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Intratumoral gene therapy of malignant brain tumor in a rat model with angiostatin delivered 2Graduate Institute of Medical Science and Department of Neurologic Surgery, Tri Service General Hospital, National Defense Medical Center, Taipei, Taiwan 3Department of Microbiology and Immunology, National Defense Medical Center, Taipei, TaiwanCorrespondence to: X Xiao, Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA 15213, USAWe have utilized a recombinant adeno associated viral (AAV) vector carrying the angiostatin gene as an anti angiogenesis strategy to treat the malignant brain tumor in a C6 glioma/Wistar rat model.

Angiostatin, as a potent angiogenesis inhibitor, shows high promises as an anti cancer drug through the inhibition of tumor neovessel formation. However, sustained in vivo protein delivery is required to achieve the therapeutic effects. The AAV vector has been proven to be able to deliver sustained and high level gene expression in vivo, and therefore, is well suited to such a purpose. In this study, we implanted 5 x 105 C6 glioma cells into the rat brain 7 days before gene therapy. Intratumoral injection of a high titer AAV angiostatin vector has rendered efficacious tumor suppression and resulted in long term survival in 40% of the treated rats, whereas the control AAV GFP vector did not have any therapeutic benefits. In addition, we have investigated the combined gene therapy of an adenoviral vector carrying the suicidal thymidine kinase gene along with the AAV angiostatin vector. The combined therapy offered the best tumor suppressive effects and increased long term survival to 55% in the treated rats. Our study has demonstrated the potential of using AAV as a safe and effective vector for anti angiogenic gene therapy of brain tumors. The median survival time of patients with GBM is less than 2 years, despite multi modality treatments with extensive surgical resection, radiotherapy, chemotherapy or immunotherapy. In order to overcome this highly lethal neoplasm, molecular neurosurgery with gene therapy tools has been the subject of investigation since 1992,1 along with novel pharmaceutical interventions such as anti angiogenesis, and immunotherapy.2,3 Many of the gene therapy approaches are based on previously established anti neoplastic principles, including the use of prodrug/suicidal genes, tumor suppressor genes and immune enhancing cytokine genes.4,5,6,7,8,9 Recently, the anti angiogenic strategies have also been investigated for cancer gene therapy.10 Angiogenesis, the outgrowth of new blood vessels from the pre existing vasculature, is essential for a number of physiological process and pathological conditions.11,12 Low grade gliomas are moderately vascularized tumors, whereas high grade gliomas show prominent neovessel proliferation with high vascular density. It is hypothesized that solid tumors are dependent on angiogenesis for sustained growth, and that anti angiogenic treatment is a promising anti neoplastic therapy.13 Cerebral gliomas have been accordingly considered as a suitable candidate for such treatment, which attempts to eradicate or suppress the tumors by interfering with its blood supply. Although multiple factors contribute to the ultimate vascularization of tumors, some factors are especially relevant to gliomas. It is possible to effectively inhibit tumor growth by inhibiting the key factors in tumor vascularization.10 Experimental evidence indicates that blocking angiogenesis by specific inhibitory agents is associated with tumor regression in animals with different types of neoplasia. The promising anti angiogenic agents include (1) naturally occurring inhibitors of angiogenesis such as angiostatin and endostatin; (2) specific inhibitors of endothelial cell growth such as TNP 470 and thalidomide; (3) agents neutralizing angiogenic peptides such as antibodies or soluble receptors against FGF and VEGF; (4) anti adhesion molecules and antibodies. Therefore, anti angiogenic therapy represents one of the promising new approaches for anti cancer therapy and it is already in early clinical trials.12,14,15 Angiostatin is a potent inhibitor of angiogenesis in tumor growth and metastasis. It is derived from plasminogen as a biologically active fragment containing the kringle domains 1 It specifically inhibits endothelial cell growth,16,17,18 but has no direct effects on other cell types including tumor cells in vitro. Systemic administration of purified angiostatin efficiently suppresses malignant glioma growth in vivo.19 Recent studies have demonstrated that retroviral and adenoviral vectors that carry the angiostatin cDNA can be used to inhibit endothelial cell growth in vitro and angiogenesis in vivo. Vector mediated pandora bracelet gold and silver inhibition of tumor angiogenesis resulted in increased apoptosis in endothelial cells, as well as tumor cells, leading to the inhibition of tumor growth. These studies support the concept of dormancy therapy20,21,22,23,24,25,26,27 and rationalize the role of angiostatin gene transfer as a novel therapeutic strategy for malignant brain tumors. Adeno associated virus (AAV) offers many of the desirable features of retroviruses and adenovirus but without some of their potential drawbacks for application in gene therapy.28 AAV is a non pathogenic, replication defective single stranded DNA virus, capable of persisting either by integrating into cheapest place to buy pandora the host genome or retaining as an episome.29,30,31 The recombinant AAV vectors can transduce both dividing and non dividing cells in vitro and in vivo.32 Efficient and long term in vivo transduction and the lack of both cytotoxicity and cellular immune responses are the hallmark features of AAV mediated gene transfer.33,34,35,36,37 Although AAV vectors have been extensively used in gene therapy for genetic and metabolic diseases, few studies have used this vector system for cancer gene therapy.34,38,39,40,41,42 In this report, we have used the AAV vector carrying the cDNA of an anti angiogenic protein, angiostatin, to treat the highly malignant and highly angiogenic brain tumor derived from a C6 glioma cell line in a rat model. Intratumoral injection of the AAV angiostatin vector resulted in significant tumor suppressive effects. In addition, we have investigated the combined gene therapy of adenoviral vector carrying the suicidal thymidine kinase gene with the AAV angiostatin vector. The combined gene therapy offered the best tumor suppressive effects along with improved survival rate of the tumor bearing rats. In vitro biological effects of angiostatin from stably transfected and AAV angiostatin infected C6 glioma cells To demonstrate vector derived angiostatin expression in vitro or in vivo, an HA tag sequence was added to the 3' end of the angiostatin cDNA to distinguish from the endogenous protein. The angiostatin HA/pCEP4 expression plasmid was used to stably transfect the C6 glioma cells. Using lysine sepharose binding method followed by immunoblotting with an anti HA antibody, we had detected a 50 kDa protein from the culture medium, as well as cell lysate (Figure 1a, lanes 3 and 6). Due to glycosylation of the angiostatin in mammalian cells and the addtion of the HA tag, the apparent molecular weight of the angiostatin is larger than the theoretically deduced one of 38 kDa. The presence of abundant angiostatin in the conditioned medium of stably tranfected C6 glioma cells indicated its efficient secretion (Figure 1a, lane 3). In addition, no significant differences in growth rates were observed among the parental C6 glioma cells, the empty vector transfected C6/pCEP4 cells, and the C6/angiostatin HA/pCEP4 cells (data not shown). Furthermore, AAV angiostatin vector infected C6 glioma cells produced significant amounts of angiostatin protein in both conditioned medium and cell lysate (Figure 1b, lanes 3 and 6). However, the uninfected cells (Figure 1b, lanes 1 and 4) and the control AAV GFP vector infected cells (Figure 1b, lanes 2 and 5) had no detectable angiostatin HA, indicating that the AAV angiostatin vector is capable of infecting the C6 glioma cells and that the angiostatin protein can be effectively secreted. To study the biological effects of angiostatin in vitro, the conditioned different pandora bracelets media from C6 glioma cells, C6/pCEP4 cells, and C6/angiostatin HA/pCEP4 cells were add to the human umbilical vein endothelial (HUVE) cells culture to see if the HUVE cells growth could be inhibited. While the conditioned medium from both C6 and C6/pCEP4 control cells did not make significant difference, the conditioned medium from C6/angiostatin HA/pCEP4 cells caused significant growth retardation of HUVE cells (data not shown), suggesting that the secreted angiostatin was biological active in inhibiting endothelial cell growth in vitro. To test if AAV vector gene transfer can exert growth inhibition on the human HUVE cells cultures, AAV angiostatin or AAV GFP vectors were used to directly infect the HUVE cells. Surprisingly, no significant difference in growth rate was observed. This failure was later attributed to the low transduction efficiency (Figure 1c. This result confirmed the in vitro biological functionality of AAV derived angiostatin protein in inhibiting endothelial cell growth. Suppression of tumor growth by stably transfected angiostatin gene in C6 glioma cells To evaluate whether the angiostatin gene is functional in vivo in inhibiting angiogenesis of the gliomas, we initially carried out a test using the stably transfected C6/angiostatin HA/pCEP4 cells, along with the control parental C6 cells and the C6/pCEP4 cells that harbored an empty plasmid vector. The glioma cells were implanted intracranially, and their in vivo growth rates were investigated in a time course. The tumors were harvested from the rat brains at a weekly interval to measure the tumor size and vascularization, while the animals in the control groups all expired over the 3 weeks due to excessive tumor burden (Figure 2a). Histopathological analysis revealed that the gliomas derived from C6 and C6/pCEP4 cells were large, red, hypervascularized with petechia, and central necrosis. However, C6/angiostatin HA/pCEP4 derived gliomas were generally small, pale, with fewer visible surface blood vessels. The average tumor sizes at the end of third week after glioma implantation were 103.33 4.37 mm3 for C6/angiostatin HA/pCEP4 cells, 797.22 6.6 mm3 for C6 cells, and 756 24.82 mm3 for C6/pCEP4 respectively Figure 2a. In another experiment, the bystander effects were examined by mixing the C6/angiostatin HA/pCEP4 cells with either the parental C6 cells or the C6/pCEP4 cells in an equal ratio before tumor implantation. Strong in vivo inhibition of both C6 and C6/pCEP4 glioma growth was also observed in the mixing experiment by the C6/angiostatin HA/pCEP4 cells Figure 2b, suggesting that the secreted angiostatin can render the 'bystander' effect in inhibiting tumor neovessel formation. Immunohistochemical staining of the tumor thin sections revealed significantly fewer microvessels in the C6/angiostatin HA/pCEP4 glioma when compared with the C6 and C6/pCEP4 gliomas Figure 2c, as determined by direct microvessel counting of the Factor VIII receptor antigen positive vessels. On the second week after tumor implantation, the blood vessel counts in C6/angiostatin HA/pCEP4 glioma were 16.5% (13.3 of those seen in C6 glioma, and the vessel counts decreased to 13.7% (16.8 by the third week Figure 2c. In addition, TUNEL staining showed that the apoptotic indexes were higher in the C6/angiostatin HA/pCEP4 glioma than those of C6 and C6/pCEP4 gliomas at 3 weeks following intracranial injection of the tumor cells Figure 2d, supporting the observation in numerous previous studies by others.20,25,41 Suppression of tumor growth by intratumoral injection of AAV angiostatin vector To assess whether AAV angiostatin vector could inhibit angiogenesis and tumor growth in vivo, the same C6 glioma/Wistar rat model was used as in the aforementioned experiments. One week after implantation of 5 105 of the C6 glioma cells into the rat brain, either AAV angiostatin vector, PBS saline or AAV GFP vector was injected into the existing brain tumor. Starting from 2 weeks after tumor implantation, groups of rats were killed every week for evaluation. The tumor volumes on the third week after implantation were 87.2 35 mm3 in AAV angiostatin treated rats, 797.24 6.62 mm3 in PBS saline treated rats, and 741 30.24 mm3 in AAV GFP treated rats, respectively (Figure 3a and b). We have also monitored the survival time course of the tumor bearing rats. All of the Wistar rats which cheap pandora charms online received C6 glioma cell implantation and were treated with PBS saline or AAV GFP vector died within 23 days due to excessive tumor burden Figure 3c. In the same period, however, more than 60% of the AAV angiostatin vector treated rats were still alive. On the 35th day after C6 glioma cell implantation, 40% of the AAV angiostatin vector treated rats survived and continued to survive for 6 months, which was the duration of the experiments Figure 3c. These rats had tiny tumor nodules in the brains upon postmortem examination, demonstrating that the AAV angiostatin vector can efficiently suppress glioma growth in vivo. Immunohistochemical staining of the tumors with anti von Willebrand factor antibody on the first, second and third week after glioma implantation showed that the AAV angiostatin treatment significantly reduced the number of blood vessels in the tumors when compared with those seen in the PBS saline treated and AAV GFP treated ones (Figure 4a). On the second week, the blood vessel counts were 34.3% (33.4 of those in PBS saline treated C6 glioma. On the third week, the vessel counts were 25.1% (28.4 of the PBS saline treated C6 glioma Figure 4b. Consistently, the apoptotic indexes of the AAV angiostatin treated tumors were higher than those seen in the PBS saline treated and AAV GFP treated gliomas by the TUNEL staining Figure 4c. The increased tumor cell apoptosis may have contributed to the tumor shrinkage during the course of gene therapy. Combined gene therapy with adenovirus HSV tk vector and AAV angiostatin vector Anti angiogenic therapy is tumorstatic instead of tumoricidal, because it exerts its function directly on the endothelial cells and indirectly on the cancer cells. A combination of two different strategies employing distinct mechanisms, for example, anti angiogenic and suicidal gene therapies, may provide additive benefits. Based on this rationale, we investigated the combined gene therapy using both the adenoviral vector carrying the thymidine kinase (tk) gene (AdRSVtk) and the AAV vector carrying the angiostatin gene (AAV angiostatin) to treat the C6 glioma in the Wistar rat model. First, a time course of tumor growth was done on the tumor bearing rats undergoing gene therapies with the combined treatment of AdRSVtk and AAV angiostatin vectors, or with a single vector treatment and without therapeutic treatment. Three weeks after implantation, the C6 glioma tumors without any therapeutic gene treatment were very large and occupied nearly half of the cranial cavity. However, the tumors in the gene therapy groups were generally smaller and pale in color with fewer visible surface blood vessels, especially in the groups receiving anti angiogenic therapy. For example, the tumor volumes on the third week after C6 cell implantation were respectively 796 29.28 mm3 in the untreated control group, 101 1.95 mm3 in the AdRSVtk injected group, 86 7.46 mm3 in the AdRSVtk and AAV GFP co injected group, and 60 10.5 mm3 in the AdRSVtk and AAV angiostatin co injected group (Figure 5a). On the fourth week after implantation, the tumor volumes in the AdRSVtk injected, AdRSVtk and AAV GFP co injected, and AdRSVtk and AAV angiostatin co injected groups further decreased to 41 14.25 mm3, 46 12.4 mm3, and 9.2 6.0 mm3, respectively Figure 5a. On the fifth week after implantation, the tumor volumes in the surviving animals of the above groups were measured at 22 3.5 mm3, 19 1.0 mm3, and 3.3 0.

96 mm3, respectively Figure 5a. All the AdRSVtk injected animals also received prodrug ganciclovir treatment at a dosage of 7.5 mg/kg body weight twice a day to achieve the tumor killing effect.

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