Proc. Natl. Acad. Sci. USAVol. 93, pp. 8502-8507, August 1996Genetics

Suppression of glioblastoma angiogenicity and tumorigenicity byinhibition of endogenous expression of vascular endothelialgrowth factor

(tumor angiogenesis/human brain tumor/antisense)

SHI-YUAN CHENG*, H.-J. Su HUANG*t, MOTOo NAGANE*, XLANG-DONG Jit, DEGUI WANG*, CHARLES C.-Y. SHIHt,WADIH ARAP*§, CHUN-MING HUANGt, AND WEBSTER K. CAVENEE*tII1*Ludwig Institute for Cancer Research, San Diego Branch, tDepartment of Medicine, and 1Center for Molecular Genetics, University of California at San Diego,9500 Gilman Drive, La Jolla, CA 92093-0660; tPharMingen, Inc., San Diego, CA 92121; and §Cancer Biology Program, Stanford University, Stanford, CA 94305

Communicated by Raymond L. White, The University of Utah, Salt Lake City, UT, April 23, 1996 (received for review March 22, 1996)

ABSTRACT The development of new capillary networksfrom the normal microvasculature of the host appears to berequired for growth of solid tumors. Tumor cells influence thisprocess by producing both inhibitors and positive effectors ofangiogenesis. Among the latter, the vascular endothelial growthfactor (VEGF) has assumed prime candidacy as a major positivephysiological effector. Here, we have directly tested this hypoth-esis in the brain tumor, glioblastoma multiforme, one ofthe mosthighly vascularized human cancers. We introduced an antisenseVEGF expression construct into glioblastoma cells and foundthat (i) VEGF mRNA and protein levels were markedly reduced,(ii) the modified cells did not secrete sufficient factors so as to bechemoattractive for primary human microvascular endothelialcells, (iii) the modified cells were not able to sustain tumorgrowth in immunodeficient animals, and (iv) the density ofin vivoblood vessel formation was reduced in direct relation to thereduction of VEGF secretion and tumor formation. Moreover,revertant cells that recovered the ability to secrete VEGF re-gained each of these tumorigenic properties. These results sug-gest that VEGF plays a major angiogenic role in glioblastoma.

The progressive growth of solid tumors is dependent on theprocess of neovascularization to develop the blood vessels thatprovide nutrients to and remove waste products from the interiorregions of neoplasms; in its absence, tumors generally cease togrow beyond a few cubic millimeters in volume (1). Tumor cellsplay an active role in angiogenic homeostasis by producing andsecreting a number of angiogenic factors, including basic fibro-blast growth factor (bFGF), angiogenin, tumor necrosis factor a,and vascular endothelial growth factor (VEGF) (2-4), as well asinhibitors of the process (5-7). VEGF, also described as vascularpermeability factor, is a specific mitogen for vascular endothelialcells in vitro and can be an angiogenic factor for neovasculariza-tion in vivo (8,9). VEGF is a 34-42 kDa heparin-binding, dimeric,disulfide-bound glycoprotein and exists as four isoforms having121, 165, 189, and 206 amino acids, respectively. In contrast tobFGF, another major angiogenic factor, it has a typical signalpeptide composed of 26 amino acids and is efficiently secretedfrom cells (10). VEGF exerts its function through binding to itsidentified receptors,flt-1 and flk-1, on the surface of endothelialcells (11, 12).

Several lines of evidence have suggested that VEGF may be amajor factor in the neovascularization and growth of humancancers. First, VEGF and its receptors are expressed at high levelsin many types of human tumors (2-4). Second, forced overex-pression of VEGF by tumor cells has, in some cases, enhancedtheir tumorigenic behavior (13). Third, the intraperitoneal injec-tion of a specific anti-VEGF mouse mAb into nude mice inhibitedtumor growth in vivo, (14). Finally, dominant-negative flk-1

mutants lacking their C termini, and which presumably formheterodimers with wild-type flk-1 upon VEGF binding but nolonger transduce signals, cause tumor growth suppression in vivo(15). However, these interpretations are complicated by thepresence of another high affinity receptor for VEGF, flt-1 (16),and by other ligands such as placental growth factor, which canalso bind toflt-1 (17).

Astrocytomas are an attractive model for the investigation ofthe role of VEGF in tumor angiogenesis. They are among themost dramatically neovascularized neoplasms with respect tovasoproliferation, endothelial cell cytology, and endothelial cellhyperplasia (18). The malignant progression of astrocytomatoward end-stage glioblastoma is well characterized in his-topathological, clinical, and molecular genetic terms (19). Studieson glioblastoma angiogenesis have revealed that expression ofVEGF and its two receptors, fit-1 and flk-1, are up-regulated inthe tumors (4). In addition, an intracerebral model of humanglioblastoma tumorigenesis has been established in the nudemouse (20). Several approaches could be envisioned to poten-tially impede VEGF expression in tumor cells. First, VEGFdisplays a limited but significant homology to that of the platelet-derived growth factor (PDGF) superfamily (10). A dominant-negative PDGF mutant that is able to form heterodimers with thewild-type PDGF but unable to bind its receptor, thereby inhibitedPDGF function (21). While VEGF contains the eight conservedcysteines found in PDGF, there are eight additional cysteineresidues within the carboxyl-terminal region that would make asimilar strategy for inhibition of VEGF activity difficult. Second,antisense oligonucleotides might be used to target VEGF andreduce its expression (22). This approach suffers from the ob-servation that irrelevant oligonucleotides can inhibit tumor-derived cell growth in vitro (23), and is further complicated by therapid degradation of synthetic oligonucleotides in cells and bio-logical fluids (22). Third, the forced expression of antisensemRNA for a given gene to analyze its biological role has beenwidely used (24, 25).

In this report, we applied the strategy of exogenous expressionof an antisense VEGF construct in a highly tumorigenic humanglioblastoma cell line to directly test the role of VEGF in tumorpathophysiology. Several single cell clones showed decreasedVEGF protein secretion, decreased ability to stimulate humanmicrovascular endothelial cell migration, and dramatic suppres-sion of subcutaneous and intracerebral in vivo tumor growth. Theattribution of these physiological effects to specific inhibition ofVEGF levels was confirmed by analysis of a revertant clone thatregained high expression of VEGF, could stimulate endothelial

Abbreviations: VEGF, vascular endothelial growth factor; bFGF,basic fibroblast growth factor; HMVEC, human microvascular endo-thelial cells; RT-PCR, reverse transcription-PCR; CM, conditionedmedium.'Towhom reprint requests should be addressed at: Ludwig Institute forCancer Research, San Diego Branch, 9500 Gilman Drive, La Jolla, CA92093-0660.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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cell migration in vitro, and showed enhanced tumorigenicity invivo. These data directly indicate that VEGF plays a critical rolein glioblastoma angiogenesis.

MATERIALS AND METHODSCell Lines and Tissue Culture. The human glioblastoma cell

line U87MG was obtained from the American Type CultureCollection. The cells were maintained in culture in Dulbecco'smodified Eagle's medium (DMEM; Life Technologies, GrandIsland, NY) supplemented with 10% heat-inactivated cosmicbovine serum (HyClone) and antibiotics. Human microvascularendothelial cells (HMVEC; Cell Systems, Kirkland, WA) were

cultured in serum free medium supplemented with 20 ng/mlbFGF and 20 ng/ml epidermal growth factor. For migrationassays, cells at passage 5 were used.

Cloning and Sequencing of VEGF cDNAs. The VEGF121,VEGF165, and VEGF189 isoforms were cloned by reverse tran-scription-PCR (RT-PCR) using primers that flank 5' and 3' endsof the VEGF cDNA coding region, respectively: GAAACCAT-GAACTTTCTGC (sense, primer 1) and CGCCTCGGC7T-GTCA (antisense, primer 2), modified to containBamHI (primer1) and XbaI (primer 2) sites. RT-PCRs were done according toinstructions of AmpliTaq RT-PCR Kit from Cetus with minormodifications in a model 9600 Thermal cycler. The VEGF2o6isoform was constructed by inserting the second part of exon 6 (51nt present only in the largest form) into a VEGF189 cDNA cloneby recombinant PCRs using two insertion primers: CTGTCTAA-TGCCCTGGAGCCTCCCTGGCCCCCATCCCTGTGGGC-CTTGCTCAG (sense, primer 3) and CAGGGCATTAGACA-GCAGCGGGCACCAACGTACACGCTCCAGGACTTAT-ACCG (antisense, primer 4). The cDNA for each of the fourisoforms of VEGF had the expected lengths of 470, 602, 674 and725 bp, respectively, and their sequences were identical to thosepreviously reported (26).

Selection of Single Cell Clones from the U87MG Cells Trans-fected with the VEGF or LacZ Expression Constructs. VEGF189cDNA was subcloned in an antisense orientation into the mam-malian cytomegalovirus-promoter expression vector, pCEP4 (In-vitrogen). The U87MG cells were transfected using the calciumphosphate precipitation method. The pool of clones was thenharvested and sorted with a fluorescence-activated cell sorter intoone cell per well in 96-well flat bottom plates with 200 ,ulDMEMcontaining 15% cosmic bovine serum per well. The survivingclones were grown and expanded, and those that expressedexogenous VEGF mRNA or LacZ enzyme activities were iden-tified either by Northern blot analysis or LacZ staining.RNA Isolation and Northern Blot Analysis. Total RNA was

isolated from the cells by using the Trizol reagent (GIBCO/BRL). Total RNA (12 ,g) were size fractionated and blottedonto a nylon membrane (Hybond N+; Amersham), fixed by UVcross-linking (Stratagene), and baked at 80°C for 1 hr. Prehy-bridization and hybridization were carried out with a 32P-labeledVEGF165 cDNA probe (0.6 kb). The membrane was washed andexposed to x-ray film. ForbFGF hybridization, the membrane wasstripped and rehybridized with a 32P-labeled bFGF DNA frag-ment (0.8 kb). Differences in RNA loading were normalized byrehybridizing the membrane with a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase probe (1.25 kb) followed by densi-tometry.

Conditioned Medium (CM) and VEGF ELISA. Secretion ofVEGF protein into CM was analyzed by ELISA with a humanVEGF Quantikine Kit from R& D Systems. To generate CM, theparental U87MG or its derivatives were grown to 70% conflu-ence. The cells were harvested, counted, and sLcded at 3.0 x 105in 12-well plates. On the next day, the med.a was changed toDMEM/0.5% bovine serum albumin/1% dialyzed fetal calfserum for another 24 hr. Then the media was replaced by the freshmedia and cells were allowed to grow for another 48 hr. The CMwas cleared by centrifugation at 14,000 rpm at 4°C for 15 min andthen stored at -80°C for ELISA analysis.

VEGF ELISA analysis was carried out according to themanufacturer's instructions with minor modifications. TheVEGF concentrations in the CM were calculated from astandard curve derived by using recombinant human VEGF165proteins (R & D Systems).

Endothelial Cell Migration Assay. The migration ofHMVECin response to purified human recombinant VEGF16s proteins (R& D Systems) and CM was assayed in 48-well blind-well chemo-taxis chambers by using gelatin-coated polycarbonate filters of 5.0,im pore size (Costar). Starved HMVEC cell suspension (50 ptl)at a concentration of 1 X 106 cells/ml was placed in the top wellof the chamber while the lower well contained either 25 p,l ofCMor 10 mg/ml of the purified human recombinant VEGF165proteins in PBS, in the absence or presence of either 1 mg/ml ofa neutralizing anti-VEGF antibody (clone MAB 293; R & DSystems) or 1 mg/ml of an isotype matched control anti-mouseantibody (IgG2b.K, clone 01951D, 8202-04; PharMingen). Afterincubation at 37°C for 2.5 hr in a humidified atmosphere of airwith 5% C02/95% air, the filters were fixed and stained withDiff-Quik (Baxter Healthcare, McGaw Park, IL) and mounted onslides with the migrated cells down. Nonmigrating cells on theupper surface were carefully removed with a cotton swab.

Tumorigenicity. Subcutaneous inoculation and tumor growthmeasurements were carried out as described (20). For intrace-rebral stereotactic implantation, 5 x 105 cells of the parentalU87MG or cells of admixture of the LacZ- or antisense VEGF-expressing clones in 5 ,l of PBS were inoculated into the corpusstriatum in the right hemisphere (20). Brains were removed andstored at -80°C. Thin cryostat sections (5 ,um) were stained withhematoxylin and eosin, and tumor size was microscopicallydetermined.Primary Tumor Cell Culture. Primary cells from a mouse

subcutaneous tumor inoculated by the U87MG VEGF antisense(revertant 6-1/R) cells were cultured as follows. The tumors weredissected, and the tumor fragments were transferred to a T-25flask containing DMEM/15% cosmic bovine serum. Several dayslater, fresh medium was changed to allow cells to grow to nearconfluence. The cells were then passaged and expanded inDMEM/10% cosmic bovine serum with hygromycin B (200pkg/ml).

Immunohistochemical Analysis of Tumors. Histological anal-ysis of subcutaneously or intracerebrally inoculated tumors wasperformed by using an anti-VEGF mAb (clone G153-694;PharMingen) as described (20). The cryostat sections were alsostained in similar ways with an anti-CD31 mouse mAb (cloneMEC 13.3; PharMingen) and an IgG2b.K isotype control mAb(clone 01951D, 8202-04; PharMingen). All sections were coun-terstained with hematoxylin. Quantitative analysis of the bloodvessel densities oftumor samples was done using the METAMORPHIMAGE SYSTEM for Microsoft Windows, Version 2.0.1. (UniversalImaging, West Chester, PA).

RESULTSStable Transfection and Expression of Antisense VEGF

Causes Reduction in Endogenous VEGF Levels with Little Effecton Cell Growth in Viro. The human glioblastoma cell line,U87MG, expresses high levels of VEGF mRNA, secretes theprotein, and expresses three of the four VEGF splice variants,VEGF121, VEGF165, and VEGF189 (data not shown). Since theVEGF189 isoform contains all of coding sequence of the smallerforms, a VEGF189 antisense construct and, to control for clonalvariation, a construct containing the bacterial ,B-galactosidase(LacZ) gene under human cytomegalovirus promoter controlwere assembled and each separately transfected into U87MGcells. Single hygromycin B-resistant cells were isolated using afluorescence-activated cell sorter and the resultant colonies wereexpanded. Sixteen clones expressing VEGF189 antisense messageand 4 clones expressing the LacZ gene were identified either byNorthern blot analysis (Fig. 1A) or by staining for ,3-galactosidaseactivity; three of each were chosen for further analysis. As

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FIG. 1. VEGFRNA expression in the parental U87MG, the LacZ-,and antisense VEGF-expressing cells. (A) Northern blot analysis.Lanes: 1, parental U87MG cells; 2-4, antisense VEGF-expressingclones 10, 13, and 17; 5-7, LacZ-expressing clones 4, 22, and 27.Symbols *, 0, and * are used to distinguish different RNA species;their descriptions are in the text. This analysis was repeated at leastthree times with similar results. (B) VEGF ELISA analysis. CM wascollected after the cells were cultured for 48 hr. Each bar represents themean ± SEM of three replicates, and the assay was repeated at least fourtimes with similar results with cells of various passage numbers.

demonstrated in Fig. 1A, the antisense VEGF-expressing clones,10, 13, and 17 each expressed VEGF189 antisense message (0, 1.0kb) with a concomitant reduction in endogenous VEGF tran-scripts (-, 3.8 and 1.4 kb). In comparison, the controls (eitherthe parental U87MG cells or the three LacZ-expressing clones,4, 22, and 27) had comparable endogenous VEGF levels withno other mRNA species detected, indicating that the reductionof steady-state VEGF mRNA was due to antisense expressionand not to intrinsic clonal variation in the parental population.The decreased endogenous VEGF message levels in theantisense VEGF-expressing clones are likely due to theirantisense RNA targeted degradation (27). To estimatewhether the reduction of VEGF mRNA expression was spe-cifically targeted by this approach, the Northern blots werestripped and rehybridized to a probe for another potentangiogenic factor, bFGF. No difference in bFGF expressionwas detected among the parental U87MG, the LacZ-, orantisense VEGF-expressing clones (Fig. 1A, m).We next performed ELISA to analyze the amounts of

secreted VEGF proteins in CM collected after cells werecultured for 48 hr. Whereas the parental U87MG and theLacZ-expressing clones 4, 22, and 27 cells secreted VEGF atconcentrations of 15 to 22 ng/ml per 106 cells, the antisense

VEGF-expressing clones 10, 13, and 17 produced VEGF atlevels that were 3- to 6-fold less (Fig. 1B). ELISA analysis ofbFGF in CM from each of cultures above showed that secre-tion was low in each of them and that these amounts weresimilar and consistent with the unaltered RNA levels (data notshown). Thus, the antisense VEGF-expressing clones exhibitedsubstantial inhibition of both VEGF mRNA and proteinsecretion, whereas the LacZ-expressing clones and the paren-tal U87MG cells expressed the expected high levels.We then sought to determine if the reduced expression of

VEGF in the antisense RNA-expressing clones was the trivialresult of cellular growth suppression as it has been observed forother genes in other systems (23). The parental U87MG, theLacZ-, and antisense VEGF-expressing clones were each seededas six replicates at two different densities and their growth ratesover 5 days were determined as described (28). The resultsshowed minimal reductions (15% or less) in the efficiencies of cellseeding or growth rates of either type of transfectants comparedwith the uncloned parental cells, suggesting that the antisense-mediated reduction in VEGF expression was not growth-related.

Cells Expressing Antisense VEGF mRNA Have DiminishedAbility to Stimulate Human Microvascular Endothelial CellMigration in Vitro. One of the initial steps in the biologicalprocess of tumor-induced neovascularization is endothelial cellmovement toward the chemoattractive stimuli produced by thetumor cells (1). We thus sought to determine if the expression ofantisense VEGF mRNA affected the ability of the U87MG cellsto secrete factors into their CM. The data shown in Fig. 2 indicatethat CM from either the parental U87MG or the LacZ-expressing clones 4, 22, and 27 were highly stimulatory forHMVEC migration, while CM from the antisense VEGF-expressing clones 10, 13, and 17 were greatly diminished in thisregard. The remainingHMVEC migration in the presence ofCMfrom the latter clones is likely due to other stimulatory substances,such as bFGF, produced by the tumor cells (29). The notion thatVEGF is the major element in the CM that promoted HMVECmigration was tested by including a VEGF neutralizing mAb inthe CM during the migration assay (Fig. 2). This antibody blockedthe stimulation of cell migration elicited by relatively high con-centrations (10 ,ug/ml) of purified human recombinant VEGF165and also by CM from the parental U87MG cells and the LacZ-expressing clones. In contrast, it had no effect on the limited

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FIG. 2. Cells expressing antisense VEGF mRNA have diminishedability to elicit HMVEC migration. In vitro migration of HMVEC inresponse to purified human recombinant VEGF (10 ng/ml; R & DSystems) and to CM was measured in modified Boyden chambers asdescribed. CM from the parental U87MG or their derivatives werediluted 1:10 with PBS and tested in the absence (solid bars) or in thepresence of either 1 mg/ml of a neutralizing anti-VEGF antibody(stippled bars) or 1 mg/ml of an IgG2b.K isotype control mAb (hatchedbars). Migrated cells were counted in 10 high powered fields (HPF,400x total magnification) per filter. All samples were examined intriplicate and data are shown as the mean ± SEM. The assay wasperformed in triplicate on three sets of CM at least twice each withsimilar results.

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FIG. 3. Cells expressing antisense VEGF mRNA have diminishedabilities to form tumors in immunodeficient animals. Cells (1 x 106)expressing antisense VEGF mRNA or LacZ were injected into theright flanks of nude mice (three to four mice in each group) while thesame number of the parental U87MG cells were implanted into the leftflank of the same animals. Tumor volumes were estimated at theindicated times after implantation, and data are shown as the mean ±SEM. The experiment was repeated at least two separate times byusing either double or single side implantation; similar results wereobtained each time.

promotion of the chemotactic response induced by the CM fromthe antisense VEGF-expressing clones. The specificity of thisblockage was demonstrated by the absence of the effects on thecell migrations by an isotype matched control mAb. These dataindicate that VEGF was the major component in the CM fromthe parental U87MG and the LacZ-expressing cells that stimu-lated HMVEC migration and that the expression of an antisenseVEGF gene construct in the U87MG cells effectively reduced

Proc. Natl. Acad. Sci. USA 93 (1996) 8505

VEGF secretion to levels below those required for one of itsprimary biological functions.

Inhibition of Endogenous VEGF Expression Suppresses theTumorigenic Ability of Glioblastoma Cells in Vtvo. Having dem-onstrated that a diminution ofVEGF secretion had substantial invitro effects, we next sought to determine its effects on in vivobehavior. We first simultaneously implanted the parentalU87MG cells and the LacZ- or antisense VEGF-expressingclones subcutaneously into alternate flanks of immune-deficientnude mice. The data in Fig. 3 show that cells from the parentalU87MG and the LacZ-expressing clones 4, 22, and 27 formedtumors with similar kinetics and of similar sizes, while tumorgrowth was remarkably suppressed when the same numbers ofcells from the antisense VEGF-expressing clones 10, 13, and 17were injected. The latter cells formed small, but palpable, massesin the mice at the same time as the parental U87MG cells, but thetumors did not progress in size. To ensure that there was noinfluence on the growth of the antisense VEGF-expressing clonesby the more quickly growing parental cells on the opposite flanksof the mice, we also individually implanted the cells into only oneflank. In these analyses, similar suppressions oftumor growth wereobserved in the mice injected with the antisense VEGF-expressingclones 10, 13, and 17 while comparable rapid tumor growth wasobserved in mice inoculated with either the parental U87MG orthe LacZ-expressing clones 4, 22, and 27 (data not shown).We then examined whether the tumor growth suppression

observed in heterotopic subcutaneous inoculations was alsoapparent with ectopic stereotactic implantation to the brain (20).Comparable numbers of cells of these types were implanted: theparental U87MG; an equal admixture of the LacZ-expressingclones 4, 22, and 27; and, an equal admixture of the antisenseVEGF-expressing clones 10, 13, and 17. The results are summa-rized in Table 1, and examples are illustrated in Fig. 4. One animalin each group that received the parental U87MG cells or theLacZ-expressing clone admixture died after 5 weeks from over-growth of the tumors, which reached volumes greater than 60cubic millimeters. The remainder of the mice in these two groups

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FIG. 4. Immunohistochemistry ofintracerebral tumors formed by theparental U87MG cells (a-c), the anti-sense VEGF-expressing cell admix-tures (d-f), the LacZ-expressing celladmixtures (g-i), and the revertant6-1/R cells (i-1). (a, d, g, andj) Stain-ing with an anti-VEGF mAb. (b, e, h,and k) Staining with an IgG2b.K isotypecontrol mAb. (c, f, i, and 1) Stainingwith an anti-CD31 mAb. (X100.)

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Table 1. Tumorigenicity of intracerebrally implanted the parental U87MG cells and of equalnumbers of admixtures of the LacZ- or antisense VEGF-expressing clones

Total no. Tumor volume, mm3 Days postimplantationTumor cells of mice (mean ± SEM) (mean ± SEM)

U87MG 8 50.9 ± 6.6 39 ± 1.4VEGF antisense 6 6.7 ± 4.5 41 ± 0.9LacZ 6 50.5 ± 17.9 35 ± 1.7

Cells (5 x 105) of U87MG or cells of a mix of the LacZ- or antisense VEGF-expressing clones in 5 ,ilof PBS were stereotactically implanted into the corpus striatum in the right hemisphere of the nude mousebrain. The brains were removed, treated, and analyzed as described. The experiments were repeated atleast two times with similar results.

each developed tumors of 45 cubic millimeters or larger. Incontrast, five of six nude mice implanted with the same numbersof the antisense VEGF-expressing clones developed masses ofless than 5 cubic millimeters; the remaining mouse only devel-oped a small tumor of less than 15 cubic millimeters at the timeof sacrifice. Thus, suppression of tumorigenicity expression wasapparent at both heterotopic and ectopic sites and was elicited bylevels of inhibition ofVEGF secretion attainable by expression ofthe antisense constructs.

Immunohistochemical analysis of the various subcutaneousand intracerebral tumors using a monoclonal anti-VEGF anti-body showed substantial VEGF protein expression in the diffusepattern expected for a secreted protein in the tumors formed byeither the parental U87MG cells or the LacZ-expressing cloneadmixture Fig. 4 a and g), whereas very little immunoreactivitywas apparent with the isotype matched control antibody (Fig. 4b and h). In contrast, little VEGF could be detected in theantisense VEGF-expressing samples (Fig. 4d) in comparison tothe signal obtained with the matched isotype control mAb (Fig.4e). To determine whether the reduced levels ofVEGF secretionand tumorigenicity were associated with a reduction in the abilityof the cells to induce neovascularization, we determined therelative blood vessel densities by immunostaining the tumorsamples with a mAb against the endothelial cell surface proteinmarker, CD31 (30). Computerized quantitative analysis showedthat blood vessel densities were reduced 3-fold in those tumorsformed by the antisense VEGF-expressing cells (Fig. 4f) ascompared with either the parental U87MG or the LacZ-expressing cells (Fig. 4 c and i).

Revertant Cells That Secrete VEGF Regain Their Angiogenicand Tumorigenic Properties. We were able to take advantage ofthe well-described phenomenon whereby exogenous gene expres-sion systems tend to lose efficiency after transfer into cells oranimals (31). One mouse in each group which had been inocu-lated with the antisense VEGF-expressing clones 10 and 17developed subcutaneous tumors that appeared to be slow grow-ing but then grew with kinetics similar to tumors caused byinoculation of the parental U87MG cells. One possibility for theseobservations was that some subset of the cells had genetically orepigenetically reverted so that they again secreted VEGF atwild-type levels. We characterized these two revertants by VEGFELISA, and selected one such tumor formed by cells from theantisense VEGF-expressing clone 17, termed as 6-1/revertant

(6-1/R), to test the possibility. At the end of 5 weeks fromsubcutaneous inoculation, the 6-1/R tumor was removed andcultured under hygromycin B-selection. Resultant cells hadgrowth rates similar to their immediate parents, the antisenseVEGF-expressing clone 17 cells, as well as to the original parentalU87MG cells. ELISA analysis showed that the concentration ofVEGF protein secreted by the 6-1/R cells had recovered to levelssimilar, or even somewhat higher, than those of the parentalU87MG cells (Table 2). The recovery of VEGF secretion alsoaccompanied the ability of the 6-1/R cells to stimulate endothe-lial cell migration, and this ability was diminished by inclusion ofthe neutralizing anti-VEGF antibodies in the CM from 6-1/Rcells, similar to results obtained with the parental U87MG cells(Table 2). Moreover, the 6-1/R cells formed tumors that greweven faster than tumors formed by the parental U87MG cellsboth in subcutaneously and intracerebrally inoculated nude mice,suggesting a strong selection in vivo for expression of VEGF.

Immunohistochemical analysis of these tumors showed highlevels of diffuse secreted VEGF in the tumors formed by the6-1/R cells compared with those of tumors formed by theantisense VEGF-expressing parental clone 17 cells (Fig. 4 j andd). The 6-1/R tumors also showed bloodvessel densities that were3-fold greater, as determined by computerized quantitative anal-ysis, than those in tumors formed by their antisense VEGF-expressing parental clone 17 cells (Fig. 41 andf). Moreover, theseeffects were observed while the expression of mRNA or secretedforms of another potential major regulator of neovascularization,bFGF, were unchanged (data not shown). Thus, these 6-1/R cellsrecovered their tumorigenic and angiogenic capacities concomi-tantly with their reversion to the parental VEGF expressionphenotype.

DISCUSSIONThe process of tumor-induced neovascularization appears toresult from a homeostatic balance between angiogenic stim-ulators and inhibitors produced by tumor cells, macrophagesrecruited by the tumor, and proliferating endothelial cells (1,32, 33). Candidates as major physiological stimulators includeVEGF, bFGF, angiogenin, and tumor necrosis factor a (2-4).Several potential angiogenic inhibitors have also been recentlyidentified as thrombospondin (5), angiostatin (6), and theglioma-derived angiogenic inhibitor (7).

Table 2. Characterization of parental U87MG, anti-17, and revertant 6-1/R cells

- Tumorigenesis

VEGF ELISA, HMVEC migration, no. of cell migrated Subcutaneous Intracerebralng/ml per CM + Tumor Tumor

Tumor cells 106 cells CMs CM + mAb IgG2b.K volume, mm3 P.O.D. volume, mm3 P.O.D.U87MG 18.7 ± 0.6 224.0 ± 3.6 102.0 ± 9.5 220.0 ± 4.1 1013.0 ± 102.2 34.0 ± 0.0 51.0 ± 6.6 39.0 ± 1.4Anti-17 5.3 ± 1.1 67.3 ± 3.0 55.0 ± 2.8 73.3 ± 2.3 7.2 ± 2.6 56.0 ± 0.0 6.7 ± 4.5 41.0 ± 1.26-1/R 23.3 ± 1.9 223.0 ± 8.2 107.0 ± 1.8 238.0 ± 11.5 2500.0 ± 149.1 34.0 ± 0.0 84.6 ± 10.9 28.0 ± 1.5

All values are ±SEM. All experiments were done as described in Figs. 1B, 2, and 3 and Table 1. They were repeated at least two to four timeswith similar results. Four to six nude mice in each group were implanted for subcutaneous and intracerebral tumors, respectively. IgG2b.K, isotypecontrol monoclonal antibody. P.O.D., postoperation days.

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Here, we applied an antisense RNA strategy to test thehypothesis that endogenously produced VEGF is a major stim-ulatory factor secreted by the U87MG glioblastoma cells. Ourresults show that a diminution of VEGF secretion by tumor cellscaused marked decreases in their tumorigenic and angiogenicbehaviors in vitro and in vivo, indicating that VEGF is the majorpositive regulator of neovascularization in these cells. This con-clusion is strongly supported by the observation that a revertantregained greater than wild-type levels of VEGF secretion in vitro,tumorigenicity in subcutaneous and intracerebral sites in vivo,endothelial cell chemoattraction in vitro, and neovascularizationin vivo. These latter results are consistent with a variety of otherstudies showing that a correlation between enhanced levels ofVEGF expression and tumor progression (3, 4). Overexpressionof VEGF in different cell lines has also been shown to to confera growth advantage in vivo (34) or enhance tumor growth (13).

It has been previously reported that another potent angiogenicgrowth factor, bFGF, as well as its receptors, are expressed inmany types of human tumors and tumor-derived cell lines in-cluding glioblastoma (29, 35), and a role for bFGF in neovascu-larization of these tumors has been suggested (36). Instead, theresults of our present studies suggest that bFGF does not play amajor role in tumor growth of the U87MG glioblastoma cells. Wedetected little bFGF in the growth media of the parental U87MG,the LacZ-, or antisense VEGF-expressing clones or tumorigenicrevertant 6-1/R cells, and these levels were unaltered in concertwith their respective tumorigenic and angiogenic behaviors. How-ever, we cannot rule out the possibility that the levels of bFGFexpressed by any of the cells are saturating with respect to asynergistic role with threshold levels of VEGF, or that other cellsmay use bFGF rather than VEGF as their major angiogenicstimulator.

In summary, we have demonstrated that suppression of en-dogenous VEGF expression and secretion of a human glioblas-toma cell line, U87MG, is sufficient to suppress its tumorigenicityin nude mice. In contrast, the parental U87MG and controlLacZ-expressing clones still retained their tumorigenic proper-ties. Immunohistochemical analysis revealed that expression ofVEGF proteins as well as blood vessel densities in these tumorscorrelated with the levels of VEGF production in both theparental U87MG and various clones. A revertant, which regainedhigh levels of VEGF expression in vitro, also regained tumorige-nicity accompanied by high expression of VEGF and increasedneovascularization in vivo. The ability of cells to overcome thesuppression ofVEGF expression by the antisense constructs maylimit the clinical utility of this approach. These results do,however, provide prima facie evidence that VEGF is the majorpositive angiogenic stimulus in these tumor cells. Support for thisconclusion has been recently gained in rat C6 cells which areanalogous to human gliomas but which also display substantialdifferences such as immunogenicity. These latter conclusionswere also limited by the potential of clonal variation (37).Nonetheless, these results, together with others modulating an-giogenesis with the inhibitors, angiostatin (6), thrombospondin (5),and antagonists for integrin acr33 (38), as well as tumor adminis-tration of amAb against VEGF (14) and dominant negative formsof the VEGF receptor flk-1 (15), strongly point to VEGF as anopportune and important target for anti-tumor therapy.

We would like to thank Dr. Sujay Singh (PharMingen) for purifyingbacterial expressed VEGF proteins and Dr. Judith Abraham (Scios-Nova, Inc., Mountain View, CA) for providing the bFGF probe.S.-Y.C. is a recipient of a Research Training Fellowship from theAmerican Lung Association of California. M.N. is a awardee of afellowship from YASUDA Medical Research Foundation, Osaka,

Japan. D.W. is a fellow of the Robert Steel Foundation for PediatricCancer Research. W.A. was supported by the Conselho Nacional dePesquisa/Brazilian National Research Council.

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