Home Duke University Press
QUICK SEARCH:   [advanced]


     
  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

First published on May 23, 2007
This version was published on July 1, 2007
Neuro Oncol 2007 9(3):280-290; DOI:10.1215/15228517-2007-017
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
9/3/280    most recent
15228517-2007-017v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Van Houdt, W. J.
Right arrow Articles by Haviv, Y. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Duke University Press

Basic and Translational Investigations

Gene delivery into malignant glioma by infectivity-enhanced adenovirus: In vivo versus in vitro models

Winan J. Van Houdt, Hongju Wu, Joel N. Glasgow, Martine L. Lamfers, Clemens M. Dirven, G. Yancey Gillespie, David T. Curiel and Yosef S. Haviv

Department of Neurosurgery, VU University Medical Center, 1007 MB Amsterdam, The Netherlands (W.J.V.H., M.L.L, C.M.D.); Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, and Surgery (H.W., J.N.G., D.T.C.), Gene Therapy Center (H.W., J.N.G., D.T.C.), Division of Cardiovascular Disease (J.N.G.), and Department of Neurosurgery (G.Y.G.), University of Alabama at Birmingham, Birmingham, AL 35294-2172, USA; and Department of Medicine and Gene Therapy Institute, Hadassah-Hebrew University Medical Center, Jerusalem, Israel, 91120 (Y.S.H.)

Address correspondence to Yosef S. Haviv, Department of Medicine and the Gene Therapy Institute, Hadassah-Hebrew University Medical Center, P.O. Box 12000, Jerusalem, Israel, 91120 (yhaviv{at}hadassah.org.il).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adenoviral (Ad) vectors demonstrate several attributes of potential utility for glioma gene therapy. Although Ad infection is limited in vitro by low expression levels of the coxsackie-adenoviral receptor (CAR), in vivo studies have shown the efficacy of Ad vectors as gene delivery vectors. To evaluate the in vivo utility of CAR-independent, infectivity-enhanced Ad vectors, we employed genetically modified Ad vectors in several experimental models of human gliomas. We used three capsid-modified Ad vectors: (1) a chimeric Ad vector with a human Ad backbone and a fiber knob of a canine Ad, (2) an Ad vector with a polylysine motif incorporated into the fiber gene, and (3) a double-modified Ad vector incorporating both an RGD4C peptide and the polylysine motif. These three modified Ad vectors target, respectively, the putative membrane receptor(s) of the canine Ad vector, heparan sulfate proteoglycans (HSPGs), and both integrins and HSPGs. Our in vitro studies indicated that these retargeting strategies all enhanced CAR-independent infectivity in both established and primary low-passage glioma cells. Enhancement of in vitro gene delivery by the capsid-modified vectors correlated inversely with the levels of cellular CAR expression. However, in vivo in orthotopic human glioma xenografts, the unmodified Ad vector was not inferior relative to the capsid-modified Ad vector. Although genetic strategies to circumvent CAR deficiency in glioma cells could reproducibly expand the cellular entry mechanisms of Ad vectors in cultured and primary glioma cells, these approaches were insufficient to confer in vivo significant infectivity enhancement over unmodified Ad vectors. Other factors, probably the extracellular matrix, stromal cells, and the three-dimensional tumor architecture, clearly play important roles in vivo and interfere with Ad-based gene delivery into glioma tumors.

Key Words: adenovirus • coxsackie-adenovirus receptor • gene therapy • glioma • infectivity enhancement


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Currently available therapeutic modalities for high-grade malignant gliomas often fail to improve patient prognosis. Standard therapy is limited by a low therapeutic index1 and by the cross-resistance between chemotherapeutic drugs.2 Within this context, gene therapy is a promising therapeutic strategy for malignant gliomas, potentially offering both tumor-targeting and novel cell-killing mechanisms. Recombinant adenoviruses appear promising as gene therapy vectors for glioma, on the merit of their ability to infect both dividing and quiescent tumor cells, genome plasticity, mild pathogenicity in humans, and safety shown in gene therapy clinical trials for glioma.3-6 While the efficiency of Ad-based gene delivery into glioma cells may depend on the expression levels of the coxsackie-adenoviral receptor (CAR), the primary Ad receptor,7-10 other reports have found that capsid-unmodified Ad is a highly efficient vector in the context of glioma.11-13 Another factor confounding the use of Ad vectors for glioma involves the dose of vector inoculum, which can be potentially toxic to neighboring brain cells.14 Clearly, both CAR-dependent and CAR-independent glioma cell infection by Ad vectors needs to be further explored. In this study, we evaluated the utility of CAR-independent genetic retargeting of Ad vectors in glioma models. Genetic retargeting is generally superior to antibody-conjugated Ad with respect to easier administration and the potential for tropism modification in the progeny of conditionally replicative Ad viruses.15

Genetic modification of Ad capsid for CAR-independent infection may involve either replacement of the entire Ad fiber or the fiber knob, or the insertion of heterologous ligands into the fiber knob of Ad5.16-20 Recent genetic approaches developed by our group for CAR-independent Ad infection include insertion of a polysine (pK7) motif into the C-terminal end of the Ad5 fiber gene, or insertion of an RGD4C (RGD) motif into the HI loop of the fiber knob, or both.21 Another CAR-independent approach employed display of a xeno-fiber knob on a human Ad capsid fiber.22 These genetic approaches to alter vector tropism all proved highly efficient in cancer cells grown in monolayer culture. In this study, we investigated whether the utility of tropism modification of Ad vectors attained in vitro is maintained in solid glioma tumor xenografts. This issue is critical for cancer gene therapy endeavors because tumor components may impose several levels of blockage on vector distribution and gene delivery. In addition to cancer cells, tumors consist of stroma comprising reactive fibroblasts and extracellular matrix, basement membranes, abnormal blood vessels, necrotic regions, and infiltrating cells of the immune system. Standard cancer therapy may further accentuate necrosis, apoptosis, and fibrosis within the tumor, thereby potentially confounding the potential distribution of viral vectors. Our results indicate that while tropism-modified Ad vectors could dramatically enhance infectivity in low-CAR glioma cells in vitro, Ad capsid modification was not the sole determinant of in vivo gene delivery in glioma xenografts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Glioma Cells
Both established and primary human malignant glioma cell lines were used in this study. The glioma cell lines U-118MG and M59K were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). D-65MG, U-251MG, U-87MG, and D-54MG cells were provided by Dr. Darell D. Bigner (Duke University Medical Center, Durham, NC, USA). U87 cells were cultured with Dulbecco's modified Eagle's medium (Mediatech, Herndon, VA, USA) with 15% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 2 mM L-glutamine, penicillin (100 IU/ml), streptomycin (100 µg/ml), and amphotericin B (2.5 µg/ml). U251, U118, M59K, D65, and D54 cells were cultured in Dulbecco's modified Eagle's/F12 medium (50:50) with 7%-10% FBS, L-glutamine, and antibiotics (Mediatech), as above. All cells were cultured at 37°C in a 5% CO2 humidified atmosphere. The primary glioma cells VU-15, VU-28, VU-78, and VU-84 were obtained and used before the eighth passage, as previously described.20 Primary malignant glioma cells were cultured in Dulbecco's modified Eagle's medium with 10% FBS, L-glutamine, and antibiotics as above.

Adenoviral Vectors
All Ad vectors used in this study were replication deficient (E1 deleted) and based on the serotype-5 Ad genome. The construction of Ad5, Ad5.pK7, Ad5.RGD, and Ad5.RGD.pK7 has been described previously.21 Ad5 is an untargeted Ad vector, used as an isogenic control for Ad5.pK7, Ad5.RGD, and Ad5.RGD.pK7. Ad5. pK7 and Ad5.RGD differ from Ad5 by the respective insertion of a polysine (pK7) or an RGD motif into the C-terminal end of the Ad5 fiber gene or the HI loop of the fiber knob. Ad5.RGD.pK7 contains an RGD motif in the HI loop of the fiber knob in addition to the pK7 motif at the C-terminal end. These Ad vectors all include two identical bicistronic reporter gene cassettes—firefly luciferase and green fluorescent protein (GFP)—each driven by a separate cytomegalovirus promoter.

A different approach for Ad capsid modification used Ad5Luc1-CK (also termed AdCK-2)22 displaying genetic xeno-fiber knob replacement via the fiber knob of the canine Ad serotype 2. The cell entry mechanism of Ad5Luc1-CK involves both the putative cellular receptor(s) of the canine Ad serotype 222 and CAR. The control Ad vector for Ad5Luc1-CK was the isogenic, capsid-unmodified Ad5Luc1. Thus, we used two groups of viral vectors, whereby a distinct untargeted Ad5 vector served as an isogenic control to the corresponding retargeted Ad vectors (Fig. 1). All viruses were rescued on 293 cells and purified by using a standard CsCl gradient protocol. The number of viral particles was determined using a conversion factor of 1.1 x 1012 viral particles (vp) per absorbance unit at 260 nm. The titers of these viruses were as follows: Ad5, 3.1 x 1012 vp/ml; Ad5.RGD, 2.1 x 1012 vp/ml; Ad5.RGD.pK7, 7.8 x 1011 vp/ml; Ad.pK7, 1.0 x 1012 vp/ml; Ad5Luc1, 3.7 x 1012 vp/ml; Ad5Luc1-CK, 9.7 x 1011 vp/ml.


Figure 1
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 1. Diagram of the various capsid-modified Ad vectors used in the study.

 
Flow Cytometry
Glioma cells were rinsed with phosphate-buffered saline (PBS), harvested by versene incubation, and resuspended in PBS containing 1% bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA). For antibody incubation, 2 x 105 cells (for VU-28, 1 x 105 cells) were incubated with the monoclonal mouse antihuman CAR antibody RmcB (produced by Dr. J.T. Douglas from the hybridoma CRL-2379 cell line, ATCC) at 1:80 for 1 h at 4°C. An isotype-matched normal mouse immunoglobulin G1 (IgG1; 1:80) was used as a negative control. After incubation, cells were rinsed with PBS-bovine serum albumin and incubated with a 1:100 dilution of fluorescein isothiocyanate (FITC)-labeled goat antimouse IgG (Alexa 488, Molecular Probes, Eugene, OR, USA) for 1 h at 4°C. After another PBS rinse, labeled cells were fixed with 1% paraformaldehyde solution and analyzed by flow cytometry.

Relative mean fluorescence intensity was calculated as the ratio of the mean fluorescence intensity of the sample of interest to that of the corresponding negative control. The FITC-positive cell population for each cell line was determined by gating cells incubated with 1% buffer only (negative control).

Fluorescence Microscopy
U118 glioma cells were plated in six-well plates (105 cells/well). Two days later, cells were infected for 1 h with either the control Ad5 vector or the retargeted vector Ad5.pK7 or Ad5.RGD.pK7, at a multiplicity of infection (MOI) of 1,000 vp/cell. Fluorescence microscopy was performed two days later with an Olympus IX70 inverted epifluorescence microscope. All samples were photographed and processed under the same conditions.

Luciferase Assays
Cells were seeded in 24-well plates (50,000 cells/well, 25,000 cells/well for VU-28), with each well containing 0.5 ml of growth medium. After 24 h, cells were infected at an MOI of 40, 200, and 1,000 in 500 µl of infection medium. After 1 h, the infection medium was replaced with growth medium. Twenty-four hours later, the growth medium was aspirated, and the cells were washed and lysed with 100 µl of lysis buffer (Promega, Madison, WI, USA) and mechanically harvested. Ten microliters of each sample was then mixed with 50 µl of luciferase assay reagent (Promega) and measured for relative light units (RLU) with a Berthold Lumat LB9501 luminometer. Standardization between cell lines and MOIs was accomplished by setting the values obtained with the relevant Ad5 control vector as 100%.

Competitive Inhibition Assays
U118 cells were plated in 12-well plates at a density of 105 cells/well the day before infection. An MOI of 200 was used for each infection. Ad5.pK7 and Ad5.RGD.pK7 were preincubated with unfractionated heparin (heparin sodium salt from porcine in intestinal mucosa [Sigma], 0.1, 1, or 10 µg/ml), to block the heparan sulfate proteoglycan (HSPG) cellular entry mechanism, as described previously.21

To block the putative cellular receptor for canine Ad vector, U87 cells were preincubated with recombinant canine knob (0.5, 5, or 50 µg/ml) and then infected with Ad5Luc1-CK at an MOI of 200. Luciferase assays were used to evaluate the efficiency of infection.

In Vivo Glioma Xenograft Model and Luciferase Imaging
All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham and performed according to their guidelines. The glioma xenograft models were established in female athymic nude mice (National Cancer Institute-Frederick Animal Production Area, Frederick, MD, USA) by stereotactical injection of glioma cells into the mouse brain at the region of the caudate-putamen nucleus. Glioma cells that stably express firefly luciferase (Luc+ U87MG), and regular glioma cells, including high-CAR D54MG cells and low-CAR U87MG cells, were used in the experiments. For each mouse, 5 x 105 glioma cells in a 5-µl volume were injected into the brain. Two weeks later, 1010 vp of each Ad vector was injected into the brain tumors that were established with regular U87MG or D54MG cells. Noninvasive, live luciferase imaging was performed at different days after viral injection to follow the transgene (luciferase reporter) expression. The experimental design of the in vivo glioma xenograft model is depicted in Table 1. Luciferase imaging was performed using a custom-built noninvasive optical imaging system similar to the ChemiPro imaging system (Roper Scientific, Tucson, AZ, USA). For live imaging, the mice were anesthetized with isoflurane inhalation. Following intraperitoneal injection of D-luciferin (2.5 mg/100 µl per mouse), the bioluminescent signals in live mice were captured with a highly sensitive back-illuminated VersArray:1KB charge-coupled device (CCD) camera (Princeton Instruments, Trenton, NJ, USA) that was equipped with a Nikkor f/1.2 lens. Images were acquired with WinView/32 (Roper Scientific) and analyzed with ImageTool 3.0 (University of Texas Health Science Center, San Antonio, TX, USA) and Photoshop 7.0 (Adobe Systems Incorporated, San Jose, CA, USA).


View this table:
[in this window]
[in a new window]

  		Table 1. Experimental design for in vivo evaluation of infectivity-modified Ad5 vectors

 

Statistical Methods
Student's t-test was used to compare the efficiency of gene delivery by the various Ad vectors in glioma cells in vitro or in the in vivo glioma tumor models; p < 0.05 was considered significant. The Wilcoxon two-sample test was used to confirm statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Heterogeneous CAR Expression in Established Glioma Cells
CAR expression is a critical determinant of the efficiency of Ad entry into glioma cancer cells grown in monolayer.23-25 Therefore, to evaluate the utility of our retargeting strategies, we first measured CAR expression in a panel of established glioma cells. We found that CAR expression in glioma cells was heterogeneous and could be grouped into three major groups: low-, medium-, or high-CAR expression (Fig. 2). Of note, the medium level of CAR expression in the established glioma cell lines D65 and M59K was derived from two distinct populations: low- and high-CAR cells. Thus, within the established glioma cell lines, there was considerable variation in CAR expression.


Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 2. Coxsackie-adenoviral receptor (CAR) expression in established glioma cells. Expression of CAR (gray) was measured in the established human glioma cell lines U87, U118, D65, M59K, D54, and U251 with indirect flow cytometry. Cells were first incubated with the monoclonal anti-CAR antibody RmcB, followed by detection with fluorescein isothiocyanate-labeled secondary antibody. An immunoglobulin G-matched isotype antibody was used as a control (black).

 
Superiority of Retargeted Ad Vectors in Established Low-CAR Glioma Cell Lines
To evaluate the hypothesis that the novel genetic strategies for tropism modification of Ad vectors could circumvent the resistance of low-CAR glioma cells to Ad5 infection, we employed three retargeted Ad vectors. The first strategy was based on a chimeric Ad vector composed of the human Ad5 shaft flanked by the canine Ad knob (Ad5Luc1-CK). The second Ad retargeting strategy was based on incorporation of a polylysine motif into the C-terminus of the Ad5 fiber (Ad5.pK7). The third Ad vector was based on dual targeting, with both a polylysine motif and an RGD peptide inserted into the Ad5 C-terminus fiber gene and HI loop of the knob, respectively (Ad5.RGD.pK7). Ad5Luc1-CK, Ad5.pK7, and Ad5.RGD.pK7 were designed to target the respective alternative Ad receptors, the putative receptor for canine Ad, HSPG, or both HSPG and cell-surface integrins. Of note, an isogenic Ad.RGD vector was less efficient in enhancing gene delivery into glioma cells and was further used only as a specific control for Ad5.RGD.pK7 (data not shown).

When low-CAR glioma cell lines were tested for susceptibility to Ad infection, the three retargeting strategies proved superior to regular Ad5 vectors with no capsid modification (Fig. 3A-C). Specifically, in the low-CAR U118 and U87 cells, substantially higher levels of gene delivery could be achieved by all three retargeted vectors. In the medium-CAR glioma cell lines D65 and M59K, the infectivity enhancement of the retargeted Ad vectors was lower than in the low-CAR cells. In high-CAR cells, there was no consistent advantage for CAR-independent infection. Of note, the degree of infectivity enhancement of the retargeted Ad vectors varied in the D65 and M59K cell lines, in accordance with the two distinct cell populations in regard to CAR expression, as shown in Fig. 2. While the double-modified Ad5.RGD.pK7 showed more efficient CAR-independent cell entry in the low-CAR primary glioma cells and in U118 cells, Ad5.pK7 infection was more efficient in the low-CAR U87 cells, possibly indicating variable HSPG and integrin cell-surface expression in different glioma cell lines. In low-CAR cells, Ad5.RGD.pK7 was superior to Ad5.RGD vector (Fig. 3C), which was previously shown to enhance infectivity in glioma cells.23,25 Thus, the novel retargeted Ad vectors Ad5Luc1-CK, Ad5.pK7, and Ad5. RGD.pK7 augment Ad-based gene delivery into established low-CAR glioma cell lines.


Figure 3
View larger version (36K):
[in this window]
[in a new window]

  		Fig. 3. Retargeted adenoviral (Ad) vectors confer infectivity enhancement in established glioma cells expressing low or medium coxsackie-adenoviral receptor (CAR) levels. (A) Analysis of low-CAR glioma cells infected by Ad vectors expressing luciferase. Glioma monolayers were infected at a multiplicity of infection (MOI) of 200 with either the retargeted Ad5Luc1-CK and its corresponding control vector Ad5Luc1 or with the retargeted Ad5 vector Ad5.pK7 or Ad5.RGD.pK7 and their corresponding control vector Ad5. Note that the mean relative light units (RLU) are shown graphically as the percentage of the relevant capsid-unmodified control Ad5 vector. Results are representative of multiple experiments with MOIs of 40, 200, and 1,000. *p < 0.0005, **p < 0.005, ***p < 0.05 (retargeted Ad vectors compared with their corresponding Ad5 controls). (B) Analysis of low-CAR glioma cells infected by Ad vectors expressing green fluorescent protein (GFP). U118 cells were infected at an MOI of 1,000 with either the control Ad5 vector or with the retargeted vector Ad5.pK7 or Ad5.RGD.pK7. Fluorescence microscopy was performed two days later. Nuclei were stained with Hoechst (red). The fluorescent signal is green (weak signal) to yellow (very strong signal). (C) Ad5.RGD.pK7 confers higher levels of infectivity enhancement than the previously reported retargeted vector Ad5.RGD in established low-CAR glioma cells. U118 cells were infected at MOIs of 40, 200, or 1,000 with either the control Ad5 vector; the novel retargeted vector Ad5Luc1-CK, Ad5.pK7, or Ad5.RGD.pK7; or the previously reported retargeted vector Ad5.RGD. Mean RLU is shown graphically as the percentage of the relevant Ad5 control vector. *p < 0.005 for Ad5.RGD.pK7 versus Ad5.RGD.

 
Capsid-Modified Ad Vectors Infect Glioma Cells via Specific, Non-CAR-mediated, Cell Entry Pathways
To evaluate the relevant cell entry mechanism into glioma cells, competitive blocking assays were performed for Ad5.pK7, Ad5.RGD.pK7, and Ad5Luc1-CK. To this end, we employed heparin or the recombinant knob protein of the canine Ad serotype 2 to block either the HSPG pathway or the putative cellular receptor for canine 2 Ad, respectively. Our results show that specific blockers of these cellular entry pathways could inhibit the infection of glioma cells by the capsid-modified vectors (Fig. 4).


Figure 4
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 4. Competitive inhibition can partially block infection with retargeted adenoviral (Ad) vectors in low-CAR glioma cells. (A) U118 cells were infected with the control Ad5 vector, Ad5.pK7, or Ad5.RGD.pK7 at a multiplicity of infection (MOI) of 200, after preincubation of the viral vectors with the indicated concentrations of heparin. The infectivity was assessed by luciferase activity. Similar results were obtained with U87 cells. (B) U87 cells were infected with the control Ad5Luc1 vector or the retargeted Ad5Luc1-CK at an MOI of 200, following incubation of the cells with the canine Ad knob at the indicated concentrations. Similar results were obtained with U118 cells.

 
Specifically, heparin could block Ad5.pK7 efficiently but could block Ad5.RGD.pK7 only to a much lower extent (Fig. 4A), indicating the significance of integrins in the CAR-independent cell entry of Ad5.RGD.pK7. The canine knob protein could partially block Ad5Luc1-CK (Fig. 4B). We have previously shown that blockade of the Ad5/CAR cellular entry pathway with Ad5 knob does not inhibit infection with Ad5Luc1-CK, Ad5.pK7, or Ad5.RGD.pK7.21 These previous findings, together with our present findings of the specific inhibition of the respective cellular entry mechanisms, seem to indicate that Ad5Luc1-CK, Ad5.pK7, and Ad5.RGD.pK7 infect low-CAR glioma cells via CAR-independent cellular entry pathways. Of note, Ad5.RGD.pK7 seems to have a broader spectrum for cell entry in consideration of its relative resistance to heparin and its promiscuous infection profile, unless both the integrins and HSPG path-ways are blocked, as previously demonstrated.21

Retargeting Ad Vectors Enhances Infectivity in Primary, Low-Passage Glioma Cells
Glioma cells derived before the eighth passage from tumor samples of patients (referred to here as primary glioma cells) are often considered to better reflect the true nature of tumor cells. Therefore, we further evaluated the utility of the three retargeting strategies in four low-passage, primary glioma cell lines. First, we classified the primary glioma cells on the basis of their CAR expression (Fig. 5A). Next, we evaluated the utility of the various retargeted Ad vectors as gene delivery vehicles into the primary glioma cells. Our findings indicate that in low-CAR primary glioma cells, all three retargeting strategies could augment gene delivery. Of note, the efficiency of Ad5.RGD.pK7 was substantially higher than that of the other vectors (Fig. 5B). In parallel with the established glioma cell lines, infectivity enhancement correlated inversely with the level of CAR expression in primary glioma cell lines.


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 5. Retargeted adenoviral (Ad) vectors confer infectivity enhancement in primary, low-passage coxsackie-adenoviral receptor (CAR)-deficient glioma cells. (A) Expression of CAR (gray) was measured in the primary human glioma cells VU-78, VU-84, VU-28, and VU-15 with indirect flow cytometry. Cells were first incubated with the monoclonal anti-CAR antibody RmcB, followed by detection with fluorescein isothiocyanate-labeled secondary antibody. Immunoglobulin G-matched isotype antibody was used as a control (black). (B) Analysis of primary glioma cells infected by Ad vectors expressing luciferase. The primary human glioma cells VU-78, VU-84, and VU-15 were infected at multiplicities of infection (MOIs) of 40, 200, and 1,000, whereas VU-28 cells were infected at MOIs of 40 and 200 with either the retargeted Ad5Luc1-CK and its corresponding control vector Ad5Luc1, or with the retargeted Ad5 vector Ad5.pK7 or Ad5.RGD.pK7 and the corresponding control vector Ad5. Mean relative light units (RLUs) are shown graphically as the percentage of the relevant Ad5 control vector. *p < 0.0005, **p < 0.005.

 
However, although the level of infectivity enhancement obtained by Ad5.RGD.pK7 in established low-CAR glioma cell lines was approximately 30-fold (Fig. 3A), in primary low-CAR glioma cells it was up to 270-fold (Fig. 5B), thereby showing the relevance of CAR-independent infection in glioma cells in monolayer culture.

Infectivity Enhancement of Ad Vectors Is Compromised In Vivo in Glioma Xenografts
We next examined whether the CAR-independent Ad infection attained in vitro may predict in vivo gene delivery. To this end, we preestablished orthotopic human glioma xenografts in mice, followed by direct intracranial injection of the various Ad vectors. To directly evaluate gene delivery in real time, we studied live animals in which luciferase activity in the tumors was imaged using a CCD camera to quantitatively measure light emission. To validate transcranial measurement of gene expression, we first confirmed intracranial light signaling from glioma xenograft tumors consisting of glioma cells constitutively expressing luciferase (Fig. 6A). The increasing luciferase signals at day 15 compared with day 10 after tumor inoculation were compatible with active tumor growth, thereby confirming the establishment of the glioma xenograft model. Next, we preimplanted two different types of orthotopic human glioma xenografts (Luc-) in nude mice, followed by intratumoral injection of either the three retargeted Ad vectors or the unmodified Ad5 isogenic control vector (Table 1). The in vivo gene delivery efficiency was evaluated by noninvasive luciferase imaging since the vectors contained the firefly luciferase reporter. Of note, the two xenograft types were composed of human glioma cells with either low (U87) or high (D54) levels of CAR expression.


Figure 6
Figure 6
View larger version (78K):
[in this window]
[in a new window]

  		Fig. 6. Retargeted adenoviral (Ad) vectors do not confer infectivity enhancement in glioma xenografts in mice. (A) Noninvasive imaging to establish the mouse xenograft glioma model. Fifty thousand U87MG glioma cells constitutively expressing luciferase (Luc+-U87MG) were stereotactically injected into the brain of a nude mouse. Luciferase imaging was performed at days 10 and 15 after tumor inoculation. (B) In vivo evaluation of gene transfer efficacy of the retargeted Ad5 vectors in high-coxsackie-adenoviral receptor (CAR) glioma xenografts. The glioma models were preestablished as above with the high-CAR D54MG cells (Luc-) in nude mice. Two weeks later, 1010 vp of the Ad5 vectors was stereotactically injected into each tumor. Five mice were used in each group. Noninvasive imaging was used to follow transgene expression. Shown here are representative images taken at day 3 after viral injection. Quantitative imaging signals (with integrated density) showed that Ad5Luc1-CK was significantly less efficient in gene delivery into the D54 glioma xenografts than was the unmodified control vector (p < 0.05), whereas Ad5.pK7 and Ad5.RGD.pK7 showed no significant differences relative to the corresponding control Ad5 vector (p > 0.05). *p < 0.05. (C) In vivo evaluation of gene transfer efficacy of the retargeted Ad5 vectors in low-CAR glioma mouse models. The glioma models were preestablished with the low-CAR U87MG cells (Luc-) in nude mice, and experiments were performed as described for Fig. 6B. Representative images taken at day 6 after viral injection are shown. Statistical analysis of the quantified imaging signals indicated that there was no significant difference between the modified Ad5 vectors and their unmodified Ad5 control vectors. Quantified data are presented individually for each vector.

 

As also found under the in vitro conditions, in high-CAR glioma tumors we observed no infectivity enhancement: the capsid-modified Ad5.pK7 and Ad5.RGD.pK7 vectors showed no significant difference from the control Ad5 vector with regard to in vivo gene delivery efficiency (p = 0.657 and p = 0.372, respectively), whereas the Ad5Luc1-CK vector appeared significantly less efficient than the isogenic control vector (p = 0.020; Fig. 6B). Unlike in the two-dimensional glioma cell monolayers, however, in low-CAR glioma tumors in vivo, the unmodified Ad vectors were not inferior to the capsid-modified Ad (p = 0.239 for Ad5.pK7, p = 0.208 for Ad5.RGD.pK7, and p = 0.447 for Ad5Luc1-CK; Fig. 6C). Thus, in human glioma xenografts, in vitro CAR independence does not necessarily predict enhanced gene delivery in vivo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of Ad-based gene therapy against gliomas may be feasible for several reasons. First, local intracranial injection is feasible using surgical and stereotactic techniques. Second, prognosis of aggressive glioma is dismal. Third, clinical gene therapy studies have shown that Admediated brain toxicity is very low, despite the capacity of Ad5 vectors to infect a number of normal brain cell populations, such as neurons, astrocytes, and ependymal cells.4,26 It is therefore not surprising that Ad vectors have been previously found to be efficient for gene delivery in several glioma models.11-13 However, a major factor limiting the assessment of Ad-based gene therapy strategies for glioma is the inadequacy of currently available human tumor models. For example, data derived from monolayer cell cultures clearly do not reflect the tumor microenvironment.

Previously, genetic strategies to modify the Ad fiber have been reported to augment infectivity in glioma cells.23,24 In this study, we revisited the hypothesis that Ad-based gene delivery into gliomas could be enhanced by genetic retargeting of the vectors to alternative cellular Ad receptors. To address this issue, we performed in vitro and in vivo studies with three distinct approaches for Ad retargeting. In vitro, we first showed that CAR expression varies considerably among glioma cell lines. CAR expression was quantified as low, medium, or high. We next demonstrated that in established and primary low-CAR glioma cell lines, all three genetic approaches could enhance Ad infection, independently of CAR.

Of note, in the low-CAR U118 glioma cell line and in primary low-CAR glioma cells, double targeting of HSPG and integrins could augment Ad infectivity (Figs. 3A, 5B), whereas in the low-CAR U87 glioma cell line, double targeting of HSPG and integrins was less efficient than HSPG-based cell entry alone. Furthermore, as shown in Fig. 4A, competitive inhibition of HSPG-mediated cell entry with heparin in U118 cells could efficiently block Ad5.pK7 infection but not Ad5.RGD.pK7 infection. These results indicate that, in U118 glioma cells, integrins may play an important role as mediators of cell entry for Ad5.RGD.pK7 but not for Ad5. pK7. Because Ad5.RGD.pK7 can potentially utilize both HSPG and integrins for cell entry, variable expression of these cell-surface receptors in distinct cell lines may account for the different capacity to achieve CAR-independent infectivity enhancement. In this regard, it has been suggested that double modification of the Ad fiber may cause conformational ligand changes21 that may affect Ad cell entry differentially in different cell lines.

A major finding of our in vivo studies in glioma xenografts was the lack of infectivity enhancement of Ad5. RGD.pK7 (Fig. 6C), in contrast to results under monolayer conditions. This observation is compatible with results of previous in vivo studies in which, in an intraperitoneal ovarian cancer model, Ad5.RGD.pK7 was less efficient than Ad5.pK7 and did not enhance infectivity over Ad5. In contrast, in a subcutaneous ovarian tumor xenograft, Ad5.RGD.pK7, but not Ad5.pK7 or Ad5.RGD, enhanced infectivity.27 The variable nature of tumors was also demonstrated in vivo in a syngeneic melanoma tumor model in which another capsid-modified Ad vector incorporating both RGD and pK7 ligands could confer infectivity enhancement in vitro but not in vivo.28 Thus, in addition to the levels of CAR expression, other factors clearly play significant roles in the susceptibility of glioma tumors to Ad vectors.

These other factors may include intratumoral restriction of vector distribution within the tumor mass by (1) high tumor-cell density and intratumoral connective tissue29; (2) limited availability of vectors within the "infection zone," corresponding to up to five layers of tumor cells around the needle track30; (3) size of virions29; (4) host immune response, augmented by display of charged ligands31; (5) tumor inactivation of viral DNA after inoculation32; (6) charged ligands promoting binding to extracellular matrix proteins, for example, integrins and HSPGs33,34; (7) a gradient of viral infection that results in a high MOI primarily around the needle track; and (8) attenuation of vectors for safety reasons. Altogether, inefficient intratumoral diffusion probably accounts for the limited distribution of Ad-mediated gene delivery in human glioma tumors, as observed in previous human clinical trials.35 Some of these obstacles may be addressed by replication-competent oncolytic viruses or via combined therapeutic modalities.36,37

In conclusion, although novel strategies for CAR-independent Ad-mediated cell entry could confer infectivity enhancement of primary and established low-CAR glioma cell lines in vitro, these approaches were less efficient in vivo. Thus, gene delivery into glioma cell monolayers, inclusive of primary glioma cells, cannot predict gene delivery in complex, heterogenic three-dimensional tumors. Further research is required to characterize and overcome the factors impeding gene delivery into glioma tumors.


   Acknowledgments
 
W.J.V.H. was supported by the Dr. Saal van Zwanenberg Foundation, the Dutch Cancer Society, the Brain Foundation of The Netherlands, and the Utrecht University Talma Eykman Award for medical students. M.L.L. is supported by Dutch Cancer Society grant VU2002-2594. Y.S.H. is supported by the Israeli Science Foundation and the B. de Rothschild Foundation. H.W. and G.Y.G. are supported by NIH Brain Tumor SPORE grant P50 CA097247. D.T.C. is supported by NIH grants P01CA104177 and R01CA083821.

Received for publication May 29, 2006. Accepted for publication October 12, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  • Elliott WL, Roberts BJ, Howard CT, Leopold WR. Chemotherapy with [Sp-4-3-(R)]-[1,1-cyclobutanedicarboxylato(2-)](2-methyl-1, 4-butanediamine-N,N')platinum (Ci-973, Nk121) in combination with standard agents against murine tumors in-vivo. Cancer Res. 1994;54: 4412-4418.[Abstract/Free Full Text]

  • Schmitt CA, Fridman JS, Yang M, et al. A senescence program controlled by p53 and p16(INK4a) contributes to the outcome of cancer therapy. Cell. 2002;109: 335-346.[CrossRef][ISI][Medline]

  • Eck SL, Alavi JB, Alavi A, et al. Clinical protocol—treatment of advanced CNS malignancies with the recombinant adenovirus H5.010RSVTK: a phase I trial. Hum Gene Ther. 1996;7: 1465-1482.[ISI][Medline]

  • Puumalainen AM, Vapalahti M, Agrawal RS, et al. Beta-galactosidase gene transfer to human malignant glioma in vivo using replication-deficient retroviruses and adenoviruses. Hum Gene Ther. 1998;9: 1769-1774.[ISI][Medline]

  • Trask TW, Trask RP, Aguilar-Cordova E, et al. Phase I study of adenoviral delivery of the HSV-tk gene and ganciclovir administration in patients with recurrent malignant brain tumors. Mol Ther. 2000;1: 195-203.[CrossRef][ISI][Medline]

  • Sandmair AM, Vapalahti M, Yla-Herttuala S. Adenovirus-mediated herpes simplex thymidine kinase gene therapy for brain tumors. Cancer Gene Ther. 2000;465: 163-170.[ISI]

  • Miller CR, Buchsbaum DJ, Reynolds PN, et al. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer. Cancer Res. 1998;58: 5738-5748.[Abstract/Free Full Text]

  • Asaoka K, Tada M, Sawamura Y, Ikeda J, Abe H. Dependence of efficient adenoviral gene delivery in malignant glioma cells on the expression levels of the coxsackievirus and adenovirus receptor. J Neurosurg. 2000;92: 1002-1008.[ISI][Medline]

  • Staba MJ, Wickham TJ, Kovesdi I, Hallahan DE. Modifications of the fiber in adenovirus vectors increase tropism for malignant glioma models. Cancer Gene Ther. 2000;7: 13-19.[CrossRef][ISI][Medline]

  • Douglas JT, Kim M, Sumerel LA, Carey DE, Curiel DT. Efficient oncolysis by a replicating adenovirus (Ad) in vivo is critically dependent on tumor expression of primary Ad receptors. Cancer Res. 2001;61: 813-817.[Abstract/Free Full Text]

  • 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.[CrossRef][ISI][Medline]

  • Tsugawa T, Kuwashima N, Sato H, et al. Sequential delivery of interferon-alpha gene and DCs to intracranial gliomas promotes an effective antitumor response. Gene Ther. 2004;11: 1551-1558.[CrossRef][ISI][Medline]

  • Immonen A, Vapalahti M, Tyynela K, et al. AdvHSV-tk gene therapy with intravenous ganciclovir improves survival in human malignant glioma: a randomised, controlled study. Mol Ther. 2004;10: 967-972.[CrossRef][ISI][Medline]

  • Parr MJ, Manome Y, Tanaka T, et al. Tumor-selective transgene expression in vivo mediated by an E2F-responsive adenoviral vector. Nat Med. 1997;3: 1145-1149.[CrossRef][ISI][Medline]

  • Franklin RJM, Quick MM, Haase G. Adenoviral vectors for in vivo gene delivery to oligodendrocytes: transgene expression and cytopathic consequences. Gene Ther. 1999;6: 1360-1367.[CrossRef][ISI][Medline]

  • Krasnykh VN, Mikheeva GV, Douglas JT, Curiel DT. Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism. J Virol. 1996;70: 6839-6846.[Abstract/Free Full Text]

  • Krasnykh V, Dmitriev I, Mikheeva G, Miller CR, Belousova N, Curiel DT. Characterization of an adenovirus vector containing heterologous peptide epitope in the HI loop of the fiber knob. J Virol. 1998;72: 1844-1852.[Abstract/Free Full Text]

  • Dmitriev I, Krasnykh V, Miller CR, et al. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol. 1998;72: 9706-9713.[Abstract/Free Full Text]

  • Dirven CMF, Grill J, Lamfers MLM, et al. Gene therapy for meningioma: improved gene delivery with targeted adenoviruses. J Neurosurg. 2002;97: 441-449.[ISI][Medline]

  • Lamfers MLM, Grill J, Dirven CMF, et al. Potential of the conditionally replicative adenovirus Ad5-delta 24RGD in the treatment of malignant gliomas and its enhanced effect with radiotherapy. Cancer Res. 2002;62: 5736-5742.[Abstract/Free Full Text]

  • Wu HJ, Seki T, Dmitriev I, et al. Double modification of adenovirus fiber with RGD and polylysine motifs improves coxsackievirus-adenovirus receptor-independent gene transfer efficiency. Hum Gene Ther. 2002;13: 1647-1653.[CrossRef][ISI][Medline]

  • Glasgow JN, Kremer EJ, Hemminki A, Siegal GP, Douglas JT, Curiel DT. An adenovirus vector with a chimeric fiber derived from canine adenovirus type 2 displays novel tropism. Virology. 2004;324: 103-116.[CrossRef][ISI][Medline]

  • Yoshida Y, Sadata A, Zhang WP, Saito K, Shinoura N, Hamada H. Generation of fiber-mutant recombinant adenoviruses for gene therapy of malignant glioma. Hum Gene Ther. 1998;9: 2503-2515.[CrossRef][ISI][Medline]

  • Shinoura N, Yoshida Y, Tsunoda R, et al. Highly augmented cytopathic effect of a fiber-mutant E1B-defective adenovirus for gene therapy of gliomas. Cancer Res. 1999;59: 3411-3416.[Abstract/Free Full Text]

  • Grill J, Van Beusechem VW, Van de Valk P, et al. Combined targeting of adenoviruses to integrins and epidermal growth factor receptors increases gene transfer into primary glioma cells and spheroids. Clin Cancer Res. 2001;7: 641-650.[Abstract/Free Full Text]

  • Alemany R, Gomez-Manzano C, et al. Gene therapy for gliomas: molecular targets, adenoviral vectors, and oncolytic adenoviruses. Exp Cell Res. 1999;252: 1-12.[CrossRef][ISI][Medline]

  • Wu H, Han T, Lam JT, et al. Preclinical evaluation of a class of infectivity-enhanced adenoviral vectors in ovarian cancer gene therapy. Gene Ther. 2004;11: 874-878.[CrossRef][ISI][Medline]

  • Koizumi N, Mizuguchi H, Utoguchi N, Watanabe Y, Hayakawa T. Generation of fiber-modified adenovirus vectors containing heterologous peptides in both the HI loop and C terminus of the fiber knob. J Gene Med. 2003;5: 267-276.[CrossRef][ISI][Medline]

  • Sauthoff H, Hu J, Maca C, et al. Intratumoral spread of wild-type adenovirus is limited after local injection of human xenograft tumors: virus persists and spreads systemically at late time points. Hum Gene Ther. 2003;14: 425-433.[CrossRef][ISI][Medline]

  • Jia W, Zhou Q. Viral vectors for cancer gene therapy: viral dissemination and tumor targeting. Curr Gene Ther. 2005;5: 133-142.[ISI][Medline]

  • Liu Q, Muruve DA. Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther. 2003;10: 935-940.[CrossRef][ISI][Medline]

  • Nemunaitis J, Khuri F, Ganly I, et al. Phase II trial of intratumoral administration of ONYX-015, a replication-selective adenovirus, in patients with refractory head and neck cancer. J Clin Oncol. 2001;19: 289-298.[Abstract/Free Full Text]

  • Haviv YS, Curiel DT. Conditional gene targeting for cancer gene therapy. Adv Drug Deliv Rev. 2001;53: 135-154.[CrossRef][ISI][Medline]

  • Thomas CE, Edwards P, Wickham TJ, Castro MG, Lowenstein PR. Adenovirus binding to the coxsackievirus and adenovirus receptor or integrins is not required to elicit brain inflammation but is necessary to transduce specific neural cell types. J Virol. 2002;76: 3452-3460.[Abstract/Free Full Text]

  • Pulkkanen KJ, Yla-Herttuala S. Gene therapy for malignant glioma: current clinical status. Mol Ther. 2005;12: 585-598.[CrossRef][ISI][Medline]

  • Haviv YS, Blackwell JL, Kanerva A, et al. Adenoviral gene therapy for renal cancer requires retargeting to alternative cellular receptors. Cancer Res. 2002;62: 4273-4281.[Abstract/Free Full Text]

  • Lam JT, Kanerva A, Bauerschmitz GJ, et al. Inter-patient variation in efficacy of five oncolytic adenovirus candidates for ovarian cancer therapy. J Gene Med. 2004;6: 1333-1342.[CrossRef][ISI][Medline]





    This Article
    Right arrow Abstract Freely available
    Right arrow Full Text (PDF)
    Right arrow All Versions of this Article:
    9/3/280    most recent
    15228517-2007-017v1
    Right arrow Alert me when this article is cited
    Right arrow Alert me if a correction is posted
    Right arrow Citation Map
    Services
    Right arrow Similar articles in this journal
    Right arrow Similar articles in ISI Web of Science
    Right arrow Similar articles in PubMed
    Right arrow Alert me to new issues of the journal
    Right arrow Download to citation manager
    Right arrow reprints & permissions
    Citing Articles
    Right arrow Citing Articles via ISI Web of Science (2)
    Right arrow Citing Articles via Google Scholar
    Google Scholar
    Right arrow Articles by Van Houdt, W. J.
    Right arrow Articles by Haviv, Y. S.
    Right arrow Search for Related Content
    PubMed
    Right arrow PubMed Citation

      Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents