Affiliation of authors: J. Fueyo, C. Gomez-Manzano, G. N. Fuller, C. A. Conrad, T.-J. Liu, H. Jiang, M. G. Lemoine, R. Sawaya, W. K. A. Yung, F. F. Lang (Brain Tumor Center), A. Khan (Department of Pediatrics), University of Texas M. D. Anderson Cancer Center, Houston, TX; R. Alemany, Institut Català dOncologia, Barcelona, Spain; K. Suzuki, D. T. Curiel, Gene Therapy Center, University of Alabama at Birmingham.
Correspondence to: Juan Fueyo, M.D., Department of Neuro-Oncology, Box 316, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030 (e-mail: jfueyo{at}mdanderson.org).
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ABSTRACT |
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INTRODUCTION |
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Although a comprehensive compilation of the genetic alterations involved in malignant gliomas is still in progress, the retinoblastoma (Rb) protein or its regulatory proteins (i.e., p16INK4a, cyclin-dependent kinases 4 and 6, and cyclin D1) are deregulated in most malignant gliomas (2). Because the Rb pathway is critical for maintaining a functional G1 checkpoint, inactivation of this pathway results in unregulated cell-cycle progression, abnormal cellular growth and, thus, progression of the malignant phenotype in the majority of cancers (3), including malignant gliomas (2).
Following previous studies on the transforming regions of the early 1 A adenoviral (E1A) protein (4), which identified the regions of the E1A protein that bind to Rb, we developed a tumor-selective adenovirus, Delta-24, that encompasses an eight-amino-acid residue (24 base pairs) deletion in the viral E1A region that is responsible for binding the Rb protein (5). This deletion, which we have shown (5) renders viral replication dependent on inactivation of Rb, generates a tumor-selective, replication-competent virus that induces an anticancer effect in glioma(s). Specifically, Delta-24 did not efficiently replicate in quiescent human lung fibroblasts, but infection of gliomas with Delta-24 inhibited their growth both in vitro and in vivo (5). The anticancer effect of Delta-24 is, however, not equal in every cell line because of the inability of Delta-24 to efficiently infect a broad spectrum of human glioma cells.
The ability of adenoviruses to infect tumor cells depends on anchorage to the coxsackie-adenovirus receptor (CAR) (6). Cancer cells express low levels of CAR and, as a result, constitute a difficult target for virus-based therapy (7). Therefore, Delta-24 was modified so that it is theoretically capable of infecting tumor cells via CAR-independent mechanisms. We hypothesized that an ability to circumvent CAR would improve the antitumor effect of Delta-24 and would reduce sequestration of the adenovirus by nontargeted normal cells expressing CAR. Because internalization of the adenovirus into host cells is mediated by a secondary interaction between RGD motifs on penton base protein loops and integrins v
5 and
v
3 (8), Delta-24 was modified to target integrins on the surface of cancer cells as its primary receptor. For this approach, we took advantage of the previously isolated peptide ACDCRGDCFCG (RGD-4C), which binds strongly to the
v
3 and
v
5 integrins (9,10). We inserted the RGD-4C sequence into the HI loop of the fiber knob protein of Delta-24 to generate Delta-24-RGD (11). In this study, we tested the hypothesis that Delta-24-RGD is more infective than Delta-24 in gliomas, but that it is equally selective for cancer cells.
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MATERIALS AND METHODS |
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The glioma cell lines D54 MG, U-251 MG, U-87 MG, T98G, U-138 MG, and Saos-2 were obtained from American Type Culture Collection (Manassas, VA). The SNB 19 glioma cell line was a gift from Dr. Jasti Rao (University of Illinois College of Medicine, Peoria, IL). Cell lines were maintained in Dulbeccos modified Eagle/F12 medium (DMEM/F12) (1 : 1, vol/vol) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37 °C. Normal human astrocytes (NHAs) were purchased from Clonetics/BioWhittaker (Walkersville, MD). NHA cultures were maintained in astrocyte growth medium from an AGM-Astroctye Medium BulletKit obtained from Clonetics/BioWhittaker. For serum starvation conditions, we grew NHAs at a low density (2 x 104/per well in a six-well plate) in the kits medium with 0.5% fetal bovine serum and no growth supplements. These culture conditions inhibited cell growth without evidence of cell death.
Adenovirus Construction and Infection
Construction of Delta-24 and Delta-24-RGD have been previously described (5,10). These adenoviral constructs have a 24 base-pair deletion of the E1A region that encompasses the area responsible for binding Rb protein (nucleotides 923946), corresponding to the amino acids L122TCHEAGF129. Wild-type adenovirus Ad300 (12), Delta-24-RGD inactivated by UV light, and mock-infected cells (i.e., with DMEM/F12 medium) were used as controls.
For the infectivity analyses, human glioma cells (5 x 105) were infected with the replication-deficient adenoviruses AdGFP and AdGFP-RGD (13), which express green-fluorescence protein (GFP). Forty-eight hours after infection, the cells were treated with 0.05% trypsin for 5 minutes and washed twice with phosphate-buffered saline (PBS). The cells were then counted for GFP-positive cells by flow cytometry as described below (14).
Measurement of CAR and v
Integrin Expression by Flow Cytometric Analysis
Cytometric analysis was used to measure the cell surface expression of CAR and v
integrins. Briefly, human glioma and NHA cultures (5 x 105 cells for both) were incubated with the anti-CAR monoclonal antibody RmcB [diluted 1 : 2000; provided by Dr. J. M. Bergelson (6), The Childrens Hospital of Philadelphia, Philadelphia, PA], the anti-human integrin mouse
v
5 antibody (clone P1F6, diluted 1 : 400; Invitrogen, Carlsbad, CA), or the anti-human integrin mouse
v
3 fluorescein-conjugated monoclonal antibody (diluted 1 : 200; Chemicon International, Temecula, CA) at 4 °C for 1 hour. For the visualization of the anti-CAR and anti-
v
5 antibodies, a second incubation with goat anti-mouse fluorescein isothiocyanate-conjugated secondary antibody (diluted 1 : 100; Santa Cruz Biotechnology, Santa Cruz, CA) was conducted at room temperature for 1 hour. The cells were then washed twice with PBS and stained with propidium iodide at 50 µg/mL and with RNase at 20 µg/mL for 15 minutes at room temperature. Cell samples were analyzed for fluorescence with an EPICS XL-MCL flow cytometer (Beckman Coulter, Miami, FL) using a 488-nm argon laser for excitation. Fluorescence was detected with a 520-nm band-pass filter, and all cytometric data were analyzed with System II Software (Beckman Coulter). CAR expression was defined as high when it was expressed in 50% or more of the examined cells and as low when it was expressed in less than 50% of the examined cells. This cutpoint was determined a priori based on the distribution of CAR expression shown in Fig. 1
.
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Glioma cell lines were incubated in DMEM/F12 media in 96-well plates at 37 °C for 24 hours. Recombinant adenovirus fiber knob protein at 100 µL/mL (a gift from Dr. J. Douglas, University of Alabama, Birmingham) or RGD peptide (Gly-Arg-Gly-Asp-Ser-Pro) at 1 mg/mL (American Peptide Company, Sunnyvale, CA) was then added to the culture. RGA peptide (Gly-Arg-Gly-Glu-Ser-Pro) (American Peptide Company) was used as a control for the RGD-based competitive assay. After a 10-minute incubation at 4 °C, cultures were infected with either AdGFP or AdGFP-RGD at a multiplicity of infection (MOI) of 50 for 5 minutes at 37 °C. Forty-eight hours later, infection efficiency was determined by counting the number of fluorescent cells among 500 cells by phase-contrast microscopy. Each experiment was performed in triplicate.
Cell Viability Assay
Human glioma cells were seeded (at 105 cells per well) in DMEM/F12 medium in six-well plates and allowed to grow for 20 hours at 37 °C. Cells were then infected with Delta-24, Delta-24-RGD, Ad300, or UV-inactivated Delta-24-RGD at MOIs of 0, 5, and 10 at 37 °C for 30 minutes. Experiments were concluded when an MOI of 10 for one of the adenoviral constructs produced a cytopathic effect of more than 50%. The cell monolayers were then washed twice with PBS and were fixed and stained with 0.1% crystal violet in 20% ethanol. Excess dye was removed by rinsing several times with water. Trypan blue experiments were performed to quantitate cell viability, as previously described (14). Briefly, cultures were infected with the adenoviral constructs at MOIs of 0, 0.1, 1, and 10, and then the viable cells were counted using a hemocytometer.
Viral Replication Experiments
Human glioma cells were seeded in DMEM/F12 medium (at 5 x 104 cells/well) in six-well plates and allowed to grow for 20 hours at 37 °C. Cells were then infected with Delta-24, Delta-24-RGD, Ad300, or UV-inactivated Delta-24-RGD at an MOI of 10. Forty-eight hours after infection, the cells were scraped into DMEM/F12 medium and were lysed with three cycles of freezing and thawing. The tissue culture infection dose50 method was used to determine the final viral titer according to the Kärber formula (15). Briefly, cell lysates were clarified by centrifugation at 1000g for 15 minutes at 4 °C, and the supernatants were serially diluted in DMEM/F12 medium for the infection of 293 cells in 96-well plates. The cells were analyzed for an adenoviral cytopathic effect 10 days after infection. Final viral titers were determined as plaque-forming units (pfu)/mL, according to the validated method developed by Quantum Biotechnology (Carlsbad, CA).
Infection with Exogenous Wild-Type Rb or p21
The Rb and p21 adenoviruses used in this study and their infectivity have been previously described (16,17). Briefly, after D54 MG and U-251 MG glioma cells (at 2 x 104 cells/well) were seeded in DMEM/F12 medium in six-well plates, the cultures were infected with replication-deficient adenoviral vectors expressing either Rb or p21 or with the control adenoviral vector Ad5CMV-pA (with an empty expression cassette) (16,17) at an MOI of 80 at 37 °C for 30 minutes. Seventy-two hours later, the cultures were treated with either Delta-24-RGD or UV-inactivated Delta-24-RGD at an MOI of 10 at 37 °C for 30 minutes. Cell viability was monitored daily and was quantitated using the trypan blue exclusion test.
Animal Studies
U-87 MG human glioma cells (5 x 105) were engrafted into the caudate nucleus of athymic mice using a guide-screw system, as previously described (18). We performed three independent experiments using 610 animals per group in each experiment. On days 3, 6, and 8 after implantation of tumor cells, animals were treated with 5-µL intratumoral injections of Delta-24-RGD, Delta-24, UV-inactivated Delta-24-RGD, or PBS (all 1.5 x 108 pfu/mL). Animals showing general or local symptoms of toxicity were killed. Surviving animals were killed 140 days after tumor implantation. Brains were then removed, fixed in 4% formaldehyde for 24 hours at room temperature, and embedded in paraffin. Hematoxylin-and-eosin-stained sections were evaluated (by Dr. G. Fuller, Department of Neuro-Pathology, M. D. Anderson Cancer Center) for evidence of tumor, necrosis, and viral nuclear inclusions. The largest (a) and the smallest (b) diameters of the tumors were measured, and these measurements were used in the calculation of tumor volume using the formula a x b2 x 0.4 (19). All animal studies were performed in the veterinary facilities of the M. D. Anderson Cancer Center in accordance with institutional, state, and federal laws and ethics guidelines for experimental animal care.
Immunohistochemical Analysis
To detect adenoviral E1A and hexon proteins in the tumor xenografts, paraffin-embedded sections of the mouse tumors were deparaffinized and rehydrated with xylene and ethanol following conventional procedures (20). Endogenous peroxidase activity was quenched by incubating the sections in 0.3% hydrogen peroxide in methanol for 30 minutes. The sections were then treated with either goat anti-hexon antibody (diluted 1 : 100; Chemicon) or goat anti-E1A antibody (diluted 1 : 100; Santa Cruz Biotechnology) at 4 °C overnight. For immunohistochemical staining, Vectastain ABC kits from Vector Laboratories (Burlingame, CA) were used according to the manufacturers instructions.
Statistical Analyses
For the in vitro experiments, statistical analyses were performed using a two-tailed Students t test. Data are expressed as mean ± 95% confidence intervals (CIs). The in vivo cytopathic effect of Delta-24-RGD and Delta-24 on human glioma xenografts was assessed by plotting survival curves according to the KaplanMeier method (21). Survival in different treatment groups was compared using the log-rank test.
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RESULTS |
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To determine the expression of CAR and RGD-related integrins in human glioma cells, we directly measured the cell surface expression of CAR, v
5, and
v
3 by flow cytometric analyses in six human glioma cell lines (Fig. 1, A
). CAR was expressed in more than or equal to 50% (high CAR expression) of the examined cells in U-251 MG, SNB 19, and D54 MG cultures and in less than 50% (low CAR expression) of the examined cells in U-87 MG, T98G, and U-138 MG cultures. The examination of the
v
5 and
v
3 integrin expression in the same set of cell lines showed that the majority of the assayed cells in every cell line expressed one of the integrins. Similar studies showed that NHAs express both CAR (65%, 95% CI = 51.3% to 79.7%) and integrins (
v
3: 30.4%, 95% CI = 15.7% to 45% and
v
5: 77.4%, 95% CI = 55.7% to 99.1%) (Fig. 1, A
).
The pattern of CAR and integrin expression suggests that a retargeted adenovirus that is able to use both CAR and RGD-related integrins for cell anchorage might be able to infect a broader spectrum of glioma cell lines (especially cell lines with lower CAR expression) than would an unmodified adenovirus. To test this hypothesis, we compared the transduction efficiency of two adenoviral vectors: AdGFP-RGD (which contains a GFP expression minicassette and the RGD-4C motif inserted in the fiber knob) and AdGFP (which also encodes the GFP gene but has a wild-type fiber knob). We used U-87 MG and U-251 MG cells to compare infectivity of the two adenoviruses in low- and high-CAR-expressing cultures, respectively. At an MOI of 25, AdGFP-RGD-infected cultures of U-251 MG cells showed 1.48-fold (95% CI = 1.15- to 1.65-fold) more GFP-positive cells than did AdGFP-infected cultures of U-251 MG cells. As expected, the difference in the infectivity of the two adenoviral vectors was more pronounced in the low-CAR-expressing cell line than in the high-CAR-expressing cell line, with 5.7-fold (95% CI = 5.14- to 6.26-fold) more GFP-positive cells in the U-87 MG cultures infected with AdGFP-RGD than in the U-87 MG cultures infected with AdGFP. Thus, the insertion of the RGD-4C motif into the fiber knob of the AdGFP vector increased the ability of the adenovirus to infect human glioma cells. In addition, the difference in the infectivity of the two adenoviral vectors was statistically significantly higher in the low-CAR-expressing cell line than in the high-CAR-expressing cell line (P<.001; two-tailed Students t test). Interestingly, the advantage of the RGD-4C insertion was also evident in the SNB 19 cell line, which has the highest percentage of cells expressing CAR (89.8%, 95% CI = 78.4% to 101.2%) and in which infection with AdGFP-RGD resulted in 1.5-fold (95% CI = 0.94- to 2.06-fold) more GFP-expressing cells than infection with AdGFP.
To determine the mechanism underlying the higher infectivity of AdGFP-RGD adenovirus compared with that of the AdGFP adenovirus, we blocked adenoviral infectivity via CAR by incubating U-251 MG human glioma cells with recombinant fiber knob protein before infection with AdGFP or AdGFP-RGD. Incubation of U-251 MG cells with fiber knob protein reduced AdGFP infectivity by more than 50% (P<.001; two-tailed Students t test), but did not statistically significantly reduce AdGFP-RGD infectivity (P = .26; two-tailed Students t test) (Fig. 1, B). These results indicate that the RGD-4C motif provides an alternative cell entry pathway when CAR is not available.
To ascertain whether this alternative cell entry pathway involves RGD-related integrins, we next incubated U-251 MG cells with RGD peptide before infecting them with AdGFP or AdGFP-RGD. Pretreatment with RGD peptide statistically significantly decreased the infectivity efficiency of both AdGFP (P = .009; two-tailed Students t test) and AdGFP-RGD (P<.001; two-tailed Students t test) by 60% (Fig. 1, B). Thus, the RGD-4C motif allows for an alternative binding site via RGD-related integrins and for CAR-independent glioma infectivity.
Cytopathic Effect of Delta-24-RGD in Human Glioma Cells
We next asked whether the increased infectivity of AdGFP-RGD, which suggests an increase in infectivity of Delta-24-RGD, translated into an increased cytopathic effect for Delta-24-RGD. To compare the cytopathic effects of Delta-24 and Delta-24-RGD, we performed dose-dependence assays in which both low- (U-87 MG, U-138 MG, T98G) and high-CAR-expressing cell lines (e.g., U-251 MG, SNB 19, D54 MG) were treated with Delta-24-RGD, Delta-24, or the wild-type adenovirus Ad300. In all six cell lines, Delta-24-RGD required a lower dose to induce a complete cytopathic effect than did Delta-24 (Fig. 2, A). Interestingly, two patterns of Delta-24-RGD- and Ad300-induced cytopathic effects were observed. In U-87 MG, T98G, and U-138 MG cell cultures, all of which had low CAR expression, Delta-24-RGD had a higher cytopathic effect than did Delta-24 or Ad300. However, in U-251 MG, SNB 19, and D54 MG cell cultures, all of which had high CAR expression, Ad300 was similar to or slightly more cytopathic than Delta-24-RGD. Thus, Delta-24-RGD was more potent than Ad300 in cells with low CAR expression. To more precisely quantitate the cytopathic effect of Delta-24 and Delta-24-RGD, we performed a trypan blue exclusion test in U-87 MG and U-251 MG cell cultures. The MOI to induce 50% cell death was between 0.1 and 1 for Delta-24-RGD and between 1 and 10 for Delta-24 in both cell lines (Fig. 2, B
).
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Because the Rb pathway is disrupted by the homozygous deletion of p16 in the U-87 MG and U-251 MG cell lines, we were also interested in determining whether Delta-24-RGD replicates efficiently in cells lacking expression of Rb. Therefore, we performed replication experiments in Saos-2, an Rb-deficient glioma cell line (5). Two independent experiments showed that Delta-24-RGD at an MOI of 10 achieved titers of 6 x 1011 and 5 x 1010 pfu/mL 2 days after infection (data not shown). Taken together, these experiments confirm the ability of Delta-24-RGD to replicate in cells with an abnormal Rb pathway due to abnormal expression of an Rb regulator (such as p16) or Rb protein.
Rb Pathway and the Cytopathic Effect of Delta-24-RGD
We previously showed that restoration of Rb function in D54 MG cells substantially decreased the cytopathic effects of the Delta-24 adenovirus (5). To determine whether Delta-24-RGD-mediated cell death is also dependent on the cell-cycle regulatory function of the Rb protein, we treated D54 MG cells with replication-deficient adenoviral vectors expressing either Rb or p21 (or an empty vector) and infected the cells with Delta-24-RGD (or UV-inactivated Delta-24-RGD). D54 cell cultures pretreated with Rb or p21 had less cell death (24.1%, 95% CI = 22.8% to 25.3% and 24.5%, 95% CI = 22.4% to 26%, respectively) than did cultures infected with the empty vector (50.2%, 95% CI = 50.1% to 50.3%) (Fig. 3). To confirm these findings, we performed a similar assay with U-251 MG cells. Similarly, U-251 MG cell cultures pretreated with Rb or p21 had less cell death (19.7%, 95% CI = 18.6% to 20.7% and 11.7%, 95% CI = 7.4% to 16%, respectively) than did cultures infected with the empty vector. Interestingly, the rescue of D54 MG cells from the cytopathic effect of Delta-24, which was reported previously (5), and the rescue of Delta-24-RGD by p21 were similar in terms of their inhibition of adenoviral-mediated cell death.
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Replication Profile of Delta-24-RGD in NHAs
Reduction of the cytopathic effect of Delta-24-RGD by the restoration of the Rb pathway suggested that Delta-24-RGD might be unable to replicate efficiently in nondividing normal human cells. To confirm this hypothesis, we designed a set of experiments involving the infection of NHAs with Delta-24-RGD. First, we assessed the ability of GFP-expressing adenoviruses to infect NHAs. At an MOI of 100, both AdGFP-RGD and AdGFP infected 80%100% of the cells. These data were consistent with the observation that a high percentage of NHAs express CAR (Fig. 1, A). Second, we assessed the ability of Delta-24-RGD, Delta-24, Ad300, and UV-inactivated Delta-24-RGD to replicate in serum-starved NHAs. After 3 days at an MOI of 1, wild-type adenovirus Ad300 was able to replicate; however, Delta-24-RGD and Delta-24 were unable to acquire a consistent replication phenotype under these conditions (Table 1
). As expected, no virus was detected in the lysates from cultures infected with UV-inactivated Delta-24-RGD. These experiments were consistent with our previous work showing that Delta-24 does not replicate efficiently in nondividing human lung fibroblasts (5).
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Effect of Delta-24-RGD on Gliomas In Vivo
To compare the therapeutic efficacies of Delta-24 and Delta-24-RGD in vivo, U-87 MG xenografts were grown in the brains of athymic mice. In three independent experiments, the mean survival for the control mice (i.e., mice receiving PBS and UV-inactivated Delta-24-RGD) was 19 days (95% CI = 18 to 20 days) and 18 days (95% CI = 18 to 19 days), respectively. In contrast, the mean survival for the Delta-24-treated mice was 50 days (95% CI = 30 to 70 days), which was statistically significantly longer than the mean survival in control mice (P<.001; log-rank test). Delta-24-RGD-treated mice survived statistically significantly longer (mean = 131 days, 95% CI = 100 to 162 days) than both the control mice and Delta-24-treated mice (both P<.001; log-rank test) (Fig. 4). There were no long-term survivors among the control mice; however, 15 of the 25 Delta-24-RGD-treated mice survived more than 4 months after tumor implantation: four of nine in experiment 1, four of six in experiment 2, and seven of 10 in experiment 3. By contrast, there were only four long-term survivors of a total of 26 Delta-24-treated mice: one of 10 in experiment 1, three of six in experiment 2, and zero of 10 in experiment 3. Thus, a higher percentage of mice were long-term survivors in the Delta-24-RGD-treated group than in the Delta-24-treated group (60% versus 15%, respectively; difference = 45%, 95% CI = 21% to 68%).
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Microscopic examination of the brains of control mice (with U-87 xenografts) revealed noninfiltrative tumors growing in a spherical pattern (Fig. 5, A). Histopathologic characteristics of the tumors included a dense cellular mass, hypervascularity, and no necrotic areas. The mean tumor volume was 81.6 mm3 (95% CI = 61.8 to 102.4 mm3), and all brains of mice that died naturally showed a midline shift and ventricular compression secondary to tumor-mass effects, which are characteristic features of herniation and indicate that tumor growth was probably the cause of death in mice that died naturally.
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Microscopic examination of the brains of all of the long-term survivors showed complete tumor regression. Sequelae of these tumors were identified, including dystrophic calcification and microcyst formation, at the tumor implantation site in the right caudate nucleus (Fig. 5, E and F). Immunohistochemical analyses of these brains (i.e., long-term survivors) with both anti-E1A and anti-hexon antibodies revealed no viral particles (data not shown). We did not observe either E1A expression or signs of inflammatory reaction in the normal brain tissue.
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DISCUSSION |
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Modification of the Delta-24 fiber knob with the insertion of the RGD-4C motif allowed Delta-24-RGD to overcome the low, heterogeneous expression of CAR in human glioma cells that is believed to limit adenovirus infectivity and, thus, to provide a cytopathic effect in these cells. As is the case in other cancers (7), CAR expression is low in the majority of gliomas (22). The insertion of an RGD-4C motif into the Delta-24 fiber knob has been shown to increase adenoviral infection of a variety of normal and cancer cell lines (23). Our finding that the RGD-4C motif increased the infectivity of Delta-24 supports this concept because the increased infectivity was related specifically to human glioma cells and because the RGD-4C motif increased infectivity of gliomas with low CAR expression.
The insertion of RGD-4C into replication-deficient adenoviral vectors, which was first described by Dmitriev et al. (24), is believed to enable direct binding and internalization of adenoviruses through a CAR-independent, RGD-related integrin-dependent mechanism (24). However, the relative importance of cell-surface v integrins versus CAR expression for entry of an adenovirus into target cells has not been completely defined. Adenovirus-mediated gene transfer is facilitated by an interaction between RGD on the adenovirus penton base and v integrins on the cell surface (18,25,26). Our findings shed light on the role of integrins in the adenoviral infectivity of gliomas. Specifically, they show that if an enhanced fiberintegrin interaction is available through the RGD-4C motif, adenoviruses infect glioma cells efficiently even when CAR expression is low. Therefore, insertion of the RGD motif into Delta-24 should provide substantial improvement in infectivity of gliomas in the clinical setting.
Of note is the fact that the RGD-4C modification of the fiber knob did not alter the ability of the adenovirus to bind CAR. This finding is demonstrated by the fact that, in cells with high CAR expression, the infectivity of adenoviral vectors with the RGD-4C modification was only slightly higher than that of adenoviral vectors without the RGD-4C modification. This finding is in contrast with the finding that adenoviruses in which the native tropism has been reduced after removal of both CAR and integrin interactions and replaced with selective binding motifs bind exclusively to cells expressing the receptors (27,28).
One of the main disadvantages of targeting a surface receptor in gliomas is that no glioma-specific receptor has been found to be expressed in the majority of glioma cells and/or in the majority of gliomas. Unlike the RGD-based approaches, the more selective targeting approaches may find application in a subset of gliomas, but they will probably never be considered a universal method to target the treatment of gliomas. The strategy of combining CAR and integrins as primary receptors might, however, raise the possibility that Delta-24-RGD would be more toxic than Delta-24. Thus, an important finding of our study is that the addition of the RGD-4C motif did not alter the toxicity of the Delta-24 adenovirus in cancer cells.
Our data showed that the E1A deletion safeguards normal cells from adenoviral replication and lysis, rendering Delta-24-RGD replicative only in cancer cells with an abnormal Rb pathway. Notably, like Delta-24 (5), Delta-24-RGD did not replicate efficiently in nondividing NHAs. Since our initial report (5), the highly selective, conditionally-replicative properties of mutant E1A replication-competent adenoviruses have been confirmed in several independent laboratories, which have consistently demonstrated the low toxicity of oncolytic adenoviruses that target the Rb (29,30) and/or the Rb/E2F pathways (31,32).
We elected to deliver Delta-24-RGD in the in vivo experiments using direct intratumoral injection. Although this approach requires low-risk but sophisticated experimental (18) and clinical techniques (i.e., stereotaxic surgery), we believe that the intratumoral approach is the most appropriate method of delivery of adenoviral vectors for brain tumors. In contrast, recent reports (29,31) suggest the possibility of intravenous delivery of Rb-targeted oncolytic adenoviruses to treat solid tumors. However, systemic delivery is unlikely to be successful in treating gliomas because, at least in part, of the bloodbrain and bloodtumor barrier. Moreover, most of the systemically delivered therapeutic adenovirus is lost to non-central nervous system tissues, such as hepatocytes and Kupffer cells (3336). These observations indicate that using the systemic delivery approach for gliomas may require increasing the dose of the therapeutic adenovirus, with the potential for systemic toxicities and low antiglioma effect, even if the vector is replication-selective.
In this study, the U-87 MG xenograft intracranial model is highly resistant to a variety of treatments (Table 2). However, treatment of athymic mice that had U-87 xenograft tumors with Delta-24-RGD did result in long-term survival in 60% of animals, and when these animals were killed at 140 days after implantation, there was no evidence of tumor (Fig. 5
). These data provide strong evidence for the potential clinical importance of the RGD-4C modification of adenoviral vectors in improving outcome in patients with gliomas.
|
In conclusion, oncolytic adenoviruses that target the Rb/E2F pathway are probably among the most efficient and selective oncolytic systems currently available (5,2932). In this study, we demonstrated that enhanced tropism of this system through genetic incorporation of the RGD-4C peptide into the Delta-24 adenovirus dramatically improved the antiglioma efficiency and maintained the selectivity of this adenovirus in cancer cells. The antiglioma activity of Delta-24-RGD suggests that it has the potential to be a much-needed agent in the treatment of gliomas.
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NOTES |
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Supported by grants from the Pediatric Brain Tumor Foundation and the University of Texas M. D. Anderson Cancer Center (to J. Fueyo), and from the Anthony Bullock Foundation (to R. Sawaya).
We thank M. Goode, Department of Scientific Publications, University of Texas M. D. Anderson Cancer Center, for editorial assistance and the University of Texas M. D. Anderson Centralized Histopathology Laboratory Core (Department of Pathology) and Flow Cytometry Laboratory Core (Department of Immunology). Technical assistance by Sushma L. Jasti and Frank W. Courmier (Brain Tumor Center) is also acknowledged.
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Manuscript received August 7, 2002; revised February 21, 2003; accepted March 13, 2003.
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