ARTICLES

Effect of In Situ Retroviral Interleukin-4 Transfer on Established Intracranial Tumors

Mary Saleh, Adrian Wiegmans, Quentin Malone, Stan S. Stylli, Andrew H. Kaye

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).


    ABSTRACT
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
BACKGROUND: Current therapies for malignant gliomas remain largely ineffective. We have previously demonstrated that interleukin 4 (IL-4) exhibits antitumorigenic activity in athymic nude mice by promoting both eosinophil infiltration and inhibition of tumor angiogenesis (formation of new blood vessels). In this study, we investigated treatment of established rat C6 cell gliomas by retroviral delivery of IL-4 in situ. METHODS: Tumors grown subcutaneously in athymic nude mice or implanted intracranially in immunocompetent Wistar rats were implanted with ecotropic retrovirus (i.e., will replicate only in cells of closely related species) packaging cells (RPCs) that were transfected with a retroviral vector encoding mouse IL-4 (1C5 cells) or a control vector (SV cells). For the demonstration of the long-term effects of such treatment, C6 cells were also implanted into the contralateral hemisphere of the brains of rats previously treated with 1C5 RPCs. Tumor volume measurements and immunohistochemical analyses were performed. RESULTS: Implantation of 1C5 RPCs into subcutaneous C6 cell tumors resulted in tumor growth arrest that was associated with eosinophil infiltration and inhibition of angiogenesis. When 1C5 RPCs were stereotactically implanted into established intracranial tumors in rats, tumor volumes were dramatically smaller than in control animals (approximately 1.8 mm3 versus 70-80 mm3, respectively) 7 days after treatment. All 1C5 RPC-treated rats survived to 106 days after C6 cell implantation (99 days after treatment; an arbitrary end point), whereas control rats had to be killed 14 days after C6 cell implantation because of extensive tumor growth. Histologic analysis demonstrated that treated tumors were completely eradicated, and immunohistochemical analysis revealed an inhibition of tumor angiogenesis and infiltration by CD8+ cells and macrophages. C6 cells implanted contralaterally into the brains of long-term-surviving rats treated with 1C5 RPCs were also rapidly and completely rejected. CONCLUSIONS: Implantation of packaging cells producing IL-4 retrovirus leads to rapid eradication of rat C6 cell gliomas and provides sustained protection against further intracranial challenge.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Malignant gliomas are the most common brain tumors in humans, and current treatments fail to provide long-term management of these tumors. The prognosis for patients with high-grade glioma remains poor: survival for less than 1 year, even following surgery and adjuvant therapies such as chemotherapy and radiation therapy (1). This disease clearly requires development of more effective therapeutic modalities that will improve long-term control and survival. The development of gene therapy techniques (2-4) during the past decade has provided a new and promising avenue of research into more efficacious treatments for gliomas and other cancers.

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.


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell Culture

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.


    RESULTS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Generation of mIL-4 Ecotropic Packaging Cells

The mIL-4 cDNA was cloned into the BglII cloning site of the pZIG retroviral vector (23) (Fig. 1).Go 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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Fig. 1. Schematic representation of the pZIG(mIL-4) retroviral vector that carries the complementary DNA (cDNA) for mouse interleukin 4 (mIL-4). The mIL-4 cDNA is cloned upstream of the internal ribosome entry site (IRES) element in the pZIG retroviral vector (23), with the ßgeo [marker gene encoding a fusion protein for neomycin resistance and ß-galactosidase (25)] cassette as the 3' cistron (23).

 
mIL-4 Retroviral Treatment of Established C6 Tumors in Athymic Mice

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).Go 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),Go less than one third of the size reached by the control tumors 7 days earlier.



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Fig. 2. Growth rate of subcutaneous rat C6 glioma cell tumors implanted in athymic (nu/nu) nude mice. Retroviral packaging cells (SV control retrovirus packaging cells or 1C5 mIL-4 [mouse interleukin 4] retrovirus packaging cells) were directly implanted into established rat C6 cell tumors in mice at day 14. Tumor volumes were compared over time with the volumes of C6 tumors that received no additional implants. All tumor volumes were determined by tridimensional caliper measurements and are presented as the mean in cm3 (plus 95% confidence intervals) of 16 tumors from 16 mice (two-sided P<.0001 at day 21).

 
Histologic Analysis of Subcutaneous C6 Tumors After mIL-4 Retroviral Treatment

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).Go 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).Go



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Fig. 3. Photomicrographs of subcutaneous tumor sections following retroviral packaging cell (RPC) implantation into established rat C6 glioma cell tumors. Panels a-d: Sections were stained with hematoxylin-eosin and demonstrate an increase in tumor necrosis following treatment with 1C5 RPCs, which produce an mIL-4 [mouse interleukin 4] encoding retrovirus. Panel a: parental C6 tumor (original magnification x100). Panel b: C6 tumors implanted with SV (control) RPCs (original magnification x100). Panel c: C6 tumors implanted with 1C5 RPCs (original magnification x100). Panel d: higher magnification of panel c, demonstrating a high level of eosinophil infiltration (original magnification x200). Panels e-h: Sections were stained by indirect immunoperoxidase with an anti-mouse PECAM monoclonal antibody, demonstrating a decrease in tumor vascularization following 1C5 RPC treatment. Panel e: parental C6 tumor (original magnification x100). Panel f: C6 tumors implanted with SV RPCs (original magnification x100). Panel g: C6 tumors implanted with 1C5 RPCs (original magnification x100). Panel h: representative ß-galactosidase staining of transduced vascular endothelial cells as observed in tumors treated with SV or 1C5 RPCs (original magnification x200).

 
The tumor-associated vascular endothelium in tumors 22 days after C6 cell implantation was stained by the indirect immunoperoxidase method using an anti-mouse PECAM monoclonal antibody that allowed the direct visualization and quantification of the blood vessels in the tumor sections (Fig. 3).Go The average number of vessels observed per standard field was significantly lower (P<.0001) in the established C6 tumors implanted with 1C5 RPCs than in either the parental C6 control or SV-implanted control tumors (Fig. 3).Go Parental C6 and SV-implanted tumors were observed to have an average of 10.50 (95% CI = 10.32-10.68) and 9.03 (95% CI = 8.88-9.18) vessels per standard field, respectively. In contrast, the tumors treated with 1C5 RPCs had an average of 3.01 (95% CI = 2.94-3.08) vessels per field. This result demonstrates an approximate 70% reduction in the number of vessels in established C6 tumors following treatment with 1C5 RPCs. In addition, the vessels observed in the 1C5-implanted tumors occurred in localized areas throughout the tumor, rather than being randomly distributed as in the controls (Fig. 3).Go Furthermore, ß-galactosidase staining of tumor sections revealed that retroviral transduction was limited to vascular endothelial cells (Fig. 3),Go while there was no evidence of RPCs persisting in the tumors at that time point.

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).Go 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),Go 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).Go 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),Go 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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Fig. 4. Analysis of established rat C6 glioma cells implanted intracranially in Wistar rats. Stereotactic intracranial implantation of either SV (control) or 1C5 (mIL-4 [mouse interleukin 4]) retroviral packaging cells was performed on day 7 after C6 cell implantation, and tumors were analyzed on day 14 (7 days after treatment). Hematoxylin-eosin staining of brain sections was used to determine mean tumor volumes in mm3 in eight rats per experimental group; the error bars show 95% confidence intervals. The experiment was repeated three times, and in all casesP<.0001 (two-sided Student's t test).

 


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Fig. 5. Photomicrographs of hematoxylin-eosin-stained sections of rat brains following the treatment of established rat C6 glioma cell tumors with SV (control) or 1C5 (mIL-4 [mouse interleukin 4]) retroviral packaging cells (RPCs). Analysis of brains at day 14 after C6 cell implantation (7 days after RPC treatment): Panel a shows C6 tumor implanted with SV RPCs (original magnification x20); panels b-d show C6 tumor implanted with 1C5 RPCs (original magnification x20) (arrows denote tumor/normal brain margin); panel e shows C6 cell tumor implanted with 1C5 RPCs at 106 days after C6 cell implantation (99 days after treatment) (original magnification x20); and panel f shows ß-galactosidase staining of transduced vascular endothelial cells persisting in 1C5-treated brains at 106 days after C6 cell implantation (original magnification x100).

 
Analysis of the brains from the 106-day, 1C5 RPC-treated rats failed to detect the presence of any tumor cells at the site of primary tumor implantation (Fig. 5).Go These brains were sectioned in their entirety, and there was no sign of tumor growth at any secondary sites within the brains. At that time, the brains appeared structurally normal, apart from the presence of a low level of residual gliosis at the injection tract. ß-Galactosidase activity was still detected in the vascular endothelial cells, which were mostly localized at the site of the original tumor and RPC implantation in the brains of these 1C5-treated animals (Fig. 5).Go

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)Go 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).Go 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).Go Many macrophages were also observed in and around the tumor margin (Fig. 6).Go 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).Go 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).Go



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Fig. 6. Photomicrographs of histochemical staining of 1C5 (mIL-4 [mouse interleukin 4]) retrovirus packaging cell (RPC)-treated rat C6 glioma cell tumors in rat brains at day 14 and day 106 after C6 cell implantation. Panel a: day-14 brain stained with hematoxylin-eosin, demonstrating a low level of eosinophil infiltration (arrows) and a high level of tumor necrosis. Panel b: day-14 brain following indirect immunoperoxidase staining with the mouse anti-rat macrophage monoclonal antibody ED1. Panel c: day-14 brain stained with the mouse anti-rat CD8+ monoclonal antibody Ox8. Panel d: day-14 brain stained with the mouse anti-rat CD4+ monoclonal antibody W3/25. Panel e: day-14 brain stained with the rat anti-mIL-4 monoclonal antibody 11B11. Panel f: day-106 rat brain following indirect immunoperoxidase staining with the rat anti-mIL-4 monoclonal antibody 11B11, demonstrating mIL-4 expression by vascular endothelial cells of blood vessels (panels a-f, original magnification x100).

 

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Table 1. Results of indirect immunoperoxidase staining of rat brain sections with a panel of monoclonal antibodies*

 
Intracranial C6 Challenge

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).Go 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)Go 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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Fig. 7. Photomicrographs of rat brains treated with 1C5 mIL-4 (mouse interleukin 4) retroviral packaging cells (RPCs) following contralateral rat C6 glioma cell challenge. 1C5 RPC-treated rats that survived 65 days after C6 cell implantation were implanted with C6 cells in the contralateral hemisphere, and their brains were analyzed 14 days later. The two hemispheres of the one rat C6 glioma cell-challenged rat brain are shown in panels a and b. Panel a: left hemisphere of rat brain following 1C5 RPC treatment of the original rat C6 glioma site, where the rat survived to 65 days (arrow denotes injection tract). The rat was killed 14 days after rat C6 cell challenge in the right hemisphere (see panel b). Panel b: right hemisphere of rat brain in panel a 14 days after C6 cell challenge in the right hemisphere (arrow denotes necrotic site). Panel c: control rat brain implanted with C6 cells at day 14 (arrow denotes tumor) (panels a-c, hematoxylin-eosin, original magnification x20).

 

    DISCUSSION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The development of gene therapy techniques offers the potential for more effective therapies for malignant gliomas. Of the delivery systems currently available, retroviruses have been employed in the majority of human clinical trials (2,3,29) to mediate the transfer of a variety of genes in situ, in vitro, and ex vivo (4). An area of intense investigation for brain tumor therapy has been the retroviral delivery of suicide genes (30,31). RPCs producing retroviruses carrying the herpes simplex virus-thymidine kinase (HSV-TK) gene were implanted intratumorally, and the resultant transduced cells were rendered lethally sensitive to ganciclovir. This approach was effective in inducing the regression of experimental tumors in rodent models (30,31). However, clinical trials of HSV-TK did not demonstrate the same efficacy; varying levels of regression were observed, and the effects were limited to small tumors (29). A low transduction frequency was reported in patients; thus, tumor regression was proposed to be a function of the "bystander effect" (in which nontransduced cells that touch transduced cells also become sensitive to ganciclovir) or due to ischemia following the transduction of tumor-associated vascular endothelial cells. Hence, more effective treatments are still required to mediate the regression of large tumors, even if a limited number of cells are transduced.

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 days—i.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+ cells—but 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.


    NOTES
 
Supported in part by the Anti-Cancer Council of Victoria and the National Health and Medical Research Council.

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.


    REFERENCES
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

1 Walker MD, Alexander E Jr, Hunt WEJ, MacCarty CS, Mahaley MS Jr, Mealey J Jr, et al. Evaluation of BCNU and/or radiotherapy in the treatment of anaplastic gliomas. A cooperative clinical trial. J Neurosurg 1978;49:333-43.[Medline]

2 Rosenberg SA, Anderson WF, Asher AL, Blaese MR, Ettinghaussen SE, Hwu P, et al. Immunization of cancer patients using autologous cancer cells modified by insertion of the gene for tumor necrosis factor. Hum Gene Ther 1992;3:57-73.[Medline]

3 Rosenberg SA, Anderson WF, Blaese MR, Ettinghaussen SE, Hwu P, Karp SE, et al. Immunization of cancer patients using autologous cancer cells modified by insertion of the gene for interleukin-2. Hum Gene Ther 1992;3:75-90.[Medline]

4 Lyerly HK, DiMaio JM. Gene delivery systems in surgery. Arch Surg 1993;128:1197-206.[Abstract]

5 Watanabe Y. Transfection of interferon-gamma gene in animal tumors—a model for local cytokine production and tumor immunity. Semin Cancer Biol 1992;3:43-6.[Medline]

6 Watanabe Y, Kuribayashi K, Miyatake S, Nishihara K, Nakayama E, Taniyama T, et al. Exogenous expression of mouse interferon gamma cDNA in mouse neuroblastoma C1300 cells results in reduced tumorigenicity by augmented anti-tumor immunity. Proc Natl Acad Sci U S A 1989;86:9456-60.[Abstract]

7 Bubenik J, Simova J, Jandlova T. Immunotherapy of cancer using local administration of lymphoid cells transformed by IL-2 cDNA and constitutively producing IL-2. Immunol Lett 1990;23:287-92.[Medline]

8 Tahara H, Zitvogel L, Storkus WJ, Zeh HJ 3rd, McKinney TG, Schreiber RD, et al. Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector. J Immunol 1995;154:6466-74.[Abstract/Free Full Text]

9 Tepper RI, Pattengale PK, Leder P. Murine interleukin-4 displays potent anti-tumor activity in vivo. Cell 1989;57:503-12.[Medline]

10 Golumbek PT, Lazenby AJ, Levitsky HI, Jaffee LM, Karasuyama H, Baker M, et al. Treatment of established renal cancer by tumor cells engineered to secrete interleukin-4. Science 1991;254:713-6.[Medline]

11 Yu JS, Wei MX, Chiocca EA, Martuza RL, Tepper RI. Treatment of glioma by engineered interleukin 4-secreting cells. Cancer Res 1993;53:3125-8.[Abstract]

12 Tepper RI, Coffman RL, Leder P. An eosinophil-dependent mechanism for the antitumor effect of interleukin-4. Science 1992;257:548-51.[Medline]

13 Paul WE, Ohara J. B-cell stimulatory factor-1/interleukin 4. Annu Rev Immunol 1987;5:429-59.[Medline]

14 Howard M, Farrar J, Hilfiker M, Johnson B, Takatsu K, Hamaoka T, et al. Identification of a T cell-derived b cell growth factor distinct from interleukin 2. J Exp Med 1982;155:914-23.[Abstract]

15 Tepper RI. The eosinophil-mediated antitumor activity of interleukin-4. J Allergy Clin Immunol 1994;94:1225-31.[Medline]

16 Hurford RK Jr, Dranoff G, Mulligan RC, Tepper RI. Gene therapy of metastatic cancer by in vivo retroviral gene targeting. Nat Genet 1995;10:430-5.[Medline]

17 Saleh M, Davis ID, Wilks AF. The paracrine role of tumour-derived mIL-4 on tumor-associated endothelium. Int J Cancer 1997;72:664-72.[Medline]

18 O'Reilly MS, Holmgren L, Chen C, Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med 1996;2:689-92.[Medline]

19 Saleh M, Stacker SA, Wilks AF. Inhibition of growth of C6 glioma cells in vivo by expression of antisense vascular endothelial growth factor sequence. Cancer Res 1996;56:393-401.[Abstract]

20 Saleh M, Vasilopoulos K, Stylli SS, Kaye AH, Wilks AF. The expression of antisense vascular endothelial growth factor (VEGF) sequences inhibit intracranial glioma growth in vivo by suppressing tumour angiogenesis. J Clin Neurosci 1996;3:366-72.

21 Maher DW, Davis I, Boyd AW, Morstyn G. Human interleukin-4: an immunomodulator with potential therapeutic applications. Prog Growth Factor Res 1991;3:43-56.[Medline]

22 Markowitz D, Hesdorffer C, Ward M, Goff S, Bank A. Retroviral gene transfer using safe and efficient packaging cell lines. Ann N Y Acad Sci 1990;612:407-14.[Abstract]

23 Saleh M. A retroviral vector that allows efficient co-expression of two genes and the versatility of alternate selection markers. Hum Gene Ther 1997;8:979-83.[Medline]

24 Lee F, Yokota T, Otsuka T, Meyerson P, Villaret D, Coffman R, et al. Isolation and characterization of a mouse interleukin cDNA clone that expresses B-cell stimulatory factor 1 activities and T-cell- and mast-cell-stimulating activities. Proc Natl Acad Sci U S A 1986;83:2061-5.[Abstract]

25 Mountford P, Zevnik B, Duwel A, Nichols J, Li M, Dani C, et al. Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc Natl Acad Sci U S A 1994;91:4303-7.[Abstract]

26 Graham FL, Eb AJ van der. Transformation of rat cells by DNA of human adenovirus 5. Virology 1973;54:536-9.[Medline]

27 Rongish BJ, Hinchman G, Doty MK, Baldwin HS, Tomanek RJ. Relationship of the extracellular matrix to coronary neovascularization during development. J Mol Cell Cardiol 1996;28:2203-15.[Medline]

28 Williams KC, Zhao RW, Ueno K, Huley WF. PECAM-1 (CD31) expression in the central nervous system and its role in experimental allergic encephalomyelitis in the rat. J Neurosci Res 1996;45:747-57.[Medline]

29 Ram Z, Culver KW, Oshiro EM, Viola JJ, DeVroom HL, Otto E, et al. Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells. Nat Med 1997;3:1354-61.[Medline]

30 Oldfield EH, Ram Z, Culver KW, Blaese RM, DeVroom HL, et al. Gene therapy for the treatment of brain tumors using intra-tumoral transduction with the thymidine kinase gene and intravenous ganciclovir. Hum Gene Ther 1993;4:39-69.[Medline]

31 Ram Z, Walbridge S, Shawker T, Culver KW, Blaese RM, Oldfield EH. The effect of thymidine kinase transduction and ganciclovir therapy on tumor vasculature and growth of 9L gliomas in rats. J Neurosurg 1994;81:256-60.[Medline]

32 Pericle F, Giovaarelli MP, Colombo MP, Ferrari G, Musiani P, Modesti A, et al. An efficient Th2-type memory follows CD8+ lymphocyte-driven and eosinophil-mediated rejection of a spontaneous mouse mammary adenocarcinoma engineered to release IL-4. J Immunol 1994;153:5659-73.[Abstract/Free Full Text]

33 Wei MX, Tamiya T, Hurford RK Jr, Boviatsis EJ, Tepper RI, Chiocca EA. Enhancement of interleukin-4-mediated tumor regression in athymic mice by in situ retroviral gene transfer. Hum Gene Ther 1995;6:437-43.[Medline]

Manuscript received July 24, 1998; revised November 30, 1998; accepted December 31, 1998.


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