HUMAN GENE THERAPY 18:589–602 (July 2007) © Mary Ann Liebert, Inc. DOI: 10.1089/hum.2007.002
Survivin-Driven and Fiber-Modified Oncolytic Adenovirus Exhibits Potent Antitumor Activity in Established Intracranial Glioma ILYA V. ULASOV,1 ZENG B. ZHU,2 MATTHEW A. TYLER,1 YU HAN,1 ANGEL A. RIVERA,2 ANDREY KHRAMTSOV,3 DAVID T. CURIEL,2 and MACIEJ S. LESNIAK1
ABSTRACT The poor prognosis of patients with malignant gliomas necessitates the development of novel therapies. Virotherapy, using genetically engineered adenovectors that selectively replicate in and kill neoplastic cells, represents one such strategy. In this study, we examined several oncolytic vectors with modified transcriptional and transductional control of viral replication. First, we incorporated the survivin promoter (S) to drive E1A gene expression. We then modified the adenovirus serotype 5 (Ad5) fiber protein via genetic knob switching or incorporation of peptide ligands to target the following glioma-associated receptors: the Ad3 attachment protein, or CD46, v3/v5 integrins, or heparan sulfate proteoglycans. The three conditionally replicative adenoviruses, CRAd-S-5/3, CRAd-S-RGD, and CRAd-S-pk7, were then examined in vitro with respect to transduction efficiency and tissue specificity. The most promising virus was then tested in vivo for evidence of tumor growth inhibition. CRAd-S-pk7 provided the highest level of viral replication and tumor oncolysis in glioma cell lines. At the same time, we observed minimal viral replication and toxicity in normal human brain. Injection of CRAd-S-pk7 inhibited xenograft tumor growth by more than 300% (p 0.001). Sixty-seven percent of treated mice with intracranial tumors were long-term survivors (110 days; p 0.005). Analysis of tumor tissue indicated increased adenoviral infectivity, decreased mitotic activity, and enhanced tumor apoptosis. These findings demonstrate the effectiveness of CRAd-S-pk7 and provide the rationale for further development of this novel oncolytic virus for glioma gene therapy.
INTRODUCTION
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LIOBLASTOMA MULTIFORME (GBM) represents the most aggressive form of primary brain cancer and despite surgery, radiotherapy, or chemotherapy, the median survival rate does not exceed 1 year (Lesniak et al., 2001; Lesniak and Brem, 2004; Hsieh and Lesniak, 2005; Lesniak, 2005). The invasive nature of GBM, the presence of local micrometastasis, and the inability to effectively penetrate the blood–brain barrier have successfully prevented the development of effective therapies. The use of conditionally replicative adenoviral vectors (CRAds) represents a novel strategy that can be used to treat GBM. Be-
cause these tumors rarely metastasize outside of the central nervous system (CNS) and recur in proximity to the original site, direct delivery of an oncolytic virus offers the potential to effectively target these tumors. Adenoviral vectors are especially suitable in this treatment strategy and studies have established the efficacy and safety of such vectors in the CNS. For example, Ad5-24, an oncolytic vector with pRb binding-deficient E1A, has been tested in vivo in human glioma xenografts in nude mice and found to induce inhibition of tumor growth (Fueyo et al., 2000). Two other variations of Ad5-24, Ad524-hyCD (a vector expressing the cytosine deaminase gene, hyCD) and Ad524-RGD (an adeno-
1Division
of Neurosurgery, Department of Surgery, University of Chicago, Chicago, IL 60637. of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery, and Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL 35294. 3Department of Pathology, University of Chicago, Chicago, IL 60637. 2Division
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590 viral vector with an insertion of the Arg-Gly-Asp motif into the fiber knob of the 24 vector), have been shown to further extend the survival of mice with experimental brain tumors (Lamfers et al., 2002; Chiocca et al., 2004; Conrad et al., 2005). Most recently, a phase I trial using ONYX-015, an E1B geneattenuated adenovirus, has been completed and showed that injection of up to 1010 plaque-forming units (PFU) was well tolerated in patients with malignant glioma (Chiocca et al., 2004). Clinical efficacy was also demonstrated in GBM patients treated with adenoviral vectors delivering herpes simplex virus thymidine kinase (HSV-TK) into the tumor resection cavity, with significant prolongation of survival in the treatment group (Immonen et al., 2004). Taken together, studies such as this clearly provide the scientific rationale for further development of targeted adenoviral gene therapies for GBM. The therapeutic efficacy of a CRAd relies on the ability of the vector to successfully target, transduce, and replicate in GBM. One approach that allows an adenovirus to specifically replicate in tumor tissue takes advantage of a tumor-specific promoter (TSP), which then responds to the specific cellular cues of tumor cells to mediate its replication. An attractive promoter element for glioma is survivin (S), a member of the inhibitor of apoptosis protein (IAP) family. Survivin expression in gliomas is associated with poor prognosis, increased rates of recurrence, and resistance to chemo- and radiotherapy (Chakravarti et al., 2002, 2004; Kajiwara et al., 2003; Yamada et al., 2003). We have shown that the incorporation of this promoter into the adenoviral E1A region is responsible for enhanced viral replication and an enhanced oncolytic effect in malignant glioma (Van Houdt et al., 2006; Ulasov et al., 2007b). Although the survivin promoter enhances the killing effect of glioma, there remains a need for a gene delivery component that mediates the effective binding and internalization of a CRAd to tumor cells. The wild-type human adenovirus (Ad5) has demonstrated relatively poor transduction in human tumors (Asaoka et al., 2000; Seki et al., 2002; Kanerva et al., 2004; Tango et al., 2004). The accepted rationale for this poor transduction is that tumor cells have limited surface expression of the Ad5 primary receptor, the coxsackievirus–adenovirus receptor (CAR) (Cripe et al., 2001). Therefore, in an attempt to increase tumor transduction efficiency, several fiber modifications have been made to increase adenoviral tropism. For example, we have previously shown that a chimeric Ad5/3 vector, which contains the shaft of Ad5 and knob of Ad3, effectively targets CD46 or the CD80/86 cellular receptor and exhibits increased transduction of malignant glioma compared with wild-type Ad5 (Ulasov et al., 2006, 2007a). We have also shown that incorporation of an RGD peptide motif into the Ad5/3 fiber knob enhances the efficiency of transduction of malignant glioma (Tyler et al., 2006). An alternative approach to enhancing adenoviral tropism has been the introduction of polylysine residues into the adenovirus fiber knob. These residues selectively bind heparan sulfate proteoglycans (HSPGs), which are overexpressed in glioma (Qiao et al., 2003). Staba and coworkers used a replication-deficient type 5 adenovirus with a pk7 fiber modification and showed that the pk7 modification endowed the virus with an enhanced ability to transduce glioma (Staba et al., 2000). Our previous findings as well as those of Staba and coworkers have given us reasonable cause to examine the use of conditionally replicating ade-
ULASOV ET AL. noviruses that contain the preceding fiber modifications in the context of malignant glioma. In this study, we hypothesized that transcriptional and transductional control of viral replication would enhance the oncolytic effect of a virus against malignant glioma. To test this hypothesis, we constructed CRAd-S-5/3, CRAd-S-RGD, and CRAd-Spk7, which bind to CD46, v3/v5, and HSPG, respectively. We then examined the targeting and oncolytic efficiency of our CRAds in vitro and in vivo. Our results clearly demonstrate that CRAd-S-pk7 has enhanced tumor targeting and exhibits enhanced tumor cell killing and should be considered for further preclinical development and testing in human clinical trials.
MATERIALS AND METHODS Cells and cell culture The human malignant glioma cell lines U-251 MG, U-118 MG, U-87 MG, and A172, human lung carcinoma cell line A549, and human kidney HEK293 cells were purchased from the American Type Culture Collection (Manassas, VA). Kings1 and no.10 glioma cell lines were purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). Primary human tissue samples were obtained from patients undergoing intracranial surgery according to a protocol approved by the Institutional Review Board of the University of Chicago (Chicago, IL). All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Mediatech, Herndon, VA) and incubated in 37°C and 5% CO2.
Adenoviral constructs The following four CRAds were generated by our group on the basis of wild-type adenovirus (AdWT): AdWT-S, CRAdSurvivin-RGD, CRAd-Survivin-5/3, and CRAd-Survivin-pk7 (Zhu et al., 2006) (Fig. 1). The CRAd-Survivin constructs have the following characteristics: (1) CRAd-Survivin constructs contain the human survivin promoter, termed CRAd-S, to drive E1 expression. The survivin-controlled E1 expression cassette was placed in the original E1 region of the Ad gene as previously described (Van Houdt et al., 2006). The native E1 promoter was deleted to avoid nonspecific viral replication; (2) recombinant adenoviruses were created on the basis of homologous recombination in 911 cells between a shuttle vector, pScs/PA/S, which carries a human survivin promoter, and a pVK700-based wild-type adenoviral 5 backbone containing an RGD motif incorporated into the HI loop of the adenoviral knob 5 protein (CRAd-S-RGD) (Dmitriev et al., 1998), a substitution of fiber knob for adenovirus knob 3 type (CRAd-S5/3) (Tekant et al., 2005), or a polylysine modification of the fiber knob (CRAd-S-pk7) (Wu et al., 2004); (3) the E3 gene was retained in the Ad genome to elevate oncolysis of the CRAd agents; and (4) a poly(A) signal was inserted between the inverted terminal repeat (ITR) and the survivin promoter to stop the nonspecific transcriptional activity of the ITR, and to retain the tumor specificity of the survivin promoter. Viruses were selected from single plaques on 911 cells, expanded in A549 cells, and then purified by double CsCl gradient ultracentrifugation (Graham et al., 1977).
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Structure of conditionally replicative adenoviral vectors used in this study.
In vitro gene transfer Viral cytopathic assays that were performed in this study have been previously described. Briefly, U-87 MG, Kings-1, U-251 MG, U-118 MG, A172, and no.10 cells (5 104 cells per well) were either mock infected or infected with conditionally replicative Ad vectors (CRAd-S-5/3, CRAd-S-RGD, or CRAd-S-pk7), AdWT, or replication-deficient virus (reAd) at 100, 10, or 1 VP/cell in 0.5 ml of DMEM with 2% FBS. After 1 hr infection, the medium was removed and fresh growth medium was added. Ten days later, the medium was aspirated and the cells were stained with crystal violet. Pictures were taken with a Sony digital camera.
Cell viability assay U-118 MG cells were seeded in a 96-well plate (1 104 cells per well) and infected 24 hr later with 100 VP/cell. The plates were incubated at 37°C and supplemented with 5% CO2. At various time points, the medium was removed and fresh medium containing 20 l of 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution (Promega, Madison, WI) was added to each well. The cells were then incubated for 4 hr and absorbance was recorded at 490 nm with an enzyme-linked immunosorbent assay (ELISA) plate reader.
Quantitative analysis of viral transcription and replication U-118 MG human glioma cells (5 104 cells per well) were grown to 60% confluence and seeded onto 24-well plates with 1 ml of F12–Dulbecco’s modified Eagle’s medium. The next day, medium was aspirated and the cells were infected with AdWT-S, CRAd-S-pk7, or AdWT adenoviral vectors at 100
VP/cell, and incubated at 37°C in a humidified atmosphere for 1 hr. The cells were rinsed with phosphate-buffered saline (PBS) and 10% growth medium was added. For replication analysis, on days 1, 2, 5, and 8 aliquots of medium and detached cells were subjected to DNA isolation and quantitative polymerase chain reaction (PCR). DNA was isolated from cells according to a standard protocol, using a DNeasy tissue kit (Qiagen, Valencia, CA) and a quantitative real-time PCR assay for E1 and E4 genes was performed. For quantification of E4 mRNA expression, cell pellets were collected on days 1 and 3. Isolated RNA was converted into cDNA with the SuperScript II system (Invitrogen, Carlsbad, CA) and subjected to quantification. The sequences of specific primers used for E4 amplification were as follows: sense, 5-GGAGTGCGCCGAGACAAC-3; antisense, 5-ACTACGTCCGGCGTTCCAT-3. The sequences for E1A were previously described (Rein et al., 2005). The PCR was performed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primers (TaqMan GAPDH control reagent; Applied Biosystems, Foster City, CA) to create an internal standard. Quantification using SYBR Green PCR master mix (Applied Biosystems) was performed according to vendor recommendations. Data were reported as the ratio of E4 copy number per human GAPDH copy.
Evaluation of virus replication and toxicity in normal human brain Primary brain slices were prepared as previously described (Kirby et al., 2004) and cultured for 1 hr at 37°C. The slices were then infected with 500 VP per slice of AdWT or CRAdS-pk7, or were mock infected. Four hours later, the slices were rinsed several times with PBS and fresh growth medium was added to the culture. One portion of infected slices was harvested on days 1 and 3 for further progeny titration on HEK293
592 cells (Fueyo et al., 2000); the second portion was evaluated for lactate dehydrogenase (LDH) release, using a cell membrane integrity kit (Promega).
In vivo xenograft models of U-87 MG glioma Five- to 6-week-old female athymic mice (n 6 per group, nu/nu; Charles River Laboratories, Wilmington, MA) were used for in vivo studies. Animals were cared for according to protocols approved by the Animal Use Committee of the University of Chicago. U-87 MG cells were cultured under standard conditions until they reached 70% confluence, were detached with 0.05% trypsin solution, washed with serum-enriched medium, and then centrifuged at 1200 rpm for 5 min at 4°C. U-87 MG xenografts were established by preparing 1 106 cells in RPMI (Mediatech) and then injecting them subcutaneously into the flank region. When tumors reached 0.7 cm3, mice bearing tumors were divided in three groups: two groups received intratumoral CRAd-S-pk7 or AdWT at 1 1011 VP/mouse, prepared in RPMI; mice in the control group were mock infected with RPMI alone. Tumor volume was measured every 2 days and determined by three-dimensional caliper measurements. Human glioma xenografts were also generated in the brains of athymic nude mice (n 6 per group) according to a protocol previously described by our group (Hsu et al., 2005; Lesniak et al., 2005). Briefly, 1 106 U-87 MG glioma cells were inoculated intracranially into nu/nu BALB/c mice (day 0). Seven days after tumor inoculation, mice were randomized into three groups, one receiving CRAd-S-pk7, one receiving AdWT, and one receiving RPMI. A total of 5 109 VP in a total volume of 5 l of RPMI per mouse was injected.
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Western blot analysis Protein extracts were prepared from xenograft cells 24 days after infection with conditionally replicative adenoviral vectors or after mock infection. Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA). Lysates were analyzed by Western blot analysis using 10% sodium dodecyl sulfate (SDS) gels. Lanes were loaded with 50 g of protein and electrophoresed for 2 hr at 90 V. Gels were transferred to polyvinylidene difluoride (PVDF) membrane, blocked with 3% nonfat dry milk, and incubated with primary antibodies overnight at 4°C. Primary antibodies included mouse monoclonal antibody IgG to -actin (1:2000 dilution; Abcam, Cambridge, MA) and rabbit polyclonal antibody to caspase-3 (1:1000 dilution; Cell Signaling Technology, Danvers, MA). Horseradish peroxidase (HRP)-conjugated secondary antibodies, including goat anti-mouse (ab5870, 1:2500 dilution; Abcam) and goat anti-rabbit (1858415, 1:5000 dilution; Pierce Biotechnology, Rockford, IL), were added for 1 hr and protein signals were detected with Immobilon Western substrate (Millipore, Billerica, MA). Pictures were taken with a Kodak 440 system.
Statistical analysis Statistical comparison for all experimental conditions was performed by Student t test. Survival was plotted as a Kaplan–Meier survival curve and statistical significance was determined by Kruskal–Wallis nonparametric analysis of variance followed by the nonparametric analog of the Newman–Keuls multiple comparison test. p 0.05 was considered statistically significant.
Histology and immunohistochemistry Tumors obtained from treated and control animals were immediately fixed in formalin (3.7% formaldehyde–PBS) and subsequently embedded in paraffin. Five-millimeter-thick sections were deparaffinized and stained with hematoxylin and eosin (H&E). Tumor sections underwent immunohistochemical analysis with goat anti-hexon antibodies (1:500 dilution; ViroStat, Portland, ME) recognizing adenoviral hexon, rabbit monoclonal anti Ki-67 antibodies (M7187, 1:50 dilution; Dako, Glostrup, Denmark), and mouse anti-human CD31 (BD Biosciences Pharmingen, San Diego, CA) and were processed with Histostain (Zymed; Invitrogen) and the EnVision system (Dako) according to the manufacturer’s instructions (Ulasov et al., 2006b). To quantify cells positively stained for hexon, CD31, and Ki-67 markers, an automated system was used to assess the quantity and morphology of stained cells (ACIS; Clarient, Aliso Viejo, CA). Eleven 20 fields were randomly selected and the computer software quantified the number of positive cells in each field.
RESULTS CRAd-S-pk7 effectively kills glioma cells in vitro To assess the oncolytic effect of our CRAds, U-87 MG, Kings-1, U-251 MG, A172, no.10, and U-118 MG glioma cell lines were exposed to CRAd-S-pk7, CRAd-S-5/3, CRAd-SRGD, AdWT, or replication-deficient reAd virus at 1, 10, and 100 VP/cell. Cytotoxic effect was then assessed via crystal violet staining. Of the tested vectors, CRAd-S-pk7 demonstrated a dose-dependent cytolytic effect in all human glioma cell lines (Fig. 2). The virus induced cell killing at doses as low as 1 VP/cell in U-118 MG cells and at 10 VP/cell in U-87 MG and U-251 MG cells. Kings-1, no.10, and A172 cells displayed lower cytotoxicity levels (10-fold less) than did U-118 MG cells. Of note, the oncolytic effect of CRAd-S-pk7 was superior to AdWT in five of the six tested cell lines. No cytotoxic effect was observed for the control, replication-defective reAd vector.
Quantitative analysis of nuclear and cytoplasmic intensities by automated cellular imaging system
CRAd-S-pk7 efficiently and selectively replicates in glioma cells
The intensity of Ki-67 and hexon staining was measured with the ACIS as described previously (van Gijssel et al., 2004). The density of CD31 formation was evaluated by counting the number of cells staining for endothelial antigen CD31 in 20 highpower fields and deriving an average value.
The in vitro results suggested that a pk7 knob modification improves the oncolytic capability of the survivin-modified CRAd in vitro. To evaluate the dynamics of cell killing detected by crystal violet, we performed an MTS analysis of U-118 MG cells, which were most susceptible at low viral concentrations (see Fig.
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FIG. 2. CRAd-S-pk7 exhibits selective oncolytic potential in human glioma cells. CRAds at the indicated doses were incubated with the following human glioma cell lines: U-118 MG, Kings-1, no.10, U-87 MG, U-251 MG, and A172. Lateral virus spread and oncolytic effect were visualized after staining of adherent cells with crystal violet. Experiments were repeated twice, independently.
2). As shown in Fig. 3A, treatment with CRAd-S-pk7 resulted in more efficient cell killing compared with AdWT infection. Thus, on day 8, CRAd-S-pk7 treatments induced 80% cell killing versus 60% demonstrated for AdWT. To further analyze the efficacy of transcription and replication, we measured E4 copy numbers in infected U-118 MG cells. CRAd-S-pk7 demonstrated approximately 100–1000 orders of magnitude enhanced transcription and replication as compared with cells treated with AdWT and AdWTS (p 0.05) (Fig. 3B and C). Finally, to test whether an increased E4 copy number level corresponded to greater E1A protein expression, we infected U-118 MG cells with CRAd-S-pk7, AdWTS, and AdWT. As shown in Fig. 3D, infection with CRAd-S-pk7 led to increased E1A protein expression in target cells from day 1 to day 3 and these levels corresponded to the increased level of E4 copy number demonstrated by CRAd-S-pk7 and described in Fig. 3C. To determine the potential cytotoxicity mediated by CRAdS-pk7 infection, we tested the activity of our virus in normal
human brain. Human brain slices were infected with AdWT or CRAd-S-pk7, or were mock infected. Replication was measured by titration of progeny released from slices and medium on days 1 and 3. As shown in Fig. 4A, CRAd-S-pk7 demonstrated significantly lower replication activity in normal brain tissue both on day 1 (1.77 vs. 112.02 for AdWT, p 0.05) and day 3 (1.99 vs. 199.52 for AdWT, p 0.01). To assess virally induced toxicity, we measured the expression of cellular proteins in the medium. Consistent with our replication data, CRAd-S-pk7 showed significantly less LDH release versus AdWT on day 3 (1.64 0.11 vs. 15.63 2.082, respectively; p 0.05) (Fig. 4B).
Growth suppression of U-87 glioma xenografts by intratumoral injection of CRAd-S-pk7 To determine whether our in vitro findings could be confirmed in vivo, we used a glioma xenograft model in nude mice
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FIG. 3. Kinetics of cell killing, viral replication, and gene expression mediated by CRAd-S-pk7 in U-118 MG human glioma cells. (A) Cytotoxic effects of CRAd-S-pk7 on U-118 MG cells. Human glioma cells were seeded in 96-well plates at densities described in Materials and Methods. The next day, cells were infected with either CRAd-S-pk7 or AdWT at 100 VP/cell. After 1 hr of adsorption, cells were incubated. Twenty microliters of MTS solution was added to each well on days 1, 2, 4, and 8 and then, 4 hr later, absorbance at 490 nm was recorded in a 96-well plate reader. In a separate set of experiments, U-118 MG cells were infected with CRAd-S-pk7, AdWT-S, or AdWT at 100 VP/cell. Viral transcription and replication were evaluated by measuring E4 mRNA (B) and E4 (C) and E1A (D) DNA copy number as described in Materials and Methods (*p 0.05).
and treated established tumors with adenoviral vectors. Human U-87 MG glioma cells were injected into the flank of nu/nu mice, resulting in established tumors in 95% of the animals. Once the tumors reached 0.7 cm3 (one week later), mice were divided into three groups and then treated by intratumoral injection of either RPMI (mock infection), AdWT, or CRAd-Spk7, the latter two at a dose of 1 1011 viral particles per injection. CRAd-S-pk7 suppressed tumor growth by more than 300% versus AdWT (p 0.001). Tumor volume in the RPMI, AdWT, and CRAd-S-pk7 groups at day 14 and day 24 was as follows: Day 14: 2.59 0.34, 1.63 0.27, and 1.21 0.14 cm3, respectively (p 0.05); Day 24: 3.91 0.38, 3.61 0.36, and 1.34 0.11 cm3, respectively (p 0.05) (Fig. 5A). To confirm the therapeutic efficacy of CRAd-S-pk7, we treated established intracranial tumors with the retargeted virus. In this study, established U-87 MG glioma xenografts grown in the brain of athymic mice were subjected to intracranial injections of AdWT or CRAd-S-pk7, or were mock infected (Fig. 5B). The median survival of mice treated with RPMI was 44 days. In contrast, the median survival of the AdWT-treated group was 71 days (p 0.05). Sixty-seven percent of mice
treated with CRAd-S-pk7 were long-term survivors ( 110 days) (p 0.005). For the purpose of statistical analysis and to ensure the validity and reproducibility of our results, both of the preceding experiments were repeated twice with similar results obtained in each case.
Ki-67 and hexon marker labeling in U-87 MG xenografts To confirm that the inhibition of tumor growth was due to viral replication, we performed H&E staining and hexon protein staining. The mice were killed and tumor sections were analyzed by immunohistochemistry for both Ki-67 and adenoviral hexon protein expression. Tumor proliferation was monitored by staining for Ki-67 in xenograft tissue slices. As shown in Fig. 6, untreated and AdWTtreated xenografts showed similarly high levels of Ki-67-positive staining whereas CRAd-S-pk7 significantly decreased the labeling index. In addition, viral replication was analyzed by examining hexon expression in treated tumors. The highest level of hexon protein was observed in tumors treated with CRAd-S-pk7.
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FIG. 4. Evaluation of CRAd-mediated toxicity in normal human brain slices. Human brain tissue slices were infected with Ad vectors at 500 VP/cell. Twenty-four or 72 hr postinfection, progeny (A) were isolated from medium and tissue slices and titrated in HEK293 cells. Alternatively, LDH release (B) was measured in slice medium. Results are presented as plaque-forming units per milliliter (A) and as percent toxicity (B). Compared with the positive control (AdWT), CRAd-S-pk7 demonstrated significantly lower replication activity in normal brain tissue both on day 1 (1.77 vs. 112.02, p 0.05) and day 3 (1.99 vs. 199.52, p 0.01). LDH data corroborate replication data and revealed more LDH released from AdWT-infected cells than from CRAdS-pk7-infected cells (15.63 2.082 vs. 1.64 0.11, p 0.05). To objectively characterize the delayed growth effect induced by CRAd-S-pk7, we measured hexon and Ki-67 labeling indexes (Fig. 7). We used digital analysis software (ACIS) to quantify the positively stained cells as described in Materials and Methods. The number of hexon-positive cells per 20 field was significantly lower in mice treated with AdWT than in mice treated with CRAd-S-pk7 (0.473 0.43 vs. 2.73 0.12, respectively; p 0.05) (Fig. 7A). These results were confirmed by examining integrated optical density (IOD) per square micrometer (2 2 in the case of AdWT vs. 6.4 3.5 in the case of CRAd-S-pk7; p 0.05) (Fig. 7B). The proliferation index for each target was also expressed as a percentage of pos-
itive nuclear staining with anti-Ki-67 antibodies in tumor cells (Fig. 7C and D). The density of Ki-67-positive cells seen in CRAd-S-pk7-treated tumor was significantly lower compared with cells treated with AdWT (9.18 5.34 vs. 22 7.42; p 0.05). These results were confirmed by examining the IOD per square micrometer (66 45 in the case of AdWT vs. 37 7.5 in the case of CRAd-S-pk7, p 0.05).
Mechanism of tumor growth suppression To assess the effect of adenoviral replication on tumor vessel density, tumor sections were stained for CD31, a marker of
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FIG. 5. Response of U-87 MG tumor to survivin-activated adenoviral gene transfer. (A) U-87 MG cells were injected into the flank of nude mice (n 6 per group) and allowed to grow to 0.7 cm3 in size. CRAd-S-pk7 or AdWT vector was injected intratumorally at a dose of 1 1011 VP/mouse. Results are presented as tumor volume (cubic centimeters) over time. CRAd-S-pk7 virus reduced tumor growth by at least 300% compared with vehicle- or AdWT-injected tumor (p 0.001). Data points represent means standard deviation. (B) Kaplan–Meier survival curves after intracranial injections of AdWT, CRAd-S-pk7, or RPMI into athymic mice (n 6 per group) bearing U-87 MG glioma. The median survival of mice treated with RPMI was 44 days. In contrast, the median survival of the AdWT-treated group was 71 days (p 0.05). Sixty-seven percent of mice treated with CRAdS-pk7 were long-term survivors ( 110 days; p 0.005). Both the flank and intracranial experiments were repeated twice with similar results.
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FIG. 6. Immunohistochemical analysis of flank tumors after intratumoral administration of conditionally replicative adenoviral vectors. Tumors were harvested 24 days after vector therapy (AdWT, CRAd-S-pk7, or RPRMI) and tissue sections underwent immunohistochemical staining for H&E (A–C), Ki-67 (D–F), and hexon protein (K–M). As shown, in tumors treated with the CRAd-S-pk7 vector, Ki-67 staining was dramatically reduced. AdWT showed positive Ki-67 staining similar to that of mockinfected cells (D and F). CRAd-S-pk7- and AdWT-treated tumors (L and M) showed positive adenoviral hexon protein, whereas in mock-infected tumors these signals were not observed (K). Original magnification, 40 for all samples.
angiogenesis. Consistent with Ki-67 data, we observed a significant decrease in CD31 staining in the case of CRAd-S-pk7treated tumors versus mock- or AdWT-infected tumors (Fig. 8A). To verify that CRAd-S-pk7 induced antiangiogenesis, microvessel density was determined by semiquantitative scoring of CD31 staining in 20 images (Fig. 8B). Treatment with CRAdS-pk7 significantly reduced the mean vascular density (MVD) as compared with mock- or AdWT-treated tumors. Thus, we observed a significant (p 0.05) difference in MVD between the mock-treated group (25.3 4.08) and the CRAd-S-pk7-infected group (10.8 1.75). Moreover, we also observed a significant difference between the AdWT-infected group (MVD, 18.3 1.71) and the CRAd-S-pk7-infected group (MVD, 10.8 1.75) (p 0.05). To further elucidate the mechanism responsible for the antitumor efficacy observed with CRAd-S-pk7, we examined the expression of caspase-3 in tumors infected with adenoviral vectors. As shown in Fig. 9, mock-infected or AdWT-treated cells did not induce cleavage of the caspase-3 protein and thus no cell death resulted. We did notice an increase in the amount of
cleaved caspase-3 fragments in samples treated with CRAd-Spk7, suggesting that intratumoral injections of CRAd-S-pk7 lead to suppression of tumor growth via activation of apoptosis. Consistent with these data, the treatment of glioma with CRAd-S-pk7 demonstrated significant therapeutic effect in mouse glioma xenografts.
DISCUSSION We have previously documented tumor-specific oncolytic capacity of a CRAd that used a survivin promoter element to drive viral replication in malignant glioma (Van Houdt et al., 2006). The use of CRAd-mediated gene therapy in the context of malignant brain tumors is hindered by a sufficient presence of the Ad5 fiber receptor, or CAR, on the glioma cell surface (Fuxe et al., 2003). Most adenoviral vectors therefore depend on CAR for cell attachment and internalization and thus tumor surface binding and gene transfer are limited in tumors where CAR expression is poor. Modifying the fiber region is neces-
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% of hexon-positive cells
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30 25 20 15 10 5
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Brown IOD/micron2
10 8 6 4
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AdWT
CRAd-S-pk7
FIG. 7. CRAd-S-pk7 shows significant reduction in tumor cell proliferation and increase in hexon staining compared with AdWT-treated control. Representative sections of U-87 MG xenograft tumors treated with CRAd-S-pk7, AdWT, or RPMI were subjected to digital analysis of Ki-67- and hexon-positive cells. Eleven randomly selected fields (magnification, 20) of U-87 MG xenografts were used to quantify positive cells (number of cells SD) (p 0.05). The number of hexon-positive cells per 20 field was significantly lower in mice treated with AdWT than in mice treated with CRAd-S-pk7 (0.473 0.43 vs. 2.73 0.12; p 0.05) (Fig. 7A). These results were confirmed by examining the integrated optical density (IOD) per square micrometer (2 2 in the case of AdWT vs. 6.4 3.5 in the case of CRAd-S-pk7; p 0.05). The proliferation index for each target was also expressed as a percentage of positive nuclear staining with anti-Ki-67 antibodies in tumor cells (C and D). The density of Ki-67-positive cells seen in CRAd-S-pk7-treated tumor was significant lower compared with cells treated with AdWT (9.18 5.34 vs. 22 7.42, respectively; p 0.05). These results were confirmed by examining the IOD per square micrometer (66 45 in the case of AdWT vs. 37 7.5 in the case of CRAd-S-pk7; p 0.05).
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FIG. 8. Immunohistochemical detection of microvessel density. Tumor sections were stained with CD31 antibodies as described in Materials and Methods. Results of this staining are presented as immunohistochemical images (A) with a semiquantitative analysis of CD31 density performed by quantification of CD31-stained vessels in 20 fields, at a magnification of 40 (B). Data show that CRAd-S-pk7 infection led to inhibition of vessel formation (10.8 1.75) as compared with AdWT infection (18.3 1.71) or after mock infection (25.3 4.08) (p 0.05).
sary to circumvent the low infectivity that results from the paucity of CAR expression. We have previously shown that a replication-deficient chimeric Ad5 vector that contains a serotype 3 fiber knob in place of the serotype 5 knob renders increased glioma transfection by binding to CD46 or CD80/86 (Ulasov et al., 2006, 2007a). We have also shown that RGD ligand incorporations into the fiber knob of replication-incompetent Ad5/3 chimera increase the efficiency of brain tumor transduction and transgene expression (Tyler et al., 2006). The efficacy of such fiber modifications has been applied to CRAds in other cancer models; the use of novel CRAds within the context of malignant glioma is currently under active investigation. Like RGD-mediated gene transfer, a polylysine fiber modification is an attractive approach for enhancing CRAd gene delivery to glioma because of its high affinity for heparan sulfate proteoglycans (HSPGs) and other HSPG-like polyanionic receptors (Gingis-Velitski et al., 2004). HSPGs interact with fibroblast growth factor (FGF)-2, a potent stimulator of angiogenesis in primary brain tumors (Murphy and Knee, 1995). HSPGs participate in ternary FGF receptor complexes and ensure stable binding of the FGF ligand to the receptor tyrosine
FIG. 9. Western blot analysis of tumors infected with either AdWT or CRAd-S-pk7, or after mock infection. Twenty-four days after intratumoral infection; tumor lysates were prepared and subjected to Western blot analysis. Membranes were incubated with a monoclonal antibody against -actin or with polyclonal antibody against caspase-3 proteins. CRAd-S-pk7 infection induced cleavage of caspase-3 proteins.
600 kinase (Alanko et al., 1994). Certain HSPGs have been shown to be overexpressed exclusively in glioma vessel endothelial cells, contributing to the aggressive proliferation of glioblastoma (Qiao et al., 2003). Replication-deficient adenoviruses with a pk7 fiber modification have exhibited enhanced transfection in passaged glioma cell lines (Staba et al., 2000). Moreover, Wickham and colleagues have previously shown that a replication-defective adenovirus with a pk7 modification can effectively target vascular smooth muscle (Wickham et al., 1997) and therefore modulate the antiangiogenic properties important for tumor control. Because of these findings, a pk7 fiber modification seemed suitable for achieving more efficient CRAd gene transfer to neoplastic areas, further enhancing the tumor-regressing capabilities of the CRAd. To identify which fiber modification endows the survivin promoter-driven CRAd with the best infectivity profile, we compared gene transfer capabilities of CRAd-S-5/3, CRAd-S-RGD, CRAd-S-pk7, and AdWT. In this study, we found that transcriptional and transductional modulation, in the form of survivin promoter and pk7 fiber modification, represents the most effective approach for glioma virotherapy. These findings were confirmed both in vitro and in vivo. Cytotoxic assays using passaged glioblastoma multiforme cells clearly indicate that CRAd-S-pk7 is more efficient than CRAd-S-RGD, CRAd-S-5/3, and AdWT in cell transduction and killing. Furthermore, E1A and E4 copy number assays demonstrate that the increased cytotoxic effect is a result of tumor-specific promoter and pk7 fiber modification. CRAd-S-pk7 demonstrated a superior ability to delay tumor progression compared with the wild-type adenovirus and mock infections. This result has implications as to how pk7 directs attachment to the glioma periphery via HSPG expression (Staba et al., 2000). HSPG–FGF-2 signaling complexes aggregate at the tumor periphery to promote angiogenesis and endothelial cell differentiation. The availability of the HSPG signaling complexes at the tumor margin renders the outer neoplastic region amenable to CRAd-S-pk7 infection and oncolysis. Thus, CRAd-S-pk7 may inhibit tumor migration via binding to “proproliferation” signaling systems and subsequently inducing oncolysis via its survivin-promoted replication. Although the success obtained with CRAd-S-pk7 in controlling and prolonging the survival of mice with intracranial tumors is encouraging, one of the limitations of this study is the use of oncolytic vectors in immunodeficient mice. However, there is no fully permissive animal model in which brain tumor virotherapy and the immune response can be examined at the same time. This limitation, however, has not impeded the development of oncolytic vectors for brain tumor therapy. As an example, the ONYX-015 oncolytic adenovirus has completed a phase I clinical trial (Chiocca et al., 2004). This virus was shown to exhibit significant oncolytic efficacy in preclinical studies of xenografts in nude mice (Shinoura et al., 1999; Geoerger et al., 2002, 2003); when tested in humans, injections of up to 1010 PFU were safe. Of the 24 patients tested in this phase I study, only 2 seroconverted from negative to positive for adenovirus antibodies. Other oncolytic adenovectors, such as Ad-24-RGD, have also completed promising preclinical studies in nude mice, and are about to enter a human clinical trial for glioma (Fueyo et al., 2000; Suzuki et al., 2001; Lamfers et al., 2002; Portella et al., 2002). Moreover, studies com-
ULASOV ET AL. paring replication-defective versus oncolytic viruses have shown that although preexisting antibodies significantly attenuate the activity of replication-defective adenovirus, they exhibit no effect on the activity of replicating adenovirus (Tsai et al., 2004). These are significant findings with important implications for glioma virotherapy because of the relatively immunocompromised state of these patients, who are typically receiving chronic steroid medication and have been treated with radiation and chemotherapy, and for injection of the virus within the brain, an organ that is relatively immunoprivileged. Of note, data obtained with immunodeficient mice have been critical to establishing the rationale for employment of tropism-modified adenoviral vectors by relevant federal regulatory bodies. In conclusion, our results indicate that transcriptional and transductional control of adenoviral replication can greatly enhance the virotherapy of malignant glioma. Our novel adenovirus, CRAdS-pk7, exhibited enhanced tropism, replication, and oncolytic capacity in the setting of malignant glioma. At the same time, we observed minimal, if any, toxicity to normal brain. The enhanced oncolytic effect mediated by pk7 warrants further studies of this novel CRAd in future preclinical and clinical studies.
ACKNOWLEDGMENTS The authors thank Baogen Lu and Hong Ju Wu (Gene Therapy Center, University of Alabama) for help with propagation of CRAds. The authors thank Maria Tretiakova (Department of Pathology, University of Chicago) for excellent technical assistance with automated image analysis system and software. This study was supported by a grant from the American College of Surgeons (M.L.) and the National Institutes of Neurological Disorders and Stroke (K08 NS046430; M.L.).
AUTHOR DISCLOSURE STATEMENT No competing financial interests exist.
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Address reprint requests to: Dr. Maciej S. Lesniak Division of Neurosurgery University of Chicago 5841 S. Maryland Avenue, MC 3026 Chicago, IL 60637 E-mail:
[email protected] Received for publication January 4, 2007; accepted after revision May 27, 2007. Published online: July 11, 2007.
