Download Granulocyte-macrophage colony-stimulating factor (GM-CSF)

Granulocyte-macrophage colony-stimulating factor
(GM-CSF) secreted by cDNA-transfected tumor cells
induces a more potent antitumor response than
exogenous GM-CSF
Fu-Shun Shi,1 Sharon Weber,1 Jacek Gan,2 Alexander L. Rakhmilevich,2 and David M. Mahvi1
Departments of 1Surgery and 2Human Oncology, University of Wisconsin, Madison, Wisconsin 53792.
Clinical cancer gene therapy trials have generally focused on the transfer of cytokine cDNA to tumor cells ex vivo and with the
subsequent vaccination of the patient with these genetically altered tumor cells. This approach results in high local cytokine
concentrations that may account for the efficacy of this technique in animal models. We hypothesized that the expression of certain
cytokines by tumor cells would be a superior immune stimulant when compared with local delivery of exogenous cytokines.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a nonviral expression vector was inserted into MDA-MB231 (human breast cancer), M21 (human melanoma), B16 (murine melanoma), and P815 (mastocytoma) cells by particle-mediated
gene transfer. The ability of transfected tumor cells to generate a tumor-specific immune response was evaluated in an in vitro mixed
lymphocyte-tumor cell assay and in an in vivo murine tumor protection model. Peripheral blood lymphocytes cocultured with
human GM-CSF-transfected tumor cells were 3- to 5-fold more effective at lysis of the parental tumor cells than were peripheral
blood lymphocytes incubated with irradiated tumor cells and exogenous human GM-CSF. Mice immunized with murine
GM-CSF-transfected irradiated B16 murine melanoma cells or P815 mastocytoma cells were protected from subsequent tumor
challenge, whereas mice immunized with the nontransfected tumors and cutaneous transfection of murine GM-CSF cDNA at the
vaccination site developed tumors more frequently. The results indicate that GM-CSF protein expressed in human and murine tumor
cells is a superior antitumor immune stimulant compared with exogenous GM-CSF in the tumor microenvironment.
Key words: Cytokines; granulocyte-macrophage colony-stimulating factor; gene transfer; tumor cells.
T
he development of cancer immunotherapy employing cytokines has been hampered by the inability to
achieve tumoricidal cytokine levels without dose-limiting
toxicity.1–3 However, systemic cytokine administration
may not be necessary for the destruction of metastatic
tumors. High peritumoral levels of cytokines produced
from genes introduced into tumor cells can attract
immune cells such as T lymphocytes, natural killer cells,
neutrophils, and macrophages to the tumor site without
systemic toxicity.4,5 The putative mechanism of cytokinemediated tumor destruction is not understood completely but often depends upon the generation of stimulated immune effector cells capable of tumor lysis.6 – 8 A
number of animal studies have confirmed that vaccination with tumor cells transfected with cytokine genes can
protect animals from subsequent challenge with wildtype tumors.9 –18 Thus, we19 and others20 –22 have shown
Received December 12, 1997; accepted May 4, 1998.
Address correspondence and reprint requests to Dr. David M. Mahvi,
University of Wisconsin Hospitals and Clinics, H4/726 CSC, 600 Highland Avenue, Madison, WI 53792.
© 1999 Stockton Press 0929-1903/99/$12.00/10
Cancer Gene Therapy, Vol 6, No 1, 1999: pp 81– 88
that the approach of vaccinating mice with autologous
tumor cells that express transgenic granulocyte-macrophage colony-stimulating factor (GM-CSF) results in
protection from subsequent tumor challenge. Numerous
clinical trials have been initiated recently using cytokine
gene-modified autologous tumor cells or tumor-infiltrating lymphocytes for the treatment of advanced cancer
patients.23–26 Data from these studies imply that high
local cytokine levels in the vicinity of tumor cells are as
effective as systemic cytokine administration; the concentration of the cytokine in the tumor microenvironment is the critical factor.27
Previous clinical gene therapy protocols have relied
on the delivery of cytokine DNA into tumor samples,
with subsequent placement of these altered tumor cells
as an antitumor vaccine.24 –26 This technique requires
the insertion of cytokine genes in appropriate expression
vectors into autologous tumors and has some limitations.28 An alternate method that would supply a high
local cytokine concentration in the vicinity of autologous
tumors is cytokine gene delivery to nontumor cells in the
vicinity of tumor cells, such as particle-mediated gene
transfer to the skin over a tumor vaccine with the gene gun.
The gene gun can directly deliver ;10,000 DNA copies
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SHI, WEBER, GAN, ET AL: GM-CSF SECRETED BY cDNA TUMOR CELLS
per cell into the skin with minimal tissue damage and thus
is ideal to test this alternate method. The therapeutic effect
of this approach against established intradermal (i.d.)
tumors has been demonstrated recently.29
We hypothesized that cytokine expression by the
tumor cell would be a more effective immune stimulant
than the recombinant cytokine protein delivered exogenously to the microenvironment of the tumor. We
chose to investigate this effect in a human in vitro mixed
lymphocyte-tumor cell culture system and in vivo in a
murine tumor protection model.
skin transfections. The DNA/gold particle preparation was
coated and distributed evenly onto the inner surface of Tefzel
tubing using a semiautomatic tube loader (Geniva); the tubing
was cut into 0.5-inch lengths to ensure the delivery of 0.5 mg of
gold and 1.25 mg of plasmid DNA of each cytokine per
transfection. For the transfection of tumor cell lines, cells were
g-irradiated at 10,000 rad using a 137Cs source. A total of 1.0 3
106 tumor cells in 20 mL of medium were spread into a 1.5-cm
diameter smear in a 35-mm dish and immediately transfected
with a 240-lb per square inch helium gas pulse followed by the
addition of 2 mL culture medium.
MATERIALS AND METHODS
After human tumor cells (MDA and M21) were transfected,
the medium was completely replenished with 2 mL of fresh
medium in duplicate every day for 6 days. Cell culture supernatant was collected at the indicated timepoints and frozen at
220°C until assayed. B16 cells were transfected with mGMCSF coated beads, and supernatants were collected after a
24-hour incubation. Transgenic hGM-CSF assays were performed using an hGM-CSF kit (Biosource, Camarillo, Calif)
according to the manufacturer’s instructions. The sensitivity of
the assay was 15.6 pg/mL. Additional supernatants of cells
transfected with hGM-CSF gene were also collected and
frozen at 220°C for later bioassay. For mGM-CSF, a similar
ELISA kit was prepared using commercial reagents (PharMingen, San Diego, Calif). The sensitivity of the assay was 156
pg/mL. All samples were assayed in duplicate.
Tumor cell lines
The MDA-MB-231 human breast cancer cell line (purchased
from American Type Culture Collection, Rockville, Md), M21
human melanoma cell line (obtained from Dr. R. Reisfeld, The
Scripps Research Institute, La Jolla, Calif), B16 mouse melanoma cell line,19 and P815 mouse mastocytoma cell line29 were
used in this study. All cell lines were grown and maintained in
RPMI 1640 cell culture medium (BioWhittaker, Walkersville,
Md) supplemented with 10% heated-inactivated fetal bovine
serum (Sigma, St. Louis, Mo), 2 mM L-glutamine, 1 mM
sodium pyruvate, 1% nonessential amino acids (all from
BioWhittaker), and 10 mg/mL ciprofloxacin (Miles, Kankakee,
Ill). The RPMI 1640 culture medium with supplements (referred to as complete RPMI 1640 medium) was also used for
culturing peripheral blood lymphocytes (PBLs). TF-1 human
erythroleukemia cell line was maintained in complete RPMI
1640 medium containing 5 ng/mL human GM-CSF (hGMCSF) (obtained from Immunex, Seattle, Wash).
Plasmids
The hGM-CSF coding region including the coding sequences
to nucleotide residues 27 to 1433 were amplified via polymerase chain reaction using wild-type hGM-CSF (a gift of Dr.
James S. Malter, University of Wisconsin) as a template. To
create the hGM-CSF expression plasmid pWRG3218, amplified cDNA was subcloned into a pUC19 vector containing a
cytomegalovirus (CMV) immediate/early promoter/enhancer,
a simian virus 40 intron sequence, and a bovine growth
hormone polyadenylation region. The murine GM-CSF
(mGM-CSF) expression plasmid pWRG3226 was created by
replacing the lacZ gene in pCMV-b (Clontech, Palo Alto,
Calif) with the mGM-CSF coding region from pGM3.2 (a gift
of Dr. Nicholas Gough, Walter and Elisa Hall Institute,
Melbourne, Australia). Both hGM-CSF and mGM-CSF plasmids contain the kanamycin-resistance gene. The luciferase
(Lux) vector pCMV-luc30 was used as the control DNA.
DNA transfections
A helium gas-driven gene gun designed by D. McCabe
(Geniva, presently PowderJect Vaccines, Madison, Wis) was
used for DNA transfections. The plasmid DNA of cytokines
was purified on Qiagen columns (Qiagen, Chatsworth, Calif)
and precipitated onto 2-mm diameter gold particles at a density
of 2.5 mg of DNA/mg of gold particles using the spermidine/
CaCl2 coating method.31 Particles were resuspended in a
solution of 0.015 mg of polyvinyl pyrrolidone per milliliter in
absolute ethanol for in vitro and ex vivo transfections and in 0.1
mg of polyvinyl pyrrolidone per milliliter of ethanol for direct
Measurement of transgenic cytokine levels by enzymelinked immunosorbent assay (ELISA)
hGM-CSF bioassay
To determine whether the hGM-CSF detected by ELISA in
culture supernatants from transfected human cells was biologically active, a TF-1 bioassay was performed. TF-1 cells (provided by T. Kitamura, University of Tokyo, Tokyo, Japan) were
derived from a patient with erythroleukemia and were depend
upon hGM-CSF or interleukin-3 for sustained proliferation.32
TF-1 cells were harvested from culture flasks and washed two
times with plain RPMI 1640 medium. Serial dilutions of 100
mL of recombinant hGM-CSF (rhGM-CSF) or 100 mL of
sample supernatants from the cells transfected with hGM-CSF
were mixed with 100 mL of TF-1 cell suspension (5 3 103
cells/well) in complete RPMI 1640 medium. After 48 hours, 1
mCi of [3H]thymidine was added to each well, and the cells
were incubated for an additional 18 hours at 37°C and 5%
CO2. Cells were harvested using a semiautomatic cell harvester
(Micromate 196, Packard, Downers Grove, Ill), and radioactivity was counted (Matrix 96, Packard).
In vitro activation of PBLs
Peripheral blood was collected from healthy donors and diluted with Hanks’ balanced salt solution (BioWhittaker); PBLs
were separated on a Lymphoprep (Nycomed Pharma AS,
Oslo, Norway). A total of 2 3 106 PBLs in 1 mL of complete
RPMI 1640 medium were mixed with 1 3 106 irradiated tumor
cells (MDA-MB-231 or M21) transfected with the hGM-CSF
gene or the Lux gene as described previously or nontransfected
with or without the addition of rhGM-CSF protein (65 ng); the
PBLs were subsequently cultured at 37°C with 5% CO2 for 6
days. Before performing the PBL activation experiment, we
transfected 1 3 106 irradiated tumor cells (MDA-MB-231 or
M21) and incubated the transfected tumor cells at 37°C and
5% CO2 for 24 hours. The supernatants of transfected tumor
cells were collected to determine the amount of transgenic
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SHI, WEBER, GAN, ET AL: GM-CSF SECRETED BY cDNA TUMOR CELLS
83
GM-CSF production by ELISA. The amount of exogenous
rhGM-CSF protein used to activate PBLs was based on this
measurement of the amount of transgenic GM-CSF present in
the supernatant after 24 hours of incubation per 106 transfected cells.
Cell-mediated cytotoxicity of human lymphocytes
After culture for 6 days, PBLs were collected from culture
flasks and used as effector cells in 4-hour chromium release
assays against MDA-MB-23133 or M21 targets.34 Briefly, target
cells were labeled with 250 mCi of 51Cr for 2 hours at 37°C. A
total of 105 effector cells in 100 mL of complete RPMI 1640
medium were diluted serially. A total of 5 3 103 labeled target
cells were then added to test wells in triplicate to produce
effector to target ratios of 20:1, 10:1, 5:1, and 2.5:1. Plates were
centrifuged at 100 3 g for 3 minutes and then incubated for 4
hours at 37°C. Supernatants were harvested using the Skatron
harvesting system (Skatron, Sterling, Va) and counted for 1
minute in a g counter. The percentage of cytotoxicity mediated
by the PBLs was calculated as follows: % of cytotoxicity 5
([exp. counts per minute (cpm) 2 spon. cpm]/[max. cpm 2
spon. cpm]) 3 100, where exp. was the experimental number of
counts from target cells incubated with effectors, spon. was the
spontaneously released counts obtained from targets cultured
in medium alone, and max. was the maximum counts obtained
from target cells lysed completely with a 2% centrimide
solution (Sigma). 51Cr release data from all four effector to
target ratios were also converted to lytic units (LU). In this
study, 1 LU was defined as the number of harvested effector
cells resulting in a 20% lysis of 5 3 103 target cells; LU are
expressed as LU/107 effector cells harvested.
Murine studies
Female C57BL/6 and DBA/2 female mice (6 – 8 weeks of age)
were obtained from the Harlan-Sprague-Dawley Animal Facility (Indianapolis, Ind) and Taconic Farms (Germantown,
NY), respectively. All animal experiments were conducted in
accordance with the principles stated in the Guide for the Care
and Use of Laboratory Animals. (Publication 86-23, National
Institutes of Health, Bethesda, Md, 1985).
The tumors used in this study were B16 melanoma and P815
mastocytoma, syngeneic in C57BL/6 and DBA/2 mice, respectively. For tumor vaccination, mice were injected i.d. on day 0
(P815 tumor) or on days 0 and 7 (B16 tumor) in the right
shaved abdominal area with 1 3 106 g-irradiated B16 melanoma cells or P815 mastocytoma tumor cells; B16 cells transfected with mGM-CSF were injected at a dose of 5 3 105. On
each day, mice received either (a) an i.d. injection of mGMCSF-transfected B16 or P815 tumor cells, (b) nontransfected
tumor cells administered as a vaccine with a cutaneous transfection of mGM-CSF cDNA to the skin over a vaccine site, (c)
nontransfected cells with a cutaneous transfection with Lux
cDNA, or (d) nontransfected cells without cutaneous transfections. In group 1, mice received a single injection of irradiated
mGM-CSF-transfected tumor cells on each day, whereas in
groups 2 and 3, cutaneous treatments were performed at four
sites on the abdominal vaccine injection area on each treatment day. Mice were challenged in the left side of the abdomen
with 105 B16 cells or 5 3 104 P815 cells at 2 weeks or 1 week
after the last vaccination, respectively. Tumor diameter was
measured two times per week.
Cancer Gene Therapy, Vol 6, No 1, 1999
Figure 1. hGM-CSF production by transfected, irradiated MDA-MB231 (f) and M21 cells (F). After irradiation (10,000 rad), cells were
transfected with GM-CSF expression plasmids using the gene gun.
At 24-hour intervals, supernatants were collected for ELISA and the
medium was exchanged with 2 mL of fresh complete RPMI 1640
medium. Therefore, only the production of transgenic GM-CSF from
the previous day was measured. No cells were replaced or removed
from the originally transfected dishes. The results are expressed in
nanograms of cytokine/106 initially treated cells/24 hours.
RESULTS
Transfection of human tumor cell lines results in
biologically active GM-CSF expression
The transfection of MDA-MB-231 and M21 with hGMCSF DNA resulted in GM-CSF protein expression of
72.2 and 60.0 ng/106 cells after 24 hours of culture,
respectively. Transfected cells demonstrated GM-CSF
protein expression that persisted for 6 days (Fig 1).
To confirm that the GM-CSF detected by ELISA was
biologically active, the supernatants of transfected cells
were added to a GM-CSF-dependent cell line, TF-1.
These cells, which were grown in the presence of supernatant from GM-CSF-transfected cells, proliferated as
measured by thymidine incorporation. In contrast, no
proliferation of TF-1 cells was observed in the presence
of supernatants from nontransfected cells or transfected
cells with the Lux gene (Fig 2). The mean response of
TF-1 to supernatants of hGM-CSF-transfected MDA
and M21 cells was 6600 cpm and 4300 cpm, respectively,
whereas the mean response to supernatants of untransfected or Lux-transfected MDA and M21 cells was 85
cpm and 6.1 cpm, respectively.
PBLs stimulated in vitro with GM-CSF-transfected
tumor cells killed parental tumor cells
The cytotoxic activity of human PBLs was assessed after
stimulation by hGM-CSF-transfected, Lux-transfected
tumor cells or by nontransfected tumor cells plus rhGMCSF protein. PBLs exposed to either MDA-MB-231 or
M21 tumor cells transgenic for hGM-CSF were superior
effectors when compared with the addition of exogenous
recombinant protein (Fig 3). Minimal lytic activity was
observed when PBLs were incubated with irradiated
tumor cells alone. The addition of GM-CSF protein to
the culture supernatant was similar to the tumor cells
alone without GM-CSF. The absolute lytic activity of the
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SHI, WEBER, GAN, ET AL: GM-CSF SECRETED BY cDNA TUMOR CELLS
Figure 2. Biological activity of hGM-CSF in the supernatants of
hGM-CSF-transfected MDA-MB-231 and M21 cells. Supernatants
were harvested after transfected cells had been incubated for 24
hours, and proliferation activity on TF-1 cells was analyzed using the
]3H[thymidine incorporation assay. The results indicated that supernatants from hGM-CSF-transfected cells contained biologically
active hGM-CSF, whereas Lux-transfected cells contained no hGMCSF. The values reported are the mean cpm of triplicate wells.
stimulated effector cells varied, but the PBLs stimulated
with tumor cells transfected with GM-CSF cDNA were
superior to control PBL populations in all samples
tested.
Transgenic GM-CSF was a more potent immunogen
than exogenous GM-CSF protein in vivo
The amount of GM-CSF protein in the local microenvironment was determined before the in vivo vaccination
studies. To titrate the dose of GM-CSF protein, murine
skin was evaluated 24 hours after either a single cutaneous treatment over a B16 tumor site or an injection of
B16 transfected ex vivo with an identical GM-CSF cDNA
expression vector (Fig 4). Tissue levels of mGM-CSF for
nontransfected cells (group 1) and a cutaneous transfection with the Lux DNA (group 2) over the top of
nontransfected tumor cells were ,0.1 ng of tissue per
site. Cutaneous transfection with a single dose of mGMCSF over the top of irradiated but nontransfected
tumors resulted in 1.5 ng of tissue per site (group 3). A
total of 6.5 ng of tissue per site was present after i.d.
injection of mGM-CSF-transfected B16 tumor vaccine
alone (group 4). Because the expression of transgenic
GM-CSF after cutaneous transfection (1.5 ng of tissue
per site/24 hours) seemed to be approximately four
times lower than after ex vivo tumor cell transfection, we
performed cutaneous transfections for tumor vaccination experiments at four sites, as indicated in the legend
to Figure 5B.
The results of two tumor vaccination experiments
using two different tumor models are presented in
Figure 5. In the P815 tumor model (Fig 5A), mice were
vaccinated once and challenged with the parental tumor
1 week later. The results show that delivery of the
GM-CSF gene into the skin above the nontransfected
tumor vaccination site resulted in protection in 16.6% of
Figure 3. Lytic activity of healthy donor PBLs cultured without tumor
cells (PBLs alone), cocultured with tumor cells as stimulator cells
(PBLs 1 SC), cocultured with GM-CSF-transfected tumor cells
(PBLs 1 SC-GM-CSF), or cocultured with tumor cells plus 65 ng of
exogenous rhGM-CSF (PBLs 1 rhGM-CSF). Two different tumor
cell lines (MDA-MB-231 (A) and M21 (B)) were used as both
stimulators and targets with five (A) and six (B) distinct PBL
populations.
mice, whereas 33.3% of mice immunized with ex vivo
GM-CSF-transfected tumor cells were protected from a
subsequent tumor challenge. The protection of animals
vaccinated with GM-CSF-transfected tumors was significantly (P , .022) greater than that seen in mice
vaccinated with control-transfected tumors, whereas the
delivery of GM-CSF cDNA to the skin over the tumor
was not significantly different from the control-transfected cohort (Mantel-Haenszel x2 test differences in
time until developing tumor). Comparison of ex vivo
GM-CSF-transfected tumor vaccination with GM-CSF
cDNA transfection of the skin over the tumor failed to
reach statistical significance. To confirm and extend
these results, we used a B16 melanoma model in the next
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SHI, WEBER, GAN, ET AL: GM-CSF SECRETED BY cDNA TUMOR CELLS
85
Figure 4. mGM-CSF levels detected in extracts from tissue biopsies
of vaccination sites after C57BL/6 mice were injected with a
transgenic B16 cell vaccine and/or subjected to cutaneous mGMCSF transfection. At 24 hours after the first vaccination or cutaneous transfection, two mice in each group were sacrificed for an
evaluation of the in vivo tissue level of mGM-CSF. Data show the
mean levels of mGM-CSF in a vaccination site (a standard 1.5 3 1.5
cm2 full thickness skin biopsy).
experiment.19 To augment the antitumor effect, we also
used two vaccinations instead of one; each vaccination
was performed at four sites. The results in Figure 5B
show that the nontransfected tumor vaccine alone or in
combination with cutaneous transfection with GM-CSF
cDNA did not result in protection from a B16 tumor
challenge. In contrast, vaccination with ex vivo GM-CSFtransfected tumor cells resulted in the protection of
62.5% mice from an identical B16 tumor challenge. In
this experimental system, ex vivo GM-CSF-transfected
tumor vaccination was statistically superior to GM-CSF
cDNA transfection of the skin over the tumor (P ,
.007).
DISCUSSION
Initial animal models for the therapy of malignancies by
vaccination with tumor cells transfected with cytokine
genes were based on the hypothesis that a high local
cytokine concentration in the vicinity of autologous
tumor cells was necessary for the protection of animals
from subsequent tumor challenge. This approach was
thought to be more effective than systemic cytokine
administration because higher local cytokine levels in
the vicinity of the tumor could be generated without the
toxicity of systemic cytokine administration.35 The most
direct method for cytokine delivery to the tumor microenvironment has been the in vitro transfection of tumor
cells, with subsequent placement of this “tumor vaccine”
i.d. or subcutaneously.4 –5,8,20 Consequently, the expression of the transgene by the tumor cells may be a
significant factor in the presence of stimulation of an
antitumor immune response.
Many cytokine genes have been studied in animal
tumor model systems and human cancer clinical trials.8,9,20,23,26,29,36 When different cytokine genes were
compared, GM-CSF most effectively prevented subse-
Cancer Gene Therapy, Vol 6, No 1, 1999
Figure 5. Effect of transgenic mGM-CSF production on protection
from tumor challenge after vaccination. A: Mice were not vaccinated
(F) or were injected i.d. with irradiated P815 tumor cells. The skin
above the tumor cell implants was transfected with GM-CSF cDNA
(‚) or left untreated (E). In another group, irradiated P815 tumor
cells were transfected ex vivo with mGM-CSF cDNA and injected
i.d. at a dose of 106 cells (F). At 1 week after vaccination, all mice
were challenged i.d. with unmodified P815 cells and observed for
tumor growth. Data are presented for six mice per group. B: Mice
were not vaccinated (F) or were injected i.d. with irradiated B16
tumor cells on days 0 and 7. The skin above each tumor cell implant
was transfected with GM-CSF cDNA (‚) or Lux cDNA (E). In another
group, irradiated B16 tumor cells were transfected ex vivo with
mGM-CSF cDNA right and injected on days 0 and 7 (F). At 2 weeks
after the last vaccination, mice were challenged i.d. with parental
B16 cells and observed for tumor growth. Data are presented for
eight mice per group.
quent tumor growth in a poorly immunogenic tumor
model.20 We confirmed these findings in a similar protection model.19 Therefore, the present study focused on
the most effective cytokine gene in this model system.
We demonstrated that hGM-CSF expression by human
tumor cell lines more effectively stimulated PBLs to kill
the parental tumor. This effect was consistent in PBL
populations tested. The length of expression by both
tested tumor cells lasted $6 days, which led to a more
prolonged exposure of effector cells to the cytokine. This
86
long-lasting although transient19 production of GM-CSF
by irradiated tumor cells was consistent with data reported by Rosenthal et al,37 who showed that irradiated
gene-transfected fibroblasts produced a detectable level
of G-CSF for 12 days and had systemic hematological
effects. mGM-CSF was detected at both the injection
site of i.d. tumor vaccination and at the site of direct skin
bombardment, which confirms that the particle-mediated gene transfer of nonviral expression vectors can
deliver genetic material directly into cells both in vitro
and in vivo.38 – 40 The latency of gene expression and thus
protein expression may result in more effective stimulation of the naive PBL populations studied. An alternative explanation may be that higher local GM-CSF
protein levels in the vicinity of tumor cells are superior
to a uniform level, as would be expected with systemic
protein. The in vivo vaccination model was designed to
result in consistent gene expression and consequently
focus on the effect of tumor cell expression versus
nontumor cell GM-CSF expression.
To confirm the in vitro results, we performed antitumor experiments in vivo using murine B16 and P815
tumor models with two GM-CSF gene therapy approaches. Previous results demonstrated that the immunization of mice ex vivo with irradiated interferon-gtransfected B16-F10.9 melanoma cells induced more
potent antitumor effects than did immunization with
interferon-g protein-treated tumor cells.41 We focused
on replicating similar cytokine concentrations in the
tumor microenvironment rather than on in vitro prestimulation and chose cutaneous transfection rather
than protein injection as a method to prolong gene
expression. In agreement with our in vitro results, our in
vivo data demonstrated that in both B16 and P815 tumor
models, mGM-CSF-transfected tumor cells had superior
antitumor efficacy compared with cutaneous transfections with the mGM-CSF gene at the vaccination site.
Mice vaccinated with mGM-CSF gene-transfected vaccine showed 62.5% protection against subsequent challenge with B16 tumors, whereas mice treated with
nontransfected cells with cutaneous transfections of
mGM-CSF showed no protection. These results on the
antitumor efficacy of mGM-CSF-transfected tumor cells
are consistent with our previous findings.19 We have
demonstrated that inflammatory polymorphonuclear
and mononuclear leukocytes are present at the site of
the mGM-CSF-transfected tumor cells but not at the
control DNA-transfected tumor site,19 which suggests
that the transgenic mGM-CSF is functioning locally and
that inflammatory cells are involved in the immunostimulatory process. The cutaneous treatment of the skin
over nontransfected tumors resulted in similar GM-CSF
expression in the tumor microenvironment; however, no
protection from subsequent tumor challenge was noted
in two different tumor systems. The in vivo vaccination
model suggests that latency of expression is not a factor
in the stimulation of this immune response, but that
expression by the tumor cell rather than the skin is
necessary.
SHI, WEBER, GAN, ET AL: GM-CSF SECRETED BY cDNA TUMOR CELLS
Both in vitro and in vivo results in the present study
suggest that the GM-CSF produced by transfected tumor cells has a more potent immunostimulatory effect
on immune cells than does the GM-CSF protein produced by other sources. Although the mechanism of this
enhancement is not clear, this effect may be due either to
the increased latency of hGM-CSF expression or to the
transgenic mechanism of expression. However, CD81 or
CD41 cells, dendritic cells, and B7-2 costimulatory
molecules have been reported to be involved in the
antitumor effect in GM-CSF-producing tumor cells following subcutaneous vaccination.42– 43 Whether these
mechanisms are also involved in the present study
remains to be investigated.
In summary, the hGM-CSF cDNA delivered by particle-mediated gene transfer resulted in biologically active protein expression in cultured human tumor cell
lines (MDA-MB-231 and M21). Tumor cells transfected
with the cytokine gene were more effective immune
stimulants than the exogenous rGM-CSF in the microenvironment of the tumor as measured by a mixed
lymphocyte-tumor culture followed by cytotoxicity assay.
The superior antitumor effect of tumor-secreted GMCSF was also demonstrated in vivo in B16 and P815
mouse models. Particle-mediated transfection of tumor
cells is an effective technique of DNA delivery to tumor
cells and may be a clinically attractive gene transfer
method for human cancer gene therapy.
ACKNOWLEDGMENTS
We thank PowderJect Vaccines, Inc. (Madison, Wis) for use of
the Accell gene gun in the study. This study was supported by
a grant from University of Wisconsin Surgical Associates.
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