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[Cancer Research 60, 4959-4967, September 1, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

Expression of Vascular Endothelial Growth Factor Is Necessary but not Sufficient for Production and Growth of Brain Metastasis1

Seiji Yano, Hisashi Shinohara, Roy S. Herbst, Hiroki Kuniyasu, Corazon D. Bucana, Lee M. Ellis, Darren W. Davis, David J. McConkey and Isaiah J. Fidler2

Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the molecular mechanisms of angiogenesis in experimental brain metastasis. Cells from six different human cancer cell lines (proven to produce visceral metastasis) were injected into the internal carotid artery of nude mice. Colon carcinoma (KM12SM) and lung adenocarcinoma (PC14PE6 and PC14Br) cells produced large, fast-growing parenchymal brain metastases, whereas lung squamous cell carcinoma (H226), renal cell carcinoma (SN12PM6), and melanoma (TXM13) cells produced only a few slow-growing brain metastases. Rapidly progressing brain metastases contained many enlarged blood vessels. The expression of VEGF mRNA and protein by the tumor cells directly correlated with angiogenesis and growth of brain metastasis. Causal evidence for the essential role of VEGF in this process was provided by transfecting PC14PE6 and KM12SM cells with antisense-VEGF165 gene, which significantly decreased the incidence of brain metastasis. In contrast, transfection of H226 human lung squamous carcinoma cells with sense-VEGF121 or sense-VEGF165 neither enhanced nor inhibited formation of brain metastases. Collectively, the results indicate that VEGF expression is necessary but not sufficient for the production of brain metastasis and that the inhibition of VEGF represents an important therapeutic target.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Brain metastasis, which occurs in 20–40% of all patients with cancer, is an important cause of cancer morbidity and mortality. The treatment of choice for a patient with a resected primary tumor and only a single brain metastasis is surgical excision, whereas radiation and/or chemotherapy are used for multiple brain metastases. Regardless of the treatment, the prognosis of patients with brain metastasis is poor (1 , 2) . Tumors are biologically heterogeneous and contain subpopulations of cells with different angiogenic, invasive, and metastatic properties (3) . To produce metastasis, tumor cells must complete a series of sequential and selective steps (3 , 4) . Failure to complete even one step eliminates the cells from the process (5) . Recent studies indicate that to produce brain metastasis, tumor cells must reach the vasculature of the brain, attach to the microvessel endothelial cells, extravasate into the parenchyma, proliferate (by responding to growth factors), and induce angiogenesis (6, 7, 8) .

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.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Tissue Culture.
PC14PE6 cells were isolated from pleural effusions developed in a nude mouse injected i.v. with parental PC14 cells, a heterogeneous PC14 human lung adenocarcinoma cell line (25) . PC14Br cells were isolated from brain metastases established in severe combined immunodeficient/beige mouse subsequent to intracarotid artery injection of PC14 cells. Karyotypic analysis of PC14PE6 and PC14Br cell lines ruled out contamination with murine cells.4 The human lung squamous cell carcinoma cell line H226 was the gift of Dr. J. D. Minna (University of Texas Southwestern Medical Center, Dallas, TX; Ref. 26 ). The human colon carcinoma KM12SM (27) , human renal cell carcinoma SN12PM6 (28) , and human melanoma TXM13 (29) cell lines were established at The University of Texas M. D. Anderson Cancer Center.

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 [{alpha}-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).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Production of Experimental Visceral and Brain Metastasis.
In the first set of experiments, we determined the metastatic potential of the five human cancer cell lines. KM12SM colon cancer cells were injected into the spleen of nude mice to produce experimental liver metastasis. The other five cell lines were injected i.v. into nude mice to produce experimental lung metastasis. By 7 weeks after injection, all cell lines tested produced experimental metastasis to the liver or lungs (Table 1)Citation . 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. 1Citation 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)Citation . 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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  		Table 1 Production of visceral or brain metastasis

 


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  		Fig. 1. Vascularization and VEGF production in brain metastases produced by human cancer cell lines. Note that brain metastases produced by KM12SM, PC14PE6, and PC14Br cells expressed high levels of VEGF and contained numerous CD31-positive large blood vessels (arrows, transluminal bridges consisting of endothelial cell processes, i.e., CD31-positive). Metastases produced by H226 or SN12PM6 cells expressed low levels of VEGF and did not contain vessels with large lumens.

 
Growth and Vascularization of Brain Metastases.
The progressive growth of a tumor mass is determined by the balance between cell proliferation and cell death (35 , 36) . Tumor cell proliferation was evaluated by immunochemical staining for BrdUrd (37) , and tumor cell death was evaluated by TUNEL staining (38) . Brain metastases produced by KM12SM, PC14PE6, and PC14Br cells contained numerous BrdUrd-positive cells and few TUNEL-positive cells. In contrast, brain metastases produced by H226 and SN12PM6 cells contained few BrdUrd-positive cells and many TUNEL-positive cells (Table 2)Citation . 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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  		Table 2 Histological analyses of brain metastasis of various human cancer cells in nude mice

 
The growth of tumors has been shown to depend on the development of an adequate blood supply through angiogenesis (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 34, 35, 36) . Vascularization in brain metastases was assessed by staining tissue sections with anti-CD31 antibody; vessel density and the size of the lumen in vessels were then determined. The vessel density in brain metastases was actually lower than that in nontumor brain parenchyma, irrespective of cell line (Table 2)Citation . 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)Citation .

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 2Citation , 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. 1Citation 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. 2Citation , 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)Citation . 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. 2Citation ; Table 3Citation ). TXM13 expressed low levels of IL-8 and EGF-R, whereas all other five cell lines expressed higher levels (Fig. 2)Citation . 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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  		Fig. 2. mRNA expression of angiogenic cytokines and EGF-R. Tumor cell lines were cultured under sparse (S) or confluent (C) conditions, mRNA was extracted, and Northern blot analysis was performed as described in "Materials and Methods." Densitometric quantitation was derived from the ratio comparing the signal of the specific transcripts with that of the 1.3-kb GAPDH (internal control). These are representative data of three independent experiments. n.d., not detected.

 

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  		Table 3 In vitro production of angiogenic cytokines by human cancer cells

Tumor cells (sparse, <100 cells/mm2; confluent, >1200 cells/mm2) were incubated in a 24-well plate in medium for 48 h. After being washed, resultant cell cultures were incubated for an additional 24 h in fresh medium, and culture supernatants were harvested. The concentration of angiogenic cytokines in the supernatants was determined by ELISA as described in "Materials and Methods." At the end of the cell culture, the cells were counted, and the number of cytokines produced by 106 cells were calculated. Data shown are representative of two independent experiments.

 
Transfection with Sense- or Antisense-VEGF Gene.
To provide causal evidence for the role of VEGF expression in production of brain metastasis, we transfected a sense-VEGF gene into H226 cells and antisense-VEGF gene into PC14PE6 and KM12SM cells. We first determined by RT-PCR which isoform of four VEGF isoforms (VEGF121, VEGF165, VEGF189, and VEGF206; Ref. 14 ) were predominantly expressed in these three cell lines. As shown in Fig. 3ACitation , 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)Citation . 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.



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  		Fig. 3. Expression of VEGF isoforms by human cancer cell lines and tumor cells transfected with sense- or antisense-VEGF gene. A, expression of VEGF isoforms determined by RT-PCR. RT-PCR products were electrophoresed on a 1.2% agarose gel and stained with ethidium bromide. The PCR products for the VEGF165 and VEGF121 transcripts are 457 and 303 bp, respectively. B, VEGF mRNA expression by tumor cell lines transfected with sense-VEGF165, sense-VEGF121, or antisense-VEGF165 genes. Total RNA was extracted from confluent cultures. Northern blot analysis was performed as described in "Materials and Methods." The numbers are densitometric quantitation of the ratio of the area between the specific endogenous VEGF transcripts and the GAPDH transcripts with the value for parental cells defined as 1.0. The data shown are representative of three independent experiments. C, proliferation assay for HDMECs. Culture supernatants and recombinant human VEGF165 (20 ng/ml) were pretreated for 2 h at 37°C with or without control IgG (10 µg/ml) or with anti-VEGF Ab (10 µg/ml). The proliferation of HDMECs was determined by the 3-(4,4-dimethylthiazol-2-yl)-2.5-diphenyl-tetrazolium bromide assay, as described in "Materials and Methods." The data shown are representative of three independent experiments. Bars, SD. *, P < 0.05 as compared with the value for medium alone. #, P < 0.05 as compared with the value for the respective control (Control IgG).

 
H226 cells transfected with the sense-VEGF165 or sense-VEGF121 gene produced 112.0 and 60.0 ng VEGF protein/106 cells/24 h (measured by ELISA), respectively (Table 4)Citation . Culture supernatants of these transfectants stimulated proliferation of HDMECs (Fig. 3C)Citation . 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.


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  		Table 4 Production of brain metastasis by tumor cells transfected with sense or antisense VEGF gene

 
Inhibition of Endogenous VEGF Expression Suppresses Production of Brain Metastasis.
We next injected H226 cells transfected with the sense-VEGF165 or sense-VEGF121 gene and nontransfected parental cells into the internal carotid artery of nude mice. Regardless of VEGF expression, by 12 weeks after injection, the cells did not produce brain metastases (Table 4)Citation . 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. 4Citation 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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  		Fig. 4. Production of brain metastasis by antisense VEGF-transfected KM12SM or PC14PE6 cells. Note that the large metastases produced by neo-transfected cells are red (highly vascular), whereas those produced by antisense VEGF-transfected cells are small and avascular. The gross appearance correlated with the presence of dilated blood vessels (CD31+) and expression of VEGF protein. Arrows, small brain metastases.

 
In the last set of studies, we examined the expression of VEGF and the pattern of vascularization in brain metastases produced by control or VEGF-antisense transfected PC14PE6 and KM12SM cells. Production of VEGF protein in brain metastasis produced by antisense-VEGF gene transfectants was lower than that of Neo-control cells. The vessel density in brain metastases of PC14PE6/AS cells was similar to nontumor parenchyma and contained only a few enlarged vessels. Brain metastases by KM12SM/AS cells also had fewer enlarged vessels than parental or Neo-control cells.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The process of metastasis consists of several sequential and selective steps (3, 4, 5 , 36) . In this study, six different highly tumorigenic human cancer cell lines were injected into the internal carotid artery of nude mice. Although the i.c. injection bypassed the initial steps of metastasis (separation from the primary neoplasm, invasion, and release into blood vessels or lymphatics) to produce brain metastases, all subsequent steps in the process (arrest in brain capillary bed, extravasation, growth, and angiogenesis) had to occur for metastasis to succeed. Previous data from our laboratory demonstrated that the production of progressively growing experimental brain metastasis depends on the ability of cells to grow in the brain parenchyma (7) . In our present study, the production of many large metastases in the brain by the KM12SM, PC14PE6, and PC14Br cells and few small brain metastases produced by the H226, SN12PM6, and TXM13 cells did not correlate with initial arrest of tumor cells in the brain (measured by radiolabeling of tumor cells with 125I-IdUrd) or collagenase activity (measured by gelatin zymography; Ref. 4 ; data not shown). The differences in metastatic potential were attributable to vascularization and hence growth.

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.


   ACKNOWLEDGMENTS
 
We thank Donna Reynolds and Kenneth Dunner, Jr., for technical assistance, Walter Pagel for critical editorial comments, and Lola López for expert preparation of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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. Back

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 Back

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. Back

4 S. Pathak (M. D. Anderson Cancer Center), personal communication. Back

Received 11/23/99. Accepted 6/28/00.


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