From the Geraldine Brush Cancer Research Institute at
the California Pacific Medical Center, San Francisco, California 94115, the § Department of Pathology, Northwestern University
School of Medicine, Evanston, Illinois 60201, ** Laboratory of
Angiogenesis Research, Microbiology and Tumor Biology Center,
Karolinska Institute, S-171 77, Stockholm, Sweden, and
School of Pharmacy, University of
Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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Cationic liposome-DNA complex (CLDC)-based
intravenous gene delivery targets gene expression to vascular
endothelial cells, macrophages and tumor cells. We used systemic gene
delivery to identify anti-angiogenic gene products effective against
metastatic spread in tumor-bearing mice. Specifically, CLDC-based
intravenous delivery of the p53 and GM-CSF genes were each as effective
as the potent antiangiogenic gene, angiostatin, in reducing both tumor
metastasis and tumor angiogenesis. Combined delivery of these genes did
not increase anti-tumor activity, further suggesting that each gene
appeared to produce its antimetastatic activity through a common
antiangiogenic pathway. CLDC-based intravenous delivery of the human
wild type p53 gene transfected up to 80% of tumor cells metastatic to
lung. Furthermore, it specifically induced the expression of the potent
antiangiogenic gene, thrombospondin-1, indicating that p53 gene
delivery in vivo may inhibit angiogenesis by inducing
endogenous thrombospondin-1 expression. CLDC-based delivery also
identified a novel anti-tumor activity for the metastasis suppressor
gene CC3. Thus, CLDC-based intravenous gene delivery can produce
systemic antiangiogenic gene therapy using a variety of different genes
and may be used to assess potential synergy of combined anti-tumor gene
delivery and to identify novel activities for existing anti-tumor genes.
Molecular genetics has identified a large number of genes whose
aberrant or loss of expression is correlated with the initiation and/or
progression of the malignant phenotype (1, 2). However, it has been
difficult to determine whether the overexpression of the normal gene
products can produce significant anti-tumor effects in tumor-bearing
animals for several reasons. First, it may be difficult to obtain
sufficient quantities of secreted gene products to test their potential
anti-cancer activity in tumor-bearing animals (3, 4). Second, testing
gene products that function only within cells, such as
tumor-suppressing DNA-binding nucleoproteins, often requires the use of
in vivo gene delivery, since administration of recombinant
proteins would not be expected to deliver therapeutic levels of the
active molecules to their required tissue and cellular sites of action
in tumor-bearing hosts. Similar cellular barriers have limited the
in vivo delivery of therapeutic levels of antisense and
ribozyme constructs directed against the products of overexpressed transforming genes (5, 6).
Existing in vivo gene delivery systems have been
particularly limited in their ability to treat metastatic cancer, which
is the major cause of morbidity and mortality in human cancer patients (7). We have developed a redesigned
CLDC1-based, intravenous gene
delivery approach that produces levels of systemic gene expression from
500 to 1,700-fold higher than intravenous injection of conventional
CLDC (8-10). We now show that these redesigned CLDC are able to
transfect large numbers of metastatic tumor cells with the wild type
human p53 gene following intravenous injection into C57Bl6 mice bearing
metastatic B16-F10 melanoma tumors. Furthermore, CLDC-based,
intravenous delivery of a variety of different anti-tumor genes each
produced significant anti-metastatic activity and concurrently reduced
tumor angiogenesis. This approach also permitted the assessment of
potential in vivo anti-tumor synergy between combinations of
anti-tumor genes, thus helping to determine whether genes are producing
their anti-tumor effects by common or independent pathways. It also
identified a novel anti-tumor activity for the CC3 metastasis
suppressor gene in tumor-bearing hosts.
Plasmids
p4199--
CMV-p53 was constructed by isolating a 1.4-kilobase
pair HindIII-SmaI fragment containing the wild
type human p53 cDNA (a gift from Dr. B. Vogelstein) and ligating it
into the HindIII-SmaI site of p4136 (11).
p4305--
CMV-angiostatin was constructed by isolating a
1.4-kilobase pair HindIII-XbaI fragment
containing the murine angiostatin cDNA (a gift from Dr. J. Folkman)
and then ligating it by blunt end ligation into the PvuII
site of p4109 (11).
p4442--
CMV-BCL-2 was constructed by ligating the human BCL-2
cDNA (a gift from Dr. S. Korsmeyer) into the EcoRV site
of pVR1223 (12).
p4447--
CMV-CC3 was obtained by isolating a 0.8-kilobase pair
fragment containing the human CC3 and ligating it into the
EcoRV site of pVR1223. The construction of p4119,
CMV-chloramphenicol acetyltransferase (CAT) (11), and p4241,
CMV-luciferase (8), have been described. Plasmids were purified as
described previously (11).
Preparation of Cationic Liposomes and CLDC
DOTIM:cholesterol multilamellar vesicles (MLV) and pure DOTIM
MLV were prepared as described previously (8). CLDC were prepared as
described (11).
Tumor Cells and Tumor Inoculation
Murine B16-F10 melanoma cells were grown in RPMI 1640 with 5%
fetal bovine serum at 37 °C with 5% CO2. The B16-F10
melanoma cells expressed the wild type p53 gene, as determined by both DNA damage and Western blot analyses (data not shown), and also expressed low levels of the CC3 protein, as determined by Western blot
analysis (data not shown). For tumor cell inoculation, B16-F10 cells
were trypsinized, and then 25,000 cells/mouse in 200 µl of culture
medium were injected by tail vein into 25-g female C57Bl6 mice
(Simonson, Gilroy, CA). B16-F10 melanoma is a highly metastatic
subclone of B16 melanoma (13) that kills mice approximately 35 days
following intravenous inoculation of 25,000 cells (data not shown).
In Vivo Transfections and Analysis of Anti-tumor Activity
Unless specifically indicated, each mouse received 25 µg of
plasmid DNA complexed to DOTIM:cholesterol MLV. The DNA:lipid ratio
(µg of DNA/nmol of total lipid) was 1:16 for all DOTIM:cholesterol MLV and 1:26 for pure DOTMA MLV. Each of these DNA:lipid ratios had
previously been determined to produce maximal levels of gene expression
following intravenous injection of CLDC (Ref. 8 and data not shown).
CLDC were injected into tumor-bearing mice either 3 or 10 days after
tumor cell inoculation or, in some cases, once only at day 7 following
tumor inoculation. For the immunohistochemistry studies, mice were
injected intravenously with CLDC 29 days after tumor cell inoculation.
All mice were sacrificed 30 days after tumor cell inoculation, and
lungs from each mouse were dissected out, infused transtracheally with
10% neutral buffered formalin (Fisher), and then fixed in 10% neutral
buffered formalin. The number and size of the black-appearing tumor
nodules were counted two times under a dissecting microscope by an
individual blinded to the identity of the groups. Only tumor nodules
>2 mm in diameter were included in the analysis, since tumor growth
beyond 2-3 mm appears to depend on the presence of tumor angiogenesis
(14-17). The potential statistical significance of differences between the various groups was assessed using an unpaired, two-sided Student's t test.
Immunohistochemistry and Vascular Staining
Formalin-fixed paraffin-embedded 4-µm sections were
deparaffinized and rehydrated to phosphate-buffered saline.
Pretreatments included microwave antigen retrieval in a 10 mM citrate buffer for 10 min (factor VIII, p53), 10%
sucrose at 80 °C for 2 h (thrombospondin), or 0.01% trypsin
with 0.25% protease for 10 min (Sigma; factor VIII). Endogenous
antibodies were blocked with CAS Block (Zymed Laboratories
Inc., S. San Francisco, CA; p53, thrombospondin) or normal goat
serum (1:10 dilution; Vector Laboratories, Burlingame, CA; factor
VIII). Overnight incubation at room temperature was performed using
polyclonal anti-p53 antibody (1:100 dilution, BioGenex Labs, San Ramon,
CA) or anti-thrombospondin (1:35 dilution; Biodesign International,
Westbrook, ME). Incubation using polyclonal anti-factor VIII (von
Willebrand's factor, 1:250 dilution; Dako Corp., Carpenteria, CA) was
carried out at room temperature for 1 h. All primary antibodies
were washed off with 0.2-1% Tween 20 (Fisher), and biotinylated
goat-anti-rabbit (1:500; Vector Laboratories; Factor VIII, p53) or
biotinylated rabbit-anti rat (1:200; Vector Laboratories;
thrombospondin) linker was applied for 30 min. Following additional
washes, ABC elite (factor VIII, p53, Vector Laboratories) or
streptavidin-horseradish peroxidase (1:100; Zymed
Laboratories Inc.) was applied for 30 min. After additional
washes, AEC (Biomeda Corp., Foster City, CA) was used for 20 min
followed by counterstaining with hematoxylin and coverslipping with
crystal mount (Biomeda Corp.).
Quantitating Tumor Angiogenesis and the Percentage of Cells
Expressing p53 and TSP-1
The intratumoral blood vessels were quantitated using factor
VIII, anti-von Willebrand's factor staining, as described above. For
each tumor, high power magnification was used with an intraocular grid.
The grid facilitated counting so that no vessel was scored twice.
Vessels that were visibly interconnected were scored only once.
Neovessels within each tumor >2 mm were counted in as many high
powered fields (grids) as could be placed within each tumor (usually
two or three, as high as five) based upon the size of the nodule. This
differs from vessel counting in larger human tumors (18), because areas
of high vascularity were not preselected. Rather, the entire cross
section of these tumor nodules was analyzed. The final angiogenesis
score for each nodule reflected a summation of all scores (the sum of
scores, divided by the total number of fields that were counted). Every
tumor nodule of >2 mm that was microscopically visualized from each
animal was analyzed for neoangiogenesis. A total of nine tumors from
five different mice treated with the CAT gene, nine tumors from four
different mice treated with the angiostatin gene, 12 tumors from six
different mice treated with the p53 gene, and four tumors from three
different mice treated with the GM-CSF gene were analyzed. Thus, 34 individual tumors from 18 individual mice, all derived from experiment
1a were individually counted. To quantitate p53 gene expression, only
cells showing the characteristic pattern of nuclear staining were
counted as positive, and for TSP-1, only cells showing cytoplasmic reactivity were counted. Scores for p53 and TSP-1 gene expression reflect the percentage of positive cells for each histologically identifiable tumor nodule evaluated. All evaluable tumors on the tissue
sections used for the assay were quantitated.
CLDC-mediated, Intravenous Delivery of the Murine Angiostatin Gene,
Murine GM-CSF, or the Wild Type Human p53 Gene Each Produces
Significant Anti-metastatic Activity--
We first compared the size
and number of lung metastases in the CLDC-treated and control C57Bl6
mice 30 days after intravenous injection of 25,000 B16-F10 melanoma
cells/mouse. Individual mice in groups of eight received 400 nmol of
DOTIM:cholesterol MLV complexed to 25 µg of an HCMV-driven expression
plasmid containing the murine angiostatin gene, the murine GM-CSF gene,
the human wild type p53 gene, or the CAT reporter gene (mock-treated
controls) on day 3 and again on day 10 following tumor inoculation. The control group received B16-F10 cells but no further treatment.
CLDC-mediated, intravenous delivery of the murine angiostatin gene,
murine GM-CSF, or the wild type human p53 gene each produced significant anti-metastatic effects, as determined by both the total
number of lung metastases (data not shown) and those greater than 2 mm
in diameter in tumor-bearing mice, when compared with either reporter
gene-treated (p < 0.05) or untreated
(p < 0.05) controls by a two-sided Student's
t test (Fig. 1A).
The p53-treated, angiostatin-treated, and GM-CSF gene-treated groups
each produced similar reductions in the number of lung metastases
compared with control groups. Reporter gene (mock)-treated and
untreated controls did not differ significantly in either the total
number of tumors or the number of tumors greater than 2 mm in diameter
(p < 0.375). Previously, intravenous injection of
DOTMA:DOPE small unilamellar vesicles complexed to 10 µg of control
(noncoding) DNA produced some degree of nonspecific anti-tumor activity
in SCID mice bearing a human breast cancer xenograft when compared with
nontreated controls (19). The presence or absence of nonspecific
CLDC-mediated anti-tumor activity between the two studies may be due to
differences in the tumor lines and/or mouse strains used, the size and
composition of the cationic liposomes used, the dose and schedule of
CLDC administration, and/or the time of sacrifice.
Co-injecting Combinations of These Genes Does Not Increase
Anti-tumor Activity--
We next tested whether co-injection of the
p53 and GM-CSF genes, the p53 and angiostatin genes, or the angiostatin
and GM-CSF genes into individual groups of mice produced synergistic
anti-tumor activity when compared with injection of the individual
genes themselves. CLDC-based intravenous injection of each gene
individually, as well as each of the gene combinations, reduced both
the total number of lung tumors and the numbers of tumors greater than
2 mm by comparable levels when compared with the control mice (Fig. 1B). Thus, no combination of genes enhanced the level of
anti-tumor activity produced by each gene individually, indicating a
lack of synergistic anti-tumor activity. As observed previously in Fig.
1A, the angiostatin, p53, and GM-CSF genes individually each produced similar levels of anti-tumor activity. CLDC-based, intravenous injection of 12.5 µg of CMV-angiostatin plus 12.5 µg of
CMV-luciferase produced antimetastatic effects that did not differ
significantly from those produced by 25 µg of CMV-angiostatin alone
(data not shown). Thus, the failure of angiostatin combined with either GM-CSF or p53 to produce synergistic anti-tumor effects does not appear
to be due to the reduced dose of the individual anti-tumor genes injected.
CLDC-based Intravenous Gene Delivery Permits Assessment of the
Anti-metastatic Activity of a Variety of Potential Anti-cancer
Genes--
We then compared the size and number of lung metastases
produced in groups of mice injected with CLDC containing the murine angiostatin gene, the human CC3 gene, the human BCL-2 gene, or the
luciferase gene (mock-treated controls) on days 3 and 10 after tumor
inoculation or no treatment (control group). Additional groups of
tumor-bearing mice received a single intravenous injection of CLDC
containing either the murine angiostatin gene or the luciferase gene 7 days after receiving B16-F10 melanoma cells in order to determine the
effect of delaying CLDC therapy on the level of anti-tumor activity
achieved. We also tested the CC3 gene (an apopotosis-inducing
metastasis suppressor gene (20)) and the BCL-2 gene (a potent
antagonist of apopotosis). Overexpression of BCL-2 has been linked to
the development of human tumors (21). CLDC-mediated, intravenous
delivery of the murine angiostatin gene and the human CC3 gene at days
3 and 10 each produced significant anti-metastatic effects, when
compared with BCL-2 gene-treated mice (p < 0.05), to
luciferase gene (mock)-treated (p < 0.05) or to
untreated (p < 0.05) controls (Fig.
1C).
A single intravenous injection at day 7 of CLDC containing the murine
angiostatin gene also significantly reduced tumor metastases when
compared with control groups (p < 0.05) but was
somewhat less effective than two injections of the angiostatin gene
initiated at a time when the tumor burden was less extensive.
Furthermore, although the B16-F10 line has been cloned as a
predominately lung metastatic tumor line (13), we noted the presence of
significant numbers of extrapulmonary B16-F10 metastases in this as
well as in prior experiments. Therefore, we counted the number of
extrapulmonary metastases present in this experiment. The overall
number of extrapulmonary metastases was significantly reduced in the
angiostatin-treated (a total of one metastasis) and CC3-treated groups
(no metastases) compared with control groups (a total of 8 ± 2.7 metastases) (p < 0.01 for angiostatin and
p < 0.005 for CC3). Metastases were present in the
liver, gastrointestinal tract, spinal cord, thymus, skin, and lymph
nodes. Thus, CLDC-based intravenous gene delivery of active
anti-angiogenic and tumor suppressor genes can reduce metastatic spread
to a variety of target organs to which solid tumors commonly
metastasize (7).
Increasing the Efficiency of CLDC-based Intravenous Delivery of the
Angiostatin Gene Further Increases Anti-tumor Activity--
To more
specifically address the relationship between the level of
antiangiogenic expression achieved and of the extent of the anti-tumor
activity produced, we tested whether increasing the level of
angiostatin gene expression through the use of a more efficient
cationic formulation could significantly increase anti-tumor efficacy
against B16-F10 metastases. Specifically, we compared the anti-tumor
activity against B16-F10 produced by the angiostatin gene delivered by
two different cationic liposome formulations: 1) DOTIM:cholesterol
liposomes (used in experiments 1A through 1C) or
2) pure DOTMA liposomes have been shown to produce approximately
10-fold higher levels of gene expression than DOTIM:cholesterol liposomes (Ref. 22 and data not shown). We found that intravenous injection of 25 µg of the angiostatin gene complexed to
DOTIM:cholesterol liposomes reduced metastases >2 mm by 65%
versus control mice, whereas the angiostatin gene complexed
to pure DOTMA liposomes reduced metastases by 88% versus
control mice (p < 0.025 versus DOTIM:cholesterol liposomes) (Fig. 1D). Thus, the degree of
anti-tumor activity achieved appears to depend on the efficiency of
gene transfer and expression produced. Increasing the level of
angiostatin gene expression further reduced the number of lung
metastases to quite low levels.
CLDC-based Intravenous Delivery of the p53, GM-CSF, or Angiostatin
Genes Significantly and Comparably Reduces Tumor Angiogenesis--
We
then attempted to identify a mechanism for the comparable levels of
anti-tumor activity produced by the p53, angiostatin, and GM-CSF genes.
We determined the mean number of intratumoral blood vessels in p53-,
GM-CSF-, angiostatin-, and CAT-treated mice using neoangiogenesis
counts facilitated by immunoreactivity to Factor VIII-anti-von
Willebrand factor antibody (23). CLDC-based delivery of the p53,
GM-CSF, and angiostatin genes each significantly reduced tumor
neovascularity when compared with control mice (p < 0.0005) (Table I). Delivery of the p53,
GM-CSF, and angiostatin genes each reduced tumor angiogenesis to a very
similar extent, indicating that each gene product induced
antiangiogenic activity within tumors in tumor-bearing mice (Table I).
Intravenous injection of CLDC containing the p53, angiostatin, or
GM-CSF genes reduced tumor angiogenesis by levels comparable with the
reduction in tumor angiogenesis produced by the implantation of a
murine T241 fibrosarcoma cell line stably transfected with the murine
angiostatin gene (24).
Immunohistochemistry Shows That Intravenous Injection of
p53-containing CLDC Can Transfect Large Numbers of Metastatic Tumor
Cells and Can Induce Widespread Intratumoral Expression of
TSP-1--
To assess to what extent the CLDC we used could transfect
tumor cells in mice bearing advanced metastatic disease, we assessed p53 immunoreactivity in the lungs of tumor-bearing mice previously injected with CLDC containing either the wild type p53 gene or the CAT
gene. These mice were injected with B16-F10 cells on day 0 and
subsequently with CLDC on day 29, a time when lung metastases were at
an advanced stage. Essentially all tumors examined in the lungs of
p53-treated mice showed p53 expression within the nucleus of
transfected cells (Fig. 2A).
Although the staining for p53 immunoreactivity varied in intensity
(Fig. 2A), large numbers of tumor cells examined showed some
degree of positive staining for p53. Immunopositivity of wild type p53
suggested a high level of p53 gene expression, since the wild type p53
generally does not stain positively in this assay because of rapid
degradation of the encoded protein (25). Positivity was also noted in
some macrophage and endothelial cell nuclei (Fig. 2A). p53
expression was not seen in either tumor cells or in normal cells in the
lungs of either CAT-treated (Fig. 2B) or untreated mice
(data not shown).
Since delivery of the p53 gene significantly reduced tumor vascularity
(Table I) and since p53 gene expression has been shown to induce
expression of the potent antiangiogenic factor, TSP-1, in cultured
fibroblasts (24) and epithelial cells (25), we determined whether
systemic delivery of the p53 gene altered TSP-1 expression in the lung,
using immunohistochemistry. p53-treated mice showed TSP-1 expression
within the cytoplasm of the large majority of tumor cells present
within the lung as well as in rare normal pulmonary cell types (Fig.
2C). TSP-1 expression was not detected in the lungs of
either CAT-treated (Fig. 2D) or in untreated mice (data not
shown). Thus, p53 gene delivery could specifically induce widespread
TSP-1 expression in metastatic tumors in vivo. TSP-1 may
mediate both the antiangiogenic (Table I) and antimetastatic (Fig. 1,
A and B) activity of p53 in B16-F10 tumor-bearing mice.
The Level of Induction of TSP-1 Expression in Metastatic Tumor
Cells Depends on the Level of p53 Gene Expression Produced--
To
directly assess the dose-response relationship between p53 gene
transduction and the induction of TSP-1 gene expression, we quantitated
the percentage of tumor cells expressing p53 and also TSP-1, following
intravenous injection of DOTIM:cholesterol liposomes complexed to
either 40 or 50 µg of the p53 gene. Increasing the DNA dose from 40 to 50 µg/mouse reproducibly increases the level of expression of
delivered genes between 5- and 10-fold following intravenous injection
of CLDC containing DOTIM:cholesterol liposomes (data not shown).
Injecting CLDC containing 40 µg of the p53 gene transfected 29.6 ± 3.7% of B16-F10 tumor cells metastatic to the lung with p53 and
induced TSP-1 expression in 39.2% ± 3.4% of these cells, whereas
injecting CLDC containing 50 µg of the p53 gene transduced 82.1% ± 2.7 of lung tumor cells with p53 and induced TSP-1 expression in 98.6% ± 0.6% of these cells (Table II). (No
positive staining for p53 or TSP-1 gene expression was detected in
either B16-F10 or normal cells from the lungs of mice injected with
CLDC containing 50 µg of the CAT gene (Fig. 2, B and
D). The levels of p53 and TSP-1 expression were increased by
2.8- and 2.5-fold, respectively, in the 50 µg-injected group and were
each significantly higher than in the 40 µg-injected group
(p < 0.0005). Therefore, the levels of both p53
transduction and TSP-1 induction varied directly with the dose of CLDC
injected, and the 50-µg dose of the p53 gene appeared capable of
transfecting the large majority of all B16-F10 tumor cells metastatic
to lung.
Anti-tumor genes have been classified into multiple different
functional categories, including tumor suppressor genes,
immunostimulatory genes, anti-oncogenes, and anti-angiogenic genes.
However, it has become evident that individual genes may produce
anti-tumor effects by several different mechanisms. As examples, the
wild type p53 gene has been shown to function as a potent tumor
suppressor gene (28, 29). More recently, p53 has also been shown to
exert antiangiogenic activity in tumor-bearing mice (30), as well as to
induce the production of the anti-angiogenic protein TSP-1 in cultured
cells (26, 27). Furthermore, stable transfection of tumor cells with
the cytokine gene GM-CSF has been shown to exert host-immune anti-tumor
activity, mediated by cytotoxic T lymphocytes following tumor cell
inoculation into mice (31). More recently, recombinant GM-CSF has been
shown to stimulate angiogenesis in vitro and in
vivo (32, 33). However, GM-CSF has also been shown to produce
antiangiogenic activity via the induction of a specific macrophage
metalloelastase, which cleaves plasminogen to generate angiostatin
(34). Since CLDC-based intravenous gene delivery targets gene
expression to macrophages, as well as to vascular endothelial cells (8,
29), the use of intravenous, CLDC-based GM-CSF gene delivery may shift
the balance of GM-CSF's pro- and anti-angiogenic activities toward
anti-angiogenesis when compared with the administration of recombinant
GM-CSF protein.
Our results show that CLDC-based intravenous delivery of the GM-CSF
gene, the p53 gene, and the anti-angiogenic angiostatin gene each
reduces both tumor angiogenesis and metastatic spread by comparable
levels (Fig. 1, A and B). Furthermore, CLDC-based delivery of combinations of these genes failed to significantly alter
anti-tumor activity when compared with injection of each gene
individually (Fig. 1B), suggesting that these genes are
producing their anti-tumor effects via a common pathway. Therefore, our results are consistent with the interpretation that the products of the
p53, GM-CSF, and angiostatin genes each produce anti-metastatic activity primarily as anti-angiogenic agents following intravenous, CLDC-based delivery. In support of this hypothesis, the level of
reduction of tumor angiogenesis produced by CLDC-based intravenous injection of the murine angiostatin gene was similar to that produced by implantation of murine fibrosarcoma cells stably transfected with
the murine angiostatin gene (Table I and Ref. 22). In addition, we have
also shown that CLDC-based intravenous delivery of the wild type human
p53 gene can specifically induce the production of TSP-1 in metastatic
tumor cells in tumor-bearing mice (Fig. 2), indicating that
p53-mediated antiangiogenesis in vivo may operate at least
in part through the induction of TSP-1 expression (26, 27).
Vascular endothelial cells, macrophages, and tumor cells have been
shown to be the three principal cell types involved in controlling the
angiogenic phenotype (14-17, 36). Previously, CLDC-based intravenous
gene delivery has been shown to target the expression of transferred
genes to vascular endothelial cells (8, 33) and macrophages (8, 35).
Our results now indicate that intravenously injected CLDC containing
the p53 gene can transfect up to 80% of all lung tumor cells in mice
bearing advanced metastatic disease (Table II). Thus, the use of CLDC
may produce high levels of antiangiogenic gene products at their
specific cellular sites of action. However, although CLDC-based
delivery of the angiostatin gene reduced lung metastases by as much as
90% compared with control mice (Fig. 1D) and brought about
significant reductions in tumor vascularity (Table I), it did not cause
complete tumor regression, as previously reported following twice daily
administration of high doses of recombinant angiostatin or implantation
of tumor cells stably transfected with the angiostatin gene (3, 4, 24).
Since high levels of angiostatin protein must be administered on a
daily basis for prolonged periods in order to achieve complete tumor
regression (3, 4), the degree of anti-tumor activity produced by only
one or two doses of CLDC over the 35-day period of this study is encouraging.
Our results also suggest that the overexpression of selected genes,
which are aberrantly expressed only in a limited number of tumor types,
may exert more generalized anti-tumor activity via CLDC-based, systemic
gene delivery. For example, highly metastatic small cell lung carcinoma
cells often lack CC3 expression. Stable transfection of these highly
aggressive small cell lung cancer cells with the CC3 gene was shown to
suppress their metastasis in mice (20). We found that CLDC-based
systemic delivery of the CC3 gene produced significant antimetastatic
activity against B16-F10 melanoma, which expresses low levels of CC3
protein (Fig. 1C). This finding indicates that enforcing
expression of higher levels of CC3 in metastatic cells that are not
originally CC3-negative can produce significant antimetastatic effects.
CLDC-based delivery of other such genes into tumor-bearing hosts
may reveal novel tumor targets not identified by more traditional
molecular genetic approaches. Furthermore, similar to existing
chemotherapy of cancer, the combined anti-tumor activities of several
different gene products, expressed simultaneously, may be required for
therapeutic efficacy, particularly in patients with extensive tumor
burdens. CLDC-based intravenous gene delivery may also be used to
assess whether combinations of putative anti-cancer genes can produce
synergistic anti-tumor activities in tumor-bearing hosts.
Metastatic spread causes death in the overwhelming majority of patients
dying from the most common forms of human cancer (7). Significant
reductions in cancer mortality will thus require the development of new
systemic therapies that can prevent and/or reverse the metastatic
spread of cancer. The ability to transfer and express anti-tumor genes
by intravenous administration offers a novel approach to the treatment
of cancer. In addition, it may permit the identification of novel
anti-cancer genes as well as reveal anti-cancer genes that act
synergistically in tumor-bearing hosts. Ultimately, it may create the
possibility of treating metastatic cancer by specifically correcting
the molecular alterations responsible for producing the malignant phenotype.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Intravenous injection of CLDC
containing the angiostatin gene, the GM-CSF gene, the CC3 gene, or the
p53 gene each produces significant antimetastatic effects in
tumor-bearing mice (25,000 B16-F10 melanoma cells/mouse intravenously
into groups of eight C57BL6 mice on day 0). All mice were
sacrificed at day 30 following tumor inoculation, and lung metastases
>2 mm were counted using a dissecting microscope. Potential
significance of differences between the various groups was determined
using a two-sided Student's t test. a,
individual mice in groups of eight were injected intravenously on day 3 and again on day 10 with CLDC containing 400 nmol of DOTIM:cholesterol
MLV complexed to 25 µg of a CMV-driven expression plasmid encoding
either the murine angiostatin gene, the murine GM-CSF gene, the human
wild type p53 gene, the CAT gene (mock-treated controls), or no
treatment (control group). b, individual mice in groups of
eight were injected intravenously on day 3 and again on day 10 with
CLDC containing 400 nmol of DOTIM:cholesterol MLV complexed to 25 µg
of CMV-angiostatin, CMV-GM-CSF, or CMV-p53 or to 12.5 µg plus 12.5 µg of CMV-angiostatin plus CMV-p53, CMV-angiostatin plus CMV-GM-CSF,
or CMV-p53 plus CMV-GM-CSF, respectively. c, individual mice
in groups of eight were injected intravenously on day 3 and again on
day 10 with CLDC containing 400 nmol of DOTIM:cholesterol MLV complexed
to 25 µg of either CMV-angiostatin, CMV-CC3, CMV-BCL-2,
CMV-luciferase, or CMV-p53. Individual mice in groups of eight were
also injected intravenously on day 7 only with CLDC containing 400 nmol
of DOTIM:cholesterol MLV complexed to 25 µg of either CMV-angiostatin
or CMV-luciferase. d, individual mice in groups of eight
were injected intravenously on day 7 only with CLDC containing either
400 nmol of DOTIM:cholesterol MLV complexed to 25 µg of either
CMV-luciferase or CMV-angiostatin or with CLDC containing 650 nmol of
pure DOTMA MLV complexed to 25 µg of CMV-angiostatin.
Intravenous, CLDC-based injection of the angiostatin, p53, or
GM-CSF genes each significantly reduces tumor vascularity
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Fig. 2.
Intravenous injection of CLDC containing the
p53 gene transfects large numbers of metastatic tumor cells and induces
expression of the endogenous TSP-1 gene. Groups of four mice that
had received 25,000 B16-F10 cells intravenously on day 0 received CLDC
containing 400 nmol of DOTIM:cholesterol MLV complexed to 50 µg of
either CMV-p53 or CMV-CAT intravenously on day 29 and were sacrificed
24 h later. The lungs were infused with 10% neutral buffered
formalin, quick frozen, and then processed as described under
"Experimental Procedures." A, high power (40×)
magnification of metastatic tumor in p53-treated mouse immunostained
for p53. Over 90% of nuclei from this pulmonary tumor nodule
demonstrate nuclear reactivity for p53, with 10% of nuclei intensely
positive, 10% moderately positive, and another 70% weakly positive
for p53 expression. B, high power (40×) magnification of a
metastatic pulmonary nodule from CMV-CAT-injected mouse, immunostained
for p53. No specific nuclear stain for p53 is shown, similar to all
other tumor nodules in both CAT-treated and untreated (data not shown)
control animals. The counter stain faintly demonstrates the tumor cell
outlines. C, high power (× 40) magnification of a
metastatic pulmonary nodule (center of field) surrounded by pulmonary
tissue from a p53-treated mouse. This tissue has been immunostained for
thrombospondin-1 and demonstrates cytoplasmic immunoreactivity
(red) in greater than 90% of the tumor cells in this
nodule. D, high power (× 40) magnification of a metastatic
pulmonary nodule from a CAT gene-treated mouse that has been
immunostained for TSP-1. No cytoplasmic immunoreactivity for TSP-1 is
detectable.
Increasing p53 gene expression increases the induction of endogenous
TSP-1 gene expression in metastatic tumors
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank S. Edgerton for excellent technical assistance, Dr. M. Kashani-Sabet for helpful suggestions, and Dr. D. Marieval for p53 analyses.
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FOOTNOTES |
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* This work was supported by the California Pacific Medical Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by National Institutes of Health (NIH) Grant CA58207 and the Carol Gollob Foundation.
Supported by NIH Grant CA71422.
§§ Supported by NIH Grants CA58914, DK45917, DK49550, and HL53762, XeneX, LLC, and by the State of California, Breast Cancer Research Program. To whom correspondence should be addressed: California Pacific Medical Research Inst., 2330 Clay St., Stern Bldg., San Francisco, CA 94115. Tel.: 415-561-1704; Fax: 415-561-1725; E-mail: debs{at}cooper.cpmc.org.
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ABBREVIATIONS |
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The abbreviations used are: CLDC, cationic liposome-DNA complex; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; MLV, multilamellar vesicle; GM-CSF, granulocyte-macrophage colony-stimulating factor; TSP-1, thrombospondin-1; DOTMA, N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride; DOTIM, 1-[2-(9(2)-octadecenoyloxy)ethyl]-2-(8(2)-heptadecenyl)-3-(2-hydroxyethyl)midizolinium chloride.
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REFERENCES |
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