inhibition of fibroblast growth factor/fibroblast growth ... · binding of 125i-labeled fgf-2 and...

[CANCER RESEARCH 61, 1717–1726, February 15, 2001]

Inhibition of Fibroblast Growth Factor/Fibroblast Growth Factor Receptor Activityin Glioma Cells Impedes Tumor Growth by Both Angiogenesis-dependent and-independent Mechanisms1

Patrick Auguste,2 Demirkan B. Gursel,2 Sylvie Lemiere, Diana Reimers, Pedro Cuevas, Fernando Carceller,James P. Di Santo, and Andreas Bikfalvi3

Growth Factor and Cell Differentiation Laboratory, University Bordeaux I, 33 405 Talence, France [P. A., D. B. G., S. L., A. B.]; Department of Histology, Hopital Ramon YCajal, 28006 Madrid, Spain [D. R., P. C., F. C.]; and Unite des Cytokines et Developpement Lymphoide, Departement d’Immunologie, Institut Pasteur, 75 724 Paris, France[J. P. D. S.]

ABSTRACT

We undertook a series of systematic studies to address the role offibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR)activity in tumor growth and angiogenesis. We expressed dominant-neg-ative FGFR2 (FGFR2-DN) or FGFR1 (FGFR1-DN) in glioma C6 cells byusing constitutive or tetracycline-regulated expression systems. Anchor-age-dependent or independent growth was inhibited in FGFR-DN-expressing cells. Tumor development after xenografting FGFR-DN-expressing cells in immunodeficient mice or after transplantation in ratbrain was strongly inhibited. Quantification of microvessels demonstrateda significant decrease in vessel density in tumors derived from FGFR-DN-expressing cells. Furthermore, in a rabbit corneal assay, the angiogenicresponse after implantation of FGFR-DN-expressing cells was decreased.In tumors expressing FGFR-DN, vascular endothelial growth factor ex-pression was strongly inhibited as compared with control tumor. Theseresults indicate that inhibition of FGF activity may constitute a dominanttherapeutic strategy in the treatment of FGF-producing cerebral malig-nancies and may disrupt both angiogenesis-dependent and -independentsignals required for glioma growth and invasion.

INTRODUCTION

FGFs4 are a large family of regulatory molecules (1, 2). They havebeen demonstrated to stimulate growth, survival, and/or differentia-tion of a number of mesenchyme-derived cells such as fibroblasts,smooth muscle cells, epithelial cells, endothelial cells, and cellsderived from the nervous system.

FGFs interact with four prototypes of tyrosine kinase receptors (1).These include FGFR1 (flg), FGFR2 (bek), FGFR3, and FGFR4.These receptors have common features including a cytoplasmic con-served tyrosine kinase domain, a transmembrane domain, and anextracellular ligand binding domain, which may contain two or threeimmunoglobulin-like domains. A number of splice variants withinthese different receptor families have been described. Each member ofthe FGF family preferentially binds to specific receptor splice vari-ants. For instance, FGF-2 or FGF-4 preferentially associates with theIIIc variants, whereas FGF-7 binds to IIIb variants (1).

FGFs and their receptors are thought to be implicated in thedevelopment of a number of malignant tumors such as melanoma (3,4) or glioma (5). Morissonet al. (5) reported that glioma cell growth

can be inhibited by antisense oligonucleotides to FGF-2. Furthermore,Wang and Becker (4) demonstrated that antisense targeting of FGF-2or FGFR1 in human melanoma inhibits tumor growth. Moreover, aFGF-2 binding protein (FGF-BP) that mobilizes FGF-2 from theextracellular matrix was expressed after malignant progression incarcinoma. Depletion of human squamous cell carcinoma (SCC ME-180) and colon carcinoma (LS174T) cell lines of their endogenousFGF-BP by targeting with specific ribozymes leads to inhibition oftumor cell growthin vitro and in vivo (6). These results suggest thatFGF is implicated in tumor growthin vitro and in vivo. However,antisense strategies are questionable because of inhibition of bothnuclear and extracellular FGF isoforms. Furthermore, the strategiesdescribed above did not clearly answer the question whether FGFsignaling is implicated at the level of the tumor cell or at the level ofthe surrounding stroma, and whether FGF receptor deregulation hasany incidence on tumor-stroma cell interactions such as angiogenesis.Receptor expression has also been demonstrated to be modulatedduring malignant progression. Low-grade astrocytoma or normalwhite matter exhibit FGFR2 and only low levels of FGFR1. Malignantastrocytomas acquire FGFR1-b (2 immunoglobulin loop form) ex-pression and loose FGFR2 expression (7). This may also contribute tothe growth advantage of malignant cells.

We undertook a series of systematic studies to clearly evaluate theinvolvement of FGF/FGFR activity in glioma development. We choseto disrupt FGF activity at the cell surface level by using a FGFR-DNstrategy (8–10). Furthermore, we used a tetracycline-regulated ex-pression system to control FGFR2-DN and FGFR1-DNin vitro andinvivo. We herein present evidence that disruption of FGF activity leadsto the inhibition of glioma growth by both angiogenesis-dependentand -independent mechanisms and may therefore constitute a domi-nant strategy for the treatment of FGF-producing cerebral tumors.

MATERIALS AND METHODS

Cells. Rat C6 glioma cells (kindly donated by Dr. Paul Canioni, UniversityBordeaux II, Bordeaux, France) were grown in DMEM (Life Technologies,Cergy-Pontoise, France) containing 7.5% FCS (Life Technologies) and anti-biotics in a 5% CO2 atmosphere.

Transfection of C6 Glioma Cells. For stable constitutive expression ofFGFR2-DN in C6 glioma cells, C6 glioma cells were cotransfected with thepRK5 expression vector containing human FGFR2-DN cDNA (tyrosine kinasedomain deleted, 3 immunoglobulin-like loop form, IIIc splicing variant; kindlydonated by Dr. Joseph Schlessinger, Department of Pharmacology, New YorkUniversity Medical Center, New York, NY) and the pCEP4 vector containinga hygromycin-resistant gene at a ratio of 1:10 using Superfect (Qiagen,Courtaboeuf, France). Hygromycin B (400mg/ml) resistant cell clones wereselected, amplified, and tested for their FGFR2-DN expression by cross-linking 125I-labeled FGF-2 to cell surface FGFRs.

For the tetracycline-regulated expression system, human FGFR2-DN ormouse FGFR1-DN (tyrosine kinase domain deleted, 2 immunoglobulin-likeloop form, IIIc splice variant; kindly provided by Dr. L. Williams, Universityof California San Francisco, San Francisco, CA) cDNA were cloned at the

Received 7/28/00; accepted 12/13/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Association de la Recherche sur leCancer and the Ministere de la Science et de la Recherche (to A. B.).

2 These authors have contributed equally to this study.3 To whom requests for reprints should be addressed, at Growth Factor and Cell

Differentiation Laboratory, University Bordeaux I, Avenue des Facultes, 33 405 Talence,France.

4 The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; DN,dominant negative; CMV, cytomegalovirus; VEGF, vascular endothelial growth factor;VEGFR, VEGF receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDGF,platelet-derived growth factor.

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EcoRI or EcoRV site into tetracycline-regulated pTet Splice vector (LifeTechnologies). C6 glioma cells were then cotransfected with pTet Splicevector containing FGFR2-DN or FGFR1-DN cDNA, the pTet-tTAk vector(Life Technologies) containing the transactivator gene, and the pCEP4 vectorcontaining a hygromycin B-resistant gene using Superfect (Qiagen). Hygro-mycin-resistant cell clones were then selected with 400mg/ml hygromycin Band 1 mg/ml tetracycline or 50 ng/ml doxycycline. Resistant clones wereamplified and tested in the absence of tetracycline for their FGFR expressionby cross-linking125I-labeled FGF-2 to cell surface receptors.

Cell Proliferation Experiments. Proliferation assays were performed asdescribed (8). Briefly, cells were seeded at 3000 cells on 3.5-cm diameterdishes in complete DMEM containing 7.5% FCS, 1% glutamine, and antibi-otics. After overnight attachment, the cells were washed with serum-freeDMEM, and test medium containing 1% FCS was added. Cells were countedat specified days with a Coulter counter (Coultronics, Margency, France).

Soft Agar Assay. FGFR-DN-expressing cells or control cells (20,000)were put in DMEM containing 7.5% FCS and 0.2% agar (overlay) onto the topof an agar underlay (DMEM containing 7.5% FCS and 0.4% agar). Cells werefed twice a week with 1.5 ml of overlay, and the colonies (.5 cells) werecounted after 2 and 3 weeks. Twenty different fields were scored from eachwell by two independent investigators. The experiments were done in dupli-cate, and the results were expressed as mean6 SD from three independentexperiments.

Binding of 125I-Labeled FGF-2 and Cross-Linking to Receptors.FGF-2was labeled with125I-Na using iodogen (Pierce Corp., Rockford, IL) ascoupling agent according to the manufacturer’s indications and according toMoscatelliet al. (11). The specific activity of125I-labeled FGF-2 was 80,000–200,000 cpm/ng. FGF-2 binding experiments to high- and low-affinity siteswere performed essentially as described by Moscatelliet al. (11). Cross-linking experiments of125I-labeled FGF-2 to receptors were performed andanalyzed as described by Bikfalviet al. (8). The gels were dried and analyzedby a PhosphorImager equipped with Image Quant software (Molecular Dy-namics, Sunnyvale, CA).

Western Blotting. Cell or tissue extracts (100 or 150mg) were loaded ontoa 15% SDS-PAGE. After electrophoresis, proteins were transferred onto aHybond-S membrane (Amersham, les Ulis, France). Then membranes wereincubated with primary antibodies (polyclonal rabbit antihuman FGF-2 AB;polyclonal goat antihuman FGF-4 AB; Santa Cruz Biotechnology, Santa Cruz,CA), washed four times, and incubated with secondary antirabbit or goatantibodies coupled to peroxidase (Dako Corp., Trappes, France). The blotswere visualized using ECL (Amersham).

Xenografting of Tumor Cells in Immunodeficient Mice. Transfected orcontrol cells (500,000) were injected in DMEM s.c. in RAG 2/gc mice. Tumorgrowth was monitored over 27 days. Mice xenografts with Tet FGFR-DN cellswere fed every day with 2 mg/ml doxycycline in 5% sucrose (Tet1 clones) orwith 5% sucrose solution alone (Tet2clones). Mice xenografted with CMVFGFR2-DN or hygromycin-resistant control cells were fed with water alone.Ten animals were analyzed for each cell clone. Tumor measurements weremade in two directions using calipers, and tumor volume was calculated byusinga2 3 b/2, wherea is the width andb the length of the tumor. At the endof the experiment, animals were sacrificed, and tumor weight was determined.Tumor tissue was then processed for histology or immunohistology (seebelow).

Northern Blot Analysis. RNA (30 mg) extracted from tumors derivedfrom FGFR-DN or control cells was run on a 1% formaldehyde-agarose geland transferred to a positively charged nylon membrane (Amersham LifeTechnologies). After baking the membrane for 2 h at 80°C, the membrane wasprehybridized in a solution containing 50% formamide, 53SSPE, 53Den-hardt’s solution, 0.5% SDS and denatured salmon sperm DNA (100 ng/ml) for4 h at 42°C. Hybridization was done overnight at 42°C with Megaprimerandom-labeled (32a-ATP) probes (Amersham). The following probes wereused: 1100-bp truncated mouse FGFR1 or 1300-bp human FGFR2 probes,576-bp human VEGF165 full-length probe, or a full-length 1300-bp ratGAPDH probe. After hybridization, membranes were washed twice in 13SSPE, 0.1% SDS at 42°C for 20 min. For high stringency, a third wash wasperformed twice in 0.13SSPE, 0.1% SDS at 55°C for 10 min. The resultswere analyzed with a PhosphorImager and Image Quant software.

Implantation of Tumor Cells into the Rat Brain. A midline skin incisionwas made, and a small burr hole was drilled in the skull 3 mm lateral to the

bregma in anesthetized male Sprague Dawley rats (250–300 g). Animals wereplaced into a stereotaxic frame, and 2ml of cell suspension, containing 100,000C6 glioma cells, were injected into the right caudate-putamen placed at thefollowing stereotaxic coordinates from bregma (nose bar at15): ML, 3 mm;AP-, 0.2 mm; and DV, 5 mm. Groups of four rats were used for each type ofC6 glioma clone. Thirty days after surgery, animals were used intraaorticperfusion with 4% paraformaldehyde, and the whole brains were dissected andphotographed. Serial 1-mm transversal sections were cut, and tumor longitu-dinal extension was measured by adding serial sections where macroscopicalterations could be detected under a surgical microscope. Selected sectionswith tumor alterations displaying maximal transversal extensions were pro-cessed for paraffin embedding. Subsequently, 8-mm sections were cut andprocessed for both immunohistochemistry and histological analysis. StainedH&E sections were used to measure tumor maximal transversal surface, andimmunostained brain sections were used for the quantification of tumor an-giogenesis.

Histology and Immunohistochemistry. Paraffin-embedded tumor tissuewas cut into 5-mm sections, rehydrated, and processed for histology or immu-nohistochemistry. For labeling with anti-CD31 antibody, sections were alsopreincubated with 0.1% trypsin in PBS. Blocking was done for 2 h in PBScontaining 0.1% Tween 20 and 1 mg/ml BSA (buffer A). Slides were thenincubated with anti-CD31 antibody (MEC 13; Becton Dickinson, le Pont deClaix, France) at 1:500 dilution in buffer A. After washing, the slides wereincubated with biotinylated anti-rat antibody (Dako) in buffer A. Subsequently,the slides were washed again and incubated with ABC reagent (Vectastain;Valbiotech, Paris, France) and 3,39-diaminobenzidine (Dako). Counterstainingwas done with Harris hematoxylin.

For von Willebrand factor staining, 8-mm sections were rehydrated andboiled for 10 min in 10 mM citrate buffer (pH 6) in a microwave oven.Incubation with primary antibodies (rabbit polyclonal antihuman von Wille-brand factor antibody; Dako), secondary antibodies (biotinylated goat antirab-bit polyclonal antibody; Vectastain), and revelation were done as describedabove. Vessel density was quantified as described (12).

Rabbit Corneal Assay for Angiogenesis.Five ml of C6 cell suspensionwere injected into the cornea of New Zealand albino rabbit eyes using a 10-mlHamilton syringe. The injection was done 2 mm away from the limbal marginof the cornea. The potency of angiogenic activity was evaluated after 2 weeksas length of vessel extension (in mm) centrally from the limbus and as sectorialcircumferential (in clock hours) involvement (13). Ten rabbit eyes wereanalyzed for each condition.

RESULTS

Overexpression of FGFR2-DN or FGFR1-DN in Rat GliomaCells. To investigate the role of FGF/FGFR activity in tumor growthand angiogenesis, we transfected rat glioma C6 cells with expressionvectors containing cDNAs encoding FGFR2-DN or FGFR1-DN re-ceptors. Two expression systems were used. In the first case, we useda CMV promoter-driven expression vector (pRK5) cotransfected witha vector encoding a hygromycin-resistant gene (pCEP4). In the secondcase, we used a regulated system under the control of tetracycline ordoxycycline. In this system, FGFR-DN expression is activated in theabsence of tetracycline or doxycycline and turned off in the presenceof 1 mg/ml tetracycline or 50 ng/ml doxycycline (tetoffsystem). Anumber of clones were isolated and analyzed for expression ofFGFR2-DN or FGFR1-DN. Cells expressing FGFR2-DN under thecontrol of the CMV promoter are designated as CMV FGFR2-DNcells. Cells transfected with the tetracycline-regulated system aredesignated as Tet1FGFR2-DN or R1-DN cells when exposed totetracycline or doxycycline and as Tet2 FGFR2-DN or R1-DN cellsin the absence of tetracycline or doxycycline. The results from twoclones are shown for CMV FGFR2-DN cells (clones 18 and 2A7) andfrom two clones for Tet FGFR2-DN (clone 5A7 and 5B1) and TetFGFR1-DN (clones 4A8 and 4A11).

Fig. 1 shows the expression of FGFR2-DN or FGFR1-DN of anumber of clones examined. Cross-linking of125I-labeled FGF-2 to

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FGF, ANGIOGENESIS, AND BRAIN MALIGNANCIES



FGF-receptors (Fig. 1) revealed a classical pattern consisting of aband ofMr 90,000 for FGFR2-DN,Mr 60,000 for FGFR1-DN, andhigher molecular weight bands corresponding to multimeric com-plexes. CMV or tetracycline-regulated cell clones showed differentdegrees of FGFR2-DN or FGFR1-DN expression. No “leakiness” inFGFR2-DN or FGFR1-DN expression was detected in Tet1FGFR2-DN or Tet1FGFR1-DN cells in cross-linking experiments.

Effect of FGFR-DN Expression on Tumor Cell PhenotypesinVitro. We next investigated the effects of inhibition of FGF/FGFRactivity on the cell phenotype. We performed proliferation experi-ments with the different cell clones as indicated in “Materials andMethods.” CMV FGFR2-DN cells grew significantly slower thanuntransfected control cells (Fig. 2A). At day 8, the following values ofinhibition (mean6 SD) were observed: 77.9%6 0.03 (clone 18);74.8% 6 0.13 (clone 2A7). Similarly, Tet2 FGFR2-DN or Tet2FGFR1-DN were also growth inhibitedin vitro when compared withTet1 FGFR2-DN or Tet1FGFR1-DN (Fig. 2,C andD). At day 8,the following values of inhibition (mean6 SD) were observed forTet2 cells: 64.3% 6 0.09 (Tet2 FGFR2-DN; clone 5A7),56.5% 6 3.8 (Tet2 FGFR2-DN; clone 5B1), 34.2%6 3.4 (Tet2FGFR1-DN; clone 4A8), and 47.9%6 0.7 (Tet2FGFR1-DN; clone4A11). Empty pTet Splice vector transfected cells grew similarly inthe presence and absence of doxycycline (Fig. 2B). These resultsindicate that cell proliferation is inhibited in FGFR-DN-expressing C6glioma cells.

Furthermore, anchorage-independent growth was inhibited in CMVFGFR2-DN, Tet2FGFR2-DN or Tet2FGFR1-DN cells (Fig. 3).The following values (mean6 SD) of inhibition in comparison tocontrol were observed in the different clones: 19.8%6 0.4 (CMVFGFR2-DN; clone 3B8), 26.7%6 1 (CMV FGFR2-DN; clone 2A7),58.7% 6 3.3 (CMV FGFR2-DN; clone 18), 98.4%6 1.2 (Tet2FGR2-DN; clone 5A7), 96.7%6 0.6 (Tet2FGFR2-DN; clone 5B1),64.1%6 7.7 (Tet2FGFR1-DN; clone 4A8), and 60.6%6 9.6 (Tet2FGFR1-DN; clone 4A11).

Fig. 2. Effect of FGFR2-DN or FGFR1-DN expression on the proliferation of C6 cells.Proliferation experiments were carried out as indicated in “Materials and Methods.”A,CMV FGFR2-DN (f, clone 18;Œ, clone 2A7) or control cells (l, clone BH2).B, emptypTet Splice control cells, clone CA8 (l, with doxycycline;f, without doxycycline).C,Tet FGFR2-DN cells, clone 5A7 (l, with doxycycline;f, without doxycycline).D, TetFGFR1-DN cells, clone 4A11 (l, with doxycycline;f, without doxycycline). A repre-sentative experiment is shown.Bars, SD; significant difference was based on theMANOVA test, followed by a test of Tukey.pp, P , 0.01;p, P , 0.05versuscontrol.

Fig. 1.A andB, expression of FGFR2-DN or FGFR1-DN in C6 tumor cells. Binding studies and cross-linking experiments with125I-labeled FGF-2 performed in CMV FGFR2-DN,Tet FGFR2-DN, Tet FGFR1-DN, or control (Hygro) cells were as indicated in “Materials and Methods.”A, CMV FGFR2-DN cells (clones 18 and 2A7) in comparison withhygromycin-resistant control cells (clone BH2).B, Tet FGFR2-DN cells (clones 5A7 and 5B1) in the presence (1) or absence (2) of tetracycline, Tet FGFR1-DN cells (clones 4A8and 4A11) in the presence (1) or absence (2) of tetracycline, and empty pTet Splice vector transfected control cells (clone CA8) in the presence (1) or absence (2) of tetracycline.

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Effect of the Inhibition of FGF/FGFR Activity in Mice Injectedwith Tumor Cells. We then investigated the growth of the differentcell clonesin vivo in mice (Fig. 4). The different clones were xeno-grafted into RAG 2/gc mice, and tumor growth was measured for 27days. RAG 2/gc mice are an alymphoid mouse strain lacking T, B, andnatural killer cells (14). Initial experiments have shown that tumortake and growth were similar in nude and RAG 2/gc mice (data notshown). Control cells transfected with the hygromycin-resistant gene(Hygro cells) alone or Tet1FGFR2-DN or R1-DN cells grew activelywhen xenografted in immunodeficient mice.

CMV FGFR2-DN cells grew much slower than Hygro cells andwere growth inhibited by 60–80% (Fig. 4A and Table 1). Further-more, Tet2FGFR2-DN (clone 5A7) and Tet2FGFR1-DN (clone4A11) are also strongly inhibited in comparison with control (60–80% of inhibition; Fig. 4,C andD). The stronger inhibitory effectinvivoobserved for tumors derived from FGFR2-DN-expressing cells incomparison with tumors derived from FGFR1-DN cells correlatedwith higher FGFR-DN expression levels observedin vitro. At day 27,tumor size was significantly smaller in tumors derived from FGFR-DN-expressing cells in comparison with control (Fig. 5; Table 1). Inaddition, tumor weight was decreased in CMV FGFR2-DN, Tet2FGFR2-DN or Tet2FGFR1-DN by 50–77% in comparison withcontrol (Table 1).

Quantification of FGFR expression in tumors by Northern blottingshowed that FGFR2-DN and R1-DN mRNA were expressed in all ofthe tumors derived from FGFR-DN-expressing cells (Fig. 6). Further-more, FGFR-DN mRNA expression was regulated in tumors derived

from Tet FGFR-DN-expressing cells when mice were fed with doxy-cycline. In addition, only endogenous FGFR1 but not FGFR2 ispresent in tumors derived from FGFR-DN-expressing cells, as shownfor human gliomas (7).

Effect of Inhibition of FGF/FGFR Activity after IntracerebralTransplantation of FGFR-DN-expressing Cells.We next evaluatedtumors generated after intracranial transplantation of transfected cellsin Sprague Dawley rats (Fig. 7 and Table 2). In the control groups(rats with control C6 glioma cell implantations), the longitudinalextension of macroscopic alterations in the right brain hemisphere(column 2 of Table 2) ranged from 3 to 5 mm. In three animals, theseaffected areas comprised the whole extension of the caudate putamennucleus and an important part of the thalamic nuclei (controls 1, 2, and

Fig. 3. Effect of FGFR2-DN or FGFR1-DN expression on growth in soft agar of C6cells. FGFR2-DN, FGFR1-DN, or control cells were plated at 20,000 cells/dish in 0.2%of agar overlay, and experiments were performed as indicated in “Materials and Meth-ods.” Colonies (.5 cells) were counted in 20 different fields by two different investiga-tors. The results are expressed as means from experiments done in duplicates;bars,SD.A, CMV FGFR2-DN-expressing cells (clones 3B8, 2A7, and 18) or control cells (cloneBH3). B, Tet FGFR2-DN (clones 5A7 and 5B1), Tet FGFR1-DN cells (clones 4A8 and4A11), or empty pTet Splice vector-transfected cells (clone CA8).f, with doxycycline;M, without doxycycline. Significant difference was based on Student’st test. pp,P , 0.01; p, P , 0.05versuscontrol.

Fig. 4. Growth of tumors derived from s.c.-implanted, FGFR-DN-expressing cells orcontrol cells in immunodeficient mice. Cells expressing FGFR2-DN or control cells wereimplanted s.c. in RAG 2/gc mice, and tumor size was measured twice a week for 4 weeksas indicated in “Materials and Methods.”A, tumors derived from CMV FGFR2-DN cells(F, clone 3B8;Œ, clone 2A7;f, clone 18) or control cells (l, clone BH2).B, tumorsderived from empty pTet Splice vector-transfected control cells, clone CA9 (l, withdoxycycline;f, without doxycycline).C, tumors derived from Tet FGFR2-DN cells,clone 5A7 (l, with doxycycline;f, without doxycycline).D, tumors derived from TetFGFR1-DN cells, clone 4A11 (l, with doxycycline;f, without doxycycline). Significantdifference was based on Student’st test at days 24 and 27 for CMV FGFR-DN-expressingtumors and at days 22, 26, and 27 for Tet FGFR-DN-expressing tumors.pp, P , 0.01;p,P , 0.05versuscontrol (pp for clone 18 at day 27).

Table 1 Reduction of volume and weight of tumors derived from FGFR-DN-expressingcells

Tumor volume and weight were determined at day 27 as indicated in “Materials andMethods.” The results are expressed in percentages (mean6 SD) of inhibition incomparison with control. Ten animals were used for each condition.

Clone Volume Weight

CMV FGFR2-DN 18 74%6 10.1 60%6 22.82A7 71.6%6 7 59.8%6 193B8 61.6%6 21.4 72%6 12.3

Tet 2 FGFR2-DN 5A7 79%6 2 76.6%6 2.1Tet 2 FGFR1-DN 4A11 65%6 31 51.9%6 19

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3), and they displaced other ipsilateral and contralateral brain struc-tures (Fig. 7A). In another rat (control 4), only the caudate-putamenwas affected, and the thalamus was spared.

In rats implanted with clone 18 and clone 2A7, the longitudinalextension of macroscopic alterations was significantly smaller than inthe control group, ranging from 1 to 2.5 mm and 1 to 1.5, respectively(Fig. 7, B andC; Table 2). One of the animals implanted with clone2A7 was discarded, because the brain showed a tumor formation onthe parietal cortex, because of glioma cell spreading after implanta-tion. Another animal of the same group did not show any morpho-logical alteration in the right brain hemisphere. In most animals, onlysome areas of the caudate-putamen and/or thalamic nuclei were af-fected, and contralateral brain structures were less displaced than inthe control group.

After histological staining of coronal brain sections, we measuredthe maximal transversal surface of these alterations (column 3 ofTable 2), which varied between 9.5 and 23.75 mm2 in the controlgroup. However, in rats implanted with clone 18 and clone 2A7,maximal transversal surfaces were in all cases notably smaller than in

control groups, varying between 3 and 8 mm2 in rats implanted withclone 18 and from 0 to 5.9 mm2 in rats implanted with clone 2A7.Hemorrhagic areas were not macroscopically evident in tumors de-veloping after implantations of clones 18 and 2A7, except for smallfoci seen in two animals. As shown in column 4 of Table 2, theaffected brain regions were completely replaced by glioma cells intwo control animals. In the other two control rats, glioma cells wereonly detected in the periphery of tumors surrounding wide areas withnecrotic brain tissue (controls 2 and 4). One brain showed severallarge tumor-associated hemorrhagic areas, whereas another tumor hadrelatively small hemorrhagic foci.

Effect of the Expression of FGFR-DN on the Angiogenic Re-sponse.We first analyzed vessel distribution and density by immu-nohistochemistry in mice with tumors from control, CMV FGFR2-DN, Tet1 FGFR2-DN or Tet1FGFR1-DN cells using anti-CD31antibodies (Fig. 8). In tumors derived from s.c. implanted control,Tet1FGFR2-DN or Tet1FGFR1-DN cells in RAG 2/gc mice, manyvessels of different sizes were visible at the tumor margins. In con-trast, tumors derived from CMV FGFR2-DN, Tet2 FGFR2-DN or

Fig. 5. Macroscopic analysis of tumors derived from s.c. implantedFGFR-DN or control cells in RAG 2/gc mice. FGFR-DN-expressingcells or control cells were injected s.c. in RAG 2/gc mice, and tumorswere analyzed at day 27.A: Right panel,CMV FGFR2-DN (clone2A7); left panel,control (BH2).B: Tet FGFR2-DN (clone 5A7).Rightpanel, without doxycycline; left panel, with doxycycline. C, Tet-FGFR1-DN (clone 4A11).Right panel, without doxycycline; leftpanel,with doxycycline.

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Tet2 FGFR1-DN cells xenografted into immunodeficient mice ex-hibited much fewer blood vessels than control tumors. Quantificationof CD31-positive cells demonstrated an inhibition of 34%6 3.8(clone 18) and 41%6 3.2 (clone 2A7) for tumors derived from CMVFGFR2-DN cells (Fig. 8A). Furthermore, tumors derived from Tet2FGFR2-DN (clone 5A7) or FGFR1-DN (clone 4A11) demonstratedan inhibition of 83%6 3.4 and 41%6 3.5, respectively (Fig. 8,C andD). No differences in vessel density was observed for control Tetclones in the presence or absence of doxycycline (Fig. 8B). Vesseldensity was also analyzed in intracranially implanted tumors usinganti-von Willebrand antibody staining instead of anti-CD31 becauseof high background staining. In this case, labeling was also signifi-

cantly decreased with inhibition values of 69.4%6 10.9 (CMVFGFR2-DN; clone 18) and 67.8%6 6.6 (CMV FGFR2-DN; clone2A7; Fig. 8E).

Cells expressing FGFR2-DN or control cells were also implantedinto the rabbit cornea. Control cells induced a strong angiogenicresponse (Fig. 9,A andC). Cells expressing FGFR2-DN (clone 2A7)showed a marked decrease in the induction of blood vessel growth(Fig. 9, B andC).

Effect of FGFR-DN on the Expression of VEGF, FGF-2, orFGF-4 in Tumor Cells. To get insight into the potential factorsdown-regulated in tumors derived from FGFR-DN expressing cells,we analyzed VEGF, FGF-2, or FGF-4 transcripts or proteins. In

Fig. 6. Northern blot analysis of FGFR-DN-expressing cells. RNA was extracted from tumors derived from FGFR-DN-expressing cells or control cells, and Northern blotting wasperformed as indicated in “Materials and Methods.” For tumors derived from Tet FGFR-DN cells, animals were fed with (1) or without (2) doxycycline, and total RNA was extractedfrom the tumors at day 27.A, hybridization with FGFR2 probe;B, hybridization with FGFR1 probe.

Fig. 7. Intracranial transplantation of FGFR-DN cells and controlcells. FGFR2-DN (B,clone 18;C, clone 2A7) or control cells (A,clone BH3) were injected into the caudate nucleus of Wistar rats asindicated in “Materials and Methods.”Arrows,sites of tumor devel-opment after 30 days of cell implantation.34.

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tumors derived from CMV FGFR2-DN cells, VEGF mRNA expres-sion was reduced by 52–65% (Fig. 10A). In tumors derived fromTet2 FGFR2-DN cells or Tet2FGFR1-DN cells, inhibition valuesfor VEGF mRNA expression of 47 and 64.2% were observed (Fig.10B). In contrast, FGF-2 and FGF-4 expression was not modulated intumors derived from FGFR-DN-expressing cells when expression wasanalyzed by Northern or Western blotting (data not shown).

Taken together, these results indicate that cells expressingFGFR-DN exhibit a significant decrease in their angiogenic responseand strongly support the involvement of FGF ligands such as FGF-2or FGF-4 in tumor angiogenesis. The effect of FGF on tumor angio-genesis seems to be, at least partially, indirect and involves VEGF,because inhibition of FGF activity in tumors derived from FGFR-DN-expressing cells down-regulates VEGF expressionin vivo.

DISCUSSION

To identify roles for FGF/FGFR activity in glial tumor develop-ment, we took advantage of the DN receptor strategy. Tyrosine kinasedomain-deleted FGFR-DNs dimerize with endogenous receptors andthus inhibit FGF signaling (15). To ascertain that the phenotypes weobserved are not related to clonal variation but to the expression of theFGFR-DNs, we used both constitutive and tetracycline-regulated ex-pression vector systems. The tetracycline-regulated expression systemhas been demonstrated to be useful forin vitro and in vivo control ofgene expression (16). A number of cell clones with constitutive andtetracycline-regulated expression were isolated that exhibited differ-ent FGFR2-DN or FGFR1-DN expression levels. In the presence oftetracycline, no leakiness of FGFR2-DN or FGFR1-DN expressionwas observed in cells with tetracycline-regulated expression. FGFR2-DN-expressing cells exhibited higher FGFR-DN amounts thanFGFR-DN expressing cells, and this occurred in cells transfected withCMV or tetracycline-regulated expression systems. The reasons forthese differences are not known.

C6 rat glioma cells express high molecular weight (nuclear,Mr

20,000 andMr 21,000) and low molecular weight FGF (cytosolic andextracellular,Mr 18,000) isoforms (17). Time course experimentsmeasuring cell proliferation in 1% FCS revealed an inhibitory effectin FGFR-DN-expressing cells. This indicates that extracellular FGF isinvolved in the autonomous proliferation of glioma cells. In contrastto these observations, it has been shown previously in NIH 3T3 cellsthat low serum growth is induced by intracellular high molecularweight FGF-2 forms but not byMr 18,000 FGF-2 (8). In addition,supertransfection of FGFR-DN receptors does not inhibit low serumgrowth in NIH 3T3 cells expressing all FGF-2 isoforms. The reasonfor these differences is not understood but may reside in the different

cell types used in these studies (transformed cells in this study andimmortalized cells in the previous study).

Anchorage-independent growth was inhibited in cells expressingFGFR2-DN or FGFR1-DN. This indicates that some of the propertiesrelated to cell transformation are inhibited by the expression ofFGFR-DN in tumor cells. This may include effects on integrins orprotease production that are involved in interactions with the extra-cellular matrix. This suggests that signaling through FGFRs modu-lates adhesive interactions with the extracellular matrix. A number ofobservations are in support of this contention. FGF-2 modulatesintegrin expression and adhesion to the extracellular matrix in endo-thelial cells (18, 19). Furthermore, endogenousMr 18,000 FGF-2 hasbeen demonstrated to interfere with the expression and function ofa4b1 anda5b1 integrins in NIH 3T3 cells (20).

In vivo growth was strongly impaired when cells with constitutiveor tetracycline-regulated FGFR2-DN or FGFR1-DN expression werexenografted in immunodeficient mice. This demonstrates that FGFsare important for glioma developmentin vivo. This is in agreementwith Yayon et al. (21), who also demonstrated the participation ofautocrine FGF in melanoma cell growth. The strong inhibitionin vivoobserved for cells expressing FGFR2-DN or FGFR1-DN under thecontrol of tetracycline or doxycycline clearly demonstrates again that

Fig. 8. Effect of FGFR-DN expression in C6 cells on the angiogenic phenotype intumors: quantification of vessel density. Tumors derived from s.c.-injected CMV FGFR2-DN, Tet FGFR2-DN, Tet FGFR1-DN, or control cells (A–D) in RAG 2/gc mice wereanalyzed by immunohistochemistry using anti-CD31 antibodies.A, tumors derived fromCMV FGFR2-DN cells (clone 2A7 and clone 18) or control (clone BH2).B, tumorderived from Tet control cells, clone CA9, with or without doxycycline.C, tumors derivedfrom Tet FGFR2-DN cells, clone 5A7 with or without doxycycline.D, tumors derivedfrom Tet FGFR1-DN, clone 4A11, with or without doxycycline. Five fields were analyzedfor each cell clone.3400. E, immunostaining of von Willebrand factor in intracerebraltumors derived from FGFR2-DN-expressing cells or control cells. Control (clone BH3);tumors derived from CMV FGFR2-DN cells (clone 18; clone 2A7) are shown. Five fieldswere analyzed for each cell clone.3400. The data are presented as means;bars, SD.Significant difference was based on Student’st test.pp, P , 0.01; p, P , 0.05 versuscontrol.

Table 2 Quantitative analysis of tumors derived from FGFR2-DN cells transplanted inthe rat brain

The experiments were performed as indicated in “Materials and Methods.”

RatsLongitudinal

extension (mm)Maximal transversal

surface(mm2)Replacement by glioma

cellsa

Control 1 3.5 20 111Control 2 5 21 11Control 3 5 23.75 111Control 4 3 9.5 11Clone 18, rat 1 2.5 7 11Clone 18, rat 2 1.5 7.9 1Clone 18, rat 3 1 3.14 1Clone 18, rat 4 1 2.9 111Clone 2A7, rat 1 0 0 0Clone 2A7, rat 2 1 5.9 111Clone 2A7, rat 3 1.5 0.8 111

a Complete (111), moderate (11), and scarce (1) glioma replacement of alteredbrain regions as visualized in histological cross-sections.

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this is not attributable to clonal variation but to a true DN effect.Inhibition was stronger to some extent for FGFR2-DN-expressingcells than for FGFR1-DN-expressing cells. This is most certainlybecause of higher FGFR-DN expression levels achieved in FGFR2-DN-expressing cells as shownin vitro. The angiogenic response wasfound to be significantly inhibited in FGFR-DN-expressing cells. Toexplain our results, different possibilities may be considered: (a) theexpression of FGF-2 or of another FGF family member is down-regulated in cells expressing FGFR-DN; (b) FGFR-DN may inhibitFGF-2 release into the extracellular compartment; (c) FGFR-DN mayimmobilize FGF at the cell surface, thereby sequestering extracellularFGF; (d) FGFR-DN could inhibit expression of another angiogenicfactor, such as VEGF. We herein demonstrate for the first time thatinhibition of endogenous FGF/FGFR activity leads to down-regula-tion of VEGF expression in tumors. This strongly supports the con-tention that FGF/FGFRs are inducing angiogenesis in tumors by anindirect mechanism that is mediated via VEGF. This is in agreementwith the observations of Seghezziet al. (22), who reported thatexogenous FGF-2 induces VEGF expression in endothelial cells. Inaddition, FGFR-DN may not only impede the activity of angiogenicfactors at a transcriptional level but may instead inhibit the mobiliza-tion of angiogenic growth factors from the tumor cell or extracellularmatrix to the endothelium by inhibiting the expression or activation ofproteases such as matrix metalloproteinases or plasminogen activa-tors. This possibility is currently being investigated. Inhibition of FGFactivity in human glioblastomas also down-regulates VEGF expres-

sion and inhibits tumor growth.5 This indicates that our results have amore general significance and also apply to human tumors. In the lightof the results described herein, endogenous FGF may be part of theangiogenic switch in glial tumors, allowing tumors to progress fromdormancy to invasiveness.

The implication of FGF/FGFR activity in angiogenesis has been amatter of debate. Knocking-out the gene for FGF family members,such asFGF-1, FGF-2,or FGF-8,did not reveal defects in embryonicvascular development (23–25). Furthermore, disruption of some of theFGFR family members is early lethal (26). This does not allow theanalysis of the embryonic vasculature. We have recently generatedtransgenic mice with targeted expression of FGFR1-DN in the retinalpigmented epithelium (27). These mice display a severe defect in thedevelopment of choroidal blood vessels. This observation stronglyindicates that FGF ligands are important in vascular developmentalprocesses. The present data support an involvement of FGF in theangiogenic response in tumors. This is further supported by Fuldhamet al. (28), who recently demonstrated un impairment in the angio-genic response in aFGF-2 transgenic mice. Taken together, theseobservations indicate that FGF may play a significant role in bothdevelopmental and repair-associated angiogenic processes.

The C6 rat glioma cells that we used as a model in our studiesexpress a number of growth factor or growth factor receptors otherthan FGF ligands or FGFRs. These include VEGF and VEGFRs

5 Unpublished results.

Fig. 9. Effect of FGFR2-DN expression in C6 cells on theangiogenic response in the rabbit cornea assay. Cells expressingFGFR2-DN (clone 2A7) or hygromycin-resistant control cells(clone BH3) were implanted into the rabbit cornea. Corneal neo-vascularization was assessed as the length of the vessel extensioncentrally from the limbus and the sectorial circumferential involve-ment. A, control (clone BH3).B, FGFR2-DN-expressing cells(clone 2A7).C, quantification of vessel density (mean values;bars,SD. Ten eyes were analyzed for each condition.

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(29–31), interleukin 6 (32, 33), tumor necrosis factor-a (32), glial cellline-derived neurotrophic factor (34), epidermal growth factor recep-tors (35), or PDGF receptors (36). Overexpression of VEGF in C6glioma cells has shown that this factor is critical for the developmentand maintenance of the tumor vasculature. Benjamin and Keshet (37)have used the tetracycline-regulated expression system to show thatVEGF has an important role as a survival factor for tumor vesselsinvivo. Blocking experiments have contributed to understand the in-volvement of different receptors or ligands in tumor growth. Plateetal. (29) and Millaueret al.(30) have shown that tumor growth in nudemice xenografted with C6 glioma cells was markedly inhibited whenVEGFR2-DN was retrovirally targeted into animals. Furthermore,VEGFR2-DN directly expressed on tumor cells was able to inhibittumor growth and angiogenesis (38). The inhibitory effect ofVEGFR2-DN on tumor angiogenesis may be explained by adsorptionof extracellular ligands. Strawnet al. (36) introduced a truncatedPDGF-b receptor into C6 rat glioma cells and showed that PDGF-BB-induced tyrosine phosphorylation of the endogenous receptor wassignificantly reduced. In addition, these cells were growth inhibitedinvitro and grew slower when xenografted in nude mice. This indicatesthat multiple growth factors are critical for glioma growth that may actdirectly on tumor cells and/or at the level of the tumor environment.

In summary, our data clearly demonstrate that FGF/FGFR activityis involved in glioma growthin vitro and in vivo. A therapeuticstrategy based on the inhibition of FGFR function may be useful forthe treatment of solid FGF-producing tumors, such as gliomas, andmay disrupt both angiogenesis-dependent and -independent signaling.This is similar to the effect of maspin, a recently discovered angio-genesis inhibitor, which also directly impedes tumor growth in addi-tion to its effect on angiogenesis (39). Thus, inhibition of the FGFsignal transduction pathway may constitute an interesting target fortherapeutic intervention.

ACKNOWLEDGMENTS

We thank Xavier Canron (Growth Factor and Cell Differentiation Labora-tory) for helpful technical assistance, Mylene Cibenel (Growth Factor and CellDifferentiation Laboratory) for preliminary work, Dr. Herve Prats (INSERM U

344, Toulouse, France) for providing recombinant human FGF-2, Dr. JosephSchlessinger (Department of Pharmacology, New York University MedicalCenter, New York, NY) for providing human FGFR2-DN, Dr. Lewis Williams(University of California San Francisco, San Francisco, CA) for providingmouse FGFR1-DN, Dr. Paul Canioni (Universite Bordeaux II) for providingC6 glioma cells, Pierre Costet (Unite de Transgenose, Universite Bordeaux II)for helpful assistance, Drs. Sophie Javerzat and Ijsbrand Kramer (GrowthFactor and Differentiation Laboratory) for critical reading of the manuscript,and Dr. P. Mora (Laboratoire de Physico-Toxico Chimie des Systemes Na-turels, Universite Bordeaux I) for helpful advice in statistical analysis.

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2001;61:1717-1726. Cancer Res Patrick Auguste, Demirkan B. Gürsel, Sylvie Lemière, et al. MechanismsGrowth by Both Angiogenesis-dependent and -independentFactor Receptor Activity in Glioma Cells Impedes Tumor Inhibition of Fibroblast Growth Factor/Fibroblast Growth

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