The FASEB Journal express article 10.1096/fj.03-0271fje.Published online December 4, 2003.

Combined inhibition of VEGF- and PDGF-signaling en-forces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms Ralf Erber,* Andreas Thurnher,* Alice D. Katsen,† Gesine Groth,‡ Heinz Kerger,‡ Hans-Peter Hammes,§ Michael D. Menger, † Axel Ullrich,║ and Peter Vajkoczy* *Department of Neurosurgery, Medical Faculty of the University of Heidelberg, Mannheim; †Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Ger-many; ‡Department of Anesthesiology and Intensive Care Medicine; §Vth Medicine Clinic, Medical Faculty of the University of Heidelberg, Mannheim; and ║Department of Molecular Biology, Max-Planck-Institute for Biochemistry, Martinsried, Germany

Corresponding author: Dr. P. Vajkoczy, Department of Neurosurgery, Klinikum Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany. E-mail: peter.vajkoczy@nch.ma.uni-heidelberg.de

ABSTRACT

Destruction of existing tumor blood vessels may be achieved by targeting vascular endothelial growth factor (VEGF) signaling, which mediates not only endothelial cell proliferation but also endothelial cell survival. In this study, however, intravital microscopy failed to demonstrate that targeting of VEGFR-2 (by the tyrosine kinase inhibitor SU5416) induces significant regression of experimental tumor blood vessels. Immunohistochemistry, electron microscopy, expression analyses, and in situ hybridization provide evidence that this resistance of tumor blood vessels to VEGFR-2 targeting is conferred by pericytes that stabilize blood vessels and provide endothelial cell survival signals via the Ang-1/Tie2 pathway. In contrast, targeting VEGFR-2 plus the plate-let-derived growth factor receptor (PDGFR)-β system (PDGFR-β) signaling (by SU6668) rap-idly forced 40% of tumor blood vessels into regression, rendering these tumors hypoxic as shown by phosphorescence quenching. TUNEL staining, electron microscopy, and apoptosis blocking experiments suggest that VEGFR-2 plus PDGFR-β targeting enforced tumor blood vessel regression by inducing endothelial cell apoptosis. We further show that this is achieved by an interference with pericyte-endothelial cell interaction. This study provides novel insights into the mechanisms of how 1) pericytes may provide escape strategies to anti-angiogenic therapies and 2) novel concepts that target not only endothelial cells but also pericyte-associated pathways involved in vascular stabilization and maturation exert potent anti-vascular effects.

Key words: angiogenesis ● glioma ● resistance ● tyrosine kinase inhibitors ● vessel maturation

he concept of treating tumors by interfering with their vascularization is based on the premise that tumor growth is dependent on angiogenesis and that tumor progression is suppressed if neovascularization is prevented. Over the past years, this concept has been

repeatedly proven in animal studies, either by interfering with positive regulators of angiogenesis T

or by taking advantage of negative regulators of angiogenesis. Vascular endothelial growth fac-tor (VEGF) has been shown to be the central positive regulator of tumor angiogenesis and, there-fore, has become the primary target when exploiting anti-angiogenic strategies. As a result, both antibodies and inhibitors specifically directed against VEGF or its receptor VEGFR-2 have been raised and have been demonstrated to potently prevent vascularization and growth of a large number of experimental tumor types (1–4). Successful treatment of established human tumors, however, might require not only prevention of further angiogenesis but also destruction of already existing tumor blood vessels to reduce the already existing tumor mass. In line with this, VEGF has been shown to not only act as an an-giogenic factor but also as a survival factor for endothelial cells and newly formed blood vessels. In the developing retina, hyperoxia-induced vessel regression has been correlated with inhibition of glial VEGF release (5). Furthermore, with the use of a tetracycline-regulated expression sys-tem, it has been demonstrated that VEGF withdrawal can force newly formed tumor blood ves-sels into regression (6). Finally, in vitro studies have demonstrated that VEGF mediates this sur-vival function for endothelial cells through VEGFR-2 and its downstream signal transduction pathways (7), suggesting that interference with VEGF/VEGFR-2 signaling may exert not only anti-angiogenic but also anti-vascular efficacy.

Recently, however, it has been realized that the susceptibility of established tumor blood vessels to an interference with VEGF/VEGFR-2 signaling may be restricted to a fraction of immature vessels that lack colocalization with pericytes (8). This is supported by the observation that con-tact between endothelial cells and periendothelial support cells, such as pericytes or smooth muscle cells, stabilizes new blood vessels, promotes endothelial survival, and inhibits endothe-lial cell proliferation (9). The fact that only immature vessels may be forced into regression by interference with VEGF/VEGFR-2 signaling is of therapeutic relevance since the literature on the pericyte coverage on tumor vessels suggests that the pericyte-free fraction of tumor blood vessels may be little or none for a significant number of both experimental and human tumor types (10–14). As a consequence, additional targeting of pericyte recruitment and peri-cyte/endothelial cell interaction may enhance tumor vessel destruction after interference with VEGF/VEGFR-2 signaling.

Although the detailed molecular basis of how pericytes confer vessel stabilization remains un-known, studies of embryogenesis have highlighted the involvement of the angiopoetin (Ang)-1/Tie2 system and the platelet-derived growth factor (PDGF)-B/PDGF receptor (PDGFR)-β sys-tem. Transgenic mice lacking adequate Ang-1/Tie2 or PDGF-B/PDGFR-β signaling failed to adequately recruit pericytes to newly formed blood vessels, which was associated with severe perturbation of blood vessel stabilization and maturation (15–18). Furthermore, interference with PDGF-B/PDGFR-β signaling resulted in disruption of already established endothelial/pericyte associations and vessel destabilization during retinal development (19). Therefore, the use of inhibitors of the PDGFR-β tyrosine kinase should provide a novel means to interfere with peri-cyte function during tumor angiogenesis (20–22).

To test our hypothesis that simultaneous targeting of endothelial cells and pericytes in tumors may enhance enforced tumor vessel regression, we compared the effects of selective VEGFR-2 inhibition vs. VEGFR-2 plus PDGFR-β inhibition on the established microvasculature of C6

tumor xenografts. We show that VEGFR-2 targeting results in activation of endothelial cell sur-vival mechanisms by pericytes, thereby, providing escape strategies from enforced tumor vessel regression. However, this resistance to tumor vessel regression can be overcome when both VEGFR-2 plus PDGFR-β are simultaneously inhibited, resulting in tumor specific endothelial cell apoptosis, blood vessel destabilization and regression, and, finally, tissue hypoxia. Here, we provide evidence that the pericyte/endothelial cell interaction represents a dynamic process that may counteract anti-vascular interventions targeting endothelial cell survival.

MATERIALS AND METHODS

Mice

Athymic nude mice (nu/nu; male/female) were bred and maintained within a specific pathogen germ-free environment and were used at 6-10 wk of age. Experiments were performed in accor-dance with the approved institutional protocol and the guidelines of the Institutional Animal Care and Use Committee.

Experimental tumor model

C6 rat tumor cells were grown and stained with DiI as described previously (23). As implanta-tion sites for the C6 cells, we used the striated skin muscle within the dorsal skin fold chamber preparation of nude mice (23–25). C6 cells were implanted as cell suspensions (5x105 tumor cells in 1.5 µl PBS; ref 24).

Intravital fluorescence videomicroscopy

Intravital fluorescence videomicroscopy was performed as described previously in detail (23, 24, 26). DiI-labeling of glioma cells allowed for precise delineation of the tumor mass from the ad-jacent host tissue applying green light epi-illumination (520-570 nm). Contrast enhancement with FITC-conjugated dextran (molecular weight=150,000; 0.1 ml iv via the tail vein) and use of blue light epi-illumination (450-490 nm) allowed us to visualize individual tumor blood vessels. Intravital microscopic evaluations included angiogenic sprouts, newly formed perfused and non-perfused microvessels, and microvascular extravasation of FITC-conjugated dextran. For quanti-tative analysis, the vascular network of the tumor was assessed in six to nine separate microvas-cular regions that were evenly distributed within the tumor (24). To assess the extent of tumor vessel regression, the identical microvascular regions were compared before and over the time course after initiation of treatment.

PO2 measurements by phosphorescence quenching

Noninvasive intravascular and interstitial PO2 measurements were performed in vivo using a novel automated system for oxygen tension analysis based on the phosphorescence quenching methodology (27). The system provides instantaneous digital readings of phosphorescence life-time and corresponding PO2, whose relationship is described in the Sten-Volmer-Eq. (28). Phos-phorescence was exited by a xenon strobe arc (Strobe MVS-2614 E, EG&G Optoelectronics, Salem, MA) attached to the Zeiss microscope. PO2 measurements were made using a water im-mersion objective (x63, Carl Zeiss GmbH, Jena, Germany) and trans-illumination technique

(Schott KL 750, Leica, Bensheim, Germany). For each individual measurement, a total of 60 light flashes at a rate of 30 Hz were used. A filter of 630 nm was used to provide maximum por-phyrin excitation. Emitted phospherescence signals passed a filter of 420 nm and were measured by a photomultiplier (Hamamatsu Photonics, Herrsching, Germany). Averaged signals were also visualized on an oscilloscope (Tektronix TDS 210, Tektronix Inc., Beaverton, OR) and trans-ferred to an automated PO2 analyzer (model 802, Vista Electronics Company, Ramona, CA), which directly converted phosphorescence decay rates into corresponding PO2 readings. For PO2 measurements, the anesthetized and spontaneously breathing mice were temperature controlled between 36 and 37°C using a heating pad. Palladium-meso-tetra (4-carboxyphenyl) porphyrin was injected at a dose of 15 mg/kg body weight via the tail vein. Intravascular oxygen tension measurements were initiated ~1-2 min after injection, while interstitial tumor tissue PO2 was measured after a period of 10-15 min, allowing enough porphyrin to extravasate for sufficient extravascular phosphorescence signal strength (27). According to the different dimensions of blood vessels, the sampling size for intravascular measurements varied between 5 to 20 × 30 µm. For interstitial PO2 tissue measurements, a sampling area of 100 × 100 µm was chosen. Thus, interstitial PO2 reflected the mean interstitial oxygen tension within the tumor mass. Each PO2 measurement in the selected microvessels and interstitial tissue segments was repeated five times. Per tumor, 10-15 different intravascular and interstitial sites were chosen for PO2 meas-urements.

Inhibition of VEGFR-2 and PDGFR-β signaling

Signaling of VEGFR-2 and PDGFR-β was inhibited in vivo by the small molecule tyrosine kinase inhibitors SU5416 (3) and SU6668 (21) (both SUGEN, Inc., South San Francisco, CA). Although SU5416 has been shown to selectively inhibit the function of VEGFR-2 [IC50 in cellu-lar assays: VEGFR-2=1 µM; PDGFR-β=20 µM; FGFR-1>50 µM; IGFR>100 µM; Her2>100 µM), SU6668 potently inhibits the function of both VEGFR-2 and PDGFR-β (IC50 in cellular assays: VEGFR-2=2.6 µM; PDGFR-β=1 µM; FGFR-1=10 µM; IGFR>100 µM; Her2>100 µM (3, 21, 29, 30) and SUGEN, unpublished data]. In pharmacokinetic studies, the drugs have a short (30 min) plasma half-life in both mice and rats (29). In contrast, they are characterized by long-lasting (at least 72 h) inhibition of the VEGF-signaling after a short exposure, suggesting a much longer biological than pharmacological half-life (29). At the applied doses, both inhibitors have been shown to comparably inhibit VEGF-dependent signaling in vivo and to block forma-tion of new tumor vessels in mice with comparable efficacy (21, 31). Consequently, the distinct target profiles of the inhibitors as well as their pharmacokinetics enabled us to compare targeting of endothelial cells via VEGFR-2 (SU5416) vs. targeting of endothelial cells plus pericytes via VEGFR-2 and PDGFR-β (SU6668).

Experimental protocol

After implantation of tumor cells into the dorsal skin fold chamber preparation, we allowed the tumors to establish their neovasculature and to initiate growth (24). From day 10 to day 18 after implantation, animals were treated with SU5416 (n=10 animals; 25 mg/kg/d in 50 µl DMSO ip) (3) or SU6668 (n=5 animals; 75 mg/kg/d in 50 µl DMSO ip). Animals receiving the solvent only served as controls (n=4; 50 µl DMSO ip). To study tumor vessel regression, intravital fluores-

cence microscopy was performed before and at 12, 24, 36, and 48 h and 4, 8, and 12 days after treatment initiation.

In a separate group (n=4 animals), mice treated with SU6668 additionally received the active caspase inhibitor ZVAD-fmk (BACHEM Biochemica, Heidelberg, Germany) (32, 33) to study the involvement of endothelial cell apoptosis in tumor vessel regression. ZVAD-fmk was admin-istered daily intraperitoneal at a dose of 0.25 mg dissolved in DMSO.

To analyze the consequence of tumor vessel regression on vascular oxygen supply and tumor oxygenation, intravascular and interstitial PO2 measurements were performed in tumors treated for 4 days with SU6668 (n=4) or solvent (n=4) applying the phosphorescence quenching tech-nique.

Tissue preparation

In parallel to the intravital microscopic experiments, additional animals from all experimental groups were killed and their tumors as well as control skin fold tissue were processed for histo-logical analysis. The tumor containing skin fold and distant, nontumor bearing skin fold tissue were dissected free en bloc. Specimens for electron microscopic analysis were fixed with 2% glutaraldehyde buffered with 0.12 M sodium cacodylate (pH 7.4). For the other histological and immunohistochemical analyses, specimens were snap-frozen in liquid nitrogen-cooled isopen-tane. Sections were cut, mounted on slides precoated with silane (Sigma, Munich, Germany), and acetone-fixed (10 min at 20°C).

Immunohistochemistry

Five micromolar cryosections were stained with hematoxylin and eosin (Merck, Darmstadt, Germany); 6–8 µm cryosections were stained for PDGFR-β (Santa Cruz Biotech., Heidelberg, Germany) or desmin (DAKO, Hamburg, Germany) to detect pericytes and for CD31 to identify endothelial cells. Desmin staining was performed as double fluorescence immunostaining in combination with the CD31 staining. For conventional immunohistochemsitry, sections were incubated with the primary antibodies at 4°C overnight (anti-PDGFR-β: 1:50, PECAM 1:100) after nonspecific reactivity was blocked with goat serum (DAKO). Then, biotinylated goat anti-rabbit serum (DAKO) was added for 60 min at room temperature. After being washed, the re-agents of the ABC kit (Vectastain, Vector, Burlingame, CA) were added. Finally, sections were mounted in glycerin gelatine. Double fluorescent immunostaining was performed sequentially. After nonspecific reactivity was blocked with 0.5% casein in PBS, sections were incubated with the primary antibody against desmin (1:50) at 4°C overnight. After being washed repeatedly, sections were incubated with biotinylated secondary antibody and diluted 1:200 (Vector) and subsequently with Avidin-FITC, 1:400 (Vector). The antibody against CD31 was applied for 1 h at room temperature, and a Cy3-conjugated secondary antibody, diluted 1:100 (Chemicon, Hof-heim, Germany), was used for detection. Sections were mounted in MOWIOL with DAPI as a counterstain.

TUNEL staining

Cryosections were permeabilized with Triton X100 (Sigma-Aldrich, Taufkirchen, Germany) at 0.1% in PBS for 10 min at room temperature. After a 30-min incubation at room temperature with 3% BSA in PBS, sections were covered with the 50 µl TUNEL-Mix (0.2M Kaliumcacody-late, 25 mM Tris-HCl, pH 6.6; 0.25 mg/ml BSA, 1 mM CoCl, 25 U TdT (Roche, Mannheim, Germany); 0.006 mM fluorescein d-UTP (AP-Biotec, Freiburg, Germany); 0.6 mM dATP) for 1 h at 37°C. After another saturation for 30 min at room temperature with 3% BSA in PBS, the sections were treated with an anti-fluorescein-Fab′-fragment, POD-conjugated (Roche). Reaction with 0.05% 3-3′-diaminobenzidine (DAB) was monitored under the microscope until sufficient staining was achieved. Sections were counterstained with hematoxylin and mounted with el-vanol.

Electron microscopy

The postfixation was performed in three steps using 1% osmium tetroxide, 1% tannic acid, and 1% uranyl acetate (34). After dehydration in ethanol, specimens were critical-point-dried and sputtered with gold (5 nm thick). Specimens were finally embedded in Araldite 502. The ultra-thin sections were stained with lead citrate and examined in a Zeiss EM 10 CR transmission electron microscope at 60 kV.

RT-PCR

Semiquantitative evaluation of rVEGF, rPDGF-B, rbFGF, and mVEGFR-2, mVEGFR-1, mTie1, mTie2, mAng-2, mFGFR-2, and mPDGFR-β expression was assessed by means of RT-PCR, using species-specific primers. The rationale behind using species specific primers was to differ-entiate mRNA expression of the host tissue (mouse) from the tumor tissue (rat). In addition, RT-PCR was also performed to assess Ang-1 expression. In this case, however, we were not able to generate a species-specific probe. Total RNA was isolated from tumor tissue using the RNAEasy-Kit (Qiagen; Hilden, Germany) and subjected to reverse transcription using poly-dT-Primers. Single-stranded cDNA was used for PCR amplification, according to standard protocols (for annealing temperatures and MgCl2-concentrations see primer list), to detect the expression of receptors and ligands. PCR products were separated on 1.5% agarose gels and stained with ethidium bromide. Serial dilutions of the cDNA template were used to evaluate PCR conditions to assure nonsaturating PCR amplifications. Quantification of PCR products was performed by densitometric analysis on ethidium bromide stained agarose gels using a Fuji LAS scanner and the AIDA 2.11 software package. Relative levels of the expression of a specific RT-PCR product were determined by taking the ratio between the densitometric unit of the specific product and that of the internal control, GAPDH and/or aldolase. Graphical representations are given as rela-tive changes to the expression observed in untreated tumors. The RT-PCR experiments were performed in triplicates.

The primer sequences were as follows (target, sequence 5′-3′, annealing temperature, MgCl2):

rVEGF fw: ACC TCC ACC ATG CCA AGT, rv: TAG TTC CCG AAA CCC TGA, 59°, 1.5 mM; rPDGF-B fw: GAA GCC AGT CTT CAA GAA GGC CAC, rv: AAC GGT CAC CCG AGT TTG AGG TGT, 48°C, 1.5 mM; rbFGF-2 fw: TGT TTC TTC TTT GAA CGC CT, rv:

GCT AGG CTA CTA CTA TAC GG, 57°C, 1.5 mM; mVEGFR-2 fw: GGA ATT CAG GCA TTG TAC TGA GAG, rv: CGG ATC CAA GTT GGT CTT TTC CTG, 62°C, 2.5 mM; mVEGFR-1 fw: GGA AAC CAC AGC AGG AAG ACG, rv: GTC AGC CAC CAC CAA TGT GC, 60°C, 2.0 mM; mTie1 fw: GAA CCT GAA GCC AAA GAC AGG, rv: CTG TCT ACA TCC ATC CAC TGT GG, 62°C, 1.5 mM; mTie2 fw: CAA CAG CGT CTA TCG GAC TCC, rv: GAA AAG GCT GGG TTG CTT GAT C, 64°C, 2.0 mM; Ang-1 fw: CCA CCA TGC TTG AGA TAG GAA CC, rv: CTG TGA GTA GGC TCG GTT CCC, 64°C, 1.5 mM; mAng-2 fw: GTG GTG CAG AAC CAG ACA GCT G, rv: CAC TTC CTG GTT GGC TGA TGC, 64°C, 2.0 mM; mFGFR-2 fw: ATG TTG AAG TCC GAC GCA AC, rv: TTG CCT TTG GAG GCG TAC TC, 53°C, 2.0 mM; mPDGFR-β fw: ACA GTG AGG ACA GAC GTC CC, rv: AAT GTG GGT TAT CTC GAG TC, 57°C, 2.0 mM.

Northern blotting for Ang-1

Total RNA was isolated by using the RNA-easy-Kit (Qiagen, Hilden; Germany). For Northern blotting, 2.0 µg of total RNA was electrophoresed in 1% formaldehyde-denatured agarose gel in 1 × 3-(N-morpholino) propanesulfonic acid buffer, transferred onto Hybond-N membrane (Am-ersham Pharmacia Biotech, Buckinghamshire, UK) by vacuum blotting, and fixed with UV-Stratalinker (Stratagene, La Jolla, CA). A mouse cDNA fragment of Ang-1 (790 kb, Ncts.: 680-1470, Gene Bank Accession No.: NM_009640) was cloned into the pCR4-Topo-vector (Invitro-gen, Karlsruhe, Germany). Plasmids were linearized by digestion with the appropriate restriction enzymes, and in vitro transcription was performed using a DIG RNA labeling Mix (Roche). Un-incorporated labeled-rUTP was removed by LiCl-precipitation. Blots were prehybridized with Dig Easy Hyb Solution (Roche) at 68°C for 15 min and hybridized with specific probes at 68°C overnight. After hybridization, the filters were washed twice with 2x SSC at room temperature for 15 min and twice with 0.1x SSC/0.1% sodium dodecyl sulfate (SDS) at 68°C for 15 min. Chemiluminescent detection was performed using CSPD (Roche) as a substrate with a Fuji LAS scanner. The equality of loading for Northern blotting was confirmed by visualizing 28S and 18S rRNA.

In situ hybridization

In situ hybridization was performed on 8-10 µm cryosections. Probes were labeled with DIG-11-UTP (Roche, Mannheim, Germany) by in vitro transcription using a 312 bp-cDNA-fragment of Ang-1 (Ncts.: 3155-3467, Gene Bank Accession No. U83508). Labeled cRNA probes were used at a concentration of 100 ng RNA/µl. Hybridization with sense probes served as a control. Sec-tions were fixed for 20 min at room temperature in 4% paraformaldehyde (freshly prepared) buffered with 2x SSC, followed by dehydration through ethanol (60, 80, 95, and 100% ethanol, 5 min each). Pretreatment with proteinase K (Roche) at 0.1–0.2 µg/ml in 2x SSC, 0.1% SDS was for 30 min at 37°C. Digestion was stopped with 0.2 M glycine/2x SSC, and the sections were postfixed for 5 min in 4% PFA. After being washed, an acetylation step was introduced to de-crease background hybridization (0.1 M triethanolamin, 0.25% acetic anhydride; Fluka, Taufkirchen, Germany). Hybridization took place in a humified chamber at 50°C for 12-36 h, and sections were covered with parafilm. After hybridization, the sections were washed two times for 30 min at 50°C with 50% formamide, 2x SSC, RNase-A digested (20 µg/ml in 2x SSC, 0.1% SDS, 30 min; 37°C), and finally washed with 50% formamide, 0.2x SSC. Detection of hy-

bridized probes was performed using an anti-digoxygenin antibody conjugated to alkaline phos-phatase (Roche; 1:250, 1 h at 37°C) and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution as substrate (Roche). Color development was for 2 to 12 h. Finally, sections were rinsed in PBS and counterstained with methyl green, rinsed in aqua dest., and mounted in glycerin gelatine.

Statistics

Quantitative data are given as mean values ± SD. Mean values from the intravital microscopic experiments, PO2 measurements, and RT-PCR experiments were calculated from the average values in each animal. For analysis of differences between the groups, one-way ANOVA (ANOVA) followed by the appropriate post hoc test for individual comparisons between the groups was performed. Results with P < 0.05 were considered significant and are marked with an asterisk.

RESULTS

Selective VEGFR-2 inhibition fails to induce regression of tumor vessels

Although SU5416 has been previously shown to effectively prevent neovascularization in the C6 xenograft model (31), it failed to induce regression of already established tumor blood vessels (Fig. 1A-C). In fact, we did not observe changes in blood vessel density, blood vessel diameter, permeability, or microvascular architecture of SU5416-treated tumors. In contrast, SU5416 sig-nificantly suppressed further neovascularization and, concomitantly, tumor expansion (Fig. 1C). These results clearly indicate that selective targeting of VEGFR-2 represents a potent means to prevent new blood vessel formation but fails to force already established tumor vessels into re-gression.

Pericytes protect tumor vessels from enforced regression

To address the mechanisms underlying the resistance of established tumor blood vessels to re-gression after interference with VEGF signaling, we first determined the fraction of mature tu-mor blood vessels, i.e., blood vessels associated with pericytes, using double fluorescence im-munostaining for the endothelial cell marker CD31 and the pericyte marker desmin. As shown in Fig. 2A and C, the majority of blood vessels in the control tumors was associated with pericytes, resulting in a maturation index of 80%. Treatment with SU5416 slightly further increased this fraction of mature vessels (Fig. 2B and C). This abundant association between pericytes and en-dothelial cells was confirmed by electron microscopy (Fig. 2D and E). Furthermore, electron microscopy revealed an important difference between control and treated tumors. Whereas blood vessels of control tumors were characterized by a loose and improper coverage of blood vessels by pericytes, blood vessels of SU5416-treated tumors demonstrated an intimate pericyte-endothelial cell association. In view of the fact that SU5416 treatment did not induce regression of immature vessels (≈20% in control tumors), these results indicate that, as a consequence of VEGFR-2 targeting, pericytes were recruited to the tumor and intensified their coverage and, thus, stability to tumor blood vessels. With the aim to further characterize these pericytes and to identify a tyrosine kinase-based target for a selective anti-pericyte intervention, we performed an

immunostaining for PDGFR-β, which confirmed expression of PDGFR-β in perivascular cells but not in tumor cells (Fig. 2F).

To gain insight into the signaling mechanisms underlying tumor blood vessel stabilization and pericyte recruitment, the expression of angiogenic growth factors and their cognate receptors in control and SU5416-treated tumors was analyzed by RT-PCR and Northern blotting. Whereas expression of the VEGF and FGF systems remained largely unchanged, expression of PDGFR-β and Tie2 were nearly doubled and expression of Ang-1 was increased by ninefold in SU5416-treated tumors (Fig. 3A-C). This change in the angiogenic expression profile is also reflected in the Ang-1:Ang-2 ratio, which was 1:11 in control tumors and turned into 8:1 in SU5416-treated tumors. By using species-specific primers (except for Ang-1), the increased expression of PDGFR-β and Tie2 was clearly attributable to pericytes and endothelial cells (i.e., cells of mouse origin), respectively. In contrast, based on the PCR data (due to an inability of the Ang-1 mRNA probe to differentiate mouse from rat), the source of Ang-1 expression remained equivocal. To further localize the increased Ang-1 expression, we performed in situ hybridizations of control and SU5416-treated tumors. These experiments demonstrated that Ang-1 was predominately expressed by perivascular cells and only to a lower extent by tumor cells (Fig. 3D and E). Con-sequently, pericytes not only stabilized tumor blood vessels by cell-to-cell contact but also pro-tected tumor blood vessels from regression by providing Ang-1 signaling, which mediates endo-thelial cell survival (35).

Next, we addressed the question whether this up-regulation of Ang-1 in pericytes after VEGFR-2 targeting was tumor specific or whether a similar increase in Ang-1 mRNA expression could be observed in normal skin also. In parallel to the expression anaylsis on tumor specimens, we therefore performed RT-PCR and in situ hybridization analysis for Ang-1 on tumor-free skin fold preparations of mice that were treated with the vehicle alone or with SU5416. However, by none of these means we could detect significant Ang-1 mRNA levels in normal skin, neither in control nor in treated tissue.

Inhibition of VEGFR-2 plus PDGFR-β induces tumor-specific blood vessel regression

At this point, we hypothesized that a simultaneous targeting of endothelial cells and pericytes may provide more effective means to enforce regression of mature tumor blood vessels. To test this, we treated C6 xenografts with SU6668, a tyrosine kinase inhibitor that not only potently targets VEGFR-2 (like SU5416) but also PDGFR-β. Under these conditions, tumor blood vessels rapidly regressed within 24 h (Fig. 4A-C). As demonstrated by sequential intravital fluorescence microscopy, vascular sprouts were most susceptible to SU6668 treatment, showing disintegration and destruction within a few hours after initiation of treatment (Fig. 4D). This was followed by destabilization and regression of already established and perfused tumor blood vessels (Fig. 4E). As demonstrated by Fig. 4A-E, regression of tumor blood vessels was accompanied by an in-crease in microvascular permeability and by microvascular hemorrhage, characterized by the extravasation of the high molecular weight FITC-dextran and the escape of red blood cells from blood vessels, respectively. In contrast to the tumor blood vessels undergoing rapid regression, blood vessels of the host tissue were unaffected by the treatment with SU6668 (Fig. 4F and G). The quantitative assessment of tumor vessel density revealed that inhibition of VEGFR-2 plus

PDGFR-β forced 40% of tumor blood vessels into regression without changing the morphology of the surviving vessels (Fig. 4H).

To test the relevance of tumor blood vessel regression in terms of interference with nutritional blood supply, we next addressed changes in tumor tissue oxygenation after SU6668 treatment. Therefore, intravascular and interstitial PO2 levels were noninvasively assessed (Table 1). Using the phosphorescence quenching technique, we were able to show that whereas the vascular oxy-gen supply remained unchanged upon SU6668 treatment (PO2=24.7±3.0 vs. 23.9±8.0 mmHg in controls), oxygenation of the tumor tissue was found significantly reduced in SU6668-treated tumors (PO2=10.1±1.8 vs. 17.2±3.3 mmHg in controls, P<0.05), reflecting a substantial mi-crovascular perfusion and nutritional supply deficit after tumor vessel regression.

Due to the combination of these anti-vascular and anti-angiogenic effects of SU6668, tumor growth was significantly suppressed compared with controls (Fig. 4H). It is noteworthy that these results were obtained only by VEGFR-2 plus PDGFR-β targeting. In contrast, inhibition of VEGFR-2 plus FGFR-1 using the tyrosine kinase inhibitor SU5402 (36) failed to induce regres-sion of tumor blood vessels (data not shown).

Regression of tumor blood vessels involves endothelial cell apoptosis and detachment

Previous studies have demonstrated that SU6668 regresses tumor blood vessels by inducing en-dothelial cell apoptosis and detachment (30). The results of our TUNEL staining and electron microscopy demonstrated that this also applied to our tumor model (data not shown). To further confirm this mechanism of tumor blood vessel regression, SU6668-treated animals received the active caspase inhibitor ZVAD-fmk, which abrogated regression of tumor blood vessels (vessel density on day 8 after initiation of treatment: 220±100 cm-1 in treated animals vs. 225±53 cm-1 in controls). Interestingly, this result not only confirmed that regression of tumor blood vessels in-volves endothelial cell apoptosis but also demonstrated that blood vessel regression after with-drawal of VEGF signaling can be pharmacologically prevented.

Inhibition of VEGFR-2 plus PDGFR-β interferes with pericyte-mediated endothelial cell survival mechanisms

To understand the distinct effects of selective VEGFR-2 targeting vs. VEGFR-2 plus PDGFR-β targeting, we next studied the pericyte-endothelial cell interaction in SU6668-treated tumors. Double fluorescence immunostaining for CD31 and desmin showed that the majority of SU6668-treated tumor blood vessels (93±6%, i.e., slightly increased compared with SU5416-treated tu-mors) remained in association with pericytes (Fig. 5A). Noteworthy, our experiments did not reveal a pericyte drop-off from tumor blood vessels as previously suggested as a mechanism of vessel destabilization after SU6668 treatment (37). Immunohistochemistry further confirmed that these pericytes expressed PDGFR-β at a level comparable to SU5416-treated tumors. Also, the angiogenic expression profile after SU6668 treatment was comparable to SU5416-treated tumors (Fig. 5B and C vs. Fig. 3A-C). Again, RT-PCR analysis and Northern blotting revealed that ex-pression of PDGFR-β and Tie2 were nearly doubled and expression of Ang-1 was increased by sevenfold. These results suggest that the amount of PDGFR-β-positive perivascular cells was comparable in SU5416-treated and SU6668-treated tumors and that perivascular cells continued

to express increasing amounts of Ang-1 mRNA despite the additional inhibition of PDGFR-β by SU6668. The latter was confirmed by in situ hybridization results.

Finally, the clue of how VEGFR-2 plus PDGFR-β targeting forced tumor blood vessels into re-gression came from our electron microscopic studies. As shown in Fig. 5D and E, these studies demonstrated that although pericytes were still present, their association with endothelial cells in SU6668-treated tumors was less intimate than in the SU5416 group, suggesting that SU6668 negatively influenced the pericyte-endothelial cell interaction. Further evidence for a negative influence of VEGFR-2 plus PDGFR-β targeting on the pericyte-endothelial cell interaction could be derived from studying the endothelial cell morphology in these tumors. Similar as previously demonstrated for transgenic mouse embryos that lack pericyte-endothelial cell interaction during vascular development due to a defect in PDGF/PDGFRR-β signaling (18), tumor blood vessels in SU6668-treated tumors presented with endothelial hyperplasia and an increased number of luminal folds (Fig. 5C and D; compare with Fig. 2D). Ultrastructural analysis of normal blood vessels in the nontumor bearing skin fold confirmed that SU6668 had no effect on the physio-logical pericyte/endothelial cell association.

DISCUSSION

In the present study, we demonstrate that selective interference with VEGFR-2 signaling fails to enforce regression of established tumor blood vessels. This resistance of tumor blood vessels to selective VEGFR-2 inhibition is conferred by pericytes that 1) initially stabilize matured vessels, 2) are secondarily recruited to immature vessels upon therapy, and 3) express compensatory en-dothelial cell survival factors (e.g., Ang-1). In contrast, inhibition of VEGFR-2 plus PDGFR-β, which simultaneously targets endothelial cells and pericytes, acts as a potent anti-vascular strat-egy, inducing endothelial cell apoptosis, tumor vessel destabilization and regression, and finally tumor hypoxia. Beyond these therapeutic aspects, our study provides novel insights into the mechanisms of how pericytes differentially promote blood vessel stabilization and maturation via the Ang-1/Tie2 and PDGF-B/PDGFR-β systems.

The C6 tumor model has been extensively characterized with respect to tumor angiogenesis and tumor microcirculation. Recent work has shown that C6 tumors are highly angiogenic and that their growth depends on the formation of new tumor blood vessels (24, 38). After implantation, C6 cells induce vascularization via sprouting angiogenesis within a few days and establish a functional microvasculature within 1 wk after implantation (24). Expression analyses have shown that C6 tumor angiogenesis is primarily mediated by VEGF, which is expressed by the tumor cells and which activates VEGFR-2 on endothelial cells of host and tumor blood vessels. Simultaneously, in a coordinate fashion with VEGFR-2 expression, Ang-2 is up-regulated by activated host and tumor endothelial cells, rendering the blood vessels into a state of pro-angiogenic plasticity and VEGF-responsiveness (25). At the same time, Ang-1 expression is 11-fold lower (as demonstrated in this study). In accordance with the predominant expression of the VEGF/VEGFR-2 system, selective inhibition of VEGFR-2 function using SU5416 has been shown to efficiently suppress C6 tumor angiogenesis and C6 tumor initiation (31).

In contrast to the successful prevention of tumor angiogenesis and tumor growth, selective inhi-bition of VEGF/VEGFR-2 signaling using SU5416 failed to interfere with endothelial cell sur-

vival and to enforce regression of already established tumor blood vessels in the present study. This was unexpected, because VEGFR-2 not only mediates endothelial cell proliferation but also endothelial cell survival (7) and VEGF withdrawal has been previously shown to ablate imma-ture tumor vessels using a tetracycline-regulated expression system for VEGF (6). This resis-tance to vessel regression could in part be explained by the high maturation index of C6 tumor blood vessels (≈80%), defined as the fraction of vessels that are associated with pericytes. It should be noted that the maturation index in our C6 tumors was much higher when compared with previous studies where the maturation index for C6 xenografts was determined to be as low as 30% (6). This difference may be attributable to the use of different pericyte markers and the strong overexpression of VEGF in C6 cells using the tetracycline-regulated expression system (6), which may lead to a disbalanced rate of vessel formation and vessel maturation when com-pared with our C6 wild-type cells.

The findings of our study are in agreement with a previous report on the beneficial effects of targeting endothelial cells and pericytes using SU6668 and SU5416 (37). In this report, using the spontaneous pancreatic islet tumor model of RIPTag2 mice, the authors demonstrated that a combined targeting of VEGF and PDGF signaling disrupts the association of pericytes with en-dothelial cells, reduces the tumor vascularity, and results in improved tumor control compared with targeting of VEGF signaling alone. However, the mechanisms of how pericytes protect en-dothelial cells from VEGFR-2 targeting have remained unclear.

Our study now extends these previous findings and provides the cellular and molecular mecha-nisms of how pericytes may protect tumor endothelial cells during targeting of VEGF signaling. As demonstrated, vessel maturation at the initiation of treatment is not the sole determinant of resistance to enforced tumor blood vessel regression. Our study revealed at least two further mechanisms of how pericytes may confer resistance to targeted tumor blood vessels in vivo. First, pericytes were recruited to immature vessels and formed an intimate association with endo-thelial cells, thereby stabilizing these vessels through an enhanced cell-to-cell contact. Second, our expression analyses using RT-PCR and in situ hybridization suggested that the pro-apoptotic effect of selective VEGFR-2 targeting may be overwhelmed by the increased activity of endothe-lial cell survival factors other than VEGF. Interestingly, this strategy has recently been proposed as one potential mechanism of acquired resistance to anti-angiogenic therapies (39).

The Ang-1/Tie2 system, which has been up-regulated during SU5416 treatment, seems to play a central role in both ways. On the one hand, Ang-1 overexpression enhances the pericyte cover-age and increases the vessel maturation index in experimental tumors by a yet unknown mecha-nism (40). On the other hand, Ang-1 dependent activation of Tie2 has been shown to exert simi-lar anti-apoptotic properties as VEGF, acting as an alternative survival factor for endothelial cells via the same signaling cascade, i.e., PI3K and Akt (35). Our finding that pericytes are the primary source of Ang-1 expression is further supported by a recent study demonstrating Ang-1 expression by pericytes during vessel maturation (41).

Only little is known about the molecular interplay of VEGFR-2 and Ang-1 as well as the mecha-nism underlying the up-regulation of Ang-1 in pericytes after VEGFR-2 inhibition in endothelial cells as observed in our study. Recently, hypoxia has been suggested as one putative stimulator of Ang-1 and Tie2, but not of Ang-2, expression in pericytes (42). If this holds true, an initial

induction of hypoxia after delayed anti-angiogenic therapy may be sufficient to stimulate peri-cytic Ang-1 expression. On the other hand, these findings are contradictory to the concept that a simultaneous expression of VEGFR-2 and Ang-2, but not Ang-1, represents the angiogenic phe-notype of tumors (25). Therefore, an alternative hypothesis would be that activation of VEGFR-2 results in a (yet unidentified) negative signaling to pericytes suppressing their expression of Ang-1. In this case, inhibition of VEGFR-2 would result in interruption of this inhibitory signal-ing pathway.

Besides Ang-1/Tie2, the PDGF-B/PDGFR-β is the second important signaling system mediating pericyte-endothelial cell interaction. So far, it remains unknown how Ang-1/Tie2 and PDGF-B/PDGFR-β exactly interact in mediating vessel maturation. The phenotypic similarities of transgenic mice lacking either one of these signaling pathways suggest that they most likely act in concert to realize stabilization and maturation of newly formed blood vessels during vascular development (15–18). The massive vessel regression that we observed after VEGFR-2 plus PDGFR-β targeting indicates that this may also apply to tumor angiogenesis and that Ang-1/Tie2 alone, in the absence of PDGF-B/PDGFR-β signaling, is not sufficient to maintain endothelial cell survival and tumor vessel maturation. Based on our results, PDGF-B/PDGFR-β signaling seems to be detrimental for an adequate pericyte-endothelial cell assembly, which in turn repre-sents the prerequisite for mediating the Ang-1/Tie2 survival signal to the endothelial cell.

Pericytes are believed to be either generated by in situ differentiation from mesenchymal cells (43) or by migration and dedifferentiation of arterial smooth muscle cells (19, 44). With the in-creasing awareness that they may play a central role in tumor angiogenesis and that they may determine the success of anti-angiogenic therapies, a great interest in identifying pericytes in tumor tissue specimens has developed. So far, pericytes have been either identified by their dis-tinctive shape and perivascular localization or by specific molecular markers such as α smooth muscle actin (α-SMA), desmin, NG2, nestin, or PDGFR-β (6, 14, 41, 45, 46). The major obsta-cle in pericyte identification has been, however, that expression of these markers can vary mark-edly in different tissue, vessel, and tumor types. Thus, lack of immunoreactivity for one single marker cannot be equated with a lack of pericytes. This notion is in line with our experience that staining for α-SMA and NG2, in contrast to the desmin and PDGFR-β staining, has proven in-sufficient for a reliable detection of pericytes in our C6 xenograft model when compared with the gold standard technique electron microscopy (unpublished data). Due to this heterogeneity in marker expression, the amount of pericyte coverage on tumor blood vessels (both under experi-mental and clinical conditions) has been reported to range from extensive (10–12) to little or none (8). Only recently, this controversy was convincingly addressed when pericyte-tumor blood vessel association was investigated by electron microscopy in combination with various immu-nohistochemical techniques, clearly demonstrating that the maturation index in experimental and human tumors may be as high as >95% (14). Interestingly, the same study has underscored the relevance of a proper physical pericyte-endothelial cell interaction (14).

It is important to note that combined targeting of VEGFR-2 and PDGFR-β had no effect on nor-mal blood vessels of the host tissue. Intravital flourescence videomicroscopy and electron mi-croscopy have demonstrated that in contrast to the tumor vasculature the integrity and function of host blood vessels as well as their pericyte-endothelial cell interaction remained unaffected after treatment with SU6668. Similar observations have been noted previously (37). Although

the explanation for this observation has to remain speculative, the resistance of normal blood vessels to VEGFR-2/PDGFR-β targeting points to a fundamental difference between tumor blood vessels and normal blood vessels. This is in line with our previous observation that physio-logical angiogenesis is resistant to blockade of in vivo VEGF/VEGFR-2 signaling (47). In con-trast to normal blood vessels, tumor blood vessels remain in a continuous state of plasticity and remodeling due to the steady release of pro-angiogenic growth factors (e.g., VEGF, Ang-2) that prevent final vessel maturation (25, 48). Our and Morikawa et al.’s (14) studies have shown that in contrast to normal blood vessels, pericytes of tumor vessels are only loosely associated with endothelial cells, therefore potentially failing to provide vessel maturation. Moreover, tumor blood vessels and normal blood vessels are characterized by a distinct gene expression pattern, suggesting a distinct target profile when considering anti-angiogenic therapies (49, 50). Taken together, these facts may explain the differential susceptibility of tumor and host blood vessels to anti-angiogenic therapies.

Consequently, an extended anti-angiogenic strategy, targeting endothelial cells and pericytes, represents an attractive approach in treating patients presenting with advanced tumors with es-tablished blood vessels. Especially because many of the endothelial-specific anti-angiogenic compounds seem to be effective in rather preventing tumor initiation than in successfully inter-fering with the progressed tumor mass (51–54). The VEGFR-2 plus PDGFR-β inhibitor SU6668 represents one example of a potent polyvalent tyrosine kinase inhibitor that not only prevents further angiogenesis but also provides a potent anti-vascular efficacy even resulting in regression of selected experimental tumor types (30). The present study, however, has not only provided insights into the mechanisms of enforced tumor vessel regression by VEGFR-2 plus PDGFR-β targeting but has also illustrated the microvascular consequences of its anti-vascular effects. Clearly, an important caveat to using similar inhibitors in a clinical setting is the acute and rapid destabilization of tumor blood vessels, which may lead to edema formation and vascular hemor-rhage. This may be especially detrimental in the treatment of brain tumors or in combining VEGFR-2 plus PDGFR-β targeting with radiation therapy, which already leads to transient ves-sel destabilization per se.

In summary, the results of this study suggest that a successful intervention in advanced tumors and long-term disease control with anti-angiogenic compounds can be best achieved with a com-bination therapy, targeting not only endothelial cells but also pericytes. Therefore, besides selec-tively targeting VEGF/VEGFR-2 as the pivotal pathway for angiogenesis, additional pathways involved in vascular stabilization and maturation should be included into the target profile.

ACKNOWLEDGMENTS

We thank S. Mohr, V. Powajbo, T. Korn, and N. Dietrich for excellent technical assistance. We are grateful to Peter Hirth, Laura Shawver, Gerald McMahon, and Julie Cherrington (all SUGEN Inc.) for providing us with the tyrosine kinase inhbitors. This study was supported by grants from the German Research Foundation (DFG SPP1069: VA151/4-2, UL 60/4-2, Ha 1755/3-2) and the European Union (BMH4-CT95-0875 to Peter Vajkoczy).

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Received April 29, 2003; accepted October 27, 2003.

Table 1 Noninvasive intravascular and interstitial PO2 measurements in C6 xenografts in controls and SU6668-treated animals, applying the phosphorescence quenching technique

Experimental Group Intravascular PO2 Interstitial PO2 Control 23.9 ± 8.0 mmHg 17.2 ± 3.3 mmHg SU6668 24.7 ± 3.0 mmHg 10.1 ± 1.8 mmHg*

Animals were treated with DMSO (control) or SU6668 for 8 days, starting on day 10 after implantation; n = 4 animals per group; *P < 0.05 vs. control.

Fig. 1

Figure 1. Selective targeting of VEGFR-2 fails to enforce regression of tumor blood vessels. A and B) Identical C6 tumor vasculature before (A) and 48 h after (B) initiation of SU5416 treatment. Bars = 50 µm. C) Quantitative analysis of tumor vessel density, tumor vessel diameter, and tumor size. Animals were treated with DMSO (n=4; 50 µl DMSO ip) or SU5416 (n=10 animals; 25 mg/kg/d in 50 µl DMSO ip). All parameters were analyzed off-line using a computer-assisted image analysis system. Means ± SD values are represented. Statistical analysis was performed using ANOVA followed by unpaired Student’s t test.

Fig. 2

Figure 2. Control and SU5416-treated tumors demonstrate a large fraction of mature blood vessels. A and B) Double fluoresecence immunostaining for CD31 (red) and desmin (green) in control (A) and SU5416-treated C6 tumors (B). Counterstain of tumor cell nuclei with DAPI (blue). Bars = 100 µm. C) Quantitative analysis of C6 tumor vessel maturation index, defined as the fraction of vessels (identified by CD31 staining) that are associated with pericytes (identified by desmin staining). Animals were treated with DMSO (n=4; 50 µl DMSO ip) or SU5416 (n=4 animals; 25 mg/kg/d in 50 µl DMSO ip). D and E) Ultrastructural analysis of pericyte-endothelial cell association in control (D) and SU5416-treated (E) C6 tumors. Whereas the pericyte-endothelial cell contact appeared to be relatively loose in control tumors, SU5416-treated tumors were charaterized by an intimate cell-to-cell contact. P, pericyte; EC, endothelial cell; T, tumor cell; RBC, red blood cell. F) Immunohistochemistry demonstrates expression of PDGFR-β by pericytes in SU5416-treated tumors. Inset shows tumor blood vessels associated with PDGFR-β-positive pericytes at higher magnification. Arrows indicate blood vessels with positive staining for PDGFR-β. Bar = 50 µm.

Fig. 3

Figure 3. Selective targeting of VEGFR-2 leads to upregulation of ANG-1 and PDGFR-β by pericytes. A) rt-PCR for ANG-1, PDGFR-b and GAPDH mRNAs from C6 tumors. Representative gel image of a control and SU5416-treated tumor. Experiments were performed in triplicate. B) Northern blot for ANG-1 mRNA from C6 tumors. Representative gel image of a control and SU5416-treated tumor. C) Angiogenic expression profile of SU5416-treated tumors relative to control tumors as assessed by rt-PCR using species-specific primers (m, mouse; r, rat). rt-PCR reactions were performed in triplicate and evaluated densitometrically. Quantification of PCR products was performed by densitometric analysis on ethidium bromide stained agarose gels. Graphical representations are given as relative changes to the expression observed in untreated tumors. D and E) In situ hybridization of control (D) and SU5416-treated C6 tumors (E) for ANG-1 mRNA demonstrates an increase in signal in pericytes after SU5416 treatment. In contrast, expression of ANG-1 in tumor cells is less pronounced. Arrows indicate detection of ANG-1 mRNA expression in perivascular cells covering two tumor blood vessels. Bars = 20 µm.

Fig. 4

Figure 4. Targeting of VEGFR-2 plus PDGFR-b induces enforced regression of tumor blood vessels. A-C) Overwiew of rapid regression of C6 tumor blood vessels within 24 h after initiation of SU6668 treatment. Bars = 100 µm. D) Vascular sprouts were most susceptible to SU6668 treatment, disrupting within 6 h after initiation of SU6668 treatment. Arrow indicates small hemorrhage from disrupted sprout. Bar = 50 µm. E) Destabilized tumor blood vessel 18 h after initiation of SU6668 treatment. Note extravasation of high molecular weight fluorescent marker and hemorrhage (indicated by arrow). Bar = 50 µm. D and E) Host blood vessels within the adjacent skin muscle (F) and subcutaneous tissue (G) remained largely unaffected by 18 h of SU6668 treatment, revealing tumor specificity of vessel regression. Arrows in F indicate perfused and still functional skin muscle capillaries. G) Capillary and postcapillary venule within the subcutaneous tissue. Bar = 50 µm. H) Quantitative analysis of vessel density, vessel diameter, and tumor size. Animals were treated with DMSO (n=4; 50 µl DMSO i.p.) or SU6668 (n=5 animals; 75 mg/kg/d in 50 µl DMSO i.p.). All parameters were analyzed off-line using a computer-assisted image analysis system. Means ± SD values are represented. Statistical analysis was performed using ANOVA followed by unpaired Student’s t test. *P < 0.05.

Fig. 5

Figure 5. Targeting of VEGFR-2 plus PDGFR-b interferes with pericyte-mediated endothelial cell survival mechanisms. A) Double fluoresecence immunostaing for CD31 (red) and desmin (green) in SU6668-treated C6 tumors reveals a preserved high maturation index of tumor blood vessels. Counterstain of tumor cell nuclei with DAPI (blue). Bars = 100 µm. B) rt-PCR for ANG-1, PDGFR-b, and GAPDH mRNAs from control and SU6668-treated C6 tumors. Gel image illustrates experiments performed in triplicate. C) Northern blot for ANG-1 mRNA from C6 tumors. Representative gel image of a control and SU6668-treated tumor. D and E) Ultrastructural analysis of pericyte-endothelial cell association in a SU6668-treated tumor reveals loose cell-to-cell contact as an indicator of disturbed pericyte-endothelial cell interaction. Also, note hyperplasia of endothelial cells and increased number of luminal folds (indicated by arrows).