Tissue Inhibitor of Metalloproteinase-3 Expression from an Oncolytic ...

Research Article

Tissue Inhibitor of Metalloproteinase-3 Expression from an Oncolytic Adenovirus Inhibits Matrix Metalloproteinase Activity In vivo without Affecting Antitumor Efficacy in Malignant Glioma 1,2

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Martine L.M. Lamfers, Davide Gianni, Ching-Hsuan Tung, Sander Idema, 2 2 5 2 Frederik H.E. Schagen, Jan E. Carette, Paul H.A. Quax, Victor W. Van Beusechem, 1 1 3,6 W. Peter Vandertop, Clemens M.F. Dirven, E. Antonio Chiocca, 2 and Winald R. Gerritsen 1

Department of Neurosurgery; 2Department of Medical Oncology, Division of Gene Therapy, VU University Medical Center, Amsterdam, the Netherlands; 3Molecular Neuro-Oncology Laboratories, Neurosurgery Service; 4Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts; 5Gaubius Laboratory TNO-PG, Leiden, the Netherlands; and 6Department of Neurological Surgery, Dardinger Center for Neuro-oncology, The Ohio State University Comprehensive Cancer Center, Columbus, Ohio

Abstract

Introduction

Oncolytic adenoviruses exhibiting tumor-selective replication are promising anticancer agents. Insertion and expression of a transgene encoding tissue inhibitor of metalloproteinase-3 (TIMP-3), which has been reported to inhibit angiogenesis and tumor cell infiltration and induce apoptosis, may improve the antitumor activity of these agents. To assess the effects of TIMP-3 gene transfer to glioma cells, a replication-defective adenovirus encoding TIMP-3 (Ad.TIMP-3) was employed. Ad.TIMP-3 infection of a panel of glioma cell cultures decreased the proliferative capacity of these cells and induced morphologic changes characteristic for apoptosis. Next, a conditionally replicating adenovirus encoding TIMP-3 was constructed by inserting the TIMP-3 expression cassette into the E3 region of the adenoviral backbone containing a 24-bp deletion in E1A. This novel oncolytic adenovirus, Ad#24TIMP-3, showed enhanced oncolytic activity on a panel of primary cell cultures and two glioma cell lines compared with the control oncolytic virus Ad#24Luc. In vivo inhibition of matrix metalloproteinase (MMP) activity by Ad#24TIMP-3 was shown in s.c. glioma xenografts. The functional activity of TIMP-3 was imaged noninvasively using a near-IR fluorescent MMP-2–activated probe. Tumoral MMP-2 activity was significantly reduced by 58% in the Ad#24TIMP-3–treated tumors 24 hours after infection. A study into the therapeutic effects of combined oncolytic and antiproteolytic therapy was done in both a s.c. and an intracranial model for malignant glioma. Treatment of s.c. (U-87MG) or intracranial (U-87DEGFR) tumors with Ad#24TIMP-3 and Ad#24Luc both significantly inhibited tumor growth and prolonged survival compared with PBS-treated controls. However, expression of TIMP-3 in the context of Ad#24 did not significantly affect the antitumor efficacy of this oncolytic agent. (Cancer Res 2005; 65(20): 9398-405)

Requests for reprints: Martine L.M. Lamfers, Neurosurgery, VU University Medical Center, Boelelaan, Amsterdam, the Netherlands 1007 MB. Phone: 31-20-444-8411; Fax: 31-20-444-8168; E-mail: [email protected]. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-04-4264

Cancer Res 2005; 65: (20). October 15, 2005

Oncolytic viruses are receiving widespread attention as novel therapeutic agents for the treatment of various malignancies (1, 2). Currently, further development of these agents includes combining the oncolytic effects of the virus with the added antitumor activity of a therapeutic transgene. Such ‘‘armed’’ therapeutic viruses carrying a variety of transgenes encoding prodrug-converting enzymes, immune stimulatory molecules, or apoptosis-enhancing proteins have already been shown to exhibit increased antitumor efficacy (3–12). However, contradictory evidence exists regarding the value of additional prodrug-converting enzymes or cytotoxic genes in the context of an oncolytic virus, as they may induce premature killing of the infected tumor cells, thereby limiting the production of progeny virus and counteracting virus-induced oncolysis (13–15). An alternative approach is to insert transgenes that exert their inhibitory effects on the tumor indirectly. This will ensure that sufficient viral replication and enzyme production is maintained. Matrix metalloproteinases (MMP) offer an interesting target for such an approach. MMPs belong to a family of structurally related proteolytic enzymes that mediate degradation of components of the extracellular matrix and basement membrane. Moreover, they mediate the release of sequestered latent growth and angiogenic factors and the activation of latent growth factors (16). Upregulation of MMP activity has been linked to tumor growth and invasion and tumor-associated angiogenesis. Malignant gliomas constitute a relevant target for anti-MMP treatment. Their highly invasive and vascularized nature contributes to the poor prognosis of glioma patients. Immunohistochemical localization studies in surgical specimens have confirmed that MMPs are overexpressed in these tumors (17–19) and correlated to malignant progression (20, 21). Interference with these proteases might therefore inhibit local tumor dissemination and neovascularization. Tissue inhibitors of metalloproteinases (TIMP) are a family of natural inhibitors that control the activity of MMPs and are often concomitantly expressed. Four members of this family have been characterized thus far (i.e., TIMP-1, TIMP-2, TIMP-3, and TIMP-4), which are expressed by a variety of cell types and can be found in most tissues throughout the body (22). In human gliomas, TIMP expression is reported to be down-regulated with increasing malignancy, resulting in an imbalance between the production of MMPs and TIMPs and a shift towards a proproteolytic state with invasive phenotype (21, 23, 24). TIMP-3 is unique within the TIMP

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TIMP-3 Expression from an Oncolytic Adenovirus

family as it remains closely associated to the extracellular matrix after being secreted by the cell. Overexpression of TIMP-3 has been shown to restrict invasiveness of various tumor cell types in vitro (25, 26). In addition, TIMP3 overexpression showed inhibitory effects on tumor growth and angiogenesis in neuroblastoma, melanoma, and hepatocellular carcinoma tumor models (27–30). Moreover, evidence is accumulating for a role of TIMP-3 in inducing apoptosis in various cell types by stabilization of death receptors such as Fas on the cell surface (31–33). Taken together, these characteristics make TIMP-3 an interesting candidate as therapeutic transgene in the context of replication-competent, tumor-selective adenoviruses for the treatment of malignant glioma. In this report, the effects of TIMP-3 overexpression on proliferation of low-passage primary and established glioma cells was studied. In addition, the in vitro and in vivo antitumor effects of concomitant adenoviral replication and TIMP-3 expression were studied using the novel tumor-selective adenovirus encoding TIMP-3, AdD24TIMP-3. We show for the first time that the effects of TIMP-3 expressed from an oncolytic virus can be directly imaged using an optical noninvasive in vivo imaging technique for MMP-2 activity.

Materials and Methods Cell culture. The Ad5 E1-transformed human embryonal kidney cell line 293, the human lung carcinoma cell line A549, and the glioma cell lines U-87MG and U-251MG were purchased from the American Type Culture Collection (Manassas, VA). U-87yEGFR was a generous gift of Dr. H. J. Su Huang (University of California at San Diego) and colo205 colon carcinoma cells were a kind gift of Dr. E. Boven (VU University Medical Center, Amsterdam, the Netherlands). This cell line was established by retroviral transfer of mutant epidermal growth factor receptor (EGFR) into the parental glioma cell line, enhancing its tumorigenic capacity (34). Primary glioma cell cultures were derived by mechanical dissociation from fresh tumor material collected during brain tumor surgery at the Department of Neurosurgery of the VU University Medical Center and the Academic Medical Center according to the method of Darling (35). Primary cell cultures are designated as VU or AMC and used before passage 8. All tumor samples included in this study were classified as astrocytoma grade 3 or glioblastoma multiforme. All cells were cultured in DMEM supplemented with 10% FCS and antibiotics (Life Technologies, Paisley, United Kingdom). Recombinant adenoviruses. A recombinant E1–deleted, replicationdeficient adenovirus expressing cytomegalovirus (CMV) immediate-early promoter-driven luciferase, Ad.Luc, was provided by Dr. R.D. Gerard (University of Texas Southwestern Medical Center, Dallas, TX). The replication-deficient adenoviral vector that expresses CMV promoter–driven enhanced green fluorescent protein (Ad.GFP) has been described previously (36). The replication-deficient adenoviral vector that expresses CMV promoter–driven TIMP-3 (Ad.TIMP-3) has been characterized previously (37) and the conditionally replicative adenovirus with an expression cassette for luciferase in the E3 region, AdD24Luc, has been described elsewhere (38). To construct the conditionally replicative adenovirus with an expression cassette for TIMP-3 in the E3 region, the CMV-TIMP-3 expression cassette was released from an adenoviral shuttle vector encoding TIMP-3 under the CMV promoter pCMV-TIMP-3 by digestion with AvrII and BglII. The 1.75-kb fragment was inserted into BamHI/XbaI-digested pABS.4 (Microbix Biosystems, Toronto, Canada). The resulting construct, pABS.4-TIMP-3, was digested with PacI, and the 3.1-kb fragment carrying the CMV-TIMP-3 cassette and kanamycin resistance gene was inserted into PacI-digested pBHG11 (Microbix Biosystems). A clone with the CMV-TIMP-3 insert was isolated and the kanamycin resistance gene was removed by digestion with SwaI followed by self-ligation, yielding pBHG11-TIMP-3. The conditionally replicative adenovirus was made by homologous recombination in 293 cells between the pXC1 (Microbix Biosystems) derivative pXC1-D24, which carries a 24-bp deletion corresponding to amino acids 122 to129 in the CR2


domain of E1A necessary for binding to the Rb protein (ref. 39; a kind gift of Dr. R. Alemany, Gene Therapy Center, UAB, Birmingham, AL), with pBHG11TIMP-3, resulting in the conditionally replicative adenovirus AdD24TIMP-3 with the E1A CR2 mutation and the TIMP-3 expression cassette in E3. Viruses were plaque purified, propagated on 293 cells for replicationdeficient vectors or on A549 cells for replicative viruses, and purified by CsCl gradient according to standard techniques. The E1D24 mutation and CMV-TIMP-3 insertion were confirmed by PCR on the final products. Particle titers of all adenoviruses were determined by absorbance measurements at 260 nm, and functional titers (PFU) were determined by end point dilution titration on 293 cells according to standard techniques. Particle/PFU ratios were 4 for Ad.GFP, 12 for Ad.Luc, 30 for Ad.TIMP-3, 11 for AdD24Luc, and 15 for AdD24TIMP-3. In all experiments, infections were normalized based on PFU titers. Proliferation assays. Primary and established glioma cells were plated subconfluently at 5 103 cells per well in 96-well plates. Infection was done in triplicate with an multiplicity of infection (MOI) range of Ad.TIMP-3 or as control Ad.GFP to allow visualization of infection efficiency. Proliferation was quantified using the tetrazolium salt WST-1 (Roche Diagnostics, Mannheim, Germany) as described by the manufacturer. WST-1 assays were done on day 10 after infection on slow-growing primary cells and on day 6 after infection on the cell lines. Proliferation is expressed as a percentage of Ad.GFP-infected controls. Hoechst staining. Hoechst staining was done on VU-135 primary glioma cells. Cells were cultured on glass slides and infected at MOI 300 with Ad.Luc or Ad.TIMP-3. On day 3 after infection, cells were fixed with 1% formaldehyde in PBS. Apoptotic cells were visualized by staining with 10 Ag/mL Hoechst-33342 (Sigma-Aldrich, Zwijndrecht, the Netherlands) and fluorescent microscopy. Cytotoxicity assays. Glioma cells were plated confluently at 104 cells per well in 96-well plates and infected in triplicate with a dose range of AdD24TIMP-3 or AdD24Luc. Viability was assessed by WST-1 assay at day 8 after infection for the cell lines and at day 12 after infection for the primary cell cultures. For coinfection experiments, U-87yEGFR cells were plated at 104 cells per well and infected with combinations of Ad.Luc with AdD24Luc or Ad.TIMP-3 with AdD24Luc. Viability was assessed daily by WST-1 assay for five consecutive days. Results are presented as percentage of uninfected controls. Immunohistochemistry. Immunohistochemical staining for TIMP-3 protein was done on formaldehyde-fixed U-251MG cells. Furthermore, staining for TIMP-3, adenovirus, and vasculature was done on acetone-fixed cryosections obtained from U-87MG s.c. tumors and brains of U-87yEGFR tumor-bearing mice. Briefly, immunohistochemical staining for TIMP-3 was done using the rabbit-anti-human TIMP-3 antibody AB802 (Chemicon International, Temecula, CA); staining for vasculature was done using the rabbit anti-von Willebrand Factor antibody (DAKO, Glostrup, Denmark); and staining for adenovirus was done using the goat anti-adenoviral hexon protein antibody 1056 (Chemicon International). Bound primary antibodies were detected using horseradish peroxidase–conjugated goat anti-rabbit or rabbit-anti-goat antibodies and exposure to the chromogens 3-amino-9ethylcarbazole or diaminobenzidine followed by counterstaining with Mayer’s hematoxylin. Negative controls were assessed by omitting the primary antibody. In addition, uninfected tumors were used as negative controls for hexon staining and colo205 colon carcinoma cells were used as a negative control for anti-TIMP-3 staining. In vivo near-IR fluorescence imaging of matrix metalloproteinase-2 activity. For imaging of inhibitory activity of AdD24TIMP-3 on MMP-2 activity in vivo, 1 106 U-87yEGFR cells were injected s.c. into both mammary pads of adult female athymic mice (CD1 nude, Charles River, Wilmington, MA). Tumors were allowed to grow to 4- to 6-mm diameter upon which the right tumor of each of six animals was intratumorally injected with 3 108 plaque-forming unit (pfu) AdD24TIMP-3 and the contralateral tumors received PBS injections. As an additional control, three animals were treated with the same dose of control virus, AdD24Luc, and PBS in contralateral tumors. After 24 hours, the imaging probe (2 nmol per animal) containing a preferential MMP-2 peptide substrate that was synthesized and characterized as described previously (40) was injected i.v. MMP-2

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Cancer Research enzymatic activity results in cleavage of the substrate and conversion of the probe into a fluorescent product and strongly increases in near-IR fluorescence signal. The near-IR fluorescence signal was imaged 15 hours after i.v. administration. Near-IR fluorescence reflectance acquisition was obtained with 615- to 645-nm excitation and 680- to 720-nm emission using a previously described imaging system (41). After data acquisition, quantification and visualization of fluorescent signals were done with a home-written program with image display and analysis suite developed in IDL (Research Systems, Boulder, CO). Regions of interest were defined and the sum and SD of the photon counts within these regions were then calculated. For visualization purposes, the bioluminescence images were fused with the corresponding white light surface images as a transparent pseudocolor overlay. Tumors were removed and frozen directly after imaging for histologic analysis. In vivo adenovirus anti-glioma efficacy studies. For assessing the therapeutic effects of AdD24TIMP-3, studies were done in both s.c. and intracranial models for glioma. S.c. tumors were established on the flanks of adult female athymic nu/nu mice (Harlan, Horst, the Netherlands) by s.c. injection of 5 106 U-87MG cells. When tumors reached an average of 150 to 175 mm3, mice were randomly divided over three groups of nine animals and received intratumoral injections on days 0, 2, and 4 of 3 108 pfu AdD24Luc or AdD24TIMP-3 in 50 AL PBS. The third group received equal volumes of PBS injections. Tumors were measured thrice weekly with a digital caliper and tumor volumes were calculated from the average of tumor width and length according to the formula 4/3pr 3. Mice were euthanized when the tumor reached a volume of 2,000 mm3. Tumor specimens were removed and frozen for histologic analysis. Intracranial glioma xenografts were established in adult female athymic mice (CD1 nude, Charles River) by stereotactic injection of 9 104 U87yEGFR cells in 3 AL Hanks buffer into the right frontal lobe, 2.5 mm lateral to the bregma at a depth of 3 mm. Injections were done under anesthesia by i.p. injection of 2 mg ketamine and 0.4 mg of xylazine in 0.9% saline. After 4 days, 2 108 pfu AdD24TIMP-3 (n = 10) or AdD24Luc (n = 9) in 3 AL PBS was inoculated stereotactically into the same coordinates. Control animals (n = 9) received stereotactic inoculation of 3 AL PBS containing 3% glycerol that corresponds to the glycerol concentration in the virus dilutions. Animals were monitored daily and sacrificed upon appearance of symptoms evident for moribund decline due to intracerebral tumor growth, such as paralysis and lethargy. Brains were removed and frozen for histologic analysis. Statistical analysis. Data from in vitro experiments are presented as means F SD. Data from MMP-2 imaging study are shown in a box plot indicating 25th and 75th percentiles with lines indicating median and mean. Statistical analysis between groups was conducted with the two-tailed Student’s t test. Tumor growth rates of s.c. tumors were determined from time of individual tumors to reach thrice initial tumor volume. Statistical significance between treatment groups and controls in their tumor growth rates was estimated by the two-tailed nonparametric Mann-Whitney test. For mice with intracerebral tumors, statistical significance of differences in survival between treatment groups and controls was assessed by the Wilcoxon test.

compared with Ad.GFP-infected controls, with the inhibitory effect ranging from 19% to 71%. Hoechst staining in parallel cultures of VU-135 showed the significantly increased presence of condensed nuclei, suggestive for apoptosis, in Ad.TIMP-3-infected cells compared with Ad.Luc controls (P = 0.013; Fig. 1B). Characterization of tissue inhibitor of metalloproteinase-3– encoding oncolytic adenovirus. To study the combined effects of adenoviral oncolysis and TIMP-3 overexpression, a conditionally replicating adenovirus was constructed encoding TIMP-3 (AdD24TIMP-3). The adenoviral backbone AdD24 carries a mutation encoding a deletion of eight amino acids in the pRbbinding CR2 domain of E1A, which induces the tumor selectivity of the virus (39) and the TIMP-3 expression cassette was inserted into the E3 region. To visualize the expression of recombinant TIMP-3 expression in vitro, U-251MG cells were infected with Ad.TIMP-3 or AdD24TIMP-3 at MOI 50 and immunohistochemical staining for TIMP-3 was done 24 hours later. As depicted in Fig. 2A, infection with Ad.TIMP-3 led to detectable levels of TIMP-3 production in f10% of cells. Infection with AdD24TIMP3 led to higher levels of TIMP-3 expression in infected cells and rounding of the infected cells, a characteristic early cytopathic effect of the oncolytic virus. Next, the oncolytic activity of AdD24TIMP-3 was compared with the control virus encoding luciferase, AdD24Luc, on a panel of

Results Effects of tissue inhibitor of metalloproteinase-3 on proliferation of glioma cells. TIMP-3 has been reported to inhibit proliferation and induce apoptosis in various tumor cells (31, 32). To study these effects of TIMP-3 in malignant glioma cells, a panel of low-passage primary glioma cell cultures obtained from patient tumor material and three established glioma cell lines (U-251MG, U-87MG, and U-87yEGFR) were infected with a concentration range of Ad.TIMP-3 or Ad.GFP. Proliferation, as measured by WST-1 assay, was assessed on day 10 after infection of slow-growing primary cells and day 6 after infection of cell lines. In Fig. 1A the effects of TIMP-3 overexpression on glioma cell proliferation are shown at the MOI that gave 80% to 100% infection. Ad.TIMP-3 infection significantly inhibited the proliferation of four of five primary glioma cell cultures and of all three cell lines


Figure 1. A, effect of Ad.TIMP-3 on primary glioma cell proliferation was determined using the WST-1 metabolic assay. Columns, mean percentages of Ad.GFP-infected controls; bars, FSD. *, P < 0.05 compared with Ad.GFP-infected controls. B, Hoechst staining of VU- 135 primary glioma cells infected with Ad.Luc or Ad.TIMP-3 at MOI 300. Columns, mean percentages of apoptotic nuclei; bars, FSD. *, P < 0.05 compared with Ad.Luc-infected controls.

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Figure 2. A, immunohistochemical staining for TIMP-3 in U-251MG cells 24 hours after mock, Ad.TIMP-3, or AdD24TIMP-3 infection. B-C, oncolytic effect of dose escalation of AdD24Luc (dashed lines ) and AdD24TIMP-3 (solid lines ) on primary glioma cell cultures and on glioma cell lines U-87MG and U87yEGFR was determined using WST-1 assay. Points, mean percentages of untreated controls; bars, FSD. D, viability of U- 87yEGFR cells infected with Ad.Luc ( + ), Ad.TIMP-3 ( . ), or coinfected with Ad.Luc and AdD24Luc ( n ), or Ad.TIMP-3 and AdD24Luc ( E ) was determined using WST-1 assay on five consecutive days after infection. Points, mean percentages of untreated controls; bars, FSD.

glioma cells. Confluent cultures of three primary low-passage glioma cell cultures were infected with a concentration range of AdD24Luc or AdD24TIMP-3 and viability was determined by WST1 assay 12 days after infection. As shown in Fig. 2B, a modest increase in oncolytic activity was seen in AdD24TIMP-3-infected cells compared with the control virus AdD24Luc-infected cells in all three cell cultures tested. In addition, the efficacy of these oncolytic viruses was tested in two glioma cell lines, U-87MG and U-87yEGFR, which are frequently used for in vivo tumor growth (Fig. 2C). Similar to the results found in primary cell cultures, AdD24TIMP-3 was more potent than the control virus AdD24Luc in both of these cell lines. In parallel cultures of AdD24Luc- and AdD24TIMP-3-infected U-87yEGFR cells, total virus production was determined by end point titration and showed that viral replication speed was not hampered by the concomitant TIMP-3 production during the lytic cycle (data not shown). To verify that the superior oncolytic efficacy of AdD24TIMP-3 compared with AdD24Luc resulted from TIMP-3 overexpression, a mix experiment was done using the replication-defective Ad.TIMP-3 in combination with replication-competent AdD24Luc. U-87yEGFR cells were coinfected with Ad.TIMP-3 or the control vector Ad.Luc at MOI 200, in combination with AdD24Luc at MOI 2. Viability was determined by WST-1 assay on days 1, 2, 3, 4, and 5 after infection.


As an additional control, the individual effects of Ad.TIMP-3 and Ad.Luc were also assessed. As shown in Fig. 2D, infection with Ad.TIMP-3 alone decreased viability up to 53% on day 5 after infection, whereas Ad.Luc had no significant effect. Coinfection of Ad.TIMP-3 with AdD24Luc led to significantly more cell kill than AdD24Luc in combination with Ad.Luc (76.7 F 3.5% versus 24.3 F 6.9% on day 5). In vivo imaging of tissue inhibitor of metalloproteinase-3 activity. Using an optical noninvasive imaging technique for recording in vivo MMP-2 activity (40), we determined whether the exogenous TIMP-3 expression from AdD24TIMP-3 could reach sufficiently high production levels to inhibit endogenous MMP activity in tumors. Nude mice bearing bilateral s.c. U87yEGFR tumors between 4 and 6 mm in diameter received three injections of PBS into their left tumor and three injections of 108 pfu AdD24TIMP-3 or 108 pfu AdD24Luc into their right tumor. After 24 hours, the MMP-2-sensitive near IR fluorescence probe (2 nmol/ animal) was systemically injected. This early time point was chosen to exclude differences in tumor size resulting from viral oncolysis and because TIMP-3 is abundantly produced by the infected cells at this time point, as shown in vitro (Fig. 2A). The mice were imaged 15 hours after probe administration. Figure 3A shows the white light, near-IR fluorescence, and color-encoded images of

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representative examples of AdD24Luc- and AdD24TIMP-3-treated animals. Quantitative analysis was done by determining total fluorescence within regions of interest in all treated animals and results are summarized in Fig. 3B. As readily visible from the nearIR fluorescence images, there was significantly less MMP-2 near-IR fluorescence signal in AdD24TIMP-3-treated tumors compared with PBS-treated contralateral controls. The difference in MMP-2 near-IR fluorescence signal between these two groups was statistically significant and resulted in an average inhibition by AdD24TIMP-3 of 58.3 F 13.5% (P = 0.007). No significant inhibition was seen in the AdD24Luc-treated tumors compared with PBS-treated contralateral controls (P = 0.34). Tumors were removed after imaging and tumor cryosections were stained for hexon and TIMP-3 (Fig. 3C). Widespread staining for hexon proteins was found in both AdD24Luc- and AdD24TIMP-3-infected tumors (top). Although endogenous TIMP-3 expression was detected in U-87MG tumors, a strong increase in TIMP-3 staining can be observed in the AdD24TIMP-3-infected tumors (bottom). Therapeutic evaluation of Ad#24TIMP-3. The therapeutic effects of AdD24TIMP-3 in vivo were assessed in both a s.c. and an intracranial xenograft model for malignant glioma. In the first experiment, established U-87MG s.c. xenografts were treated with a total dose of 9 108 pfu AdD24Luc or AdD24TIMP-3 or with the same volume of PBS. Treatment with intratumoral injections of AdD24Luc or AdD24TIMP-3 both significantly suppressed tumor growth compared with PBS controls with a tumor growth delay of

7.1 days (P = 0.007) and 10.1 days (P = 0.001), respectively (Fig. 4A). Interestingly, in two of nine AdD24TIMP-3-treated tumors, complete regression and long-term survival was observed, whereas none of the AdD24Luc- or PBS-treated tumors regressed. However, mean tumor growth rate of AdD24TIMP-3-treated mice was not significantly reduced compared with AdD24Luc-treated mice (P = 0.31). Tumors removed on day 10 after infection for early time point analysis showed widespread areas of adenoviral replication as detected by anti-adenovirus hexon staining. Parallel sections stained for TIMP-3 showed high level TIMP-3 expression in infected cells (Fig. 4B). Staining of cryosections of AdD24Lucand AdD24TIMP-3-infected U-87MG tumors removed at time of sacrifice for adenoviral hexon proteins and TIMP-3 displayed only very few hexon-positive cells per section and high levels of endogenous TIMP-3 expression (data not shown). Moreover, vWF staining for endothelial cells and quantification of vascular density showed no significant differences in vascularization among the three groups (data not shown). A second in vivo experiment was done in an intracranial brain tumor model in nude mice using U87yEGFR cells. These tumors overexpress mutant EGFR, which has been shown to result in a highly vascularized and more aggressive phenotype compared with U-87MG tumors. In addition, these tumors have been shown sensitive to antiangiogenic treatment (42, 43). Mice were treated with intratumoral injection of PBS or 2 108 pfu AdD24Luc or AdD24TIMP-3. As shown in Fig. 4C, treatment with AdD24Luc or AdD24TIMP-3 both significantly

Figure 3. A, in vivo near-IR fluorescence imaging of MMP-2 activity in U-87yEGFR tumor-bearing mice treated with AdD24Luc (top row ) or AdD24TIMP-3 (bottom row). Tumors (encircled with dotted lines ) were injected with PBS or virus (arrows ) 39 hours before imaging. White-light images (left ), raw image acquisition of near-IR fluorescence signal obtained at 700-nm emission (middle ), and the color-coded near-IR fluorescence signal superimposed onto white-light images (right ). B, quantitative image analysis of tumoral near-IR fluorescence signals are shown in a box plot indicating 25th and 75th percentiles. Solid line, median; dashed line, mean fluorescence as percentage of PBS controls. *, P < 0.01 compared with AdD24Luc-infected controls. C, immunohistochemical staining for adenoviral hexon proteins and TIMP-3 expression in AdD24Luc- and AdD24TIMP3-infected U-87yEGFR tumors excised after imaging. Original magnification, 20.


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Figure 4. A, effect of AdD24Luc and AdD24TIMP-3 on growth of subcutaneous U-87MG xenografts. Mice received intratumoral injections of either PBS (--) or 3 108 pfu AdD24Luc ( n ) or AdD24TIMP-3 ( E ) on days 0, 2, and 4 (arrows ). Points, mean tumor volumes (tumor growth); bars, FSE. B, immunohistochemical staining for adenoviral hexon and TIMP-3 expression in AdD24TIMP-3-infected s.c. U-87MG tumor excised on day 10 after infection. Original magnification, 40. C, effect of AdD24Luc and AdD24TIMP-3 on survival of mice bearing intracranial U-87yEGFR xenografts. Mice received intratumoral injection of PBS (--) or 2 108 pfu AdD24Luc ( n ) or AdD24TIMP-3 ( E ) on day 4 after tumor cell injection (arrow ). % Survival of initial number of animals per group. D, immunohistochemical staining for TIMP-3, showing border between tumor and normal brain, and vWF, showing intratumoral vasculature, in cryosections of brains of PBS-treated U-87yEGFR tumor-bearing mice (left). Original magnification, 20. H&E and vWF staining of brains of long-term survivors showing small tumor remnant with extensive calcification surrounded by remaining vasculature (right ). Original magnifications, 10 and 40.

prolonged survival of tumor-bearing mice compared with PBStreated mice, which all perished by day 18 (P < 0.005). Moreover, treatment with AdD24Luc or AdD24TIMP-3 resulted in 33% and 20% long-term survivors (up to day 120), respectively. However, no significant difference in survival was found between the two viral treatments (P = 0.78). Staining of cryosections of U87yEGFR tumors removed at time of sacrifice for TIMP-3 and vWF showed a high level of endogenous TIMP-3 expression and the highly vascularized nature of these tumors (Fig. 4D). No change in TIMP-3 expression was observed in the AdD24Luc- or AdD24TIMP3-treated tumors. Moreover, quantitative analysis of vessel density showed no significant difference in vessel density between AdD24Luc- or AdD24TIMP-3-treated tumors (data not shown). Analysis of cryosections of brains of long-term survivors showed the presence of a small tumor remnant (f1 2 mm) with extensive calcification. Stainings for TIMP-3 and adenovirus hexon were negative (data not shown) and staining for vWF showed the presence of few remaining vessels surrounding the tumor remnant (Fig. 4D).

Discussion Overexpression of TIMP-3 in tumor cells has been shown to suppress tumor growth in experimental models for melanoma and neuroblastoma and in squamous cell, breast, and colon carcinoma (27, 29, 44, 45). The proposed mechanisms by which these effects are achieved include the ability of TIMP-3 to induce apoptosis directly (32), by inhibition of tumor angiogenesis (28), and by its


ability to inhibit remodeling of tumor extracellular matrix thereby preventing tumor growth and expansion as well as invasion and metastasis (25, 26). In the present study, we have examined the effect of TIMP-3 overexpression on patient-derived primary glioma samples and three established glioma cell lines. In addition, we studied the effects of TIMP-3 in the context of the tumor selective oncolytic adenovirus AdD24, allowing virus-induced oncolysis to be combined with local TIMP-3 treatment in in vitro and in vivo models of malignant glioma. Our data show that overexpression of TIMP-3 inhibited the proliferative potential of three cell lines (36-71%) and four of five low-passage primary glioma cell cultures (19-54%) and induced morphologic changes suggestive for apoptosis. These results are in accordance with previous studies, which have suggested that TIMP-3 can induce apoptotic cell death and inhibition of monolayer cell growth in various tumor cell lines (25, 26, 45, 46). Based on these results as well as the reported inhibitory effects of TIMP-3 on angiogenesis (27, 28, 44, 47), we proceeded to construct a conditionally replicating adenovirus encoding TIMP-3. With this novel oncolytic adenovirus, AdD24TIMP-3, the combined effects of oncolytic viral therapy and local TIMP-3 production could be studied. AdD24TIMP-3 showed enhanced oncolytic activity on a panel of primary glioma cell cultures and glioma cell lines when compared with the control virus AdD24Luc. In vivo functional activity of TIMP-3 expressed in the context of a replicative virus was shown using a novel optical imaging method using an MMP-2-sensitive probe, which produces a near-IR fluorescent signal (40). Although established U-87yEGFR

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tumors express endogenous TIMP-3, as noted by immunohistochemical staining for TIMP-3, intratumoral injection of AdD24TIMP-3 strongly reduced the MMP-2 activity in these tumors. Herewith, we were able to show that functional transgene expression from a replicative adenovirus can reach sufficiently high levels to achieve a biological effect that can be imaged noninvasively. To assess whether this inhibition would lead to antitumor effects in vivo, AdD24TIMP-3 and AdD24Luc were tested in both a s.c. and an intracranial mouse xenograft model for malignant glioma. In both models, treatment with these oncolytic viruses significantly inhibited tumor growth and prolonged survival of these animals compared with PBS-treated controls. In the s.c. U-87MG tumors, a trend toward enhanced antitumor efficacy of AdD24TIMP-3 compared with AdD24Luc was observed; however, the mean tumor growth rates of AdD24Luc- and AdD24TIMP-3-treated tumors did not differ significantly. Furthermore, no significant difference in the survival rate of mice bearing intracranial U87yEGFR tumors treated with the luciferase- or TIMP-3-encoding viruses was found. Various issues can be considered to explain this lack of in vivo efficacy. First, the question arises whether exogenous TIMP-3 production in the context of a replicating adenovirus is maintained sufficiently long enough in vivo to achieve a therapeutic effect. Parallel studies in mice bearing intracranial U-87yEGFR tumors injected with AdD24Luc showed strong expression of luciferase at 24 hours and a gradual, up to 30-fold, decrease during the course of 15 days after infection.7 A longer follow-up could not be done due to animals becoming symptomatic. However, these results do suggest that transgene expression from an oncolytic adenovirus is not limited to a single round of replication of the virus. Whether the TIMP-3 production is maintained high enough to significantly affect tumoral and stromal MMP activity during the time span of the in vivo experiments remains unresolved. Second, reported studies describing the effects of TIMP-3 gene delivery on s.c. tumor growth were done by ex vivo infection of tumor cells before implantation (28, 44, 45) or by injection of TIMP3 expressing vectors into small established tumors (25-100 mm3; ref. 29). The s.c. tumors in our study were treated at a mean volume of 150 to 175 mm3, at which time the tumors may be less susceptible to TIMP-3 treatment. Indeed, Spurbeck et al. showed that small established tumors (50 mm3) were susceptible to retroviral TIMP-3 treatment, whereas large established tumors (500 mm3) were not (27). It remained unclear, however, whether this was due to lack of efficient tumor cell transduction in the large tumors or the fact that larger, older tumors have already established a mature neovasculature that is not reversed by TIMP-3 treatment. The latter seems more likely as others have reported tumor size dependent effects of (synthetic) MMP inhibitors as well (48). Third, treatment of small intracranial tumors by intratumoral injection of the oncolytic viruses improved survival but also failed

7 Unpublished data.

to show additional antitumor activity of TIMP-3. Contrary to s.c. models for malignant glioma, effective treatment of intracranial tumors by MMP inhibitors has only been reported scarcely (49). Moreover, clinical experience with monotherapy of synthetic MMP inhibitors such as Marimastat, Metastat, and Prinomastat in glioma patients has showed little to no tumor response in these studies (50–52). This suggest that although MMPs are up-regulated in malignant gliomas and correlate with their malignant progression (18, 21, 53), the complexity of the MMP system and redundancy in proteolytic enzyme cascades may play a role in circumventing the effects of MMP inhibitors such as TIMP-3. Moreover, in vitro and s.c. tumor models may not accurately predict the outcome of anti-MMP treatment in intracranial tumors due to differences in microenvironment such as type of extracellular matrix and specific characteristics of the endothelial cells in the brain. Finally, the lack of enhanced in vivo efficacy of TIMP-3 insertion into the AdD24 backbone may reside in the fact that the viral oncolysis itself already produces a very potent anticancer effect, which is not significantly affected by additional indirect effects on the tumor. This becomes particularly evident when considering the fact that treatment with an oncolytic adenovirus, with or without TIMP-3, cures up to 33% of mice bearing intracranial U-87yEGFR gliomas, an enhanced tumorigenic and notoriously difficult to treat tumor (34). Among the various treatments studied in this tumor model (42, 43, 54, 55), only one report described the occurrence of cures (up to 20%) after combined gene and radiation therapy (56). Therefore, the impressive antitumor effect achieved after a single injection of our oncolytic adenoviruses in these tumors may be difficult to enhance by combining with the relatively modest antitumor effects that TIMP-3 may confer. Such effects are perhaps more readily detected using tumor models of slow-growing invasive phenotypes. Overall, we found that TIMP-3 gene transfer was capable of inducing apoptosis in primary glioma cells in vitro. Although expression of TIMP-3 in the context of a conditionally replicating adenovirus also led to enhanced cell killing of primary and clonal human glioma cells in vitro, these properties did not result in significantly greater antitumor activity when combined with adenoviral oncolysis in in vivo models for malignant glioma. Nevertheless, our imaging data do show that AdD24TIMP-3-induced TIMP-3 expression can reach sufficiently high levels to inhibit in vivo tumoral MMP activity, indicating that AdD24TIMP-3 may be of potential interest in more invasive/metastatic tumor models.

Acknowledgments Received 12/6/2004; revised 5/23/2005; accepted 7/14/2005. Grant support: Dutch Cancer Society grant VU2002-2594, Ter Meulen Fund of the Royal Netherlands Academy of Arts and Sciences, and Oncology Research Institute VU University. 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. We thank Prof. Dr. Victor van Hinsbergh for critical reading of the article; Wei-Tsung Chen, Eveline Hoebe, and Ida van der Meulen for technical assistance; and Ted Graves for assistance in quantitative analysis of imaging data.

1. Dobbelstein M. Replicating adenoviruses in cancer therapy. Curr Top Microbiol Immunol 2004;273:291–334. 2. Lou E. Oncolytic viral therapy and immunotherapy

of malignant brain tumors: two potential new approaches of translational research. Ann Med 2004; 36:2–8. 3. Wildner O, Morris JC, Vahanian NN, et al. Adenoviral vectors capable of replication improve the efficacy of


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References

HSVtk/GCV suicide gene therapy of cancer. Gene Ther 1999;6:57–62. 4. Aghi M, Chou TC, Suling K, Breakefield XO, Chiocca EA. Multimodal cancer treatment mediated by a replicating oncolytic virus that delivers the oxazaphosphorine/


TIMP-3 Expression from an Oncolytic Adenovirus rat cytochrome P450 2B1 and ganciclovir/herpes simplex virus thymidine kinase gene therapies. Cancer Res 1999; 59:3861–5. 5. Zhang JF, Hu C, Geng Y, et al. Treatment of a human breast cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regression due to viral oncolysis and gene therapy. Proc Natl Acad Sci U S A 1996;93:4513–8. 6. Chase M, Chung RY, Chiocca EA. An oncolytic viral mutant that delivers the CYP2B1 transgene and augments cyclophosphamide chemotherapy. Nat Biotechnol 1998;16:444–8. 7. Freytag SO, Rogulski KR, Paielli DL, Gilbert JD, Kim JH. A novel three-pronged approach to kill cancer cells selectively: concomitant viral, double suicide gene, and radiotherapy. Hum Gene Ther 1998;9:1323–33. 8. Parker JN, Gillespie GY, Love CE, et al. Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors. Proc Natl Acad Sci U S A 2000;97:2208–13. 9. van Beusechem VW, van den Doel PB, Grill J, Pinedo HM, Gerritsen WR. Conditionally replicative adenovirus expressing p53 exhibits enhanced oncolytic potency. Cancer Res 2002;62:6165–71. 10. Sova P, Ren XW, Ni S, et al. A tumor-targeted and conditionally replicating oncolytic adenovirus vector expressing TRAIL for treatment of liver metastases. Mol Ther 2004;9:496–509. 11. Bristol JA, Zhu M, Ji H, et al. In vitro and in vivo activities of an oncolytic adenoviral vector designed to express GM-CSF. Mol Ther 2003;7:755–64. 12. Zhang ZL, Zou WG, Luo CX, et al. An armed oncolytic adenovirus system, ZD55-gene, demonstrating potent antitumoral efficacy. Cell Res 2003;13:481–9. 13. Lambright ES, Amin K, Wiewrodt R, et al. Inclusion of the herpes simplex thymidine kinase gene in a replicating adenovirus does not augment antitumor efficacy. Gene Ther 2001;8:946–53. 14. Yoon SS, Carroll NM, Chiocca EA, Tanabe KK. Cancer gene therapy using a replication-competent herpes simplex virus type 1 vector. Ann Surg 1998;228:366–74. 15. Morris JC, Wildner O. Therapy of head and neck squamous cell carcinoma with an oncolytic adenovirus expressing HSV-tk. Mol Ther 2000;1:56–62. 16. Rao JS. Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer 2003;3: 489–501. 17. Nakada M, Nakamura H, Ikeda E, et al. Expression and tissue localization of membrane-type 1, 2, and 3 matrix metalloproteinases in human astrocytic tumors. Am J Pathol 1999;154:417–28. 18. Yamamoto M, Mohanam S, Sawaya R, et al. Differential expression of membrane-type matrix metalloproteinase and its correlation with gelatinase A activation in human malignant brain tumors in vivo and in vitro . Cancer Res 1996;56:384–92. 19. Forsyth PA, Wong H, Laing TD, et al. Gelatinase-A (MMP-2), gelatinase-B (MMP-9) and membrane type matrix metalloproteinase-1 (MT1-MMP) are involved in different aspects of the pathophysiology of malignant gliomas. Br J Cancer 1999;79:1828–35. 20. Lampert K, Machein U, Machein MR, et al. Expression of matrix metalloproteinases and their tissue inhibitors in human brain tumors. Am J Pathol 1998; 153:429–37. 21. Kachra Z, Beaulieu E, Delbecchi L, et al. Expression of matrix metalloproteinases and their inhibitors in human brain tumors. Clin Exp Metastasis 1999;17:555–66. 22. Gomez DE, Alonso DF, Yoshiji H, Thorgeirsson UP. Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol 1997;74: 111–22. 23. Mohanam S, Wang SW, Rayford A, et al. Expression of tissue inhibitors of metalloproteinases: negative regu-


lators of human glioblastoma invasion in vivo . Clin Exp Metastasis 1995;13:57–62. 24. Nakagawa T, Kubota T, Kabuto M, et al. Production of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 by human brain tumors. J Neurosurg 1994;81:69–77. 25. Ahonen M, Baker AH, Kahari VM. Adenovirusmediated gene delivery of tissue inhibitor of metalloproteinases-3 inhibits invasion and induces apoptosis in melanoma cells. Cancer Res 1998;58:2310–5. 26. Baker AH, George SJ, Zaltsman AB, Murphy G, Newby AC. Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP-3. Br J Cancer 1999;79:1347–55. 27. Spurbeck WW, Ng CY, Vanin EF, Davidoff AM. Retroviral vector-producer cell-mediated in vivo gene transfer of TIMP-3 restricts angiogenesis and neuroblastoma growth in mice. Cancer Gene Ther 2003;10: 161–7. 28. Spurbeck WW, Ng CY, Strom TS, Vanin EF, Davidoff AM. Enforced expression of tissue inhibitor of matrix metalloproteinase-3 affects functional capillary morphogenesis and inhibits tumor growth in a murine tumor model. Blood 2002;100:3361–8. 29. Ahonen M, Ala-Aho R, Baker AH, et al. Antitumor activity and bystander effect of adenovirally delivered tissue inhibitor of metalloproteinases-3. Mol Ther 2002;5:705–15. 30. Tran PL, Vigneron JP, Pericat D, et al. Gene therapy for hepatocellular carcinoma using non-viral vectors composed of bis guanidinium-tren-cholesterol and plasmids encoding the tissue inhibitors of metalloproteinases TIMP-2 and TIMP-3. Cancer Gene Ther 2003;10: 435–44. 31. Bond M, Murphy G, Bennett MR, Newby AC, Baker AH. Tissue inhibitor of metalloproteinase-3 induces a Fas-associated death domain-dependent type II apoptotic pathway. J Biol Chem 2002;277:13787–95. 32. Ahonen M, Poukkula M, Baker AH, et al. Tissue inhibitor of metalloproteinases-3 induces apoptosis in melanoma cells by stabilization of death receptors. Oncogene 2003;22:2121–34. 33. Wetzel M, Rosenberg GA, Cunningham LA. Tissue inhibitor of metalloproteinases-3 and matrix metalloproteinase-3 regulate neuronal sensitivity to doxorubicin-induced apoptosis. Eur J Neurosci 2003;18: 1050–60. 34. Nishikawa R, Ji XD, Harmon RC, et al. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci U S A 1994;91:7727–31. 35. Darling JL. The in vivo biology of human brain tumors. In: Thomas DGT, editor. Neurooncology: primary malignant brain tumors. Baltimore (MD): Johns Hopkins University Press; 1990. p. 1–25. 36. van Beusechem VW, van Rijswijk AL, van Es HH, et al. Recombinant adenovirus vectors with knobless fibers for targeted gene transfer. Gene Ther 2000;7:1940–6. 37. van der Laan WH, Quax PH, Seemayer CA, et al. Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3. Gene Ther 2003;10:234–42. 38. Carette JE, Graat HCA, Schagen FHE, et al. Replication-dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice acceptor site. J Gene Med 2005;7: 1053–62. 39. Fueyo J, Gomez-Manzano C, Alemany R, et al. A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo . Oncogene 2000;19: 2–12. 40. Bremer C, Tung CH, Weissleder R. In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat Med 2001;7:743–8.

41. Mahmood U, Tung CH, Bogdanov A, Jr., Weissleder R. Near-infrared optical imaging of protease activity for tumor detection. Radiology 1999;213:866–70. 42. Mishima K, Johns TG, Luwor RB, et al. Growth suppression of intracranial xenografted glioblastomas overexpressing mutant epidermal growth factor receptors by systemic administration of monoclonal antibody (mAb) 806, a novel monoclonal antibody directed to the receptor. Cancer Res 2001;61:5349–54. 43. Abe T, Terada K, Wakimoto H, et al. PTEN decreases in vivo vascularization of experimental gliomas in spite of proangiogenic stimuli. Cancer Res 2003;63:2300–5. 44. Anand-Apte B, Bao L, Smith R, et al. A review of tissue inhibitor of metalloproteinases-3 (TIMP-3) and experimental analysis of its effect on primary tumor growth. Biochem Cell Biol 1996;74:853–62. 45. Bian J, Wang Y, Smith MR, et al. Suppression of in vivo tumor growth and induction of suspension cell death by tissue inhibitor of metalloproteinases (TIMP)3. Carcinogenesis 1996;17:1805–11. 46. Baker AH, Zaltsman AB, George SJ, Newby AC. Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro . TIMP-3 promotes apoptosis. J Clin Invest 1998;101: 1478–87. 47. Collen A, Hanemaaijer R, Lupu F, et al. Membranetype matrix metalloproteinase-mediated angiogenesis in a fibrin-collagen matrix. Blood 2003;101:1810–7. 48. Price A, Shi Q, Morris D, et al. Marked inhibition of tumor growth in a malignant glioma tumor model by a novel synthetic matrix metalloproteinase inhibitor AG3340. Clin Cancer Res 1999;5:845–54. 49. Lakka SS, Gondi CS, Yanamandra N, et al. Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis. Oncogene 2004;23:4681–9. 50. Phuphanich SLV, Yung W, Forsyth P, Maestro R, Perry J. A multicenter, randomized, double-blind, placebo-controlled trial of Marimastat in patients with glioblastoma multiforme or gliosarcoma following completion of conventional first-line treatment. Proc Am Soc Clin Oncol 2001;20:52a. 51. New PMT, Phuphanich S. A phase I/II study of COL-3 administered on a continuous oral schedule in patients with recurrent high grade glioma. Preliminary results of the NABTT 9809 clinical trial. Neuro-oncol 2002;4:373. 52. Levine PPS, Glantz MJ. Randomized phase II study of temozolomide with and without the metalloprotease inhibitor prinomastat in patients with glioblastoma multiforme following best surgery and radiation therapy. Proc Annu Meet Am Soc Clin Oncol 2002; 21:100. 53. Sawaya RE, Yamamoto M, Gokaslan ZL, et al. Expression and localization of 72 kDa type IV collagenase (MMP-2) in human malignant gliomas in vivo . Clin Exp Metastasis 1996;14:35–42. 54. Klingler-Hoffmann M, Fodero-Tavoletti MT, Mishima K, et al. The protein tyrosine phosphatase TCPTP suppresses the tumorigenicity of glioblastoma cells expressing a mutant epidermal growth factor receptor. J Biol Chem 2001;276:46313–8. 55. Nagane M, Narita Y, Mishima K, et al. Human glioblastoma xenografts overexpressing a tumor-specific mutant epidermal growth factor receptor sensitized to cisplatin by the AG1478 tyrosine kinase inhibitor. J Neurosurg 2001;95:472–9. 56. Nestler U, Wakimoto H, Siller-Lopez F, et al. The combination of adenoviral HSV TK gene therapy and radiation is effective in athymic mouse glioblastoma xenografts without increasing toxic side effects. J Neurooncol 2004;67:177–88.

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