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Tumor Biology |
Max-Planck-Institute for Physiological and Clinical Research, 61231 Bad Nauheim, Germany
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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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VEGF5 and its high-affinity tyrosine kinase receptor Flk-1/KDR (VEGFR-2) are central regulators of both physiological and pathological angiogenesis. Gene targeting experiments showed that early embryonic vascular development is dependent on VEGF function (2 , 3) . Flk-1 is expressed in endothelial cells or their precursors and is essential for endothelial cell differentiation, vasculogenesis, and hematopoiesis (4) . Consistent with a signaling function in angiogenesis, Flk-1 is down-regulated in most adult vascular beds. However, VEGF and Flk-1 are up-regulated in a variety of human tumors, including glioma and carcinoma of several tissues (5 , 6) . The expression of VEGF in the tumor cells and Flk-1 in the tumor endothelium indicates that this signal transduction system stimulates the proliferation and the survival of tumor vessels by a paracrine mechanism (5, 6, 7) . Direct evidence for this hypothesis was provided by the inhibition of tumor growth in animal models by the application of VEGF neutralizing antibodies (8 , 9) or by the gene transfer of dominant-negative Flk-1 receptor mutants (10 , 11) . In the latter experiments, the cotransplantation of retrovirus producer cells encoding a signaling-defective Flk-1 mutant with tumor cells resulted in the suppression of tumor growth and angiogenesis in intracerebral implants of rat gliosarcoma cells or in xenografts of these and various other tumors. These studies revealed the Flk-1/KDR receptor as central target for antiangiogenic tumor therapy and encouraged the development of low molecular weight inhibitors of Flk-1 signaling that prevented tumor growth in experimental systems (12) . Moreover, these results support the validity of gene therapy approaches for the antiangiogenic treatment of tumors. To minimize the risk of inherent side effects, however, it is highly desirable to restrict the expression of therapeutic genes to the tumor endothelium by using transcriptional control elements of endothelium-specific genes. Although evidence for endothelium-specific gene expression after retrovirus-mediated gene transfer in vitro has been presented (13) , the promoter sequences used in these studies are not sufficient to direct selective expression of the transgene to the tumor endothelium in vivo. In contrast to other endothelial genes that are expressed constitutively, gene regulatory sequences of the Flk-1/KDR gene are ideally suited for the delivery of therapeutic genes to the tumor endothelium because Flk-1 is absent from most vascular beds of the adult organism but strongly and specifically up-regulated in tumor endothelium (14) .
We have recently established transgenic mouse lines that express the LacZ reporter gene under the control of a 939-bp fragment of the 5'-flanking region of the Flk-1 gene, in combination with an enhancer element located in a 2.3-kb fragment of the first intron (15) . In these mice, high-level, endothelium-specific expression of the LacZ reporter gene is observed throughout vascular development but is down-regulated in most adult vessels. Here, we have analyzed whether the Flk-1 promoter/enhancer elements are also active during tumor angiogenesis in three different tumor models in vivo. Transplants of B16 melanoma and BFS-1 fibrosarcoma in syngeneic mice and polyoma middle T-induced mammary adenocarcinoma were analyzed in the transgenic reporter mice. In all three tumor types, endothelium-specific transgene expression was detected, indicating that the gene regulatory sequences sufficient for up-regulating the Flk-1 expression in tumor endothelium are localized in the characterized promoter/enhancer fragments. This should allow the study of the transcriptional control mechanisms involved in the induction of Flk-1 expression in vascular development and tumor angiogenesis. Moreover, these Flk-1 regulatory elements should represent a powerful tool to target the expression of therapeutic genes specifically to the tumor vasculature, opening new strategies for an antiangiogenic tumor therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Generation of Tumors.
Media and supplements were obtained from Life Technologies, Inc.
(Eggenstein, Germany). B16 melanoma cells (American Type Culture
Collection) were cultured in DMEM+ containing
10% FCS (PAA Laboratories, Linz, Austria), 1%
penicillin/streptomycin, and 2% glutamine. BFS-1 fibrosarcoma cells
(17)
were grown in RPMI containing 10% FCS, 1%
penicillin/streptomycin, 1% pyruvate, and 2% glutamine. Confluent
monolayers were washed two times with PBS and trypsinated, and the cell
suspension was collected by centrifugation (1200 rpm for 5 min at room
temperature). The cells were resuspended in
DMEM+, and 1.5 x 106
cells/50 µl were injected s.c. into adult
mice. Tumors were harvested at 4, 7, or 12 days p.i. for histological
analysis.
Double transgenic animals, which express the LacZ reporter gene and the polyoma middle T oncogene, were generated by cross-breeding male MMTVPyVT transgenic mice with female mice of line 2603 or Flk-1 +/LacZ knock-in mice, respectively. Mammary tumors developed between 11 and 16 weeks after birth. Genotyping was performed by PCR analysis as described (18) using the primer pairs LacZP1/LacZP2 and PyVTforw/PyVTrev: LacZP1, 5'-ATCCTCTGCATGGTCAGGTC-3'; LacZP2, 5'-CGTGGCCTGATTCATTCC-3'; PymTforw, 5'-GCAGGCATAT AAGCAGCAGTCAC-3'; and PymTrev, 5'-TGTCGGGTTGCTCAGAAGACTC-3'.
Histological Analysis.
Tumor-bearing mice were perfused with 1% PFA. The tumors were removed
and postfixed for 4 h at 4°C in 1% PFA, incubated overnight in
18% sucrose in PBS, embedded in Tissue Tek (Sakura), and stored at
-80°C. For immunohistochemical analysis, 14-µm frozen sections
were prepared, air dried, and stained. Double staining for
ß-galactosidase and PECAM-1 and staining for Flk-1 was performed on
serial sections. For the ß-gal/PECAM-1 double staining, sections were
first incubated with a monoclonal rat-anti-PECAM-1 antibody
(19)
, rinsed two times in PBS, fixed in 1% PFA, and
stained for ß-gal as described (20)
. Afterward, the
slides were washed in PBS, fixed in 1% PFA, and rinsed two times with
PBS. The sections were then incubated with a biotinylated
rabbit-antirat secondary antibody (Vectastain Elite ABC kit; Vector
Laboratories, Inc., Burlingame, VT) that was subsequently detected with
avidin-biotinylated horseradish peroxidase complex (Vectastain Elite
ABC kit). The color was developed with an AEC kit (Sigma, Deisenhofen,
Germany). The Flk-1 immunostaining was performed using a rat monoclonal
anti-Flk-1 antibody (21)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Frozen sections of all tumors were prepared and stained
in parallel for ß-gal expression and for the endothelial marker
PECAM-1 (19)
to visualize the blood vessels. To analyze
the endogenous Flk-1 expression, we performed immunohistological
staining with a monoclonal anti-Flk-1 antibody (21)
on
adjacent tumor sections. Endothelium-specific reporter gene expression
was detected at all three time points investigated (Fig. 1, B–D
; Table 1A
; and data not shown). Tumors
isolated after 4 days were already palpable (
1 mm in diameter) but
were mostly avascular. The LacZ reporter gene was weakly
expressed in 50% of the capillaries in the tumor periphery, which
showed Flk-1 expression (data not shown). Tumors harvested at day
7 p.i. were highly vascularized, and uniform LacZ
reporter gene expression was seen in the majority of the tumor blood
vessels (Fig. 1B
; Table 1A
). No obvious
differences in the patterns of endogenous Flk-1 expression (Fig. 1E
; Table 1A
) and of LacZ reporter
gene expression was detectable. Tumors that had developed central
necrosis showed an accumulation of small, Flk-1-positive vessels in
viable tumor tissue at the borderline of necrosis (Fig. 1, C and F)
. Strong reporter gene expression was detected in most
if not all of these vascular structures (Fig. 1C)
. In the
majority of the melanomas examined at 12 days p.i., the Flk-1
expression corresponded well with the reporter gene expression (Fig. 1, D and G
; Table 1A
).
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; Table 1A
; and data not shown). In contrast
to the 4-day tumors in the transgenic line 2603, all melanomas in the
Flk-1 +/LacZ knock-in mice analyzed were already vascularized. This
difference might be attributable to differences in the genetic
background of the reporter mice. In three of four 4-day p.i. tumors, as
well as in 7-day p.i. and 12-day p.i. tumors, the majority of the blood
vessels was LacZ positive (Fig. 1
H–J; Table 1A
),
corresponding to the Flk-1 expression pattern (Table 1A)
.
Necrotic areas that had developed in half of the tumors at day 7 p.i. were surrounded by blood vessels expressing the LacZ
reporter gene (Fig. 1I)
and Flk-1 (Table 1A)
, similar to the melanomas grown in line 2603. These results indicate that Flk-1 regulatory sequences that are capable of inducing endothelium-specific reporter gene expression in the vasculature of B16 melanoma are contained in the characterized Flk-1 promoter/enhancer sequences. Moreover, transgene expression was induced in parallel with Flk-1 in blood vessels in the periphery of prevascular tumors, which are recruited by the growing tumor.
Flk-1 Regulatory Sequences Are Active in BFS-1
Fibrosarcoma.
We next investigated whether the Flk-1 promoter/enhancer
sequences are also functional in other tumor types. BFS-1 fibrosarcoma
cells were injected s.c. into transgenic mice of line 2603, and tumors
were harvested 4 and 7 days p.i. and analyzed as described above. We
observed strong reporter gene expression in the tumor vasculature at
both time points (Fig. 2, A and B)
. At day 4 p.i., when the tumors
were already palpable, four of six fibrosarcomas were highly
vascularized and showed a strong reporter gene expression in the
majority of the tumor blood vessels (Fig. 2A
; Table 1B
). In most of these tumors (three of four), the Flk-1
staining pattern (Fig. 2C)
resembled the ß-gal staining.
At 7 days p.i., the tumors showed a dense vascular network and strong
ß-gal staining in most of the vessels (Fig. 2B
; Table 1B
), resembling the Flk-1 expression pattern (Fig. 2, B and D)
. Similar observations were made in four
fibrosarcomas at day 7 p.i. grown in a second independent
transgenic Flk-1/LacZ line (line 2610), indicating that the
LacZ reporter gene expression is not dependent on a
specific integration site of the transgene (data not shown). In
comparison, we analyzed fibrosarcomas grown in the Flk-1 +/LacZ
knock-in mice. Most of the tumors examined at day 4 p.i. and day
7 p.i. were highly vascularized and showed a strong
LacZ reporter gene expression in the majority of the tumor
blood vessels (Fig. 2, E and F
; Table 1B
), corresponding to the Flk-1 expression (Table 1B)
. Thus, in experimental BFS-1 fibrosarcoma, the Flk-1
expression pattern corresponded well with the LacZ reporter
gene expression in both the transgenic mouse line 2603 and the Flk-1
+/LacZ knock-in mice. These data demonstrate that the Flk-1
promoter/enhancer sequences are sufficient to confer a strong
endothelium-specific LacZ reporter gene expression also in
experimental fibrosarcoma.
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. The tumors differed in size, measuring 3, 4, and 10 mm in
diameter, respectively. Although the two smaller tumors showed more
Flk-1-positive than LacZ-positive capillaries, the majority of tumor
blood vessels in the largest tumor was LacZ positive, and no
significant difference in the Flk-1 and the LacZ reporter
gene expression was detectable. In whole-mount-stained tumors of Flk-1
LacZ/+ knock-in mice, the LacZ reporter gene was also
expressed in vessel-like structures (data not shown).
Immunohistological analysis of three tumors revealed that the Flk-1
expression corresponded well with the LacZ reporter gene
expression (Fig. 3B)
in all tumors investigated.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the involvement of the VEGF/VEGFR signal transduction system in tumor angiogenesis is well established, the mechanisms of Flk-1 up-regulation in tumor endothelial cells is still to be elucidated. In a cerebral slice culture system, it has been shown that Flk-1 expression is up-regulated indirectly in response to hypoxia via its ligand VEGF (24) . VEGF itself is strongly up-regulated in hypoxic regions in several tumor types (25 , 26) and perhaps also by tumor-associated cells and stromal cells surrounding the tumor (27) . To gain more insight into the transcriptional mechanisms of Flk-1 up-regulation in the tumor endothelium, we analyzed the activity of Flk-1 gene regulatory elements in experimental melanoma, fibrosarcoma, and mammary adenocarcinoma. The tumors were grown in transgenic mice that express the LacZ reporter gene under control of Flk-1 promoter/enhancer elements (line 2603). In these mice, a 939-bp fragment of the Flk-1 promoter, in combination with an enhancer element located in the first intron, mediated endothelium-specific LacZ reporter gene expression in transgenic mouse embryos (15) . As described for the endogenous Flk-1 gene (28) , transgene expression was down-regulated in most vascular beds of adult mice (15) . In this study, we demonstrate that the analyzed Flk-1 promoter/enhancer sequences are also active during tumor angiogenesis in vivo. To our knowledge, this is the first description of gene regulatory sequences that specifically target gene expression to the tumor endothelium. The Flk-1 gene regulatory sequences were activated relatively independently of the tumor type. Endothelium-specific reporter gene expression was also observed in fibrosarcomas grown in a second independent transgenic line (line 2610), demonstrating that the transgene expression is not dependent from a specific integration site. Whether the copy number of the transgene has an influence on the strength of reporter gene expression remains to be determined.
Interestingly, prevascular melanomas in line 2603 at day 4 p.i. showed LacZ reporter gene expression and Flk-1 staining in blood vessels surrounding the tumor tissue. This observation is consistent with the idea that tumor neovascularization is initiated in the periphery of the tumor, through the up-regulation and activation of Flk-1 in endothelial cells of normal vessels. Later on, the melanomas of line 2603 and of the Flk-1 +/LacZ knock-in mice showed similar vascularization and a similar LacZ staining pattern. These data demonstrate that the Flk-1 promoter/enhancer elements used are sufficient for a strong, endothelium-specific LacZ reporter gene expression during B16 melanoma angiogenesis. Because fibrosarcomas also grown in line 2603 showed endothelium-specific LacZ reporter gene expression, the activity of the regulatory Flk-1 gene sequences is not restricted to the endothelium in a single tumor type.
In polyoma middle T oncogene-induced mammary adenocarcinoma, strong endothelium-specific LacZ reporter gene expression was detectable in all tumors analyzed, demonstrating that the Flk-1 gene sequences are not only active in transplanted tumors but also in a tumor that arises in vivo. The incomplete reporter gene expression in the two smaller carcinomas of line 2603 suggests that additional Flk-1 regulatory sequences might be required to induce early angiogenesis in this tumor model. However, because the more advanced tumor showed an almost homogeneous vascular LacZ staining, the analyzed Flk-1 gene sequences appear to be sufficient for a strong reporter gene expression in the vasculature of expanding adenocarcinoma.
The Flk-1 promoter, which contributes to a strong gene
expression, is activated in vitro by Sp-1 (29)
and by hypoxia-inducible factor-2
(15)
, a basic
helix-loop-helix/PAS-domain transcription factor that is prominently
expressed in the embryonic endothelium (30)
and in
hemangioblastoma (31)
. On the basis of these observations,
it seems likely that hypoxia-inducible factor-2 stimulates
transcription of Flk-1 during embryonic vascular development
and in hemangioblastoma. The endothelial-specific promoter/enhancer
sequences of the Flk-1 gene used in this study also contain
binding sites for transcription factors of the Tal1, GATA, and c-ets
families (15)
. Members of these families are expressed in
endothelial cells or their precursors (30
, 32, 33, 34, 35, 36, 37)
and are
likely to mediate the endothelium specificity of Flk-1
expression during embryonic development in a combinatorial
fashion.6
During neovascularization of benign and malignant tumors, endothelial
cells express c-Ets1, whereas no expression is detected in mature
capillaries and larger vessels without angiogenic activity
(33)
. Our recent observation that c-Ets1 stimulates the
Flk-1 promoter6
supports the
hypothesis that c-Ets1 regulates Flk-1 expression in tumors. The nature
of transcription factors that up-regulate Flk-1 in tumor endothelium,
however, remains to be determined.
On the basis of the results of this study, the further functional analysis of the Flk-1 promoter/enhancer sequences allows an unraveling of the signaling pathways that lead to the up-regulation of Flk-1 in the endothelium of tumor blood vessels. Insights into the mechanisms that control Flk-1 transcription might open up new strategies to modulate Flk-1 expression during tumor angiogenesis and endothelial cell survival. Previous gene therapy protocols relied mainly on rather unspecific expression systems (10 , 11 , 38) . In contrast, the isolated regulatory elements could be used to develop vectors that allow targeting of the expression of therapeutic genes specifically to tumor endothelium. For example, the thymidine kinase gene of the herpes simplex virus that activates the prodrug gancyclovir was recently used successfully to target tumor cells in vivo (38) . The inhibition of the VEGF/VEGFR signal transduction system by a dominant-negative Flk-1 mutant that is specifically targeted to the tumor endothelium would be an alternative strategy for the antiangiogenic tumor therapy. Flk-1 promoter/enhancer sequences may thus turn out to become a powerful tool for antiangiogenic tumor therapy and vascular targeting.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants of the
Bundesministerium für Bildung und Forschung and the
Deutsche Krebshilfe. 
2 In memoriam: Werner Risau (1953–1998).
3 Present address: Aventis Research and
Technologies, 66926 Frankfurt/Main, Germany.
4 To whom requests for reprints should be
addressed, at Max-Planck-Institute for Physiological and Clinical
Research, Parkstrasse 1, D-61231 Bad Nauheim, Germany. Phone:
49-6032-705293; Fax: 49-6032-72259; E-mail: g.breier{at}kerckhoff.mpg.de
5 The abbreviations used are: VEGF, vascular
endothelial growth factor; VEGFR, VEGF receptor; p.i., postinjection;
PECAM, platelet/endothelial cell adhesion molecule; PFA,
paraformaldehyde; ß-gal, ß-galactasidase.
6 Kappel, A., Schlaeger, T. M., Flamme, I.,
Orkin, S. H., Risau, W., and Breier, G. Role of SCL/Tal-1, GATA, and
Ets transcription factor binding sites for the regulation of
Flk-1 expression during murine vascular development. Blood,
in press, 2000.
Received 1/28/00. Accepted 8/24/00.
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