The Role of Interleukin-2 in Combination Adenovirus Gene Therapy for Head and Neck Cancer
Bert W. OMalley, Jr.,
Duane A. Sewell,
Daqing Li,
Ken-ichiro Kosai,
Shu-Hsia Chen,
Savio L.C. Woo and
Ling Duan
Department of Otolaryngology Head & Neck Surgery (B.W.O.,
D.A.S., D.L., L.D), Johns Hopkins University, Baltimore, Maryland
21203,
Department of Cell Biology (S.L.C.W., S.-H.C.),
Baylor College of Medicine, Houston, Texas 77030,
Department of Pathology (K.-i.K.), Osaka University Medical
Center, Osaka 565, Japan
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ABSTRACT
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Interleukin-2 (IL-2) gene therapy alone and in
combination with the herpes thymidine kinase gene (tk) was used to
evaluate immunological responses and antitumor effects in head and neck
cancer. Established floor of mouth squamous cell carcinomas in C3H/HeJ
mice were directly injected with recombinant adenoviral vectors
carrying both therapeutic and control genes. One week after adenoviral
gene transfer, only the animals treated with combination IL-2+tk or tk
alone demonstrated significant tumor regression. Residual tumors were
harvested for microscopic evaluation and immunohistochemistry staining,
which revealed a predominance of CD8+ lymphocytes in the tumor beds of
the animals treated with IL-2. To evaluate the systemic immune effects
of IL-2, animals treated with single or combination gene therapy
received a second site challenge with parental tumor cells or a
heterologous but syngeneic sarcoma cell line. Mice treated with
combination IL-2 and tk demonstrated a protective systemic immunity
specific to the parental tumor cell line, whereas no systemic immune
response was evident in mice receiving IL-2 alone. In a separate
experiment, a range of concentrations of the adenovirus IL-2 vector
were used to treat established tumors. Even with the maximal
single-dose adenovirus concentration, IL-2 alone was ineffective as a
single therapy. These results support the use of adenovirus-mediated
gene transfer of IL-2 as an effective immunotherapy when used
adjuvantly with the tk "suicide gene".
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INTRODUCTION
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The idea of treating cancer with immune stimulating factors is not
new, but the understanding of the immune systems role in allowing
tumor formation as well as in treating established tumors is in
evolution. A recent hypothesis is that tumor-specific antigens may
actually be expressed in many, if not all, human tumors, and the immune
system fails because of an inadequate or incomplete antitumor response
(1). Based on this hypothesis, the prospect of using cytokines to
enhance the natural immune response is encouraging. One cytokine, in
particular, that has shown promise in the treatment of cancer is
interleukin-2 (IL-2) (2, 3).
IL-2 is produced by stimulated T lymphocytes (4) and is a known T cell
growth factor as well as T cell activation factor. IL-2 also appears to
be a potent growth and activation factor for natural killer (NK) cells
(5, 6, 7). In experimental animal tumor studies, IL-2 has been shown to
augment the effect of concurrently administered cytotoxic T cells and
has restored normal proliferative responses in patients with cancer and
acquired immunodeficiency syndrome (8). Phase I clinical trials using
systemically administered IL-2 have demonstrated some success in tumor
regression (2, 3). A major limitation of systemic IL-2, however, is the
severe toxicity, which includes fever, chills, headaches, and capillary
leak syndrome (2, 3, 9).
In addition to the toxicity, the systemic administration of cytokines
to stimulate an immunological response bypasses a critical principle in
lymphokine physiology. This principle is that lymphokines act in a
paracrine fashion to generate and maintain the specificity of the
immunological response (10). Under physiological circumstances,
specific cytokines are produced locally in high concentrations at the
antigen site. These cytokines act locally in concert with
antigen-driven signals to generate effector responses. Pharmacological
doses of cytokines administered systemically, however, result in high
concentrations in the vasculature at sites distant from the antigen but
often in suboptimal levels in tissues at the site of antigen.
Considering both the toxicity and the lack of paracrine function, local
delivery of cytokines appears to be a safer and more physiological
approach to cytokine-based cancer therapy. Important to this concept
are studies that have shown that local cytokine delivery can produce
dramatic inflammatory effects without significant systemic toxicity (1, 11, 12).
A pilot clinical trial for inoperable squamous cell cancer of the head
and neck was reported by Forni and co-workers (13). In this study,
patients received daily local injections of recombinant IL-2 around the
regional draining lymph nodes for 10 days, which were repeated on a
monthly basis for 1 yr. Twenty five percent of the patients
demonstrated partial or complete tumor regression after local IL-2
therapy. Although disease-free survival was increased compared with
nontreated controls, the responding patients developed a recurrence
35 months after treatment. These and other promising results of local
cytokine delivery have led to the interest in developing cancer vaccine
strategies.
A notable limitation to local delivery of the recombinant protein is
the quick clearance of the protein and the need for multiple closely
spaced injections to maintain an antitumor response. A solution to this
limitation may lie in the application of gene transfer of cytokines
such as IL-2 to provide local sustained release of the therapeutic
protein. The replication-defective adenovirus is a widely studied
vector for gene transfer and has many important features that are
useful for cancer therapy strategies. Adenoviral vectors can carry
therapeutic genes at titers of up to 1011 plaque-forming
units (pfu)/ml, which is significantly greater than retroviral vectors
(14). The adenoviral isolate can also be injected alone and directly
into tumors with resulting effective gene expression. Furthermore, the
adenovirus genome remains episomal rather than integrating into the
chromosome as occurs with retroviruses. The adenovirus is also being
used in multiple safety studies and human clinical trials for various
human diseases (15, 16, 17, 18).
We have developed a head and neck cancer model to study gene transfer
strategies and have demonstrated antitumor efficacy in both single and
combination gene therapy treatments (19, 20). Our investigations center
on combining a popular "suicide gene", the herpes virus thymidine
kinase gene (tk) with the gene for IL-2 for the treatment of squamous
cell carcinoma of the head and neck. Delivery of the tk gene coupled to
systemic administration of the nucleoside analog ganciclovir (GCV)
results in necrosis and direct cytotoxicity to dividing cells. When
local IL-2 expression is combined at the site of tumor necrosis, we
propose that a synergistic antitumor response results (20, 21). With
respect to the safety of this strategy, we have not detected any local
or distant pathological effects either from a direct adenovirus vector
toxicity or from a secondary inflammatory response in the brain, liver,
or floor of mouth and neck (19, 20, 22). The immune response after gene
transfer of IL-2 in the head and neck cancer model, however, has yet to
be evaluated. The following study investigates the immunological role
IL-2 plays in single or combination gene therapy and provides insight
into the limitations of adenoviral-mediated cytokine delivery to head
and neck cancer.
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RESULTS
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Effects of Adenoviral Transduction in Vitro
Adenovirus (ADV)/Rous sarcoma virus (RSV)-murine (m)IL-2 was
delivered to squamous cell carcinoma (SCC) VII cells in
vitro to determine the presence of direct cytotoxicity from the
vector and to confirm functional transduction of the tumor cells. The
ADV/RSV-mIL-2 vector had no effect on cell viability (P
> 0.4, Students t test) compared with the control (Fig. 1A
). Previous in vitro studies have
demonstrated no toxic effects of tk or ß-gal control adenovirus
vectors (19, 20).

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Figure 1. In Vitro Response to Adenovirus Transduction
with mIL-2
A, ADV/RSV-mIL-2 transduction of SCC VII squamous carcinoma cells
in vitro at MOIs ranging from 0 (PBS) to 50. Cell
survival was assessed 3 days after transduction. There was no evidence
of direct cytotoxicity from the IL-2 vector compared with the PBS
control (P = 0.4). B, PCR confirming the presence
of vector within the cells at 72 h posttransduction. Note the
bands at 207 bp, which represent the region specific to the
ADV/RSV-mIL-2 vector. Lanes 14 represent cells that have been
transduced at descending MOIs of 50, 20, 15, and 10.
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To demonstrate the presence of the IL-2 gene within the transduced
cells, PCR amplification was performed using primers specific to the
ADV/RSV-mIL-2 vector. The vector was amplified in the 1050
multiplicity of infection (MOI) treatment groups, but not in the 0 MOI
control (Fig. 1B
). Functional transduction was confirmed by assaying
the tumor cell culture supernatant for IL-2. Using an IL-2-specific
enzyme-linked immunosorbent assay kit, IL-2 protein was detected in
supernatants at both 24 and 72 h after transduction (950
pg/liter x 106 cells).
Characterization of Immune Infiltrates after Adenovirus Treatment
in Vivo
The purpose of this experiment was to evaluate the role of
designated lymphocytes in the therapeutic effect of adenovirus gene
transfer. Twenty mice received floor of mouth injections with the
squamous carcinoma cells as described. At the time of adenovirus
injection, tumor sizes ranged from 70100 mm3. There were
no significant differences in pretreatment tumor sizes between
experimental groups. In the four experimental groups (tk+IL-2, tk
alone, IL-2+ ß-gal, and ß-gal control), each tumor was treated with
a total of 1 x 109 pfu in a total volume of 50 µl.
For animals receiving combined therapy, 2 x 108 pfu
of ADV/RSV-mIL-2 was delivered. All animals subsequently received
intraperitoneal administration of GCV at 25 mg/kg twice daily for six
days and were killed on day 7. Consistent with our previous findings,
only the groups treated with tk + IL-2 and tk alone demonstrated
significant tumor regression compared with the ß-gal alone control
animals (P = 0.0004; Mann Whitney analysis). Also
consistent was the finding that the tk + IL-2-treated animals
demonstrated significant regression as compared with the tk
alone-treated animals (P = 0.0006).
Immunohistochemical analysis was performed on all tumor specimens, and
positive staining cells were counted per ten high-powered fields (Fig. 2
). There were no statistical differences between groups
for CD4 staining, but CD8 staining in the tk+IL-2 group was
statistically greater than in the tk or ß-gal alone group. Although
CD8 staining in the tk+IL-2 group was higher than in the IL-2+ß-gal
group, significant differences were not evident. Notable was the fact
that all samples showed a minimum staining for both CD4 and CD8
lymphocytes, whereas the residual tumors treated with IL-2 alone or in
combination with tk showed an average of 2 to 5 times more positive CD8
cells. The enhanced CD8 lymphocyte tumor infiltration appears to be a
direct result of adenoviral gene transfer of IL-2. Despite the
increased CD8 lymphocytes in the IL-2+ß-gal group, however, no
therapeutic benefit was seen. This finding indicates a lack of
significant tumor recognition by these cytotoxic T lymphocytes in the
groups not treated with tk.

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Figure 2. Summary of Immunohistochemical Staining on Residual
Tumor Specimens 1 Week after Adenoviral Treatment
Five residual tumors from each animal group were randomly selected and
stained for CD8, CD4, and NK lymphocytes with the mean staining and
range depicted for each group. The CD8 staining in the tk+IL-2 group
was statistically greater than the tk or ß-gal alone group
(P = 0.009 to 0.016; Mann-Whitney analysis).
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Systemic Antitumor Immunity in Animals Receiving mIL-2 and tk
Adenoviral Therapy
Head and neck tumors were established in 44 mice. After 5 days,
neck skin flaps were raised surgically, and tumors were identified in
all animals. Tumor sizes ranged from 8 to 24 mm3 based on
caliper measurements. Although the animals were randomly placed into
four experimental groups, there were some significant differences in
pretreatment tumor sizes. The pretreatment tumor sizes for both the
IL-2+ß-gal and the ß-gal alone groups were significantly smaller
than the tk+IL-2 and tk alone groups (P = 0.0003 and
0.0019; Mann-Whitney analysis). There were no significant differences
in pretreatment sizes when the IL-2+ß-gal vs. the ß-gal
alone group or the tk+IL-2 vs. the tk alone group
(P = .2.9) were compared. Animals received GCV at the
same dosing scheme as before, and tumor sizes were calculated on live
animals by external caliper measurements in three dimensions. The
external measurements were performed because of the need for the
survival experiment with the second tumor challenge. Posttreatment
tumor sizes ranged from 0800 mm3. Despite the larger
pretreatment size, both the tk+IL-2 and the tk groups demonstrated
significant tumor regression compared with both IL-2+ß-gal and
ß-gal alone groups (P = 0.00010.007; Mann-Whitney
analysis) (Fig. 3
). Although the IL-2+ß-gal group had
the widest range of posttreatment tumor sizes, the mean value was very
close to the ß-gal alone group, and there was no significant
difference from the control (P = 0.7). Based on
previous experience, tumors less than 10 mm3 do not project
from the floor of mouth enough to allow identification and caliper
measurement. Therefore, tumors less than 10 mm3 could have
been present in the tk+IL-2 and tk animals that received a tumor size
calculation of 0 mm3.

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Figure 3. Box and Whisker Plot of Residual Tumor Sizes
(ranging from 0800 mm3) after Various Adenoviral
Treatments in 44 C3H/HeJ mice with Established Floor of Mouth Tumors
The full range of data points is depicted by the vertical
lines, and each subsequent horizontal line
depicts percentiles in descending order of 90, 75, 50, 25, and 10th
percentile where applicable. The geometric shape within the
box represents the median. Only the tk+IL-2 and tk groups
demonstrate a significant antitumor response (P =
0.0003 and 0.0019).
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After the floor of mouth tumors were measured, the mice received
opposite flank injections of tumorigenic doses of SCC VII cells and
control RIF-1 sarcoma cells. Animals were killed after 14 days, and the
flanks were examined externally as well as incised and dissected to
assess second-site tumor growth. The time point of 14 days was chosen
because of the limitation in survival for the control groups and animal
care and use committee restrictions on tumor burden. As compared with
the control RIF-1 flank, 100% of the mice treated with tk+IL-2 and
25% of the mice treated with tk alone failed to develop a SCC VII
flank tumor. For the IL-2+ß-gal and ß-gal alone groups, only 15%
of the animals failed to develop a parental SCC VII tumor (Fig. 4
). All tumors at second sites were greater than 100
mm3 in size. The protective systemic antitumor immunity is
dependent on the combination of tk+IL-2 as this group was significantly
different from all other groups (P = 0.003; Fisher
Exact analysis).

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Figure 4. Systemic Immunity against the SCC VII Squamous
Carcinoma at a Distant Site Challenge
Tumorigenic doses of SCC VII and the heterologous, but syngeneic,
fibrosarcoma RIF-1 tumor line were injected into flanks of C3H/HeJ mice
that received previous adenoviral treatment to their primary floor of
mouth tumors. Palpable SCC VII tumors were present in all groups except
the tk+IL-2 combination group at the 2 week sacrifice point.
Subcutaneous dissection confirmed the lack of SCC VII tumor growth in
the tk+IL-2 animals. Tumor-specific immunity was supported by the
failure to suppress RIF-1 tumor growth in all groups.
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Dose Response to ADV/RSV-mIL-2 in Vivo
The experimental results have shown no significant antitumor
effect after treatment with adenovirus vector containing IL-2 at 2
x 108 pfu. A dose response experiment was performed in 35
animals to determine whether an antitumor effect could be generated
with either more or less total adenovirus delivered to established
tumors. Floor of mouth tumors were generated as before, and
pretreatment tumor sizes ranged from 2060 mm3 (no
significant pretreatment size differences). Five groups were divided as
follows: group 1, 5 x 108 ADV/RSV-mIL-2; group 2,
2.5 x 108 pfu; group 3, 1 x 108
pfu; group 4, 5 x 107 pfu; and group 5, PBS-treated
control. One week after adenovirus or PBS control treatment, animals
were killed and tumors were measured. Large tumors grew in all groups
with no significant differences between any group (P =
0.94; Mann-Whitney analysis) (Fig. 5
). Microscopic
evaluation of all residual tumors was performed after routine
hematoxylin and eosin staining. A minimal inflammatory infiltrate was
detected equally in each group, and no tumor necrosis or evidence of
cytotoxic effect was seen (data not shown). These results suggest that
adenovirus delivery of IL-2 alone is ineffective as a single injection
in the established tumors.

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Figure 5. Dose Response to ADV/RSV-mIL-2 Treatment in
Established Tumors
No significant antitumor effect was noted up to the maximum
plaque-forming units obtainable with this vector in a single controlled
injection (P = 0.94). Solid bars
represent the mean tumor sizes with SD shown.
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DISCUSSION
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Although both CD4 and CD8 lymphocytes appear to be important in
the generation of a synergistic antitumor response after combination
gene therapy, it is the specificity of the lymphocytes for the parental
tumor that appears most important. This concept is supported by the
immunohistochemistry results of the treated tumors. Both the
combination tk+IL-2- and IL-2+ß-gal-treated animals revealed both CD4
and CD8 infiltration with an increase in CD8 vs. the other
non-IL-2-containing groups (only the tk+IL-2 group was significantly
increased by statistical analyses). Despite the similar total numbers
and distribution of CD4 and CD8 lymphocytes in these two groups, only
the tk+IL-2 group had significant tumor regression compared with the
ß-gal alone control. Also, nonspecific tumor killing from NK cells
stimulated by IL-2 does not appear to be present as no NK-positive
staining was seen in tumor specimens. We thus conclude that the
combination of tk+IL-2 creates a local atmosphere conducive to
generating tumor-specific immune responses.
Tumor regression was significant in the tk alone group, but the
combination tk+IL-2 therapy was more effective. Inasmuch as CD8
infiltration was 2 to 5 times more intense in the tk+IL-2 group, local
IL-2 production appears to be a critical component in increasing
tumor-specific lymphocytes and inducing synergistic effects on tumor
regression when combined with tk treatment. It is possible that the CD8
cell infiltrate may also reflect, to some degree, an immune response
against tk or ß-gal proteins. However, the CD8 values are
significantly lower than the IL-2-containing groups. In ongoing work
with these vectors, we have never seen a significant antitumor response
to tumors treated with tk or tk+IL-2 without GCV or to tumors treated
with ß-gal with or without GCV (Refs. 19, 20 and our unpublished
data). Furthermore, when established tumors are treated with
ADV/RSV-mIL-2 alone (without ß-gal), there is still an increased CD8
infiltrate vs. those tumors treated with ADV/RSV-ß-gal
alone (our unpublished data). These findings collectively support the
importance of the IL-2 and not tk or ß-gal on both the extent and
tumor specificity of inflammatory infiltrate.
The results of the second tumor challenge experiment support the
hypothesis that combination therapy creates a tumor-specific immune
response. Effective tumor regression was seen in both tk+IL-2 and tk
alone groups; however, only the combination therapy provided a
tumor-specific immunity. After the various adenoviral treatments to
floor of mouth tumors, SCC VII cells injected in the flanks grew as
tumors in all groups except those treated with combination tk+ IL-2.
The specificity of this antitumor immunity was further illustrated by
the significant growth of a heterologous sarcoma cell line (RIF-1)
injected concurrently in the opposite flank. Therefore, the systemic
immunity in the tk+IL-2-treated animals was specific for the parental
SCC VII tumor cells.
The in vitro experiments reveal a lack of direct
cytotoxicity ADV/RSV-mIL-2 up to MOI values well above those achievable
in vivo and demonstrate effective IL-2 expression in
transduced cells. To determine whether our lack of efficacy for IL-2
alone was simply a matter of concentration of adenovirus delivered to
the floor of mouth tumors, the dose-response experiment was performed.
Adenovirus containing a range of IL-2 up to the maximum possible dose
for 50 µl (5 x 108 pfu) was delivered to
established tumors. No significant tumor regression was seen as
compared with controls, and microscopic examination revealed no
necrosis or other pathological effects. The combination therapy data
coupled to the dose-response results strongly support the adjuvant role
of IL-2 in conjunction with adenovirus tk therapy.
Based on the above results, it appears that direct tumor killing from
tk (and GCV administration) provides a medium that enables enhanced
local IL-2 expression to generate tumor-specific immune responses. We
hypothesize that the necrosis and cellular debris from tks direct
tumor killing results in release or concentration of tumor-specific
antigens. Local antigen-presenting cells present the tumor antigens to
CD8 cells and stimulate a tumor-specific immune response. The increased
local production of IL-2 after intratumor adenovirus delivery enhances
the complete immune response, providing both effective local tumor
regression and systemic antitumor immunity. Although the data are
consistent with such a conclusion, more extensive immune studies are
needed to provide stronger support of this hypothesis in the head and
neck tumor model.
The notable weakness of this system is a lack of complete cure despite
effective tumor regression, immune system stimulation, and increased
survival. We have thus far been unable to consistently prevent tumor
recurrence with this strategy. The majority of animals develop
recurrent floor of mouth tumors within 14 weeks after the response
(20). As in most animal tumor models, recurrence signifies persistence
of the original tumor. The persistence of tumor may be a result of
incomplete tumor transduction with adenovirus vector or possibly
immunoselection. Future studies will address these issues and will
include both repeat adenovirus injection and the addition of other
cytokines, such as granulocyte-macrophage colony-stimulating factor,
which may enhance antigen presentation as well as long-term antitumor
effects. The adenovirus gene therapy strategy is still in its infancy,
but the findings of effective tumor regression and tumor-specific
immune stimulation support the need for continued work toward future
clinical application in the treatment of head and neck cancer.
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MATERIALS AND METHODS
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Construction of Recombinant Adenoviral Vectors
Construction of a replication-defective adenoviral vector
containing the tk gene under transcriptional controls of the Rous
sarcoma virus (RSV) long-terminal repeat (ADV/RSV-tk) has been reported
previously (19, 22). A replication-defective adenoviral vector
containing the mIL-2 cDNA under the transcriptional control of the RSV
long-terminal repeat promoter (ADV/RSV-mIL-2) was similarly constructed
and plaque purified. The viral titer (plaque-forming units/ml) was
determined by plaque assay.
In Vitro Experiments
The squamous carcinoma cell line SCC VII was used in all
experiments. Originally, the SCC VII squamous cell carcinoma arose
spontaneously in C3H/HeJ mice and has subsequently been propagated
in vivo (23). The cells were cultured in T-75 tissue culture
flasks (Corning Glass Works, Corning, NY) containing 30 cc RPMI 1640
media (Sigma Chemical Co., St. Louis, MO), 12% bovine calf serum, 1%
penicillin-streptomycin, and 1% L-glutamine. Cells were
maintained in 5% CO2 incubators. The recombinant
adenovirus containing IL-2 was added at MOI values ranging from 050
to the different wells just after the cells were plated. Seventy-two
hours after plating, the cells were trypsinized and counted with a
hemacytometer.
PCR reaction was performed to confirm the presence of the vector
construct within the cells. Cells were digested with 1% SDS-proteinase
K at 42 C for 12 h. DNA was extracted by ethanol precipitation.
PCR amplification was performed with the primers RSV 270A
(GACTCCTAACCGCGTACA) and ADV 3205 (GTGTTACTCATAGCGCGTAA), which are
specific for the ADV/RSV-mIL-2 vector, with the following PCR protocol:
95 C for 1 min, 5x for 1 min, and 7x for 1 min for 35 cycles, and
then 7x for 5 min. Five microliters of the 50-µl reaction was run on
a 1% agarose gel.
In Vivo Experiments
All animal experiments were performed on C3H/HeJ mice (Jackson
Laboratories, Bar Harbor, ME) using sterile technique under a laminar
flow hood in accordance with the Johns Hopkins Animal Care and Use
Committee regulations. Mice 610 weeks old were anesthetized using the
inhalational agent Metophane, and a 0.1 cc suspension of 5 x
105 SCC VII cells in HBSS was injected directly into the
floor of the mouth. The animals were then maintained in standard
housing conditions.
Five days after cell implantation, mice were anesthetized with 0.5 cc
avertin at a concentration of 20 mg/ml with the depth of anesthesia
determined by toe pinch. A skin incision was made in the lower neck,
and surgical dissection revealed the established floor of mouth tumors.
Tumors were measured in three dimensions with calipers. Using a
100-µl syringe (Hamilton, Reno, NV) and 26-gauge needle, 1.0 x
109 total pfu of either ADV/RSV-tk, ADV/RSV-ß-gal
control, ADV/RSV-tk+ADV/RSV-mIL-2 (2.0 x 108), or
ADV/RSV-mIL-2 (2.0 x 108) + ADV/RSV-ß-gal in 50
µl solution were injected directly into the tumors. Neck incisions
were closed with 40 silk suture (Ethicon, Somerville, NJ). Eighteen
hours after adenoviral injection, the mice were administered GCV ip at
a regimen of 25 mg/kg twice daily for 6 days.
For the second tumor challenge experiments, tumor sizes were assessed
on the seventh day after adenoviral treatment by external caliper
measurements. The right and left flanks were then injected separately
with tumorigenic doses of either SCC VII or the syngeneic fibrosarcoma
cell line RIF-1. Tumor growth was evaluated 1 and 2 weeks after
injection.
Immunohistochemistry
For the immunohistochemistry studies, mice were killed 1
week after adenoviral treatment of the floor of mouth tumors. Tumor
sizes were measured, and the fluorescein anti-fluorescein system was
used to identify infiltrating inflammatory cells in residual tumor
masses. Frozen tissues were sectioned at 4 µm and placed on
silane-coated slides. Endogenous peroxidase activity in the tissue was
blocked by H2O2 treatment. Nonspecific binding was blocked
with PBS containing 0.3% BSA. Fluorescein-conjugated primary
monoclonal antibodies used in the assay were as follows: rat anti-mouse
CD4 (L3T4) (GIBCO BRL, Grand Island, NY), rat anti-mouse CD8a (Ly-2)
(GIBCO, BRL), mouse anti-mouse NK (5E6) (Pharmingen, San Diego, CA).
After reaction with primary antibodies, the sections were rinsed and
incubated with peroxidase-conjugated rabbit anti-fluorescein
isothiocyanate (DAKO, Carpinteria, CA) for 2 h at room
temperature. After rinsing, the slides were incubated in chromogen
solution (diaminobenzidine, 3 mg; PBS, 10 ml; 8% NiCl, 50 µl; 30%
H2O2, 1 µl) for 10 min. The reaction was stopped in
running distilled water for 1 min, and the slides were counterstained
with Nuclear Fast Red for 5 min.
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ACKNOWLEDGMENTS
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This work was supported in part by the Johns Hopkins Clinician
Scientist Award and Grant 1 R29 Grant DE 1177201 from the National
Institute of Dental Research (to B.W.O. Jr.).
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FOOTNOTES
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Address requests for reprints to: Bert W. OMalley, Jr., M.D., Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins University, P.O. Box 41402, Baltimore, Maryland 21203-6402.
Received for publication January 29, 1997.
Accepted for publication March 21, 1997.
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