ARTICLE

Regression of Tumor Growth and Induction of Long-Term Antitumor Memory by Interleukin 12 Electro-Gene Therapy

Shulin Li, Xinjian Zhang, Xueqing Xia

Affiliation of authors: Department of Otolaryngology/Head and Neck Surgery, Henry Ford Health System, Detroit, MI.

Correspondence to: Shulin Li, Ph.D., Department of Otolaryngology/Head and Neck Surgery, Henry Ford Health System, 2799 W. Grand Blvd., Detroit, MI 48202 (e-mail: li200248083{at}yahoo.com).


    ABSTRACT
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Interleukin 12 (IL-12) is a proinflammatory cytokine with antitumor activity. Plasmid-based intratumoral gene therapy for treating malignancy with IL-12 or other genes is safe, inexpensive, and simple to carry out. However, effective delivery methods for injecting DNA plasmid into a tumor to generate therapeutic levels of gene product are lacking. To overcome this obstacle, we used electroporation to deliver the IL-12 gene intratumorally in a murine squamous cell carcinoma (SCC) model, SCCVII. Methods: Plasmids with or without mouse IL-12 gene were injected into SCCVII tumors of C3H/HeJ mice (n = 5 per group). Electric pulses were then applied to the tumors, a process termed electro-gene therapy. The first treatment was administered when the tumor reached 4–6 mm in diameter, and the second treatment followed a week later. The tumor size, survival, and ability to generate systemic antitumor memory were assessed at various time intervals. Changes in gene expression were measured using northern blot analysis, and vessel density and T-cell infiltration were examined by immunostaining. The results were analyzed by two-sided Student's t tests. Results: Electroporation of 20 µg or 40 µg of IL-12 DNA plasmid eradicated tumors in 40% of mice (P = .031 and .022, respectively). A total of six mice from two separate experiments with regressed tumors were challenged with homologous SCCVII tumor cells multiple times; three of six mice showed no tumor growth for more than 11 months and thus indicated the generation of antitumor memory in these mice. IL-12 electro-gene therapy was associated with increased expression of IL-12, interferon-{gamma}, monokine induced by interferon-{gamma}, and interferon-inducible protein 10. IL-12 electro-gene therapy was also associated with decreased vessel density and increased infiltration of CD8+ T cells after the second administration (P = .02 and .03, respectively). Conclusion: IL-12 electro-gene therapy appears to be effective in reducing tumor growth by triggering both antiangiogenic effects and an immune response. The antitumor memory was seen to last more than 11 months. Because IL-12 electro-gene therapy is easy to administer and is effective, it could potentially be applicable in the treatment of electrode-accessible malignancies, such as head and neck SCCs.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interleukin 12 (IL-12), a heterodimeric proinflammatory cytokine, is produced primarily by dendritic cells, monocytes/macrophages, and neutrophils. It plays a critical role in producing an immune response, as indicated in many ways; for example, induction of interferon-{gamma} (IFN-{gamma}) and tumor necrosis factor-{alpha} (TNF-{alpha}) (13), differentiation of naive T cells into the Th1 phenotype that is important for initiating cell-mediated antitumor effect (4, 5), and augmentation of the cytotoxic activity of resting natural killer (NK) cells and activated T cells (6). More importantly, IL-12 elicits a potent systemic antitumor response in a variety of tumors (7, 8).

Although IL-12 protein therapy was effective in multiple tumor models and clinical studies (911), preclinical studies and early clinical trials have demonstrated that it causes systemic toxicity. Also, it requires daily injections (12, 13). Multiple preclinical models demonstrated the effectiveness of viral IL-12 gene therapy (14, 15), but there are concerns about the immunogenicity and complexity in manufacturing the viral particles. In contrast, plasmid containing IL-12 gene is simple to manufacture, and plasmid-based IL-12 gene therapy is easy to execute. However, this approach is limited by a shortage of effective delivery methods that can be used to achieve therapeutic levels of gene expression. As a result, only small-size tumors can be controlled by most of the nonviral IL-12 gene therapy approaches, such as a polymer-based gene delivery (16). Thus, it is critical to enhance the efficiency of the gene delivery method for nonviral gene therapy to make this approach more applicable in a clinical setting.

Electroporation is a very powerful technique for delivering DNA to many tissues in vivo (17, 18). It uses an electric pulse to create transient aqueous pathways (or pores) in the cell membrane, through which the plasmid can gain entry into the cell. We have previously demonstrated (19) that delivery of a reporter gene by electroporation into the muscle enhances gene transfection efficiency and the level of gene expression by approximately 100-fold to 1000-fold. We have also demonstrated inhibition of remote tumor growth after intramuscular injection of IL-12 DNA plasmid by electroporation (20), which demonstrates a potential of preventing recurrence or inhibiting microscopic malignancies after surgery. However, to date, it has not been determined whether delivery of IL-12 plasmid DNA directly into tumors via electroporation will regress relatively large squamous cell carcinoma (SCCVII) tumors and induce long-term systemic antitumor protection, as compared with what is achievable by viral IL-12 gene therapy (2123).

Here, we hypothesize that intratumoral IL-12 electro-gene therapy given in optimal doses and at a specified delivery schedule will eradicate relatively large SCCVII solid tumors and will create long-term immune protection by inducing the expression of antitumoral genes.


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Experimental Animals

We purchased 6-week-old to 8-week-old female C3H/HeJ mice weighing 18–20 g from the Jackson Laboratory (Bar Harbor, ME) and maintained them under National Institutes of Health guidelines that were approved by Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences where the work began.

Preparation of IL-12 DNA for Animal Application

Valentis, Inc. (The Woodlands, TX) provided the IL-12 gene construct. Interferon-inducible protein 10 (IP-10) was cloned into a backbone (24) after obtaining it by reverse transcription–polymerase chain reaction (RT–PCR) technique. The control construct for IL-12 gene therapy was prepared by deleting the DNA fragment encoding mouse IL-12 from the IL-12 DNA plasmid (25). We manufactured all plasmids using the QIAGEN Endo-Free Prep Kit (Valencia, CA) and performed quality control such as gel electrophoresis analysis and restriction enzyme digestion analysis, as described previously (24).

Generation of SCCVII Tumors in Mice and Monitoring Tumor Growth

SCCVII is a spontaneously arising murine squamous cell carcinoma. We obtained this cell line from Dr. Candice Johnson's laboratory at the University of Pittsburgh (Pittsburgh, PA) and maintained the cells in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS; Life Technologies, Rockville, MD). We inoculated mice subcutaneously with 1 x 105 SCCVII cells in a 30 µL volume. When the established tumors reached a measurable size (4–6 mm) in diameter, we randomly divided the animals into groups of five mice each for efficacy experiments, five to eight mice for vessel density and T-cell infiltration studies, and three to four mice for gene expression experiments. After initiation of IL-12 DNA treatment, we monitored tumor growth every 3 days for 3 weeks. We measured tumor diameters with a caliper and calculated the tumor volume with the formula: V = {pi}/8(a x b2), where V is the tumor volume, a is the maximum tumor diameter, and b is the diameter at 90 ° to a (26). At the completion of each experiment, we killed the mice with CO2 narcosis.

Electroporation of DNA Plasmid into Tumors

We formulated purified DNA plasmids in 150 mM NaCl solution (24). After anesthetizing animals by intraperitoneal administration of a mixture of ketamine (42.8 mg/mL), xylazine (8.6 mg/mL), and acepromazine (1.4 mg/mL) at a dose of 1.8–2.0 mL/kg, we injected the tumors using a 28.5-gauge needle with a 50-µL formulation containing 20 µg or 40 µg DNA plasmid. We then applied electric pulses to the tumors by positioning two heads of a caliper electrode to two sides of the tumor at a distance of 5 mm, keeping contact with the skin. A power supply device (ECM 830; Genetronics, San Diego, CA) applied square wave pulses to the tumor through the electrode with the following parameters: two 20-msec pulses at 400 V/cm and a 200-µsec interval between the pulses (the square wave pulses stop immediately after the pulsing is over and cause no postpulse heating). We established this pulse protocol on the basis of maximum level of expression of reporter gene luciferase (data not shown).

Protocol for IL-12 DNA Treatment

The protocol to establish tumors in mice and to deliver DNA into the tumor by electroporation is described above. The initial DNA administration was performed when the tumor reached 4–6 mm in diameter, and the second administration was performed 1 week later. The subsequent administrations were performed every 4 days. In all experiments, we used a 20-µg DNA plasmid dose unless specified otherwise.

To determine whether electroporation of IL-12 DNA plasmid led to a high level and long duration of IL-12 expression, we compared IL-12 expression levels in the tumor extracts and in sera of mice treated with 20 µg of IL-12 DNA plasmid or the control plasmid given via injection with or without electroporation following injection. Four mice were used for each treatment or control group. The initial treatment was performed when the tumor reached 4–6 mm in diameter, and the second administration was performed 1 week after the first administration. Two days after the second administration of the DNA plasmid, we obtained blood from the retro-orbital plexus for gene expression analysis and then killed the mice and harvested the tumors for additional analyses.

To determine the effect of IL-12 electro-gene therapy on tumor growth, we treated mice (n = 5 per group) with IL-12 DNA or control DNA plasmid (with or without electroporation) a total of four times, beginning 12 days after tumor inoculation, when the tumor diameter reached approximately 5 mm. Because preliminary studies indicated negligible differences between the control plasmid delivery by injection and control plasmid delivery by electroporation, we discontinued the former control group in an effort to reduce the number of animals used. The different times when plasmid DNA was administered is indicated by arrows in the appropriate figures (see Fig. 2, A and BGo).



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Fig. 2. Interleukin 12 (IL-12) electro-gene therapy induces statistically significant therapeutic effects in mice (n = 5 in each group). Error bars represent mean ± 95% confidence intervals. The initial administration was performed when tumors reached 4–6 mm in diameter, and arrows indicate the times of administration. E+ = DNA delivered into tumors by electroporation; pCtrl and pIL-12 represent control and IL-12 DNA plasmids, respectively. A) Inhibition of tumor growth by IL-12. The tumor growth was statistically significantly inhibited by the IL-12 electro-gene therapy at the end of 3 weeks following the treatment (P = .025). B) Dose-dependent inhibition of tumor growth by IL-12. C) Increase in survival rate after IL-12 treatment (P = .031 for 20-µg pIL-12 dosage, and P = .022 for 40-µg pIL-12 dosage, respectively). Mice bearing tumors bigger than 2 cm in diameter were considered as dead mice. The average survival rate (95% confidence interval) for pIL-12 treatment groups and control group during the period of day 25 to 80 days after inoculation of tumor cells are 69.4 (53.7 to 85.1) for the dosage of 20 µg of pIL-12, 86.1 (71.8 to 100.4) for the dosage of 40 µg of pIL-12, and 27.7 (2.5 to 52.7) for the dosage of 40 µg of pCtrl, respectively. The mice at risk from groups receiving 20 µg of pIL-12, 40 µg of pIL-12, and 40 µg of pCtrl (control DNA) on day 30 were four of five, four of five, and four of five, respectively; on day 60: two of five, four of five, and zero of five, respectively; and on day 90: two of five, two of five, and zero of five, respectively. For both pIL-12 DNA treatment groups, there were 40% of mice with tumor regression, and the same mice survived for 365 days (data not shown).

 
Analysis of IL-12, IFN-{gamma} and Vascular Endothelial Growth Factor (VEGF) by Enzyme-Linked Immunosorbent Assay (ELISA)

To obtain serum for gene expression analysis, we centrifuged the blood at 4000 rpm (2500g) for 5 minutes at room temperature, using serum separators (BD Biosciences, Franklin Lakes, NJ) for separation. We also harvested tumors from the killed animals. The tumor tissues were snap-frozen in liquid nitrogen and lyophilized overnight. The tumor tissues were homogenized with the use of a Mini-Bead Beater (BioSpec Products, Bartlesville, OK) in the presence of 2-mm silica beads (19). We assayed the collected sera and the supernatants of the tumor extracts for VEGF, IL-12, and IFN-{gamma} expressions with an ELISA kit (R&D Systems, Minneapolis, MN), using a precision microplate reader to detect the color changes (Molecular Devices, Menlo Park, CA). We assayed total protein as previously described (27). VEGF, IL-12, and IFN-{gamma} were expressed as picograms/milligrams total protein or picograms/milliliters serum.

Immunostaining Analysis

We performed immunostaining to determine vessel density and T-cell infiltration. The vessels in the tumor tissues were stained using an antibody to CD31, an endothelial cell marker (1 : 200; BD Biosciences; Los Angeles, CA), and the infiltrated CD8+ T cells were stained with an anti-CD8 antibody (1 : 50; BD Bioscience) as previously described in detail (27). The number of vessels and the CD8+ T cells were scored from a minimum of four microscopic fields from five independent tumors treated with IL-12 or control DNA plasmid injected by electroporation as described above. The average number of vessels per field was determined under a microscope at x20 magnification, and the average number of CD8+ T cells per field was determined under a light microscope at x10 magnification.

Analysis of Expression IP-10 and Monokine Induced by Interferon-{gamma}

The expression of IP-10 and monokine induced by interferon-{gamma} (Mig) was determined by northern blot analysis, as described in our previous publication (19). The level of IP-10 and Mig expression was quantified by scanning the signal intensity with a PhosphorImager analyzer (Model 445 SI; Molecular Dynamics, Sunnyvale, CA). To simplify the results, the level of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was artificially defined as 1.

Statistical Analysis

Tumor growth; the number of vessels and CD8+ T cells; and the expression levels of IL-12, VEGF, and IFN-{gamma} were the primary outcomes measured. We used a one-way analysis of variance (ANOVA) to analyze experimental data and the two-sided Student's t test to compare means of individual treatments when the primary outcome was statistically significant. The survival analysis was performed using chi-square test. P values less than .05 were considered statistically significant.


    RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Enhancement of Functional IL-12 Expression in Tumors by Electroporation of IL-12 DNA into Tumors

Analysis of the tumor extract from mice treated with IL-12 DNA plasmid (20 µg) via electroporation showed on day 2—after the second administration—an average of 143 pg/mg total protein (95% confidence interval [CI] = 69.9 to 216.1) of IL-12 expressed in tumors. This expression was about ninefold higher than IL-12 expression in tumors receiving control plasmid DNA in the same manner (Fig. 1, AGo). The level of IL-12 detected from injection of IL-12 DNA plasmid without electroporation was similar to that of the control plasmid and was 15.7 pg/mg total protein (95% CI = 11.0 to 20.4). Thus, electroporation of IL-12 DNA resulted in a statistically significantly (P = .015) higher level of IL-12 expression compared with the control groups; that is, electroporation of control DNA plasmid or injection of IL-12 DNA plasmid alone without electroporation.



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Fig. 1. Electroporation of interleukin 12 (IL-12) DNA compared with control DNA increases the level of IL-12 expression (picograms/milligrams total protein) in tumors (A; P = .015) and induces interferon-gamma (IFN-{gamma}) (picograms/milligrams total protein) (B; P = .041) in vivo. pCtrl and pIL-12 represent control and IL-12 DNA plasmids, respectively. Each bar represents an individual animal (n = 4 per group). The control DNA plasmid was prepared by deletion of the DNA fragment encoding IL-12 from the IL-12 DNA plasmid. Twenty micrograms of DNA plasmid was injected into each tumor by electroporation as described in "Materials and Methods," and tumors were harvested 2 days after the second administration.

 
To determine whether the IL-12 protein produced in tumors after electroporation of the IL-12 DNA plasmid was biologically functional, we analyzed the level of IFN-{gamma} induction. Determination of IFN-{gamma} was chosen because the induction of IFN-{gamma} expression by IL-12 is a hallmark of IL-12 function. Indeed, IFN-{gamma} production was statistically significantly increased (P = .041) after electroporation of the IL-12 DNA plasmid into the tumors. The average value for the IFN-{gamma} level in tumor tissue was 197 pg/mg total protein (95% CI = 52.8 to 341.2) on day 2 after the second administration of IL-12 DNA plasmid by electroporation. This level was 12-fold higher than the level detected in tumors injected with the control plasmid DNA (16.4; 95% CI = 9.5 to 23.3) (Fig. 1Go, B). Thus, the IL-12 product generated after electroporation of the DNA plasmid into tumor was biologically functional.

Inhibition of SCCVII Tumor Growth by IL-12 Electro-Gene Therapy

Tumor growth was statistically significantly reduced by the end of 3 weeks (P = .025) in animals receiving IL-12 DNA via electroporation compared with the groups receiving control plasmid DNA (n = 5 in each group). On day 4 after the fourth administration or 31 days after the inoculation of tumor cells, a 15-fold reduction in tumor size compared with that of the control group was observed in the group treated with IL-12 electro-gene therapy (Fig. 2, AGo).

To determine whether the therapeutic effect was dose dependent, and to determine whether the survival was prolonged, in a separate experiment we treated tumors in animals (n = 5 per group) with either 20 µg or 40 µg of IL-12 DNA plasmid or 40 µg of control DNA plasmid via electroporation, for a total of four administrations at the schedules indicated by the arrows in Fig. 2Go, B. The higher dose of IL-12 DNA demonstrated a more statistically significant inhibition of tumor growth than the low-dose treatment (Fig. 2, BGo). However, tumors were eradicated in 40% of the animals treated with either dosage of IL-12 DNA plasmid delivered by electroporation, and their survival was prolonged beyond 80 days, compared with the control mice, which died by day 40 after tumor cell inoculation (Fig. 2, CGo).

We rechallenged the tumor-free mice obtained from two separate experiments (total n = 6) with 5 x 105 SCCVII tumor cells, which was five times the number of cells used for the initial tumor inoculation, and three of the six mice remained tumor free. These three mice were rechallenged once per month for 11 months and remained tumor free, demonstrating that a long-term antitumor memory was elicited by intratumoral electroporation of IL-12 DNA plasmid. After 12 months, these mice were killed.

Increased Infiltration of T Cells into Tumors and Inhibition of Angiogenesis by IL-12 Electro-Gene Therapy

To determine the cellular mechanism(s) responsible for the increased antitumor efficacy exhibited by the intratumoral electroporation of IL-12 DNA (20 µg), we analyzed the infiltration of T cells and the vessel density of the tumors using antibodies against the T cells and endothelial cell marker CD31, respectively (Table 1Go). The immunostaining results demonstrated that the vessel density was reduced statistically significantly (P = .001 and .02, respectively) by IL-12 electro-gene therapy compared with injection of control DNA plasmid after the first and second administration. However, the increase in CD8+ T-cell infiltration between control and IL-12-treated groups was statistically significant (P = .03) only after the second administration (Table 1Go). There was no statistically significant increase in the infiltration of CD4+ T cells (data not shown). These results suggest that the increased antitumor effect of IL-12 electro-gene therapy is caused by both an enhanced antiangiogenesis effect and possibly a T-cell-mediated immune response.


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Table 1. Vessel density and CD8+ T-cell infiltration 2 days after each interleukin 12 (IL-12) administration*
 
Enhanced Expression of IP-10 and Mig in Tumor Tissue by IL-12 Electro-Gene Therapy

The increase in CD8+ T-cell infiltration and decrease in vessel density observed in our study implicated the involvement of molecules that can chemoattract T cells and inhibit angiogenesis. It has been shown that IP-10 and Mig are regulated by IFN-{gamma} to chemoattract immune cells and inhibit angiogenesis (28, 29) and that IL-12 augments IFN-{gamma} production (30). To determine whether IP-10 and Mig showed increased expression by IL-12 treatment, we analyzed their expression levels in tumors harvested from three individual mice 2 days after the second delivery of 20 µg (lanes 4–6) or 40 µg (lanes 7–9) of IL-12 DNA plasmid or 40 µg of control DNA plasmid (lanes 1–3) via electroporation (Fig. 3Go). Northern blot analysis showed an increase in the expression levels of IP-10 and Mig in all the tumor samples receiving IL-12 gene therapy. The levels of Mig and IP-10 expression increased 15-fold and fivefold, respectively, on the second day after the second administration compared with the injection of control DNA plasmid (40 µg).



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Fig. 3. Interleukin 12 (IL-12) electro-gene therapy induces expression of monokine induced by interferon-{gamma} (Mig) and interferon inducible protein 10 (IP-10) in tumors (n = 3) as determined by northern blot analysis. Twenty micrograms of IL-12 DNA (lanes 4–6), 40 µg of IL-12 DNA (lanes 7–9), and 40 µg of control DNA (lanes 1–3) plasmids were injected into the tumors by electroporation. pCtrl and pIL-12 represent control and IL-12 DNA plasmids, respectively. Two days after the second administration, total RNA was prepared from tumor tissues and the expression of IP-10 and Mig were determined by northern blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as an internal control (housekeeping gene) for the expression level of IP-10 and Mig. Each bar represents an individual animal (n = 3 per group). The level of IP-10 and Mig expression was quantified by scanning the expression signal intensity with a PhosphorImager analyzer (Model 445 SI; Molecular Dynamics, Sunnyvale, CA). To simplify the results, the level of GAPDH expression (control) was artificially defined as 1, and then the mean expression values (95% confidence interval) for Mig and IP-10 in the IL-12 DNA-treated groups were 15.4 (8.6 to 22.2) and 5.1 (2.7 to 7.5), respectively, and for the control groups were 1 (0.6 to 1.4) and 1 (0.7 to 1.3), respectively.

 
Expression of VEGF in Tumors After Administration of IL-12 DNA by Electroporation

To determine whether the antiangiogenic effect observed with IL-12 electro-gene therapy was a result of decreased expression of VEGF, we analyzed VEGF expression levels in both the serum and tumor tissue from treated animals (electroporation of IL-12 DNA or control DNA plasmids). Results showed that 2 days after the first administration of IL-12 DNA plasmid (20 µg), the level of VEGF expression in either the tumor or serum from the two treatment groups was similar (Fig. 4, A and BGo). However, the expression level was decreased in the tumor tissue after the second administration in animals receiving IL-12 DNA by electroporation compared with injection of control DNA plasmid, although it was not statistically significant (P = .07), whereas the serum expression levels remained the same (P = .218) (Fig. 4, A and BGo).



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Fig. 4. Vascular endothelial growth factor (VEGF) expression in the tumor (A) and serum (B) 2 days following the second administration of interleukin 12 (IL-12) gene (pIL-12) or control DNA (pCtrl). The second administration was performed 1 week after the first administration. Twenty micrograms of DNA plasmid was delivered into tumors by electroporation. Each bar represents an individual animal (n = 4 and n = 3 per group for the first and second administration, respectively).

 

    DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The electroporation technique creates transient pores in the cell membrane through which DNA plasmid molecules can gain entry into the cell. This has been tested in multiple tissues in vivo, including tumors (18, 31). Our results clearly demonstrate the ability of electroporation in vivo to achieve an adequate expression level for IL-12 following electroporation-based gene therapy, resulting in an efficacious treatment of SCCVII tumors. An increased level of IL-12 expression and enhanced antitumor effect by IL-12 electro-gene therapy was also reported in other tumor models this year (3235) while this manuscript was in preparation, demonstrating that intratumoral IL-12 electro-gene therapy has become a hot spot of nonviral gene therapy. As far as we know, this was the first demonstration of the generation of long-term systemic antitumor protection (more than 11 months) following IL-12 electro-gene therapy.

Tumor eradication was observed in 40% of mice that received IL-12 electro-gene therapy in independent experiments (Fig. 2, CGo); the same tumor-free mice lived for 365 days after tumor inoculation (data not shown). To reduce the potential suffering of patients who in a clinical setting may receive too frequent administrations, we recently tested the following options in mice: We increased the electroporation voltage from the previous 400 V/cm to 500 V/cm and the interval to once every 10 days at a dose of 40 µg of IL-12 DNA plasmid. As a result, 80% of the mice became tumor free after two administrations (data not shown). Because we initiated gene therapy when the tumor was approximately 5 mm in diameter (the size reported in the literature for viral-based transfer of IL-12), our results demonstrated the same effectiveness as viral IL-12 gene therapy (21, 22). Eradication of relatively large tumors by IL-12 electro-gene therapy lately has been observed in other tumor models (34, 35). Thus, IL-12 electro-gene therapy was comparable to some viral gene therapies in the animal models. In addition, others have demonstrated that intratumoral electroporation of IL-12 gene directly into melanoma in a mouse model has less risk of toxicity than adenovirus IL-12 gene therapy. No increased level of IL-12 expression was detected systemically after electroporation (32); we observed similar results in our study (data not shown). Thus, less toxicity was expected with the IL-12 electro-gene therapy approach.

The mechanism by which IL-12 gene therapy elicits an antitumoral activity is complex. The results determined in other tumor models suggest that the mechanism of IL-12 gene action is dependent on the specific model examined. For melanoma, the antitumor effect elicited may be exclusively caused by its antiangiogenic effect (36), whereas for other tumor models, augmentation of CD8+ T-cell cytotoxic activity may be the major mechanism (9, 16, 37). The role of CD4+ T cells in tumor inhibition by IL-12 is not clearly understood and may depend on the tumor model and on the amount and timing of IL-12 production (26). Our data suggest that both antiangiogenic effect and CD8+ T-cell response may be responsible for the antitumor effect elicited by IL-12 electro-gene therapy. This is because there was a decreased vessel density and an increased infiltration of CD8+ T cells (Table 1Go) in tumors from mice receiving the intratumoral injection of IL-12 DNA plasmid by electroporation, and long-term antitumor immune protection was generated in 50% of tumor-free mice. The dual effect of IL-12 electro-gene therapy was also observed in hepatocellular carcinoma, in which the decreased vessel density and generation of the cytotoxic T lymphocyte response were observed (33).

Our data also suggest that a two-phase mechanism for IL-12 antitumor action may exist: an antiangiogenic phase (phase I) and an antitumor immune response phase (phase II). In phase I, there was no net increase in CD8+ T-cell infiltration, only decreased vessel density, after the first administration of IL-12 DNA by electroporation (Table 1Go). In phase II, there was both an increased infiltration of CD8+ T cells and a decreased vessel density after the second administration (Table 1Go). Because the increased expression of IP-10 and Mig inhibits endothelial cell proliferation and vessel formation (28, 29, 3841) and is chemotactic for NK cells, monocytes, and activated T cells (42, 43), we can infer that the therapeutic effect seen in our tumor model was manifested in two phases at the cellular level. The dual effects of therapy, that is, inhibition of angiogenesis and CD8+ T-cell response, may have been a result of the increased expression of Mig and IP-10 in the tumor tissue (Fig. 3Go).

Although the expression of VEGF was not statistically significantly decreased in the tumor treated with IL-12 electro-gene therapy (P = .07), it tended to be lower in the tumors treated with IL-12 electro-gene therapy (Fig. 4, A and BGo) than in the tumors treated with control DNA plasmid by electroporation. However, this was probably not the cause of the reduced vessel density observed after electroporation of the IL-12 gene but, rather, a result of a reduction in endothelial cells that produce VEGF. This is based on the timing of the observed effect: The decreased vessel density was detected after the first administration of IL-12 DNA by electroporation, but the reduced VEGF expression was observed only after the second administration (Fig. 4Go). We speculate that the reduction of VEGF expression after the second administration may be caused by accumulation of VEGF inhibitory factors, and the exact mechanism is not clear.

In conclusion, delivery of the IL-12 gene by electroporation into tumors is a simple and effective method of therapy against SCCVII tumors in a murine model. The mechanism of the antitumor effect was likely caused by IL-12-induced increased expression of IFN-{gamma}, Mig, and IP-10, which trigger both the immune response and antiangiogenic response. These results could rapidly be translated into the clinical trial, particularly for the treatment of SCC of the head and neck, because this type of cancer is more accessible for the needle electrode, and electroporation chemotherapy is already under evaluation in the clinical setting (44).


    NOTES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Supported by a grant from the Elsa Pardee Foundation. We express our appreciation to Valentis, Inc. for allowing us to use their gene constructs.


    REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received October 23, 2001; revised February 26, 2002; accepted March 22, 2002.



             
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