Affiliations of authors: M. Saleh, A. Wiegmans, Q. Malone, A. H. Kaye (The Molecular Neuroscience and Gene Therapy Laboratories), S. S. Stylli (Cell Biology Laboratory), The Department of Surgery, The University of Melbourne, and The Royal Melbourne Hospital, Parkville Victoria, Australia.
Correspondence to: Mary Saleh, Ph.D., The Molecular Neuroscience and Gene Therapy Laboratories, The Department of Surgery, The University of Melbourne, Clinical Sciences Bldg. Level 5, Royal Parade Parkville, The Royal Melbourne Hospital, Victoria 3050, Australia (e-mail: mary.saleh{at} nwhcn.org.au).
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ABSTRACT |
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INTRODUCTION |
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The use of cytokines in immunotherapy has been investigated as a potential therapy for gliomas. Several cytokines, such as interferon gamma (5,6), interleukin 2 (IL-2) (7), IL-12 (8), and IL-4 (9-12), have been demonstrated to mediate tumoricidal activities in vivo. IL-4 (13) is a multifunctional lymphokine produced by the TH2 subset of helper T lymphocytes and has a broad range of activities on B and T lymphocytes in vitro (14). IL-4 has been demonstrated to have antitumorigenic actions against cancers of diverse histologic origin in vivo (9-12) Tumor cells that have been engineered to express IL-4 failed to form tumors when implanted in immunocompetent mice but formed tumors after a period of inhibition in immunocompromised mice (9-11). Histologic analysis of the tumor sites revealed that the initial inhibition of tumor growth was due to a profuse eosinophil infiltrate (9-12,15,16) but that T-cell-mediated immunity was required for permanent tumor suppression (12).
We have previously reported that rat C6 glioma cells engineered to express mouse IL-4 (mIL-4) in a tetracycline-responsive expression system failed to form tumors when the cells were implanted in athymic nude (nu/nu) mice (17). Analysis of the mechanism of tumor inhibition revealed that IL-4 has an antitumorigenic activity mediated through two processes. The expression of IL-4 by C6 cells resulted in eosinophil infiltration as previously reported, as well as inhibition of tumor angiogenesis (17). This inhibition of tumor angiogenesis may be mediated by changes in the expression of vascular endothelial growth factor (VEGF) receptor (VEGFR), as we could demonstrate that exogenous mIL-4 can suppress the expression of VEGFR2 in cultured mouse endothelial cells (17). The double action of IL-4 suggests that it is an ideal candidate for gene therapy of solid tumors because it both elicits a host-mediated immune response and inhibits tumor angiogenesis. Both of these pathways have been demonstrated to be appropriate targets for effective treatment of solid tumors (6,7,18-20). Clinical trials that address the systemic administration of IL-4 have reported that patients suffer severe side effects in response to the high doses of IL-4 required to achieve tumor inhibition (21). Thus, for IL-4 to be used effectively as an antitumor therapy, it must be expressed only at high local concentrations within the tumor.
We have investigated the potential therapeutic efficacy of using retroviruses to deliver IL-4 intratumorally to eradicate established C6 gliomas. The advantage of using retroviral gene transfer over other viral delivery systems is that retroviruses will only integrate their DNA into dividing cells. The rapidly dividing cells in the brain are primarily the tumor cells and the tumor-associated vascular endothelium, while the vast majority of normal brain cells are quiescent. Therefore, retroviral transduction can be effectively targeted to the tumor site.
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MATERIALS AND METHODS |
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The rat C6 glioma cell line was obtained from American Type Culture Collection (ATCC), Manassas, VA, and were cultured as previously described (19). The ecotropic retroviral packaging cell line GP+E-86 (NIH/Swiss embryo) (22) (a gift from the Rotary Bone Marrow Research Institute, Melbourne, Australia) and the NIH 3T3 cell line (NIH/Swiss embryo fibroblast; ATCC) were cultured as described previously (23). Packaging cells and retroviral infected NIH 3T3 cells resistant to geneticin G418 (Life Technologies Pty Ltd., Melbourne, Australia) were maintained in medium supplemented with this antibiotic at concentrations of 1 mg/mL and 0.4 mg/mL of medium, respectively (23).
mIL-4 Retroviral Construct
The details of the pZIG(SV) retroviral vector have been reported previously (23). The mIL-4 complementary DNA (cDNA) (24) was introduced by ligation into the BglII cloning site of the pZIG(SV) vector upstream of the internal ribosome entry site element (Fig. 1). The ßgeo marker gene, which encodes a fusion protein for neomycin resistance and ß-galactosidase (25), is contained in the pZIG(SV) vector as the 3' cistron. Restriction mapping and nucleotide sequencing were used to confirm the correct orientation of the mIL-4 cDNA in pZIG(mIL-4).
Isolation of Retrovirus-Producing GP+E-86 Clones
Transfection of the GP+E-86 ecotropic (capable of transducing mouse and rat dividing cells) retroviral packaging cells (RPCs) (22) with 20 µg of pZIG(mIL-4) DNA or pZIG(SV) DNA (a retroviral control) was performed by calcium phosphate transfection (19,26). Transfected cells were selected in complete culture medium supplemented with 1 mg/mL G418. Individual G418-resistant packaging cell clones were grown to subconfluence in the absence of G418. Dilutions of these supernatants were used to transduce NIH 3T3 cells as described previously (23), and NIH 3T3 cells were then cultured in 0.4 mg/mL G418 to determine viral titers. We then cultured 106 transduced NIH 3T3 cells of each selected clone for 48 hours in normal medium at 37 °C, and we tested supernatants by enzyme-linked immunosorbent assay (R and D Systems, Minneapolis MN) for mIL-4 production. The expression of ß-galactosidase by transfected packaging cells and infected NIH 3T3 cells was measured as described previously (23).
In Vivo Tumor Analysis
Adult male or female athymic (nu/nu) mice (ARC, Perth, Australia) were subcutaneously implanted with 106 C6 cells in 100 µL of serum-free culture medium. Tumor volumes were determined every 2nd day by tridimensional caliper measurements (length x width x height) throughout the experiments. Fourteen days after implantation, when tumor volumes were between 0.15 and 2.0 cm3, 106 SV (retrovirus control packaging cells) or IC5 (mIL-4 retrovirus packaging cells) cells in 100 µL of serum-free medium were implanted directly into the established subcutaneous C6 tumors. The sizes of 16 tumors for each cell line, implanted in 16 individual mice for each cell line, were calculated. Tumors were excised at the end of the experiments and were analyzed histologically.
Adult male or female Wistar rats were anesthetized, and 105 rat C6 glioma cells (resuspended in 10 µL of serum-free cell culture medium) were implanted stereotactically into the rat brain through the coronal suture left of the midline, to a depth of 5 mm. Seven days after implantation, the procedure was repeated on the same rats except that 106 SV control RPCs or 1C5 mIL-4 RPCs were stereotactically implanted directly into the tumor implantation site, and the incision was closed. This experiment was performed on eight rats per experimental group and C6 parental controls, for each time point in three sets of experiments. The brains of the rats were analyzed histologically at 7, 14, and 106 days after C6 cell implantation, and tumor volume was calculated as length x width x depth (mm3), as had been done for the subcutaneous tumors, in order to overcome differences in tumor shape.
A second group of eight rats that were treated with 1C5 RPCs and that survived to 65 days after C6 cell implantation into their left hemispheres were further challenged with 105 C6 cells stereotactically implanted into the contralateral hemisphere of the brain. Previously unimplanted control rats were also implanted with C6 cells in a similar manner also in their right hemispheres. Fourteen days after the C6 challenge, their brains were subjected to detailed immunohistochemical analysis.
The animals were cared for in accordance with the guidelines put forth by the National Health and Medical Research Council and The University of Melbourne.
Histologic Analysis of Tissue
Serial frozen sections (7 µm) of subcutaneous tumor or whole brains were stained in the following ways: 1) hematoxylin-eosin for tumor cell volumes and infiltrating eosinophils; 2) indirect immunoperoxidase with the rat anti-mouse PECAM monoclonal antibody (PharMingen, San Diego, CA) or mouse anti-rat PECAM monoclonal antibody (Endogen, Woburn, MA) for vascular endothelial cells in mouse (17,19,20) or rat (27,28) tissue, respectively; 3) indirect immunoperoxidase with the rat anti-mIL-4 monoclonal antibody 11B11, the mouse anti-rat CD3+/TcR monoclonal antibody 453(MCA), the mouse anti-rat CD4+ monoclonal antibody W3/25, the mouse anti-rat CD8+ monoclonal antibody Ox8, or the mouse anti-rat macrophage monoclonal antibody ED1 (gifts from P. Motram, Department of Surgery, The University of Melbourne); and 4) ß-galactosidase staining for visualization of RPCs and transduced cells (23). We calculated vascular density by counting the number of vessels in 10 random standard fields (0.55 x 0.4 mm) in five sections per tumor.
Statistical Analyses
A two-sided Student's t test (mice paired with one another) was used to calculate the level of statistical significance for the tumor growth rates derived from parental C6 tumors and from SV- or 1C5-treated tumors. For effects on angiogenesis, a two-sided Student's t test (paired) was used to calculate the level of statistical significance for the number of vessels observed in control C6 tumors and in SV-treated tumors and 1C5-treated tumors. P values less than .05 were considered statistically significant.
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RESULTS |
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The mIL-4 cDNA was cloned into the BglII cloning site of
the pZIG retroviral vector (23) (Fig. 1).
The retrovirus packaged in GP+E-86 ecotropic cells was determined to
have titers of 5 x 105 to 1.35 x 106
colony-forming units (CFUs)/mL, and the transduced NIH 3T3 cells
produced 225-454 ng/mL of mIL-4 (see "Materials and
Methods" section). The 1C5 mIL-4 RPC line used in this study has a
titer of 1.3 x 106 CFU/mL, and the shuttle vector alone
control cell line (SV) has a titer of 1.4 x 106 CFU/mL.
NIH 3T3 cells transduced with 1C5-derived retrovirus expressed 454
ng/mL of mIL-4 in 48 hours (see "Materials and Methods"
section), whereas mIL-4 expression was not detected in SV transduced
NIH 3T3 cells. The ecotropic retroviruses produced (SV or 1C5) failed
to effectively transduce rat C6 cells in culture (<80 CFU/mL) or C6
tumor cells in vivo, presumably because of the expression of a
low number of appropriate receptors on this C6 cell line. Expression of
the ß-galactosidase marker gene in tumors was observed only in the
vascular endothelium, indicating that the transduction of these
ecotropic retroviruses is targeted to the dividing mouse or rat
vasculature and not to the tumor cells (see below).
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C6 tumors were established subcutaneously in athymic nude (nu/nu)
mice. When tumor volumes were between 0.15 and 0.2 cm3, the
mIL-4/GP+E-86 cell line (1C5) or shuttle vector alone/GP+E-86 cell line
(SV) was implanted intratumorally, and tumor growth was monitored for
the duration of the experiments. Tumors arising from untreated parental
C6 cells or C6 cells treated with SV RPCs continued to grow
exponentially and had volumes in excess of 1.2 cm3 at 21 days
after C6 cell implantation (Fig. 2). In contrast, the growth rate of
established C6 tumors implanted with 1C5 RPCs was arrested
(P<.0001). At 21 days after C6 cell implantation (7 days
after 1C5 RPC implantation), the tumors began to grow very slowly but
never entered an exponential growth phase. By day 28, the tumors
appeared to have an arrested growth rate and had reached a volume of
only 0.48 cm3 (95% confidence interval [CI] =
0.40-0.56 cm3) (Fig. 2),
less than one
third of the size reached by the control tumors 7 days earlier.
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Frozen sections of experimental or control subcutaneous C6 tumors at
day 22 after C6 cell implantation were stained with hematoxylin-eosin
to analyze the degree of tumor necrosis and granulocyte infiltrate.
This analysis revealed that the parental C6 tumors or tumors implanted
with SV RPCs had a low rate of necrosis (10%-15%) and a high
index of viable mitoses, but they were devoid of a granulocyte
infiltrate (Fig. 3, a). In contrast, the established
tumors implanted with 1C5 RPCs had a higher level of necrosis (60%)
and a higher degree of eosinophil infiltration compared with findings in
either parental C6 control tumors or tumors implanted with SV RPCs (Fig. 3).
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Eradication of Established Intracranial Tumors in Immunocompetent Rats by mIL-4 Retroviral Therapy
C6 glioma cells were stereotactically implanted into the brains of immunocompetent Wistar rats, and tumors were allowed to grow for 7 days. Histologic analysis of tumors of one set of rats at day 7 after implantation confirmed the presence of an established tumor in their brain; the average tumor volume was 6.59 mm3 (95% CI = 6.44-6.74 mm3). Either SV or 1C5 RPCs were then stereotactically implanted directly into the intracranial tumors of another set of C6-implanted rats, and the rats were allowed to recover. Rats implanted with control parental C6 cells alone, or SV RPCs appeared lethargic 14 days after C6 cell implantation (i.e., 7 days after RPC implantation), whereas rats implanted with 1C5 cells appeared healthy and very active. Thus, the experimental end point for all of the control rats was determined to be day 14 after C6 cell implantation. The control rats were killed at that time. The rats treated with 1C5 were continually monitored for any signs of lethargy or distress. A number of the 1C5-treated rats were also culled at day 14 after C6 cell implantation for histologic analysis of their brains. All of the rats treated with 1C5 cells appeared healthy and active for the duration of the experiment, which was arbitrarily terminated at day 106 after C6 cell implantation, and demonstrated 100% survival compared with control SV-treated rats. This represents a dramatic increase in survival for 1C5-treated rats over control rats.
Histologic analysis of the rat brains at day 14 after C6 cell implantation revealed that the tumors
implanted with the 1C5 RPC cells were significantly smaller in volume (P<.0001)
than the untreated parental C6 tumors or the tumors in animals implanted with SV RPCs (Fig. 4). Parental C6 tumors and SV RPCs-implanted tumors had similar mean
volumes of 72.11 mm3 (95% CI = 70.29-73.93 mm3)
and 79.87 mm3 (95% CI = 76.4-83.33 mm3),
respectively. However, the tumors implanted with 1C5 RPCs had a mean volume of 1.84 mm3 (95% CI = 1.04-2.64 mm3) (Fig. 4),
which is 2.4% of the control tumor volume at day 14 and 30% of the
tumor volume observed at the time of 1C5 implantation (day 7 after C6). In addition, the 1C5
RPC-implanted tumors were often very necrotic (Fig. 5).
ß-Galactosidase staining of day-14 brains revealed that the only transduced cells in the
brains were the vascular endothelial cells (as observed at day 106; see Fig. 5),
which were predominantly localized at the site of the tumor, and that the
majority of RPCs had died and were no longer present at the site of implantation.
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IL-4-Mediated Inflammatory Response
Hematoxylin-eosin staining of brain tumor tissue at day 14 after C6
cell implantation revealed that there was a low level of eosinophil
infiltration in the 1C5-treated tumors (Fig. 6) that
was absent in C6 and SV controls, as expected. Indirect
immunoperoxidase staining with a panel of monoclonal antibodies was
used to further differentiate the different T-cell populations
infiltrating the tumor sites (Table 1).
At day 14 ,
control C6 tumors were observed to contain a very low number of
CD4+ cells, while SV-treated tumors were also infiltrated
with CD8+ T cells. In contrast, the 1C5-treated tumors had a
high level of CD8+ cells and to a lesser degree
CD4+ and CD3+ cells infiltrating both the tumor and
the normal brain tissue immediately surrounding the tumor (Fig. 6).
Many macrophages were also observed in and around the tumor margin
(Fig. 6).
Macrophages and CD8+ T cells were also
observed around clusters of C6 cells that had migrated a short distance away
from the primary tumor site (data not shown). Immunoperoxidase staining
with a rat anti-mIL-4 monoclonal antibody (11B11) demonstrated that
mIL-4 was being expressed at high levels at day 14 in the transduced
vascular endothelial cells in and around the tumor margin in the
1C5-treated tumors (Fig. 6).
Moreover, mIL-4 was still being expressed
in the vascular endothelium at the site of original implantation in the
brains of 1C5-treated rats at day 106 (Fig. 6).
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An additional set of eight rats that had been treated with 1C5 RPCs
and that survived to 65 days after C6 cell implantation was
subsequently challenged by the contralateral implantation of C6 cells.
We fully analyzed the brains of these challenged rats and control rats
as described above, at 14 days following this challenge. This further
study revealed an absence in these rat brains of the initial C6 tumor
that had been implanted in the left hemisphere, confirming the results
of our experiments described above (Fig. 7). In addition, the C6 cells
implanted in the right contralateral hemisphere did not form tumors, in
contrast to their control counterparts (control tumor volume,
71.3 mm3 [95% CI = 69.9-72.7 mm3]).
Either a complete absence of tumor or an area of necrosis accompanied by an
infiltration of macrophages, CD4+ cells, and CD8+
cells was observed at the site of implantation (Fig. 7)
in all of the treated
rat brains. Furthermore, we failed to observe eosinophils at the site of implantation (data not
shown). There was no sign of tumor growth or clusters of viable tumor
cells at the site of tumor challenge in the 1C5-implanted rats. This
result was consistent in all of the treated rats challenged.
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DISCUSSION |
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We have previously demonstrated (17) that IL-4 mediated a tumoricidal activity via (at least) two biologic mechanisms. As other laboratories have reported, IL-4 elicits an initial eosinophil infiltration into the tumor (9-12,15-17), with the subsequent recruitment of the T-cell arm of the immune response, which is required for long-term tumor inhibition (12). In addition, we have also demonstrated that IL-4 inhibits tumor angiogenesis, which greatly contributes to tumor inhibition in athymic mice (17). We have now investigated the effectiveness of retroviral delivery of IL-4 in eradicating established intracranial tumors and the mechanisms that underpin this response.
When 1C5 RPCs were implanted into established, subcutaneous C6 tumors in nu/nu mice, tumor growth was arrested after an initial decrease in the tumor growth rate compared with that in controls. Analysis of 1C5-treated tumors revealed a high level of eosinophil infiltration, a high degree of necrosis, and a statistically significant reduction in the level of tumor vascularization. In addition, the vessels were observed in localized patches and were not randomly distributed as in control tumors. The localized patches of vessels most likely represent vessels that existed prior to IL-4 expression in the tumor. Although the proliferating tumor edge has continued to expand and VEGF is expressed by the tumor cells, tumor angiogenesis does not occur because IL-4 expression has disrupted the VEGF paracrine pathway possibly via the suppression of VEGFR expression. This growing tumor edge becomes necrotic and cannot continue to grow further without vasculature support. These results demonstrate that IL-4 has a tumoristatic effect in immunocompromised mice within the duration of our experiments, predominantly through inhibition of further tumor angiogenesis. The inhibition of angiogenesis and the presence of eosinophils were not sufficient to eradicate established tumors in our nude mouse experiments.
The stereotactic implantation of 1C5 RPCs into established C6 intracranial tumors in rats resulted in a rapid eradication of the existing tumors, and suppression of tumor growth was sustained throughout our experiments. IL-4 treatment dramatically increased survival of the rats, inasmuch as all of the treated rats survived to 106 days, in contrast to 14 days for the control rats. Because these experiments were terminated solely for the purposes of histologic analysis, this represents the absolute minimum increase in median survival. Seven days after 1C5 RPC implantation, the tumors were 30% of the size they were in the animals killed at the time of RPC implantation and 2.4% of the size of tumors at that time in the control rats. A low level of eosinophil infiltration was observed and was accompanied by reduced vascularization and high levels of necrosis in the 1C5-treated tumors. In contrast to the tumors in control rats, these tumors also contained substantial numbers of CD8+ T cells and macrophages and, to a lesser degree, CD4+ cells. This finding supports previous reports (10,32) stating that CD8+ cells predominate in IL-4-mediated T-cell responses. The expression of high levels of mIL-4 was also observed at day 14 in transduced vascular endothelial cells at the site of the tumor in 1C5-treated rats. Some CD8+ cells were observed in the SV-treated tumors, which may have been in response to the implantation of mouse RPCs into rat brains. A low number of packaging cells were still present in the treated tumors at that time.
Analysis of rat brains that exhibited long-term tumor inhibition (at 106 daysi.e., 99 days after 1C5 RPC implantation) revealed a complete absence of tumor cells at the site of primary implantation or at secondary sites. It is interesting that blue (ß-galactosidase-stained) vessels were still observed predominantly at the site of the original tumor and RPC implantation, although they were found in lower numbers than in the day-14 brains. Immunohistochemical staining with an anti-mIL-4 monoclonal antibody demonstrated that mIL-4 was still being expressed in these transduced endothelial cells (at lower levels than in day 14) but was not associated with a persistent eosinophil or T-cell inflammatory response. This result demonstrates that IL-4 expression is stable in transduced cells and is sustained for a substantial period of time. Retroviral transduction was not observed in any other organs examined in the rats at either day 14 or day 106 after C6 cell implantation, and there was no sign of eosinophilia or mIL-4 expression in the blood of 1C5-treated rats at day 14 or day 106 (data not shown). It does not appear that persisting IL-4 expression in the brain caused side effects or other complications because the rats were healthy and active throughout the 99 days after 1C5 RPC treatment. Furthermore, we demonstrated that IL-4-treated rats had sustained, long-term immune memory that results in the rejection of a subsequent C6 cell challenge. The rejection of implanted tumor cells was rapid and was associated with an infiltration predominantly of macrophages and CD8+, CD4+, and CD3+ cellsbut few eosinophils. The sustained inhibition of C6 tumor growth in these rats, therefore, would suggest that the probability of tumor recurrence in animals treated with IL-4 is substantially reduced, if not eliminated. This is an important factor in glioma treatment because tumor cells often infiltrate the normal brain and are not removed surgically with the primary tumor, which can lead to tumor recurrence. Clinically, 1C5 RPC treatment could rapidly eradicate the tumor and provide the patient with sustained immunity against regrowth of the tumor at the primary site or secondary sites. In addition, because of the sustained immunity that is produced, we believe that it is highly unlikely that 1C5 RPC therapy would need to be administered more than once. Therefore, the potential of a human anti-mouse RPC immune response, which would develop with repeated implantations of packaging cells, would not be a factor in this treatment protocol.
It has been previously reported that the implantation of 1C4 RPCS into nu/nu mouse brain tumors resulted in a median survival of 38 days compared with 25 days for C6 controls (33). IL-4 treatment in this model was performed when tumors would have been very small, inasmuch as RPC implantation was performed 3 days after 103 C6 cells were implanted intracranially. The failure of IL-4 to completely eradicate tumors in immunocompromised mice is confirmed by our findings, in which the T-cell arm of the immune system appears necessary for complete and sustained tumor regression. In athymic animals, the suppression of tumor angiogenesis by IL-4 inhibits any further increase in tumor volume, and the growing tumor margin becomes necrotic. The antitumorigenic role of eosinophils is still unclear; they may phagocytose tumor cells, which by itself is not sufficient to completely eradicate existing tumors, particularly those of larger volumes. In immunocompetent animals, IL-4 appears to induce a complete host-mediated immune response that serves to reduce tumor burden in a manner similar to that of surgery. It is the interplay of the two mechanisms mediated by IL-4 that allows not only tumor inhibition but also tumor eradication. Although patients with cerebral gliomas demonstrate varying levels of T-cell suppression, which increases after surgery and corticosteroid therapy, this suppression is not complete and is mostly transient. Because we find that IL-4 expression is sustained in transduced cells, expression levels should still be high enough to elicit a T-cell inflammatory response in these patients once steroid therapy has ceased, allowing the eradication of arrested tumors.
Our results demonstrate the efficacy of IL-4 in eradicating existing tumors and in providing long-term, continued suppression. Tumor eradication by this strategy depends on the high local concentration of IL-4 expression within the tumor rather than on the very high frequency of transduction that is required for antisense or suicide gene expression. A transduced cell can provide a constant source of IL-4 production that, in turn, influences a large number of neighboring cells. Thus, this approach should be far more effective than approaches reliant on very high transduction levels, such as those that employ suicide genes. Another advantage of this strategy is that it targets for therapeutic intervention biologic systems that are common to all gliomas. Targeting broadly based pathways, such as tumor angiogenesis and host-mediated immune responses, provides therapies that overcome the heterogeneity of malignant gliomas. It is this heterogeneity that makes targeting a treatment to specific receptors or growth factors ineffective. Also, it is important to note that we achieved complete eradication of established C6 gliomas in our model by using ecotropic retroviruses that only transduced the dividing vascular endothelium. We are now developing an amphotropic retroviral IL-4 model (i.e., the retroviruses produced can transduce dividing cells of a variety of species, including humans) to determine the ratio of RPCs to tumor volume required for treatment of large tumors. In an amphotropic model, the retroviruses should also transduce the C6 tumor cells, resulting in even higher transduction frequencies. It is hoped that the 1C5 RPC strategy can be developed for testing in clinical trials as a primary therapy for inoperable tumors or as an adjuvant therapy following surgery.
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NOTES |
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We express our sincere gratitude to Dr. A. F. Wilks for invaluable critical discussion, support, and creativity during the development of this project and for critical review of the manuscript. We also thank Drs. P. Motram and L. Murray-Segal for antibodies. We are extremely grateful for the generous and continuing support of Health Care of Australia (Mayne Nickless P/L ) and The Friends of the Royal Melbourne Hospital Neuroscience Foundation.
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Manuscript received July 24, 1998; revised November 30, 1998; accepted December 31, 1998.
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