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

Antiangiogenic combination tumor therapy blocking a v -integrins and VEGF-receptor-2 increases therapeutic effects in vivo

Sebastian Strieth 1,2 * , Martin E. Eichhorn 1,3 , Arne Sutter 4 , Alfred Jonczyk 4 , Alexander Berghaus 2 and Marc Dellian 1, 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 cur- rently evaluated in clinical trials. The aim of the study was to investigate the effects of an antiangiogenic combination therapy inhibiting a v -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 skin- fold chamber preparations of Syrian golden hamster s (60 6 5 g ) bearing A-Mel-3 tumors. From day 3–10 after tumor-cell implan- tation, animals (n 5 6 per group) were treated by monotherapies using the cRGD-pep tide (114 mg/kg/day; i.p.), the VEGFR-2 an-

tagonist (6 mg/kg/day; i.p.) or by the combination of both mono- therapies. A control group received only the solvent DMSO. Using intravital micro scopy parameters of intratumoral microcircula- tion were analyzed on day 5, 7 and 10. In separate experiments subcutaneous tumor growth and metastasis formation was eval- uated starting therapy on day 0. Functional vessel density was sig- nificantly 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 tu- mor growth and metastasis formation using the combination ther- apy. 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 metas- tasis 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 microvascu- lature. 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 microves- sels and acquired drug resistance is less likely to occur in the ge- netically stable endothelial cell. 1 Nevertheless, extensive experi- mental 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 Consid- ering 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 combina- tion therapies. Conventional cancer chemotherapy today is rely- ing on combination schedules of single cytotoxic substances to optimize efficacy. Comparably, anti-angiogenesis might benefit from the combination of monotherapies. Redundancy of proangio- genic growth factors expressed by tumor cells or antiapoptotic sig- naling 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 effi- cacy of anti-angiogenic treatment and favors the search for ef- fective 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 microcircu-

3

induces functional changes of tumor microcircu- 3 P u b l i c a t io

P u b l i c a t io n o f t h e I n t e r n a t i o n a l U n i o n A g a i n s t C a n c e r

lation, resulting in a transient ‘‘normalization’’ of tumor perfusion 6 responsible for synergistic effects after combination with radiation therapy. Comparably, it has been shown in clinical trials that anti- angiogenesis can act synergistically with conventional chemother- apy. The VEGF/VEGF-receptor-2-system appears to be crucial in the regulation of tumor angiogenesis. One of the most effective ki- nase inhibitors selective for VEGFR-2 used to be the small molec- ular substance SU5416. 9 But considering the rather disappointing outcome of phase II clinical trials using SU5416 as antiangiogenic

monotherapy

apy resulting even in thromboembolic complications 12,13 or even combined with another antiangiogenic substance such as inter- feron a (with at least evidence of biological activity 14 ) i t 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 monothera- pies. Among possible strategies combining blockage of quite inde- pendent angiogenic pathways or combining ‘‘direct’’ and ‘‘indi- rect’’ inhibition of angiogenesis 15 the following are most promis- ing: ‘‘direct’’ angiogenesis inhibition interferes straightforward with the tumor endothelial cell, while ‘‘indirect’’ inhibition blocks proangiogenic signaling between the tumor cell and the tumor en- dothelial-cell compartment. Accordingly, SU5416 is to be classi- fied as an ‘‘indirect’’ inhibitor. In contrast, cellular adhesion to a variety of extracellular matrix proteins containing Arg-Gly-Asp

or in combination with conventional chemother-

7

8

10,11

sequences mediated by a -integrins has been shown to prevent ap- optosis/anoikis among tumor endothelial cells. 16,17 These so- called RGD sequences ( e.g. EMD121974 5 Cilengitide, EMD270179 as a cyclic compound) can be used to effectively in- hibit tumor angiogenesis and progression 17 19 and can thus be classified as ‘‘direct’’ inhibitors of angiogenesis.

Furthermore, the regulation of angiogenesis by a v -integrins and VEGF has been described in detail and reflects 2 distinct angio- genic pathways. 20,21 Both monotherapeutic options blocking ei-

ther pathway have shown efficacy in our tumor model.

aim of this study was to investigate the effects of a combined anti-

angiogenic treatment using inhibition of a v -integrins by a cyclic RGD-peptide (EMD270179) and functional blockage of the VEGFR-2 by SU5416 on tumor angiogenesis, growth and metas- tasis in just the same and thus comparable in vivo tumor model. Our data clearly support the concept of antiangiogenic combi- nation therapy because tumor angiogenesis in vivo is significantly inhibited by the combination of cRGD peptides (EMD270179) and SU5416. Our findings suggest that the combined antiangio- genic therapy using the inhibition of a v -integrins and VEGF/ VEGFR-2 signaling may be especially beneficial when scheduled

So, the

v

18,22

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 angiogenesis- dependent tumor and metastasis initiation or tumor recurrence.

STRIETH ET AL.

height ( h) o f each tumor nodule were measured and tumor volume was calculated according to the formula:

V tumor ¼ 0: 837 3 l 3 w 3 h

Metastases of the animals were determined by daily palpation of axillar or inguinal lymph nodes.

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 microcir- culation in vivo , a dorsal skinfold chamber consisting of 2 sym- metrical titanium frames was surgically implanted into the dorsal

skin as described earlier in detail.

Following a recovery period

of at least 24 hr from anesthesia and microsurgery, chamber prepa- rations fulfilling the criteria of microscopically intact microcircu-

lation were inoculated with 2 l l o f dense tumor-cell suspension ( 2 3 10 5 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., Ketavet 1 ; Parke-Davis, Berlin, Germany) and xylazine (10 mg/kg b.w. i.p., Rompun 1 ; Bayer, Leverkusen, Germany).

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 os-

motic pumps (Alzet

2001, ALZA, Palo Alto, CA) intraperitone-

ally 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 quantifi- cation for determination in plasma of 5 ng/ml.

1

23,24

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 anesthe- sia, 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 sub- sequent 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.

and Klyscz

et al.

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 Wayland :

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 RGD- peptide (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 combina- tion of both monotherapies. The control group received intraper- itoneal 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 ran- domly assigned to the 4 groups ( n 5 6). Starting on the day of tu- mor-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 antago-

nist (6 mg/kg b.w. daily; i.p. bolus injection) or by the combina-

tion 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 subcu-

taneous 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 pal- pable axillar or inguinal lymph node metastases (verified by au- topsy) beyond day 16.

25

26

27

v

RBC

1:6

3

2

d

2

Q ¼

The microvascular perfusion index was calculated as follows:

microcirculatory perfusion index ¼ 10 3 fvd 3Q

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 10 6 ) were suspended in a 1 0 l l volume RPMI-1640 medium (Biochrom, Berlin, Germany) and injected subcutaneously in the back of the animal. After tumor-cell inocu- lation the longer (l ) and the shorter ( w) perpendicular axes and the

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; Jan- del Scientific, San Rafael, CA). Metastasis analysis ( i.e. metasta- sis-free intervals) was performed according to the Kaplan–Meier method and the differences among the groups were compared for

ANTIANGIOGEN IC COMBINAT ION TUMOR THERAP Y BLOCKIN G

V -INTEGRINS AND VEGF-RE CEPTOR-2

425

statistical significance using the Cox-Mantel test (Statistica, Stat- Soft 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 a v -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 ob- servation time comparable to animals receiving no osmotic pumps. In animals receiving no osmotic pumps cRGD plasma lev- els 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 con- cerning the continuous i.p. application regimen (Table I).

Effects on tumor microcirculation by combined inhibition of a v -integrins and VEGFR-2 Tumors were allowed to grow for 3 days and induce angiogene- sis as seen light microscopically before we randomly assigned ani- mals 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 micros- copy. 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 im- plantation intact tumor microcirculation was observed in DMSO controls (Figs. 1 a and 1b). Comparing with these controls (Fig. 1a) and with the antiangiogenic monotherapies (Figs. 1 c and 1e ) reduced intratumoral functional vessel density was observed after treatment with the combinatory regimen (Fig. 1 g). Detailed analy- sis revealed impaired microvessel sprouting following monothera- peutic treatment (Figs. 1 d and 1f). In contrast, after combined anti- angiogenic 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. 2 a). Mono- therapy 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 combi- natory 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 sig- nificant 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 i n central regions of interest after combined antiangiogenic therapy. Further intratumoral microcirculatory parameters as red blood cell velocity ( v RBC ) 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 flow segmental ) revealed that this parameter did not equally increase as observed in DMSO-treated tumors at the end of the observation time fol- lowing any antiangiogenic therapy with lowest values found in the combination therapy group (Fig. 3c ). This resulted finally in a sig- nificantly 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 level cRGD (ng/ml)

1

2,670 6 564

4

1,429 6 357

6

1,523 6 240

7

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).

T h e a n t i a n g i og e ni c c o m bi n a t i on w a s s u pe r i o r t o bo t h m on o-

t h e r a p i e s , r e s ul t i ng i n a r e m a r k a b l e i n h i bi t i o n of t um o r g r o w t h .

A f t e r 5 d a y s o f t r e a t m e n t , t u m o r s t r e a t e d b y t h e c om b i n a t i o n

t h e r a p y w e r e s i g n i fi c a n t l y s m a l l e r t h a n t h o s e t r e a t e d b y D MS O

( c on t r o l s ) a n d t hi s w a s m a i nt a i n e d up t o t h e e n d of t he ob s e r v a - t i o n p e r i od o n d a y 1 6. A r ou n d t h i s d a y m e t a s t a t i c d i s e a s e

o

c c ur r e d i n c o nt r ol s a nd a n i m a l s w e r e s u bs e q u e n t l y s a c r i fi c e d

b

e y o n d d a y 16 a s s o o n a s fi r s t pa l p a bl e l y m p h n o d e m e t a s t a s e s

w

e r e f ou n d ( F i g . 5 ) . W h i l e i n c i d e n c e o f m e t a s t a s e s i n c on t r o l s

w

a s o n da y s 1 3 a n d 1 4 , r e s p e c t i ve l y, i n a n i m a l s t r e a t e d w i t h t h e

a n t i a n g i o g e n i c c o m b i n a t i o n t h e r a p y fi r s t l y m p h no d e m e t a s t a s e s

w e r e f o u n d 7 d a y s l a t e r — i . e . o n d a y 2 1 ( p < 0 . 0 5 ) . A t t h e v e r y

e n d o f t h e o b s e r v a t i o n t i m e o n d a y 4 8 t h e r e w a s s t i l l o n e a n i m a l a f t e r a n t i a n g i o g e n i c c om b i n a t i o n t h e r a p y e xh i b i t i n g n o m e t a s t a - s e s a t a ut o p s y .

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). Intri- guingly, 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 me- tastasis 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—Bevacizumab 8 —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 tu- mor angiogenesis in vivo and is superior—although not in a classi- cal synergistic manner—to monotherapies with the single agents. Moreover, tumor growth and the formation of metastases can sig- nificantly 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 dis-

appointing experiences in clinical studies revealing some adverse side effects of SU5416, ranging from headache, phebitis, fatigue and lymphopenia to even accumulated thrombembolic complica-

tions after combination with cisplatin/gemcitabine. Nevertheless, dose-descalating regimens using combination thera- pies may overcome these toxicity problems. We used SU5416 at a significantly lower dose (6 mg/kg/day) compared to previous pre- clinical studies (25 mg/kg/day ). It may be possible that dose-

10–14,30 ,31

22

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STRIETH ET AL.

426 STRIETH ET AL. F IGURE 1 – In vivo fluorescence microscopy of tumor angiogenesis. I.v.

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 ). Compar- ing with the antiangiogenic monotherapies ( c, e ) reduced intratumoral functional vessel density was observed after treatment with the combina- tion ( g). Detailed analysis revealed immature sprouts following monotherapeutic treatment (arrows in d, f) while combined antiangiogenic treat- ment induced microvessel regression (arrows in h ). (a, b ) DMSO; (c, d ) cRGD; ( e, f ) VEGFR-2 ant.; ( g, h ) comb.

ANTIANGIOGEN IC COMBINAT ION TUMOR THERAP Y BLOCKIN G

V -INTEGRINS AND VEGF-RE CEPTOR-2

427

FIGURE 2 – Quantitative analy-

sis of intratumoral functional ves- sel density (fvd). ( a ) There was a significant reduction of intratu- moral functional vessel density among animals treated with the combination therapy on day 7 compared to that on day 5 o f tumor growth. At the end of the observa- tion period reduction of fvd in the combination group reached statisti- cal significance compared to all controls. (b ) Separate analysis of fvd in peripheral and central tumor regions revealed an overall tend- ency to smaller fvd values in the tumor center. Comparing to con- trols reduction of fvd by combined antiangiogenic therapy was first observed on day 7 i n 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 <

$, p < 0.05 vs. DMSO, VEGFR-2 ant.; #, p < 0.05 vs. all other groups;
$, p < 0.05 vs. DMSO, VEGFR-2 ant.; #, p < 0.05 vs. all other groups;

0.05

vs. all other groups; &, p <

0.05

vs. cRGD, comb.; §, p < 0.05

vs. tumor periphery (Kruskal–

Wallis ANOVA on ranks).

vs. tumor periphery (Kruskal– Wallis ANOVA on ranks). reduction alone may not be the solution for

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 angiogenesis 15 using func- tional blockage of a v -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 damage

‘‘direct’’ and ‘‘indirect’’ inhibitors of angiogenesis. 15 In considera-

tion of the short half-lives of cRGD peptides and the fact that the angiogenic phenotype is the result of a continuous balance of acti- vating 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 den- sity are dynamic in nature, the chronic model used in this study relying on functional microvessels is considered to be superior to

as well as by the successful combination of

32,33

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 tran- sitory ‘‘normalization’’ o f tumor microvessels, thus enabling syn- ergism with perfusion-dependent treatment modalities ( e.g. radia- tion 3739 ). 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 intravi- tal microscopic findings using SU5416 and endostatin, repec-

tively.

Estimating tumor perfusion with respect to changes in

functional vessel density thus resulted in a significantly reduced microcirculatory perfusion index in our study.

6

15

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STRIETH ET AL.

428 STRIETH ET AL. F IGURE 3 – Quantitative analy- sis of further intratumoral microcir- culatory

F IGURE 3 – Quantitative analy- sis of further intratumoral microcir- culatory parameters. ( a ) Red blood cell velocity ( v RBC ): v RBC was not significantly affected by any treat- ment. ( b) Vessel diameter: at the end of the observation time vessel diameters increased significantly in all groups. ( c) Blood flow in ves- sel segments (blood flow segmental ):

while we found improved blood flow segmental among DMSO-tre ated tumors at the end of the observa- tion time, blood flow segmental did not equally increase using any antiangiogenic therapy with lowest values after comb inatory treat- ment. ( d ) Microvascular perfusion index: at the end of the observation time the microvascular perfusion index was significantly lower in tumors treated with the antiangio- genic combination therapy com- pared 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).

vs. all other groups (Kruskal–Wallis ANOVA on ranks). For n ov el va sc ul ar

For n ov el va sc ul ar targeting sub st an ce s or ant ibody-ba se d appr oach es di re ct ing t oxins or coa gulation protei ns to angiog en ic tumor m icrovessels, it is reported that cent ra l r ath er than peripheral

tumor a re as are aff ec te d. 40 So these a ge nt s l ea ve a v ia ble rim at the tumor p er ip he ry responsibl e f or tumor r egrowth. Moreov er, since hypoxia i n t he tumor c en te r is a key regul at in g f actor i n a ngioge ne - sis, we analyzed tumor m ic rocircul at io n in c en tral and p er ip hera l tumor reg ions separately. I n our stud y, fvd w as fir st reduce d i n c en- tral tumor a re as by monotherapy w it h c RG D peptides a s well a s by the com bi na ti on but not by SU5416. Nev er theless, a m or e t ha n 3- fold hi gher mono therapeutic dose of S U5 41 6 i nduced a l os s of the

peri-to-

in tr atum or al vascul ar dens it y gradient in a comparable

chamber model. 4 1 In our s tudy, i n c ontrast to pre fe re nt ia l e ff ec ts on tumor subregions by monothe ra pi es the c om bina tion therapy a lo ne finally redu ce d f vd not only i n cen tr al bu t e ven i n peripheral tum or areas. Consequently these c ha nges resulted in enhanc ed starvation effects o n the tumor a s d oc um en te d by a redu ce d overall pe rf us io n inde x a ft er 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

In line with these con-

intervention or second-line prophylaxis.

siderations 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

Comparably,

angiogenesis, thus retarding tumor progression.

there is evidence that SU5416 is rather capable of retarding initial

tumor growth than of inhibiting tumor expansion at later stages. 44

42

18

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 inocu-

lation.

Accordingly, we found a significant inhibition of tumor

growth in subcutaneous tumors initiating antiangiogenic combina- tion therapy right on the day of tumor-cell inoculation. Timing of antiangiogenesis is crucial for therapeutic efficacy as shown ear- lier by Bergers et al. comparing the effectiveness of antiangio- genic drugs at different tumor stages. 45 Certain antiangiogenic agents may enable tumor regressions even after initiation of ther- apy at late stages. 45 However, angiogenesis is pathophysiologi- cally considered an early event during carcinogenesis. Conse- quently, interventions may be most effective if therapy starts early—or even prophylactically as ‘‘angioprevention.’’ 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 pre- vious experiments with local tumor therapy have shown appear-

ance and growth of metastasis of A-Mel-3 tumors is closely

related to the time after subcutaneous inoculation and less depend- ent on the burden of the primary tumor. 48 Thus, we interpret the observed delay of incidence of palpable lymph node metastasis as

22

46

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V -INTEGRINS AND VEGF-RE CEPTOR-2

429

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

A-Mel-3 tumors on day 16 after tumor-cell inoculation. F IGURE 5 – Kaplan-Meier analysis of metastasis-free
A-Mel-3 tumors on day 16 after tumor-cell inoculation. F IGURE 5 – Kaplan-Meier analysis of metastasis-free

FIGURE 5 – Kaplan-Meier analysis of metastasis-free intervals. The onset of palpable lymph node metastases was significantly de- layed in animals treated with the antiangiogenic combination therapy compared to all other groups; * p < 0.05. DMSO, ; cRGD, ; VEGFR-2 ant., – – – ; Comb. , – .

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.

a v -integrins have been reported to be expressed on melanoma cells

themselves.

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 ves- sel 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 angiogene- sis in vivo is significantly improved combining a cRGD peptide (EMD270179) and SU5416. Our findings suggest that the com- bined antiangiogenic therapy using the inhibition of a v -integrins and VEGF/VEGFR-2 signaling may be beneficial when scheduled as prophylaxis or early intervention, thus avoiding angiogenesis- dependent tumor and metastasis initiation or tumor recurrence.

Consequently, the question can be raised whether

49

Acknowledgements

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

430

STRIETH ET AL.

References

1. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2002;2:727–39.

2. Klement G, Baruchel S, Rak J, Man S, Clark K, Hicklin DJ, Bohlen P, Kerbel RS. Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression with- out overt toxicity. J Clin Invest 2000;105:R15–R24.

3. Eskens FA. Angiogenesis inhibitors i n clinical development; where are we now and where are we going? Br J Cancer 2004;90:1–7.

4. Rak J, Yu JL, Kerbel RS, Coomber BL. What do oncogenic mutations have to do with angiogenesis/vascular dependence of tumors? Cancer Res 2002;62:1931–4.

5. Kerbel RS, Kamen BA. The anti-angiogenic basis of metronomic chemotherapy. Nat Rev Cancer 2004;4:423–36.

6. Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005;307:58–62.

7. Wachsberger P, Burd R, Dicker AP. Tumor response to ionizing radi- ation combined with antiangiogenesis or vascular targeting agents:

exploring mechanisms of interaction. Clin Cancer Res 2003;9:1957–

71.

8. Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004;350:2335–42.

9. Fong TA, Shawver LK, Sun L, Tang C, App H, Powell TJ, Kim YH, Schreck R, Wang X, Risau W, Ullrich A, Hirth KP, et al. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res

1999;59:99–106.

10. Stadler WM, Cao D, Vogelzang NJ, Ryan CW, Hoving K, Wright R, Karrison T, Vokes EE. A randomized Phase II trial of the antiangio- genic agent SU5416 in hormone-refractory prostate cancer. Clin Can- cer Res 2004;10:3365–70.

11. Zangari M, Anaissie E, Stopeck A, Morimoto A, Tan N, Lancet J, Cooper M, Hannah A, Garcia-Manero G, Faderl S, Kantarjian H, Cherrington J, et al. Phase II study of SU5416, a small molecule vas- cular endothelial growth factor tyrosine kinase receptor inhibitor, in patients with refractory multiple myeloma. Clin Cancer Res

2004;10:88–95.

12. Kuenen BC, Levi M, Meijers JC, van Hinsbergh VW, Berkhof J, Kak- kar AK, Hoekman K, Pinedo HM. Potential role of platelets in endo- thelial damage observed during treatment with cisplatin, gemcitabine, and the angiogenesis inhibitor SU5416. J Clin Oncol 2003;21:2192–8.

13. Kuenen BC, Rosen L, Smit EF, Parson MR, Levi M, Ruijter R, Huis- man H, Kedde MA, Noordhuis P, van der Vijgh WJ, Peters GJ, Cropp GF, et al. Dose-finding and pharmacokinetic study of cisplatin, gemci- tabine, and SU5416 in patients with solid tumors. J Clin Oncol

2002;20:1657–67.

14. Lara PNJ, Quinn DI, Margolin K, Meyers FJ, Longmate J, Frankel P, Mack PC, Turrell C, Valk P, Rao J, Buckley P, Wun T, et al. SU5416 plus interferon-a in advanced renal cell carcinoma: a phase II Califor- nia Cancer Consortium Study with biological and imaging correlates of angiogenesis inhibition. Clin Cancer Res 2003;9:4772–81.

15. Abdollahi A, Lipson KE, Sckell A, Zieher H, Klenke F, Poerschke D, Roth A, Han X, Krix M, Bischof M, Hahnfeldt P, Grone HJ, et al. Combined therapy with direct and indirect angiogenesis inhibition results in enhanced antiangiogenic and antitumor effects. Cancer Res

2003;63:8890–8.

16. Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integ- rin-a v b 3 for angiogenesis. Science 1994;264:569–71.

17. Brooks PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T, Klier G, Cheresh DA. Integrin-a v b 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994;79:

1157–64.

18. Buerkle MA, Pahernik SA, Sutter A, Jonczyk A, Messmer K, Dellian M. Inhibition of the a m -integrins with a cyclic RGD peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. Br J Cancer 2002;86:788–95.

19. DeNardo SJ, Burke PA, Leigh BR, O’Donnell RT, Miers LA, Kroger LA, Goodman SL, Matzku S, Jonczyk A, Lamborn KR, DeNardo GL. Neovascular targeting with cyclic RGD peptide (cRGDf-ACHA) to enhance delivery of radioimmunotherapy. Cancer Biother Radio- pharm 2000;15:71–9.

20. Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA, Cheresh DA. Definition of two angiogenic pathways by distinct a v - integrins. Science 1995;270:1500–2.

21. Soldi R, Mitola S, Strasly M, Defilippi P, Tarone G, Bussolino F . Role of a v b 3 -integrin in the activation of vascular endothelial growth factor receptor-2. EMBO J 1999;18:882–92.

22. Pahernik S, Harris AG, Schmitt-Sody M, Krasnici S, Goetz AE, Del- lian M, Messmer K. Orthogonal polarisation spectral imaging as a

new tool for the assessment of antivascular tumour treatment in vivo:

a validation study. Br J Cancer 2002;86:1622–7.

23. Asaishi K, Endrich B, Goetz A, Messmer K. Quantitative analysis of microvascular structure and function in the amelanotic melanoma A- Mel-3. Cancer Res 1981;41:1898–904.

24. Endrich B, Hammersen F, Goetz A, Messmer K. Microcirculatory blood flow, capillary morphology and local oxygen pressure of the hamster amelanotic melanoma A-Mel-3. J Natl Cancer Inst 1982;

68:475–85.

25. Klyscz T, Junger M, Jung F, Zeintl H. Cap image—a new kind of computer-assisted video image analysis system for dynamic capillary microscopy. Biomed Tech Berl 1997;42:168–75.

26. Zeintl H, Sack FU, Intaglietta M, Messmer K. Computer assisted leu- kocyte adhesion measurement in intravital microscopy. Int J Micro- circ Clin Exp 1989;8:293–302.

27. Baker M, Wayland H. On-line flow rate and velocity profile measure- ment for blood in microvessels. Microvasc Res 1974;7:131–43.

28. Minischetti M, Vacca A, Ribatti D, Iurlaro M, Ria R, Pellegrino A, Gasparini G, Dammacco AF. TNP-470 and recombinant human inter- feron-a2a inhibit angiogenesis synergistically. Br J Haematol 2000;

109:829–37.

29. Parangi S, O’Reilly M, Christofori G, Holmgren L, Grosfeld J, Folkman J, Hanahan D. Antiangiogenic therapy of transgenic mice impairs de novo tumor growth. Proc Natl Acad Sci USA 1996;93:

2002–7.

30. Kuenen BC. Analysis of prothrombotic mechanisms and endothelial perturbation during treatment with angiogenesis inhibitors. Pathophy- siol Haemost Thromb 2003;33 (Suppl 1):13–4.

31. Stopeck A, Sheldon M, Vahedian M, Cropp G, Gosalia R, Hannah A. Results of a phase I dose-escalating study of the antiangiogenic agent, SU5416, in patients with advanced malignancies. Clin Cancer Res

2002;8:2798–805.

32. Browder T, Butterfield CE, Kraling BM, Shi B, Marshall B, O’Reilly MS, Folkman J. Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer Res 2000;

60:1878–86.

33. Klement G, Huang P, Mayer B, Green SK, Man S, Bohlen P, Hicklin D, Kerbel RS. Differences in therapeutic indexes of combination met- ronomic chemotherapy and an anti-VEGFR-2 antibody in multidrug- resistant human breast cancer xenografts. Clin Cancer Res 2002;

8:221–32.

34. Mendel DB, Schreck RE, West DC, Li G, Strawn LM, Tanciongco SS, Vasile S, Shawver LK, Cherrington JM. The angiogenesis inhibi- tor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phosphorylation and function. Clin Cancer Res 2000;

6:4848–58.

35. Hlatky L, Hahnfeldt P, Folkman J. Clinical application of antiangio- genic therapy: microvessel density, what it does and doesn’t tell us.

J Natl Cancer Inst 2002;94:883–93.

36. Jain RK, Munn LL, Fukumura D. Dissecting tumour pathophysiology using intravital microscopy. Nature Rev Cancer 2002;2:266–76.

37. Fenton BM, Paoni SF, Ding I. Pathophysiological effects of vascular endothelial growth factor receptor-2-blocking antibody plus fractio- nated radiotherapy on murine mammary tumors. Cancer Res 2004;

64:5712–9.

38. Kozin SV, Boucher Y, Hicklin DJ, Bohlen P, Jain RK, Suit HD. Vas- cular endothelial growth factor receptor-2-blocking antibody potenti- ates radiation-induced long-term control of human tumor xenografts. Cancer Res 2001;61:39–44.

39. Lee CG, Heijn M, di Tomaso E, Griffon-Etienne G, Ancukiewicz M, Koike C, Park KR, Ferrara N, Jain RK, Suit HD, Boucher Y. Anti- vascular endothelial growth factor treatment augments tumor radia- tion response under normoxic or hypoxic conditions. Cancer Res

2000;60:5565–70.

40. Halin C, Zardi L, Neri D. Antibody-based targeting o f angiogenesis. News Physiol Sci 2001;16:191–4.

41. Vajkoczy P, Menger MD, Vollmar B, Schilling L, Schmiedek P, Hirth KP, Ullrich A, Fong TA. Inhibition of tumor growth, angiogenesis, and microcirculation by the novel Flk-1 inhibitor SU5416 as assessed by intravital multi-fluorescence videomicroscopy. Neoplasia 1999;1:

31–41.

42. Eichhorn ME, Strieth S, Dellian M. Anti-vascular tumor therapy:

recent advances, pitfalls and clinical perspectives. Drug Resist Updat

2004;7:125–38.

43. Li Y, Wang MN, Li H, King KD, Bassi R, Sun H, Santiago A, Hooper AT, Bohlen P, Hicklin DJ. Active immunization against the vascular endothelial growth factor receptor flk1 inhibits tumor angiogenesis and metastasis. J Exp Med 2002;195:1575–84.

44. Erber R, Thurnher A, Katsen AD, Groth G, Kerger H, Hammes HP, Menger MD, Ullrich A, Vajkoczy P . Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering

ANTIANGIOGEN IC COMBINAT ION TUMOR THERAP Y BLOCKIN G

V -INTEGRINS AND VEGF-RE CEPTOR-2

431

with pericyte-mediated endothelial cell survival mechanisms. FASEB J 2004;18:338–40.

45. Bergers G, Javaherian K, Lo KM, Folkman J, Hanahan D. Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. Science

1999;284:808–12.

46. Bisacchi D, Benelli R, Vanzetto C, Ferrari N, Tosetti F, Albini A. Anti-angiogenesis and angioprevention: mechanisms, problems and perspectives. Cancer Detect Prev 2003;27:229–38.

47. Saaristo A, Karpanen T, Alitalo K. Mechanisms of angiogenesis and their use in the inhibition of tumor growth and metastasis. Oncogene

2000;19:6122–9.

48. Dellian M, Walenta S, Gamarra F, Kuhnle GE, Mueller-Klieser W, Goetz AE. High-energy shock waves enhance hyperthermic response of tumors: effects on blood flow, energy metabolism, and tumor growth. J Natl Cancer Inst 1994;86:287–93.

49. Seftor RE, Seftor EA, Hendrix MJ. Molecular role(s) for integrins in human melanoma invasion. Cancer Metastasis Rev 1999;18:359–

75.

50. Allman R, Cowburn P, Mason M. In vitro and in vivo effects of a cyclic peptide with affinity for the a m b3-integrin in human melanoma cells. Eur J Cancer 2000;36:410–22.