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Clinical Cancer Research Vol. 11, 6270-6279, September 1, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Inhibition of {alpha}vß3 Integrin Survival Signaling Enhances Antiangiogenic and Antitumor Effects of Radiotherapy

Amir Abdollahi1, David W. Griggs3, Heike Zieher1, Alexandra Roth1, Kenneth E. Lipson4, Rainer Saffrich1, Hermann-Josef Gröne2, Dennis E. Hallahan5, Ralph A. Reisfeld6, Juergen Debus1, Andreas G. Niethammer1 and Peter E. Huber1

Authors' Affiliations: Departments of 1 Radiation Oncology and 2 Pathology, German Cancer Research Center, Heidelberg, Germany; 3 Pfizer Global Research and Development, Chesterfield, Missouri; 4 SUGEN, Inc., South San Francisco, California; 5 Department of Radiation Oncology, The Vanderbilt Clinic, Vanderbilt University, Nashville, Tennessee; and 6 Department of Immunology, The Scripps Research Institute, La Jolla, California

Requests for reprints: Peter E. Huber, Department of Radiation Oncology, German Cancer Research Center (DKFZ), 280 Im Neuenheimer Feld, 69120 Heidelberg, Germany. Phone: 49-6221-42-2515; Fax: 49-6221-42-2514; E-mail: p.huber{at}dkfz.de.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The involvement of {alpha}vß3 and {alpha}vß5 integrins in angiogenesis and the use of integrin antagonists as effective antiangiogenic agents are documented. Radiotherapy is an important therapy option for cancer. It has been shown that ionizing radiation exerts primarily antiangiogenic effects in tumors but has also proangiogenic effects as the reaction of the tumor to protect its own vasculature from radiation damage. Here, we show that combined treatment with S247, an Arg-Gly-Glu peptidomimetic antagonist of {alpha}vß3 integrin, and external beam radiotherapy are beneficial in local tumor therapy. We found that radiation up-regulates {alpha}vß3 expression in endothelial cells and consecutively phosphorylates Akt, which may provide a tumor escape mechanism from radiation injury mediated by integrin survival signaling. In the presence of S247, the radiation-induced Akt phosphorylation is strongly inhibited. Our studies on endothelial cell proliferation, migration, tube formation, apoptosis, and clonogenic survival show that the radiosensitivity of endothelial cells is enhanced by the concurrent administration of the integrin antagonist. The in vitro data are successfully translated into human glioma (U87), epidermoid (A431), and prostate cancer (PC3) xenograft models growing s.c. on BALB/c-nu/nu mice. In vivo, the combination of S247 treatment and fractionated radiotherapy (5 x 2.5 Gy) leads to enhanced antiangiogenic and antitumor effects compared with either monotherapies. These results underline the importance of {alpha}vß3 integrin when tumors protect their microvasculature from radiation-induced damage. The data also indicate that the combination of integrin antagonists and radiotherapy represents a rational approach in local cancer therapy.

Radiotherapy is an important nonsurgical treatment for cancer. Recent studies have shown that tumor vasculature and, in particular, the vascular endothelial cells are important targets of radiotherapy (1–3). A substantial body of data now exists that supports the view that integrins, such as {alpha}vß3, {alpha}vß5, and {alpha}5ß1, play a critical role in tumor angiogenesis and tumor growth (4–10). Likewise, high-level expression of integrin {alpha}vß3 was reported to be characteristic of several invasive tumor types, including glioma, prostate, and breast cancer, where it contributes to their malignant phenotype (4, 8, 11). Consequently, integrin antagonists have been successfully applied as antitumor agents (4, 7, 9, 10). However, the effects of integrin signaling on angiogenesis pathways are not completely understood and conflicting results have been reported (12–15).

It is conceivable that to be effective in the clinical setting, integrin antagonists, like other antiangiogenic agents, will be used in combination with standard anticancer regimen such as chemotherapy or radiotherapy; however, it is unclear which combination will be most promising. It has been shown in an experimental breast cancer model in mice that administration of a cyclic Arg-Gly-Glu (RGD) peptide (Cilengitide) resulted in increased efficacy of radioimmunotherapy using yttrium-90 (a ß-emitter) labeled antitumor antibody (16). Whereas Cilengitide alone did not alter tumor growth, the up to 50% increase in tumor uptake of radiolabeled anti–breast cancer antibody after treatment with the cyclic RGD peptide was suggested as a possible mechanism for the enhanced antitumor efficacy of radioimmunotherapy (16, 17).

Here, we were interested in the interaction of standard external beam radiotherapy using high-energy photons and small-peptide integrin antagonism. One rationale for combining external beam radiotherapy with integrin antagonism is based on reports showing that the combination of angiogenesis inhibitors and radiotherapy results in increased antiangiogenic and antitumor efficacy (18–20). This concept has been extensively investigated, e.g., for the combination of vascular endothelial growth factor (VEGF) signaling inhibitors with radiotherapy but also for VEGF inhibitors with chemotherapy. Both combination concepts have recently entered clinical trials (1, 21–26). One explanation for the benefit of combining VEGF inhibitors with radiation is the finding that radiation itself up-regulates VEGF expression in tumors and VEGF receptor 2 (VEGFR2) expression in endothelial cells (1, 25). This suggests a radiation-inducible escape mechanism by which tumors protect their associated vasculature. Thus, coadministration of VEGF inhibitors is one possible option to circumvent this escape mechanism. However, the VEGF system is probably not the only survival signal after radiation damage (1, 27). In fact, it is known that integrin-related survival pathways are very similar to those triggered by growth factors, such as VEGF, and that they are coupled. We hypothesize here that integrin survival signaling may play an important role in endothelial cell sensitivity to ionizing radiation.

Here, we investigate the benefits of combining S247, a recently described RGD-based synthetic peptidomimetic antagonist of {alpha}v-integrins (28, 29), with external beam radiotherapy. To study the mechanisms of the cooperative action of both agents, we focused on three issues: First, we investigated the role of integrins in the direct response of endothelial cells to radiation. Second, we explored the role of integrins in the radiation-induced communication between tumor and its microenvironment. Third, we focused on effects induced by abrogating integrin survival signaling in combination with radiotherapy on tumor growth and tumor angiogenesis in vivo.

We found that radiation induces expression of {alpha}vß3 in human endothelial cells, suggesting that a concurrent integrin antagonist might attenuate integrin survival signaling. We then tested the combined in vitro effects of radiotherapy and S247 by measuring endothelial cell apoptosis, adhesion, proliferation, migration/invasion, tube formation, and survival. To analyze the role of integrin signaling in endothelium-tumor interactions, we established a coculture system consisting of prostate cancer cells (PC3) and endothelial cells [human umbilical vascular endothelial cell (HUVEC)]. Finally, we investigated the combined effects of S247 and fractionated radiation (5 x 2.5 Gy) in our three s.c. human tumor models in BALB/c-nu/nu mice.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents and cell culture. Primary isolated HUVECs and human dermal microvascular endothelial cells (HDMEC; Promocell, Heidelberg, Germany) were cultured up to passage 5. Cells were maintained in culture at 37°C with 5% CO2 and 95% humidity in serum reduced (5% FCS) modified Promocell medium supplemented with 2 ng/mL VEGF and 4 ng/mL basic fibroblast growth factor (bFGF; refs. 1, 30, 31). Human prostate (PC3), glioma (U87), and vulva (A431) tumor cells (Tumorbank DKFZ, Heidelberg, Germany) were cultured in DMEM medium (10% FCS). All experiments were carried out with HUVEC (up to passage 5) and a selection of experiments was confirmed using HDMEC (up to passage 6). Antibody against {alpha}vß3 (clone LM609) was purchased from Chemicon, Inc. (Temecula, CA).

The novel nonpeptidic compound (S247) was chosen from a library of chemically synthesized RGD peptide mimetics. S247, a highly potent antagonist that selectively inhibits the activities of the {alpha}v subunit–containing family of integrins (7, 28, 29), was obtained from Pharmacia Corporation (Oncology Pharmacology, Discovery Research, Pharmacia Corporation, St. Louis, MO).

Adhesion, proliferation, and clonogenic and tube formation assay. Tests were done as previously described (1, 30). Briefly, to measure effects on cell proliferation, 5 x 104 endothelial cells were seeded on 25 cm2 collagen-coated flasks overnight at standard conditions. The cells were treated with or without S247 for 1 hour, thereafter incubated for another 72 hours, and counted. For adhesion tests, HUVEC and/or HDMEC endothelial cells were incubated with S247 and seeded in flasks coated with different extracellular matrix components (Becton Dickinson, Heidelberg, Germany). Cells were washed and counted after 2 hours. For the clonogenic assay, increasing numbers of cells (102 to 5 x 104) were plated in 25 cm2 flasks, incubated with S247, and returned to the incubator for 14 to 17 days, after which they were stained with crystal violet (Sigma, Munich, Germany). For the tube formation assays, 24-well plates were coated with 300 µL Matrigel (Becton Dickinson), cells were plated, S247 added, and after 6 hours incubation cells were fixed and stained with Diff-Quik II reagents (Dade Behring, AG, Marburg, Germany).

Matrigel invasion, migration, and coculture experiments. Invasion of HUVEC and HDMEC in vitro was measured on Matrigel-coated (0.78 mg/mL) transwell inserts with 8 µm pore size (Becton Dickinson, Heidelberg, Germany). Cells were trypsinized and 200 µL of cell suspension (3 x 105 cells/mL) per experiment were added to transwells in triplicate. Chemoattractant medium containing VEGF and bFGF (500 µL) was added to the lower wells. For coculture studies, PC3 cells were seeded in 24-well plates and, after irradiation of PC3 cells, Matrigel-coated transwells with endothelial cells were added to the upper compartment. After 18 hours of incubation, endothelial cells that had invaded the underside of the membrane were fixed and stained with Diff-Quik II solution (Dade Behring) and sealed on slides. Migrating cells were counted by microscopy.

Flow cytometry. Apoptosis was determined by sub-G1 analysis by fluorescence-activated cell sorting. Up to 72 hours after therapy, fluorescence-activated cell sorting analysis (Becton Dickinson FACScans, San Jose, CA) for DNA content was done as previously described (1, 30). Briefly, cells were fixed in Hank's solution and 70% ethanol, centrifuged, washed in PBS, and the supernatant removed. These cells were then resuspended in the staining solution of PBS, RNase, and propidium iodide.

Immunocytochemistry. HUVEC and HDMEC were grown on round, 11 mm glass coverslips and fixed 2.5 hours following irradiation with 0, 2, 5, 10, 15, and 20 Gy. After fixation in 3.7% paraformaldehyde, cells were incubated with a monoclonal {alpha}vß3-specific primary antibody (LM609, Chemicon, Hofheim, Germany) followed by incubation with the Alexa-488–conjugated antimouse secondary antibody (Molecular Probes, Leiden, the Netherlands). To detect Akt phosphorylation, rabbit anti-Akt, phosphospecific (Ser473) primary antibody (Santa Cruz, Heidelberg, Germany) was used. Cells were counterstained with propidium iodide for nuclear staining. Finally, cells were washed, mounted with Mowiol on microscope slides, and observed on a Zeiss Axiovert 10 inverted microscope with a 20x objective. For each radiation dose, at least five slides were analyzed. Images from six representative high-power fields per slide were acquired. A cooled charged-coupled device camera (Photometrics CH250) was used and fluorescence excitation was detected with an FITC filter set (acquisition time = 5 seconds). Images were stored as TIFF files on a Sun SparcStation 20 Unix workstation. Image processing and analysis was done with programs written for the Khoros Software package. The settings and image processing procedures were constant for all analyzes.

Animal studies. Animal studies were done according to the rules for care and use of experimental animals and approved by the local and governmental Animal Care Committee instituted by the German government (Regierungspraesidium, Karlsruhe). For tumor growth experiments with s.c. growing human xenotransplants, athymic 8-week-old, 20 g BALB/c-nu/nu mice were obtained from Charles River Laboratories (Sulzfeld, Germany). Human PC3 prostate carcinoma cells, U87 glioblastoma cells, and A431 vulva carcinoma cells were injected s.c. into the right hind limb (1-5 x 106 cells in 100 µL PBS). Animals were randomized for therapy when tumor volume reached 200 mm3 as determined thrice weekly by direct measurement with calipers and calculated by the formula volume V = length x width x width x 0.5. Starting on day 0, S247 was administered s.c. in PBS bid at 25 mg/kg until the end of observation. Radiotherapy (5 x 2.5 Gy) was delivered on 5 consecutive days using a Co-60 source (Siemens, Gammatron, Erlangen, Germany).

Immunohistochemistry and terminal deoxynucleotidyl transferase–mediated nick end labeling. For histologic analysis, tumors were harvested from three animals 10 days after the start of S-247 therapy and at the end of the observation period, fixed in buffered formalin, and embedded in paraffin. Tissue slices (5 µm) were stained with H&E and general tissue morphology was visualized and photographed with a camera (Nikon Super Coolscan ED 4000, Tokyo, Japan) mounted on a Zeiss microscope (Carl Zeiss, Jena, Germany). Tumor cell proliferation was assessed by percentage Ki-67–positive cells determined by immunohistochemical staining with the MIB-1 monoclonal mouse anti-human Ki-67 antibody (DAKO, Hamburg, Germany). Sections were counterstained with H&E. Ki-67 staining was quantified by counting the number of positively stained nuclei of 200 to 250 cells in 10 randomly chosen fields at x100 magnification. To quantify tumor vessel counts, frozen sections were fixed and stained with primary antibody to CD31 (Becton Dickinson, Heidelberg, Germany) and 10 random fields at x100 magnification were chosen. To detect the phosphorylated Akt status in vivo, paraffin-embedded tissue sections were stained using rabbit antiphosphorylated Akt (Ser473) antibody (immunohistochemistry specific, Cell Signaling Technology, Beverly, MA) and Signal Stain phosphorylated Akt (Ser473) immunohistochemistry detection kit (Cell Signaling Technology) according to the instructions of the manufacturer (32). To identify apoptotic cells in vivo, terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) assay was done using In situ Cell Death Detection kit (Roche Diagnostics, Mannheim, Germany) as described (22, 33). Slides were counterstained with hematoxylin and mounted. Stained tissue sections were analyzed under a light microscope. The number of TUNEL+ cells was counted at x400 magnification and related to the size of one high-power field. At least 10 high-power field per section were analyzed.

Statistical analysis. The tumor volume V was calculated (0.5 x a x b2) and normalized to V0 at the onset of treatment (day 0). Statistical evaluation of tumor growth was undertaken by daily comparisons of the volumes. In addition, the general response to treatment was assessed on the basis of the time, Tn, required to reach n times the initial tumor volume. For multiple comparisons, the Kruskall-Wallis ANOVA was used for nonparametric variables. For parametric variables, ANOVA was used along with Fisher's least significant difference. All analyses were two-tailed.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Radiation up-regulates {alpha}vß3 integrin in human endothelial cells. To investigate the role of integrins in endothelial cell response to ionizing radiation, we treated HUVEC and HDMEC with ionizing radiation from 2 to 20 Gy. {alpha}vß3 expression was measured 2.5 hours after irradiation by immunocytochemistry with {alpha}vß3-specific monoclonal antibody (Fig. 1A). In HUVEC, the expression of {alpha}vß3 increased with radiation dose, starting at the typical clinical radiation daily fraction of 2 Gy and reaching a plateau at 15 Gy (Fig. 1B). Results for HDMEC were qualitatively similar. This radiation-induced enhancement of {alpha}vß3 expression in primary human endothelial cells was similar to that observed in a recent report describing in vivo phage display of a peptide library in mice with tumors (34). There, the integrin family was proposed as a potential mediator of radiation-inducible targeted therapy. Therefore, our data on regulation of {alpha}vß3 integrin by radiation suggests, therefore, that this integrin may also have a role in radiation response of human endothelium.



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  		Fig. 1. Radiation-induced up-regulation of {alpha}vß3 integrin in HUVEC 2.5 hours after irradiation. A, immunocytochemistry of HUVEC irradiated with 0, 2, 5, 10, 15, and 20 Gy, respectively. Cells were stained with {alpha}vß3 receptor–specific antibody and photographed under the microscope. Photos represent typical fields on each slide. B, image analysis of {alpha}vß3 receptor–specific antibody staining from (A). Points, means from averaged intensities over six high-power fields per slide from five slides per radiation dose as described in Materials and Methods; bars, SD.

 
S247 is a potent peptidomimetic integrin antagonist. To investigate the role of integrins in response of endothelial cells to radiation, we used S247, a novel synthetic low-molecular-weight peptidomimetic compound of the ligand amino acid sequence RGD. This compound has high potency against the family of {alpha}v subunit–containing integrins. In binding assays with HEK293 cells transfected with human {alpha}vß3 and {alpha}vß5, S247 inhibited adhesion with an IC50 of 0.4 ± 0.24 and 1.5 ± 1.26 nmol/L, respectively. However, the compound was significantly less potent against two other integrins that also utilize the RGD sequence motif for ligand recognition, {alpha}IIbß3 (IC50, 380 ± 92 nmol/L) and {alpha}5ß1 (IC50, 64 ± 6 nmol/L). Thus, S247 is >3 orders of magnitude more selective for {alpha}vß3 than for {alpha}IIbß3. The degree of activity against the former integrin was considered physiologically insignificant because there was a complete lack of effect on platelet aggregation at concentrations as high as 500 µmol/L. Furthermore, such concentrations were not achieved in the plasma in any of our animal studies, and no notable differences in bleeding or wound healing were observed in animals after surgery (26). S247 reduced HUVEC cell adhesion, especially to vitronectin (Fig. 2A). S247 had only a minor effect on the attachment of cells to laminin or collagen, indicating that it does not interfere with interactions that do not require the RGD ligand recognition motif (Fig. 2A). Results for HDMEC were qualitatively similar as for HUVEC (data not shown).



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  		Fig. 2. S247 alone is a potent inhibitor of HUVEC adhesion, proliferation, and survival. A, S247 inhibits HUVEC adhesion dependent on different extracellular matrix components; points, mean (n = 6); bars, SD. B, HUVEC proliferation in response to S247; points, mean (n = 4); bars, SD. C, clonogenic HUVEC survival in response to S247; points, mean (n = 5); bars, SD.

 
Effects of S247 and radiation on endothelial cell proliferation. First, we assessed the potential antiangiogenic effects of the integrin antagonist alone on human endothelial cells. To this end, we did proliferation, tube formation, and migration/invasion assays. S247 was found to be a potent inhibitor of HUVEC proliferation with an IC50 of 1.5 µmol/L (Fig. 2B). Next, we found that the combination of S247 with radiation significantly enhanced the antiproliferative effects in HUVEC for all radiation doses tested between 0.5 and 10 Gy (P < 0.05; Fig. 3A). S247 also reduced significantly (P < 0.05; Fig 3B) cell count in U87 human glioblastoma, A431 human epidermoid, and PC3 prostate cancer cells. The combination with radiation was able to further decrease tumor cell number versus monotherapies (P < 0.05). Analogous combination experiments showed that S247 also reduced clonogenic survival of HUVEC and tumor cells, an effect that was significantly enhanced by radiation (P < 0.05; Fig. 3C and D). Experiments using HDMEC showed qualitatively similar results in cell count and clonogenic survival assays as HUVEC (data not shown).



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  		Fig. 3. Combined effects of S247 and radiation on endothelial cells and tumor cells. A, HUVEC proliferation in response to S247 alone and in combination with radiation; points, mean (n = 5); bars, SD. B, tumor cell proliferation in response to S247 (1 µmol/L) alone and in combination with 2 Gy radiation; columns, mean (n = 4); bars, SD. *P < 0.05 versus control, **P < 0.05 versus each respective monotherapy. C, HUVEC clonogenic survival in response to S247 alone and in combination with radiation; points, mean (n = 5); bars, SD. D, clonogenic tumor cell survival in response to S247 (1 µmol/L) alone and in combination with 2 Gy; columns, mean (n = 5); bars, SD. *P < 0.05 versus control, **P < 0.05 versus each respective monotherapy. E, isobologram analysis for IC50 of HUVEC compared with control. Diagonal line, theoretical line of additivity. Area under the line, synergism for HUVEC proliferation inhibition.

 
We next did isobolographic analysis to determine whether combinations exhibited additive or synergistic antiproliferative activity in HUVEC (35). Endothelial cells were treated with different combinations of S247 concentrations and radiation doses and we estimated, by graphical interpolation from the dose-response curves, the concentration at which the proliferation was inhibited by 50% of control (IC50). Figure 3E depicts the isobologram generated from these data. When compared with the theoretical (diagonal) line representing additive effectivity, all values from the combination treatment S247 + radiation fall below the line, indicating synergistic activity for the end point proliferation inhibition.

Effects of S247 and radiation on endothelial cell apoptosis in vitro. Previous findings suggested that antiangiogenic effects may be attributable to the ability of {alpha}vß3 antagonists to induce apoptosis in proliferating blood vessels (7, 36). We measured the apoptosis rate of HUVEC 4 hours after treatment with S247 (1 µmol/L) and 2 Gy radiation by sub-G1 peak (fluorescence-activated cell sorting) analysis. Figure 4B shows that incubation of endothelial cells with 1 µmol/L S247 induced ~15% of apoptotic cells compared with 5% in nontreated controls (P < 0.01), whereas 9% apoptotic endothelial cells were observed after 2 Gy (P < 0.05 versus control). The combination of S247 and radiation resulted in ~25% apoptotic cells, an effect that was significantly greater than each treatment alone (P < 0.05). We have previously shown that ionizing radiation induced phosphatidylinositol 3'-kinase signaling leads to the activation of a key antiapoptic protein kinase Akt (also known as protein kinase B) in endothelial cells (37). Here, we found that the integrin antagonist S247 can interfere with this survival mechanism by inhibiting Akt activity as shown by immunohistochemistry (Fig. 4C and D). Thus, we could show that S247 potently inhibited Akt phosphorylation induced by 2 Gy irradiation in human endothelial cells. These observations suggest that apoptosis can contribute to the suppression of endothelial cell proliferation and clonogenic survival upon treatment with the S247 integrin antagonist.



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  		Fig. 4. S247 increases radiation-induced HUVEC apoptosis and migration inhibition. A, migrated endothelial cells through Matrigel matrix after treatment of cells as indicated. Columns, mean (n = 6); bars, SD. *P < 0.05 versus control, **P < 0.05 versus each respective monotherapy. B, apoptotic fraction of HUVEC 4 hours after treatment with S247 (1 µmol/L) and 2 Gy radiation determined by fluorescence-activated cell sorting analysis as described in Materials and Methods. Columns, mean (n = 6); bars, SD. *P < 0.05 versus control, **P < 0.05 versus each respective monotherapy. C, immunocytochemistry of phosphorylated Akt protein 45 minutes postradiation shows that S247 attenuates radiation-induced activation of Akt. HUVECs were treated with S247 in the presence and absence of 2 Gy irradiation. The cells were stained with specific antibody against phosphorylated Akt (Ser473; green). For nuclear localization, cells were counterstained with propidium iodide (red). D, diagram of radiation-induced survival signaling. Radiation induces up-regulation of VEGF in tumor cells and up-regulates VEGFR2 and {alpha}vß3 in endothelium. The intricate cell signaling leads to activation of antiapoptotic Akt protein. Thus, the tight convergence of cell-matrix and cell-growth factor interactions results in radioresistance of endothelial cells, which can be reversed by S247.

 
Effects of S247 and radiation on endothelial cell migration/invasion. Next, we did migration/invasion assays and found that S247 (1 µmol/L) strongly inhibited HUVEC migration through the extracellular matrix (Matrigel) with ~80% reduction (P < 0.05; Fig. 4A). Radiation alone only modestly inhibited migration. However, the combination of the integrin antagonist with radiation further reduced the antimigratory effects significantly compared with single treatment (P < 0.05). Migration experiments using HDMEC showed qualitatively similar results (data not shown).

Effects of S247 and radiation on endothelial cell tube formation. In addition to proliferation and migration, the sprouting of endothelial cells and formation of tubes are crucial steps in the angiogenic process. In tube formation assays, we observed that both S247 and radiation alone could inhibit the HUVEC tube formation process. This inhibition was significantly increased if both modalities were applied simultaneously (Fig. 5A and B). These data suggest that a combination of ionizing radiation with an integrin antagonist that inhibits integrins relevant to angiogenesis, such as {alpha}vß3, leads to strong antiangiogenic effects in vitro.



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  		Fig. 5. Effect of S247 and radiation on HUVEC tube formation and invasion. A, tube formation done as described in Materials and Methods. Combination of S247 and radiation significantly inhibits the ability of endothelial cell to form tubes. B, quantitative analysis of tube length. Columns, mean (n = 5); bars, SD. *P < 0.05 versus control, **P < 0.05 versus monotherapy. C, analysis of paracrine prosurvival effects of radiation using a coculture of HUVEC and PC3 cells. Visualization of migrating endothelial cells that were chemically attracted by irradiated PC3 tumor cells. HUVECs were pipetted into inserts of Matrigel-coated transwells with the lower chambers containing PC3 cells that were irradiated immediately before seeding. Endothelial cells were supplemented with either control media or control media containing S247. D, coculture invasion/migration quantitative analysis. The bottom PC3 prostate cancer cell compartment was irradiated with the indicated dose. Immediately thereafter, HUVECs were seeded in the top compartment and incubated with S247 or control. Migrated HUVECs were counted. Columns, means (n = 7); bars, SD. *P < 0.05 versus control, **P < 0.05 versus monotherapy.

 
S247 and radiation in tumor-endothelium coculture model. Here, we sought to analyze the proangiogenic effects of radiation mediated by integrin signals. In an effort to mimic in vivo conditions, we used a coculture system utilizing transwell chambers in which PC3 tumor cells were placed in the bottom compartment and endothelial cells were placed in the top compartment, both separated by a Matrigel matrix. Recently, we reported that radiation of PC3 tumor cells up-regulates VEGF and bFGF and that these angiogenic growth factors may activate and attract endothelial cells toward the tumor cell compartment (1). It has also been shown that this activation can be suppressed in part by small molecule inhibitors of VEGFR2 receptor tyrosine kinases. Here, we used a similar approach to determine if S247 could also inhibit endothelial cell migration when activated via tumor cell irradiation in the coculture model. We found that the integrin antagonist potently inhibited HUVEC migration through the Matrigel matrix following its activation via irradiation of tumor cells (Fig. 5C and D). This interruption of the radiation-tumor-endothelial cell communication may play a role in enhancement of the radiation therapy effect observed in our tumor models in vivo. Therefore, the combination of an integrin antagonist with radiation in the treatment of cancer may contribute to the prevention of radiation-induced angiogenic effects by functional inhibition of activated endothelium.

S247 and radiation in human xenograft tumors. We next investigated whether the findings of antiangiogenic activity in vitro translated to in vivo antitumor efficacy in s.c. growing human tumor xenografts in nude mice. In all three models used (U87, PC3, and A431 tumors), S247 monotherapy, as well as radiation monotherapy, induced significant delays in tumor growth (Fig. 6A-C; Table 1). The combination of radiation (5 x 2.5 Gy) with concurrent S247 administration resulted in a significantly greater delay in tumor growth than that produced by either therapy alone, and in some animals was able to mediate tumor regression. All combination treatments were well tolerated, and no difference in animal behavior or weight was found between experimental groups that indicated low general toxicity of the monotherapies and the combination therapy.



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  		Fig. 6. S.c. tumor growth in vivo on BALB/c nude mice after treatment with S247 and radiation and the combination of both. Tumors were treated as described in Materials and Methods. Points, mean of tumor volume V normalized to the initial volume V0; bars, SE. A, U87 human glioblastoma. By-day comparison shows S247 versus control, P < 0.05 from day 4 on; radiotherapy (RT) versus control, P < 0.05 from day 6 on; S247 + RT versus each monotherapy, P < 0.05 from day 6 on (except day 18). B, PC3 human prostate cancer. S247 versus control, P < 0.02 from day 6 on; radiotherapy versus control, P < 0.02 from day 6 on; S247 + radiotherapy versus S247, P < 0.05 from day 23 on; S247 + radiotherapy versus radiotherapy, P < 0.05 from day 35 on. C, A431 human vulva carcinoma. S247 versus control, P < 0.02 from day 3 on; radiotherapy versus control, P < 0.05 from day 3 on; S247 + radiotherapy versus S247, P < 0.02 from day 7 on; S247 + radiotherapy versus radiotherapy, P < 0.05 from day 7 on.

 

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  		Table 1. General tumor response in vivo

 
Histologic examination of tumor xenografts showed marked differences in tumors from animals given S247, radiation, or the combination therapy versus controls (Fig. 7). Tumor cellular proliferation (%Ki-67 positivity) was reduced after S247 or radiation treatment in U87 and PC3 tumors and this growth inhibition was markedly enhanced after combination of both therapies (Fig. 7C and D). In addition, immunohistochemical staining for CD31 revealed a decrease in vessel count after the RT/S247 combination treatment. Again, the combination therapy was more effective in decreasing tumor vessel growth when compared with the monotherapies (Fig. 7A and B). To confirm our data on S247-mediated inhibition of Akt signaling and enhancement of endothelial cells apoptosis in vivo, phosphorylated Akt immunostaining and TUNEL assay were done on paraffin-embedded tissue sections (Fig. 7E and F). Treatment with S247 resulted in reduced phosphorylation of phosphorylated Akt and increased endothelial cell apoptosis, in particular in irradiated tumors.



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  		Fig. 7. Immunohistochemical analysis of human xenograft tumors from Fig. 6 treated with S247 and/or radiation. A, vascular density shown by CD31 staining as described in Materials and Methods. Combined treatment with S247 and fractionated irradiation (5 x 2.5 Gy) significantly reduce the vascularization of A431 tumor xenografts. Representative areas are shown for all four treated groups. B, vessel count in PC3 tumors after CD31 staining. Columns, means; bars, SD. *P < 0.05 versus control, **P < 0.05 versus monotherapies. C, Ki-67 immunohistochemistry in PC3 tumors 10 days after therapy start. D, quantitative analysis of Ki-67 staining in PC3 and U87 tumors on day 10 after therapy start. Columns, mean; bars, SD. *P < 0.05 versus control, **P < 0.05 versus monotherapies. E, phosphorylated Akt immunostaining. Paraffin-embedded tissue sections of control (Ctrl), integrin antagonist (S247), external beam radiation (RT), and combined administration of integrin antagonist and radiotherapy (RT + S247) treated A431 tumor xenografts were stained using rabbit antiphosphorylated Akt (Ser473) antibody. Intense positive staining of endothelial cells are shown in control and irradiated tumor (black arrows with solid line), whereas treatment with S247 resulted in reduced phosphorylation of phosphorylated Akt (black arrows with dot lines). *Erythrocytes within the vessels. F, apoptotic endothelial cells detected by TUNEL assay. Paraffin-embedded tissue sections of control, integrin antagonist, external beam radiation, and combined administration of integrin antagonist and radiotherapy treated A431 tumor xenografts. Black arrows with solid lines, TUNEL + apoptotic endothelial cells and tumor cells. Black arrows with dot lines, TUNEL-negative endothelial cells. Left pictures, x100 view; right pictures, the square box within the x100 view magnified to x400. *Erythrocytes within the vessels.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Here, we show that integrin antagonists and external beam radiotherapy can be favorably combined in local cancer therapy. We found that the RGD peptidomimetic integrin antagonist S247 enhanced radiation-induced antiangiogenic effects in human endothelial cells in vitro and significantly augmented the local antitumor effect of fractionated radiation in tumor models in vivo without apparent increased toxicity. S247 also significantly interrupted the proangiogenic/prosurvival signaling via radiation-induced up-regulation of {alpha}vß3 expression and consecutive Akt activation.

Our data show that S247 and radiation have both marked antiangiogenic and antitumor activities as monotherapies, which was significantly (P < 0.01) enhanced when these modalities were combined. In human glioblastoma (U87), epidermoid (A431), and prostate (PC3) carcinoma xenograft models, the concurrent administration of S247 and fractionated irradiation (5 x 2.5 Gy) significantly increased tumor growth delay compared with either treatment alone.

To understand the underlying mechanism of this effective regimen, we could provide several lines of evidence: First, radiation up-regulates {alpha}vß3 expression and activates Akt in endothelial cells, thus forming a defense mechanism and survival signal against radiation damage—which is counteracted by S247. Second, treatment with S247 resulted in an inhibition of radiation-induced Akt phosphorylation and enhancement of radiation-induced endothelial cell apoptosis in vitro and in vivo. Third, the combined S247/radiation treatment further impaired vascular morphogenesis significantly more than each treatment alone. Fourth, selective radiation of tumor cells induced enhanced endothelial cell invasiveness through the Matrigel matrix in the coculture model. This invasion of endothelial cells was markedly attenuated in the presence of the integrin antagonist in the endothelial cell compartment. From these data, it is conceivable that {alpha}vß3 integrin–mediated survival mechanisms after radiation toxicity can effectively be interrupted by coadminstration of an integrin antagonist.

Integrin-stimulated pathways are similar to those triggered by growth factors and are intimately coupled with them. For example, {alpha}vß3 integrin physically associates with VEGFR2 and enhances VEGF signaling. Furthermore, integrin-mediated outside-in signals cooperate with VEGFR2 to promote proliferation, migration, and survival in endothelial cells (Fig. 4D; refs. 38, 39). In addition, integrin blocking experiments have shown that angiogenesis induced by bFGF is dependent on {alpha}vß3 (39). Furthermore, it has been reported that ionizing radiation–induced phosphatidylinositol 3'-kinase signaling leads to the activation of the key antiapoptic protein kinase Akt in endothelial cells (18, 37). In this context, it is of interest that radiation has been shown to induce VEGF and bFGF expression in tumor cells and VEGFR2 up-regulation in the endothelium (1, 25). Likewise, it has been shown that the proapoptotic effects of radiation on endothelial cells are suppressed by the presence of VEGF and bFGF (1, 3, 22, 31). High local concentrations of VEGF and bFGF in the tumor microenvironment might, therefore, promote radioresistance by inducing a highly specific radioprotective effect toward the VEGFR2- and {alpha}vß3 integrin–positive endothelial cells of the tumor. Thus, the combination of radiotherapy with a drug that blocks bFGF, VEGF or VEGFR2, or, as shown here, {alpha}vß3 integrin should selectively amplify the proapoptotic effects of radiotherapy against activated endothelial cells (Fig. 4D). In contrast, it may not work against other types of dividing normal cells and thus improve the therapeutic index. In conclusion, inhibition of VEGF or integrin survival signaling may lead to the reversal of endothelial cell resistance to radiotherapy, which would be a way to enhance antitumor effects of radiotherapy (18).

In support of this view of interrelated signals, a highly interactive angiogenic signaling network in human endothelial cells has been described (40). Genes, including Id-1, Stat3, and {alpha}v and ß3 integrins, were collectively down- or up-regulated depending on the angiogenic status of the cells. Independent analysis of the human endothelial transcriptome after ionizing radiation revealed an up-regulation of integrins and proangiogenic transcription factors, such as Id-1 and Stat3, in the acute phase, hours after radiation insult (41). Together with data on coregulation and communication of Ids, Stats, and integrins (4, 42, 43), our data indicate that inhibition of integrin signaling by S247 may lead to a broader down-regulation of angiogenic and survival factors, which is needed to block the undesirable proangiogenic effects induced by radiation.

Besides the signaling aspects, the increased tumor penetration of radiolabeled antibodies after cyclic RGD peptide further indicates a potential improvement of the pathophysiologic conditions by integrin antagonists with potential implications for subsequent radiotherapy and/or chemotherapy (16, 17). However, the potential beneficial effects of integrin antagonists on tumor vascular permeability, perfusion, and interstitial pressure remain to be elucidated.

Taken together, our data imply that the release of paracrine growth factor by the tumor and up-regulation of {alpha}vß3 integrin in the endothelium may represent a coordinated mechanism by which primary radiation-induced antivascular effects are attenuated. These results support the idea that the combined use of radiotherapy and integrin antagonists may potentially allow a lowering of the radiation doses required to achieve local tumor control and thus spare healthy tissue from unnecessary high radiation doses. Our findings suggest that the pharmacologic antagonism of integrin functions attained with low-molecular-weight RGD peptidomimetic agents, in combination with radiotherapy, may represent a rational approach to the treatment of cancer.


   Acknowledgments
 
We thank Thuy Trinh, Ute Haner, Conny Leimert, and Alexandra Tietz for excellent technical assistance; Peter Peschke and Klaus Weber for scientific suggestions; and Michael Wannenmacher for his general support.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/23/04; revised 5/17/05; accepted 6/ 3/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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