Suppression of Lung Tumor Growth and Metastasis in Mice by Adeno-Associated Virus-Mediated Expression of Vasostatin Ke Xia Cai, 1 LaiYingTse, 3 Carly Leung, 1 PaulK.H.Tam, 2 Ruian Xu, 3,4 and Mai Har Sham 1 Abstract Purpose: Angiogenesis inhibitors have strong therapeutic potential as antitumor agents in suppressing tumor growth and metastatic progression.Vasostatin, the N-terminal domain of calreticulin, is a potent angiogenesis inhibitor. In this study, we determined the effectiveness of vasostatin delivered by recombinant pseudotype adeno-associated virus 2/5 (rAAV2/5-VAS) as a gene therapy approach forlung cancer treatment. Experimental Design: WeusedrAAV2/5todeliver vasostatinintratumorallyorsystemicallyin different mouselungtumormodels ö subcutaneous, orthotopicxenograft, and spontaneous metastasislungtumormodels.ThetherapeuticefficacyofrAAV2/5-VASwasdeterminedbymoni- toringtumorvolume,survivalrate,anddegreeofneovascularizationaftertreatmentinthesemodels. Results: Mice bearing subcutaneous tumor of rAAV2/5-VAS pretreated Lewis lung carcinoma cells showed >50% reduction in primary tumor volume and reduced spontaneous pulmonary metastases.The tumor-suppressive action of rAAV2/5-VAS in subcutaneous human lung tumor A549xenograftcorrelatedwithareducednumberofcapillaryvesselsintumors.Intheorthotopic xenograft model, rAAV2/5-VAS suppressed metastasis of A549 tumors to mediastinal lymph nodes and contralateral lung. Furthermore, treatment of immunocompetent mice in the spontaneous lung metastases model with rAAV2/5-VAS after primary tumor excisionprolonged their median survival from 21to 51.5 days. Conclusion: OurresultsshowtheeffectivenessofrAAV2/5-VASasanangiogenesisinhibitorin suppressingtumorgrowthduringdifferentstagesoftumorprogression,validatingtheapplication of rAAV2/5-VAS gene therapy intreatment against lung cancer. Angiogenesis is a major feature of tumor growth and metastasis. As such, targeting the tumor neovasculature is an attractive strategy for effective cancer therapy. A number of endogenous angiogenesis inhibitors have been identified and tested in preclinical models (1), and several inhibitors, including endostatin and angiostatin, have been introduced into clinical trials (2). Although existing antiangiogenesis therapies show lower toxicity than conventional treatments, such as radiotherapy, they are often associated with limited tumor regression and other clinical effects (3). Therefore, much effort has been focused on identifying novel angiogenesis inhibitors and evaluating new approaches to maximize the effects of antiangiogenesis therapies (4). Vasostatin, a 180–amino acid fragment from the N-terminal domain of calreticulin, is a potent endogenous angiogenesis inhibitor (5). Daily administration of recombinant vasostatin, a small soluble molecule, was shown to reduce xenograft growth in mice (5, 6). Repeated vasostatin gene delivery by DNA i.m. injection or adenoviral vectors i.t. injection also suppressed subcutaneous tumor growth in mice (7, 8). The biosafety of vasostatin has been recognized, as vasostatin treatment did not affect physiologic angiogenesis at tumor-inhibiting doses (9). However, previous reports on the therapeutic effects of vaso- statin were based solely on subcutaneous tumor models, whereas the key challenge in cancer therapy lies in eradicating metastases at the subclinical and advanced stages, wherein the tumor microenvironment affects induction of tumor angiogen- esis (10). Given the strong therapeutic potential of this molecule, evaluation of the efficacy of vasostatin in more relevant orthotopic and metastatic models of tumorigenesis is duly warranted. The method used for delivering angiogenesis inhibitors greatly affects their efficacy (3, 11). Maintaining sustained levels of angiogenesis inhibitors is crucial for prolonged suppression of angiogenesis. To target tumor angiogenesis, gene therapy vectors are required to offer long-term expression without vector-associated toxicity or immunity. Adeno-associ- ated viruses (AAV) have been previously used with acceptable safety (12, 13) and are attractive vectors for angiogenesis inhibitor gene transfer as they offer stable and persistent gene expression in transduced cells (14). Recombinant AAV (rAAV) vectors are effective in using normal tissues as a platform for Cancer Therapy: Preclinical Authors’ Affiliations: Departments of 1 Biochemistry and 2 Surgery, Li Ka Shing Faculty of Medicine and 3 Institute of Molecular Biology,The University of Hong Kong, Pokfulam, Hong Kong, China and 4 Institute of Molecular Medicine, Huaqiao University, Fujian, China Received8/8/07;revised10/11/07;accepted10/11/07. Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage charges.This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solely toindicatethisfact. Note: Supplementary data for this article are available at Clinical Cancer Research Online(http://clincancerres.aacrjournals.org/). Requests for reprints: Mai Har Sham, Department of Biochemistry, Li Ka Shing Faculty of Medicine,The University of Hong Kong, 3rd Floor Laboratory Block, 21Sassoon Road, Pokfulam, Hong Kong, China. Phone: 852-28199195; Fax: 852-28551254;E-mail:mhsham@hkucc.hku.hk. F 2008AmericanAssociationforCancerResearch. doi:10.1158/1078-0432.CCR-07-1930 www.aacrjournals.org Clin Cancer Res 2008;14(3) February 1, 2008 939 Research. on May 20, 2020. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from