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MOLECULAR THERAPY
Vol. 5, No. 6, June 2002
Copyright © The American Society of Gene Therapy
1525-0016/02 $35.00
668
ARTICLE
doi:10.1006/mthe.2002.0601, available online at http://www.idealibrary.com on IDEAL
IL-12 Plasmid Delivery by in Vivo Electroporation for
the Successful Treatment of Established Subcutaneous
B16.F10 Melanoma
M. Lee Lucas,
1
Loree Heller,
2
Domenico Coppola,
3
and Richard Heller
1,2,4,*
1
Department of Medical Microbiology and Immunology/Institute for Biomolecular Science, University of South Florida, Tampa, Florida 33612, USA
2
Center for Molecular Delivery, University of South Florida, Tampa, Florida 33612, USA
3
Interdisciplinary Oncology Program, H. Lee Moffitt Cancer Center, University of South Florida, Tampa, Florida 33612, USA
4
Department of Surgery, University of South Florida, Tampa, Florida 33612, USA
*To whom correspondence and reprint requests should be addressed. Fax: (813) 974-3339. E-mail: rheller@hsc.usf.edu.
Interleukin-12 (IL-12) has been used in numerous immunotherapy protocols against melanoma.
However, delivery of IL-12 in the form of recombinant protein can result in severe toxicity, and
gene therapy has had limited success against B16.F10 murine melanoma. The purpose of this
study was to examine the effectiveness of in vivo electroporation for the delivery of plasmid
DNA encoding IL-12 as an antitumor agent against B16.F10 melanoma. We treated mice bear-
ing established B16.F10 melanoma tumors with intratumoral (i.t.) or intramuscular (i.m.) injec-
tions of a plasmid encoding IL-12, followed by in vivo electroporation. For i.t. treatments, we
used an applicator containing six penetrating electrodes to deliver 1500-V/cm, 100-s pulses.
We administered i.m. pulses with an applicator containing four penetrating electrodes deliv-
ering 100-V/cm, 20-ms pulses. The i.t. treatment resulted in the cure of 47% of tumor-bear-
ing mice, and 70% of cured mice were resistant to challenge with B16.F10 cells. The i.m. treat-
ment did not result in tumor regression. We found that i.t. treatment resulted in increased levels
of IL-12 and interferon-␥ (IFN-␥) within the tumors, the influx of lymphocytes into the tumors,
and reduction in vascularity. Neither i.m. nor i.t. treatment was successful against B16.F10
tumors in a nude mouse model, supporting a role for T cells in regression of this tumor model.
Key Words: IL-12, electroporation, melanoma, plasmid DNA, B16.F10, gene therapy
INTRODUCTION
The antitumor potential of IL-12 has been reported in
numerous immunotherapy studies [1–11]. The proposed
antitumor mechanisms of IL-12 include effects on the
immune system such as the induction of IFN-␥, upregula-
tion of T cells, and proliferation of natural killer (NK) cells.
In addition, IL-12 inhibits angiogenesis, the formation of
new blood vessels [1,10]. This wide range of effects on the
immune system as well as antiangiogenic properties results
in a potentially potent antitumor treatment.
Unfortunately, preclinical and clinical trials using
systemic administration of recombinant IL-12 demon-
strated potential adverse side effects [11,12]. The use of
gene therapy for the delivery of IL-12, by gene gun,
resulted in fewer side effects than recombinant protein
therapy [11]. Several studies using viral and nonviral gene
delivery techniques have reported success in slowing
and/or preventing tumor growth [6–11]. However, these
studies have had limited success in demonstrating
complete regression of the poorly immunogenic B16.F10
melanoma and subsequent resistance to challenge.
In vivo electroporation is a gene delivery technique
that has been used successfully for efficient delivery of
plasmid DNA to many different tissues [13–26]. We
recently reported the expression of IL-12 and IFN-␥ in the
serum of mice after intramuscular delivery of a plasmid
encoding IL-12 with electroporation [18], and other stud-
ies have reported the administration of in vivo electro-
poration for delivery of plasmid DNA to B16 melanomas
[22–26]. Although systemic administration of recombi-
nant IL-12 revealed its antitumor potential [1], expression
of IFN-␥ at the tumor site has been shown to be critical
for successful tumor regression [4,9]. Systemic and local
expression of a gene or cDNA encoded by a plasmid can
be obtained with administration of in vivo electropora-
tion. Use of in vivo electroporation enhances plasmid
DNA uptake in tumor tissue, resulting in expression
within the tumor [22–25], and delivers plasmids to
MOLECULAR THERAPY
Vol. 5, No. 6, June 2002
Copyright © The American Society of Gene Therapy
669
ARTICLE
doi:10.1006/mthe.2002.0601, available online at http://www.idealibrary.com on IDEAL
RESULTS
Intratumoral Delivery of IL-12 by Electroporation
Results in Tumor Regression, Long-Term Animal
Survival, and Resistance to Challenge
To explore the antitumor potential of IL-12 delivered by
in vivo electroporation, we treated C57BL/6 mice with
established subcutaneous B16.F10 melanoma by injecting
50 g (1 g/l) of plasmid DNA encoding IL-12 (pIRES IL-
12) in sterile saline into the tumor (i.t.) or the gastrocne-
mius muscle (i.m.), followed by electroporation. An appli-
cator containing six penetrating electrodes was used to
deliver 1500-V/cm, 100-s pulses i.t.; this protocol is sim-
ilar to those used in gene delivery and electrochemother-
apy protocols that resulted in successful tumor regression
[23,27,28]. For i.m. delivery, an applicator, specifically
designed for the mouse gastrocnemius muscle and con-
taining four penetrating electrodes, was used to adminis-
ter 100-V/cm, 20-ms pulses, a protocol shown to result in
high systemic IL-12 and IFN-␥ expression [18]. A single
treatment did not result in long-term animal survival (data
not shown). Therefore, in the following experiments we
administered a second treatment 7 days (day 7) after the
initial treatment (day 0). Tumor size was evaluated
throughout the experiment, and the results are presented
as the fold increase over day 0 tumor volume for each
treatment group (Fig. 1A). Treatment with pIRES IL-12
injected i.t. followed by electroporation slowed tumor
growth, with nearly half (8/17) of the mice showing com-
plete regression of their tumors. Progressive tumor growth
was observed in mice receiving i.m. injections of plasmid
muscle tissue, resulting in systemic cytokine expression
[18].
Of the studies in the B16 melanoma model, only that
of Heller et al. showed cures of established tumors with
resistance to challenge [23]. In that study, tumor regres-
sion resulted from a combination of electrochemother-
apy and gene delivery by in vivo electroporation using a
pulse protocol delivering microsecond (s) pulses [23].
Lohr et al. compared delivery by electroporation with
adenoviral vectors and found that electroporation was
effective in delivering plasmid coding for IL-12 and,
unlike adenoviral delivery, did not result in toxic side
effects [25]. In addition to these studies, Kishida et al.
also used electroporation for delivery of IL-12 and IL-18
to a B16 model [26]. Millisecond (ms) pulses were admin-
istered in these two studies, and unfortunately, neither
protocol resulted in complete regression of established
tumors and long-term survival of animals [25,26].
Its wide range of effects on the immune system and
its antiangiogenic properties make IL-12 an excellent
candidate for use as an immunotherapeutic agent.
Because of its potential toxicity, it is important to give
careful consideration to the delivery method of IL-12. In
vivo electroporation is a safe, nontoxic delivery system
and has been used for efficient delivery of chemothera-
peutic agents and plasmid DNA, including plasmids
encoding IL-12 [18,23,27]. Therefore, we hypothesized
that administration of an electroporation protocol for
delivery of IL-12 will result in regression of B16.F10
melanoma tumors and long-term animal survival.
FIG. 1. Administration of plasmid DNA encoding IL-12 followed by electroporation results in complete tumor regression. (A) Fold increase over day 0 tumor vol-
ume following treatment. P, pIRES IL-12; V, control plasmid, pND2Lux; E, electroporation. Treatment mode of delivery: i.t., intratumor; i.m., intramuscular. A
plus sign indicates treatment was administered; a minus sign indicates treatment was not administered. Initial treatment day is day 0; mice were treated again
on day 7. Results for all groups (except P
–
E
+
i.t. and V
+
E
+
i.t.) represent the combined data from three replicate experiments, and error bars represent the stan-
dard error of the mean. The P
–
E
+
i.t. and V
+
E
+
i.t. treatment groups were tested in one experiment because existing data in our lab showed these treatments to
be ineffectual. Error bars for these two groups represent standard deviation. The total number of samples for each treatment group are as follows: P
–
E
–
, n = 16;
P
–
E
+
i.t. and V
+
E
+
i.t., n = 8; and for the remainder of groups, n = 17. Mice were killed when tumor volume exceeded 1000 mm
3
. Data are expressed for sur-
viving mice on each day. (B) Percentage survival of mice represented in (A). Mice either succumbed to disease or were killed when tumor volume exceeded
1000 mm
3
.
AB
MOLECULAR THERAPY
Vol. 5, No. 6, June 2002
Copyright © The American Society of Gene Therapy
670
ARTICLE
doi:10.1006/mthe.2002.0601, available online at http://www.idealibrary.com on IDEAL
encoding IL-12 followed by electroporation. Mice not
receiving electrical pulses, (P
+
E
–
), showed continued tumor
growth until all mice were killed or succumbed to the
tumor burden. Neither the administration of electropora-
tion alone (P
–
E
+
) nor i.t. delivery of a control vector
(pND2Lux) with electroporation (V
+
E
+
) decreased tumor
growth. These results provide evidence that neither elec-
trical pulses alone nor plasmid DNA is responsible for
tumor regression. None of the treatment groups except
the P
+
E
+
i.t. group showed tumor regression, although P
+
E
–
i.t. did show slower tumor growth than P
–
E
–
through day
14 (P < 0.05).
Evaluation of mice 100 days after the initial treatment
showed that 47% of mice (8/17) receiving i.t. delivery of
IL-12 with electroporation were tumor-free (Fig. 1B).
These mice were considered cured. All mice receiving i.t.
treatment with IL-12 and electroporation experienced
prolonged survival compared with animals in other treat-
ment groups. None of the mice in control groups sur-
vived longer than 35 days. Specifically, if left untreated
or treated with pulses alone, mice did not survive longer
than 21 days.
We challenged seven of the animals that showed com-
plete regression and remained disease-free for 50 days in
the right flank with B16.F10 tumor cells. No additional
treatments were administered. Of the seven challenged,
five were resistant to tumor growth on the right flank,
while tumors grew in 100% of naive mice. This result sug-
gests the development of an immune memory response
following treatment of the initial subcutaneous tumor
established on the left flank.
Intratumoral Administration of IL-12 with
Electroporation Results in Cytokine Expression
within the Tumor
As mentioned earlier, IL-12 induces several effects on the
immune system. To evaluate the cytokine expression
induced by either i.m. or i.t. treatment, we analyzed
serum and tumor levels of IL-12 and IFN-␥. Serum levels
of both cytokines were highest after i.m. injection
followed by electroporation (Fig. 2A). Serum IL-12 peaked
at 320 pg/ml 10 days after treatment, whereas serum
IFN-␥ induced by IL-12 expression peaked at 177 pg/ml
on day 14. Serum levels of both cytokines were signifi-
cantly greater from mice treated i.m. with electroporation
than other treatments on days 5, 10, and 14 (P < 0.05).
Serum levels of these cytokines in mice treated with i.t.
injection followed by electroporation were not signifi-
cantly greater than expression in mice that received no
treatment (P > 0.05).
Analysis of IL-12 and IFN-␥ expression within the
tumors revealed that i.t. treatment with electroporation
resulted in the presence of these cytokines at the tumor
site (Fig. 2B). Intratumoral IL-12 reached 3 pg/mg of tumor
tissue on day 5 and remained at that level through day 10,
whereas IFN-␥ levels peaked at 8.16 pg/mg of tumor on day
5. Treatment with pIRES IL-12 injected i.t. followed by
electroporation produced significantly higher (P < 0.05)
IFN-␥ levels than other treatment groups on days 5 and 10.
Although tumor expression of IL-12 reached 3 pg/mg of
tumor with i.t. treatment, as opposed to 0.64 pg/mg of
tumor with i.m. treatment, these levels were not signifi-
cantly greater (P > 0.05) as a result of a wide spectrum of
expression levels in these tumors after i.t. treatment
(0.5–6.9 pg/mg of tumor tissue).
Treatment with i.m. injection followed by electropo-
ration did not result in significant (P > 0.05) cytokine
expression within the tumors (Fig. 2B). Following i.m.
treatment the highest IFN-␥ expression measured was 1
pg/mg of tumor on day 17 (Fig. 2B). Therefore, treatment
protocols that did not result in tumor regression also did
not produce intratumoral IL-12 or IFN-␥ expression. These
results support previous reports on the critical need for
cytokine expression within the tumor [4,9].
FIG. 2. Analysis of serum and tumor tissue for IL-12 and IFN-␥ expression. P, pIRES IL-12; E, electroporation. Mode of delivery: i.t., intratumor; i.m., intramus-
cular. (A) Serum levels of IL-12 and IFN-␥ in tumor-bearing mice. For each treatment group on each day tested, n = 4 mice. Error bars represent standard devi-
ation. (B) Mean tumor expression of IL-12 and IFN-␥. For each treatment group on each day tested, n = 4 mice. Error bars represent standard deviation.
AB
MOLECULAR THERAPY
Vol. 5, No. 6, June 2002
Copyright © The American Society of Gene Therapy
671
ARTICLE
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Tumor Regression Is Characterized by Lymphocytic
Infiltrate at the Tumor Site
Resistance to challenge following successful tumor regres-
sion suggests the development of an immune memory
response. We examined the tumors histologically 5 days
after initial treatment to evaluate the influx of immune
cells to the tumor. We stained tumor sections with hema-
toxylin and eosin (H&E) to distinguish infiltrating
immune cells from tumor cells. The H&E-stained sections
showed infiltration of lymphocytes into the tumors of
mice 5 days after receiving i.t. injection of pIRES IL-12 fol-
lowed by electroporation (Fig. 3C). In contrast, mice not
treated or receiving i.m. treatment with electroporation
did not display a great influx of lymphocytes (Figs. 3A and
3B). Treatment protocols not including in vivo electropo-
ration (P
+
E
–
either i.t. or i.m.) also did not result in the
influx of lymphocytes (data not shown).
By immunohistochemical phenotyping, we demon-
strated that the lymphocytes observed in tumors follow-
ing i.t. treatment with IL-12 and electroporation were
CD4
+
and CD8
+
T cells (Figs. 4C and 4D). In comparison,
lymphocytes were observed in limited numbers in
untreated tumors (Figs. 4A and 4B). Treatment of mice
with i.m. injection followed by electroporation also
resulted in limited lymphocytic infiltrate, similar to that
characterizing the untreated control group (Figs. 4E and
4F). Additionally, mice receiving injection of plasmid
encoding IL-12 (P
+
E
–
i.t. or i.m.) or control plasmid with
electroporation (V
+
E
+
i.t.) did not show infiltrating lym-
phocytes (data not shown).
Treatment Does Not Result in Tumor Regression in a
Nude Mouse Model
To further evaluate the need for T lymphocytes in tumor
regression, we used athymic nude mice deficient in T cells
as the mouse model in place of C57BL/6 mice. We injected
B16.F10 tumor cells subcutaneously and began treatment
when tumors reached 3–5 mm in diameter. Mice received
i.t. treatments as explained earlier: i.t. injections of plas-
mid encoding IL-12 without electroporation, i.t. injection
of a control plasmid followed by electroporation, or i.t.
injections of plasmid encoding IL-12 followed by electro-
poration. Because of the lack of successful response in
C57BL/6 mice following i.m. injection, we administered
only i.t. treatments. None of the treatments in the nude
mouse model resulted in tumor regression (Fig. 5A). In
addition, no mice in any treatment group survived longer
than 30 days (Fig. 5B). This observation further suggests
the necessity of a T-cell response for successful regression
of B16.F10 melanoma tumors.
Intratumoral Administration of IL-12 with
Electroporation Results in an Antiangiogenic Effect
Another potential role of IL-12 on tumor regression is its
effect on angiogenesis. To assess the antiangiogenic role
of IL-12 on B16.F10 tumors in C57BL/6 mice, we stained
representative sections of three tumors from each treat-
ment group with anti-CD31 antibodies, marking endothe-
lial cells. Five different areas of highest vascularity were
examined at a magnification of ⫻400 for each group (Fig.
6). A representative section of the vessels in an untreated
tumor on day 0 is shown in Fig. 6A. Figures 6B and 6C
show the large number of vessels present within untreated
tumors or tumors from mice receiving i.m. injection fol-
lowed by electroporation on day 5. In contrast, Fig. 6D
shows the reduction of blood vessels after i.t. injection
and electroporation on day 5. Tumors from mice receiv-
ing injection of plasmid encoding IL-12 without electro-
poration (P
+
E
–
i.t. or i.m.) or control plasmid with elec-
troporation (V
+
E
+
) did not show a reduction in vasculature
(data not shown).
In addition, we counted vessels in each of the three
tumors excised from untreated mice, mice receiving i.m.
IL-12 and electroporation, and mice receiving i.t. IL-12
and electroporation. Table 1 shows the number of blood
vessels counted in the field of highest vascularity at a mag-
nification of ⫻400 for each of the three excised tumors.
Only i.t. injection followed by electroporation (P
+
E
+
i.t.)
resulted in significant (P < 0.05) vessel reduction compared
with untreated animals. Although an antiangiogenic effect
was observed following i.t. treatment with electropora-
tion, the lack of response in the nude mouse model
FIG. 3. Representative sections of tumor tissue, 5 days after treatment, analyzed by H&E staining for infiltrating immune cells. Three sections per tumor were
examined. All sections are shown at ⫻250 magnification. An area containing immune cells is marked by a box. (A) No treatment. (B) Administration of IL-12
i.m. with electroporation. (C) Administration of IL-12 i.t. with electroporation.
ABC
MOLECULAR THERAPY
Vol. 5, No. 6, June 2002
Copyright © The American Society of Gene Therapy
672
ARTICLE
doi:10.1006/mthe.2002.0601, available online at http://www.idealibrary.com on IDEAL
The data reported here support
earlier studies suggesting that suc-
cessful tumor regression is depend-
ent on local rather than systemic
expression of IL-12 and IFN-␥
[4,9,10]. Both of these cytokines
induce numerous factors that may
account for their antitumor activ-
ity. Interleukin-12 upregulates T
and NK cells and stimulates IFN-␥
production, which in turn initiates
the release of antiangiogenic factors
and the possible upregulation of
major histocompatibility complex
I (MHC I) [1,10]. Direct treatment
of the tumor ensures that IL-12 is
readily available at that specific site.
Therefore, the resulting antitumor
effects are directed at the tumor site
and not diluted systemically.
Administration of pIRES IL-12
by i.t. injection followed by elec-
troporation resulted in increased
IL-12 and IFN-␥ expression at the
tumor site. Variations in tumor
size at the time of treatment may
explain the wide range of IL-12
expression observed within the
tumors after i.t. in vivo electropo-
ration. Larger tumors can more
readily accommodate the injected
volume, thereby reducing the
possibility of decreased levels of
plasmid DNA in a small tumor
resulting from fluid leakage. A
greater number of tumor cells are
potentially targeted in larger
tumors as opposed to those that
are smaller. The surrounding epi-
dermal cells of a smaller tumor
may also receive the plasmid.
However, these cells have a high
turnover rate, and therefore sustained plasmid DNA
expression is unlikely.
The highest expression of IFN-␥ at the tumor site fol-
lowing i.m. injection and electroporation was measured at
1 pg/mg of tumor on day 17 (Fig. 2B). However, this find-
ing was not constant and was usually observed within very
large tumors undergoing ischemic necrosis. It is possible
that IFN-␥ expression in these tumors resulted from the
treatment or was due to infiltrating macrophages attracted
to tissue necrosis.
In vivo electroporation following i.m. administration of
pIRES IL-12 induced systemic expression of the cytokines
IL-12 and IFN-␥. An earlier report from our laboratory
showed that i.m. injection of a plasmid encoding IL-12
FIG. 4. Representative sections of tumor tissue, 5 days after treatment, analyzed by immunohistochemistry for the
presence of CD4
+
and CD8
+
lymphocytes. Three sections per tumor were examined. All sections are shown at ⫻400
magnification. Positive cells are stained brown. An arrow in (B) points to a cell representative of positive staining.
(A, B) Staining for CD4
+
lymphocytes and CD8
+
lymphocytes, respectively, from untreated tumors. (C, D) Staining
for CD4
+
lymphocytes and CD8
+
lymphocytes, respectively, from tumors receiving i.t. injection of plasmid DNA
encoding IL-12 followed by electroporation. (E, F) Staining for CD4
+
lymphocytes and CD8
+
lymphocytes, respec-
tively, from tumors following i.m. administration of plasmid DNA encoding IL-12 with electroporation.
A
B
CD
EF
suggests that T cells may be a critical factor for obtaining
regression of B16.F10 melanoma. An antiangiogenic
response may, however, contribute to stabilization of
tumor size while an immune response is mounted.
DISCUSSION
This report has demonstrated that IL-12 delivered in the
form of plasmid DNA with the aid of electroporation can
result in successful regression of B16.F10 tumors. The ani-
mals remain disease-free and are resistant to challenge at
a distant site. This is the first report to demonstrate nearly
a 47% survival rate following gene therapy treatment of
established subcutaneous B16.F10 melanoma tumors.
MOLECULAR THERAPY
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followed by the administration of ms pulses resulted in
long-term expression of IL-12 and IFN-␥ in the serum of
mice. In this report systemic levels of IL-12 and IFN-␥ did
not peak until day 10 and 14 after the initial i.m. treatment.
The untreated mice began to succumb to tumor burden by
this time period. In contrast, the i.t. treatment has the
advantage of potentially inducing high levels of
IFN-␥–dependent antitumor factors directly within the
tumor and within 5 days of treatment. These results support
previous reports that intratumor expression of cytokines are
related to successful tumor regression [4,9,10].
Administration of IL-12 to the tumor with electropo-
ration also induced an infiltration of immune cells, par-
ticularly lymphocytes, to the tumor site. The lymphocytes
were identified as CD4
+
and CD8
+
by immunohistochem-
istry. Very few lymphocytes were observed in untreated
animals, animals receiving i.t. injection without electro-
poration, animals receiving i.t. injection of control plas-
mid with electroporation, as well as animals receiving i.m.
administration of IL-12 and electroporation. The absence
of tumor regression and animal survival in the nude mouse
model further suggests the critical need for lymphocytes
in the treatment of this tumor model.
Another effect of IL-12 is inhibition of angiogenesis.
Staining of tumor sections with anti-CD31 antibodies
showed the reduction of vascularity following i.t. treat-
ment with IL-12 and electroporation. Again, these effects
were not seen in untreated animals or those receiving i.m.
treatment. Therefore, an antiangiogenic effect is observed
following the same treatment protocol that results in
tumor regression.
Although this report investigated numerous effects of
IL-12 in relation to tumor regression, other potential influ-
ences cannot be ruled out. Schultz et al. reported no effects
of CpG motif on tumor regression from the IL-12 cDNA
in this plasmid but did not determine effects from CpG
motifs within the plasmid backbone [8]. Furthermore, IL-
12 itself may increase the expression of accessory mole-
cules such as MHC I [10]. These variables and others may
also have a function in successful tumor regression.
As mentioned earlier, two other studies have used elec-
troporation for delivery of IL-12 to B16 melanomas but
were unsuccessful in obtaining complete regression and
disease-free or cured mice [25,26]. In both of these stud-
ies, ms pulses were delivered to tumors. In contrast, Heller
et al. administered µs pulses for delivery of plasmids encod-
ing IL-2 or granulocyte/macrophage colony-stimulating
factor (GM-CSF) by in vivo electroporation along with elec-
trochemotherapy and demonstrated successful tumor
regression [23]. Although different therapeutic molecules
were delivered in these studies, this report shows the most
substantial therapeutic effect to date by delivering a plas-
mid encoding IL-12 using s pulses.
One advantage of electroporation is the ability to tai-
lor the pulse protocols to different tissues. Millisecond
pulses are more successful than s pulses in i.m. delivery
for obtaining sustained systemic cytokine expression [18].
Other tissue types, such as skin, are more successfully
transfected with s pulses [14]. The rounder morphology
of the B16.F10 tumor cell is more similar to that of a skin
cell than the elongated muscle fiber. Therefore, the vari-
ances in tumor regression following ms or s pulse pro-
tocols may be the result of not applying the appropriate
pulse conditions for the tissue type.
The potential for clinical success makes in vivo electro-
poration a promising treatment modality. Localized gene
therapy reduces the risk of adverse side effects associated
with systemic recombinant protein therapy. In mice, local
delivery of IL-12 by gene gun resulted in similar tumor
regression rates as recombinant IL-12 treatment but with
fewer adverse side effects [11]. In addition, Lohr et al. found
that delivery of IL-12 by electroporation did not result in
FIG. 5. Administration of IL-12 followed by electroporation does not result in tumor regression in a nude mouse model. (A) Fold increase over day 0 tumor vol-
ume following treatment. P, pIRES IL-12; V, control plasmid, pND2Lux; E, electroporation. Mode of delivery: i.t., intratumor. Initial treatment day is day 0; mice
were treated again on day 7. The data represent two experiments each, with four mice in each group. Error bars represent standard deviation. Mice were killed
when tumor volume exceeded 1000 mm
3
. Data are expressed for surviving mice on each day. (B) Percentage survival of mice represented in (A). Mice either
succumbed to disease or were killed when tumor volume exceeded 1000 mm
3
.
AB
MOLECULAR THERAPY
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the side effects observed following adenoviral delivery of IL-
12 [25]. However, complete regression of established
B16.F10 melanoma resulting in disease-free survival was not
achieved by either of these treatment modalities.
Electroporation is an effective, yet safe, delivery system as
shown in electrochemotherapy clinical trials, which used
electroporation to target tumors with chemotherapeutic
drugs [27]. The delivery conditions used for i.t. treatment
in the experiments reported here are similar to those used
for electrochemotherapy clinical trials. Those studies
revealed these conditions to be tolerable for patients and did
not result in damage to normal tissue [27].
In summary, we report a treatment modality that can
eradicate established B16.F10 melanoma tumors and result
in resistance to renewed tumor growth following chal-
lenge. After i.t. delivery of plasmid DNA encoding IL-12
by in vivo electroporation, 47% of mice showed complete
regression of their tumors and remained disease-free. These
mice were challenged with B16.F10 tumor cells, and five
of seven remained tumor-free for an additional 100 days,
after which they were humanely killed. We also show that
i.t. injection of plasmid DNA encoding IL-12 and electro-
poration is more effective than i.m. delivery for promot-
ing tumor regression and prolonging animal survival. The
success of this treatment in this tumor model stems from
the local expression of IL-12 and IFN-␥, infiltrating lym-
phocytes, and inhibition of angiogenesis within the
treated tumor.
MATERIALS AND METHODS
Tumor cells and mice. B16.F10 murine melanoma cells (CRL 6475;
American Type Culture Collection, Rockville, MD) were maintained in
Dulbecco’s minimal Eagle’s medium (DMEM) supplemented with 10% FCS
and 0.2% gentamicin. Cells were trypsinized and washed in sterile PBS before
injection. The left flank of C57BL/6 mice (National Cancer Institute, Bethesda,
MD) was shaved and 1 ⫻ 10
6
cells in 50 l of sterile PBS were injected sub-
cutaneously. When challenged, mice were injected with 5 ⫻ 10
5
B16.F10 cells
in the right flank. Tumors were measured using digital calipers, and treatment
was begun when tumors reached 3–5 mm in diameter, ~ 7–10 days after injec-
tion. Tumor volume (v) was calculated using the formula v = a
2
b/6, where
a = the smallest diameter and b = the perpendicular diameter. Mice were
housed in accordance with AALAM guidelines.
Plasmid DNA. pIRES IL-12 was a gift from Karin Moelling (University of
Zurich, Zurich, Switzerland). Briefly, pIRES IL-12 contains both subunits joined
by an internal ribosomal entry site (IRES) behind a single cytomegalovirus
(CMV) promoter [8]. Robert Malone (Gene Delivery Alliance, Inc., Rockville,
MD) donated the pND2Lux, which encodes the reporter gene luciferase.
Qiagen Mega Kits (Qiagen, Valencia, CA) were used for plasmid preparations.
pIRES IL-12 was prepared with an endotoxin-free kit.
Intratumor treatment. Mice were anesthetized using 97% oxygen and 3%
isoflurane. Tumors were injected with 50 l (1 g/l) plasmid DNA in sterile
saline using a tuberculin syringe with a 25-gauge needle. A circular applica-
tor containing six penetrating electrodes ~ 1 cm in diameter [15,28] was
inserted into the tumor. Briefly, six rotating pulses were delivered at 1500
V/cm (99 s, 1 Hz) using a BTX T820 pulse generator and autoswitcher (BTX,
San Diego, CA) [15,28].
Intramuscular treatment. Mice were anesthetized as described earlier. The
skin surrounding the gastrocnemius muscle was shaved. Plasmid DNA diluted
FIG. 6. Immunohistochemical analysis
of tumor tissue for the presence of
blood vessels. Representative sections
rich in vessels are depicted for each
treatment. Three sections per tumor
were examined. All sections are shown
at ⫻400 magnification. An arrow in (A)
points to a representative blood vessel.
(A) Presence of blood vessels within
tumors on day 0, before treatment. (B)
Untreated tumors on day 5. (C) Tumors
on day 5 from mice receiving i.m. injec-
tion of plasmid DNA encoding IL-12 fol-
lowed by electroporation. (D) Blood
vessels on day 5 from mice receiving i.t.
administration of plasmid DNA encod-
ing IL-12 followed by electroporation.
A
C
D
B
TABLE 1: Tumor blood vessel counts from C57BL/6 mice in
each treatment group
P
–
E
–
P
–
E
–
i.t. P
+
E
–
i.m. V
+
E
+
i.t. P
+
E
+
i.m. P
+
E
+
i.t.
Tumor 1 24 10 27 20 17 6
Tumor 2 32 21 32 28 38 12
Tumor 3 49 28 39 39 38 18
P
–
, No plasmid; E
–
, no electroporation; P
+
, pIRES IL-12; E
+
, electroporation; V
+
, control
plasmid, pND2Lux; i.t., intratumoral; i.m., intramuscular.
MOLECULAR THERAPY
Vol. 5, No. 6, June 2002
Copyright © The American Society of Gene Therapy
675
ARTICLE
doi:10.1006/mthe.2002.0601, available online at http://www.idealibrary.com on IDEAL
in sterile saline (50 l, 1 g/l) was injected into the gastrocnemius muscle
using a tuberculin syringe and a 25-gauge needle. An applicator specially
designed for the mouse gastrocnemius containing four penetrating electrodes
in a rectangular pattern (2 ⫻ 5 mm) was inserted into the muscle surround-
ing the injection site. A total of 12 pulses was delivered segmentally at 100
V/cm (20 ms, 1 Hz) using a BTX T820 pulse generator. A manual switch was
used to administer three pulses in each of four directions as follows, with num-
ber of electrodes active given in parentheses: across 2 mm distance (4); first
diagonal (2); second diagonal (2); and across 5 mm distance (4). The first
treatment (day 0) was delivered to the left leg and the second treatment (day
7) was delivered to the right leg.
ELISA. Mice were humanely killed using CO
2
asphyxiation, and then blood
and tumors were collected on each day from four mice per treatment group.
For detection of cytokines in the serum, blood was collected by cardiac punc-
ture and stored at 4⬚C overnight. Serum was extracted from blood samples by
centrifugation (3 minutes at 5000 rpm) at 4⬚C, and stored at –20⬚C until ana-
lyzed. To measure cytokine levels within the tumor tissue, the tumors were
removed, frozen immediately on dry ice, weighed, and then stored at –80⬚C.
For analysis, the tumors were thawed, and 1 ml of a solution containing PBS
and 10% protease inhibitor cocktail (P8340; Sigma, St. Louis, MO) was added.
The tissues were kept on ice, homogenized using a PowerGen 700 (Fisher
Scientific, Pittsburgh, PA), centrifuged for 3 minutes at 5000 rpm at 4⬚C, and
then supernatants were assayed by ELISA. Both serum and tumor samples were
analyzed using murine IFN-␥ and IL-12 p70 ELISA kits (R&D Systems,
Minneapolis, MN). Serum levels were calculated as pg of cytokine per ml of
serum. Cytokine levels in the tumor were calculated as pg of cytokine per mg
of tumor.
Histology. Mice were humanely killed by CO
2
asphyxiation. Tumors were
excised and placed in 50-ml conical tubes containing 10 ml of 10% forma-
lin. The tissue was stained with H&E after fixation, as follows: after fixation
in 10% neutral buffered formalin for 6 hours, representative tissue samples
were processed into paraffin blocks using a Miles VIP tissue processor (Miles
Inc., Mishawaka, IN). Briefly, tissues were dehydrated in ascending grades of
ethanol, cleared in xylene, and infiltrated in paraffin (Tissue Prep 2; Fisher
Scientific). Following embedding, tissues were sectioned on a standard rota-
tory microtome and 4-m sections were retrieved from a waterbath and
mounted on glass slides. Three sections per tumor were examined. Sections
were heat-dried and stained with H&E (Richard-Allan Scientific, Kalamazoo,
MI) using standard histologic techniques. Using a synthetic mounting
medium, coverslips were then placed.
Immunohistochemistry. Immunohistochemical staining was conducted to
examine the tumors for the presence of CD4
+
lymphocytes, CD8
+
lympho-
cytes, and blood vessels using the following antibodies: rat anti-mouse CD4,
rat anti-mouse CD8a (Ly2), and rat anti-mouse CD31 (PECAM-1), respectively
(PharMingen, Cambridge, MA). Mice were humanely killed by CO
2
asphyxi-
ation. Tumors were excised with scissors and the skin removed, then imme-
diately frozen in a mixture of dry ice and ethanol, and stored at (80⬚C. Frozen
sections of 5 m were obtained. For immunohistochemical analysis, rat anti-
mouse CD4, rat anti-mouse CD8a (Ly2), or rat anti-mouse CD31 (PECAM-1)
was applied to tissue sections at a dilution of 1:50 and incubated for 30 min-
utes, followed by detection with the Vector Elite Rat IgG–Peroxidase kit at 2⫻
concentration (15 minutes each in biotinylated anti-rat IgG and ABC com-
plex). Immunostaining was carried out on the Dako autostainer. Sections
were analyzed at ⫻400 magnification.
Treatment of nude mice. BALB/c athymic nude mice were obtained from
the National Cancer Institute and used at 7 weeks of age. B16.F10 cells were
prepared as described earlier. Mice were injected subcutaneously in the left
flank with 1 ⫻ 10
6
B16.F10 cells in 50 l of sterile PBS. Treatment was begun
when the tumors reached 3–5 mm in diameter. Mice received intratumor
therapy as described earlier.
Statistical methods. Statistical analysis was performed by ANOVA or two-
tailed Student’s t-test.
ACKNOWLEDGMENTS
We thank Karin Moelling (University of Zurich, Zurch, Switzerland) for donat-
ing pIRES IL-12; Robert Malone (Gene Delivery Alliance, Inc., Rockville, MD) for
the pND2Lux; and Carlos Pottinger and Sandra Livingston (University of South
Florida, Tampa, Florida) for technical assistance. This research was supported
by the Center for Molecular Delivery, University of South Florida, Tampa, Florida.
RECEIVED FOR PUBLICATION NOVEMBER 16, 2001;
ACCEPTED MARCH 20, 2002.
REFERENCES
1. Brunda, M., et al. (1993). Antitumor and antimetastatic activity of interleukin 12 against
murine tumors. J. Exp. Med. 178: 1223–1230.
2. Brunda, M., et al. (1995). Role of interferon-␥ in mediating the antitumor efficacy of inter-
leukin-12. J. Immunother. 17: 71–77.
3. Harada, M., et al. (1998). Role of the endogenous production of interleukin 12 in
immunotherapy. Cancer Res. 58: 3073–3077.
4. Yu, W., et al. (1997). IL-12-induced tumor regression correlates with in situ activity of IFN-
␥ produced by tumor-infiltrating cells and its secondary induction of anti-tumor pathways.
J. Leukoc. Biol. 62: 450–457.
5. Dabrowska, A., Giermasz, A., Marczak, M., Golab, J., and Jakobisiak, M. (2000). Potentiated
antitumor effects of interleukin 12 and matrix metalloproteinase inhibitor batimastat against
B16.F10 melanoma in mice. Anticancer Res. 20: 391–394.
6. Nanni, P., et al. (1998). Interleukin 12 gene therapy of MHC-negative murine melanoma
metastases. Cancer Res. 58: 1225–1230.
7. Egilmez, N., et al. (2000). In situ tumor vaccination with interleukin-12-encapsulated
biodegradable microspheres: induction of tumor regression and potent antitumor immu-
nity. Cancer Res. 60: 3832–3837.
8. Schultz, J., Pavlovic, J., Strack, B., Nawrath, M., and Moelling, K. (1999). Long-lasting anti-
metastatic efficiency of interleukin 12-encoding plasmid DNA. Hum. Gene Ther. 10:
407–417.
9. Toda, M., Martuza, R., Kojima, H., and Rabkin, S. (1998). In situ cancer vaccination: an IL-
12 defective vector/replication-competent herpes simplex virus combination induces local
and systemic antitumor activity. J. Immunol. 160: 4457–4464.
10. Colombo, M., et al. (1996). Amount of interleukin 12 available at the tumor site is critical
for tumor regression. Cancer Res. 56: 2531–2534.
11. Rakhmilevich, A., et al. (1999). Gene gun-mediated IL-12 gene therapy induces antitumor
effects in the absence of toxicity: a direct comparison with systemic IL-12 protein therapy.
J. Immunother. 22: 135–144.
12. Marshall, E. (1995). Cancer trial of interleukin-12 halted. Science 268: 1555.
13. Titomirov, A., Sukharev, S., and Kistanova, E. (1991). In vivo electroporation and stable
transformation of skin cells of newborn mice by plasmid DNA. Biochim. Biophys. Acta 1088:
131–134.
14. Heller, R., et al. (2001). Intradermal delivery of interleukin-12 plasmid DNA by in vivo elec-
troporation. DNA Cell Biol. 20: 21–26.
15. Heller, R., Jaroszeski, M., and Gilbert, R. (1996). In vivo gene electroinjection and expres-
sion in rat liver. FEBS Lett. 389: 225–228.
16. Aihara, H., and Miyazaki, J. (1998). Gene transfer into muscle by electroporation in vivo.
Nat. Biotechnol. 16: 867–870.
17. Mir, L., et al. (1999). High-efficiency gene transfer into skeletal muscle mediated by elec-
tric pulses. Proc. Natl. Acad. Sci. USA 96: 4262–4267.
18. Lucas, M., and Heller, R. (2001). Immunomodulation by electrically enhanced delivery of
plasmid DNA encoding IL-12 to murine skeletal muscle. Mol. Ther. 3: 47–53,
doi:10.1006/mthe.2000.0233.
19. Heller, L., Jaroszeski, M., Coppola, D., Pottinger, C., and Heller, R. (2000). Electrically medi-
ated plasmid DNA delivery to hepatocellular carcinomas in vivo. Gene Ther. 7: 826–829.
20. Wells, J., Li, L., Sen, A., Jahreis, G., and Hui, S. (2000). Electroporation-enhanced gene deliv-
ery in mammary tumors. Gene Ther. 7: 541–547.
21. Goto, T., et al. (2000). 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 97: 354–359.
22. Rols, M., et al. (1998). In vivo electrically mediated protein and gene transfer in murine
melanoma. Nat. Biotechnol. 16: 168–171.
23. Heller, L., Pottinger, C., Jaroszeski, M., Gilbert, R., and Heller, R. (2000). In vivo electro-
poration of plasmids encoding GM-CSF or interleukin-2 into existing B16 melanomas com-
bined with electrochemotherapy induces long-term antitumor immunity. Melanoma Res.
10: 577–583.
24. Niu, G., et al. (1999). Gene therapy with dominant-negative Stat3 suppresses growth of
the murine melanoma B16 tumor in vivo. Cancer Res. 59: 5059–5063.
25. Lohr, F., et al. (2001). Effective tumor therapy with plasmid-encoded cytokines combined
with in vivo electroporation. Cancer Res. 61: 3281–3284.
26. Kishida, T., et al. (2001). In vivo electroporation-mediated transfer of interleukin-12 and
interleukin-18 genes induces significant antitumor effects against melanoma in mice. Gene
Ther. 8: 1234–1240.
27. Heller, R., Gilbert, R., and Jaroszeski, M. (1999). Clinical applications of electrochemother-
apy. Adv. Drug Deliv. Rev. 35: 119–129.
28. Gilbert, R., Jaroszeski, M., and Heller, R. (1997). Novel electrode designs for elec-
trochemotherapy. Biochim. Biophys. Acta. 1334: 9–14.
