In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.
Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.
1. Cell Culture
2. Nude Rat Model of Bone Metastasis
3. Magnetic Resonance Imaging (MRI)
4. Volumetric Computed Tomography (VCT)
5. Ultrasound (US)
6. Postprocessing of Imaging Data
7. Representative Results
Following intraarterial injection of MDA-MB-231 cells into the SEA (Figure 1), site-specific bone metastases develop in the respective hind leg of the nude rat. Osteolytic lesions confined to the femur, tibia and fibula can be imaged non-invasively by MRI, VCT and US (Figure 2) beginning approximately 25-30 days post injection and followed-up for several weeks. When combining MRI, VCT and US including native and contrast-enhanced techniques, complementary information can be assessed in bone metastases that are composed of a soft tissue tumor (tumor cells and stroma) and the respective osteolytic lesion (bone destruction). For comparison of the respective data between the techniques, all three imaging modalities can be used sequentially in the same rat. MRI displays morphology of the bone metastatic soft tissue that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone in the course of development. Functional parameters such as regional blood volume, perfusion and vessel permeability can be obtained from DCE-MRI and quantified (Figure 3). Bone structure, and in particular osteolytic changes in the metastases are assessed in high resolution by VCT. Complementary to MRI findings, osteolytic lesions are located adjacent to intramedullary tumor growth. VCT angiography reveals the altered macrovessel architecture of bone metastases, and DCE-VCT displays respective aspects of microcirculation (Figure 4). Due to local destruction of cortical bone in metastatic lesions, US is applicable to assess morphological and functional features of the soft tissue tumor by the use of B-mode and Doppler techniques. Upon application of microbubbles, DCE-US allows for real-time imaging of vascularization in bone metastases (Figure 5).
Figure 1. Hind leg of a nude rat prepared for tumor cell inoculation as imaged through an operation microscope. A, branching pattern of the femoral artery (FA) including the superficial epigastric artery (SEA), descending genicular artery (DGA), popliteal artery (PA) and saphenous artery (SA). Arterial clips placed on the SA, PA and proximal FA as well as ligation of SEA; B, SEA was cut proximal of the ligation; C, muscle relaxation of the SEA after addition of papaverine; D, incision of the SEA (taken up by a forceps); E, insertion of needle into SEA; F, fixated needle in the SEA (external fixating device) and injection of MDA-MB-231 tumor cells via the SEA into the DGA and PA by virtue of the clips.
Figure 2. A, human MR system (Symphony, Siemens, Germany) and a home-built coil for radiofrequency excitation and detection placed in the scanner; B, flat panel equipped volumetric computed tomograph (Volume CT, Siemens, Germany); C, clinical ultrasound system Acuson Sequioa 512 (Siemens-Acuson, Mountain View, CA).
Figure 3. Axial MR sections. Left panel, T2w MRI; middle panel, amplitude A (DCE-MRI); right panel, exchange rate constant kep (DCE-MRI). Arrows point at bone metastases. The color map for DCE-MRI data ranges from red (high values) to blue (low values).
Figure 4. 3D VCT reconstructions of the osteolytic bone metastasis (left panel) and an angiography (middle panel) as well as an DCE-VCT section in axial orientation from the parameter peak enhancement (right panel). The color map for DCE-VCT data ranges from red (high values) to blue (low values).
Figure 5. US images from B-mode (morphology, left panel), Doppler (perfusion, middle panel) and CEUS (right panel, peak enhancement after injection of microbubbles from real-time imaging of vascularization) of a bone metastasis.
Supplemental movie 1. Click here to view supplemental movie.
The method of inducing experimental bone metastases presented here in combination with the imaging procedures enable to follow-up osteolytic lesions in nude rats longitudinally. In our model, MDA-MB-231 human breast cancer cells are injected into the SEA which is an anastomosis between the iliac artery via the pudendoepigastric trunk and the femoral artery. Consequently, the blood flow into the supplied region of the knee joint is maintained after ligation of the SEA. The advantages of this model as compared to established models of bone metastasis are the site-specific appearance of bone metastases as compared with the intracardiac injection model11 and the inclusion of the pathogenic processes of tumor cell extravasation and migration to the target tissue as compared to the tibia injection model12. Further, in this model a systemic tumor burden, in particular visceral dissemination, is omitted which allows for longitudinal studies over several weeks, and thus allows the reduction of required animals1,13.
The role of angiogenesis as essential process to promote tumor cell proliferation and induce bone resorption in the pathogenesis of bone metastasis was previously demonstrated in ex vivo studies14,15. Here, we present in vivo imaging techniques to non-invasively assess angiogenesis in these lesions applying MRI, VCT and US. Using a nude rat model, complementary information of vascularization including functional information on blood volume and vessel permeability/perfusion (DCE-MRI, DCE-VCT), vessel morphology in high resolution (VCT angiography), perfusion (US Doppler) and real-time imaging of vascularization (DCE-US) can be obtained1-7,16.
Imaging of angiogenic parameters using MRI, VCT and US enables the elucidation of the pathogenic role of angiogenesis in skeletal metastasis non-invasively and in vivo without the need of repetitive histological evaluations3,4,6. Another application for the above-mentioned imaging techniques is the investigation of therapeutic effects in longitudinal studies upon anti-angiogenic or standard therapies for skeletal metastases. For demonstration of pharmacological response, longitudinal studies covering up to 70 days after tumor cell inoculation with group sizes between 8 and 17 rats were performed to demonstrate anti-tumor, anti-angiogenic and anti-resorptive effects2-7,17. Due to the application of imaging methods on scanners for human use in a clinically relevant animal model, the presented procedures are of high translational value for assessment of treatment response in patients with bone metastases16.
In conclusion, using this site-specific animal model of breast cancer bone metastases, morphological and functional aspects of angiogenesis can be imaged non-invasively and in vivo using MRI, VCT and US.
The authors have nothing to disclose.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB-TR 23 and SFB-TR 79, T.B. and D.K.). The authors would like to thank Renate Bangert, Karin Leotta and Lisa Seyler for excellent technical assistance.
Name of the reagent | Company | Catalogue number |
MDA-MB-231 human breast cancer cells | American Type Culture Collection (ATCC) | HTB-26 |
RPMI-1640 | Invitrogen | 61870 |
FCS | Invitrogen | 10270 |
Trypsin-EDTA | Invitrogen | 25300 |
Carprofen Rimadyl | Pfizer | PZN 110208 |
Magnevist | Bayer-Schering | PZN 6961516 |
Imeron 400 MCT | Bracco | PZN 228654 |
SonoVue | Bracco | PZN 1567358 |
Papaverin | Alfa Aesar | L 04152 |
Isofluran | Baxter | HDG 9623 |
Symphony (Magnetic resonance imaging) | Siemens | |
Volume CT (Volumetric computed tomography) | Siemens | |
Acuson Sequioa 512 (Ultrasound) | Siemens-Acuson |