SEARCH

SEARCH BY CITATION

Keywords:

  • bone metastases;
  • integrins;
  • osteolysis;
  • volumetric computed tomography;
  • dynamic contrast-enhanced MRI

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The aim of this study was to investigate the effect of inhibiting αvβ3vβ5 integrins by cilengitide in experimentally induced breast cancer bone metastases using noninvasive imaging techniques. For this purpose, nude rats bearing established breast cancer bone metastases were treated with cilengitide, a small molecule inhibitor of αvβ3 and αvβ5 integrins (75 mg/kg, five days per week; n = 12 rats) and compared to vehicle-treated control rats (n = 12). In a longitudinal study, conventional magnetic resonance imaging (MRI) and flat panel volumetric computed tomography were used to assess the volume of the soft tissue tumor and osteolysis, respectively, and dynamic contrast-enhanced (DCE-) MRI was performed to determine functional parameters of the tumor vasculature reflecting blood volume and blood vessel permeability. In rats treated with cilengitide, VCT and MRI showed that osteolytic lesions and the respective bone metastatic soft tissue tumors progressed more slowly than in vehicle-treated controls. DCE-MRI indicated a decrease in blood volume and an increase in vessel permeability and immunohistology revealed increased numbers of immature vessels in cilengitide-treated rats compared to vehicle controls. In conclusion, treatment of experimental breast cancer bone metastases with cilengitide resulted in pronounced antiresorptive and antitumor effects, suggesting that αvβ3vβ5 inhibition may be a promising therapeutic approach for bone metastases.

Bone metastases occur frequently in many human malignancies including breast, prostate and lung carcinoma. The stimulation of osteoclasts by tumor cells proliferating within the bone marrow is a feature of the pathogenesis of bone metastases, and both the tumor and the bone microenvironment must be considered when strategies for therapy of bone metastases are developed.1 Bisphosphonates are potent inhibitors of osteoclast function that have been used over the last decades to treat patients with bone metastases. However, they do not induce regression of bone metastases. This, together with the adverse effects associated with bisphosphonate therapy such as osteonecrosis of the jaw and renal toxicity, emphasize the urgent need for the development of novel therapies that can be applied alternatively and as combination partners to target bone metastases more effectively.

Integrins are a family of 24 transmembrane proteins that integrate extracellular and intracellular activities. Besides their role in promoting physical adhesion, integrin signaling can induce cell spreading, migration, survival, proliferation and differentiation.2 The αvβ3 integrin interacts with several extracellular matrix (ECM) proteins including vitronectin, fibronectin, osteopontin, bone sialoprotein (BSP) and fibrinogen.3, 4 It is strongly expressed on activated tumor endothelial cells, whereas on resting endothelial cells in nondiseased tissues, its expression is generally low.5–7 In the pathogenesis of bone metastases, osteoclasts too express αvβ3 integrin, and selective αvβ3 inhibitors have been shown to inhibit osteoclast-mediated bone resorption in experimental prostate carcinoma bone metastases.8 Furthermore, αvβ3 integrin overexpression on tumor cells stimulated metastasis to bone in experimental models.9, 10 The closely related integrin αvβ5 is also a vitronectin receptor involved in breast cancer cell migration and invasion, but is less studied in the pathogenesis of bone metastasis, although it is overexpressed by osteoclasts and a wide range of cancer cells.11, 12 Together with αvβ5, αvβ3 integrin recognizes the arginine-glycine-aspartic acid (RGD) peptide sequence of extracellular ligands.13 Cilengitide (EMD 121974) is a cyclic pentapeptide containing the sequence RGDf(N-Me)V with high affinity for αvβ3 and αvβ5, which inhibits αvβ3vβ5-dependent cellular processes.14–17 As cilengitide inhibits αvβ3 and αvβ5 integrin from human, bovine and rat origin, it can be appropriately used in both experimental and clinical studies.15, 16 In recent phase II trials for treatment of glioblastoma multiforme, cilengitide has shown promising results including indications of antitumor activity and a good safety profile.13, 18 Cilengitide has antiangiogenic activity in model systems, correlating with its inhibition of attachment, migration, sprouting, differentiation, and in the induction of anoikis in those endothelial angiogenic cells whose adhesion and survival is dependent on αvβ3vβ5.15, 19, 20 Nevertheless, targeting αv integrins for therapy remains contentious, and for some tumors growth is accelerated in mice lacking αvβ3 and αvβ5, whereas in others, tumor growth and angiogenesis are accelerated by cilengitide.21, 22

In this study, we have used noninvasive imaging techniques to examine the dynamics of metastatic lesion development under therapy with cilengitide. Computed tomography (CT) and magnetic resonance imaging (MRI) are currently used to determine the extent of the osteolysis and the respective soft tissue component (STC) of bone metastases. For in vivo imaging of angiogenesis in bone metastases, dynamic contrast-enhanced (DCE-) MRI allows assessment of functional parameters associated with blood volume and vessel permeability in these skeletal lesions.23 We recently introduced an in vivo model of experimental breast cancer bone metastasis in which angiogenesis, soft-tissue lesion size, and extent of osteolysis can be monitored simultaneously and longitudinally by volumetric computed tomography (VCT), morphologic MRI and DCE-MRI.23, 24 Here we have used this model to noninvasively assess the treatment effects of cilengitide inhibiting αvβ3 and αvβ5 integrins in breast cancer bone metastases.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell lines and culture conditions

The human estrogen-independent breast cancer cell line MDA-MB-231 was purchased from American Type Culture Collection. Cells were cultured routinely in RPMI-1640 (Invitrogen, Karlsruhe, Germany) and supplemented with 10% FCS (Sigma, Taufkirchen, Germany). All cultures were kept under controlled conditions (humidified atmosphere, 5% CO2 and 37°C) and passaged two to three times a week to keep them in logarithmic growth.

Flow cytometry

The integrin expression profile of MDA-MB-231 human breast cancer cells was characterized using flow cytometry. Surface integrin staining on live cells was performed as described with minor modifications.25 Briefly, cells were harvested, rinsed, suspended in PBS-BSA (containing divalent cations) and sequentially incubated with mouse anti-αvβ3 (LM60926), mouse anti-αvβ5 (P1F627; Millipore, Schwalbach, Germany) or mouse anti-αv (17E625) followed by staining with fluorescinated goat-anti-mouse IgG and propidium iodide (5 μg/μl). Incubations used 10 μg/μl primary antibody concentrations and were for 45 min on ice. Flow cytometry was performed on a FACScan instrument (Becton-Dickinson, Heidelberg, Germany), gating for viable cells, and collecting 10,000 events per staining. The mean fluorescence intensity of the integrin staining was normalized using the staining intensity of the second layer reagent as background.

Animal model and therapy application

Experiments performed in this study were approved by the Local Governmental Animal Ethics Committee. Nude rats (RNU strain) were obtained from Harlan-Winkelmann GmbH (Borchen, Germany) at the age of 6 weeks and housed in a specific pathogen-free environment in a mini barrier system of the central animal facility of the DKFZ. Animals were kept under controlled conditions (21 ± 2°C room temperature, 60% humidity and 12 hr light-dark rhythm) and offered autoclaved food and water ad libitum. Subconfluent MDA-MB-231 cells were harvested using 0.05% Trypsin-EDTA (Gibco®; Invitrogen, Karlsruhe, Germany) counted on a Neubauer's chamber and resuspended in RPMI-1640 to a final concentration of 105 cells in 200 μl. Rats were anesthetized using a mixture of nitrous oxide (1 l/min), oxygen (0.5 l/min) and isoflurane (1.5 vol %). Arterial branches of the right hind leg were dissected and 105 cells injected into the superficial epigastric artery as described previously.28 Bone metastases were established and observed exclusively in the femur, tibia and fibula of the right hind leg. After 30 days of cancer cell transplantation, rats (n = 24) were randomly divided into two groups, one group receiving the cyclic RGD-peptide inhibitor of αvβ3vβ5 integrins (cilengitide, EMD 12197414, 17, 29; Merck, Darmstadt, Germany) intraperitoneally five times per week in isotonic saline (75 mg/kg; n = 12 rats) and the other, sham-treated group, serving as a control (n = 12 rats). The observation period of all animals was 55 days, and no rat in the study died ahead of schedule.

In vivo imaging

After the inoculation of cancer cells, each rat was imaged at days 30, 35, 45, and 55 using (i) a flat-panel equipped volumetric computed tomography (Volume CT; Siemens, Germany) and (ii) a 1.5-T clinical magnetic resonance scanner (Symphony; Siemens, Erlangen, Germany) equipped with a home-built receive-transmit coil (cylindrical volume resonator with an inner diameter of 83 mm and a usable length of 120 mm). Prior to in vivo imaging with VCT and MRI, rats were anesthetized with nitrous oxide, oxygen and isoflurane as described above.

Volumetric computed tomography

VCT imaging was obtained using the following parameters: tube voltage 80 kV, tube current 50 mA, scan time 51 sec, rotation speed 10 sec, frames per second 120, matrix 512 × 512, and slice thickness 0.2 mm. Image reconstructions were performed using a modified FDK (Feldkamp Davis Kress) cone beam reconstruction algorithm (kernel H80a; Afra, Erlangen, Germany).

Magnetic resonance imaging

The T2-weighted imaging was performed using a turbo spin echo sequence (orientation axial, TR 3240 ms, TE 81 ms, matrix 152 × 256, FOV 90 × 53.4 mm2, slice thickness 1.5 mm, 3 averages and scan time 3 min 40 sec). For DCE-MRI, a saturation recovery turbo flash sequence through the largest diameter of the tumor (orientation axial, TR 373 ms, TE 1.86 ms, matrix 192 × 144, FOV 130 × 97.5 mm, slice thickness 5 mm, measurements 512, averages 1, and scan time 6 min 55 sec) was used. After 20 sec baseline, 0.1 mmol/kg Gd-DTPA (Magnevist; Bayer Schering Pharma, Berlin, Germany) was intravenously infused for a time period of 10 sec.

Postprocessing

Unenhanced VCT images and MRI-acquired T2-weighted images were analyzed using the Medical Imaging Interaction Toolkit (Heidelberg, Germany) to determine volumes of osteolytic lesions (OL) and (STCs), respectively. DCE-MRI acquired data was analyzed using the Dynalab workstation (Mevis Research, Bremen, Germany) according to the two-compartment model of Brix to determine the parameters amplitude A and exchange rate constant kep, as described.23, 30 Briefly, the injected contrast media is distributed in both compartments (intravascular space and extravascular, interstitial space). The accumulation of contrast agent in these compartments over time is characterized by the amplitude A (associated with blood volume), whereas the exchange of contrast agent between the intravascular space and the interstitial space is characterized by the exchange rate constant kep (associated with vessel permeability). For determination of the respective values of the amplitude A and kep of bone metastases in our study, a region of interest was placed around the STC on color maps for A and kep, respectively, using the Dynalab workstation (Mevis Research, Bremen, Germany).

Histology

At the end of the observation period, lower limbs of each animal were amputated and muscular tissue removed. Bones with surrounding soft tissue tumors were stored in 70% ethanol and embedded in a methylmethacrylat-based compound (Technovit 9100 NEU, Heraeus Kulzer, Hanau, Germany) according to the instructions of the manufacturer. The 5-μm thick sections were cut (Microm HM340e microtome; Thermo Scientific, Waltham, MA), mounted on SuperFrost Plus microscope slides and dried overnight at 60°C. In addition freshly removed soft tissue tumors were embedded in optimum cutting temperature (OCT) compound (TissueTec, Sakura, Japan) and stored at −80°C. The 7-μm thick cryosections (obtained on a Leica CM 3050S) were thaw-mounted, fixed in methanol and acetone and washed in PBS. For immunostaining, the Technovit-embedded sections were incubated overnight at 4°C with primary antibodies in PBS containing 12% bovine serum albumin. The following primary antibodies were used: rabbit anti collagen IV polyclonal antibody (1:50; Progen Biotechnik GmbH, Heidelberg, Germany) and mouse anti smooth muscle actin (SMA) polyclonal antibody (1:400; Sigma Aldrich, Saint Louis, MO). After washing in PBS, sections were incubated with secondary antibodies for 1 hr at room temperature as follows: Texas Red dye-conjugated donkey anti-rabbit IgG (1:100; Jackson Immunoresearch, Suffolk, UK) and Cy2-conjugated goat anti-mouse IgG (1:50; Jackson Immunoresearch, Suffolk, UK). Cryosections were incubated overnight at 4°C with the following antibodies: mouse anti-human integrin αvβ3 Alexa Fluor 488 conjugated monoclonal [LM609] antibody (1:100; Millipore GmbH, Schwalbach, Germany) and mouse monoclonal [P1F6] antibody to integrin αvβ5 (Phycoerythrin) (1:100; Abcam, Cambridge, UK). After a nuclear staining step with DAPI (4′,6-diamidino-2-phenylindole, Serva, Heidelberg, Germany) sections were mounted in Fluoromount G (Southern Biotech, USA). Sections were examined using a Leica microscope (DMRE Bensheim, Germany) equipped with a digital camera (F-view XS; Soft Imaging System, Münster, Germany). Mean positive area fractions of SMA and collagen IV (in percent) as well as mean vessel diameters (in μm) were determined from four representative animals of each group analyzing 10 fields of view chosen randomly from each rat using Analysis Software (cell; Olympus Soft Imaging Solutions, Münster, Germany). Immunostainings for CD 31 (endothelial cells) and collagen IV (basal lamina) on tumor vessels were seen to be strongly positively correlated in STCs of bone metastases (data not shown).

For light microscopical analysis, sections were stained with Mayer's hematoxylin (Carl Roth, Karlsruhe, Germany) and eosin (Merck, Darmstadt, Germany), mounted using Eukitt mounting medium (O. Kindler, Freiburg, Germany) and analyzed using a microscope (DM LB; Leica, Wetzlar, Germany) equipped with a digital camera (DFC 320; Leica, Wetzlar, Germany).

Statistical analyses

For each animal, volumes of the osteolysis and STC, amplitude A and exchange rate constant kep were plotted vs. time after tumor cell inoculation (due to technical reasons one animal of the control group could not be evaluated for the amplitude A and kep). Normalization of the data to the corresponding initial value at Day 30 for each animal was performed, and changes were expressed in percent. For statistical comparisons of data from noninvasive imaging and histological analysis, the respective values were compared between the control and treatment groups using the two-sided Wilcoxon test; p values < 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

MDA-MB-231 human breast cancer cells express αvβ5 but only low levels of αvβ3 integrins in vitro

The entire population of MDA-MB-231 cells in vitro expressed αv integrins as detected by the pan alpha-v reagent 17E6 (Fig. 1a). They showed low-cell surface expression of αvβ3 integrins by flow cytometry using the standard defining antibodies in the literature (36% of the cells were gated; median intensity 3-fold background), whereas staining strongly for αvβ5 integrins (100% cells gated; median intensity 10-fold background) (Figs. 1b and 1c). MDA-MB-231 also expressed α2, α3, α5, α6 and β1, β4, but not α4 or β6 chains (data not shown). In situ immunohistochemistry showed that soft tissue tumors stained strongly and quite uniformly for αvβ5 but had only weak patches of staining for αvβ3 (Fig. 1d).

thumbnail image

Figure 1. Expression of integrins of MDA-MB-231 cells in vitro (ac) and in bone metastases (d). MDA-MB-231 cells were stained with antibodies recognizing the αv chains (17E6; a), αvβ3 (LM609; b) or αvβ5 (P1F6; c) integrin complexes, and expression was evaluated by flow cytometry (open curves), staining due to the second layer reagent was minimal (closed curves). The raw data curves have been smoothed for presentation. Immunohistology section (d) of the STC from a control animal staining for αvβ3 (red), αvβ5 (green) and DAPI (blue). A merged image (αvβ3, αvβ5 and DAPI) is shown and single channels for αvβ3 and αvβ5. Bar, 50 μm.

Download figure to PowerPoint

Treatment with cilengitide reduces the volume of OL and STCs in experimental bone metastases as assessed in vivo with VCT and MRI

Tumor bearing animals were randomly assigned to two groups before therapy was begun at Day 30. The mean relative volumes of the OL and the STCs of bone metastases (STC) increased continuously in untreated rats until the end of the observation time (Day 55 post tumor cell injection) compared to the initial values at Day 30 after cancer cell injection (Fig. 2a). Mean relative values of the OL volumes increased by 1.9-, 4.5- and 9.7-fold in the control group and by 1.5-, 2.4- and 3.5-fold in the treatment group (at Days 35, 45 and 55, respectively) when compared to initial values at Day 30 (Fig. 2a, Fig. 3a). Significant differences between the groups were found at Days 45 (p < 0.05) and 55 (p < 0.01) for the OL (Fig. 2a). The mean volume of STC had increased by 2.3-, 10.4- and 22.5-fold in controls at Days 35, 45 and 55, respectively (Fig. 2a). However, the increase in mean relative STC values in bone metastases of the treatment group increased only by 2.2-, 4.9- and 6.3-fold for the volume of STC compared to initial values (Fig. 2a, Fig. 3b). Significant differences between the control and on-therapy groups were recorded at Days 45 (p < 0.05) and 55 (p < 0.01; Fig. 2a) for the STC. In the treatment group, three rats (25%) showed new bone formation under therapy with cilengitide as imaged by VCT (Fig. 3c). This bone formation was confined to the OL, and no excessive increase in bone mass beyond the osteolysis was observed. Such a de novo bone formation further confirmed by histology did not occur in control animals.

thumbnail image

Figure 2. Volumetric analyses of OLs and soft tissue tumors (a) as well as quantification of mean relative values of parameters A (associated with blood volume) and kep (associated with vessel permeability) (b) from experimental bone metastases: comparison between untreated and cilengitide-treated rats. Values are given in percent and are presented as mean values relative to initial values determined at Day 30 after cancer cell inoculation at which time cilengitide therapy was started. The y axis, mean relative values in percent (×100); x axis, days (d) after cancer cells inoculation (d35, d45 and d55); error bars, SEM; *p < 0.05; **p < 0.01.

Download figure to PowerPoint

thumbnail image

Figure 3. Morphological characteristics of vehicle treated and cilengitide-treated experimental bone metastases. Volumes of the OLs (a, c) and soft tissue tumors (b) were determined by the analysis of images acquired by VCT and MRI, respectively, at Days 30, 35, 45 and 55 after cancer cell injection. Therapy with cilengitide commenced after imaging on Day 30. Compare differences in bone loss and soft tumor burden between vehicle treated (a, b: upper rows) as well as cilengitide-treated animals resulting in inhibition of osteolysis and bone formation (a, b: lower rows; c). Representative VCT images: 3D bone surface reconstructions, and MRI: axial slices from T2-weighted imaging. Arrows, proximal tibia of the hind leg.

Download figure to PowerPoint

Experimental breast cancer bone metastases treated with cilengitide reveal changes in DCE-MRI derived parameters for both, relative blood volume and vessel permeability

For the mean relative values of the DCE-MRI parameter amplitude A, a significant decrease was found in animals treated with the αvβ3vβ5 inhibitor at Days 45 (102% of initial value; p < 0.05) and 55 (93% of initial value; p < 0.05) as compared to controls (Day 45, 125% and Day 55, 105% of initial values) but not on Day 35 postinoculation (106% in controls vs. 97% in treated rats; p > 0.05) (Fig. 2b, Fig. 4a). DCE-MRI parameter exchange rate constant kep also revealed significant differences at Day 55 postinoculation with increased values in treated animals (72% of initial value; p < 0.05) compared to controls (40% of initial value) but not on Days 35 (controls, 86% and treated animals, 69%; p > 0.05) or 45 (controls, 63% and treated animals, 88%; p > 0.05) (Fig. 2b, Fig. 4b).

thumbnail image

Figure 4. DCE-MRI-acquired color maps depicting functional parameters of bone metastases amplitude A (associated with blood volume) (a) and exchange rate constant kep (associated with vessel permeability) (b): comparison between untreated and cilengitide-treated rats at Days 30, 35, 45 and 55 after cancer cell inoculation. Cilengitide treatment began following imaging at Day 30. Rats bearing MDA-MB-231 bone metastases were imaged at Day 30 and then following control (upper rows) or cilengitide (lower rows) treatment. These color maps were calculated by the use of DynaLab software, red color denotes high (h) values for the given parameter and blue color denotes low (l) values. The same scaling ranges were used to produce these images for experimental and control animals.

Download figure to PowerPoint

Histological analysis reveals new bone formation, decreased vessel diameter and reduced colocalization of SMA and collagen IV in blood vessels of animals after treatment with cilengitide when compared to untreated controls

In control rats, bone metastases contained tumor cells (representing the soft tissue tumor) within areas of bone resorption corresponding to VCT and MR imaging (Fig. 5a). After treatment with cilengitide, newly formed bone was confirmed on hematoxylin/eosin-stained sections (Fig. 5b) taken from the proximal tibia of the animal, as shown in Fig. 3c. Immunofluorescence analysis in control animals revealed irregular vessels with small diameters, indicated by collagen IV staining in the basal lamina of vessels, which were not colocalized with SMA, along with larger vessels showing collagen IV/SMA colocalization (Fig. 5c). After 4 weeks treatment with cilengitide essentially only small and mesh-like vessels were seen without clear co-localization of SMA and collagen IV (Fig. 5d). Quantification of the immunofluorescent analysis resulted in significantly decreased mean positive area fractions of SMA (p < 0.05) and significantly increased area fractions of collagen IV (p < 0.01) in treated animals as compared to controls (Fig. 6a). The ratio of SMA and collagen IV (treated rats: 0.60/3.32; control rats: 0.83/2.37) was significantly decreased in animals after 4 weeks treatment with cilengitide (p < 0.01), and the mean vessel diameter in cilengitide-treated bone metastases (6.6 μm) was significantly smaller than in control rats (8.8 μm, p < 0.01; Fig. 6b).

thumbnail image

Figure 5. Histological analysis of experimental breast cancer bone metastases of untreated and cilengitide-treated rats. Hematoxylin/eosin-stained sections of an OL in a control rat (a; t: tumor cells; b: bone; arrow: osteoclast) and new bone formation in a treated rat (b; b: bone; arrows: osteoblasts). Immunohistology sections of the STC from a control animal (c) and a cilengitide-treated rat (d). Green color shows collagen IV staining whereas red denotes structures staining for SMA; blue, cell nuclei. Arrows point at larger vessels with partial colocalization of smooth muscle actin and collagen IV, whereas double arrows indicate smaller vessels without clear colocalization of green and red staining. Enlarged images of the highlighted structures are shown below (a′, b′, c′, c″, d′, d″). a-d: bar 100 μm; a′–d″: bar 50 μm.

Download figure to PowerPoint

thumbnail image

Figure 6. Quantification of histological analysis. Values of fractional mean area stained for SMA and collagen IV (Col. IV) are expressed as percent total area examined (a), while the blood vessel diameters are presented as mean values in μm (b). Error bars, SEM; *p < 0.05; **p < 0.01.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The aim of this study was to assess the effects of the αvβ3vβ5 integrin inhibitor cilengitide on breast cancer bone metastases in nude rats transplanted with human MDA-MB-231 breast cancer cells. We used the noninvasive imaging techniques VCT, morphological MRI and DCE-MRI to follow-up longitudinal progression. Our primary findings are that cilengitide treatment, begun a month after tumors have been allowed to implant into bone, decreases osteolysis of breast cancer metastases in nude rats and the volume of the soft tissue tumor components. Cilengitide increases intratumoral vascular permeability, reduces the apparent numbers of mature intratumoral vessels and unexpectedly causes an resumption of bone formation in a quarter of the animals under therapy.

We found a significant decrease in osteolysis using VCT during therapy with cilengitide in nude rats. Several studies have reported a decrease of bone resorption in breast cancer bone metastases after inhibition of the integrin αvβ3.9, 31, 32 However, these groups have used MDA-MB-231 cells engineered and cloned to overexpress αvβ3 or breast cancer cell lines such as MDA-MB-435 that strongly express this integrin. As the MDA-MB-231 cells we used only express low levels of αvβ3, the antiresorptive effect observed here may not have been primarily due to the inhibition of this integrin on tumor cells, but also of αvβ3 on osteoclasts and on the intratumoral vasculature and αvβ5 integrin on all three compartments.12, 33 In previous studies, osteoclasts that express high levels of the αvβ3 integrin, bind several RGD-containing ECM proteins including vitronectin, osteopontin and BSP.34 By these interactions, αvβ3 is involved in the regulation of osteoclast activity, and the inhibition of this integrin was found to reduce osteoclast-mediated bone resorption.35 Furthermore, as angiogenesis is required for initiation and maintenance of osteoclastic bone resorption, its inhibition by cilengitide might have contributed to the observed decrease of osteolysis we observed after cilengitide treatment.36 As cilengitide cross reacts with human and rat αv integrins, the observed effects in our study are because of the inhibition of αvβ3 and αvβ5 integrins on both, MDA-MB-231 and host cells in particular of the vascular and bone compartments. Which compartments are targeted to produce the effects we report here is under investigation.

Interestingly, three animals (25%) treated with cilengitide here showed an increase in bone matrix, i.e., new bone formation in the OLs, which was not seen in control animals. There are no known therapies in use today for patients suffering from bone metastases, where such an effect is seen. After treatment with bisphosphonates, a sclerotic rim around OLs is a common sign for treatment response indicating local bone mineralization, but new bone formation is not seen after this therapy.37 Both integrins, αvβ3 and αvβ5, are expressed by osteoblasts and are associated with osteoblast migration, adhesion and activity.38 We have previously shown in this model of breast cancer bone metastases that the inhibition of BSP also resulted in decreased bone resorption and new bone formation.28, 39 As BSP binds αvβ3 integrin, the inhibition of either factors, BSP or αvβ3, might result in osteoblastic bone formation via the same pathway.40 However, the exact mechanism inducing bone regrowth must still be elucidated.

Not only were there antiresorptive effects but also the respective STCs had a lower volume than in the control animals, indicating an antitumor effect of cilengitide. Cilengitide inhibits the growth of several experimental tumors including melanomas and glioblastomas.41, 42 Because of the high expression of αvβ5 and the low expression of αvβ3 of MDA-MB-231 cells, the antitumor effect we report here may be a consequence of directly inhibiting αvβ5 on the surface of the breast cancer cells, combined with the antiangiogenic effects of inhibiting αvβ3 and αvβ5 on the endothelia of tumor vessels.15 However, this hypothesis was based only on the integrin expression of MDA-MB-231 cells observed in our study and has to be verified experimentally in further studies. Chen et al.43 previously observed that MDA-MB-231 cells expressed αvβ3 and αvβ5 integrins at similar levels suggesting that treatment effects of cilengitide might vary depending on the expression pattern of the respective cell clone used.

Antiangiogenic effects of cilengitide have been described previously in vitro and in vivo.15, 19, 41, 44 In our study, cilengitide treatment of experimental breast cancer bone metastases resulted in a decrease of the amplitude A and an increase of the exchange rate constant kep as assessed by DCE-MRI. These results indicated a decrease in blood volume and an increase of vessel permeability in these skeletal lesions, compatible with an “antiangiogenic” effect. In experimental glioblastomas and melanomas, a decrease in tumor vascularization and tumor growth followed treatment with cilengitide.21, 29 It is generally assumed that the antiangiogenic activity of cilengitide and related inhibitors is due to the experimentally observable inhibition of sprouting and differentiation and the induction of anoikis of angiogenic endothelial cells relaying on αvβ3 and αvβ5 for adhesion and survival.15, 45 In our immunohistological analysis, we observed vessel remodeling after cilengitide treatment including significantly decreased mean vessel diameter and SMA/collagen IV ratio, indicating that smaller vessels lacking pericyte and smooth muscle cells occurred more frequently in these animals than in untreated controls. These results of vessel remodeling rather than complete regression of tumor vessels upon cilengitide treatment are in good agreement with the moderate changes of DCE-MRI parameters A and kep. Taken together, we conclude that cilengitide triggered a decrease in blood volume (assessed by the amplitude A) due to smaller and partly nonfunctional blood vessels and increased vessel permeability (assessed by the exchange rate constant kep) was observed due to the increased number of immature vessels that arose after treatment with cilengitide.

Increased vessel permeability as seen in our study was previously reported by Alghisi et al.,46 who reported VE-cadherin delocalization from cell-cell contact sites on cilengitide treatment leading to a loss of cellular contacts and an increase of endothelial monolayer permeability. In bone metastases, this effect might improve local drug delivery to these lesions when combining cilengitide with standard treatments such as bisphosphonates or chemotherapy. In comparison with bisphosphonates showing predominantly antiosteoclastic and chemotherapy exhibiting mainly cytotoxic effects in bone metastases, cilengitide shows antiresorptive, antitumor and antiangiogenic efficacy in our study. Because of the favorable safety profile of this drug and the alternative mechanism of action compared to currently used treatments, cilengitide emerges as a promising novel therapy for breast cancer metastasis to bone and could be validated either as a single agent or in combination with bisphosphonates and chemotherapy in further experimental and clinical studies. Cilengitide might also be a suitable combination partner for ionizing radiation in the treatment of skeletal lesions due to its previously reported radio sensitizing effects in various tumors including breast cancer.47–49 In some rodent tumor models, a lack of αvβ3 and αvβ5 integrins or an inhibition by low concentrations of cilengitide stimulate tumor growth.50, 51 This seems not to be the case in the breast tumor-to-bone model we report here. Whether one or other of these experimental contexts better reflects the response of human pathologies to αv integrin inhibitors, however, must remain to be proven by clinical trial.19

In conclusion, treatment of well-established experimental breast cancer bone metastases with cilengitide resulted in an inhibition of bone resorption and soft tissue tumor growth in these osseous lesions and partial regrowth of bone. Although further experimental and clinical studies are required, cilengitide is a possible option for breast cancer patients suffering from metastases to bone.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Karin Leotta, Renate Bangert, Lisa Seyler and Catherine Eichhorn for excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Guise TA. Breaking down bone: new insight into site-specific mechanisms of breast cancer osteolysis mediated by metalloproteinases. Genes Dev 2009; 23: 211723.
  • 2
    Schwartz MA. Integrin signaling revisited. Trends. Cell Biol 2001; 11: 46670.
  • 3
    Varner JA, Brooks PC, Cheresh DA. The integrin alpha V beta 3: angiogenesis and apoptosis. Cell Adhes Commun 1995; 3: 36774.
  • 4
    Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002; 110: 67387.
  • 5
    Max R, Gerritsen RR, Nooijen PT, Goodman SL, Sutter A, Keilholz U, Ruiter DJ, De Waal RM. Immunohistochemical analysis of integrin alpha v beta3 expression on tumor-associated vessels of human carcinomas. Int J Cancer 1997; 71: 3204.
  • 6
    Nemeth JA, Nakada MT, Trikha M, Lang Z, Gordon MS, Jayson GC, Corringham R, Prabhakar U, Davis HM, Beckman RA. Alpha-v integrins as therapeutic targets in oncology. Cancer Invest 2007; 25: 63246.
  • 7
    Mulder WJ, Castermans K, van Beijnum JR, Oude Egbrink MG, Chin PT, Fayad ZA, Löwik CW, Kaijzel EL, Que I, Storm G, Strijkers GJ, Griffioen AW, et al. Molecular imaging of tumor angiogenesis using alphavbeta3-integrin targeted multimodal quantum dots. Angiogenesis 2009; 12: 1724.
  • 8
    Nemeth JA, Cher ML, Zhou Z, Mullins C, Bhagat S, Trikha M. Inhibition of alpha(v)beta3 integrin reduces angiogenesis, bone turnover, and tumor cell proliferation in experimental prostate cancer bone metastases. Clin Exp Metastasis 2003; 20: 41320.
  • 9
    Pecheur I, Peyruchaud O, Serre CM, Guglielmi J, Voland C, Bourre F, Margue C, Cohen-Solal M, Buffet A, Kieffer N, Clezardin P. Integrin alpha(v)beta3 expression confers on tumor cells a greater propensity to metastasize to bone. FASEB J 2002; 16: 12668.
  • 10
    Sloan EK, Pouliot N, Stanley KL, Chia J, Moseley JM, Hards DK, Anderson RL. Tumor-specific expression of alphavbeta3 integrin promotes spontaneous metastasis of breast cancer to bone. Breast Cancer Res 2006; 8: R20.
  • 11
    Silvestri I, Longanesi Cattani I, Franco P, Pirozzi G, Botti G, Stoppelli MP, Carriero MV. Engaged urokinase receptors enhance tumor breast cell migration and invasion by upregulating alpha(v)beta5 vitronectin receptor cell surface expression. Int J Cancer 2002; 102: 56271.
  • 12
    Inoue M, Ross FP, Erdmann JM, Abu-Amer Y, Wei S, Teitelbaum SL. Tumor necrosis factor alpha regulates alpha(v)beta5 integrin expression by osteoclast precursors in vitro and in vivo. Endocrinology 2000; 141: 28490.
  • 13
    Reardon DA, Nabors LB, Stupp R, Mikkelsen T. Cilengitide: an integrin-targeting arginine-glycine-aspartic acid peptide with promising activity for glioblastoma multiforme. Expert Opin Investig Drugs 2008; 17: 122535.
  • 14
    Dechantsreiter MA, Planker E, Matha B, Lohof E, Holzemann G, Jonczyk A, Goodman SL, Kessler H. N-Methylated cyclic RGD peptides as highly active and selective alpha(v)beta(3) integrin antagonists. J Med Chem 1999; 42: 303340.
  • 15
    Nisato RE, Tille JC, Jonczyk A, Goodman SL, Pepper MS. Alpha v beta 3 and alphav beta 5 integrin antagonists inhibit angiogenesis in vitro. Angiogenesis 2003; 6: 10519.
  • 16
    Patsenker E, Popov Y, Sickel F, Schneider V, Ledermann M, Sägesser H, Niedobitek G, Goodman SL, Schuppan D. Pharmacological inhibition of integrin alphavbeta3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis. Hepatology 2009; 50: 150111.
  • 17
    Xiong JP, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman SL, Arnaout MA. Crystal structure of the extracellular segment of integrin alpha V beta3 in complex with an Arg-Gly-Asp ligand. Science 2002; 296: 1515.
  • 18
    Reardon DA, Fink KL, Mikkelsen T, Cloughesy TF, O'Neill A, Plotkin S, Glantz M, Ravin P, Raizer JJ, Rich KM, Schiff D, Shapiro WR, et al. Randomized phase II study of cilengitide, an integrin-targeting arginine-glycine-aspartic acid peptide, in recurrent glioblastoma multiforme. J Clin Oncol 2008; 26: 56107.
  • 19
    Buerkle MA, Pahernik SA, Sutter A, Jonczyk A, Messmer K, Dellian M. Inhibition of the alpha-nu integrins with a cyclic RGD peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. Br J Cancer 2002; 86: 78895.
  • 20
    Strieth S, Eichhorn ME, Sutter A, Jonczyk A, Berghaus A, Dellian M. Antiangiogenic combination tumor therapy blocking alpha(v)-integrins and VEGF-receptor-2 increases therapeutic effects in vivo. Int J Cancer 2006; 119: 42331.
  • 21
    Hodivala-Dilke K. Alphavbeta3 integrin and angiogenesis: a moody integrin in a changing environment. Curr Opin Cell Biol 2008; 20: 5149.
  • 22
    Taverna D, Moher H, Crowley D, Borsig L, Varki A, Hynes RO. Increased primary tumor growth in mice null for beta3- or beta3/beta5-integrins or selectins. Proc Natl Acad Sci USA 2004; 101: 7638.
  • 23
    Bäuerle T, Bartling S, Berger M, Schmitt-Gräff A, Hilbig H, Kauczor HU, Delorme S, Kiessling F. Imaging anti-angiogenic treatment response with DCE-VCT, DCE-MRI and DWI in an animal model of breast cancer bone metastasis. Eur J Radiol 2010; 73: 2807.
  • 24
    Bäuerle T, Hilbig H, Bartling S, Kiessling F, Kersten A, Schmitt-Gräff A, Kauczor HU, Delorme S, Berger MR. Bevacizumab inhibits breast cancer-induced osteolysis, surrounding soft tissue metastasis, and angiogenesis in rats as visualized by VCT and MRI. Neoplasia 2008; 10: 51120.
  • 25
    Mitjans F, Sander D, Adan J, Sutter A, Martinez JM, Jaggle CS, Moyano JM, Kreysch HG, Piulats J, Goodman SL. An anti-alpha v-integrin antibody that blocks integrin function inhibits the development of a human melanoma in nude mice. J Cell Sci 1995; 108( Pt 8): 282538.
  • 26
    Cheresh DA, Spiro RC. Biosynthetic and functional properties of an Arg-Gly-Asp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen, and von Willebrand factor. J Biol Chem 1987; 262: 1770311.
  • 27
    Weinacker A, Chen A, Agrez M, Cone RI, Nishimura S, Wayner E, Pytela R, Sheppard D. Role of the integrin alpha v beta 6 in cell attachment to fibronectin. Heterologous expression of intact and secreted forms of the receptor. J Biol Chem 1994; 269: 69408.
  • 28
    Bäuerle T, Adwan H, Kiessling F, Hilbig H, Armbruster FP, Berger MR. Characterization of a rat model with site-specific bone metastasis induced by MDA-MB-231 breast cancer cells and its application to the effects of an antibody against bone sialoprotein. Int J Cancer 2005; 115: 17786.
  • 29
    Yamada S, Bu XY, Khankaldyyan V, Gonzales-Gomez I, McComb JG, Laug WE. Effect of the angiogenesis inhibitor cilengitide (EMD 121974) on glioblastoma growth in nude mice. Neurosurgery 2006; 59: 130412.
  • 30
    Brix G, Semmler W, Port R, Schad LR, Layer G, Lorenz WJ. Pharmacokinetic parameters in CNS Gd-DTPA enhanced MR imaging. J Comput Assist Tomogr 1991; 15: 6218.
  • 31
    Harms JF, Welch DR, Samant RS, Shevde LA, Miele ME, Babu GR, Goldberg SF, Gilman VR, Sosnowski DM, Campo DA, Gay CV, Budgeon LR, et al. A small molecule antagonist of the alpha(v)beta3 integrin suppresses MDA-MB-435 skeletal metastasis. Clin Exp Metastasis 2004; 21: 11928.
  • 32
    Zhao Y, Bachelier R, Treilleux I, Pujuguet P, Peyruchaud O, Baron R, Clement-Lacroix P, Clezardin P. Tumor alphavbeta3 integrin is a therapeutic target for breast cancer bone metastases. Cancer Res 2007; 67: 582130.
  • 33
    Eliceiri BP, Puente XS, Hood JD, Stupack DG, Schlaepfer DD, Huang XZ, Sheppard D, Cheresh DA. Src-mediated coupling of focal adhesion kinase to integrin alpha (v) beta5 in vascular endothelial growth factor signaling. J Cell Biol 2002; 157: 14960.
  • 34
    Duong LT, Rodan GA. Integrin-mediated signaling in the regulation of osteoclast adhesion and activation. Front Biosci 1998; 3: d757d768.
  • 35
    Nakamura I, Duong le T, Rodan SB, Rodan GA. Involvement of alpha(v) beta3 integrins in osteoclast function. J Bone Miner Metab 2007; 25: 33744.
  • 36
    Andersen TL, Sondergaard TE, Skorzynska KE, Dagnaes-Hansen F, Plesner TL, Hauge EM, Plesner T, Delaisse JM. A physical mechanism for coupling bone resorption and formation in adult human bone. Am J Pathol 2009; 174: 23947.
  • 37
    Hamaoka T, Madewell JE, Podoloff DA, Hortobagyi GN, Ueno NT. Bone imaging in metastatic breast cancer. J Clin Oncol 2004; 22: 294253.
  • 38
    Lai CF, Cheng SL. Alphavbeta integrins play an essential role in BMP-2 induction of osteoblast differentiation. J Bone Miner Res 2005; 20: 33040.
  • 39
    Bäuerle T, Peterschmitt J, Hilbig H, Kiessling F, Armbruster FP, Berger MR. Treatment of bone metastasis induced by MDA-MB-231 breast cancer cells with an antibody against bone sialoprotein. Int J Oncol 2006; 28: 57383.
  • 40
    Karadag A, Ogbureke KU, Fedarko NS, Fisher LW. Bone sialoprotein, matrix metalloproteinase 2, and alpha(v)beta3 integrin in osteotropic cancer cell invasion. J Natl Cancer Inst 2004; 96: 95665.
  • 41
    Mitjans F, Meyer T, Fittschen C, Goodman S, Jonczyk A, Marshall JF, Reyes G, Piulats J. In vivo therapy of malignant melanoma by means of antagonists of alphav integrins. Int J Cancer 2000; 87: 71623.
  • 42
    MacDonald TJ, Taga T, Shimada H, Tabrizi P, Zlokovic BV, Cheresh DA, Laug WE. Preferential susceptibility of brain tumors to the antiangiogenic effects of an alpha(v) integrin antagonist. Neurosurgery 2001; 48: 1517.
  • 43
    Chen Q, Manning AD, Millar H, McCabe FL, Ferrante C, Sharp C, Shahied-Arruda L, Doshi P, Nakada MT, Anderson GM. CNTO 95, a fully human anti αv integrin antibody, inhibits cell signalin, migration, invasion, and spontaneous metastasis of human breast cancer cells. Clin Exp Metastasis 2008; 25: 13948.
  • 44
    Patsenker E, Popov Y, Stickel F, Schneider V, Ledermann M, Sagesser H, Niedobitek G, Goodman SL, Schuppan D. Pharmacological inhibition of integrin alphavbeta3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis. Hepatology 2009; 50: 150111.
  • 45
    Brooks PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T, Klier G, Cheresh DA. Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994; 79: 115764.
  • 46
    Alghisi GC, Ponsonnet L, Ruegg C. The integrin antagonist cilengitide activates alphaVbeta3, disrupts VE-cadherin localization at cell junctions and enhances permeability in endothelial cells. PLoS One 2009; 4: e4449.
  • 47
    Abdollahi A, Griggs DW, Zieher H, Roth A, Lipson KE, Saffrich R, Gröne HJ, Hallahan DE, Reisfeld RA, Debus J, Niethammer AG, Huber PE. Inhibition of alpha(v) beta3 integrin survival signaling enhances antiangiogenic and antitumor effects of radiotherapy. Clin Cancer Res 2005; 11: 62709.
  • 48
    Mikkelsen T, Brodie C, Finniss S, Berens ME, Rennert JL, Nelson K, Lemke N, Brown SL, Hahn D, Neuteboom B, Goodman SL. Radiation sensitization of glioblastoma by cilengitide has unanticipated schedule-dependency. Int J Cancer 2009; 124: 271927.
  • 49
    Albert JM, Cao C, Geng L, Leavitt L, Hallahan DE, Lu B. Integrin alpha v beta 3 antagonist cilengitide enhances efficacy of radiotherapy in endothelial cell and non-small-cell lung cancer models. Int J Radiat Oncol Biol Phys 2006; 65: 153643.
  • 50
    Reynolds LE, Wyder L, Lively JC, Taverna D, Robinson SD, Huang X, Sheppard D, Hynes RO, Hodivala-Dilke KM. Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins. Nat Med 2002; 8: 2734.
  • 51
    Reynolds AR, Hart IR, Watson AR, Welti JC, Silva RG, Robinson SD, Da Violante G, Gourlaouen M, Salih M, Jones MC, Jones DT, Saunders G, et al. Stimulation of tumor growth and angiogenesis by low concentrations of RGD-mimetic integrin inhibitors. Nat Med 2009; 15: 392400.