Int. J. Cancer: 119, 423–431 (2006) ' 2006 Wiley-Liss, Inc.

Antiangiogenic combination tumor therapy blocking av-integrins and VEGF-receptor-2 increases therapeutic effects in vivo
Sebastian Strieth1,2*, Martin E. Eichhorn1,3, Arne Sutter4, Alfred Jonczyk4, Alexander Berghaus2 and Marc Dellian1,2 1 Institute for Surgical Research, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany 2 Department of Otorhinolaryngology, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany 3 Department of Surgery, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany 4 Merck KGaA, Darmstadt, Germany
Anti-angiogenesis is a promising strategy for cancer therapy currently evaluated in clinical trials. The aim of the study was to investigate the effects of an antiangiogenic combination therapy inhibiting av-integrins by a c(yclic)RGD-peptide (EMD270179) and blocking VEGFR-2 by SU5416 on tumor angiogenesis and progression in vivo. Experiments were performed in dorsal skinfold chamber preparations of Syrian golden hamsters (60 6 5 g) bearing A-Mel-3 tumors. From day 3–10 after tumor-cell implantation, animals (n 5 6 per group) were treated by monotherapies using the cRGD-peptide (114 mg/kg/day; i.p.), the VEGFR-2 antagonist (6 mg/kg/day; i.p.) or by the combination of both monotherapies. A control group received only the solvent DMSO. Using intravital microscopy parameters of intratumoral microcirculation were analyzed on day 5, 7 and 10. In separate experiments subcutaneous tumor growth and metastasis formation was evaluated starting therapy on day 0. Functional vessel density was significantly reduced by the combination therapy compared to that by all other groups on day 10. Although intratumoral red blood cell velocity and vessel diameters were less affected, blood flow in vessel segments and the microcirculatory perfusion index were lower after combined therapy compared to controls. In addition, we observed a significantly stronger inhibition of subcutaneous tumor growth and metastasis formation using the combination therapy. These data clearly support the concept of antiangiogenic combination therapy and demonstrate that it may especially be effective when scheduled as an early or prophylactic treatment regimen, thus avoiding angiogenesis-dependent tumor and metastasis initiation or tumor recurrence. ' 2006 Wiley-Liss, Inc. Key words: antiangiogenic combination therapy; RGD; SU5416; tumor microcirculation; intravital microscopy

Tumor growth has been shown to depend on angiogenesis, the formation of new blood vessels from the existing host microvasculature.1 Anti-angiogenesis as tumor therapy strategy promises to overcome some of the problems of conventional chemotherapy, since angiogenesis is a rather specific feature of tumor microvessels and acquired drug resistance is less likely to occur in the genetically stable endothelial cell.1 Nevertheless, extensive experimental efforts have proven evidence that in spite of initial tumor dormancy and continuous therapy, tumors treated by an effective anti-angiogenic monotherapy finally ended up in relapse.2 Considering numerous effective antiangiogenic monotherapies—many of them currently in clinical trials—it is rather surprising that there is still need of experimental evaluation of anti-angiogenic combination therapies.3 Conventional cancer chemotherapy today is relying on combination schedules of single cytotoxic substances to optimize efficacy. Comparably, anti-angiogenesis might benefit from the combination of monotherapies. Redundancy of proangiogenic growth factors expressed by tumor cells or antiapoptotic signaling to endothelial cells by certain of these factors may evoke resistance even to anti-angiogenic monotherapy. In addition, heterogenous vascular dependence of tumor cells may limit efficacy of anti-angiogenic treatment and favors the search for effective combination therapies.4 In line with these considerations are findings of additive effects of ‘‘metronomic’’ low-dose and thus antivascular chemotherapy and antiangiogenesis (for review see ref. 5). Furthermore, it is an ongoing matter of debate whether anti-angiogenesis induces functional changes of tumor microcircuPublication of the International Union Against Cancer

lation, resulting in a transient ‘‘normalization’’ of tumor perfusion6 responsible for synergistic effects after combination with radiation therapy.7 Comparably, it has been shown in clinical trials that antiangiogenesis can act synergistically with conventional chemotherapy.8 The VEGF/VEGF-receptor-2-system appears to be crucial in the regulation of tumor angiogenesis. One of the most effective kinase inhibitors selective for VEGFR-2 used to be the small molecular substance SU5416.9 But considering the rather disappointing outcome of phase II clinical trials using SU5416 as antiangiogenic monotherapy10,11 or in combination with conventional chemotherapy resulting even in thromboembolic complications12,13 or even combined with another antiangiogenic substance such as interferon a (with at least evidence of biological activity14) it may appear to be mandatory to preclinically modify antiangiogenic combination strategies or alter the dosage of this drug. Detailed knowledge of cellular mechanisms regulating tumor angiogenesis enables reasonable combinations of antiangiogenic monotherapies. Among possible strategies combining blockage of quite independent angiogenic pathways or combining ‘‘direct’’ and ‘‘indirect’’ inhibition of angiogenesis15 the following are most promising: ‘‘direct’’ angiogenesis inhibition interferes straightforward with the tumor endothelial cell, while ‘‘indirect’’ inhibition blocks proangiogenic signaling between the tumor cell and the tumor endothelial-cell compartment. Accordingly, SU5416 is to be classified as an ‘‘indirect’’ inhibitor. In contrast, cellular adhesion to a variety of extracellular matrix proteins containing Arg-Gly-Asp sequences mediated by av-integrins has been shown to prevent apoptosis/anoikis among tumor endothelial cells.16,17 These socalled RGD sequences (e.g. EMD121974 5 Cilengitide, EMD270179 as a cyclic compound) can be used to effectively inhibit tumor angiogenesis and progression17–19 and can thus be classified as ‘‘direct’’ inhibitors of angiogenesis. Furthermore, the regulation of angiogenesis by av-integrins and VEGF has been described in detail and reflects 2 distinct angiogenic pathways.20,21 Both monotherapeutic options blocking either pathway have shown efficacy in our tumor model.18,22 So, the aim of this study was to investigate the effects of a combined antiangiogenic treatment using inhibition of av-integrins by a cyclic RGD-peptide (EMD270179) and functional blockage of the VEGFR-2 by SU5416 on tumor angiogenesis, growth and metastasis in just the same and thus comparable in vivo tumor model. Our data clearly support the concept of antiangiogenic combination therapy because tumor angiogenesis in vivo is significantly inhibited by the combination of cRGD peptides (EMD270179) and SU5416. Our findings suggest that the combined antiangiogenic therapy using the inhibition of av-integrins and VEGF/ VEGFR-2 signaling may be especially beneficial when scheduled
Grant sponsor: Merck KGaA, Darmstadt, Germany. *Correspondence to: Department of Otorhinolaryngology, Klinikum Grosshadern, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich, Germany. Fax: 149-89-2180-76532. E-mail: sebastian.strieth@med.uni-muenchen.de Received 2 August 2005; Accepted 12 December 2005 DOI 10.1002/ijc.21838 Published online 13 February 2006 in Wiley InterScience (www.interscience. wiley.com).

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as prophylaxis or early intervention, thus avoiding angiogenesisdependent tumor and metastasis initiation or tumor recurrence. Material and methods Dorsal skinfold chamber preparation Experiments were performed with male Syrian golden hamsters (6–8-week old, 60 6 5 g b.w.). The animals were housed in single cages and had free access to tap water and standard laboratory food (ssniff, Spezialdiaeten GmbH, Soest, Germany) throughout the experiments, according to institutional and governmental guidelines. To permit quantitative fluorescence analysis of tumor microcirculation in vivo, a dorsal skinfold chamber consisting of 2 symmetrical titanium frames was surgically implanted into the dorsal skin as described earlier in detail.23,24 Following a recovery period of at least 24 hr from anesthesia and microsurgery, chamber preparations fulfilling the criteria of microscopically intact microcirculation were inoculated with 2 ll of dense tumor-cell suspension ($2 3 105 cells) of the A-Mel-3 amelanotic melanoma of the hamster onto the striated skin muscle layer. All surgical procedures were performed under anesthesia with ketamine (100 mg/kg b.w. i.p., Ketavet1; Parke-Davis, Berlin, Germany) and xylazine (10 mg/kg b.w. i.p., Rompun1; Bayer, Leverkusen, Germany). In vivo fluorescence microscopy Permanently indwelling fine polyethylene catheters (PE10, inner diameter 0.28 mm) were implanted into the right jugular vein on day 3 after tumor-cell implantation under general anesthesia, as described above. For intravital microscopy the awake chamber-bearing hamster was immobilized in a Perspex tube on an individually designed stage (Effenberger, Munich, Germany) under a modified Leitz microscope (Orthoplan; Leitz, Munich, Germany). FITC-labeled dextran (MW 500,000; 0.05–0.1 ml of a 5% solution in 9% NaCl; Sigma, Deisenhofen, Germany) as a plasma marker was injected intravenously to visualize tumor microcirculation. Selective observation of FITC-labeled plasma was possible using epiillumination with a 100 W mercury lamp attached to a Ploemopack illuminator with a Leitz I2/3 filter block (excitation 450–490 nm, emission ! 515 nm). In vivo fluorescence microscopy was performed on day 5, 7 and 10 after implantation of tumor cells. Six regions of interest per animal were randomly selected in the center and in the periphery of the tumor. Images were acquired by a SIT video camera (C2400-08; Hamamatsu, Herrsching, Germany) and recorded on S-VHS video tape for subsequent offline analysis. Analysis of microcirculatory parameters was performed by an image analysis system (Cap Image; Zeintl, Heidelberg, Germany). This system was described in detail by Zeintl et al.25 and Klyscz et al. 26 and allows measurement of functional vessel density (fvd) as a parameter of angiogenic activity. The blood flow in vessel segments (Q) was calculated according to the equation first described by Baker and Wayland27:  2 vRBC d 3 Q¼ 1:6 2 The microvascular perfusion index was calculated as follows: microcirculatory perfusion index ¼ 10À3 fvd3Q Subcutaneous tumor growth and onset of metastatic disease The dorsal skin of male Syrian golden hamsters (70 6 5 g b.w.) under general anesthesia was shaven and chemically depilated. A-Mel-3 cells ($5 3 106) were suspended in a 10 ll volume RPMI-1640 medium (Biochrom, Berlin, Germany) and injected subcutaneously in the back of the animal. After tumor-cell inoculation the longer (l) and the shorter (w) perpendicular axes and the

height (h) of each tumor nodule were measured and tumor volume was calculated according to the formula: Metastases of the animals were determined by daily palpation of axillar or inguinal lymph nodes. Plasma pharmacokinetics of cRGD delivered by osmotic pumps The cRGD peptide (EMD270179) and SU5416 we used in this study was generously provided by Merck KGaA (Darmstadt, Germany). Application of the drug was performed by placing osmotic pumps (Alzet1 2001, ALZA, Palo Alto, CA) intraperitoneally following median laparotomy under general anesthesia. In pilot studies we compared effective plasma levels of the cRGD peptide (EMD270179) after i.p. and s.c. application of the osmotic pumps in male Syrian golden hamsters (n 5 3). Therefore, cRGD peptides were dissolved in 50% DMSO and titrated with 0.5 M HCl to pH 4 (final concentration, 285 mg/ml). The osmotic pumps were filled and placed i.p. and s.c. (in the lumbosacral region), respectively. According to the manufacturer’s description osmotic pumps release their load continuously, delivering 1 ll/day for 7 days, thus yielding an effective daily dose of 114 mg/kg b.w. Fine polyethylene catheters (PE10, inner diameter 0.28 mm) were permanently inserted into the right carotid artery on the day of pump implantation. Blood was drawn and effective plasma levels of the cRGD peptide were measured on days 1, 4, 6 and 7 after pump implantation by liquid chromatography mass spectrometry (LC-MS). This bioanalytical method has a lower limit of quantification for determination in plasma of 5 ng/ml. Treatment and experimental groups Three days after tumor implantation into the dorsal skinfold chamber preparation, animals were randomly assigned to 4 groups: From day 3–10 after tumor-cell implantation, animals (n 5 6 per group) were treated by monotherapies using the cyclic RGDpeptide (initial loading i.p. bolus of 30 mg/kg b.w., then 114 mg/kg b.w. daily by osmotic pump i.p.), the VEGFR-2 antagonist (6 mg/kg b.w. daily by bolus injection i.p.) or by the combination of both monotherapies. The control group received intraperitoneal injections of the solvent DMSO alone. Using intravital microscopy, intratumoral functional vessel density and other microcirculatory parameters were analyzed on days 5, 7 and 10 after tumor-cell implantation. At the end of the observation time (day 10) animals were killed by an overdose of pentobarbital (Nembutal, Sanofi-Leva, Hannover, Germany). In subcutaneous tumor growth experiments, animals were randomly assigned to the 4 groups (n 5 6). Starting on the day of tumor-cell inoculation, animals were treated by monotherapies using the cyclic RGD-peptide (initial i.p. bolus of 30 mg/kg b.w., then 98 mg/kg b.w. daily by osmotic pump i.p.), the VEGFR-2 antagonist (6 mg/kg b.w. daily; i.p. bolus injection) or by the combination of both monotherapies. The control group received only the solvent DMSO. Tumor volume was measured on days 2, 5, 7, 9, 12, 14 and 16, and regional lymph nodes were palpated daily. In additional experiments we started treatment after 2 days of subcutaneous tumor growth (cyclic RGD-peptide: initial i.p. bolus of 30 mg/kg b.w., then 98 mg/kg b.w. daily by osmotic pump i.p.; the VEGFR-2 antagonist: 12 mg/kg b.w. daily; i.p. bolus injection). Experiments were terminated as soon as animals exhibited palpable axillar or inguinal lymph node metastases (verified by autopsy) beyond day 16. Statistical analysis Results are presented as mean 6 SEM. Data were evaluated using the Friedman repeated measures ANOVA on ranks and Kruskal–Wallis ANOVA on ranks, respectively (SigmaStat; Jandel Scientific, San Rafael, CA). Metastasis analysis (i.e. metastasis-free intervals) was performed according to the Kaplan–Meier method and the differences among the groups were compared for Vtumor ¼ 0:837 3 l 3 w 3 h

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statistical significance using the Cox-Mantel test (Statistica, StatSoft Inc., Tulsa, OK). p values smaller than 5% were considered to be significant. Results Plasmapharmacokinetics of cRGD delivered by osmotic pumps To ensure pump function plasma levels of the av-integrin antagonizing cyclic RGD peptide (EMD270179) were measured in a pilot study comparing i.p. and s.c. implanted osmotic pumps, respectively. All animals tolerated pump placement well. There were no signs of discomfort and all animals gained weight during the observation time comparable to animals receiving no osmotic pumps. In animals receiving no osmotic pumps cRGD plasma levels were below the limit of quantification of 5 ng/ml. While s.c. implanted osmotic pumps yielded lower or varying plasma levels of the cRGD peptide (data not shown), in all animals receiving i.p. osmotic pumps we found quite consistent plasma levels on days 1, 4, 6 and 7 after pump implantation so that we were confident concerning the continuous i.p. application regimen (Table I). Effects on tumor microcirculation by combined inhibition of av-integrins and VEGFR-2 Tumors were allowed to grow for 3 days and induce angiogenesis as seen light microscopically before we randomly assigned animals into the 4 groups and treatment started. On day 5 after tumor-cell implantation and thus after 2 days of treatment, tumor angiogenesis was observed by intravital microscopy. Characteristics of tumor angiogenesis as chaotic young microvessel sprouts with varying diameters forming loops and anastomoses were observed in all groups at this early timepoint. At the end of the observation period on day 10 after tumor-cell implantation intact tumor microcirculation was observed in DMSO controls (Figs. 1a and 1b). Comparing with these controls (Fig. 1a) and with the antiangiogenic monotherapies (Figs. 1c and 1e) reduced intratumoral functional vessel density was observed after treatment with the combinatory regimen (Fig. 1g). Detailed analysis revealed impaired microvessel sprouting following monotherapeutic treatment (Figs. 1d and 1f). In contrast, after combined antiangiogenic treatment stagnant fluorescent dyes and fluorescence contrast interruptions along vessel runs indicative of regression of formerly perfused microvessels were observed (Fig. 1h). Quantitative analysis of intratumoral functional vessel density among the 4 groups confirmed these observations (Fig. 2a). Monotherapy with the cRGD peptide and the combination treatment showed effects on fvd already on day 7. But when comparing with the initial observation timepoint there was a significant reduction of intratumoral fvd only in tumors treated according to the combinatory regimen. In the group treated with the combination, this low niveau of fvd was maintained up to the end of the observation period on day 10 after tumor-cell implantation, thus yielding a significant difference to all the other groups. Separate analysis of fvd in peripheral and central tumor regions revealed an overall tendency to smaller fvd values in the tumor center (Fig. 2b). Reduction of fvd was first observed on day 7 in central regions of interest after combined antiangiogenic therapy. Further intratumoral microcirculatory parameters as red blood cell velocity (vRBC) and vessel diameters were less affected by any treatment (Figs. 3a and 3b). At the end of the observation period vessel diameters increased in all the groups. Calculating blood flow in vessel segments (blood flowsegmental) revealed that this parameter did not equally increase as observed in DMSO-treated tumors at the end of the observation time following any antiangiogenic therapy with lowest values found in the combination therapy group (Fig. 3c). This resulted finally in a significantly lower microvascular perfusion index in tumors treated

TABLE I – PLASMAPHARMACOKINETICS OF cRGD PEPTIDES (EMD 270179) DELIVERED CONTINUOUSLY BY OSMOTIC PUMPS (n 5 3) Time (days after i.p. pump implantation) Plasma levelcRGD (ng/ml)

1 4 6 7

2,670 6 564 1,429 6 357 1,523 6 240 1,612 6 148

with the antiangiogenic combination therapy compared to that in the controls (Fig. 3d). Subcutaneous tumor growth and onset of metastatic disease To test the effects of a combined antiangiogenic therapy on tumor growth, we treated subcutaneously growing tumors starting therapy right on the day of tumor-cell inoculation (Figs. 4a and 4b). The antiangiogenic combination was superior to both monotherapies, resulting in a remarkable inhibition of tumor growth. After 5 days of treatment, tumors treated by the combination therapy were significantly smaller than those treated by DMSO (controls) and this was maintained up to the end of the observation period on day 16. Around this day metastatic disease occurred in controls and animals were subsequently sacrificed beyond day 16 as soon as first palpable lymph node metastases were found (Fig. 5). While incidence of metastases in controls was on days 13 and 14, respectively, in animals treated with the antiangiogenic combination therapy first lymph node metastases were found 7 days later—i.e. on day 21 (p < 0.05). At the very end of the observation time on day 48 there was still one animal after antiangiogenic combination therapy exhibiting no metastases at autopsy. In contrast to this early intervention strategy we treated s.c. tumors at later stages (i.e. after 2 days of s.c. tumor growth). Intriguingly, there is only a transitory statistically significant benefit of the combination therapy concerning tumor growth (up to day 5), which is not detectable any more at a later timepoints (after 7 days, data not shown). In contrast, the effect on the onset of tumor metastasis is maintained starting the combination therapy at this later tumor stage (data not shown).

Discussion After successfull combination with conventional therapy and approval of the first antiangiogenic drug—Bevacizumab8—it becomes obvious that the establishment of further antiangiogenic drugs in clinic may be reached by variations in scheduling and by regimens combining agents in upcoming antiangiogenic trials.6,1 The data presented here clearly show that the combination of cRGD-peptides and a VEGFR-2 antagonist effectively inhibits tumor angiogenesis in vivo and is superior—although not in a classical synergistic manner—to monotherapies with the single agents. Moreover, tumor growth and the formation of metastases can significantly be delayed when therapy is initiated at an early tumor stage. There are preclinical studies showing efficacy of antiangiogenic combinations using TNP-470 and interferon-a,28,29 as well as using endostatin and SU5416.15 Unfortunately, the hope of having a highly specific antiangiogenic substance was contrasted by disappointing experiences in clinical studies revealing some adverse side effects of SU5416, ranging from headache, phebitis, fatigue and lymphopenia to even accumulated thrombembolic complications after combination with cisplatin/gemcitabine.10–14,30,31 Nevertheless, dose-descalating regimens using combination therapies may overcome these toxicity problems. We used SU5416 at a significantly lower dose (6 mg/kg/day) compared to previous preclinical studies (25 mg/kg/day22). It may be possible that dose-

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FIGURE 1 – In vivo fluorescence microscopy of tumor angiogenesis. I.v. injected FITC-dextran highlights perfused microvessels. At the end of the observation period on day 10 after tumor-cell implantation an intact tumor microcirculation was observed in DMSO controls (a, b). Comparing with the antiangiogenic monotherapies (c, e) reduced intratumoral functional vessel density was observed after treatment with the combination (g). Detailed analysis revealed immature sprouts following monotherapeutic treatment (arrows in d, f) while combined antiangiogenic treatment induced microvessel regression (arrows in h). (a, b) DMSO; (c, d) cRGD; (e, f) VEGFR-2 ant.; (g, h) comb.

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FIGURE 2 – Quantitative analysis of intratumoral functional vessel density (fvd). (a) There was a significant reduction of intratumoral functional vessel density among animals treated with the combination therapy on day 7 compared to that on day 5 of tumor growth. At the end of the observation period reduction of fvd in the combination group reached statistical significance compared to all controls. (b) Separate analysis of fvd in peripheral and central tumor regions revealed an overall tendency to smaller fvd values in the tumor center. Comparing to controls reduction of fvd by combined antiangiogenic therapy was first observed on day 7 in central tumor regions. DMSO, ; cRGD, ; VEGFR-2 ant., ; comb., . n 5 6 per group; mean 6 SEM. *, p < 0.05 vs. prior day of measurement (Friedman repeated measures ANOVA on ranks); $, p < 0.05 vs. DMSO, VEGFR-2 ant.; #, p < 0.05 vs. all other groups; &, p < 0.05 vs. cRGD, comb.; §, p < 0.05 vs. tumor periphery (Kruskal– Wallis ANOVA on ranks).

reduction alone may not be the solution for the serious toxicity problems of SU5416. Thus, aim of the study was less to exculpate SU5416 than to conceptually investigate the combination of ‘‘direct’’ and ‘‘indirect’’ inhibition of angiogenesis15 using functional blockage of av-integrin and VEGFR-2 signaling. We were inspired by reports of a successful combination of VEGFR-2 inhibition and continuous low-dose ‘‘metronomic’’ scheduling of conventional chemotherapy yielding antivascular collateral damage32,33 as well as by the successful combination of ‘‘direct’’ and ‘‘indirect’’ inhibitors of angiogenesis.15 In consideration of the short half-lives of cRGD peptides and the fact that the angiogenic phenotype is the result of a continuous balance of activating and inhibiting agents we decided to apply antiangiogenic cRGD peptides continuously by osmotic pumps. On the other hand, recent findings of long-lasting effects of SU5416 in spite of a rather short plasma half-life implicated no need of continuous application of SU5416.31,34 Histologically assayed vascular density may not reliably reflect antiangiogenic effects. Since changes in absolute microvessel density are dynamic in nature, the chronic model used in this study relying on functional microvessels is considered to be superior to

histological microvessel density.35,36 Intravital microscopically assayed fvd thus appears as the gold standard for the evaluation of effectiveness of an antiangiogenic therapy.36 Concerning intratumoral microcirculatory changes it is intriguing that most recent reports indicate that antiangiogenesis may result in a transitory ‘‘normalization’’ of tumor microvessels,6 thus enabling synergism with perfusion-dependent treatment modalities (e.g. radiation37–39). Our experimental setup is methodologically sufficient to even reflect such transient microcirculatory changes during therapy before finally an inadequate tumor microcirculation might remain. Starting treatment at a rather early tumor stage detailed analysis of tumor microcirculation revealed no dramatic changes in intratumoral red blood cell velocity and vessel diameters and there was a tendency to reduced blood flow in vessel segments at the end of the observation period. But fvd was significantly reduced by our combined antiangiogenic therapy at the end of the observation period. These findings are in accordance with intravital microscopic findings using SU5416 and endostatin, repectively.15 Estimating tumor perfusion with respect to changes in functional vessel density thus resulted in a significantly reduced microcirculatory perfusion index in our study.

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FIGURE 3 – Quantitative analysis of further intratumoral microcirculatory parameters. (a) Red blood cell velocity (vRBC): vRBC was not significantly affected by any treatment. (b) Vessel diameter: at the end of the observation time vessel diameters increased significantly in all groups. (c) Blood flow in vessel segments (blood flowsegmental): while we found improved blood flowsegmental among DMSO-treated tumors at the end of the observation time, blood flowsegmental did not equally increase using any antiangiogenic therapy with lowest values after combinatory treatment. (d) Microvascular perfusion index: at the end of the observation time the microvascular perfusion index was significantly lower in tumors treated with the antiangiogenic combination therapy compared to that of the controls. DMSO, ; cRGD, ; VEGFR-2 ant., ; comb., . n 5 6 per group; mean 6 SEM. *, p < 0.05 vs. prior day of measurement (Friedman repeated measures ANOVA on ranks); #, p < 0.05 vs. all other groups (Kruskal–Wallis ANOVA on ranks).

For novel vascular targeting substances or antibody-based approaches directing toxins or coagulation proteins to angiogenic tumor microvessels, it is reported that central rather than peripheral tumor areas are affected.40 So these agents leave a viable rim at the tumor periphery responsible for tumor regrowth. Moreover, since hypoxia in the tumor center is a key regulating factor in angiogenesis, we analyzed tumor microcirculation in central and peripheral tumor regions separately. In our study, fvd was first reduced in central tumor areas by monotherapy with cRGD peptides as well as by the combination but not by SU5416. Nevertheless, a more than 3fold higher monotherapeutic dose of SU5416 induced a loss of the peri-to-intratumoral vascular density gradient in a comparable chamber model.41 In our study, in contrast to preferential effects on tumor subregions by monotherapies the combination therapy alone finally reduced fvd not only in central but even in peripheral tumor areas. Consequently these changes resulted in enhanced starvation effects on the tumor as documented by a reduced overall perfusion index after combination therapy. Among antitumor strategies and in contrast to novel vascular targeting agents aiming at the established tumor microcirculation, antiangiogenesis appears to be conceptually suitable for an early intervention or second-line prophylaxis.42 In line with these considerations are reports that show that an active immunization against VEGFR-2 inhibits tumor growth and metastasis in mice challenged with B16 melanoma or Lewis lung carcinoma cells.43 We demonstrated earlier that cRGD preferentially abrogated early angiogenesis, thus retarding tumor progression.18 Comparably, there is evidence that SU5416 is rather capable of retarding initial tumor growth than of inhibiting tumor expansion at later stages.44

This is again in accordance with our own findings in a previous study showing that SU5416 resulted in a potent inhibition of early angiogenesis and a nearly complete suppression of tumor growth starting therapy right on the day of subcutaneous tumor-cell inoculation.22 Accordingly, we found a significant inhibition of tumor growth in subcutaneous tumors initiating antiangiogenic combination therapy right on the day of tumor-cell inoculation. Timing of antiangiogenesis is crucial for therapeutic efficacy as shown earlier by Bergers et al. comparing the effectiveness of antiangiogenic drugs at different tumor stages.45 Certain antiangiogenic agents may enable tumor regressions even after initiation of therapy at late stages.45 However, angiogenesis is pathophysiologically considered an early event during carcinogenesis. Consequently, interventions may be most effective if therapy starts early—or even prophylactically as ‘‘angioprevention.’’46 This is in line with observations we made starting therapy at later stages of tumor growth (i.e. after 2 days of s.c. tumor growth) in contrast to an early intervention strategy. Only a transient effect on tumor growth by the antiangiogenic combination therapy was detectable at this later stage. Nevertheless, the effect on the onset of tumor metastasis remained unaffected starting combination therapy at this later tumor stage (data not shown). It is well accepted that not only tumor progression but also metastastic spread and growth depends on angiogenesis.47 As previous experiments with local tumor therapy have shown appearance and growth of metastasis of A-Mel-3 tumors is closely related to the time after subcutaneous inoculation and less dependent on the burden of the primary tumor.48 Thus, we interpret the observed delay of incidence of palpable lymph node metastasis as

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FIGURE 4 – Tumor growth of subcutaneously inoculated A-Mel3 tumor cells. (a) Analysis of s.c. tumor growth kinetics. Starting therapy right on the day of tumorcell inoculation, tumor growth was significantly delayed only by treatment with the antiangiogenic combination therapy comparing with monotherapies and DMSO-treated controls. DMSO (s); cRGD (m); VEGFR-2 ant. (u); comb. (Ä). #, p < 0.05 comb vs. DMSO; n 5 6 per group; mean 6 SEM. (Kruskal–Wallis ANOVA on ranks.) (b) Representative A-Mel-3 tumors on day 16 after tumor-cell inoculation.

a consequence of combined antiangiogenic treatment. However, further studies with alternate tumor metastasis models allowing detailed analysis are needed to clarify the exact interference of antiangiogenic therapies on this critical step in tumor progression. av-integrins have been reported to be expressed on melanoma cells themselves.49 Consequently, the question can be raised whether cRGD peptides inhibit tumor growth by direct tumor-cell toxicity. Allman addressed this issue and found such a cytotoxic effect against tumor cells per se in vitro but not in vivo.50 Moreover, the transparent window model allows the direct visualization of tumor microvessels as the therapeutic target and antiangiogenic effects on functional vessel density in vivo. Nevertheless, we can not completely rule out an additional direct effect of the cRGD peptide on tumor cells. In conclusion, the data presented here support the concept of antiangiogenic combination therapy. The inhibition of angiogenesis in vivo is significantly improved combining a cRGD peptide (EMD270179) and SU5416. Our findings suggest that the combined antiangiogenic therapy using the inhibition of av-integrins and VEGF/VEGFR-2 signaling may be beneficial when scheduled as prophylaxis or early intervention, thus avoiding angiogenesisdependent tumor and metastasis initiation or tumor recurrence.
FIGURE 5 – Kaplan-Meier analysis of metastasis-free intervals. The onset of palpable lymph node metastases was significantly delayed in animals treated with the antiangiogenic combination therapy compared to all other groups; * p < 0.05. DMSO, ; cRGD, ; VEGFR-2 ant., – – –; Comb., – Á Á.

Acknowledgements The authors thank B. Ladstetter and B. Neuteboom from Merck, Grafing (Germany), for technical assistance.

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