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Tumor Biology |
Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The growth and spread of neoplasms is dependent on the establishment of adequate blood supply, i.e., angiogenesis (9, 10, 11, 12) . The onset of angiogenesis is determined by the balance between proangiogenic and antiangiogenic molecules at the local tissue level (9, 10, 11, 12) . Angiogenesis can occur by either sprouting or nonsprouting processes (13) . Sprouting angiogenesis occurs by branching (true sprouting) of new capillaries from preexisting vessels. Nonsprouting angiogenesis results from the enlargement, splitting, and fusion of preexisting vessels produced by the proliferation of endothelial cells within the wall of a vessel. Transcapillary pillars (or transluminal bridges) are sometimes observed in enlarged vessels produced by nonsprouting angiogenesis (13) . This type of angiogenesis can concurrently occur with sprouting angiogenesis in the vascularization of organs or tissues such as the lung, heart, and yolk sac during development (13) . The mechanism of nonsprouting angiogenesis in metastasis is not yet known, but VEGF,3 also called vascular permeability factor, which plays a pivotal role in developmental, physiological, and pathological neovascularization (14, 15, 16, 17) , is a candidate effector. VEGF stimulates the proliferation and migration of endothelial cells and induces the expression of metalloproteinases and plasminogen activity by these cells (18, 19, 20, 21) . Moreover, overexpression of VEGF in tumor cells enhances tumor growth and metastasis in several animal models by stimulating vascularization (increased microvessel density; Refs. 22, 23, 24 ).
The purpose of this study was to determine some of the molecular determinants of vascularization, including VEGF, in brain metastases particularly in relation to angiogenesis. We show that formation of brain metastasis by metastatic human cancer cells correlates with VEGF expression and that the growing brain metastases contain numerous enlarged blood vessels. Moreover, reduction of VEGF expression (by antisense-VEGF gene transfection) suppressed formation of enlarged blood vessels and inhibited brain metastasis. VEGF thus plays a crucial role in the stimulation of angiogenesis and formation of brain metastasis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All tumor cell lines were maintained as adherent monolayer cultures in Eagle’s MEM supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine, 2-fold vitamin solution, and penicillin-streptomycin (CMEM; Flow Laboratories, Rockville, MD), and incubated at 37°C in 5% CO2-95% air. All tumor cell cultures were free of Mycoplasma and pathogenic murine viruses (assayed by Microbiological Associates, Bethesda, MD). HDMECs (Cascade Biologicals, Portland, OR) were cultured in medium 131 with microvascular growth supplement (Cascade Biologicals). For proliferation assays, the endothelial cells were used at passages 2–5.
Reagents.
Recombinant human VEGF165 protein and antihuman VEGF polyclonal Ab were
purchased from R&D Systems (Minneapolis, MN). BrdUrd was purchased
from Sigma Chemical Co. (St. Louis, MO).
Mice.
Athymic Ncr-nu/nu male mice were purchased from the Animal
Production Area, National Cancer Institute-Frederick Cancer Research
Facility (Frederick, MD). The mice were maintained in a barrier-type
facility approved by the American Association for Accreditation of
Laboratory Animal Care and in accordance with current regulations and
standards of the United States Department of Agriculture, Department of
Health and Human Services, and NIH.
Production of Visceral Metastasis.
Cultured PC14PE6 and PC14Br cells were harvested by pipetting. KM12SM,
H226, SN12PM6, and TXM13 cells were harvested by a 2-min exposure to
0.25% trypsin-0.02% EDTA solution. The cells were washed twice in
HBSS and resuspended in Ca2+- and
Mg2+-free HBSS. Cell viability was determined by
the trypan blue exclusion test, and only single-cell suspensions of
>90% viability were used for in vivo studies.
To produce experimental lung metastasis, tumor cells (1 x 106/300 µl HBSS) were injected into the lateral tail vein of unanesthetized mice (25) . To produce experimental liver metastasis, tumor cells (1 x 106/50 µl HBSS) were injected into the spleen of nude mice after laparotomy under methoxyflurane anesthesia (30) . At different times after tumor cell injection, the mice were euthanized by methoxyflurane, and the subclavian artery was severed. The lungs and liver were rinsed in HBSS and fixed in Bouin’s solution. The experimental lung and liver metastases were counted with the aid of a dissecting microscope.
Production of Brain Metastases.
To produce brain metastases, mice were anesthetized by methoxyflurane,
restrained on a cork board on the back, and placed under a dissecting
microscope as described previously (7)
. The carotid artery
was prepared for an injection distal to the point of division into the
internal and external carotid arteries. A ligature of 4-0 silk suture
was placed in the distal part of the common carotid artery. A second
ligature was placed and loosely tied proximal to the injection site. A
sterile cotton tip applicator was inserted under the artery just distal
to the injection site to elevate the carotid artery. This procedure
controlled bleeding from the carotid artery by regurgitation from
distal vessels. The artery was nicked with a pair of microscissors,
and a <30-gauge glass cannula was inserted into the lumen. To assure
proper delivery, the cells (2.5 x 104
/100 µl HBSS) were injected slowly, the
second ligature was tightened, and the skin was closed with sutures
(7)
. The mice were killed at different times, and the
brain was removed and cut into 2–3-mm sections. The presence of
visible brain metastases was confirmed by histology (7)
.
In Situ Fluorescent TUNEL Assay.
Brain tissues were fixed in 10% buffered formalin solution and then
embedded in paraffin. Thin sections (4 µm) were prepared, and the
TUNEL assay was performed using a commercial kit according to the
manufacturer’s protocol (Promega Corp., Madison, WI). Briefly, tissue
sections were deparaffinized and fixed at room temperature for 5 min in
4% paraformaldehyde. Cells were stripped of proteins by incubation for
10 min with 20 µg/ml proteinase K. The tissue sections were then
permeabilized by incubating with 0.5% Triton X-100 in PBS for 5 min at
room temperature. After being rinsed twice with PBS for 5 min, the
slides were incubated with terminal deoxynucleotidyl transferase buffer
for 10 min. Terminal deoxynucleotidyl transferase and buffer were then
added to the tissue sections and incubated in a humid atmosphere at
37°C for 1 h. The slides were washed for 5 min three times with
PBS. Prolong solution (Molecular Probes, Eugene, OR) was used to mount
the coverslips. Immunofluorescence microscopy was performed using a
x40 objective (Zeiss Plan-Neofluar) on an epifluorescence
microscope equipped with narrow bandpass excitation filters mounted on
a filter wheel (Ludl Electronic Products, Hawthorne, NY) to select for
green fluorescence. Images were captured using a cooled CCD camera
(Photometrics, Tucson, AZ) and SmartCapture software (Digital
Scientific, Cambridge, England) on a Macintosh computer. Images were
further processed using Adobe PhotoShop software (Adobe Systems,
Mountain View, CA).
In Situ mRNA Hybridization Technique.
Specific oligonucleotide DNA probes in the antisense orientation were
designed complementary to the mRNA transcripts based on published
reports of the cDNA sequence: VEGF,
TGG'TGA'TGT'TGG'ACT'CCT'CAG'TGG'GCU; bFGF,
CGG'GAA'GGC'GCC'GCT'GCC'GCC'; IL-8, CTC'CAC'AAC'CCT'CTG'CAC'CC; and
EGF-R, GGA'GCG'CTG'CCC'CGG'CCG'TCC'CGG (31
, 32)
.
The specificity of the oligonucleotide sequence was initially
determined by a Gene Bank European Molecular Biology Library database
search with the use of the Genetics Computer Group sequence analysis
program (Genetics Computer Group, Madison, WI) based on the FastA
algorithm. All DNA probes were synthesized with six biotin molecules
(hyperbiotinylated) at the 3' end via direct coupling, with the use of
standard phosphoramidite chemistry (Research Genetics, Huntsville, AL).
The staining for in situ mRNA hybridization was performed as
described previously (32)
. Tissue sections of
formalin-fixed, paraffin-embedded specimens were mounted on
silane-treated ProbeOn slides (Fisher Scientific Co.). The slides were
placed in the Microprobe slide holder (Fisher Scientific Co.), dewaxed,
and rehydrated with Autodewaxer and Autoalcohol (Research
Genetics), digested with pepsin, and then hybridized by use of the
Microprobe manual staining system (Fisher Scientific Co.). The probes
were hybridized for 45 min at 45°C, and the samples were then washed
three times for 2 min each time with 2x SSC at 45°C. RNase-free
water was used to make up Tris buffer and 2x SSC solutions. The
samples were then incubated with alkaline phosphate-labeled avidine for
30 min at 45°C, rinsed in 50 nmol/l Tris buffer (pH 7.6), rinsed with
alkaline phosphate enhancer for 1 min, and incubated with a chromogen
substrate for 20 min at 45°C. Additional incubation with fresh
chromogen was done if it was necessary to enhance a weak reaction. A
positive enzymatic reaction in this assay stained red. Known positive
controls were used in each hybridization reaction. A
poly(dT)20 oligonucleotide was used to verify the
integrity of mRNA in each sample. Controls for endogenous alkaline
phosphate included treatment of the sample in the absence of the
biotinylated probe and use of chromogen alone.
Image Analysis to Quantify Intensity of Color Reaction.
Stained sections were examined in a Zeiss photomicroscope (Carl Zeiss,
Thornwood, NY) equipped with a three-chip, charge-coupled device color
camera (model DXC-960 MD; Sony Corp., Tokyo, Japan). The images were
analyzed with the use of Optimas software (Optimas Corp., Bothell, WA),
as described previously (32)
. Images covering the range of
staining intensities were captured electronically, a color bar
(montage) was created, and a threshold value was set in the red, green,
and blue modes of the color camera. All subsequent images were
quantified on the basis of this threshold. The integrated absorbance of
the selected fields was determined on the basis of its equivalence to
the mean log inverse gray value multiplied by the area of the field.
The samples were not counterstained; therefore, the absorbance was
attributable solely to the product of the in situ mRNA
hybridization reaction. Five different fields in each sample were
quantified to derive an average value. We determined the intensity of
staining by standardizing the value with the integrated absorbance of
poly d(T)20, which was set at 100.
Histology and Immunohistochemistry.
For BrdUrd staining, the mice were injected i.v. with 250 µg of
BrdUrd. One h later, the mice were killed, and the brain was collected
and cut into 2–3-mm sections, which were placed into either buffered
10% formalin solution or OCT compound (Miles Laboratories, Elkhart,
IN) to be snap-frozen in liquid nitrogen. For staining for BrdUrd and
VEGF, tissue sections of formalin-fixed, paraffin-embedded specimens
were deparaffinized in xylene, rehydrated in graded alcohol, and
transferred to PBS. For BrdUrd staining, the slides were then treated
with 2 N HCl for 30 min at 37°C. For VEGF staining, the
slides were treated with pepsin for 20 min at room temperature. For
CD31 staining, sections of frozen tissues (8 µm thick) were fixed
with cold acetone and transferred to PBS. The slides were then rinsed
twice with PBS, and endogenous peroxidase was blocked by the use of 3%
hydrogen peroxide in PBS for 12 min. The samples were then washed three
times with PBS and incubated for 10 min at room temperature with a
protein-blocking solution consisting of PBS (pH 7.5) containing 5%
normal horse serum and 1% normal goat serum. Excess blocking solution
was drained, and the samples were incubated for 18 h at 4°C with
a 1:100 dilution of monoclonal mouse anti-BrdUrd Ab (Becton Dickinson,
Mountain View, CA), a 1:400 dilution of rabbit polyclonal anti-VEGF Ab
(Santa Cruz Biotechnology, Santa Cruz, CA), or a 1:100 dilution of
monoclonal rat anti-CD31 Ab (PharMingen, San Diego, CA). The samples
were then rinsed four times with PBS and incubated for 60 min at room
temperature with the appropriate dilution of peroxidase-conjugated
antimouse IgG1, antirabbit IgG, or antirat IgG. The slides were rinsed
with PBS and incubated for 5 min with diaminobenzidine (Research
Genetics, Huntsville, AL). The sections were then washed three times
with distilled water and counterstained with Gill’s hematoxylin.
Sections (4 µm thick) of formalin-fixed, paraffin-embedded tumors
were also stained with H&E for routine histological examination.
Vascular Density.
Blood vessels in solid tumors growing in the brain of nude mice were
counted under a light microscope after immune staining of sections with
anti-CD31 Ab. Areas containing the highest number of capillaries and
small venules were identified by scanning the tumor sections at low
power (x40). Individual vessels were counted in x100 fields [x10
objective and x10 ocular (0.145-mm2/field)]. A
structure was classified as a vessel on the basis of the criteria of
Weidner et al. (33)
, which do not require
observation of a vessel lumen for this classification.
mRNA Analysis.
Polyadenylated mRNA was extracted from 107 to
108 cultured cells growing under sparse (<100
cells/mm2) and confluent (>1200
cells/mm2) conditions using the FastTrack mRNA
isolation kit (Invitrogen Co., San Diego, CA). In some experiments,
total RNA was extracted from 1 x 106 cells in confluent cultures using TRI Reagent
(Molecular Research Center, Inc., Cincinnati, OH). Total RNA (10 µg)
or mRNA (3 µg) were electrophoresed on 1% denaturing
formaldehyde/agarose gel, transferred to a GeneScreen nylon membrane
(DuPont Co., Boston, MA), and UV cross-linked with 120,000
µJ/cm2 using a UV Stratalinker 1800
(Stratagene, La Jolla, Ca). Hybridizations were performed as described
previously (31)
. Nylon filters were washed two times at
60–65°C with 0.1% SSC and 0.1% SDS. The cDNA probes used in this
analysis were a 0.204-kb BamHI/EcoRI cDNA
fragment of human VEGF (from Dr. B. Berse, Harvard Medical School,
Boston, MA), a 1.4-kb EcoRI cDNA fragment of bovine bFGF, a
0.5-kb EcoRI cDNA fragment of human IL-8 (provided by Dr. K.
Matsushima, University of Tokyo, Tokyo, Japan), a 3.8-kb
XhoI cDNA fragment of human EGF-R (provided by Dr. F. Kern,
Washington, DC), and a 1.3-kb PstI cDNA fragment of rat
GAPDH. Each cDNA fragment was purified by agarose gel electrophoresis,
recovered by use of GeneClean (BIO 101, Inc., La Jolla, CA), and
radiolabeled by the use of the random primer technique with
[
-32P]deoxyribonucleotide triphosphates.
Expression of the mRNA was quantified by densitometry of autoradiographs with the use of Image Quant software program (Molecular Dynamics, Sunnyvale, CA); each sample value was calculated from the ratio of the average areas between the specific mRNA transcripts and the 1.3-kb GAPDH mRNA transcript in the linear range of the film.
Determination of VEGF, bFGF, and IL-8 Protein Levels.
The levels of VEGF, bFGF, and IL-8 proteins in culture supernatants
were determined using an ELISA kit according to the manufacturer’s
instructions (R&D Systems, Minneapolis, MN).
VEGF Isoforms.
First-strand cDNA was synthesized from 1 µg of total RNA using a
First Strand Synthesis kit (Pharmacia, Piscataway, NJ) in 33 µl of
reaction mixture, according to the manufacturer’s instructions. The
synthesized first-strand cDNA (1 µl) was amplified by PCR in a final
volume of 50 µl containing 10 mM Tris-HCl, 3
mM MgCl2, 50 mM KCl,
0.01% gelatin, 200 mM deoxynucleotide triphosphate, 50
pmol of each primer, and 2.5 units of AmpliTaq Gold Taq polymerase
(Perkin-Elmer, Foster City, CA). Sequences of VEGF primers used were
sense 5'-TCCAGGAGTACCCTGATGAG-3' and antisense
5'-CTTTCCTGGTGAGAGATCTGG-3' immediately flanking the region of the VEGF
open reading frame involved in the alternative splicing of several
exons. PCR amplification of VEGF cDNA was performed under the following
conditions: 30 cycles, 1 min at 94°C, 1 min at 58°C, and 1 min at
72°C. Before the first cycle, a denaturation step of 8 min at 95°C
was included, and after 30 cycles, the extension step was prolonged for
7 min at 72°C (34)
.
Subcloning of VEGF165 Gene into pcDNA3 and DNA
Transfection.
The full-length cDNA for VEGF165, a gift from Dr. J. Abraham (Scios
Nova, Mountain View, CA), was subcloned into the BamHI site
of pcDNA3, a eukaryotic expression vector driven by the human
cytomegalovirus promoter (Invitrogen). Subcloning into the
BamHI restriction site yielded an insert in either the sense
or antisense orientation. The orientation and proof of completeness of
the insert were determined by DNA sequencing (Core Sequencing Facility,
M. D. Anderson Cancer Center; data not shown). The sense-VEGF121
expression vector was obtained as described previously
(34)
.
For transfection, tumor cells were plated at a density of 2–5 x 105 cells/100-mm dish. H226 cells were transfected with the sense-VEGF165 or sense-VEGF121 genes, and KM12SM cells were transfected with antisense-VEGF165 gene using a stable mammalian transfection kit (Stratagene, La Jolla, CA). PC14PE6 cells were transfected with antisense-VEGF165 gene using a LipofectAMINE Reagent kit (Life Technologies, Inc., Gaithersburg, MD). The cultures were incubated for 12 h and then washed and fed with fresh CMEM. Drug selection started 48 h after transfection by adding 250-1000 µg/ml G418 (Life Technologies) in CMEM. This medium was replaced every 3 days; and 3 weeks later, G418-resistant colonies were separately transferred to individual wells of a 48-well plate. The expression of VEGF in individual clones was determined by Northern blot analysis.
In Vitro Endothelial Cell Proliferation Assay.
HDMECs (5 x 103
/38
mm2 well) were plated in triplicate in 96-well
plates precoated with 1.5% gelatin. The cells were incubated overnight
in supplemented M131 medium, washed, and incubated for 72 h with
MEM containing test samples. The proliferative activity was determined
by the 3-(4,4-dimethylthiazol-2-yl)-2.5-diphenyl-tetrazolium bromide
assay using an MR5000 96-well microtiter plate reader set at 570 nm
(21)
. The percentage of increase in proliferation of
HDMECs was calculated by the formula [B - A/A] x 100, where A is the absorbance
of the control cultures and B is the absorbance of the
treated cultures (14)
.
Statistical Analysis.
The significance of differences in microvessel density and size of
blood vessel lumen was analyzed by Student’s t test
(two-tailed).
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RESULTS |
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. Next, we injected the cell lines into the right internal carotid
artery (i.c.) of nude mice. The KM12SM colon cancer cells and PC14PE6
and PC14Br lung adenocarcinoma cells produced a high incidence of
rapidly progressive lesions (see Fig. 1
for examples) associated with deleterious systemic effects, such as
cachexia, listlessness, and the protrusion of the right eye bulb (the
side of i.c. injection). The mice became moribund within 7–8 weeks
after tumor cell injection. In contrast, mice injected i.c. with H226
lung squamous cell carcinoma, SN12PM6 renal cell carcinoma, or TXM13
melanoma cells had a low incidence of brain metastasis. The few brain
lesions surrounded small capillaries in the brain parenchyma (Fig. 1)
.
The mice survived more than 9 weeks without exhibiting any systemic
symptoms. Taken together, the results suggest that regardless of
production of visceral metastasis, KM12SM, PC14PE6, and PC14Br cells
produce a higher incidence of rapidly progressing brain metastases than
H226, SN12PM6, and TXM13 cells.
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. Because TXM13 cells produced few small experimental metastases in the
brain, we could not perform immunohistochemistry. These results
correlated with the rate of growth in the brain parenchyma.
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. Interestingly,
brain metastases produced by KM12SM, PC14PE6, and PC14Br had an even
lower vessel density than those produced by H226 and SN12PM6 cells.
However, brain metastases produced by KM12SM, PC14PE6, or PC14Br cells
contained a larger proportion of blood vessels with large lumens (large
enough for several erythrocytes) than did metastases produced by H226
or SN12PM6 cells or the nontumor parenchyma. Transluminal bridges of
endothelial cell processes, which are hallmarks of nonsprouting
angiogenesis (13)
, were observed frequently in the
enlarged vessels found in brain metastases produced by KM12SM, PC14PE6,
and PC14Br cells (Fig. 1)
.
In Vitro and in Vivo Expression of
Angiogenic Cytokines.
Because numerous cytokines, including VEGF, bFGF, and IL-8, play a
major role in angiogenesis (39)
, the mRNA expression of
these molecules in brain metastasis was examined by in situ
mRNA hybridization. As shown in Table 2
, brain metastases produced by
KM12SM, PC14P6, and PC14B4 cells expressed more VEGF mRNA than brain
metastases produced by H226 or SN12PM6 cells. No significant
differences in expression of bFGF, IL-8, or EGF-R (40)
were found among the lines. Consistent with mRNA expression, brain
metastasis produced by highly metastatic cells produced a higher level
of VEGF protein than those by low metastatic cell lines (see Fig. 1
for
examples).
In the next set of studies, we determined the constitutive expression
of several angiogenic cytokines and EGF-R in six tumor cell lines
in vitro. Because expression of VEGF and bFGF is cell
density dependent (41
, 42)
, tumor cells were cultured
under both sparse and confluent conditions. All six cell lines tested
expressed higher VEGF under confluent culture conditions than under
sparse conditions. As shown in Fig. 2
, KM12SM, PC14PE6, and PC14Br cells (highly brain metastatic) expressed
a higher level of VEGF mRNA than H226, SN12PM6, and TXM13 cells (low
brain metastatic) under either sparse or confluent culture conditions.
Consistent with the mRNA expression, highly brain metastatic cells
produced a higher level of VEGF protein than low brain metastatic cells
growing in either sparse or confluent conditions (Table 3)
. Interestingly, tumor cells producing few small brain metastases
(H226, SN12PM6, and TXM13) expressed more bFGF than cells producing
numerous large brain metastases (KM12SM, PC14PE6, and PC14B4) at
protein and/or mRNA levels (Fig. 2
; Table 3
). TXM13 expressed low
levels of IL-8 and EGF-R, whereas all other five cell lines expressed
higher levels (Fig. 2)
. VEGF expression thus directly correlated with
the potential of tumor cells to produce brain metastases containing
blood vessels with dilated lumens.
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, VEGF121 and VEGF165 were predominantly expressed in the
cell lines, although the magnitude of VEGF expression in the H226 cells
was lower than in the other cells. We therefore elected to transfect
the sense-VEGF165 and sense-VEGF121 genes into
the H226 cells. On the other hand, because the VEGF165 isoform contains
all of the coding sequence of the VEGF121 isoform (43)
,
the PC14PE6 and KM12SM cells were transfected only with the
antisense-VEGF165 gene. Cells transfected with only pcDNA3
vector served as a control (Neo-control). Stable transfectants were
obtained subsequent to selection and cloning in medium containing G418.
In each cell line, the in vitro proliferation of
transfectants (sense or antisense) did not differ from parental or
Neo-control cells (data not shown). Northern blot analysis showed that
H226 cells transfected with the sense-VEGF165 or
sense-VEGF121 gene (H226/V165 and H226/V121, respectively)
expressed exogenous VEGF mRNA at 0.6 and 0.45 kb, respectively (Fig. 3B)
. Likewise, antisense-VEGF165 transfectants (PC14PE6/AS
and KM12SM/AS) expressed exogenous antisense-VEGF mRNA (0.6 kb). In
addition, endogenous VEGF mRNA expression (3.7 and 1.4 kb) in these
clones was inhibited by 60–80% as compared with parental and
Neo-control cells.
|
. Culture supernatants of these transfectants stimulated proliferation
of HDMECs (Fig. 3C)
. Pretreatment of the supernatants with
anti-VEGF Ab abrogated this activity, indicating that the VEGF produced
by sense-VEGF gene transfectants was biologically active.
Transfection of cells with the antisense-VEGF gene reduced
VEGF protein secretion and VEGF activity (stimulation of HDMEC
proliferation) by 50–80% as compared with control cells.
|
. We also injected
PC14PE6 and KM12SM cells into the internal carotid artery of nude mice
and evaluated their metastatic potential to the brain. By day 34, all
mice injected with parental or Neo-control PC14PE6 or KM12SM cells
developed brain metastases >2.5 mm in diameter, and many of these
metastases were associated with cerebral hemorrhage (see Fig. 4
for examples). Transfection of PC14PE6 or KM12SM with VEGF-antisense
decreased the incidence of brain metastasis by 66 or 25%,
respectively. In addition, the diameter of brain metastases developed
by PC14PE6/AS and KM12SM/AS cells was <1.5 mm. No hemorrhage was
observed in these brain metastases.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Consistent with other reports showing that continuous infusion of VEGF into the brain induced tortuous and dilated vessels in the brain of adult mice (44) , the level of VEGF expression by human cancer cells directly correlated with the development of large blood vessels in the experimental brain metastasis. To produce brain metastasis, malignant tumor cells must complete all of the steps in this selective process (3, 4, 5) . Production of VEGF and vascularization are but one essential step in metastasis (36) . Indeed, transfection of antisense-VEGF gene into highly brain metastatic cells reduced the diameter/number of enlarged vessels and inhibited metastasis. In contrast, transfection of sense-VEGF gene into tumor cells with low metastatic potential did not facilitate brain metastasis. Collectively, these experiments conclude that VEGF expression is essential but not sufficient for production of progressively growing brain metastasis.
The formation of enlarged tumor vessels in other organs has been reported. Vessels in the peritoneal wall enlarge during the formation of malignant ascites, in direct correlation with VEGF/vascular permeability factor expression (45) , and abnormally large vessels form in s.c. tumors produced by glioma cells overexpressing VEGF (46) . Collectively, the data suggest that VEGF plays a pivotal role in tumor-associated nonsprouting angiogenesis.
Several lines of evidence indicate that VEGF levels must reach a threshold before it functions in physiological and pathological conditions (22 , 47) . For example, a decrease in VEGF secretion to 20–30% of parental glioma cells inhibits in vivo growth (47) . Because angiogenesis consists of several distinct steps (21 , 48) that can be regulated by VEGF (14 , 21) , a decrease in VEGF could inhibit angiogenesis in the brain. Indeed, our data show that decreasing VEGF production (by antisense transfection) to 20–50% of parental cell level, which is accompanied by reduced biological activity (measured by stimulation of HDMEC proliferation) was associated with inhibition of both angiogenesis and brain metastasis formation.
VEGF had been shown to promote tumor growth in previously described primary brain tumor models (Refs. 22 , 23 ; tumor cells were stereotaxically injected into the brain parenchyma), but they were different from our metastasis model for brain lesions. VEGF overexpression can enhance growth of melanoma in the brain (23) . Moreover, reduction of VEGF expression by antisense-VEGF gene transfection inhibits growth of glioma (47) and melanoma (23) . In this study, we demonstrate that reduction of VEGF secretion (by antisense gene transfection) inhibits brain metastasis, suggesting its value as a therapeutic target. Newer and more efficient vector systems for optimizing gene delivery and gene expression will be necessary to deliver the antisense-VEGF gene therapies. Among these are various inhibitors of VEGF and its receptors (Flt-1 and Flk-1/KDR), such as humanized neutralizing antibody for VEGF (49) , dominant-negative VEGF (50) , VEGF receptor tyrosine kinase inhibitors (51) , soluble Flt-1 (52) , and dominant-negative Flk-1 (53) . In any case, future antiangiogenesis therapy targeting VEGF may be useful for the control and treatment of brain metastasis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by Cancer Center
Support Core Grant CA16672 and Grant R35-CA42107 (to I. J. F.) from
the National Cancer Institute, NIH. 
2 To whom requests for reprints should be
addressed, at Department of Cancer Biology, Box 173, University of
Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,
TX 77030. Phone: (713) 792-8577; Fax: (713) 792-8747; E-mail: ifidler{at}notes.mdacc.tmc.edu
3 The abbreviations used are: VEGF, vascular
endothelial growth factor; HDMEC, human dermal microvascular
endothelial cell; Ab, antibody; TUNEL, terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end labeling; bFGF, basic
fibroblast growth factor; EGF-R, epidermal growth factor-receptor; i.c.
intracarotid; BrdUrd, 5-bromo-2'-deoxyuridine; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
4 S. Pathak (M. D. Anderson Cancer Center),
personal communication.
Received 11/23/99. Accepted 6/28/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S. Yano, H. Muguruma, Y. Matsumori, H. Goto, E. Nakataki, N. Edakuni, H. Tomimoto, S. Kakiuchi, A. Yamamoto, H. Uehara, et al. Antitumor Vascular Strategy for Controlling Experimental Metastatic Spread of Human Small-Cell Lung Cancer Cells with ZD6474 in Natural Killer Cell-Depleted Severe Combined Immunodeficient Mice Clin. Cancer Res., December 15, 2005; 11(24): 8789 - 8798. [Abstract] [Full Text] [PDF]
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E. I. Deryugina, L. Soroceanu, and A. Y. Strongin Up-Regulation of Vascular Endothelial Growth Factor by Membrane-type 1 Matrix Metalloproteinase Stimulates Human Glioma Xenograft Growth and Angiogenesis Cancer Res., January 1, 2002; 62(2): 580 - 588. [Abstract] [Full Text] [PDF]
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C. Jing, C. Beesley, C. S. Foster, H. Chen, P. S. Rudland, D. C. West, H. Fujii, P. H. Smith, and Y. Ke Human Cutaneous Fatty Acid-binding Protein Induces Metastasis by Up-Regulating the Expression of Vascular Endothelial Growth Factor Gene in Rat Rama 37 Model Cells Cancer Res., June 1, 2001; 61(11): 4357 - 4364. [Abstract] [Full Text] [PDF]
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R. S. Herbst and I. J. Fidler Angiogenesis and Lung Cancer: Potential for Therapy Clin. Cancer Res., December 1, 2000; 6(12): 4604 - 4606. [Full Text]
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