally, SCF induces mediator release from mouse mast cells in vitro (22) and can trigger mast cell activation and a mast cell-dependent inflammatory response in vivo (23). SCF is a product of the steel gene in mice and has two transmem- brane isoforms, SCF-1 and SCF-2 (24, 25). SCF-1, encoded by full-length mRNA, is a 248-amino acid protein that can be hydrolyzed by proteases, resulting in a soluble form of SCF. SCF-2, derived from alternatively spliced SCF mRNA, gives rise to a smaller 220-amino acid protein that lacks the same proteolytic cleavage site. It is inef- fectively cleaved at an alternative site and therefore remains almost exclusively as a cell membrane protein. Several types of tumor cells exhibit an increased production of SCF (26) in addition to other growth factors. However, the direct effect of SCF on tumor angiogenesis has not been examined. We hypothesized that large amounts of SCF released from tumor cells may account for the increased number of mast cells in tumors and may lead to an accelerated angiogenic response. In this report, we provide direct evidence that SCF expressed by mammary tumor cells modulates tumor angiogenesis by regulating mast cell activity. MATERIALS AND METHODS Cell Culture. Ethylnitrosourea-induced mammary tumor cells were gener- ated in Berlin Druckrey IV rats (27). This animal model for human breast cancer is characterized by a short latency period for tumor development, ovarian hormone dependency, high incidence of malignant tumors, and wide- spread metastases (27–29). In addition, these mammary tumors are well vascularized, and the supporting fibrovascular stroma is infiltrated with large numbers of mast cells. Numerous cell lines and clones with different tumori- genic and metastatic potentials have been isolated from these tumors (30). One cell line, designated Brc, was grown in a 37°C incubator with 10% CO 2 in complete medium consisting of DMEM with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 g/ml strep- tomycin, and 0.25 g/ml amphotericin B. Cells were given fresh medium Received 5/24/00; accepted 9/27/00. 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. 1 Supported by Interdisciplinary Research Initiatives funding from Texas A&M Uni- versity. 2 Present address: Simmons Comprehensive Cancer Center and the Department of Pharmacology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, NB4.120, Dallas, TX 75235-9111. 3 To whom requests for reprints should be addressed, at Cardiovascular Research Institute and Department of Medical Physiology, The Texas A&M University System Health Science Center, 702 SW H. K. Dodgen Loop, Medical Research Building Room 206E, Temple, TX 76504. Phone: (254) 742-7037; Fax: (254) 742-7145; E-mail: cjm@tamu.edu. 4 The abbreviations used are: SCF, stem cell factor; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; vWF, von Willebrand factor. 6757 on June 3, 2015. © 2000 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from