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Tumor Biology |
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 Y Cajal, 28006 Madrid, Spain [D. R., P. C., F. C.]; and Unité des Cytokines et Développement Lymphoide, Departement d’Immunologie, Institut Pasteur, 75 724 Paris, France [J. P. D. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 conserved tyrosine kinase domain, a transmembrane domain, and an extracellular ligand binding domain, which may contain two or three immunoglobulin-like domains. A number of splice variants within these different receptor families have been described. Each member of the FGF family preferentially binds to specific receptor splice variants. For instance, FGF-2 or FGF-4 preferentially associates with the IIIc variants, whereas FGF-7 binds to IIIb variants (1) .
FGFs and their receptors are thought to be implicated in the development of a number of malignant tumors such as melanoma (3 , 4) or glioma (5) . Morisson et 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-2 or FGFR1 in human melanoma inhibits tumor growth. Moreover, a FGF-2 binding protein (FGF-BP) that mobilizes FGF-2 from the extracellular matrix was expressed after malignant progression in carcinoma. Depletion of human squamous cell carcinoma (SCC ME-180) and colon carcinoma (LS174T) cell lines of their endogenous FGF-BP by targeting with specific ribozymes leads to inhibition of tumor cell growth in vitro and in vivo (6) . These results suggest that FGF is implicated in tumor growth in vitro and in vivo. However, antisense strategies are questionable because of inhibition of both nuclear and extracellular FGF isoforms. Furthermore, the strategies described above did not clearly answer the question whether FGF signaling is implicated at the level of the tumor cell or at the level of the surrounding stroma, and whether FGF receptor deregulation has any incidence on tumor-stroma cell interactions such as angiogenesis. Receptor expression has also been demonstrated to be modulated during malignant progression. Low-grade astrocytoma or normal white matter exhibit FGFR2 and only low levels of FGFR1. Malignant astrocytomas acquire FGFR1-ß (2 immunoglobulin loop form) expression and loose FGFR2 expression (7) . This may also contribute to the growth advantage of malignant cells.
We undertook a series of systematic studies to clearly evaluate the involvement of FGF/FGFR activity in glioma development. We chose to disrupt FGF activity at the cell surface level by using a FGFR-DN strategy (8, 9, 10) . Furthermore, we used a tetracycline-regulated expression system to control FGFR2-DN and FGFR1-DN in vitro and in vivo. We herein present evidence that disruption of FGF activity leads to the inhibition of glioma growth by both angiogenesis-dependent and -independent mechanisms and may therefore constitute a dominant strategy for the treatment of FGF-producing cerebral tumors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transfection of C6 Glioma Cells.
For stable constitutive expression of FGFR2-DN in C6 glioma cells, C6
glioma cells were cotransfected with the pRK5 expression vector
containing human FGFR2-DN cDNA (tyrosine kinase domain deleted, 3
immunoglobulin-like loop form, IIIc splicing variant; kindly donated by
Dr. Joseph Schlessinger, Department of Pharmacology, New York
University Medical Center, New York, NY) and the pCEP4 vector
containing a hygromycin-resistant gene at a ratio of 1:10 using
Superfect (Qiagen, Courtaboeuf, France). Hygromycin B (400 µg/ml)
resistant cell clones were selected, 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 or mouse FGFR1-DN (tyrosine kinase domain deleted, 2 immunoglobulin-like loop form, IIIc splice variant; kindly provided by Dr. L. Williams, University of California San Francisco, San Francisco, CA) cDNA were cloned at the EcoRI or EcoRV site into tetracycline-regulated pTet Splice vector (Life Technologies). C6 glioma cells were then cotransfected with pTet Splice vector containing FGFR2-DN or FGFR1-DN cDNA, the pTet-tTAk vector (Life Technologies) containing the transactivator gene, and the pCEP4 vector containing a hygromycin B-resistant gene using Superfect (Qiagen). Hygromycin-resistant cell clones were then selected with 400 µg/ml hygromycin B and 1 µg/ml tetracycline or 50 ng/ml doxycycline. Resistant clones were amplified and tested in the absence of tetracycline for their FGFR expression by cross-linking 125I-labeled FGF-2 to cell surface receptors.
Cell Proliferation Experiments.
Proliferation assays were performed as described (8)
.
Briefly, cells were seeded at 3000 cells on 3.5-cm diameter dishes in
complete DMEM containing 7.5% FCS, 1% glutamine, and antibiotics.
After overnight attachment, the cells were washed with serum-free DMEM,
and test medium containing 1% FCS was added. Cells were counted at
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 top of an agar
underlay (DMEM containing 7.5% FCS and 0.4% agar). Cells were fed
twice a week with 1.5 ml of overlay, and the colonies (>5 cells) were
counted after 2 and 3 weeks. Twenty different fields were scored from
each well by two independent investigators. The experiments were done
in duplicate, and the results were expressed as mean ± SD from three independent experiments.
Binding of 125I-Labeled FGF-2 and Cross-Linking to
Receptors.
FGF-2 was labeled with 125I-Na using iodogen
(Pierce Corp., Rockford, IL) as coupling agent according to the
manufacturer’s indications and according to Moscatelli et
al. (11)
. The specific activity of
125I-labeled FGF-2 was 80,000–200,000 cpm/ng.
FGF-2 binding experiments to high- and low-affinity sites were
performed essentially as described by Moscatelli et al.
(11)
. Cross-linking experiments of
125I-labeled FGF-2 to receptors were performed
and analyzed as described by Bikfalvi et al.
(8)
. The gels were dried and analyzed by a PhosphorImager
equipped with Image Quant software (Molecular Dynamics, Sunnyvale, CA).
Western Blotting.
Cell or tissue extracts (100 or 150 µg) were loaded onto a 15%
SDS-PAGE. After electrophoresis, proteins were transferred onto a
Hybond-S membrane (Amersham, les Ulis, France). Then membranes were
incubated 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 goat antibodies coupled to peroxidase (Dako Corp., Trappes, France).
The blots were visualized using ECL (Amersham).
Xenografting of Tumor Cells in Immunodeficient Mice.
Transfected or control cells (500,000) were injected in DMEM s.c. in
RAG 2/
c mice. Tumor growth was monitored over
27 days. Mice xenografts with Tet FGFR-DN cells were fed every day with
2 mg/ml doxycycline in 5% sucrose (Tet+ clones) or with 5% sucrose
solution alone (Tet- clones). Mice xenografted with CMV FGFR2-DN or
hygromycin-resistant control cells were fed with water alone. Ten
animals were analyzed for each cell clone. Tumor measurements were made
in two directions using calipers, and tumor volume was calculated by
using a2 x b/2,
where a is the width and b the length of the
tumor. At the end of the experiment, animals were sacrificed, and tumor
weight was determined. Tumor tissue was then processed for histology or
immunohistology (see below).
Northern Blot Analysis.
RNA (30 µg) extracted from tumors derived from FGFR-DN or control
cells was run on a 1% formaldehyde-agarose gel and transferred to a
positively charged nylon membrane (Amersham Life Technologies). After
baking the membrane for 2 h at 80°C, the membrane was
prehybridized in a solution containing 50% formamide, 5x SSPE, 5x
Denhardt’s solution, 0.5% SDS and denatured salmon sperm DNA (100
ng/ml) for 4 h at 42°C. Hybridization was done overnight at
42°C with Megaprime random-labeled (32
-ATP)
probes (Amersham). The following probes were used: 1100-bp truncated
mouse FGFR1 or 1300-bp human FGFR2 probes, 576-bp human
VEGF165 full-length probe, or a full-length
1300-bp rat GAPDH probe. After hybridization, membranes were washed
twice in 1x SSPE, 0.1% SDS at 42°C for 20 min. For high stringency,
a third wash was performed twice in 0.1x SSPE, 0.1% SDS at 55°C for
10 min. The results were analyzed with a PhosphorImager and Image Quant
software.
Implantation of Tumor Cells into the Rat Brain.
A midline skin incision was 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 were placed into a stereotaxic frame,
and 2 µl of cell suspension, containing 100,000 C6 glioma cells, were
injected into the right caudate-putamen placed at the following
stereotaxic coordinates from bregma (nose bar at +5): ML, 3 mm; AP-,
0.2 mm; and DV, 5 mm. Groups of four rats were used for each type of C6
glioma clone. Thirty days after surgery, animals were used intraaortic
perfusion with 4% paraformaldehyde, and the whole brains were
dissected and photographed. Serial 1-mm transversal sections were cut,
and tumor longitudinal extension was measured by adding serial sections
where macroscopic alterations could be detected under a surgical
microscope. Selected sections with tumor alterations displaying maximal
transversal extensions were processed for paraffin embedding.
Subsequently, 8-µm sections were cut and processed for both
immunohistochemistry and histological analysis. Stained H&E sections
were used to measure tumor maximal transversal surface, and
immunostained brain sections were used for the quantification of tumor
angiogenesis.
Histology and Immunohistochemistry.
Paraffin-embedded tumor tissue was cut into 5-µm sections,
rehydrated, and processed for histology or immunohistochemistry. For
labeling with anti-CD31 antibody, sections were also preincubated with
0.1% trypsin in PBS. Blocking was done for 2 h in PBS containing
0.1% Tween 20 and 1 mg/ml BSA (buffer A). Slides were then incubated
with anti-CD31 antibody (MEC 13; Becton Dickinson, le Pont de Claix,
France) at 1:500 dilution in buffer A. After washing, the slides were
incubated 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
3,3'-diaminobenzidine (Dako). Counterstaining was done with Harris
hematoxylin.
For von Willebrand factor staining, 8-µm sections were rehydrated and boiled for 10 min in 10 mM citrate buffer (pH 6) in a microwave oven. Incubation with primary antibodies (rabbit polyclonal antihuman von Willebrand factor antibody; Dako), secondary antibodies (biotinylated goat antirabbit polyclonal antibody; Vectastain), and revelation were done as described above. Vessel density was quantified as described (12) .
Rabbit Corneal Assay for Angiogenesis.
Five µl of C6 cell suspension were injected into the cornea of New
Zealand albino rabbit eyes using a 10-µl Hamilton syringe. The
injection was done 2 mm away from the limbal margin of the cornea. The
potency of angiogenic activity was evaluated after 2 weeks as length of
vessel extension (in mm) centrally from the limbus and as sectorial
circumferential (in clock hours) involvement (13)
. Ten
rabbit eyes were analyzed for each condition.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fig. 1
shows the expression of FGFR2-DN or FGFR1-DN of a number of clones
examined. Cross-linking of 125I-labeled FGF-2 to
FGF-receptors (Fig. 1)
revealed a classical pattern consisting of a
band of Mr 90,000 for FGFR2-DN,
Mr 60,000 for FGFR1-DN, and higher
molecular weight bands corresponding to multimeric complexes. CMV or
tetracycline-regulated cell clones showed different degrees of FGFR2-DN
or FGFR1-DN expression. No "leakiness" in FGFR2-DN or FGFR1-DN
expression was detected in Tet+ FGFR2-DN or Tet+ FGFR1-DN cells in
cross-linking experiments.
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. At day 8, the following values of inhibition
(mean ± SD) were observed: 77.9% ± 0.03
(clone 18); 74.8% ± 0.13 (clone 2A7). Similarly, Tet-
FGFR2-DN or Tet- FGFR1-DN were also growth inhibited in
vitro when compared with Tet+ FGFR2-DN or Tet+ FGFR1-DN (Fig. 2, C and D)
. At day 8, the following values of
inhibition (mean ± SD) were observed for Tet- cells:
64.3% ± 0.09 (Tet- FGFR2-DN; clone 5A7), 56.5% ± 3.8 (Tet- FGFR2-DN; clone 5B1), 34.2% ± 3.4 (Tet-
FGFR1-DN; clone 4A8), and 47.9% ± 0.7 (Tet- FGFR1-DN;
clone 4A11). Empty pTet Splice vector transfected cells grew similarly
in the presence and absence of doxycycline (Fig. 2B)
. These
results indicate that cell proliferation is inhibited in
FGFR-DN-expressing C6 glioma cells.
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. The following values (mean ± SD) of inhibition in
comparison to control were observed in the different clones:
19.8% ± 0.4 (CMV FGFR2-DN; clone 3B8), 26.7% ± 1 (CMV FGFR2-DN; clone 2A7), 58.7% ± 3.3 (CMV FGFR2-DN;
clone 18), 98.4% ± 1.2 (Tet- FGR2-DN; clone 5A7),
96.7% ± 0.6 (Tet- FGFR2-DN; clone 5B1), 64.1% ± 7.7 (Tet- FGFR1-DN; clone 4A8), and 60.6% ± 9.6
(Tet- FGFR1-DN; clone 4A11).
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. The different clones were xenografted into RAG
2/c mice, and tumor growth was measured for 27
days. RAG 2/c mice are an alymphoid mouse
strain lacking T, B, and natural killer cells (14)
.
Initial experiments have shown that tumor take and growth were similar
in nude and RAG 2/c mice (data not shown).
Control cells transfected with the hygromycin-resistant gene (Hygro
cells) alone or Tet+ FGFR2-DN or R1-DN cells grew actively when
xenografted in immunodeficient mice.
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and Table 1
). Furthermore, Tet- FGFR2-DN (clone 5A7) and Tet- FGFR1-DN (clone
4A11) are also strongly inhibited in comparison with control (60–80%
of inhibition; Fig. 4, C and D
). The stronger
inhibitory effect in vivo observed for tumors derived from
FGFR2-DN-expressing cells in comparison with tumors derived from
FGFR1-DN cells correlated with higher FGFR-DN expression levels
observed in 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
). In addition, tumor weight was decreased in CMV FGFR2-DN,
Tet- FGFR2-DN or Tet- FGFR1-DN by 50–77% in comparison with control
(Table 1)
.
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. Furthermore, FGFR-DN mRNA expression was regulated in tumors derived
from Tet FGFR-DN-expressing cells when mice were fed with
doxycycline. In addition, only endogenous FGFR1 but not FGFR2 is
present in tumors derived from FGFR-DN-expressing cells, as shown for
human gliomas (7)
.
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and Table 2
). In the control groups (rats with control C6 glioma cell
implantations), the longitudinal extension of macroscopic alterations
in the right brain hemisphere (column 2 of Table 2
) ranged from 3 to 5
mm. In three animals, these affected areas comprised the whole
extension of the caudate putamen nucleus and an important part of the
thalamic nuclei (controls 1, 2, and 3), and they displaced other
ipsilateral and contralateral brain structures (Fig. 7A)
. In
another rat (control 4), only the caudate-putamen was affected, and the
thalamus was spared.
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; Table 2
). One of the animals
implanted with clone 2A7 was discarded, because the brain showed a
tumor formation on the parietal cortex, because of glioma cell
spreading after implantation. Another animal of the same group did not
show any morphological alteration in the right brain hemisphere. In
most animals, only some areas of the caudate-putamen and/or thalamic
nuclei were affected, and contralateral brain structures were less
displaced than in the control group.
After histological staining of coronal brain sections, we measured the
maximal transversal surface of these alterations (column 3 of Table 2
),
which varied between 9.5 and 23.75 mm2 in the
control group. 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 with clone 18 and from 0 to 5.9
mm2 in rats implanted with clone 2A7. Hemorrhagic
areas were not macroscopically evident in tumors developing after
implantations of clones 18 and 2A7, except for small foci seen in two
animals. As shown in column 4 of Table 2
, the affected brain regions
were completely replaced by glioma cells in two control animals. In the
other two control rats, glioma cells were only detected in the
periphery of tumors surrounding wide areas with necrotic brain tissue
(controls 2 and 4). One brain showed several large tumor-associated
hemorrhagic areas, whereas another tumor had relatively small
hemorrhagic foci.
Effect of the Expression of FGFR-DN on the Angiogenic Response.
We first analyzed vessel distribution and density by
immunohistochemistry in mice with tumors from control, CMV FGFR2-DN,
Tet+ FGFR2-DN or Tet+ FGFR1-DN cells using anti-CD31 antibodies (Fig. 8)
. In tumors derived from s.c. implanted control, Tet+ FGFR2-DN or Tet+
FGFR1-DN cells in RAG 2/c mice, many vessels
of different sizes were visible at the tumor margins. In contrast,
tumors derived from CMV FGFR2-DN, Tet- FGFR2-DN or Tet- FGFR1-DN
cells xenografted into immunodeficient mice exhibited much fewer blood
vessels than control tumors. Quantification of CD31-positive cells
demonstrated an inhibition of 34% ± 3.8 (clone 18) and
41% ± 3.2 (clone 2A7) for tumors derived from CMV FGFR2-DN
cells (Fig. 8A)
. Furthermore, tumors derived from Tet-
FGFR2-DN (clone 5A7) or FGFR1-DN (clone 4A11) demonstrated an
inhibition of 83% ± 3.4 and 41% ± 3.5,
respectively (Fig. 8, C and D)
. No differences in
vessel density was observed for control Tet clones in the presence or
absence of doxycycline (Fig. 8B)
. Vessel density was also
analyzed in intracranially implanted tumors using anti-von Willebrand
antibody staining instead of anti-CD31 because of high background
staining. In this case, labeling was also significantly decreased with
inhibition values of 69.4% ± 10.9 (CMV FGFR2-DN; clone 18)
and 67.8% ± 6.6 (CMV FGFR2-DN; clone 2A7; Fig. 8E
).
|
. Cells expressing FGFR2-DN (clone 2A7)
showed a marked decrease in the induction of blood vessel growth (Fig. 9, B and C)
.
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. In tumors derived from Tet- FGFR2-DN cells or Tet-
FGFR1-DN cells, inhibition values for VEGF mRNA expression of 47 and
64.2% were observed (Fig. 10B)
. In contrast, FGF-2 and
FGF-4 expression was not modulated in tumors derived from
FGFR-DN-expressing cells when expression was analyzed by Northern or
Western blotting (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C6 rat glioma cells express high molecular weight (nuclear, Mr 20,000 and Mr 21,000) and low molecular weight FGF (cytosolic and extracellular, Mr 18,000) isoforms (17) . Time course experiments measuring cell proliferation in 1% FCS revealed an inhibitory effect in FGFR-DN-expressing cells. This indicates that extracellular FGF is involved in the autonomous proliferation of glioma cells. In contrast to these observations, it has been shown previously in NIH 3T3 cells that low serum growth is induced by intracellular high molecular weight FGF-2 forms but not by Mr 18,000 FGF-2 (8) . In addition, supertransfection of FGFR-DN receptors does not inhibit low serum growth in NIH 3T3 cells expressing all FGF-2 isoforms. The reason for these differences is not understood but may reside in the different cell types used in these studies (transformed cells in this study and immortalized cells in the previous study).
Anchorage-independent growth was inhibited in cells expressing FGFR2-DN
or FGFR1-DN. This indicates that some of the properties related to cell
transformation are inhibited by the expression of FGFR-DN in tumor
cells. This may include effects on integrins or protease production
that are involved in interactions with the extracellular matrix. This
suggests that signaling through FGFRs modulates adhesive interactions
with the extracellular matrix. A number of observations are in support
of this contention. FGF-2 modulates integrin expression and adhesion to
the extracellular matrix in endothelial cells (18
, 19)
.
Furthermore, endogenous Mr 18,000
FGF-2 has been demonstrated to interfere with the expression and
function of 4ß1 and 5ß1 integrins in NIH 3T3 cells
(20)
.
In vivo growth was strongly impaired when cells with constitutive or tetracycline-regulated FGFR2-DN or FGFR1-DN expression were xenografted in immunodeficient mice. This demonstrates that FGFs are important for glioma development in vivo. This is in agreement with Yayon et al. (21) , who also demonstrated the participation of autocrine FGF in melanoma cell growth. The strong inhibition in vivo observed for cells expressing FGFR2-DN or FGFR1-DN under the control of tetracycline or doxycycline clearly demonstrates again that this is not attributable to clonal variation but to a true DN effect. Inhibition was stronger to some extent for FGFR2-DN-expressing cells than for FGFR1-DN-expressing cells. This is most certainly because of higher FGFR-DN expression levels achieved in FGFR2-DN-expressing cells as shown in vitro. The angiogenic response was found to be significantly inhibited in FGFR-DN-expressing cells. To explain our results, different possibilities may be considered: (a) the expression of FGF-2 or of another FGF family member is down-regulated in cells expressing FGFR-DN; (b) FGFR-DN may inhibit FGF-2 release into the extracellular compartment; (c) FGFR-DN may immobilize FGF at the cell surface, thereby sequestering extracellular FGF; (d) FGFR-DN could inhibit expression of another angiogenic factor, such as VEGF. We herein demonstrate for the first time that inhibition of endogenous FGF/FGFR activity leads to down-regulation of VEGF expression in tumors. This strongly supports the contention that FGF/FGFRs are inducing angiogenesis in tumors by an indirect mechanism that is mediated via VEGF. This is in agreement with the observations of Seghezzi et al. (22) , who reported that exogenous FGF-2 induces VEGF expression in endothelial cells. In addition, FGFR-DN may not only impede the activity of angiogenic factors at a transcriptional level but may instead inhibit the mobilization of angiogenic growth factors from the tumor cell or extracellular matrix to the endothelium by inhibiting the expression or activation of proteases such as matrix metalloproteinases or plasminogen activators. This possibility is currently being investigated. Inhibition of FGF activity in human glioblastomas also down-regulates VEGF expression and inhibits tumor growth.5 This indicates that our results have a more general significance and also apply to human tumors. In the light of the results described herein, endogenous FGF may be part of the angiogenic switch in glial tumors, allowing tumors to progress from dormancy to invasiveness.
The implication of FGF/FGFR activity in angiogenesis has been a matter of debate. Knocking-out the gene for FGF family members, such as FGF-1, FGF-2, or FGF-8, did not reveal defects in embryonic vascular development (23, 24, 25) . Furthermore, disruption of some of the FGFR family members is early lethal (26) . This does not allow the analysis of the embryonic vasculature. We have recently generated transgenic mice with targeted expression of FGFR1-DN in the retinal pigmented epithelium (27) . These mice display a severe defect in the development of choroidal blood vessels. This observation strongly indicates that FGF ligands are important in vascular developmental processes. The present data support an involvement of FGF in the angiogenic response in tumors. This is further supported by Fuldham et al. (28) , who recently demonstrated un impairment in the angiogenic response in a FGF-2 transgenic mice. Taken together, these observations indicate that FGF may play a significant role in both developmental and repair-associated angiogenic processes.
The C6 rat glioma cells that we used as a model in our studies express
a number of growth factor or growth factor receptors other than FGF
ligands or FGFRs. These include VEGF and VEGFRs (29, 30, 31)
,
interleukin 6 (32
, 33)
, tumor necrosis factor-
(32)
, glial cell line-derived neurotrophic factor
(34)
, epidermal growth factor receptors (35)
,
or PDGF receptors (36)
. Overexpression of VEGF in C6
glioma cells has shown that this factor is critical for the development
and maintenance of the tumor vasculature. Benjamin and Keshet
(37)
have used the tetracycline-regulated expression
system to show that VEGF has an important role as a survival factor for
tumor vessels in vivo. Blocking experiments have contributed
to understand the involvement of different receptors or ligands in
tumor growth. Plate et al. (29)
and Millauer
et al. (30)
have shown that tumor growth in
nude mice xenografted with C6 glioma cells was markedly inhibited when
VEGFR2-DN was retrovirally targeted into animals. Furthermore,
VEGFR2-DN directly expressed on tumor cells was able to inhibit tumor
growth and angiogenesis (38)
. The inhibitory effect of
VEGFR2-DN on tumor angiogenesis may be explained by adsorption of
extracellular ligands. Strawn et al. (36)
introduced a truncated PDGF-ß receptor into C6 rat glioma cells and
showed that PDGF-BB-induced tyrosine phosphorylation of the endogenous
receptor was significantly reduced. In addition, these cells were
growth inhibited in vitro and grew slower when xenografted
in nude mice. This indicates that multiple growth factors are critical
for glioma growth that may act directly on tumor cells and/or at the
level of the tumor environment.
In summary, our data clearly demonstrate that FGF/FGFR activity is involved in glioma growth in vitro and in vivo. A therapeutic strategy based on the inhibition of FGFR function may be useful for the treatment of solid FGF-producing tumors, such as gliomas, and may disrupt both angiogenesis-dependent and -independent signaling. This is similar to the effect of maspin, a recently discovered angiogenesis inhibitor, which also directly impedes tumor growth in addition to its effect on angiogenesis (39) . Thus, inhibition of the FGF signal transduction pathway may constitute an interesting target for therapeutic intervention.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from the
Association de la Recherche sur le Cancer and the Ministère 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 Facultés, 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.
Received 7/28/00. Accepted 12/13/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R. Huang, Y. Lin, Q. Shi, L. Flowers, S. Ramachandran, I. R. Horowitz, S. Parthasarathy, and R.-P. Huang Enhanced Protein Profiling Arrays with ELISA-Based Amplification for High-Throughput Molecular Changes of Tumor Patients' Plasma Clin. Cancer Res., January 15, 2004; 10(2): 598 - 609. |
, clone 18; 
, clone 2A7) or control cells (
, clone
BH2). B, empty pTet Splice control cells, clone CA8
(
,
without doxycycline. Significant difference was based on Student’s
t test. **, P < 0.01;
*, P < 0.05 versus
control.
