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Tumor Biology |
Laboratory of the Biology and Treatment of Metastasis, Mario Negri Institute for Pharmacological Research, 24125 Bergamo [R. Gia., M. R. B. , C. F.]; Units of General Pathology and Immunology [R. Giu., D. C., M. P.], and Histology [B. S., M. P. M. T.], Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, 25123 Brescia; and Department of Experimental Medicine and Pathology, La Sapienza University, 00161 Rome [A. S.], Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After transfection, independent clones were obtained in which FGF2 mRNA and protein were up-regulated compared with parental cells. Also, the conditioned medium of Tet-FGF2 transfectants caused proliferation, urokinase-type plasminogen activator up-regulation, migration, and sprouting of cultured endothelial cells. A 3-day treatment of Tet-FGF2 cell cultures with tetracycline abolished FGF2 overexpression and the biological activity of the conditioned medium without affecting their proliferative capacity.
Tet-FGF2 cells formed tumors when nude mice received s.c. injections. The administration of 2.0 mg/ml tetracycline in the drinking water before cell transplantation, continued throughout the whole experiment, inhibited FGF2 expression in Tet-FGF2 tumor lesions. This was paralleled by a significant decrease in the rate of tumor growth and vascularization to values similar to those observed in lesions generated by parental HEC-1-B cells. Tetracycline administration 20 days after tumor cell implant, although equally effective in reducing FGF2 expression and inhibiting tumor vascularity, only minimally impaired the growth of established Tet-FGF2 tumors.
The results indicate that FGF2 expression deeply affects the initial tumor growth and neovascularization of HEC-1-B human endometrial adenocarcinoma in nude mice. On the contrary, the growth of established tumors appears to be independent of the inhibition of FGF2 expression and decreased vascular density. The possibility that a significant reduction of angiogenesis may not affect the progression of large tumors points to the use of antiangiogenic therapy in early tumor stage.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the last few years, various angiogenic factors other than FGF2 have been identified. Among them, VEGF appears to play a major role in tumor neovascularization (22) . Indeed, VEGF antagonists, which include neutralizing antibodies (23) , antisense-VEGF cDNA (24) , and dominant-negative VEGF receptor mutant (25) , can inhibit tumor growth in different experimental models. Also, VEGF levels in tumor biopsies correlate with blood vessel density of the neoplastic tissue and may be of prognostic significance (26 , 27) .
At variance with VEGF, FGF2 is released by producing cells via an alternative secretion pathway (28 , 29) , and it accumulates in the ECM, where it is mobilized by ECM-degrading enzymes (30 , 31) . Accordingly, FGF2 is detectable in the urine of patients with a wide spectrum of cancers (32 , 33) and in the cerebrospinal fluid of children with brain tumors (34) . Interestingly, the appearance of an angiogenic phenotype correlates with the export of FGF2 during the development of fibrosarcoma in a transgenic mouse model (35) . These data suggest that FGF2 production and release may occur in vivo and may influence solid tumor growth and neovascularization by autocrine and paracrine modes of action. Accordingly, neutralizing anti-FGF2 antibodies affect tumor growth under defined experimental conditions (36, 37, 38) . Relevant to this point is the recent observation that a secreted FGF-binding protein that mobilizes stored extracellular FGF2 can serve as an angiogenic switch for different tumor cell lines, including squamous cell carcinoma and colon cancer cells (39) . Interestingly, targeting of FGF-binding protein with specific ribozymes reduces significantly the growth and vascularization of xenografted tumors in mice (39) despite the high levels of VEGF produced by these cells (40) . These data suggest that modulation of FGF2 expression may allow a fine-tuning of the angiogenesis process even in the presence of significant levels of VEGF.
The tetracycline-regulated system (Tet-off system) allows the overexpression of the gene under study in the absence of tetracycline and the decrease in the basal level expression of the gene in the presence of the antibiotic (41) . The Tet-off system, therefore, may represent a unique tool to investigate the role of the expression of angiogenic growth factors on tumor growth and neovascularization. Indeed, the Tet-off system has been used to study the effect of the conditional switching of VEGF and FGF-binding protein expression on the growth of human tumor cells injected in nude mice (42 , 43) .
Recently, we have shown that constitutive FGF2 overexpression causes a significant increase in the angiogenic activity and tumorigenic capacity of the human endometrial adenocarcinoma HEC-1B cell line (44) . In the present study, to investigate the impact of the modulation of FGF2 expression on neovascularization during tumor growth, HEC-1B cells have been transfected with the single expression vector version of the Tet-off system (45) that harbors the human FGF2 cDNA. Stable transfectants were evaluated for their angiogenic and tumorigenic capacity in the absence or in the presence of tetracycline. The results indicate that the growth and vascularization of human endometrial adenocarcinoma HEC-1-B FGF2-transfected tumors strongly depend on FGF2 expression during the initial stages of tumor formation in nude mice. At variance, expansion of established tumors appears to be independent of FGF2 expression and related vascular density.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To generate tetracycline-responsive FGF2-transfectants, a 1108 bp human FGF2 cDNA that encodes for low and high molecular weight FGF2 isoforms (46) was cloned EcoRI into SIN-RetroTet vector (45) to give the expression vector pTet-FGF2. Next, HEC-1-B cells plated at 8.0 x 105 cells/100-mm plate were transduced with pTet-FGF2. Because the SIN-RetroTet vector does not harbor any antibiotic resistance gene (45) , cells were cotransfected with a calcium phosphate precipitate that contained 20 µg of the expression vector pTet-FGF2, a limiting amount (2 µg) of the pN06 plasmid that harbors the hygromycin resistance gene (kindly provided by G. Persico, I.I.G.B., Naples, Italy), and 40 µg of salmon sperm DNA as a carrier. Under these experimental conditions, pN06-transduced, hygromycin-resistant clones are anticipated to have incorporated also the pTet-FGF2 plasmid. On this basis, 70 µg/ml hygromycin were added to the culture medium 20 h after transfection. After 3 weeks of selective pressure, the hygromycin-resistant clones (Tet-FGF2 cells) were isolated, expanded, and tested for FGF2 expression and tetracycline responsiveness.
HEC-1-B, FGF2-B9, and Tet-FGF2 cells were grown in MEM supplemented with 1% nonessential amino acids, 1% sodium pyruvate, and 10% FBS. Transformed fetal bovine aortic endothelial GM 7373 cells were obtained from the National Institute of General Medical Sciences Human Genetic Mutant Cell Repository (Institute for Medical Research, Camden, NJ). They correspond to the BFA-1c multilayered transformed clone described previously (47) . GM 7373 cells were grown in Eagle’s MEM containing 10% FBS, vitamins, and essential and nonessential amino acids. BALB/c mouse aortic endothelial 22106 cells (48) were grown in DMEM added with 10% FBS.
Reagents.
Human recombinant Mr 18,000 FGF2 was
expressed in Escherichia coli and purified by
heparin-Sepharose affinity chromatography as described
previously (49)
. Rabbit polyclonal anti-FGF2
antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). The rat
monoclonal antimouse CD31 antibody MEC 13.3 was kindly provided by A.
Vecchi (Istituto Mario Negri, Milan, Italy). Tetracycline was
from Sigma Chemical Co.
FGF2 Protein Production by Tet-FGF2 Transfectants.
Transfectants were grown to confluence. Then, cells were incubated in
fresh medium for 3 days in the absence or in the presence of 10 ng/ml
tetracycline. Conditioned media were collected, and monolayers were
washed with 2.0 M NaCl in PBS to elute ECM-bound FGF2
(50)
. Then, cells were scraped with a rubber policeman and
sonicated on ice at 50 W for 20 s in PBS. Quantification of
intracellular FGF2 present in the cell lysate and extracellular FGF2,
obtained by pooling together the ECM-bound and the free growth factor,
was performed using the Quantikine FGF basic Immunoassay (R&D Systems
Inc., Minneapolis, MN). Data were normalized for the protein content of
the cell extracts.
For immunoblot analysis, 500-µg aliquots of the cell extracts were loaded onto 0.1 ml heparin-Sepharose columns. After a 0.5 M NaCl wash, resin beads were boiled, samples were run on SDS-15% PAGE, and proteins were electrophoretically transferred to nitrocellulose membranes in 20% methanol, 190 mM glycine, and 25 mM Tris-HCl. After transfer, membranes were saturated with PBS containing 3% BSA and 10% FBS and probed for 2 h at room temperature with anti-FGF2 antibody diluted 1:50 in PBS/0.5% BSA. Immunocomplexes were visualized by chemiluminescence Western blotting using the enhanced chemiluminescence Western blotting kit (Amersham Life Sciences) according to the manufacturer’s instructions.
FGF2 mRNA Expression.
Northern blot analysis of total RNA (40 µg/sample) was performed
according to standard procedures using the human 1108 bp FGF2 cDNA as a
probe (44)
. Uniform loading of the gels was assessed by
ethidium bromide staining.
Conditioned Medium of Tet-FGF2 Transfectants.
Cultures of the different Tet-FGF2 clones were grown in the absence or
in the presence of tetracycline for 3 days. Then, all of the cultures
were incubated for 3 more days in serum-free medium in the absence of
tetracycline. Conditioned media were collected, clarified by
centrifugation, concentrated 10 times with
Mr 10,000 cutoff centrifugal concentrators
(Centriplus; Amicon, Beverly, MA), and stored at -20°C until use.
Cell Growth Assay.
Cell proliferation assay on GM 7373 cells was performed as described
previously (49)
. Briefly, GM 7373 cells were seeded at
75,000 cells/cm2 in 24-well dishes. Plating
efficiency was higher than 90%. After overnight incubation, cells were
incubated for 24 h in fresh medium containing 0.4% FBS in the
absence or in the presence of aliquots (100 µg/ml) of the conditioned
medium of Tet-FGF2 clones. At the end of the incubation, cells were
trypsinized and counted in a Burker chamber. Control cultures incubated
with no addition or with 10 ng/ml of recombinant FGF2 underwent
0.1–0.2 and 0.7–0.8 cell population doublings, respectively. Cells
grown in 10% FBS underwent 1.0 cell population doublings
(49)
.
uPA Up-Regulation Assay.
Confluent cultures of GM 7373 cells were incubated for 18–20 h in
fresh medium containing 0.4% FBS and aliquots (100 µg/ml) of the
conditioned medium of Tet-FGF2 clones. After incubation, cell layers
were washed twice with PBS, and uPA activity was measured in the cell
extracts (49)
by using the plasmin chromogenic substrate
H-D-norleucyl-hexahydrotyrosil-lysine-p-nitroanilide
acetate (American Diagnostic, Greenwich, CT).
Migration Assay.
A modification of the Boyden chamber technique was used to evaluate
cell migration (49)
. Briefly, the Neuroprobe 48-well
microchemotaxis chamber was used. The two wells were separated by a
polyvinyl pyrrolidone-free Nucleopore filter, 8-µm pore size,
coated with gelatin (5 µg/ml). Aliquots (10 µg of protein) of the
conditioned medium were dissolved in fresh medium containing 0.1%
heat-inactivated FBS and placed in the lower wells, and the filters
were adjusted over the holes. After the top plate was applied and
mounted, 50 µl of GM 7373 cell suspension (106
cells/ml) were added to each upper well. After 4 h of incubation
at 37°C, the filters were removed and fixed in methanol. The cells on
the upper surface of the filter were removed with a cotton swab, and
the cells that migrated across the filter were stained with Diff-Quick
and counted in 10 different fields for each well at x 40 magnification.
Endothelial Cell-sprouting Assay.
Fibrinogen (2.5 mg/ml) was dissolved in calcium-free medium containing
aliquots (150 µg of protein) of the conditioned medium of Tet-FGF2
transfectants. Then, mouse aortic endothelial cell aggregates, prepared
as described previously(48)
, were resuspended in the
fibrinogen solution, and clotting was started by the addition of
thrombin (250 milliunits/ml). The mixture was transferred into 24-well
plates and allowed to gel at 37°C. Trasylol (200 KIU/ml) was added to
the gel and to the culture medium to prevent the dissolution of the
substrate. Cell aggregates were maintained for 2–3 days in DMEM
containing the same dilution of conditioned medium present within the
gel and were evaluated for the capacity to originate endothelial cell
sprouts radiating out from the aggregates (48)
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Tumorigenicity Assay.
Female NCr-nu/nu mice were obtained from the animal
production colony of the National Cancer Institute, Frederick Cancer
Research and Development Center (Frederick, MD) and used when 6–8
weeks of age. Cells were harvested by brief exposure to 0.25%
trypsin/0.02% EDTA, washed twice, and suspended in HBSS. Mice received
a s.c. injection in the dorsal scapular region of 1 x 106 cells suspended in 0.2 ml HBSS. The tumor
mass was measured twice a week with calipers, and tumor weight was
estimated by the formula: (length x width2)/2 (51)
. When indicated,
animals received tetracycline in the drinking water (2 mg/ml) with a
change every other day throughout the whole experimental period
starting 4 days before or 20 days after cell implantation. At
different time points, animals were sacrificed, sera were collected,
and tumors were harvested and processed for FGF2 antigen and mRNA
expression analysis and blood vessel immunostaining (see below). In
some experiments, frozen biopsies of the xenografts corresponding to
500 µg of protein were sonicated in PBS, and proteins were analyzed
for FGF2 content by the Quantikine FGF basic Immunoassay (R&D Systems
Inc., Minneapolis, MN).
FGF2 Immunohistochemistry and in Situ
Hybridization.
Xenograft specimens were embedded in OCT compound and frozen, and
5-µm sections were obtained with a cryostat microtome. Then, sections
were processed for immunohistochemical analysis by using anti-FGF2
antibody. To this purpose, sections were rinsed in PBS and incubated
for 20 min with 0.3% H2O2
in absolute methanol to block endogenous peroxidase and for 20 min
further with 0.2% Triton X-100 in PBS. Then, a 30-min preincubation
with diluted normal serum was followed by incubation at 4°C with
anti-FGF2 antibody (1:10 dilution) in a humidified chamber. Sections
were then exposed to biotinylated secondary antibody (Vector
Laboratories) and to avidin-biotin-peroxidase complex (Dako ABComplex
HRP) for 30 min. Peroxidase color reaction was developed with
3-amino-9-ethyl-carbazole (Sigma), and the sections were lightly
counterstained with Mayer’s hematoxylin.
For in situ hybridization studies, the human 1108 bp FGF2 cDNA fragment was labeled with biotin-labeled dATP using the Random Primer Biotin Labeling kit with streptavidin-horseradish peroxidase (NEN Life Science Products, Boston, MA). After rehydration, tissue sections were prepared for hybridization by flooding them with 50% formamide in 2 x SSC buffer and warming up to 70°C. FGF2 probe (20 ng) was dissolved in 100% formamide, boiled for 10 min, mixed to a final volume of 15 µl of hybridization mixture (10% dextran sulfate, 2 x SSC, 500 µg/ml tRNA, 0.2 mg/ml BSA, and 10 mM DTT in diethyl pyrocarbonate-treated water), and placed onto each tissue section. Specimens were sealed and incubated overnight at 45°C in a humidified chamber. After hybridization, specimens were stained using the Tyramide Signal Amplification-Indirect In Situ Hybridization kit (NEN Life Science Products) according to the manufacturer’s instructions, counterstained in hematoxylin, and mounted for microscopy.
Assessment of Microvessel Density.
To evaluate microvessel density, frozen sections from each tumor were
immunostained with a rat antimurine CD31 antibody for the detection of
blood vessels, according to the procedure described in detail
previously (44)
. Sections were examined at low-power
magnification to identify the areas with the highest density of
CD31-positive vessels. In each case, the most vascularized area was
selected, and microvessels in a x 400-field were
counted. Because all of the HEC-1-B-derived xenografts were
characterized by a poorly vascularized central zone of necrosis,
CD31-positive vessels were counted within the tumor parenchyma at the
periphery of the lesion where angiogenesis is more robust
(52)
. No significant differences in microvessel counts
were observed between paired sections of individual tumors.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the absence of tetracycline, Tet-FGF2 clones express high levels of
FGF2 mRNA and of low and high molecular weight FGF2 protein when
compared with parental HEC-1-B cells by Northern blot and Western blot
analysis. These levels are similar to those measured in FGF2-B9 cells.
In the presence of tetracycline, FGF2 expression is dramatically
suppressed in Tet-FGF2 25H and 15H transfectants, whereas it remains
unchanged in FGF2-B9 cells (Fig. 1A and B)
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In agreement with previous observations on FGF2-transfected HEC-1-B cells (44) , the in vitro replication rate of Tet-FGF2 transfectants did not differ from that of parental cells and was not affected by the presence of tetracycline (data not shown), thus confirming the inability of FGF2 overexpression to affect the in vitro growth of HEC-1-B cells.
Tet-FGF2 Transfectants Release Biologically Active FGF2.
Previous observations in our laboratory had shown that FGF2
transfection in HEC-1-B cells results in the production of clones
characterized by a different capacity to release FGF2 and that FGF2
export decides the biological behavior of these clones
(44)
. When FGF2-B9 cells and Tet-FGF2 25 H and 15H
transfectants were evaluated for their capacity to export FGF2 by an
ELISA, the results demonstrated that all of the tested clones release
significant amounts of FGF2 (Fig. 1C)
. In agreement with
previous observations on different FGF2 transfectants (28
, 48
, 53)
, released FGF2 represents 
2–5% of the total
cell-associated growth factor. Tetracycline treatment caused a 90–95%
reduction of the levels of released FGF2 in Tet-FGF2 cells without
affecting FGF2 release in FGF2-B9 cells (Fig. 1C)
.
To assess whether extracellular FGF2 produced by the different Tet-FGF2
clones is biologically active, cells were grown in the absence or in
the presence of tetracycline for 3 days. Then, all of the
cultures were incubated for 3 more days in serum-free medium in the
absence of the antibiotic. During this period, no significant
reexpression of FGF2 was observed in tetracycline-pretreated Tet-FGF2
cells (data not shown). The conditioned medium was collected and
evaluated for the capacity to affect endothelial cell functions
in vitro. As shown in Fig. 2A
, only the conditioned medium of the tetracycline-untreated
clones induced a significant mitogenic response in cultured GM 7373
endothelial cells. Accordingly, the conditioned medium of untreated
Tet-FGF2 clones stimulated uPA production and migration in GM 7373
cells and caused endothelial cell sprouting when added to murine aortic
endothelial cell aggregates grown in tridimensional fibrin gel (Fig. 2B
-E). Again, the conditioned medium collected
from tetracycline-pretreated Tet-FGF2 clones was ineffective on
endothelial cell response. It must be pointed out that tetracycline
pretreatment did not affect the biological activity of the conditioned
medium of B9-FGF2 cells that constitutively express the growth factor
(Fig. 2)
.
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Modulation of in Vivo FGF2 Expression by
Tetracycline.
The tumorigenicity of Tet-FGF2 and FGF2-B9 cells was evaluated in nude
mice. To this purpose, Tet-FGF2 clones 15H and 25H and FGF2-B9 cells
were injected s.c. in the flank of nude mice (five animals/group) at
1 x 106 cells/implant, and their
rate of growth was monitored. All of the cell lines grew at a similar
rate in vivo, with the median time to reach tumor weight of
200 mg being 25 days for FGF2-B9 xenografts and 24 and 26 days for
Tet-FGF2 15H and 25 H clones, respectively. Clone Tet-FGF2 15H was
chosen for additional studies.
To assess the capacity of tetracycline to suppress FGF2 expression in
Tet-FGF2 transfectants in vivo, nude mice that received s.c.
injections with 1 x 106 Tet-FGF2
15H cells were given the antibiotic dissolved in the drinking water
throughout the whole experimental period. Control animals that received
injections with the same tumor cell preparation were never exposed to
the antibiotic. Tetracycline did not affect the health status of the
mice as evaluated by lack of body weight loss compared with control
mice. After 4 weeks, animals were sacrificed, their serum was
collected, and xenograft specimens were analyzed for FGF2 expression by
in situ hybridization and immunohistochemistry. As shown in
Fig. 3A and C
, Tet-FGF2 15H cells express high levels
of FGF2 mRNA and protein in tumor tissue. At variance, FGF2 expression
in tumor parenchyma is dramatically reduced in animals given
tetracycline, with limited FGF2 immunoreactivity being detectable in
blood vessel endothelial cells (Fig. 3 B and D)
.
ELISA confirmed the significant decrease of FGF2 protein levels in
Tet-FGF2 tumor extracts from tetracycline-treated animals
(2.7 ± 0.4 and 1.1 ± 0.2 pg FGF2/µg
of tumor protein in tetracycline-untreated and -treated animals,
respectively). At variance, FGF2 mRNA and protein levels were not
affected by tetracycline treatment in tumors derived from FGF2-B9 cells
(data not shown).
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Taken together, these data demonstrate that the tetracycline regimen in the drinking water was able to cause a long-lasting decrease of FGF2 expression in Tet-FGF2 transfectants, with limited amounts of FGF2 protein being still detectable within the lesion and possibly produced by infiltrating host stromal cells, including endothelial cells.
Growth and Neovascularization of Tet-FGF2 Xenografts.
To evaluate the effect of FGF2 expression on tumor growth, Tet-FGF2 15H
and FGF2-B9 cells were transplanted in nude mice receiving either
tetracycline or regular drinking water throughout the whole
experimental period starting 4 days before tumor cell injection (Fig. 4)
. In mice that received injections with Tet-FGF2 15H cells, tumor take
at day 10 was 100%, and tumor lesions reached the size of 200 mg at
day 24 (range = 23–29 days). Tetracycline treatment
caused a 10-day delay in Tet-FGF2 15H tumor take (100% of tumors being
palpable at day 20) and a marked decrease in their growth rate with
tumor lesions reaching the size of 200 mg at day 37 (range = 28–47 days). The rate of growth of Tet-FGF2 15H tumors in the
presence of tetracycline was similar to that observed for lesions
generated by parental HEC-1-B cells that do not express FGF2 (Fig. 4B)
. In contrast, tumor take and growth of FGF2-B9 lesions
were not affected by tetracycline. In the presence or absence of the
antibiotic, tumors showed in fact a similar latency (100% of tumors
being palpable at day 10) and reached the size of 200 mg at day 25
(range = 24–36 days and 23–39 days, respectively; Fig. 4A
).
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In a second experiment, to assess the impact of the modulation of FGF2
expression on vascularization of Tet-FGF2 xenografts, nude mice given
or not given tetracycline were transplanted with Tet-FGF2 15H or
FGF2-B9 cells as above, and vascular density was evaluated in CD31
immunostained tumor sections. For all of the groups, animals were
sacrificed at 6–7 weeks when the tumors reached an average size of 800
mg, with the exception of Tet-FGF2 15H tumors from mice given
tetracycline that at the same time measured 300 mg. To avoid a bias
attributable to possible differences in vascularity relative to tumor
size, an additional group of tetracycline-treated Tet-FGF2 15H lesions
was analyzed at 9–10 weeks when their average size was 800 mg. As
shown in Fig. 5
, tetracycline treatment causes an early 65–70% decrease of the
vascular density in Tet-FGF2 15H lesions at 6–7 weeks to values
similar to those observed in parental HEC-1-B cell tumors of similar
size. Moreover, blood vessel formation remained suppressed in animals
given tetracycline in large-sized Tet-FGF2 15H lesions at 9–10
weeks. At variance, the vascularity of FGF2-B9 tumors was not affected
by the antibiotic treatment. It must be pointed out that, independent
of the levels of FGF2 expression, all of the tumors were characterized
by a poorly vascularized central zone of necrosis. Thus, CD31-positive
vessels were counted within areas of viable tumor parenchyma at the
periphery of the lesion where angiogenesis is more robust
(52)
. In agreement with previous observations
(54)
, striking differences in the morphology of
CD31-positive microvessels were observed when FGF2-overexpressing
lesions were compared with FGF2-less tumors (Fig. 3 E and F)
. Indeed, the microvasculature of all of the Tet-FGF2 15H
and FGF2-B9 lesions examined showed a remarkable heterogeneity in lumen
diameter with numerous large-caliber vessels. In contrast, all of the
tumors originated by Tet-FGF2 15H cells in tetracycline-treated animals
or by parental HEC-1-B cells in control animals were characterized by
homogenous small-caliber vessels.
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Taken together, these data indicate that the early suppression of FGF2 expression causes a significant decrease in tumor growth and vascularization in Tet-FGF2 transfectants. The extent of the inhibitory effect on blood vessel density is constant during tetracycline treatment, being therefore independent of the size of the tumor.
To assess whether FGF2 expression could also affect tumor growth
and vascularization when the lesions have been established, animals
that received injections with Tet-FGF2 15H cells but not receiving
tetracycline were randomized in two groups 20 days after cell
transplantation (mean size of the tumor lesions equal to 127 ± 51 mg). At this point, one group of mice was given tetracycline
in the drinking water, whereas the other group was left untreated. As
shown in Fig. 6A
, initiation of tetracycline treatment after tumors have
been established causes only a minimal, not statistically significant
delay of tumor growth. In contrast, evaluation of blood vessel density
in tumors isolated from animals sacrificed 6 weeks after the beginning
of tetracycline administration indicates that the late tetracycline
treatment was still able to cause a significant decrease in tumor
vascularity despite its lack of effect on the rate of tumor growth
(Fig. 6B)
. Interestingly, in this trial the decrease in
vascularity was already observed in representative tumors
(n = 4) analyzed 2 weeks after the beginning
of the tetracycline treatment (33 ± 4 blood
vessels/ x 400 field).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When injected s.c. in nude mice, Tet-FGF2 cells and FGF2-B9 cells originate highly vascularized tumors that grow at a similar rate, significantly higher than that of parental HEC-1-B cell xenografts. Tetracycline administration in the drinking water throughout the whole experimental period caused a significant delay in tumor take and a decrease in the growth rate of Tet-FGF2 xenografts during the early stages of tumor development. This was paralleled by a significant decrease in tumor vascularization observed in the early development of lesions (tumor burden = 50 mg) and maintained in large-sized tumors (800 mg). Several experimental evidences indicate that the inhibitory effect of tetracycline on early Tet-FGF2 tumor growth is because of its ability to switch off FGF2 expression with a consequent decrease in tumor vascularization: a) tetracycline administration in the drinking water suppresses FGF2 expression in Tet-FGF2 lesions at both mRNA and protein levels; b) the serum of tetracycline-treated animals is able to inhibit FGF2 expression in Tet-FGF2 cells in vitro; and c) tetracycline administration does not exert a significant effect on FGF2 expression, tumor growth, and vascularization of FGF2-B9 lesions constitutively expressing FGF2. It must be pointed out that FGF2 expression and/or tetracycline treatment do not affect Tet-FGF2 cell proliferation in vitro. This is in keeping with previous observation on the lack of effect of FGF2 overexpression on FGF2-B9 cell proliferation (44) . Nevertheless, we cannot rule out the possibility that modulation of FGF2 expression in transfected HEC-1-B cells may affect other biological properties of this cell line with possible consequences on its tumorigenic potential. HEC-1-B cells express FGFR-1 and FGFR-4, and both exogenous and endogenous FGF2 can modulate uPA production in these cells (44) .
Tet-FGF2 lesions eventually reach a large tumor burden also under the continuous presence of tetracycline, thus indicating that FGF2 up-regulation does not represent an absolute requirement for tumor growth and vascularization in this experimental system. Indeed, parental HEC-1-B cells that do not express significant levels of FGF2 are tumorigenic when injected in nude mice, with growth and vascularization of HEC-1-B xenografts being similar to that observed for tetracycline-treated Tet-FGF2 lesions (present study and Ref. 44 ). We have observed that HEC-1-B cells secrete significant amounts of VEGF (54) . VEGF levels are not affected by FGF2 expression in these cells, as shown by ELISA of the conditioned medium of parental, FGF2-B9, and Tet-FGF2 cells grown in the absence and in the presence of tetracycline. Also, ELISA of FGF2-B9 and Tet-FGF2 tumor extracts showed that intratumor VEGF levels remain constant throughout the experimental period both in the absence and in the presence of tetracycline treatment.4 These data suggest that the production of VEGF and possibly of other as yet unidentified angiogenesis factors are sufficient to allow a basal level of vascularization able to sustain tumor growth in HEC-1-B cells. FGF2 up-regulation causes a further induction of tumor vascularization that results in an earlier appearance and a faster rate of growth of the lesion during the early stages of tumor development. It is interesting to note that the levels of VEGF released by FGF2-B9 and Tet-FGF2 cells (range between 1.9 and 2.6 ng of immunoreactive VEGF/ml of conditioned medium) are significantly higher than the levels of released FGF2. Thus, in agreement with previous observations (54) , the data suggest that the release of limited amounts of FGF2 may deeply affect early tumor growth and vascularization even in the presence of a high background of secreted VEGF. This hypothesis is supported by the synergistic action exerted by the two growth factors in stimulating angiogenesis (55 , 56) and by the possibility to inhibit the in vivo growth and angiogenic activity of various human tumor cell lines by either anti-FGF2 (39) or anti-VEGF (25 , 57) approaches (see Ref. 40 for further discussion). The concomitant block of more than one angiogenic factor (e.g., VEGF and FGF2) to impair tumor progression in the model system here described deserves further investigation.
An interesting observation in our study is that the down-regulation of FGF2 expression after the tumor had reached a certain size did not affect the further growth of the lesion. Similar results had been obtained by the conditional switching of VEGF- or FGF-binding protein expression in large lesions originated by the injection of tumor cells of different origin (42 , 43) , thus suggesting that our findings are not the consequence of unique properties of the cell line here adopted. In the previous studies (42 , 43) , late down-regulation of VEGF- or FGF-binding protein expression did not affect the growth of large tumors, although it resulted in a marked inhibition of small tumors. However, the effect of VEGF expression on tumor vasculature was not investigated, and the increased expression of FGF2 and interleukin-8 after the late VEGF down-regulation was hypothesized to be sufficient to sustain tumor growth and vascularization (42) , whereas the modulation of FGF-binding protein did not appear to exert any effect on blood vessel density of the lesions (43) . In the present study, down-regulation of FGF2 expression in established lesions caused instead a rapid and significant decrease of tumor vascularization, similar to that observed when tetracycline regimen was initiated before tumor cell implantation. Thus, our data indicate that a significant decrease in blood vessel density is not paralleled by a significant decrease of the growth of established tumors. This raises the possibility that a threshold effect may exist in the relationship between tumor growth and vascularization in established cancers. According to this hypothesis, an increase in vascularization above the threshold values (e.g., by angiogenic growth factor overexpression) will not provide any advantage for established lesions, the growth of which will be inhibited only when vascularization falls below the threshold values. This hypothesis may explain the observation that early initiation of angiostatic therapies can be more efficacious than late initiation in reducing tumor growth (58, 59, 60) . The possibility also exists that tumor microcirculation not lined by endothelial cells may contribute to established tumor growth (61) . The impact of angiogenesis inhibitors on this form of tumor microcirculation remains to be established.
Although quantification of the number of CD31-positive microvessels in "vascular hot spots" represents a widely used procedure to assess tumor vascularization in experimental and clinical protocols, microvessel density may not necessarily reflect tumor perfusion and blood supply. However, we have observed that Tet-FGF2-overexpressing tumors are characterized by an increase in blood vessel density that is paralleled by a striking increase in blood vessel diameter when compared with parental HEC-1-B lesions. Remarkably, tetracycline treatment caused a significant reduction in both microvessel density and caliber. These findings support previous observations (54) on the modifications of the tumor microvascular architecture of HEC-1-B xenografts after FGF2 overexpression. Indeed, tridimensional morphometric analysis of microvascular corrosion casts indicated that blood vessels of FGF2-overexpressing tumors are characterized by a wider average vascular diameter when compared with the microvasculature of parental HEC-1-B lesions and by an extreme variability of the diameter of each individual vessel (54) . It is worth noting that the possibility to generate microvascular casts of these s.c. tumors after injection of the casting resin into the ascending aorta (54) suggests that both large- and small-caliber vessels are functional. Interestingly, VEGF overexpression also induces an increase in tumor vessel density and size (62) . Accordingly, inhibition of tumor angiogenesis by anti-VEGF antibody causes a decrease of microvessel density and diameter, with a consequent decrease in blood supply (63) . Similar conclusions have been drawn for the antiangiogenic action exerted by thrombospondin-1 or -2 overexpression in wound healing and tumor growth (64 , 65) . Additional experiments will be required to fully elucidate the impact of the modulation of FGF2 expression on tumor microvascular architecture, functional vascularity, and blood supply.
In the context of our experimental model, tetracycline mimics an antiangiogenic molecule by inhibiting FGF2 expression and exerting a significant effect on Tet-FGF2 tumor vascularization at both early and late stages of tumor progression. It is worth noting that tetracycline administration in the drinking water had no effect on FGF2-B9 tumor vascularization (see above) and on the angiogenic response elicited by human recombinant FGF2 in a murine Matrigel plug assay (66) ,5 thus indicating that the antibiotic does not exert a direct antiangiogenic effect under our experimental conditions. However, despite the ability of both early and late tetracycline regimes to inhibit tumor vascularization, only small tumors respond also with an inhibition in the rate of growth. To this respect, two points are worth noting. First, antiangiogenic therapy may be more efficacious in preventing or delaying tumor relapse and/or the appearance of metastases by inducing tumor dormancy (67 , 68) rather than in causing the regression of established tumor lesions (see also Refs. 69, 70, 71 ). Second, the evaluation of the efficacy of antiangiogenic therapy cannot be restricted to the assessment of its impact on tumor vascularization and, conversely, an antiangiogenic therapy able to significantly affect tumor vascularization may not impair the size of the tumor. Thus, our data expand previous observations on the different efficacy of antiangiogenic drugs in prevention, intervention, and regression trials in a transgenic mouse model of pancreatic islet carcinogenesis (60) .
In conclusion, the results indicate that beyond the initial phases, the progressive growth of tumors is at least partly independent of FGF2 expression and vascular density. The possibility that a significant block of angiogenesis may not affect the expansion of large cancers needs to be considered in the design of clinical trials with antiangiogenic compounds.
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FOOTNOTES |
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1 Supported in part by Grants from Fondazione
Italiana per la Ricerca sul Cancro (to R. Gia.), from
Associazione Italiana per la Ricerca sul Cancro and from Istituto
Superiore di Sanità (AIDS Project; to M. P. and
R. Gia.), and by National Research Council (Target Project on
Biotechnology), Ministero dell’Università e della Ricerca
Scientifica e Tecnologica (Cofinanziamento 1999 "Infiammazione" and
"60%"; to M. P.). 
2 To whom requests for reprints should be
addressed, at University of Brescia School of Medicine, Biomedical
Sciences and Biotechnology, via Valsabbina 19, 25123 Brescia, Italy.
Phone: 39-0303717311; Fax: 39-0303701157; E-mail: presta{at}med.unibs.it
3 The abbreviations used are: FGF2, fibroblast
growth factor-2; ECM, extracellular matrix; FBS, fetal bovine serum;
FGFR, tyrosine kinase fibroblast growth factor receptor; uPA,
urokinase-type plasminogen activator; VEGF, vascular endothelial growth
factor.
4 R. Giuliani and D. Coltrini, unpublished
observations.
5 M. R. Bani and R. Giavazzi, unpublished
observations.
Received 3/ 8/00. Accepted 10/31/00.
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). Within each group, two subgroups of
mice (n = 8) received s.c. injections
with 1 x 106 FGF2-B9 cells
(A) or with 1 x 106 Tet-FGF2
15H cells (B). One additional group of untreated animals
(n = 5) received injections with
1 x 106 parental HEC-1-B cells (
in
B).
) and one group receiving 2 mg/ml tetracycline in the
drinking water (
). In A, tumor growth was
monitored with calipers. In B, mice were sacrificed at 9
weeks after cell injection, and tumor vascularity was assessed by CD31
immunostaining of tissue sections as described in "Materials and
Methods." **, statistically different from control group
(P < 0.01, Student’s t
test).