Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

This experiment uses complimentary imaging techniques to non-invasively assess angiogenesis in a rat model of breast cancer. Bone metastasis first inject human breast cancer cells into the superficial epigastric artery to induce site-specific osteolytic bone metastases at the hind leg of nude rats. After 25 to 30 days, prepare the animal for imaging analysis and acquire data by magnetic resonance imaging, computed tomography and ultrasound scanning.

Next, use the complementary morphological and functional data from these imaging modalities to describe different aspects of vascularization in osteolytic lesions. Results obtained include macro vessel architecture, local blood volume, perfusion, and permeability, as well as real-time imaging of the vascularization in bone metastases. These methods can help to answer key questions on the role of angiogenesis in the pathogenesis of bone metastasis, and that has implications on imaging and treatment of malignant osseous lesions.

For the animal model described here, there is no need for usage of an N-D-A-M-B 2 3 1 bone specific sub cell line because the tumor take rate is over 90%Begin with a logarithmic culture of human breast cancer cells in RPMI 1640. Media supplemented with 10%fetal calf serum. After harvesting the sub confluent tumor cells with trypsin enumerate the cells using a neubauer's chamber and suspend them in RPMI 1640, all experiments were approved by the responsible Governmental Animal Ethics Committee Anesthetized the six to eight week old rat as given in the text that is enclosed for the surgical procedure.

Use an appropriate binocular operating microscope with a magnification of 16 fold. Begin by cutting the skin and the subcutaneous tissue in the indual region at a length of two to three centimeters. Now dissect all arteries that branch off the femoral artery, put clips on the femoral artery proximal of the S CA's origin, as well as on the DGA, PA, and S SA to temporarily occlude local blood flow.

Then ligate the SEA at its distal part to allow opening of this vessel without bleeding. Cut the SEA proximal of the ligation and administer a 1%pipen solution onto the SEA. Now, cut approximately half of the s e's diameter with scissors, and while holding the cut end of the vessel with a forceps, insert a needle into the lumen.

If possible, fix the needle in an external device to reduce irregular movements that could lead to a perforation of the vessel wall. Then connect a syringe to the needle. Remove the clip from the distal femoral artery and place it on the saphenous artery.

Proceed to slowly inject O 0.2 milliliters of the MDA MD 2 31 breast cancer cells into the superficial epigastric artery. Note that by virtue of the clips, the cells are directed to the DGA and pa. Remove the needle and ligate the superficial epigastric artery to prevent bleeding before taking off the arterial clips.

After closing the wound and terminating the inhalation anesthesia, allow 25 to 30 days of tumor growth in the animal. Use a dedicated experimental MR scanner or a human MR system with an appropriate animal coil like this home-built coil for radio frequency excitation and detection. After anesthetizing the rat with oxygen and ISO fluorine, place a catheter in the tail vein and fix it on the tail.

Using tape, connect a syringe containing the contrast reagent. Then place the rat in the MR system maintaining the inhalation anesthesia. Begin with a morphologic MR sequence to locate the bone metastasis.

Next, determine a slice of the bone metastasis with the largest diameter and start the sequence for D-C-E-M-R-I after approximately 30 seconds. Begin to inject the contrast agent over a time period of 10 seconds. Choose an appropriate CT system, either a human or an experimental scanner.

Here we use a prototype of a flat panel equipped volumetric computed tomograph. After anesthetizing the rat with oxygen and isof fluorine, place a catheter in the tail vein and fix it on the tail using tape. Then connect a syringe containing contrast agent.

Now place the rat on the scanner under inhalation and anesthesia. Set the scan parameters for VCT. Several experimental and clinical ultrasound systems are available for this purpose.

After anesthetizing the rat with oxygen and isof fluorine, place a catheter in the tail vein and fix it on the tail using a tape. Then connect a syringe containing a microbubble contrast agent. Fix the ultrasound transducer on the respective hind leg using a tripod and apply ultrasound gel between the transducer and the hind leg.

Next, perform B mode imaging transmission. To determine the largest diameter of the bone metastasis and fix the transducer in this position, add doppler signal on the B mode images for information on tissue perfusion. Keeping in mind that only lesions that disrupt cortical bone are accessible to ultrasound waves.

For dynamic contrast enhanced ultrasound, set the ultrasound device in cadence, contrast pulse sequencing mode, inject the microbubbles and record a thine loop of 90 seconds length. Process the morphological information derived from M-R-I-V-C-T and ultrasound to characterize the soft tissue tumor and the skeletal destruction of the bone metastases. Then determine the location and the volume of the bone metastases using a DICOM viewer For angiography.

Using eRx DICOM viewer, consider the VCT data to obtain the branching pattern of vessels in bone metastases. Reconstruct 2D or 3D images using the information of the arterial phase with or without subtraction techniques. In order to quantify the functional parameters of vascularization from D-C-M-R-I-D-C-E-V-C-T and DCE ultrasound use software tools specific for each modality.

For D-C-E-M-R, I use Dyna Lab to determine the vascular parameters associated with blood volume and exchange rate constant associated with perfusion and vessel permeability in bone metastases. To quantify D-C-E-V-C-T data, perform a descriptive analysis of data to calculate parameters such as area under the curve or peak enhancement. Also quantify information from real time DCE ultrasound, using quantitative analysis software by analyzing cine loops According to the implemented bolus injection model place, the region of interest over the bone metastasis determine either descriptive factors such as area under the curve or quantitative parameters from color coded maps.

Intraarterial injection of M-D-A-M-B 2 31 cells into the SEA was used to develop site-specific bone metastases in the respective hind leg of the nude rat. This image shows the branching pattern of the femoral artery, including the superficial epigastric artery descending ular artery, popliteal artery, and saphenous artery. The arterial clips are placed on the sa, PA and proximal fa, as well as ligation of SEA.

The SEA was then cut proximal of the ligation and after addition of papine, the muscle of the vessel wall is relaxed using forceps. The SEA is incised and a needle is inserted into the SEA using an external fixating device for the needle. Human breast cancer tumor cells are injected via the SEA and directed into the DGA and pa.

By virtue of the clipse osteolytic lesions, confined to the femur, tibia and fibula can be imaged non-invasively by M-R-I-V-C-T and ultrasound beginning approximately 25 to 30 days post-injection and followed up for several weeks. All three imaging modalities can be used sequentially in the same rat. The 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.

In these axial sections, the T two weighted MR image shows the soft tissue part of the bone metastasis and the D-C-E-M-R-I color maps display the values for amplitude A and exchange rate constant within the lesion bone structure and in particular, osteolytic changes in the metastases are assessed in high resolution by VCT complimentary to MRI findings, OSTEOLYTIC lesions are located adjacent to intramedullary tumor growth. VCT angiography reveals the altered macro vessels architecture of bone metastases and D-C-E-V-C-T displays respective aspects of microcirculation due to local destruction of cortical bone in metastatic lesions. Ultrasound is applicable to assess morphological and functional features of the soft tissue tumor by use of B mode and doppler techniques.

Upon application of microbubbles, DCE ultrasound allows for realtime imaging of vascularization in bone metastases. After watching this video, you should have a good understanding of how to inoculate tumor cells for site-specific breast cancer, bone metastases to image morphological and functional aspects of angiogenesis in these lesions.Non-invasively.