SPARC expression is associated with impaired tumor growth, inhibited angiogenesis and changes in the extracellular matrix Alexandre Chlenski 1 , Shuqing Liu 1 , Lisa J. Guerrero 1 , Qiwei Yang 1 , Yufeng Tian 1 , Helen R. Salwen 1 , Peter Zage 2 and Susan L. Cohn 2 * 1 Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA 2 Department of Pediatrics, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA Secreted protein, acidic and rich in cysteine (SPARC), is a multi- functional matricellular glycoprotein. In vitro, SPARC has antian- giogenic properties, including the ability to inhibit the prolifera- tion and migration of endothelial cells stimulated by bFGF and VEGF. Previously, we demonstrated that platelet-derived SPARC also inhibits angiogenesis and impairs the growth of neuroblas- toma tumors in vivo. In the present study, we produced rhSPARC in the transformed human embryonic kidney cell line 293 and show that the recombinant molecule retains its ability to inhibit angiogenesis. Although 293 cell proliferation was not affected by exogenous expression of SPARC in vitro, growth of tumors formed by SPARC-transfected 293 cells was significantly impaired com- pared to tumors comprised of wild-type cells or 293 cells trans- fected with a control vector. Consistent with its function as an angiogenesis inhibitor, significantly fewer blood vessels were seen in SPARC-transfected 293 tumors compared to controls, and these tumors contained increased numbers of apoptotic cells. Light microscopy revealed small nests of tumor cells surrounded by abundant stromal tissue in xenografts with SPARC expression, whereas control tumors were comprised largely of neoplastic cells with scant stroma. Mature, covalently cross-linked collagen was detected in SPARC-transfected 293 xenografts but not in control tumors. Our studies suggest that SPARC may regulate tumor growth by inhibiting angiogenesis, inducing tumor cell apoptosis and mediating changes in the deposition and organization of the tumor microenvironment. ' 2005 Wiley-Liss, Inc. Key words: tumor growth; angiogenesis; SPARC; collagen; transglutaminase; neuroblastoma SPARC, also known as osteonectin and BM-40, is a matricellu- lar glycoprotein that modulates interactions between cells and the extracellular matrix. 1 In vitro, SPARC is capable of modifying cell shape, acting as a deadhesive protein, regulating the production of several extracellular matrix proteins and inhibiting cell cycle pro- gression. Although the precise mechanisms by which SPARC mod- ulates these cell–matrix interactions remain unclear, its antiproli- ferative effect appears to be mediated, at least in part, by its ability to regulate the activity of certain growth factors (reviewed by Yan and Sage 2 ). SPARC has been shown to suppress the proliferation of human fibroblasts by directly binding to PDGF-AB or -BB. Sim- ilarly, by directly binding VEGF, SPARC inhibits microvascular endothelial cell proliferation stimulated by VEGF 165 . SPARC also blocks the mitogenic effects of the potent angiogenic stimulator bFGF, indicating that it has antiangiogenic activity. Studies in our laboratory have demonstrated that SPARC, puri- fied from human platelets, potently suppresses bFGF-induced angiogenesis in vivo and impairs the growth of NB xenografts. 3 In addition, we have shown that a SPARC peptide, FS-E, designed to represent the EGF-like module of the follistatin domain, blocked bFGF-induced angiogenesis in a rat corneal assay and inhibited neovascularization induced by NB cells. 4 However, the role endogenous SPARC plays in regulating tumor angiogenesis and growth remains unclear. In some types of cancer, high levels of SPARC expression in neoplastic cells have been correlated with disease progression and poor prognosis. 5,6 Strong expression of SPARC in stromal cells has also been observed in some malignant tumors, suggesting that it may contribute to tumor invasion. 7 In contrast, other studies suggest that SPARC is a tumor suppressor. SPARC induces apoptosis of ovarian cancer cells, 8 and antitumor effects have been observed in several types of human neoplasm in addition to NB, including pancreatic 9 and breast 10 cancers. Consistent with its role as a tumor suppressor, enhanced tumor growth has been reported in SPARC-null mice following inocula- tion of Lewis lung carcinoma 11 or pancreatic cancer cells. 12 SPARC-null mice have characteristic curled tails and lax skins consequent to altered collagen fibrillogenesis. 13 Interestingly, SP –/– mice also exhibited alterations in the production and organi- zation of the extracellular matrix components within and surround- ing the implanted tumors. Tumor capsules in SP –/– mice contained less collagen, and collagen fibers were smaller and had fewer covalent cross-links than those in tumor capsules grown in SP 1/1 mice. These observations led Brekken et al. 11 to speculate that the enhanced growth of tumors in SP –/– mice was due, at least in part, to changes in the organization of the extracellular matrix that cre- ated a less restrictive microenvironment for tumor progression. In the present study, we produced rhSPARC in adenovirus-trans- formed mammalian human embryonic kidney 293 cells 14 to further investigate its role in tumor growth and angiogenesis. We show that rhSPARC retains its ability to induce endothelial cell apoptosis and inhibit endothelial cell migration in vitro. In vivo, rhSPARC potently inhibited neovascularization in the Matrigel assay. In addi- tion, the tumorigenicity of 293 cells exogenously expressing SPARC was significantly impaired compared to parental cells and 293 cells transfected with a control empty vector. SPARC-trans- fected xenografts were less vascular than controls, and increased numbers of apoptotic cells were present in these tumors. Further- more, the assembly of the extracellular matrix within and surround- ing SPARC-transfected xenografts significantly differed from con- trol tumors. Our data suggest that in addition to blocking angiogen- esis and inducing apoptosis, rhSPARC may regulate tumor growth by mediating changes in the tumor microenvironment. Material and methods Cell culture and rhSPARC production Human SPARC cDNA was amplified as described 3 with primers 5 0 -CACTGAAGCTTCCCAGCACCATGAGGGC-3 0 and Grant sponsor: National Institutes of Health; Grant number: NS 049814; Grant sponsor: Neuroblastoma Children’s Cancer Society; Grant sponsor: Friends for Steven Pediatric Cancer Research Fund; Grant sponsor: Elise Anderson Neuroblastoma Research Fund; Grant sponsor: Robert H. Lurie Comprehensive Cancer Center, National Cancer Institute; Grant number: 5P30CA60553. *Correspondence to: Division of Hematology/Oncology, Children’s Memorial Hospital, Box #30, 2300 Children’s Plaza, Chicago, IL 60614, USA. Fax: 1773-880-3053. E-mail: scohn@northwestern.edu Received 29 March 2005; Accepted after revision 24 May 2005 DOI 10.1002/ijc.21357 Published online 28 July 2005 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: BAEC, bovine aorta endothelial cell; bFGF, basic fibro- blast growth factor; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; HUVEC, human umbilical vein endothelial cell; MVD, mean vascular density; NB, neuroblastoma; PDGF, platelet-derived growth factor; rhSPARC, recombinant human SPARC; TG2, transglutaminase II; TUNEL, TdT-mediated dUTP nick end labeling; VEGF, vascular endothe- lial growth factor. Int. J. Cancer: 118, 310–316 (2006) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer