The Oncologist, Vol. 5, Suppl 1, 16-19,
April 2000
© 2000 AlphaMed Press
Measuring VEGF-Flk-1 Activity and Consequences of VEGF-Flk-1 Targeting In Vivo Using Intravital Microscopy: Clinical Applications
Peter Vajkoczya,
Andreas Thurnhera,
K. Peter Hirthb,
Lothar Schillinga,
Peter Schmiedeka,
Axel Ullrichc,
Michael D. Mengerd
a Department of Neurosurgery, Klinikum Mannheim, University of Heidelberg, Mannheim, Germany;
b SUGEN, Inc., South San Francisco, CA, USA;
c Department of Molecular Biology, Max-Planck-Institute for Biochemistry, Martinsried, Germany;
d Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany
Correspondence:
Peter Vajkoczy, M.D., Department of Neurosurgery, Klinikum Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany. Telephone: +49-621-383-2360; Fax: +49-621-383-2004; e-mail: peter.vajkoczy{at}nch.ma.uni.heidelberg.de
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Abstract
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Vascular endothelial growth factor (VEGF)-Flk-1/KDR tyrosine kinase signaling pathway plays a pivotal role in tumor angiogenesis. Targeting this angiogenic signaling pathway presents a promising alternative for the treatment of neoplasms. However, recent experimental and clinical studies have suggested that VEGF-Flk-1/KDR activity is unevenly distributed throughout the tumor microvasculature. To further evaluate this phenomenon, the regional differences in VEGF-Flk-1/KDR signaling activities in vivo were studied using intravital fluorescence videomicroscopy in an experimental murine brain tumor model. Regional VEGF-Flk-1/KDR was assessed using the small molecule inhibitor SU5416, which selectively inhibits the tyrosine kinase receptor Flk-1. C6 glioblastoma cells were implanted into the dorsal skinfold chamber preparation of nude mice. The process of tumor vascularization was repeatedly assessed over 22 days. SU5416 treatment resulted in a significant reduction in tumor vascular density (p < 0.05). Regional microvascular evaluation indicated that the magnitude of this antiangiogenic effect was pronounced in the more angiogenic and better vascularized peritumoral areas than in the intratumoral areas of the tumor microvasculature. These results demonstrate regional differences in Flk-1 activity in vivo that may have significant impact on the susceptibility of tumors to compounds that target VEGF-Flk-1/KDR. This finding should be considered in upcoming clinical trials targeting individual signal transduction systems in cancer patients.
Key Words. Antiangiogenic therapy • Protein tyrosine kinase • Glioblastoma • Vascularization • Vascular endothelial growth factor (VEGF) • Microcirculation
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Introduction
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Angiogenesis is the continuous formation of new blood vessels from the existing vasculature. Without angiogenesis, most solid tumors would not progress to a clinically relevant size because of inadequate tissue oxygenation and nutritional supply [1]. The process of tumor angiogenesis is mediated by angiogenic growth factors and their cognate receptors via a paracrine mechanism [2]. A substantial body of evidence has emerged, suggesting that the vascular endothelial growth factor (VEGF)-Flk-1/KDR system is the dominant signal transduction pathway in regulating tumor angiogenesis [3]. Therefore, targeting VEGF-Flk-1/KDR signaling represents a promising alternative in the treatment of neoplasms that are resistant to the therapeutic armamentarium of surgery, radiotherapy, and cytotoxic chemotherapy. One promising therapeutic approach is to use selective small molecule inhibitors of tyrosine phosphorylation to inhibit the VEGF-Flk-1/KDR signaling pathway. SU5416, one of the most potent Flk-1/KDR tyrosine kinase inhibitors, exerts a strong, rapid, and long-lasting antiproliferative effect on endothelial cells without affecting tumor cell growth in vitro and inhibits growth of multiple tumor types of various tissue origins in vivo [4, 5].
Recent experimental and clinical studies have suggested that VEGF-Flk-1/KDR activity is unevenly distributed throughout the tumor microvasculature. This view is supported by the regionally heterogeneous expression of VEGF and its receptors in tumors [6, 7], findings that provide the functional basis for regional differences in tumor angiogenic activities observed in experimental solid tumors [8].
Regional variations in VEGF-Flk-1/KDR activity and angiogenic activity may have significant impact on the clinical application of therapeutic compounds targeting this angiogenic pathway. Regional differences in VEGF-Flk-1/KDR activity and angiogenic activity may result in distinct susceptibilities to this novel therapeutic strategy within the tumor. In the present study, regional VEGF-Flk-1/KDR activities were evaluated in vivo using intravital fluorescence videomicroscopy in an experimental brain tumor model. The functional consequences of SU5416 treatment on regional tumor-induced angiogenesis and tumor microcirculation were assessed to determine VEGF-Flk-1/KDR activity.
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Methods
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Cells and Cell Culture
C6 rat glioblastoma cells were cultured in Ham's F-10 culture medium in 12-well dishes at 37°C in a 5% CO2 humidified atmosphere. A suspension of 5 x 105 cells was implanted into the skin chamber for tumor growth studies as previously described [8].
Animals and Dorsal Skinfold Chamber Model
The experiments were performed in athymic, adult nude mice (nu/nu; male, 28-35 g). All operative procedures as well as intravital microscopic observations were performed under ketamine/xylazine (subcutaneous) anesthesia. The microsurgical techniques for the preparation of the dorsal skinfold chamber have been described [8].
Experimental Protocol
Animals were treated daily with an intraperitoneal bolus of SU5416 in vehicle dimethylsulfoxide (DMSO) (n = 7; 25 mg/kg in 50 µl DMSO), starting on the day of tumor cell implantation. Animals in the control groups received either the vehicle DMSO (n = 3; 50 µl) or saline (n = 3; 50 µl). Animals were weighed regularly and observed for behavioral changes. The macroscopic appearance of the skinfold chamber preparation and the implanted tumor were documented daily. Intravital fluorescence videomicroscopy studies of the tumor microcirculation were performed on days 10 and 18 after cell implantation. On these days, the newly formed microvasculature within the fluorescently labeled tumor mass (intratumoral) and at the tumor periphery (peritumoral, outside the tumor and next to the tumor edge) was assessed separately. The newly formed tumor microvessels, which could be clearly distinguished by their chaotic arrangement from the hosts' striated muscle microvessels displaying the typical parallel arrangement of the muscle capillaries, were measured. Vascular densities were measured in six to nine regions in each animal for each observation time point.
Intravital Fluorescence Videomicroscopy
After cell inoculation, glioma growth and vascularization were documented daily by photomicroscopic study. Repeated intravital fluorescence videomicroscopy (epi-illumination) was performed using a modified Axiotech Vario microscope with a blue (450-490 nm) and green (520-570 nm) filter block (Zeiss; Oberkochen, Germany) [8]. Fast blue labeling of tumor cells allowed for precise delineation of the tumor from the surrounding, unaffected host tissue as well as identification of individual tumor cells with ultraviolet light epi-illumination. Contrast enhancement with 2% fluorescein isothiocyanate-dextran150 and blue light epi-illumination allowed for visualization of angiogenic sprouts, individual microvessels, and the tumor microvasculature.
Analysis of Parameters
Quantitative analysis included the tissue area covered by the newly formed microvascular network (mm2) and the vascular density (cm–1). Vascular density was defined as the length of all newly formed microvessels in each area of interest and observation time point. Newly formed tumor microvessels were not categorized into arterioles, capillaries, and venules because this classification is based on morphological and physiological criteria for normal tissue that are not applicable to tumors.
Statistical Analyses
Quantitative data are given as mean values ± standard deviation. Mean values of microcirculatory data were calculated from the average values in each animal. For analysis of differences between the groups, posthoc unpaired t test was used following one-way analysis of variance (ANOVA). Results with p < 0.05 were considered significant.
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Results
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Control animals receiving the vehicle DMSO or saline (n = 3 each) showed no significant difference in any of the evaluated parameters and were thus grouped together (control group, n = 6) for graphic representation and statistical evaluation. No mortality was observed, and the sleeping and feeding behavior of the animals was not altered during the 22-day treatment and observation period.
Treatment with SU5416 resulted in a marked reduction of the tissue area covered by newly formed tumor microvasculature, indicating reduced initial tumor vascularization (Fig. 1
). In contrast to the control group, chamber preparations from SU5416-treated animals did not show tumor-induced hyperemia, edema formation, or major intratumoral bleeding. Quantitative analysis of the vascular density revealed marked regional differences in angiogenic activity and susceptibility to SU5416 treatment (Fig. 2). In controls, the vascular density was significantly higher in peritumoral areas than in intratumoral areas, indicating distinct regional angiogenic activities. SU5416 treatment had a greater inhibitory impact on tumor vascularization in peritumoral areas than in intratumoral areas (Fig. 2).
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Figure 1. Influence of Flk-1 tyrosine kinase inhibition on C6 glioblastoma vascularization as evaluated by monitoring the tissue area covered by tumor microvasculature. Animals were treated with saline or vehicle dimethylsulfoxide (DMSO) (50 µl/d, i.p.; n = 6; closed bars) or SU5416 (25 mg/kg/d, i.p.; n = 7; open bars). The tissue area covered by the tumor microvasculature was analyzed planimetrically off-line using a computer-assisted image analysis system. The mean ± SD values are represented. Statistical analysis was performed using ANOVA followed by unpaired Student's t test. *p < 0.05 versus control.
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Figure 2. Influence of Flk-1 tyrosine kinase inhibition on regional C6 glioblastoma angiogenesis as evaluated by analyzing the vascular density within peritumoral (A) and intratumoral (B) areas. Animals were treated with saline/dimethylsulfoxide (DMSO) (50 µl/d, i.p.; n = 6; closed bars) or SU5416 (25 mg/kg/d, i.p.; n = 7; open bars). Vascular density was analyzed offline using a computer-assisted image analysis system. The mean ± SD values are represented. Statistical analysis was performed using ANOVA followed by unpaired Student's t test. *p < 0.05 versus control.
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Discussion
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In the present study, we used a versatile in vivo tumor angiogenesis assay combined with a computer-assisted image analysis method that allows quantitative assessment of tumor vascularization in vivo. The dorsal skinfold chamber preparation used with an immunodeficient mouse strain is a well-established model for tumor implantation site [8, 9]. This model makes it possible to have a noninvasive, repetitive, and quantitative evaluation of tumor growth, tumor-induced angiogenesis, and tumor microcirculation by means of intravital fluorescence microscopy [9]. The strength of the model is its high spatial resolution in the range of less than 1 µm, permitting a precise regional analysis of the newly formed tumor microvasculature [8].
Further, the intravital fluorescence videomicroscopic technique allows quantitative evaluation of various microcirculatory and microhemodynamic parameters, which may be mandatory because nutritive tissue perfusion not only depends on morphological criteria such as vascular density but also on microcirculatory and rheological parameters.
As in previous reports, the present study confirmed the fundamental role of VEGF-Flk-1/KDR signaling in tumor angiogenesis. When VEGF binds to Flk-1/KDR, the intrinsic tyrosine kinase function is activated, resulting in phosphorylation of the receptor, initiation of an intracellular multistep-signaling pathway, and, finally, proliferation of the endothelial cells. Inhibition of this cellular signaling cascade at the level of the tyrosine kinase using small molecule inhibitors interferes with tumor-induced angiogenesis and tumor growth.
In the present study, the newly formed tumor microvasculature was analyzed separately (in peritumoral and intratumoral areas), revealing increased angiogenic activities consistently in peritumoral versus intratumoral areas. The observations agree with the hypothesis that most solid tumors grow and expand at the tumor periphery. In accordance with its inhibitory activity on endothelial cell proliferation, the most prominent antiangiogenic activity of SU5416 was seen within the peritumoral areas where most of the tumor-induced neovascularization occurs. The results of this study point to regional differences in Flk-1 activity and susceptibility of the tumor microvasculature to VEGF-Flk-1/KDR targeting in vivo—a finding that should be considered in clinical trials targeting individual signal transduction systems in cancer patients.
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Acknowledgments
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This study was supported in part by the German Research Foundation (VA 151/4-1, UL 60/4-1), the Forschungsfond Mannheim (58/96), and an EU grant (BMH4-CT95-0875).
K. Peter Hirth, Ph.D., is employed by SUGEN, Inc., and provided SU5416.
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accepted for publication February 14, 2000.
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Abstract
Introduction
