Suppression of Intracranial Human Glioma Growth After Intramuscular Administration of an Adeno-Associated Viral Vector Expressing Angiostatin1

[CANCER RESEARCH 62, 756–763, February 1, 2002] Suppression of Intracranial Human Glioma Growth after Intramuscular Administration of an Adeno-associated Viral Vector Expressing Angiostatin1

Hsin-I Ma, Ping Guo, Juan Li, Shinn-Zong Lin, Yung-Hsiao Chiang, Xiao Xiao2, and Shi-Yuan Cheng2 Cancer Institute and Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 [H-I. M., P. G., S-Y. C.]; Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 [H-I. M., J. L., X. X.]; and Department of Neurological Surgery and Tri-Service General Hospital, Taipei, Taiwan [H-I. M., Y-H. C.], Neuromedical Scientific Center, Buddhist Tzu-Chi General Hospital, Hualian, Taiwan [S-Z. L.]

ABSTRACT delivery of the recombinant antiangiogenic proteins requires enor- mous quantity as well as administration of the therapeutic products for Despite various therapeutic interventions, glioblastoma multiforme a prolonged period. To overcome the shortcomings of protein deliv- (GBM) is one of the most highly vascularized neoplasms in humans with ery, alternative approaches, such as gene delivery of the antiangio- poor prognosis. In this study, we show that a single i.m. injection of an adeno-associated viral (AAV) vector expressing angiostatin, a potent an- genic factors, have been explored. Because the presence of sustained giogenic inhibitor, effectively suppresses human glioma growth in the and high-level antiangiogenic proteins is essential to maintain tumor brain of nude mice. Approximately 40% of the tumor-bearing mice neovessel inhibition and hence tumor growth suppression, previous treated with AAV-angiostatin vector survived for >10 months (the dura- attempts of gene therapy fell short, attributable mainly to transient or tion of the experiments). In contrast, 100% of the tumor-bearing mice in insufficient gene expression of the antiangiogenic factors (10–18). the control groups, with or without i.m. injection of a control vector AAV vectors have been used widely to achieve efficient and AAV-GFP, died because of excessive tumor burden by 6 weeks. High long-term gene delivery to treat numerous genetic diseases in a wide levels of angiostatin produced by the AAV vector were detected in blood variety of animal models (19–24) as well as in human trials (25–28). circulation for >250 days after the one-time vector injection. The secreted AAV vectors are derived from the nonpathogenic, replication-defec- angiostatin specifically targeted neovessels in the brain tumors, as evi- tive parvovirus that contains a single-stranded DNA genome. The denced by the diminished vessel densities and increased apoptosis of tumor cells surrounding these neovessels. Our study thus demonstrates vectors are able to effectively transduce dividing and nondividing that AAV-mediated antiangiogenesis gene therapy offers efficient and cells both in vitro and in vivo (29), thus offering stable gene transfer sustained systemic delivery of the therapeutic product, which in turn by either integrating into the host chromosomes or persisting as an effectively suppresses glioma growth in the brain. episome (30–32). In addition, the lack of cytotoxicity and minimal cellular immune responses after AAV-mediated in vivo gene transfer also contribute to the success of long-term gene delivery in a variety INTRODUCTION of tissues including liver, brain, and muscle (22, 24, 26, 33–37). Novel strategies are needed to treat malignant GBM,3 the most In this study, we report that a single i.m. administration of an AAV common type of human brain tumor. Despite multiple therapeutic vector carrying angiostatin gene effectively suppresses human U87 approaches including surgical resection, radiotherapy, chemotherapy, MG glioma growth in the brains of nude mice. Muscle was used here and immunotherapy, the median survival time of patients with GBM as a platform to produce AAV-encoded angiostatin and to secrete the is Ͻ2 years. The malignant progression from astrocytoma to GBM is therapeutic product into the blood circulation at high levels for pro- often accompanied by increased angiogenesis and up-regulation of longed periods. Such systemic delivery of angiostatin led to the vascular endothelial growth factor and its receptors (1). Tumors regression of established i.c. gliomas by targeted inhibition of tumor modulate the angiogenesis process by producing both positive and neovessel development. No notable local or systemic toxicity was negative effectors (2–4). A number of tumor-derived circulating pro- observed during the course of gene therapy. Thus, systemic delivery teins exhibit potent antiangiogenic effects (5, 6). For example, an- of antiangiogenic proteins by AAV-mediated muscle gene transfer giostatin is derived from plasminogen as a biologically active frag- offers a novel and promising approach for brain cancer therapy. ment containing four kringle domains (5). Several reports have shown that direct administration of purified recombinant angiostatin protein MATERIALS AND METHODS through different routes in vivo can inhibit tumor growth and even cause tumor regression in animal models (7–9). Angiostatin interacts Construction of an AAV Vector Carrying the Angiostatin Gene. A cDNA coding for the mouse angiostatin was amplified by PCR using primers directly with endothelial cells as an endothelial cell inhibitor inde- corresponding to the amino acid residues 1–6 and 461–466 of plasminogen. pendent of the blood-brain-barrier because i.p. injection of angiostatin This construct has an endogenous plasminogen secretory signal, a preactiva- can inhibit i.c. glioma growth (7, 10). Although promising, direct tion peptide sequence, four kringle domains (1–4), and a short HA tag sequence (38). The cDNA fragment was then cloned into a pXX-UF1 expres- Received 9/20/01; accepted 12/3/01. sion vector (39). pXX-angiostatin-HA, pXX2, and pXX6 were cotransfected The costs of publication of this article were defrayed in part by the payment of page into 293 cells for viral vector production (40). The titer of AAV vectors was charges. This article must therefore be hereby marked advertisement in accordance with determined by a dot blot assay (40). A control AAV-GFP virus was also 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by a grant from the Brain Cancer Program of James S. McDonnell constructed using a similar approach. Foundation and a start-up fund from University of Pittsburgh Cancer Institute (to S-Y. C.). Angiostatin Protein Analyses. The U87 MG glioma cells were infected 2 To whom requests for reprints should be addressed, at University of Pittsburgh with 1 ␮l of AAV-angiostatin-HA or AAV-GFP (1 ϫ 1013 viral particles/ml) School of Medicine, Department of Molecular Genetics and Biochemistry, BST W-1244, for 72 h. The conditioned medium and cell lysates were incubated with 200 Lothrup Street, Pittsburgh, PA 15261. Phone: (412) 648-9487; Fax: (412) 624-1401; E-mail: [email protected] (X. X.); Cancer Institute and Department of Pathology, University lysine-Sepharose at 4°C overnight. The bound materials were separated on a of Pittsburgh, BST W-1055, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: (412) 648- 10% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. 3317; Fax: (412) 624-7737; E-mail: [email protected] (S-Y. C.). The membrane was blocked and probed with an anti-HA antibody (Covance 3 The abbreviations used are: GBM, glioblastoma multiforme; AAV, adeno-associated Co., Richmond, CA; Ref. (38). To examine the AAV angiostatin-HA in vivo, virus; i.c., intracranial; HA, hemagglutinin antigen; BrdUrd, bromodeoxyuridine; IHC, ␮ immunohistochemistry; GFP, green fluorescent protein; AI, apoptotic index; TUNEL, 10 l of mouse serum collected from tail veins were analyzed by immuno- terminal deoxynucleotidyl transferase-mediated nick end labeling. precipitation, followed by Western blot analysis. 756

and substrate for alkaline phosphatase that were included in the TUNEL staining kit. The AI was determined by counting 1000 to 3000 cells in each stained tissue section. The AI was calculated as a ratio of apoptotic cells (brown stained nuclei) to total tumor and endothelial cells (dark blue stained by hematoxylin) within these areas (43).

RESULTS Single Administration of AAV Vector in Muscle Resulted in a High-Level and Prolonged Secretion of Angiostatin into Blood Circulation. Muscle has been used previously to produce therapeutic proteins encoded by AAV vectors (44– 49). To determine whether AAV-encoded angiostatin can be efficiently ex- Fig. 1. Immunoblot analysis of angiostatin in mouse sera. A, representative serum pressed in mouse muscle and secreted into blood circulation over levels of angiostatin from individual mice that were i.m. treated with AAV-GFP (Lane 1) an extended period, we analyzed serum samples from mice that had or AAV-angiostatin (Lanes 2–8) at indicated times. High levels of angiostatin were found in the majority of i.m. AAV-angiostatin-treated mice (Lanes 2–8). No angiostatin was received an i.m. injection of either the AAV-angiostatin vector, the detected in AAV-GFP treated mice (Lane 1). B, serum levels of angiostatin and survival control AAV-GFP vector, or PBS. Blood samples from individual of the tumor-bearing mice. Two of the long-term survival mice were analyzed and showed mice (Fig. 1A) or from the same mice at different time points (Fig. persistent angiostatin expression from days 106 to 193 (Lanes 3–5) in one mouse and from days 69 to 193 (Lanes 6–9) in another mouse. No angiostatin was detected in PBS-treated 1B) were analyzed. In a majority of the cases, 43- to 182-fold mice (Lane 1) or in AAV-GFP-treated mice (Lane 2). However, another two separate i.m. increases and prolonged expression of AAV-angiostatin were de- AAV-angiostatin-treated mice died because of the tumor burden but showed no or low level of angiostatin expression (Lanes 10 and 11). The molecular weight of angiostatin tected. However, angiostatin was not detected in the blood samples was Mr 58,000, instead of Mr 38,000 (13). Numbers in the parentheses are the differences from the PBS (Fig. 1B, Lane 1) or AAV-GFP (Fig. 1A, Lane 1 and of angiostatin expression in folds in comparison with the controls (serum samples from Fig. 1B, Lane 2) treated mice. The expression levels of angiostatin PBS or AAV-GFP-treated mice). Their calculations are described in “Materials and Methods.” increased along with time (Fig. 1A, Lanes 2–8, and Fig. 1B, Lanes 3–5 and 6–9), consistent with previous studies using different genes carried by AAV vectors in muscle tissues (44, 45, 49). In Animal Studies. Eighteen days before i.c. implantation of the U87 MG addition, high levels of angiostatin in the serum closely correlated ␮ cells, 500 l of AAV-angiostatin-HA vector or AAV-GFP control vector with the survival time of the mice (see below). For example, a ϫ 13 (1 10 viral particles/ml) were injected into the thighs and gluteal muscles. considerable amount of angiostatin was detected (74-fold or higher In another control group, 500 ␮l of PBS were injected into the mice. On day increases) in serum from mice that had survived for Ͼ300 days zero, 5 ϫ 105 of U87 MG cells were implanted into the mouse brains (41). Mouse brain, muscle, liver, lung, heart, and kidney tissues were removed. Thin after implantation of the U87 MG cells in the brain (Fig. 1B, Lanes cryostat sections were stained with H&E. The sizes of brain tumors were 3–5 and 6–9). In contrast, some other mice that only had 2.6–10- microscopically determined (42). To evaluate the proliferative activities of fold increases of circulating angiostatin (Fig. 1B, Lanes 10 and 11) glioma and endothelial cells in vivo, 300 ␮l of BrdUrd solution (Amersham, died because of tumor burden by weeks 8 and 10 after implantation Piscataway, NJ) were injected i.p. into one group of mice 30 min before the of the U87 MG cells. These data demonstrate that high levels of animals were sacrificed. The brains and other organs of these mice were fixed angiostatin in the mouse blood circulation indeed can be achieved and embedded (43). Mouse brain, muscle, liver, heart, kidney, and lung tissue after AAV vector-mediated gene transfer in muscle. were stained with H&E, an anti-CD31 antibody (PharMingen, San Diego, CA), an anti-HA antibody, an anti-BrdUrd antibody (Amersham), and a TUNEL staining kit (Roche Diagnostics, Indianapolis, IN) as described previously (41, 43). Quantitative Analyses for Expression of Angiostatin-HA and IHC Data. We could not quantify the amounts of in vivo circulated angiostatin-HA proteins in serum samples collected from mice because of the unavailability of commercial ELISA kits. Thus, we estimated the differences of overexpressed angiostatin-HA proteins detected by the immunoprecipitation followed by Western blot analysis among the serum samples collected from various mice in different groups. Defined areas of the positive signals of angiostatin-HA in the X-ray film were scanned into a computer program (Bio-Rad Quantity One program; Bio-Rad Laboratories, Richmond, CA,) and the differences among these areas were compared using the lowest density (PBS or GFP-treated lanes, in pixels) as a numerator. The final differences in folds were then normalized to each other by the amounts of total proteins in each serum sample that were analyzed. All tumors were sectioned through their largest diameter, and then representative sections were used for quantitative immunohistochemistry. The mean values of the sections from three to five separate mouse brains in each group were used for the quantitative analyses. Quantitative analysis of the blood vessel densities of tumor samples was done as described previously using the Metamorph Image System for Microsoft Windows (Universal Imaging, West Fig. 2. Suppression of i.c. U87 MG glioma growth by AAV-angiostatin. Brain tumor Chester, PA; Ref. 41). The proliferative index of BrdUrd incorporation was growth rates were measured in mice that were i.m. treated with PBS (n ϭ 6) or AAV-GFP calculated as a percentage of positive nuclei (brown colored) to total cells (n ϭ 6) or AAV-angiostatin (n ϭ 14). Volumes of established gliomas were estimated as under light microscopy. To calculate BrdUrd labeling index, Ͼ2000 cells were described previously (42). At each time point, minimums of two mice were used to obtain examined in each tissue section. The apoptotic cells were shown as fluores- the estimated tumor volumes. Insets, H&E stains on cryostat sections of mouse brains from each group at indicated time points. Arrows, tumor sites in each brain slide. The cence-positive cells visualized by fluorescence microscopy. After the photo- experiments include 6–14 mice in each group and were performed twice with similar graphs were taken, these slides were treated with anti-fluorescein antibody-AP results; bars, SD. 757

gradually converted into a double-stranded template for transgene expression (44). Given the vicious growth of U87 MG tumors and the slow onset of AAV vector gene expression, we injected the AAV angiostatin vector into the muscle 18 days prior to tumor implantation. This measure assured sufficient AAV transgene expression upon intracerebral implantation of 5 ϫ 105 U87 MG glioma cells. Simi- larly, we injected the muscle of the two control groups either with an AAV-GFP vector or with PBS 18 days prior to glioma inoculation. To evaluate the tumor-suppressive effects of AAV-encoded angiostatin, the i.c. glioma growth rates were measured in the treatment group that received the i.m. injection of AAV-angiostatin as well as in the two control groups. Consistent with our previous studies (41, 42), brain tumors in the two control groups had extensive growth (Fig. 2). By contrast, brain tumors in the mice that received an i.m. injection of AAV-angiostatin vector experienced slower growth in the first six weeks, followed by regression 6 weeks after glioma inoculation (Fig. Fig. 3. Long-term survival of glioma-bearing mice after AAV-angiostatin i.m. injec- 2). This phenomenon of initial slow tumor growth followed by a tion. Survival analyses were done on mice that were i.m. treated with PBS (n ϭ 6) or steady regression could possibly be attributed to the initial low levels AAV-GFP (n ϭ 3) or AAV-angiostatin (n ϭ 14). At 6 weeks after implantation of U87 MG glioma cells into mouse brains. All mice in control groups (PBS or AAV-GFP IM of angiostatin expression, which were insufficient to stop the tumor treated) died because of excessive tumor growth. However, the AAV-angiostatin-treated growth. However, the gradual increase of AAV vector gene expres- tumor-bearing mice had much improved survival rates with 57% of the mice surviving sion with time was sufficient in inhibiting tumor progression (Fig. 1A, over 7 weeks and 43% of the mice surviving for long term. The data were obtained from the identical sets of animal experiments described in Fig. 2, and similar survival rate Lanes 4–8; Fig. 1B, Lanes 3–5 and 6–9). Histological analysis of curves were obtained each time. H&E staining was performed on the brain tumor samples from mice treated with AAV-angiostatin vector, AAV-GFP vector, and PBS Systemic Circulation of AAV-encoded Angiostatin Suppressed saline (Fig. 2). By the fifth week after glioma inoculation, the tumor Local Glioma Growth in Brains. Previous studies using AAV vec- volumes in the control groups reached 40.0 Ϯ 12.0 mm3 for PBS- tors for i.m. gene transfer have indicated a lag period of 2–3 weeks, treated mice and 35.0 Ϯ 8.0 mm3 for AAV-GFP treated mice. By the during which time the single-stranded AAV vector genomes were sixth week after glioma inoculation, the tumor volumes increased to

Fig. 4. Immunohistochemical analyses of various U87 MG i.c. gliomas. a–d, histological analyses on cryostat brain sections of an i.m. PBS-treated mouse sacrificed on the 6th week after implantation of U87 MG cells. e–h, an i.m. AAV-GFP-treated mouse on the 6th week. i.m. AAV-angiostatin-treated mice on the 6th (i–l) and the 10th (m–p) weeks are shown. The sections were stained with an anti-HA antibody (a, e, i, and m), an anti-CD31 antibody (b, f, j, and n), TUNEL staining (c, g, k, and o), and an anti-BrdUrd antibody (d, h, l, and p). Strong immunoactivities of the anti-HA antibody and TUNEL were found in brain tumors of mice that were treated by IM AAV-angiostatin (i/m and k/o, respectively). High levels of BrdUrd incorporation were detected in brain tumors of mice in all groups (d, h, l, and p). Arrows in j/k and n/o, corresponding vessels in these tumor tissue sections. Three to five individual tumor samples of each class were analyzed each time, and the experiments were repeated three times with similar results. 758

greater than 62.8 Ϯ 4.4 mm3 (Fig. 2), and all of the control mice succumbed to the excessive tumor burden. In contrast, volumes of the brain tumors in the AAV-angiostatin vector i.m. treated mice were 24.0 Ϯ 11.0 mm3 by week 6 and decreased to 3.0 Ϯ 2.1 mm3 (Fig. 2) by week 10 after tumor inoculation. Next, we evaluated in a separate experiment the survival of the tumor-bearing mice in the therapeutic group treated with AAV- angiostatin and the two control groups treated with AAV-GFP or PBS. Although all of the mice in the two control groups died within 6 weeks after glioma inoculation (Fig. 3), the mice in the therapeutic group, which received a one-time i.m. injection of AAV-angiostatin vector, only developed small tumors by 6 weeks, and the tumors remained small and decreased in size as time progressed (Fig. 2). Fifty-seven % (8 of 14) of the mice treated by the i.m. AAV-angiostatin injection survived 8 weeks, and 43% (6 of 14) survived Ͼ42 weeks after glioma inoculation. Another group of mice that received an additional injection of AAV-angiostatin vector i.c. 1 month after an i.m. injection of the same vector (2 weeks after glioma inoculation) exhibited essen- tially the same survival rate as the group treated with an i.m. injection alone (data not shown). It is unclear why the additional i.c. treatment of AAV-angiostatin vector did not achieve additional tumor-suppressive effect. Serum sample analysis revealed that some of the AAV- angiostatin-treated mice that succumbed to brain tumor in 8–10 weeks after glioma inoculation had lower levels of circulating angiostatin (Fig. 1A, Lanes 10 and 11), whereas the long-term survivors had high levels of serum angiostatin (Fig. 1A, Lanes 3–5 and 6–9). These results provided supportive evidence that high-level expression of the angiogenic inhibitor is critical to effectively inhibit tumor growth. Targeted Inhibition of Tumor Neovasculature by Angiostatin Led to Increased Apoptosis. To determine whether circulating angiostatin could specifically target tumor vessels and cause apoptosis in glioma, we performed immunostaining on established U87 MG gliomas from both the AAV-angiostatin i.m.-treated mice and the control mice. Because the angiostatin cDNA was tagged with a HA epitope, an antibody against the tag was used to specifically detect vector- derived product. This strategy can avoid the interference by endogenous angiostatin production associated with tumor itself. As expected, the tumor blood vessels and tumor mass in the mice (Fig. 4, i and m) that were treated by AAV-angiostatin i.m. injection were stained positive by the anti-HA antibody. In contrast, no angiostatin was detected in the nearby brain tissues or in the opposite hemisphere of the normal brain area that did not have tumor inoculation (Fig. 4, i and m, and data not shown). In addition, no angiostatin was detected in the tumor vessels of the control mice that were i.m. injected either by AAV-GFP (Fig. 4e) or by PBS (Fig. 4a). These results suggest selective enrichment of angiostatin in the tumor vasculature by targeted binding. Angiostatin might force the tumor cell population into a dormant state by directly targeting the vascular compartment of the glioma (7). The positive staining of some of the tumor mass might be attributable to the leaky nature of the glioma neovessels that leaked angiostatin into the surrounding tumor area. To score neovessel density in the tumors, we stained U87 MG glioma with an anti-CD31 antibody that recognizes blood vessels and quantified the neovessel Fig. 5. Decreased vessel densities and increased apoptotic rates in U87 MG i.c. gliomas densities in various gliomas. We observed that 80% or more decreases established in i.m. AAV-angiostatin-treated mice. Quantitative analyses of the IHC data in vessel densities in the AAV-angiostatin-treated gliomas (Fig. 4, j were done as described in “Materials and Methods.” The representative IHC stains from and n, and Fig. 5A) was accompanied by much smaller tumors (Fig. control and i.m. AAV-angiostatin-treated mice are shown in Fig. 4. A, vascular densities in various gliomas of IHC data that are shown in Fig. 4, b, f, j, and n. B, BrdUrd 4, i and m, and Fig. 2). incorporation in cells of the various gliomas of IHC data that are shown in Fig. 4, d, h, l, To further demonstrate that tumor growth suppression by AAV- and p. C, AI in various gliomas of IHC data that are shown in Fig. 4, c, g, k, and o. In each encoded angiostatin was attributable mainly to increased tumor cell analysis, five to seven different areas within the same tissue section were examined. The mean values of the sections from three to five separate mouse brains in each group were apoptosis rather than decreased proliferation, we analyzed the tumors used for the quantitative analyses. Data are means; bars, SD. Numbers above each column by TUNEL and BrdUrd labeling assays. Comparable in vivo BrdUrd are the numbers of mice analyzed in each group. Numbers in the parentheses under the X axis are the differences in folds of i.m. AAV-angiostatin-treated mice in comparison incorporation was detected in all of the U87 MG gliomas (Fig. 4, d, with the controls (PBS or AAV-GFP treated mice). h, l, and p, and Fig. 5B) after i.v. administration of BrdUrd, indicating 759

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2002 American Association for Cancer Research. AAV-ENCODED ANGIOSTATIN SUPPRESSED BRAIN GLIOMA GROWTH that angiostatin did not affect tumor cell proliferation. Nonetheless, DISCUSSION 3–4.5-fold increases of apoptotic activities were detected in the tu- In this study, we have explored a therapeutic approach using a mors of AAV-angiostatin i.m.-treated mice (Fig. 4, k and o, and Fig. single i.m. injection of an AAV vector expressing angiostatin to 5C), whereas few apoptotic cells were found in the tumors of the achieve systemic protein delivery, which thereby suppresses human control mice (Fig. 4, c and g, and Fig. 5C). In addition, apoptotic U87 MG glioma growth in the brain. AAV-encoded angiostatin, i.m. endothelial cells were also observed in the glioma vasculature of mice administered, was expressed at high concentrations in the bloodstream that were i.m. injected with AAV-angiostatin (arrows in Fig. 4, over a prolonged period of time. Fifty-seven % of the AAV-angiosta- comparing j and n with k and o). These results suggest that tumor tin-treated mice survived to the 8th week, and 43% of the treated mice endothelial cells targeted AAV-angiostatin suppressed vessel devel- survived for more than 42 weeks, whereas all of the control mice died opment and caused tumor cell death attributable to insufficient nutri- within 6 weeks after i.c. implantation of the U87 MG glioma cells. tion. Muscle-secreted circulating AAV-angiostatin both decreased Histological analysis of the tumors from i.m. AAV-angiostatin-treated tumor neovessel densities and induced apoptosis in endothelial and mice demonstrated that inhibition of the i.c. U87 MG glioma forma- glioma cells, thereby inhibiting tumor growth in the brain. tion correlated with decreased tumor vascularization and increased Lack of Local and Systemic Toxicity after AAV-Angiostatin apoptotic levels in tumor cells that are in the vicinity of these Gene Therapy. To determine whether i.m. expression of angiostatin neovessels. and its subsequent systemic secretion had any adverse effects on High-level and continuous delivery of angiostatin is required for angiogenesis of other tissues, we examined the histology, blood vessel effective suppression of tumor growth. Angiostatin does not directly density, and angiostatin distribution in muscle, liver, heart, lung, and inhibit angiogenic stimulatory pathways, such as the vascular endo- kidney of the treated and control mice. Immunofluorescent staining thelial growth factor pathway (51). Instead, it suppresses angiogenesis with the anti-HA tag antibody revealed high levels of angiostatin by inducing apoptosis of endothelial cells in vitro (52) and in vivo expression in AAV-angiostatin vector-injected muscles (Fig. 6A, i and (13). Recent studies have demonstrated that direct delivery of the l), whereas no angiostatin was detected in the muscles of the control angiostatin protein for the treatment of malignant glioma by i.p. or mice (Fig. 6A, c and f). Furthermore, no central nucleation, degener- intratumoral injection suppressed tumor growth (7, 53). However, ation/atrophy, inflammation, or change of blood vessel density was there exist formidable limitations in treating malignant brain tumors found in AAV-angiostatin vector-injected muscle (Fig. 6A, a, b, d, e, using frequent bolus injections of the purified protein (10). As a viable g, h, j, and k). In addition, immunofluorescent staining with the alternative, gene delivery or gene therapy can also achieve in vivo anti-HA tag antibody did not reveal any difference in the liver (Fig. delivery of angiostatin by means of genetically modified cells (17, 6B, c, f, i, and l), heart, lung, and kidney tissues (data not shown) 38), retroviral (13, 18) or adenoviral (12, 13, 15, 16, 54) vectors that among AAV-angiostatin vector-treated and the control mice. No express antiangiogenic genes. For example, a single systematic ad- detectable changes on tissues organization (Fig. 6B, a, d, g, and j)or ministration of adenoviral vector carrying different antiangiogenic obvious differences in blood vessel densities (Fig. 6B, b, e, h, and k) genes resulted in high-level expression of the therapeutic proteins in among AAV-angiostatin vector-treated and control mice were found mouse blood stream for 2–3 weeks (16). But the gene expression in those tissues, including the liver. Careful examination of the AAV- faded away primarily because of the immune response against ade- angiostatin vector-treated, long-term survivors did not reveal any novirus-infected cells and direct cytotoxicity of the vector. Previously, no experiments were reported using the AAV for in vivo cancer gene change of overall health or abnormal behavior. therapy with the antiangiogenesis strategies, although AAV offers It has been reported previously that endostatin did not affect wound efficient and long-term in vivo gene delivery (for Ͼ2 years) without healing in the experimental mice (50). Data from our studies seem to both cytotoxicity and cellular immune responses to the target tissues substantiate this notion. All of the AAV-angiostatin i.m. injected mice (10, 22, 24, 26, 34, 45–49, 55). Our experiments provide evidence had gone through skull skin incision (ϳ1.2 cm in length) for glioma supporting the advantage of using AAV vectors for systematic, in vivo inoculation, at which time significant levels of circulating angiostatin antiangiogenic brain cancer gene therapy. were already detectable after 18 days of vector gene expression. In our model system, skeletal muscle served as a platform for However, the skin wound healing was indistinguishable from that of continuous secretion of angiostatin. The advantage of using muscle the control mice. In addition, another group of AAV-angiostatin i.m. tissue as a gene delivery site for secreted factors is its easy accessi- injected mice received an i.c. injection of the same vector at 2 weeks bility and potential reversibility. We did not observe any adverse after i.c. glioma inoculation, 1 month after i.m. vector injection. The effects to the injected muscle and other tissues, despite high level of skin wound healing after the second incision was again indistinguish- unregulated gene expression. Thus, the i.m. injection method for able from that of the control mice. Furthermore, multiple tail clipping systemic delivery of the antiangiogenesis factors should also be very for blood sample collections did not reveal any delay in wound useful in treating other solid tumors, for example, lung, liver, prostate, healing in AAV-angiostatin-treated mice. These observations strongly and breast cancers that are heavily dependent on angiogenesis. Al- suggest that AAV vector-mediated, long-term and high-level secretion though the lack of toxicity is a general phenomenon of AAV-mediated of angiostatin had no adverse effects on skin wound healing. in vivo gene therapy (19–21), the use of a regulatory gene expression

Fig. 6. Immunohistochemical analyses of muscle tissues (A) and liver tissues (B) from various mice. A, histological analyses on cryostat muscle tissue sections of an i.m. PBS-treated mouse sacrificed on the 6th week after inoculation of U87 MG cells into the brain (a–c), an i.m. AAV-GFP-treated mouse on the 6th week (d–f), an IM AAV-angiostatin-treated mouse on the 6th week (g–i) and on the 10th week (j–l). These sections were stained with H&E (a, d, g, and j), an anti-CD31 antibody (b, e, h, and k), an anti-HA antibody on muscle tissues visualized by a secondary antirabbit antibody conjugated with Alex488 (green; Molecular Probe, Inc.; c, f, i, and l). B, histological analyses on cryostat liver tissue sections of an i.m. PBS-treated mouse sacrificed on the 6th week after inoculation of U87 MG cells into the brain (a–c), an i.m. AAV-GFP-treated mouse on the 6th week (d–f), an i.m. AAV-angiostatin-treated mouse on the 6th week (g–i) and on the 10th week (j–l). These sections were stained with H&E (a, d, g, and j), an anti-CD31 antibody (b, e, h, and k), an anti-HA antibody on liver tissues visualized by a secondary antirabbit antibody conjugated with Alex594 (red; Molecular Probe, Inc.; c, f, i, and l). The same analyses were also done on heart, kidney, and lung tissues of the treated and untreated mice (data not shown). The experiments were repeated for two additional times using various organ tissue samples from all groups of mice, and identical staining results were obtained for these samples at each time. 761

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2002 American Association for Cancer Research. AAV-ENCODED ANGIOSTATIN SUPPRESSED BRAIN GLIOMA GROWTH system can offer an added safety feature for studies in the future. For ACKNOWLEDGMENTS instance, a tetracycline-regulated, long-term gene expression system in AAV vector was successfully tested in rat brain (56), and reversible We thank G. Robertson and Michael Xiao for critical reading and editing of the manuscript. GFP gene expression was achieved by the addition or removal of doxycycline in drinking water. Similarly, AAV-mediated erythropoietin gene transfers in a single or two separate vectors carrying the tet- REFERENCES or rapamycin-dependent regulatory systems were also successfully 1. Plate, K. H., and Risau, W. Angiogenesis in malignant gliomas. Glia, 15: 339–347, tested in small and large animals (57–59). Therefore, the inducible 1995. gene expression systems should be implemented in the additional 2. 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Hsin-I Ma, Ping Guo, Juan Li, et al.

Cancer Res 2002;62:756-763.

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