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[Cancer Research 62, 756-763, February 1, 2002]
© 2002 American Association for Cancer Research

Experimental Therapeutics

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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite various therapeutic interventions, glioblastoma multiforme (GBM) is one of the most highly vascularized neoplasms in humans with poor prognosis. In this study, we show that a single i.m. injection of an adeno-associated viral (AAV) vector expressing angiostatin, a potent angiogenic inhibitor, effectively suppresses human glioma growth in the brain of nude mice. Approximately 40% of the tumor-bearing mice treated with AAV-angiostatin vector survived for >10 months (the duration of the experiments). In contrast, 100% of the tumor-bearing mice in the control groups, with or without i.m. injection of a control vector AAV-GFP, died because of excessive tumor burden by 6 weeks. High levels of angiostatin produced by the AAV vector were detected in blood circulation for >250 days after the one-time vector injection. The secreted angiostatin specifically targeted neovessels in the brain tumors, as evidenced by the diminished vessel densities and increased apoptosis of tumor cells surrounding these neovessels. Our study thus demonstrates that AAV-mediated antiangiogenesis gene therapy offers efficient and sustained systemic delivery of the therapeutic product, which in turn effectively suppresses glioma growth in the brain.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Novel strategies are needed to treat malignant GBM,3 the most common type of human brain tumor. Despite multiple therapeutic approaches including surgical resection, radiotherapy, chemotherapy, and immunotherapy, the median survival time of patients with GBM is <2 years. The malignant progression from astrocytoma to GBM is often accompanied by increased angiogenesis and up-regulation of vascular endothelial growth factor and its receptors (1) . Tumors modulate the angiogenesis process by producing both positive and negative effectors (2, 3, 4) . A number of tumor-derived circulating proteins exhibit potent antiangiogenic effects (5 , 6) . For example, angiostatin is derived from plasminogen as a biologically active fragment containing four kringle domains (5) . Several reports have shown that direct administration of purified recombinant angiostatin protein through different routes in vivo can inhibit tumor growth and even cause tumor regression in animal models (7, 8, 9) . Angiostatin interacts directly with endothelial cells as an endothelial cell inhibitor independent of the blood-brain-barrier because i.p. injection of angiostatin can inhibit i.c. glioma growth (7 , 10) . Although promising, direct delivery of the recombinant antiangiogenic proteins requires enormous quantity as well as administration of the therapeutic products for a prolonged period. To overcome the shortcomings of protein delivery, alternative approaches, such as gene delivery of the antiangiogenic factors, have been explored. Because the presence of sustained and high-level antiangiogenic proteins is essential to maintain tumor neovessel inhibition and hence tumor growth suppression, previous attempts of gene therapy fell short, attributable mainly to transient or insufficient gene expression of the antiangiogenic factors (10, 11, 12, 13, 14, 15, 16, 17, 18) .

AAV vectors have been used widely to achieve efficient and long-term gene delivery to treat numerous genetic diseases in a wide variety of animal models (19, 20, 21, 22, 23, 24) as well as in human trials (25, 26, 27, 28) . AAV vectors are derived from the nonpathogenic, replication-defective parvovirus that contains a single-stranded DNA genome. The vectors are able to effectively transduce dividing and nondividing cells both in vitro and in vivo (29) , thus offering stable gene transfer by either integrating into the host chromosomes or persisting as an episome (30, 31, 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 of tissues including liver, brain, and muscle (22 , 24 , 26 , 33, 34, 35, 36, 37) .

In this study, we report that a single i.m. administration of an AAV vector carrying angiostatin gene effectively suppresses human U87 MG glioma growth in the brains of nude mice. Muscle was used here as a platform to produce AAV-encoded angiostatin and to secrete the therapeutic product into the blood circulation at high levels for prolonged periods. Such systemic delivery of angiostatin led to the regression of established i.c. gliomas by targeted inhibition of tumor neovessel development. No notable local or systemic toxicity was observed during the course of gene therapy. Thus, systemic delivery of antiangiogenic proteins by AAV-mediated muscle gene transfer offers a novel and promising approach for brain cancer therapy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of an AAV Vector Carrying the Angiostatin Gene.
A cDNA coding for the mouse angiostatin was amplified by PCR using primers corresponding to the amino acid residues 1–6 and 461–466 of plasminogen. This construct has an endogenous plasminogen secretory signal, a preactivation peptide sequence, four kringle domains (1–4), and a short HA tag sequence (38) . The cDNA fragment was then cloned into a pXX-UF1 expression vector (39) . pXX-angiostatin-HA, pXX2, and pXX6 were cotransfected into 293 cells for viral vector production (40) . The titer of AAV vectors was determined by a dot blot assay (40) . A control AAV-GFP virus was also constructed using a similar approach.

Angiostatin Protein Analyses.
The U87 MG glioma cells were infected with 1 µl of AAV-angiostatin-HA or AAV-GFP (1 x 1013 viral particles/ml) for 72 h. The conditioned medium and cell lysates were incubated with lysine-Sepharose at 4°C overnight. The bound materials were separated on a 10% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. The membrane was blocked and probed with an anti-HA antibody (Covance Co., Richmond, CA; Ref. (38) . To examine the AAV angiostatin-HA in vivo, 10 µl of mouse serum collected from tail veins were analyzed by immunoprecipitation, followed by Western blot analysis.

Animal Studies.
Eighteen days before i.c. implantation of the U87 MG cells, 500 µl of AAV-angiostatin-HA vector or AAV-GFP control vector (1 x 1013 viral particles/ml) were injected into the thighs and gluteal muscles. In another control group, 500 µl of PBS were injected into the mice. On day zero, 5 x 105 of U87 MG cells were implanted into the mouse brains (41) . Mouse brain, muscle, liver, lung, heart, and kidney tissues were removed. Thin cryostat sections were stained with H&E. The sizes of brain tumors were microscopically determined (42) . To evaluate the proliferative activities of glioma and endothelial cells in vivo, 300 µl of BrdUrd solution (Amersham, Piscataway, NJ) were injected i.p. into one group of mice 30 min before the animals were sacrificed. The brains and other organs of these mice were fixed and embedded (43) . Mouse brain, muscle, liver, heart, kidney, and lung tissue 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 Chester, PA; Ref. 41 ). The proliferative index of BrdUrd incorporation was calculated as a percentage of positive nuclei (brown colored) to total cells under light microscopy. To calculate BrdUrd labeling index, >2000 cells were examined in each tissue section. The apoptotic cells were shown as fluorescence-positive cells visualized by fluorescence microscopy. After the photographs were taken, these slides were treated with anti-fluorescein antibody-AP 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
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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, 45, 46, 47, 48, 49) . To determine whether AAV-encoded angiostatin can be efficiently expressed in mouse muscle and secreted into blood circulation over an extended period, we analyzed serum samples from mice that had received an i.m. injection of either the AAV-angiostatin vector, the control AAV-GFP vector, or PBS. Blood samples from individual mice (Fig. 1A)Citation or from the same mice at different time points (Fig. 1B)Citation were analyzed. In a majority of the cases, 43- to 182-fold increases and prolonged expression of AAV-angiostatin were detected. However, angiostatin was not detected in the blood samples from the PBS (Fig. 1BCitation , Lane 1) or AAV-GFP (Fig. 1ACitation , Lane 1 and Fig. 1BCitation , Lane 2) treated mice. The expression levels of angiostatin increased along with time (Fig. 1ACitation , Lanes 2–8, and Fig. 1BCitation , Lanes 3–5 and 6–9), consistent with previous studies using different genes carried by AAV vectors in muscle tissues (44 , 45 , 49) . In addition, high levels of angiostatin in the serum closely correlated with the survival time of the mice (see below). For example, a considerable amount of angiostatin was detected (74-fold or higher increases) in serum from mice that had survived for >300 days after implantation of the U87 MG cells in the brain (Fig. 1BCitation , Lanes 3–5 and 6–9). In contrast, some other mice that only had 2.6–10-fold increases of circulating angiostatin (Fig. 1BCitation , Lanes 10 and 11) died because of tumor burden by weeks 8 and 10 after implantation of the U87 MG cells. These data demonstrate that high levels of angiostatin in the mouse blood circulation indeed can be achieved after AAV vector-mediated gene transfer in muscle.



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  		Fig. 1. Immunoblot analysis of angiostatin in mouse sera. A, representative serum levels of angiostatin from individual mice that were i.m. treated with AAV-GFP (Lane 1) 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 detected in AAV-GFP treated mice (Lane 1). B, serum levels of angiostatin and survival of the tumor-bearing mice. Two of the long-term survival mice were analyzed and showed 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 mice (Lane 1) or in AAV-GFP-treated mice (Lane 2). However, another two separate i.m. 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 was Mr 58,000, instead of Mr 38,000 (13) . Numbers in the parentheses are the differences of angiostatin expression in folds in comparison with the controls (serum samples from PBS or AAV-GFP-treated mice). Their calculations are described in "Materials and Methods."

 
Systemic Circulation of AAV-encoded Angiostatin Suppressed Local Glioma Growth in Brains.
Previous studies using AAV vectors for i.m. gene transfer have indicated a lag period of 2–3 weeks, during which time the single-stranded AAV vector genomes were 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 x 105 U87 MG glioma cells. Similarly, 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)Citation . 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. 2)Citation . This phenomenon of initial slow tumor growth followed by a steady regression could possibly be attributed to the initial low levels of angiostatin expression, which were insufficient to stop the tumor growth. However, the gradual increase of AAV vector gene expression with time was sufficient in inhibiting tumor progression (Fig. 1ACitation , Lanes 4–8; Fig. 1BCitation , Lanes 3–5 and 6–9). Histological analysis of H&E staining was performed on the brain tumor samples from mice treated with AAV-angiostatin vector, AAV-GFP vector, and PBS saline (Fig. 2)Citation . By the fifth week after glioma inoculation, the tumor volumes in the control groups reached 40.0 ± 12.0 mm3 for PBS-treated mice and 35.0 ± 8.0 mm3 for AAV-GFP treated mice. By the sixth week after glioma inoculation, the tumor volumes increased to greater than 62.8 ± 4.4 mm3 (Fig. 2)Citation , 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 ± 11.0 mm3 by week 6 and decreased to 3.0 ± 2.1 mm3 (Fig. 2)Citation by week 10 after tumor inoculation.



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  		Fig. 2. Suppression of i.c. U87 MG glioma growth by AAV-angiostatin. Brain tumor growth rates were measured in mice that were i.m. treated with PBS (n = 6) or AAV-GFP (n = 6) or AAV-angiostatin (n = 14). Volumes of established gliomas were estimated as described previously (42) . At each time point, minimums of two mice were used to obtain 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 experiments include 6–14 mice in each group and were performed twice with similar results; bars, SD.

 
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)Citation , 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)Citation . 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 >42 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 essentially 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. 1ACitation , Lanes 10 and 11), whereas the long-term survivors had high levels of serum angiostatin (Fig. 1ACitation , 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.



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  		Fig. 3. Long-term survival of glioma-bearing mice after AAV-angiostatin i.m. injection. Survival analyses were done on mice that were i.m. treated with PBS (n = 6) or AAV-GFP (n = 3) or AAV-angiostatin (n = 14). At 6 weeks after implantation of U87 MG glioma cells into mouse brains. All mice in control groups (PBS or AAV-GFP IM treated) died because of excessive tumor growth. However, the AAV-angiostatin-treated tumor-bearing mice had much improved survival rates with 57% of the mice surviving 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. 2Citation , and similar survival rate curves were obtained each time.

 
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)Citation 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 mCitation , 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)Citation or by PBS (Fig. 4a)Citation . 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 densities in various gliomas. We observed that 80% or more decreases in vessel densities in the AAV-angiostatin-treated gliomas (Fig. 4, j and nCitation , and Fig. 5ACitation ) was accompanied by much smaller tumors (Fig. 4, i and mCitation , and Fig. 2Citation ).



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  		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.

 


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  		Fig. 5. Decreased vessel densities and increased apoptotic rates in U87 MG i.c. gliomas established in i.m. AAV-angiostatin-treated mice. Quantitative analyses of the IHC data were done as described in "Materials and Methods." The representative IHC stains from control and i.m. AAV-angiostatin-treated mice are shown in Fig. 4Citation . A, vascular densities in various gliomas of IHC data that are shown in Fig. 4, b, f, j, and nCitation . B, BrdUrd incorporation in cells of the various gliomas of IHC data that are shown in Fig. 4, d, h, l, and pCitation . C, AI in various gliomas of IHC data that are shown in Fig. 4, c, g, k, and oCitation . In each 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 used for the quantitative analyses. Data are means; bars, SD. Numbers above each column 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 with the controls (PBS or AAV-GFP treated mice).

 
To further demonstrate that tumor growth suppression by AAV-encoded angiostatin was attributable mainly to increased tumor cell apoptosis rather than decreased proliferation, we analyzed the tumors by TUNEL and BrdUrd labeling assays. Comparable in vivo BrdUrd incorporation was detected in all of the U87 MG gliomas (Fig. 4, d, h, l, and pCitation , and Fig. 5BCitation ) after i.v. administration of BrdUrd, indicating that angiostatin did not affect tumor cell proliferation. Nonetheless, 3–4.5-fold increases of apoptotic activities were detected in the tumors of AAV-angiostatin i.m.-treated mice (Fig. 4, k and oCitation , and Fig. 5CCitation ), whereas few apoptotic cells were found in the tumors of the control mice (Fig. 4, c and gCitation , and Fig. 5CCitation ). In addition, apoptotic endothelial cells were also observed in the glioma vasculature of mice that were i.m. injected with AAV-angiostatin (arrows in Fig. 4Citation , comparing j and n with k and o). These results suggest that tumor endothelial cells targeted AAV-angiostatin suppressed vessel development and caused tumor cell death attributable to insufficient nutrition. Muscle-secreted circulating AAV-angiostatin both decreased tumor neovessel densities and induced apoptosis in endothelial and glioma cells, thereby inhibiting tumor growth in the brain.

Lack of Local and Systemic Toxicity after AAV-Angiostatin Gene Therapy.
To determine whether i.m. expression of angiostatin and its subsequent systemic secretion had any adverse effects on angiogenesis of other tissues, we examined the histology, blood vessel density, and angiostatin distribution in muscle, liver, heart, lung, and kidney of the treated and control mice. Immunofluorescent staining with the anti-HA tag antibody revealed high levels of angiostatin expression in AAV-angiostatin vector-injected muscles (Fig. 6A, i and l)Citation , whereas no angiostatin was detected in the muscles of the control mice (Fig. 6A, c and f)Citation . Furthermore, no central nucleation, degeneration/atrophy, inflammation, or change of blood vessel density was found in AAV-angiostatin vector-injected muscle (Fig. 6A, a, b, d, e, g, h, j, and k)Citation . In addition, immunofluorescent staining with the anti-HA tag antibody did not reveal any difference in the liver (Fig. 6B, c, f, i, and l)Citation , heart, lung, and kidney tissues (data not shown) among AAV-angiostatin vector-treated and the control mice. No detectable changes on tissues organization (Fig. 6B, a, d, g, and j)Citation or obvious differences in blood vessel densities (Fig. 6B, b, e, h, and k)Citation among AAV-angiostatin vector-treated and control mice were found in those tissues, including the liver. Careful examination of the AAV-angiostatin vector-treated, long-term survivors did not reveal any change of overall health or abnormal behavior.



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  		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.

 
It has been reported previously that endostatin did not affect wound healing in the experimental mice (50) . Data from our studies seem to substantiate this notion. All of the AAV-angiostatin i.m. injected mice had gone through skull skin incision (~1.2 cm in length) for glioma inoculation, at which time significant levels of circulating angiostatin were already detectable after 18 days of vector gene expression. However, the skin wound healing was indistinguishable from that of the control mice. In addition, another group of AAV-angiostatin i.m. injected mice received an i.c. injection of the same vector at 2 weeks after i.c. glioma inoculation, 1 month after i.m. vector injection. The skin wound healing after the second incision was again indistinguishable from that of the control mice. Furthermore, multiple tail clipping for blood sample collections did not reveal any delay in wound healing in AAV-angiostatin-treated mice. These observations strongly suggest that AAV vector-mediated, long-term and high-level secretion of angiostatin had no adverse effects on skin wound healing.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have explored a therapeutic approach using a single i.m. injection of an AAV vector expressing angiostatin to achieve systemic protein delivery, which thereby suppresses human U87 MG glioma growth in the brain. AAV-encoded angiostatin, i.m. administered, was expressed at high concentrations in the bloodstream over a prolonged period of time. Fifty-seven % of the AAV-angiostatin-treated mice survived to the 8th week, and 43% of the treated mice survived for more than 42 weeks, whereas all of the control mice died within 6 weeks after i.c. implantation of the U87 MG glioma cells. Histological analysis of the tumors from i.m. AAV-angiostatin-treated mice demonstrated that inhibition of the i.c. U87 MG glioma formation correlated with decreased tumor vascularization and increased apoptotic levels in tumor cells that are in the vicinity of these neovessels.

High-level and continuous delivery of angiostatin is required for effective suppression of tumor growth. Angiostatin does not directly inhibit angiogenic stimulatory pathways, such as the vascular endothelial growth factor pathway (51) . Instead, it suppresses angiogenesis by inducing apoptosis of endothelial cells in vitro (52) and in vivo (13) . Recent studies have demonstrated that direct delivery of the angiostatin protein for the treatment of malignant glioma by i.p. or intratumoral injection suppressed tumor growth (7 , 53) . However, there exist formidable limitations in treating malignant brain tumors using frequent bolus injections of the purified protein (10) . As a viable alternative, gene delivery or gene therapy can also achieve in vivo delivery of angiostatin by means of genetically modified cells (17 , 38) , retroviral (13 , 18) or adenoviral (12 , 13 , 15 , 16 , 54) vectors that express antiangiogenic genes. For example, a single systematic administration of adenoviral vector carrying different antiangiogenic genes resulted in high-level expression of the therapeutic proteins in mouse blood stream for 2–3 weeks (16) . But the gene expression faded away primarily because of the immune response against adenovirus-infected cells and direct cytotoxicity of the vector. Previously, no experiments were reported using the AAV for in vivo cancer gene therapy with the antiangiogenesis strategies, although AAV offers efficient and long-term in vivo gene delivery (for >2 years) without both cytotoxicity and cellular immune responses to the target tissues (10 , 22 , 24 , 26 , 34 , 45, 46, 47, 48, 49 , 55) . Our experiments provide evidence supporting the advantage of using AAV vectors for systematic, in vivo antiangiogenic brain cancer gene therapy.

In our model system, skeletal muscle served as a platform for continuous secretion of angiostatin. The advantage of using muscle tissue as a gene delivery site for secreted factors is its easy accessibility and potential reversibility. We did not observe any adverse effects to the injected muscle and other tissues, despite high level of unregulated gene expression. Thus, the i.m. injection method for systemic delivery of the antiangiogenesis factors should also be very useful in treating other solid tumors, for example, lung, liver, prostate, and breast cancers that are heavily dependent on angiogenesis. Although the lack of toxicity is a general phenomenon of AAV-mediated in vivo gene therapy (19, 20, 21) , the use of a regulatory gene expression system can offer an added safety feature for studies in the future. For instance, a tetracycline-regulated, long-term gene expression system in AAV vector was successfully tested in rat brain (56) , and reversible 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- or rapamycin-dependent regulatory systems were also successfully tested in small and large animals (57, 58, 59) . Therefore, the inducible gene expression systems should be implemented in the additional studies involving antiangiogenic gene therapy.

A major drawback of AAV-mediated in vivo gene transfer is the slow onset of transgene expression. One of the rate-limiting steps is the conversion of single-strand AAV DNA genomes into double-strand template for transcription (60 , 61) . This property puts a constraint on the time frame concerning injection of the vector in that sufficient gene expression needs to come into effect before the animals succumb to excessive tumor burden. That is the reason that we injected the AAV vectors 18 days prior to glioma cells implantation. Only by that time was significant expression of angiostatin observed in the mouse circulation (Fig. 1ACitation , Lane 2). However, tumors in the clinical scenario progress at a slower pace than in the animal models, thus providing a wider window of opportunity for AAV-mediated gene therapy. A recently developed new AAV vector structure that harbors a double-strand DNA genome may overcome the problem of slow onset of AAV gene expression, because the double-strand DNA template is immediately available for transcription (62 , 63) . The shortened gene expression by the double-strand DNA template of AAV genome provides an excellent opportunity to treat human astrocytomas because in clinic, progression of brain tumors always precedes cancer therapy. We have constructed several antiangiogenic or proapoptotic factors in the double-strand DNA template of the new AAV vector. In vivo experiments are under way to suppress the progressively growing and established i.c. gliomas.

In our study, we did not observe complete protection in a large portion of the tumor-bearing animals after the one-time i.m. injection of the AAV-angiostatin vector. In another experiment, we boosted the AAV-encoded gene delivery by intratumoral injection on day 14 after the establishment of an U87 MG glioma in the brain. To our surprise, no further suppression of tumor growth was observed (data not shown). Angiostatin delivered either as purified proteins (7) or by adenovirus (12 , 13) has shown effects on suppressing various established tumors including i.c. gliomas. We have also used the AAV-angiostatin vector to treat established C6 i.c. gliomas in Wistar rats intratumorally. We observed suppression of the established C6 glioma growth and increased rates of animal survival.4 A possible explanation is that although AAV-angiostatin could directly transduce U87 MG glioma tumor cells, sufficient levels of circulating angiostatin after i.m. AAV injection might already exist to inhibit tumor growth. Alternatively, the i.c. injection of AAV-angiostatin in the U87 MG glioma tumors did not generate sufficient angiostatin that could contribute additional inhibitory effect. Finally, there may be certain heterogenicity in the tumor cell population that is less sensitive to antiangiogenesis therapy (64) . Although our therapeutic results are highly encouraging, additional or synergistic efficacy may be obtained when combined with other therapeutic strategies, such as tumor suppressor, proapoptotic factors, suicidal or cytokine genes, or even with the conventional surgical, chemo-, and radiation therapies. All together, our study presents a novel approach of developing antiangiogenic therapies to treat i.c. human glioblastoma.


   ACKNOWLEDGMENTS
 
We thank G. Robertson and Michael Xiao for critical reading and editing of the manuscript.


   FOOTNOTES
 
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 in part by a grant from the Brain Cancer Program of James S. McDonnell Foundation and a start-up fund from University of Pittsburgh Cancer Institute (to S-Y. C.). Back

2 To whom requests for reprints should be addressed, at University of Pittsburgh School of Medicine, Department of Molecular Genetics and Biochemistry, BST W-1244, 200 Lothrup Street, Pittsburgh, PA 15261. Phone: (412) 648-9487; Fax: (412) 624-1401; E-mail: xiaox{at}pitt.edu (X. X.); Cancer Institute and Department of Pathology, University of Pittsburgh, BST W-1055, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: (412) 648-3317; Fax: (412) 624-7737; E-mail: chengs{at}msx.upmc.edu (S-Y. C.). Back

3 The abbreviations used are: GBM, glioblastoma multiforme; AAV, adeno-associated virus; i.c., intracranial; HA, hemagglutinin antigen; BrdUrd, bromodeoxyuridine; IHC, immunohistochemistry; GFP, green fluorescent protein; AI, apoptotic index; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling. Back

4 H-I. Ma, S-Z. Lin, Y-H. Chiang, J. Li, S-L. Chen, Y-P. Tso, and X. Xiao, unpublished results. Back

Received 9/20/01. Accepted 12/ 3/01.


   REFERENCES
Top
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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