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).
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ABSTRACT |
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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We purchased 6-week-old to 8-week-old female C3H/HeJ mice weighing 1820 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 transcriptionpolymerase chain reaction (RTPCR) 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 (46 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 = /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 1.82.0 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 46 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 46 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 B).
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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- 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-
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-
The expression of IP-10 and monokine induced by interferon- (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- 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.
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RESULTS |
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Analysis of the tumor extract from mice treated with IL-12 DNA plasmid (20 µg) via electroporation showed on day 2after the second administrationan 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, A). 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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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, A).
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. 2, B. The higher dose of IL-12 DNA demonstrated a more statistically significant inhibition of tumor growth than the low-dose treatment (Fig. 2, B
). 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, C
).
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 1). 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 1
). 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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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- to chemoattract immune cells and inhibit angiogenesis (28, 29) and that IL-12 augments IFN-
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 46) or 40 µg (lanes 79) of IL-12 DNA plasmid or 40 µg of control DNA plasmid (lanes 13) via electroporation (Fig. 3
). 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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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 B). 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 B
).
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DISCUSSION |
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Tumor eradication was observed in 40% of mice that received IL-12 electro-gene therapy in independent experiments (Fig. 2, C); 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 1) 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 1). In phase II, there was both an increased infiltration of CD8+ T cells and a decreased vessel density after the second administration (Table 1
). 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. 3
).
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 B) 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. 4
). 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-, 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).
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NOTES |
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REFERENCES |
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1 Chan SH, Perussia B, Gupta JW, Kobayashi M, Pospisil M, Young HA, et al. Induction of interferon gamma production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers. J Exp Med 1991;173:86979.[Abstract]
2
Wu CY, Demeure C, Kiniwa M, Gately M, Delespesse G. IL-12 induces the production of IFN-gamma by neonatal human CD4 T cells. J Immunol 1993;151:193849.
3 Brunda MJ. Role of IL12 as an anti-tumour agent: current status and future directions. Res Immunol 1995;146:62228.[Medline]
4 Hsieh TT, Pao CC, Hor JJ, Kao SM. Presence of fetal cells in maternal circulation after delivery. Hum Genet 1993;92:2045.[Medline]
5 Manetti R, Parronchi P, Giudizi MG, Piccinni MP, Maggi E, Trinchieri G, et al. Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells. J Exp Med 1993;177:1199204.[Abstract]
6 Gately MK, Wolitzky AG, Quinn PM, Chizzonite R. Regulation of human cytolytic lymphocyte responses by interleukin-12. Cell Immunol 1992;143:12742.[Medline]
7 Brunda MJ. Interleukin-12. J Leukoc Biol 1994;55:2808.[Abstract]
8
Robertson MJ, Cameron C, Atkins MB, Gordon MS, Lotze MT, Sherman ML, et al. Immunological effects of interleukin 12 administered by bolus intravenous injection to patients with cancer. Clin Cancer Res 1999;5:916.
9 Brunda MJ, Luistro L, Warrier RR, Wright RB, Hubbard BR, Murphy M, et al. Antitumor and antimetastatic activity of interleukin 12 against murine tumors. J Exp Med 1993;178:122330.[Abstract]
10 Tannenbaum CS, Wicker N, Armstrong D, Tubbs R, Finke J, Bukowski RM, et al. Cytokine and chemokine expression in tumors of mice receiving systemic therapy with IL-12. J Immunol 1996;156:6939.[Abstract]
11
Rakhmilevich AL, Turner J, Ford MJ, McCabe D, Sun WH, Sondel PM, et al. Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors. Proc Natl Acad Sci USA 1996;93:62916.
12 Sarmiento UM, Riley JH, Knaack PA, Lipman JM, Becker JM, Gately MK, et al. Biologic effects of recombinant human interleukin-12 in squirrel monkeys (Sciureus saimiri). Lab Invest 1994;71:86273.[Medline]
13 Tare NS, Bowen S, Warrier RR, Carvajal DM, Benjamin WR, Riley JH, et al. Administration of recombinant interleukin-12 to mice suppresses hematopoiesis in the bone marrow but enhances hematopoiesis in the spleen. J Interferon Cytokine Res 1995;15:37783.[Medline]
14 Motoi F, Sunamura M, Ding L, Duda DG, Yoshida Y, Zhang W, et al. Effective gene therapy for pancreatic cancer by cytokines mediated by restricted replication-competent adenovirus. Hum Gene Ther 2000;11:22335.[Medline]
15 Mazzolini G, Qian C, Narvaiza I, Barajas M, Borras-Cuesta F, Xie X, et al. Adenoviral gene transfer of interleukin 12 into tumors synergizes with adoptive T cell therapy both at the induction and effector level. Hum Gene Ther 2000;11:11325.[Medline]
16 Mendiratta SK, Quezada A, Matar M, Wang J, Hebel HL, Long S, et al. Intratumoral delivery of IL-12 gene by polyvinyl polymeric vector system to murine renal and colon carcinoma results in potent antitumor immunity. Gene Ther 1999;6:8339.[Medline]
17 Titomirov AV, Sukharev S, Kistanova E. In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim Biophys Acta 1991;1088:1314.[Medline]
18 Somiari S, Glasspool-Malone J, Drabick JJ, Gilbert RA, Heller R, Jaroszeski MJ, et al. Theory and in vivo application of electroporative gene delivery. Mol Ther 2000;2:17887.[Medline]
19 Li S, Zhang X, Xia X, Zhou L, Breau R, Suen J, et al. Intramuscular electroporation delivery of IFN-alpha gene therapy for inhibition of tumor growth located at a distant site. Gene Ther 2001;8:4007.[Medline]
20 Hanna E, Zhang X, Woodlis J, Breau R, Suen J, Li S. Intramuscular electroporation delivery of IL-12 gene for treatment of squamous cell carcinoma located at a distant site. CA Gene Ther 2001;8:17.
21 Bramson JL, Hitt M, Addison CL, Muller WJ, Gauldie J, Graham FL. Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12. Hum Gene Ther 1996;7:19952002.[Medline]
22 Caruso M, Pham-Nguyen K, Kwong YL, Xu B, Kosai KI, Finegold M, et al. Adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma. Proc Natl Acad Sci USA 201996;93:113026.
23
Siders WM, Wright PW, Hixon JA, Alvord WG, Back TC, Wiltrout RH, et al. T cell- and NK cell-independent inhibition of hepatic metastases by systemic administration of an IL-12-expressing recombinant adenovirus. J Immunol 1998;160:546574.
24 Li S, MacLaughlin FC, Fewell JG, Li Y, Mehta V, French MF, et al. Increased level and duration of expression in muscle by co-expression of a transactivator using plasmid systems. Gene Ther 1999;6:200511.[Medline]
25 Blezinger P, Freimark BD, Matar M, Wilson E, Singhal A, Min W, et al. Intratracheal administration of interleukin 12 plasmid-cationic lipid complexes inhibits murine lung metastases. Hum Gene Ther 1999;10:72331.[Medline]
26 Puisieux I, Odin L, Poujol D, Moingeon P, Tartaglia J, Cox W, et al. Canarypox virus-mediated interleukin 12 gene transfer into murine mammary adenocarcinoma induces tumor suppression and long-term antitumoral immunity. Hum Gene Ther 1998;9:248192.[Medline]
27 Li S, Zhang X, Xia X, Zhou L, Breau R, Suen J, et al. Intramuscular electroporation delivery of IFN-alpha gene therapy for inhibition of tumor growth located at a distant site. Gene Ther 2001;8:4007.[Medline]
28 Aihara H, Miyazaki J. Gene transfer into muscle by electroporation in vivo.Nat Biotechnol 1998;16:86770.[Medline]
29 Belardelli F, Ferrantini M, Santini SM, Baccarini S, Proietti E, Colombo MP, et al. The induction of in vivo proliferation of long-lived CD44hi CD8+ T cells after the injection of tumor cells expressing IFN-alpha1 into syngeneic mice. Cancer Res 1998;58:5795802.[Abstract]
30 Yoshida A, Koide Y, Uchijima M, Yoshida TO. IFN-gamma induces IL-12 mRNA expression by a murine macrophage cell line, J774. Biochem Biophys Res Commun 1994;198:85761.[Medline]
31
Goto T, Nishi T, Tamura T, Dev SB, Takeshima H, Kochi M, et al. Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci USA 2000;97:3549.
32
Lohr F, Lo DY, Zaharoff DA, Hu K, Zhang X, Li Y, et al. Effective tumor therapy with plasmid-encoded cytokines combined with in vivo electroporation. Cancer Res 2001;61:32814.
33
Yamashita YI, Shimada M, Hasegawa H, Minagawa R, Rikimaru T, Hamatsu T, et al. Electroporation-mediated interleukin-12 gene therapy for hepatocellular carcinoma in the mice model. Cancer Res 2001;61:100512.
34 Tamura T, Nishi T, Goto T, Takeshima H, Dev SB, Ushio Y, et al. Intratumoral delivery of interleukin 12 expression plasmids with in vivo electroporation is effective for colon and renal cancer. Hum Gene Ther 2001;12:126576.[Medline]
35 Kishida T, Asada H, Satoh E, Tanaka S, Shinya M, Hirai H, et al. In vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice. Gene Ther 2001;8:123440.[Medline]
36 Asselin-Paturel C, Lassau N, Guinebretiere JM, Zhang J, Gay F, Bex F, et al. Transfer of the murine interleukin-12 gene in vivo by a Semliki Forest virus vector induces B16 tumor regression through inhibition of tumor blood vessel formation monitored by Doppler ultrasonography. Gene Ther 1999;6:60615.[Medline]
37 Dow SW, Elmslie RE, Fradkin LG, Liggitt DH, Heath TD, Willson AP, et al. Intravenous cytokine gene delivery by lipid-DNA complexes controls the growth of established lung metastases. Hum Gene Ther 1999;10:296172.[Medline]
38 Angiolillo AL, Sgadari C, Taub DD, Liao F, Farber JM, Maheshwari S, et al. Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo.J Exp Med 1995;182:15562.[Abstract]
39
Rizzuto G, Cappelletti M, Maione D, Savino R, Lazzaro D, Costa P, et al. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci USA 1999;96:641722.
40
Sgadari C, Angiolillo AL, Cherney BW, Pike SE, Farber JM, Koniaris LG, et al. Interferon-inducible protein-10 identified as a mediator of tumor necrosis in vivo.Proc Natl Acad Sci USA 1996;93:137916.
41 Palmer K, Emtage PC, Strieter RM, Gauldie J. Transient gene transfer of non-ELR chemokines to rodent lung induces mononuclear cell accumulation and activation. J Interferon Cytokine Res 1999;19:138190.[Medline]
42 Taub DD. Chemokine-leukocyte interactions. The voodoo that they do so well. Cytokine Growth Factor Rev 1996;7:35576.[Medline]
43
Sgadari C, Farber JM, Angiolillo AL, Liao F, Teruya-Feldstein J, Burd PR, et al. Mig, the monokine induced by interferon-gamma, promotes tumor necrosis in vivo.Blood 1997;89:263543.
44 Allegretti JP, Panje WR. Electroporation therapy for head and neck cancer including carotid artery involvement. Laryngoscope 2001;111:526.[Medline]
Manuscript received October 23, 2001; revised February 26, 2002; accepted March 22, 2002.
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