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2001;61:3281-3284.Cancer ResFrank Lohr, David Y. Lo, David A. Zaharoff, et al. Electroporationin VivoCombined withEffective Tumor Therapy with Plasmid-encoded Cytokines

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[CANCER RESEARCH 61, 32813284, April 15, 2001]

Advances in Brief

Effective Tumor Therapy with Plasmid-encoded Cytokines Combined with in

Vivo Electroporation1

Frank Lohr,2 David Y. Lo, David A. Zaharoff, Kang Hu, Xiuwu Zhang, Yongping Li, Yulin Zhao,

Mark W. Dewhirst, Fan Yuan, and Chuan-Yuan Li3

Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina 27710 [F. L., D. Y. L., K. H., X. Z., Y. L., Y. Z., M. W. D., C-Y. L.], and Department

of Biomedical Engineering, Duke University, Durham, North Carolina 27708 [D. A. Z., F. Y.]

Abstract

Plasmids may have unique advantages as a gene delivery system.

However, a major obstacle is the low in vivo transduction efficiency. In

this study, an electroporation-based gene transduction approach was

taken to study the effect ofinterleukin (IL)-2 or IL-12 gene transduction on

the growth of experimental murine tumors. Significant intratumoral gene

transduction was achieved by electroporation of tumors that had been

injected with naked plasmids encoding reporter genes and cytokine genes

(IL-2 and IL-12) under the control of a constitutive cytomegalovirus

promoter. In addition, significant tumor growth delay could be achieved

in a murine melanoma line B16.F10 with the cytokine genes. Most impor-

tantly, systemic transgene levels were negligible when compared with

intratumoral adenovirus-mediated IL-12 gene delivery, which leads to

significantly higher systemic cytokine levels. Therefore, naked plasmid-

and in vivo electroporation-mediated cancer gene therapy may be thera-

peutically efficacious while maintaining low systemic toxicity.

Introduction

Recombinant viral vectors account for the majority of gene delivery

approaches used in current cancer gene therapy studies. Examples

include adenovirus, herpes virus, and retrovirus vectors. The main

advantage of viral vectors is their high efficiency of gene transduction.

However, there are also many potential problems, such as efficiency

of production and safety. For example, adenovirus is one of the most

commonly used vector systems for gene therapy. Most of the adeno-

virus vectors used are rendered replication deficient. However, it has

been reported that new unwanted variants can develop as a result of

recombination during the production process. In addition, adenovi-

ruses can elicit strong immune responses that will diminish efficacy in

later administrations and could represent a hazard for adverse reac-

tions. At higher doses, biosafety is also a big concern. For example,

even with strictly local infection, there may be systemic toxicity

because of virus leaking into the systemic circulation, then infecting

the liver with high efficiency (1, 2).

Plasmid DNA, on the other hand, is a relatively safe alternative to

viral vectors. The toxicity is generally very low, and large-scale

production is relatively easy. However, a major obstacle that has

prevented the widespread application of plasmid DNA is its relative

inefficiency in gene transduction. Therefore, most applications for

plasmid DNA have been limited to vaccine studies with a few excep-

tions (35).

Methods that can significantly enhance the efficiency of plasmid

DNA transduction efficiency will greatly extend the utility of this

promising mode of gene transfer. Electroporation has long been used

to effectively transport molecules, including DNA, into living cells in

vitro (6). More recent reports demonstrated that it can safely be used

in vivo, e.g., by enhancing the local efficacy of chemotherapeutic

agents (7, 8). When used in conjunction with DNA plasmids, it greatly

enhances the local transfection efficiency over plasmid injection

alone (9).

In this study, we applied this new approach to antitumor immuno-

gene therapy. We report the effectiveness and possible advantages of

local gene therapy with plasmid encoded mIL-24 and mIL-12 in

combination with in vivo electroporation in a murine melanoma tumor

model with particular attention to intratumoral and systemic transgene

levels.

Materials and Methods

Tumor Models/Cell Culture. B16.F10 (American Type Culture Collec-

tion, Manassas, Virginia) is a metastasizing subline of the B16 melanoma that

arose spontaneously and is syngeneic with C57BL/6 mice. In vitro it is

maintained in DMEM. Transplantation into C57BL/6 mice at 106 cells/animal

establishes tumors in 100% of mice. 293 cells (American Type Culture

Collection, Manassas, Virginia) were used for adenovirus propagation andwere maintained in DMEM as well. All cell culture media were supplemented

with 10% fetal bovine serum (Hyclone, Logan, UT) and 1 g each of penicillin/

streptomycin (Life Technologies, Inc., Grand Island, NY) per 100 ml.

Plasmids. The pEGFP-N1 plasmid was obtained from Clontech Corp.

(Palo Alto, CA). It encodes the EGFP protein under the control of a CMV

promoter. Plasmid pNGVL-mIL2 and pNGVL-mIL12 were obtained from the

NGVL at the University of Michigan (Ann Arbor, MI). They encode mIL-2

and mIL-12 genes under the control of the CMV promoters, respectively.

Plasmid pNGVL--gal was also obtained from the NGVL. It encodes a nuclear

targeted -gal gene under the control of the CMV promoter.

Adenovirus Vector. The adenoviral vector AdIL12 (kindly provided by

Dr. Frank L. Graham, McMaster University, Hamilton, Ontario, Canada) used

in this study was described previously and is based on an Ad5 recombinant

system (10). In short, both mIL-12 subunit cDNAs were inserted in the E1

region and placed under control of the murine CMV promoter. Efficientexpression of both IL-12 subunits was achieved by placing an internal ribo-

some entry site in-between. Viruses were propagated in 293 cells and purified

by CsCl banding according to a standard protocol (11).

In Vivo Electroporation and Adenovirus Infection. Animal care and

experimental procedures were in accord with institutional guidelines. All

animals were anesthetized before virus or plasmid injection/electroporation

with Ketamine/Xylazine at 1.8/0.15 mg/mouse. After tumors grew to sizes of

57 mm in diameter (or 65179 mm3 in volume), they were injected intratu-

morally either with AdIL12 (3 108 particles in 50 l of PBS) or with 50 g

Received 11/3/00; accepted 3/1/01.

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.1 This study was supported by Grant CA81512 from the National Cancer Institute, a

grant from the Komen Foundation for Breast Cancer Research (to C-Y. L.), as well asGrants CA40355 and CA42745 from the National Cancer Institute (to M. W. D.). F. L. is

supported by Grant Lo 713/1-1 from the German Research Council (Deutsche Forsch-

ungsgemeinschaft).2 Present address: Department of Radiation Oncology, INF 400, University of Heidel-

berg, 69120 Heidelberg, Germany.3 To whom requests for reprints should be addressed, at Department of Radiation

Oncology, Duke University Medical Center, Box 3455, Durham, NC 27710. Phone:

(919) 681-4721; Fax: (919) 684-8718; E-mail: [email protected].

4 The abbreviations used are: mIL, murine interleukin; EGFP, enhanced green fluo-rescent protein; CMV, cytomegalovirus; NGVL, National Gene Vector Laboratory; -gal,

-galactosidase.

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of either EGFP-, -gal-, mIL-2- or mlL12 plasmid in 50 l PBS. Animals

intended for combination treatment underwent subsequent electroporation

within 15 min. Electroporation pulses were delivered with 2 2-cm stainless

steel plates attached to a caliper electrode (Genetronics, Inc., San Diego, CA).

Electrode Gel (Signa Gel; Parker Laboratories, Inc., Fairfield, NJ) was applied

to the electrodes to reduce the interfacial resistance and maintain good elec-

trical contact between electrode and skin. The caliper electrodes were clamped

on the tumor in an approximate dorsal-ventral orientation to avoid placing any

bone within the electric field. The distance between electrodes was 6 mm.

Square wave electric pulses were generated with an Electro Square Porator

T820 (Genetronics, Inc.). Three pulses (100V/50 ms) were delivered, followed

by three more pulses at the opposite polarity. These electroporation parameters

were selected based on previous reports (1214) and our own preliminary

experiments.

Detection of Reporter Gene Expression. About 48 h after injection of

either EGFP or -gal plasmid injection with or without consecutive electro-

poration, animals were sacrificed, and tumors were harvested. Animals were

anesthetized and sacrificed by cervical dislocation. Tumors injected with -gal

plasmid were fixed in 4% paraformaldehyde/PBS for 48 h at 4C. Tumors were

sectioned at 250 m. Sections were rinsed three times for 30 min each in rinse

buffer (100 mM sodium phosphate, 2 mM MgCl2

, 0.01% deoxycholic acid, and

0.02% NP40) at room temperature. Sections were stained for 24 h at room

temperature. Staining solution was rinse buffer, 5 m M potassium ferricyanide,

5 mM potassium ferrocyanide, and 1 mg/ml 5-bromo-4-chloro-3-indolyl--D-

galactopyranoside. After 24 h, staining solution was removed, and sections

were stored in 70% ethanol before mounting (in 70% ethanol) for microscopy.

For fluorescence microscopy, EGFP-injected tumors were sectioned fresh

(without freezing or fixation) at 250 m and mounted in PBS for immediate

microscopy. To visualize EGFP, a Xenon arc lamp and a FITC filter were used

on a Zeiss Axioskop. Images were acquired with a color CCD camera and

frame-grabbing equipment at identical magnification, light intensity, and am-

plification for each sample pair of tumors from electroporated or non-electro-

porated animals, respectively. Five to seven animals were used for each

treatment group.

Measurement of IL-12 Levels. mIL-12 levels in serum samples and tumor

extracts were detected with an mIL-12 ELISA kit (R&D Systems, Minneap-

olis, MN) that detects the heterodimer of p35 and p40 (p75) with a detection

level of 8 pg/ml. Serum was obtained from blood samples drawn from the tail

vein before and at different time points until 9 days after intratumoral AdIL12

injection or plasmid injection with consecutive electroporation in two to four

animals/data point. For detection of intratumoral mIL-12, untreated control

tumors and tumors at different time points after infection or plasmid injection/

electroporation were harvested (two to four animals/data point). Tumors were

homogenized in PBS (with Complete protease inhibitor; Boehringer Mann-

heim) and spun down, and supernatant was collected for measurement.

Tumor Growth Delay Studies. About 106 B16.F10 cells in 50 l of PBS

were transplanted in the right hind limbs of C57BL/6 mice. Treatment was

initiated when the tumors had reached a mean diameter of 57 mm (corre-

sponding to a tumor volume of 65179 mm3). Each treatment group consisted

of nine animals and was treated with a single intratumoral injection of 50 g

of plasmid encoding EGFP, mIL-2, or mIL-12 with or without subsequent

electroporation, respectively. The injection was carried out in 50 l of PBS.

Tumor volume was determined by measuring the largest (L) and the smallest

(S) diameters of the tumor and calculated as V /6(L S2). Growth curves

are plotted as the mean relative treatment group tumor volume SE. Relative

tumor growth rate were calculated for the first 6 days after treatment.

Histological Examination of the Tumors. Tumors from different treat-

ment groups were excised at the end of experiments (15 days after initial

treatment). They were then deep frozen in liquid nitrogen with Tissue-Tek

OCT compound (Sakura, Torrance, CA) as embedding medium, sectioned, and

mounted. They were then stained with H&E and evaluated at 400.

Results

Intratumoral Expression of Reporter Genes in Vivo. Electropo-

ration of the plasmids were carried out according to conditions estab-

lished by previous reports (1214) and our own experience. B16.F10

tumors grown to 57 mm in diameter (or 65179 mm3) in volume

were injected with the reporter plasmids. Expression of reporter genes

(EGFP and -gal) after plasmid injection and electroporation in

tumor tissue was assessed in fresh tissue sections (at 250 m) by light

microscopy (transmission and fluorescence imaging; Fig. 1). For both

reporter systems, very few cells were positive when only naked DNA

without consecutive electroporation was injected [Fig. 1a (EGFP) and

1c (-gal)]. The combination with electroporation resulted in consis-

tently efficient transduction of a higher number of cells with both

reporter genes [Fig. 1b (EGFP) and 1d(-gal)]. In EGFP experiments,

plasmid injection with electroporation allowed 38% (as evaluated by

fluorescence-activated flow cytometry) of all of the cells in the tumor

mass to be transduced with the EGFP gene in comparison with

0.1% in tumors injected with EGFP plasmid alone.

Intratumoral and Systemic Expression of Therapeutic Genes in

Vivo. To quantitatively evaluate local and systemic transgene expres-

sion as a consequence of electroporation and control gene transfer

approaches, mIL-12 (as a heterodimer of p35 and p40 subunits) levels

were assessed in tumor and serum of untreated and treated animals.

The mIL-12 levels in the serum and tumors were below the detection

threshold (8 pg/ml) in untreated or electroporation-alone control an-

imals. Fig. 2 is a summary of the peak mIL-12 levels in treated

animals. With the mIL-12 plasmid injection alone, the cytokine level

in the tumor reached from below the level of detection (day 2) to a

peak of 0.3 ng/g (day 5), whereas the level in the serum reached from

below the level of detection (day 2) to a peak of 0.4 ng/ml (day 5). Inthose tumors that were injected with the control GFP-encoding plas-

mid, the tumor and serum levels of the cytokine were similar to those

with mIL-12 alone, indicating that the low level of cytokine expres-

sion observed with the mIL-12 plasmid injection alone were perhaps

the result of DNA injection itself rather than any specific gene

expression. When mIL-12 plasmid injection was combined with elec-

troporation, the cytokine level reached 2.6 ng/g on day 2 and 5.4 ng/g

on day 5 in the tumor, whereas the level in the serum reached from

below the level of detection (day 2) to 0.35 ng/ml, similar to that

achieved with plasmid injection alone. In comparison, in animals that

were intratumorally injected with a therapeutically effective dose

[according to previous studies in our laboratory (15)] of 3 108

plaque-forming units of AdIL12, an adenovirus encoding the mIL-12

gene under the control of the CMV promoter, the mIL-12 levels were

Fig. 1. In vivo expression of reporter genes in B16.F10 melanoma 48 h after injection

of 50 g of EGFP or -gal plasmid (in 50 l of PBS) into tumors (at 57 mm in diameter

or 65179 mm3 in volume) with or without consecutive electroporation. Photomicro-

graphs (500) of: a, EGFP plasmid injection without electroporation; b, EGFP plasmid

injection with electroporation; c, -gal plasmid injection without electroporation; and d,-gal plasmid injection with electroporation. Sections were carried out at 250 m usingfresh tumor tissues that had not been fixed or frozen. In each group of experiments, five

to seven animals were used.

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between 7.2 (day 2) and 5.7 ng/g (day 5) in the tumor, whereas the

peak value (at day 2) in the serum reached 20 ng/ml. For both

modalities, tumor mIL-12 levels returned to baseline at 9 days after

treatment. Therefore, significant tumor levels of IL-12 can be

achieved in vivo by combining mIL-12 plasmid injection and electro-

poration when compared with AdIL12 injection. Serum levels, how-

ever, were greatly elevated after local AdIL12 injection (reaching a

maximum of 20 ng/ml), while they were close to nonspecific DNA

control after the combination of plasmid injection and electroporation

(maximum of 0.4 ng/ml; Fig. 2). In addition, apparent toxicity was

observed in animals injected with AdIL12, consistent with our earlier

observations (15). The toxicities include weigh loss, apathy, and

splenomegaly (15).

Significant Tumor Growth Delay after Electro-Gene Therapy.

Experiments were then conducted to examine the antitumor efficacy

of the combined electroporation/plasmid DNA transfer approach. Fig.

3A shows the mean relative volumes ( SE) for B16.F10 tumors in

C57BL/6 mice treated with injections of control plasmid (EGFP),

mIL-2 or mIL-12 with or without in vivo electroporation. Fig. 3Bshows the relative growth rate of different groups in the first 6 days

after treatment. Electroporation in conjunction with control plasmid

(pEGFP-N1) did not result in significant growth delay over control

plasmid alone or nontreated controls. In addition, injections of naked

mIL-2 or mIL-12 plasmid alone did not result in any significant

growth delay over control plasmid. The combination of electropora-

tion with either mIL-2 or mIL-12 plasmid resulted in a significant

growth delay of approximately 515 days when compared with both

control plasmid plus electroporation (P 0.01) and the respective

naked cytokine plasmids (P 0.05), with mIL-12 plus electropora-

tion being the most effective. These experiments were conducted three

times to ensure the reproducibility of the experiments. In all three

experiments, a similar pattern of tumor growth delays was observed.

Additional experiments (with the IL-12 plasmid) indicate that it ispossible to carry out a second plasmid injection and electroporation to

extend the duration of tumor suppression (data not shown). As to the

mechanisms of the antitumor effect of IL-12, our past study (15)

indicates that stimulation of T cells, natural killer cells, and the

antiangiogenic properties of IL-12 all played important roles in sup-

pressing tumor growth in the B16.F10 model.

Discussion

IL-2 and IL-12 have shown promising antitumor effects in preclin-

ical studies by improving immune responses against tumors, although

their mechanism of action has not completely been elucidated as yet

(2, 10, 16 21). Exploitation of these antitumor properties in humans,

however, has thus far been hampered by significant systemic toxicity

(19, 2224). Confining the expression of those genes to the tumor by

using intratumoral injection of viral vectors had some success (10,

20). Even with this approach, however, dose-limiting systemic toxic-

ity has nevertheless been observed by several investigators in animal

models (1, 2, 15). Our observations of elevated serum levels of IL-12

in animals treated with intratumoral injection of adenovirus encoding

IL-12 and associated toxicities, most likely because of adenovirus

leaking into the systemic circulation and infecting parenchymatous

organs (24), fall in line with these reports.

Increasing the gene transduction efficiency of naked plasmid DNA

should alleviate some of the problems associated with virus-mediated

gene transfer. Recently, successful in vivo transfer of IL genes into

muscle (9) and of marker and therapeutic suicide genes into normal

tissues and tumors (9, 2528) have been reported. In this report, we

describe the efficient use of IL-based immunotherapy and electropo-

ration in a murine melanoma tumor. Our results support the idea that

the combination of injecting plasmids encoding secretable therapeutic

genes (such as IL-12) with electroporation leads to significant local

antitumor effects with reduced systemic cytokine levels when com-

pared with gene therapy based on an adenoviral vector. Even with the

simple electroporation protocol used in these experiments, satisfactory

local gene expression and a distinct therapeutic effect could be ob-served. Systemic transgene levels, however, were greatly reduced

with the plasmid/electroporation combination when compared with

the adenovirus approach. The observed lower systemic cytokine levels

Fig. 2. Maximum IL-12 levels in tumor and serum after intratumoral injection of IL-12

plasmid (50 g in 50 l) with consecutive electroporation or after intratumoral injectionof recombinant adenovirus encoding IL-12 (3 108 plaque-forming units of AdIL12). For

adenovirus, the samples were taken 2 days after viral injections. For plasmids, the samples

were taken 5 days after plasmid injection. The results are plotted as mean range of two

to four animals per data point; bars, SE.

Fig. 3. Results from tumor growth delay experiments. A, mean relative tumor volumes

(bars, SE) for B16.F10 in C57BL/6 mice tumors treated with injections of control plasmid

(EGFP), mIL-2 or mIL-12 with or without in vivo electroporation.f, no treatment control;

, EGFP plasmid alone; , EGFP plasmid electroporation (EP); E, mIL-2 plasmid

alone; F, mIL-2 plasmid EP;, mIL-12 plasmid alone; , mIL-12 plasmid EP. B,relative tumor growth rate in the first 6 days after treatment. The values represent the

slopes of the relative growth curve in A as calculated by linear curve fitting.

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should reduce the systemic toxicity of DNA-based cytokine therapy.

Another advantage of this approach is its inherent low immunogenic-

ity, greatly facilitating multiple, repeated applications, which may be

necessary in many circumstances.

The transfection efficiency in vivo depends on a multitude of

parameters, such as the amount of plasmid, time between plasmid

injection and electroporation, temperature during electroporation, and

above all electrode geometry and pulse parameters (field strength,

pulse length, pulse sequence, and others; Refs. 26, 28, and 29). Much

progress has been made improving the electroporation paradigm, and

it is very likely that additional improvements will be achieved. Elec-

tro-gene therapy, therefore, may emerge as a viable alternative to

virus vector-based approaches, especially in tumors that are not easily

infected with the current vectors and when secretable therapeutic

genes are used.

Acknowledgments

We thank Dr. Frank L. Graham of McMaster University for providing us

with the AdIL12 vector. We also thank the NGVL at the University of

Michigan for providing plasmids. We also express our gratitude to the anon-

ymous reviewers who provided many helpful suggestions for our manuscript.

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