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Transcriptional Targeting of Adenovirally Delivered Tumor Necrosis Factor by Temozolomide in Experimental Glioblastoma

¹ Department of Surgery, Section of Neurosurgery and ² Department of Radiation and Cellular Oncology, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois; ³ Brain Tumor Research Laboratories, Division of Neurosurgery, Department of Surgery, University of Alabama, Birmingham, Alabama; and ⁴ Dana-Farber Cancer Institute, Department of Medical Oncology, Boston, Massachusetts

	ABSTRACT

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Temozolomide is an oral alkylating agent shown to have modest efficacy in the treatment of glioblastoma multiforme. Tumor necrosis factor (TNF-) is a polypeptide cytokine with synergistic antitumor activity in combination therapy with alkylating agents. We investigated the combined use of Ad.Egr-TNF, a replication-defective adenoviral vector encoding the cDNA for TNF- under the control of chemo-inducible elements of the egr1 gene promoter, and intraperitoneal temozolomide in an intracranial human malignant glioma model. In hind limb U87MG xenografts, temozolomide produced a 6.4-fold greater induction of TNF- after infection with Ad.Egr-TNF compared with Ad.Egr-TNF alone at 96 hours (P < 0.02). TNF- and temozolomide combination leads to a synergistic decrease in U87 cell viability at 72 hours compared with either treatment alone (P < 0.001). Median survival for animals treated with Ad.Egr-TNF alone, temozolomide alone, and Ad.Egr-TNF/temozolomide was 21, 28, and 74 days, respectively (P < 0.001 by log-rank). Flow cytometric assessment of apoptosis revealed a synergistic increase in U87 cell apoptosis in vitro at 72 hours (P < 0.05), and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) evaluation of tumor sections revealed significantly increased TUNEL-positive cells after combination treatment compared with either treatment alone (P < 0.05). In conclusion, combination treatment with transcriptionally activated intratumoral TNF- and systemic temozolomide significantly prolongs survival in an experimental glioblastoma multiforme model.

	Introduction

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Despite aggressive multimodal therapy, median survival of patients with malignant glioma is less than 1 year. Chemotherapy has been shown to modestly increase survival in patients who fail surgery and radiotherapy (1) and temozolomide, an oral alkylating agent, has recently been favored in both the recurrent and upfront setting (2 , 3) . This improvement in patient survival with chemotherapy, however, remains on the order of weeks due to both an inherent chemo- and radioresistance of glioma cells and an inability to deliver high concentrations of chemotherapeutic drug because of systemic and neurologic toxicity. Chemo/radiotherapy-activated gene therapy is a developing treatment paradigm that attempts to improve the therapeutic index of a variety of potentially therapeutic compounds. Tumor necrosis factor (TNF-) is a cytokine with significant anticancer activity (4) . Although toxicity has limited its systemic use (5) , a synergistic antitumor effect is seen when it is used at high concentrations in regional perfusion strategies with alkylating agents (6) . The replication-defective adenoviral vector Ad.Egr-TNF consists of a chimeric gene encoding CArG elements of the chemoinducible/radioinducible early growth response-1 (egr1) gene promoter ligated upstream to the human TNF- cDNA. This vector has been shown to be induced to express TNF- by the drug cisplatin (7) . In this study, we show that temozolomide can induce the intratumoral (i.t.) expression of TNF- from Ad.Egr-TNF and that combination treatment with this vector and temozolomide leads to a significant prolongation in survival in an intracranial xenograft model of malignant glioma. The primary mechanism of this antiglioma effect appears likely to be due to a synergistic enhancement of tumor cell apoptosis by the combination of TNF- and temozolomide.

	Materials and Methods

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Animals, Cells, and Vector
Six- to 7-week-old female athymic nude mice (Frederick Cancer Research Institute, Frederick, MD) were used, and surgery was performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Chicago. The human glioblastoma cell line, U87 MG, was cultured in DMEM (Invitrogen Life Technologies, Inc., Carlsbad, CA) supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 µg/mL) at 37°C and 5% CO₂. Ad.Egr-TNF (ViraQuest Inc., North Liberty, IA) was stored at –80°C and diluted in formulation buffer to the appropriate concentration. Temozolomide (Schering Corp., Kenilworth, NJ) was dissolved in DMSO with the final concentration not exceeding 0.1% (v/v).

Tumor Necrosis Factor Induction In vitro
U87 cells (10⁶) were plated and incubated overnight. The cells were then infected with Ad.Egr-TNF at 100 multiplicities of infection for 3 hours at 37°C. After incubation, 3.8 mL of complete medium with or without temozolomide were added. Conditioned media were harvested at 48 hours after treatment, and human TNF- production was quantified using a Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D System, Inc., Minneapolis, MN).

Tumor Necrosis Factor Induction In vivo
U87 cells (5 x 10⁶) in 100 µL of DMEM were injected subcutaneously (s.c.) into the right hind limb of nude mice. When tumors reached an average size of 200 mm³ (length x width x thickness/2), the tumor-bearing mice were randomized into four groups: (1) untreated control, (2) Ad.Egr.TNF alone, (3) temozolomide alone, and (4) Ad.Egr-TNF and temozolomide. Ad.Egr-TNF was injected i.t. at a dose of 2 x 10⁸ particle units (pu) each day. Two doses of temozolomide were given: 2.5 and 5 mg/kg/d by intraperitoneal (i.p.) injection 3 hours after vector. Four consecutive daily i.t. and i.p. injections were given, and control animals received i.t. and i.p. serum-free medium. Animals were euthanized on days 2 and 4 (i.e., 48 and 96 hours after treatment initiation), and tumors were harvested, snap-frozen in liquid nitrogen, and homogenized in radioimmunoprecipitation assay buffer [150 mmol/L NaCl, 10 mmol/L Tris (pH 7.5), 5 mmol/L EDTA (pH 7.5), 100 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, and 2 µg/mL aprotinin]. Protein was isolated, and concentration was measured using Protein Assay reagent (Bio-Rad Laboratories, Hercules, CA). TNF- levels in the supernatants were measured as described above.

U87 Cell Viability Studies
Trypan blue dye exclusion method was used. U87 cells (10⁴) were plated and incubated at 37°C overnight. Subsequently, 10 ng/mL TNF- [dose based on Staba et al. (8) ] and/or 100 µmol/L temozolomide-containing media [dose based on Hirose et al. (9) and in vitro induction studies below] were added for 3 hours and washed. At 24, 48, and 72 hours after exposure to agent, the cells were trypsinized, and the viable cell number per well was determined using a hemocytometer. 3-(4,5-dimethylthiazol(-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt colorimetric assay, per the manufacturer’s protocol (Cell Titer 96 Aqueous, One Solution cell proliferation assay; Promega, Madison, Wisconsin), was used to verify cell viability at 72 hours. Absorbance was read at 490 nm using an ELISA microplate reader after 1.5 hour, at 37°C. All of the studies were performed in triplicate.

Xenograft Studies
U87 hind limb xenografts were established as described above. Mice were randomized into the same groups, and treatment was initiated (day 0). Ad.Egr-TNF (2 x 10⁸ pu) was injected i.t. twice a week for four total injections, and 5 mg/kg temozolomide was given i.p. 3 hours after each vector injection for a total of 20 mg/kg. The dose of temozolomide used was approximately 0.2 LD₁₀ and was chosen to have modest antitumor effect but to not be curative based on previous studies (10) and data from our lab showing LD₅₀ for i.p. temozolomide to be approximately 500 mg/kg. Tumor volume was measured every 2 to 3 days as above. Fractional tumor volume (V/V₀ where V₀ = volume on day 0) was calculated and plotted. For intracranial experiments, 5 x 10⁵ U87 cells were inoculated into the right caudate nucleus on day 0 using a screw guide technique (11) . On day 5, mice were randomized into four groups as above, and 5 x 10⁸ pu of Ad.Egr-TNF in a 5-µL volume were injected once via the screw guide directly into the tumor. Temozolomide (5 mg/kg) was given i.p. 3 hours after intracranial vector inoculation. Four consecutive daily i.p. temozolomide injections were given to a total dose of 20 mg/kg. Control animals received serum-free medium i.t. and i.p. Daily assessment of animal appearance was made. Mice were followed until death or sacrificed when moribund. Mouse brains were harvested after intracardiac perfusion and fixed with 10% neutral buffered formalin. For terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) evaluation (see below), animals were sacrificed on day 7 after treatment (n = 3 per group).

Flow Cytometric Analysis of Apoptosis
Fractional DNA Content.
U87 cells (10⁵) were plated overnight at 37°C with 5% CO₂. The cells were then treated with TNF- (10 ng/mL) and/or temozolomide (100 µmol/L). At 72 hours, the cells were washed in PBS and fixed in ice-cold 70% (v/v) ethanol. The cells were washed twice, incubated in RNase (1 mg/mL) for 30 minutes at 37°C, and then incubated in propidium iodide solution (100 µg/mL) for 30 minutes at 4°C. Flow cytometric analysis was performed on a FACSort instrument (Becton Dickinson Immunocytometry Systems, San Jose, CA), and the data were analyzed using the CellQuest software (Becton Dickinson).

Annexin V Binding.
At 72 hours, cells were washed in PBS and incubated in the dark for 15 minutes with binding buffer containing 5 µL of Annexin V-FITC and 5 µL of propidium iodide (Annexin V-FITC apoptosis detection kit II; ref. 12 ). The data were analyzed by Flowjo analysis software (Tree Star Inc., Ashland, OR).

Histologic Analysis
(1) Paraffin embedded brains were sectioned (8 µm), stained with hematoxylin and eosin, and analyzed in a blinded fashion.

(2) TUNEL assay was performed in accordance with the manufacturer’s instructions (Chemicon, Temecula, CA) and analyzed blindly at x400 magnification by use of a computer-aided light microscope with reconstruction software (Neurolucida, Microbrightfield, VT). Number of TUNEL-positive cells per 10^–6 mm² was documented.

Statistical Analysis
Results are expressed as mean value ± SD. Statistical significance was taken as P < 0.05 using a one-tailed Student’s t test. Analysis of variance (ANOVA) was also used. Kaplan-Meier survival curves were plotted for the intracranial experiment and analyzed by the log-rank method.

	Results

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Tumor Necrosis Factor Induction and Assessment of U87MG Cell Viability.
We initially investigated whether temozolomide could induce the expression of TNF- from U87 cells infected with Ad.Egr-TNF. In vitro, there was no TNF- detected in the untreated control or temozolomide alone cells, however, after Ad.Egr-TNF infection,100 µmol/L temozolomide induced a 2.3-fold increase in TNF- expression compared with cells infected with vector alone (Fig. 1A) . We confirmed induction in vivo using hind limb xenografts; again there was no TNF- detected in the untreated control or temozolomide alone animals, however, after combination treatment with Ad.Egr-TNF/temozolomide, 287 ± 111 pg TNF-/mg protein were detected at 96 hours—6.4-fold the value in the Ad.Egr-TNF alone animals (n = 3 animals per group, P = 0.02; Fig. 1B ). To determine whether this combination treatment strategy could result in a significant antitumor response, the cytotoxic effect of TNF- and temozolomide on glioma cell viability was evaluated in vitro. TNF- (10 ng/mL) and 100 µmol/L temozolomide alone had minimal effects on U87 cell viability; however, combination treatment lead to a significant reduction in cell viability, the magnitude of which was greater than the sum of the reductions of either treatment alone. Support for a synergistic interaction between TNF- and temozolomide was observed after ANOVA assessment (P = 0.0016; Fig. 2A and B ).

View larger version (24K): [in this window] [in a new window]   Fig. 1. A, in vitro tumor necrosis factor (TNF-) induction. U87 cells infected with Ad.Egr-TNF at 100 multiplicities of infection for 3 hours were treated with 100 or 200 µmol/L temozolomide. TNF- production was detected by ELISA at 48 hours in untreated controls (UTC), Ad.Egr-TNF alone, temozolomide alone, and combination Ad.Egr-TNF and temozolomide (performed in triplicate). B, in vivo TNF- induction. Hind limb xenografts were treated with a combination of 2 x 108 pu of Ad.Egr-TNF i.t. and either 2.5 mg/kg/d or 5 mg/kg/d temozolomide i.p. 3 hours after vector. Tumors were harvested on days 2 and 4 (i.e., 48 and 96 hours after treatment initiation); protein concentration was calculated, and TNF- was detected by ELISA. The values show the mean ± SD, P = 0.02 combination Ad.Egr-TNF and 5 mg/kg/d temozolomide verses Ad.Egr-TNF alone at 48 and 96 hours (n = 3 animals per group).

View larger version (18K): [in this window] [in a new window]   Fig. 2. U87 cell viability. U87 cells were treated with 10 ng/mL TNF- and 100 µmol/L temozolomide for 3 hours and cell viability was measured. Four groups were assessed: (1) untreated control, (2) TNF- alone, (3) temozolomide alone, and (4) TNF- and temozolomide (performed in triplicate and repeated). A, trypan blue dye exclusion method at 24, 48, and 72 hours after treatment. B, MTS colorimetric assay at 72 hours (performed in triplicate and repeated). Values represent mean ± SD; evidence for synergy is supported by the findings that (1) the magnitude of the reduction in viability with both treatments together is greater than the sum of the reductions for each treatment separately, and (2) ANOVA model P = 0.0016 for TNF- and temozolomide compared with temozolomide alone.

View larger version (23K): [in this window] [in a new window]   Fig. 3. A, U87 hind limb xenograft growth curves. Animals were randomized into four groups: (1) untreated control, (2) Ad.Egr-TNF alone, (3) temozolomide alone, and (4) Ad.Egr-TNF and temozolomide. Ad.Egr-TNF was injected i.t. (2 x 108 pu in 100 µL of medium) twice per week, and temozolomide was given i.p. at 5 mg/kg 3 hours after vector injection to a total of 20 mg/kg. Fractional tumor volume V/V0 (where V0 = volume on day 0) was calculated and plotted. Values represent mean V/V0± SD, P < 0.02 at day 20 of Ad.Egr-TNF/temozolomide versus temozolomide alone (n = 10 per group; repeated with similar results). B, Kaplan-Meier survival curves of nude mouse intracranial xenografts. Ad.Egr-TNF (5 x 108 pu) was injected once i.t., and four consecutive daily i.p. injections of 5 mg/kg temozolomide were given; animals were sacrificed when moribund. Statistical significance, P < 0.001 by log-rank method Ad.Egr-TNF and temozolomide compared with temozolomide alone (n = 9 per group; repeated with similar results).

View larger version (35K): [in this window] [in a new window]   Fig. 4. A and B, flow cytometric analysis of U87 cells at 72 hours after treatment with 10 ng/mL TNF- and 100 µmol/L temozolomide for 3 hours. A, analysis of DNA fragmentation after staining with propidium iodide. B, evaluation of Annexin V binding and propidium iodide staining. The magnitude of the increase in apoptosis after both TNF- and temozolomide treatment together is greater than the sum of the increases after each separately, ANOVA gives P < 0.05 Annexin V-positive cells treated with TNF- and temozolomide compared with those treated with temozolomide alone (experiments were repeated and performed in triplicate). C, TUNEL assay of U87 intracranial tumors sacrificed on day 7 post-treatment. TUNEL-positive cell number per 10–6 mm2 was recorded; statistical significance, P < 0.05 Ad.Egr-TNF/temozolomide verses TNF- or temozolomide alone (n = 3 per group).

	Discussion

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In isolated limb perfusion studies, where high dose TNF- is used with an alkylating agent, a synergistic interaction has been documented that has been shown to be due to an increase in tumor necrosis possibly due to an increase in vascular permeability leading to an increase in i.t. drug concentration (16) . A similar pattern of tumor necrosis has also been observed when radiotherapy is combined with Ad.Egr-TNF in a flank glioma model (8) . Although tumor necrosis was not observed histologically in our experiments, we did observe a significant increase in tumor cell apoptosis both in vitro and in vivo. However, we and others have shown that neither TNF- alone nor temozolomide alone results in significant apoptosis in glioma cells (9 , 17) . Considered together, these data strongly suggest that there is a direct interaction between TNF- and temozolomide in glioma cells that enhances apoptosis, resulting in the therapeutic benefit reported in our experiments.

A therapeutic increase in tumor cell apoptosis has been speculated to be a desirable goal of novel glioma therapies (18) , particularly because tumor necrosis has been associated with a significantly worse prognosis in glioblastoma multiforme patients (19) . However, additional studies are necessary to determine the mechanism involved in the induction of apoptosis and whether treatment-induced apoptosis yields a greater therapeutic ratio in malignant glioma than therapeutically induced necrosis.

Mortality from malignant glioma is related primarily to recurrent disease, which is almost universally local (nonmetastatic) in nature (20) . For this reason, these tumors are an ideal target for such a regionally activated treatment strategy. The considerable antitumor efficacy demonstrated in this series of experiments provides preliminary support for the potential use of this treatment paradigm in recurrent glioma patients. Although the animals in the treatment groups were noted to be healthy, formal toxicity studies will need to be performed before making conclusions regarding the potential toxic effects of this treatment.

	FOOTNOTES

 
Grant support: Cancer Research Foundation Young Investigator award and NIH grant 5P50CA97247.

Note: D. W. Kufe and R. R. Weichselbaum are consultants to and have an equity interest in Gen Vec Corporation, Gaithersburg, MD.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Bakhtiar Yamini, MC 4066, Section of Neurosurgery, 5841 South Maryland Avenue, University of Chicago Hospitals, Chicago, IL 60637. Phone: 773-702-2475; Fax: 773-702-5234; E-mail: byamini{at}surgery.bsd.uchicago.edu

Received 6/15/04. Revised 7/16/04. Accepted 8/ 3/04.

	REFERENCES

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