Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Longitudinal imaging is used in preclinical studies to follow the progress of a disease or measure the effect of a therapeutic. In oncology. Optical imaging methods provide rigorous tools to monitor tumor growth and deliver precise quantitation of cell growth or gene expression at each time point in such a study.

However, these optical techniques cannot directly account for changes in the underlying structure. These anatomical changes can be measured using high resolution techniques such as micro ct. For longitudinal imaging, a low x-ray dose is essential to minimize the effects of the cumulative radiation on the underlying biology.

These optical and micro CT images can then be coregistered to provide a combination of functional and anatomical data, ensuring that the maximum amount of information is extracted from the animal model. Today we'll demonstrate how to monitor tumor metastases and follow OSTEOLYTIC lesion development using optical imaging and micro ct. The quantum effects perform CT at a very low radiation dose.

This allows for longitudinal monitoring of osteolytic lesion development with a cumulative radiation dose far below LD 50 levels. In this experiment, cells from a human mammary tumor cell line expressing the luciferase gene will be used as an optical indicator of tumor agenesis in vivo. This cell line was created from a spontaneous lymph node metastasis that originated from a D three H one mammary fat pad tumor, and is known to aggressively form metastasis.

The cells readily grow in standard media with no need for selection of markers. Cells for intracardiac injection are prepared and luciferase activity is verified by standard methods as shown previously prior to intracardiac injection. The animal is anesthetized using 2%isof fluorine, and a toe pinch is performed to confirm deep sedation.

The hardest part is this procedure is the intracardiac injection of the animals. To account for any unforeseen situation during the injection is helpful to have more than the required number of mice on hand. One to 3 million cells in a volume of 20 microliters will be injected into the left ventricle of each animal.

This cell suspension also contains 150 micrograms per milliliter of d Lucifer. To validate the injection technique to inject, insert a 30 gauge needle attached to a one cc syringe into the second intercostal space. Three millimeters to the left of the sternum and aim centrally, pull back slightly on the plunger while slowly advancing the needle.

Continuous entrance of red oxygenated blood into the transparent needle hub indicates proper positioning of the needle into the left ventricle. After confirming the success of the intracardiac injection, metastasis of the tumor cells is monitored by bioluminescence imaging using the IVUS spectrum in vivo imaging system. To begin this procedure, place the anesthetized mouse in an imaging shuttle.

This shuttle helps maintain consistent positioning of the animal for simple transfer from the ivus spectrum to the quantum fx. With newly developed living image 4.1 software optical and CT images can be coregistered based on a fiducial in the CT image. Use the imaging wizard in the living image software 4.1 to automatically select the imaging parameters.

Depending on the nature of the experiment, multiple images can be acquired and compared in longitudinal studies covering seconds or months. Mice that showed bone metastases using bioluminescence imaging are imaged in the quantum FX micro CT system to monitor the development of osteolytic lesions. Since the imaging shuttle supports the mouse for data capture in both the ivus spectrum and the quantum FX micro CT system, the animal can be transferred directly from the ivus spectrum system to the Quantum FX micro CT system.

For imaging, two 60 millimeter images are stitched together to capture the whole animal view. For imaging the knee joint in high resolution, the geometry of the CT system can be changed and imaged at a smaller field of view. With a smaller voxel size, a single scan with the 60 millimeter FOV for co-registration takes just 17 seconds and delivers a dose of only approximately 13 m.

Therefore, the mice can be imaged multiple times with the micro CT system without adversely affecting the tumor growth or health of the animal. Once images have been captured on both the ivus spectrum and quantum FX micro CT system, the data is coregistered to accomplish this. Micro CT images are exported as DICOM files and loaded into the living image.

4.1 3D multimodality tool in the living image software run the D light program to obtain the 3D reconstructed optical signal. The automatic co-registration feature of the software can then be used to detect the fiducial to coregister the optical BLI signal to the micro CT image. When the intracardiac injection is performed correctly, the mouse shows bioluminescence signal throughout the body when imaged in the IVUS system.

In contrast, if the cells are not injected correctly in the heart, the bioluminescence signal will be localized as shown in this example where cells were incorrectly injected into the right ventricle. At day six post-injection of cells, bioluminescence imaging with ivus spectrum showed metastases to the bone in seven out of nine mice to obtain more anatomical details of the osteolytic activity due to tumor development, mice were imaged in the quantum FX micro CT system. On the top of this graph.

The bioluminescent images showed tumor progression longitudinally. The CT images revealed osteolytic lesion developed on day 10 and progressed rapidly during the course of the study. Coregistered datasets can be analyzed quantitatively to monitor multiple parameters during disease progression.

Here we are tracking growth of the metastatic tumor together with the development of the osteolytic lesions in the bone as the bioluminescence signals near the knee increased. We observed osteolytic lesions at the distal femur and proximal tibia. These femurs were segmented and registered using a mutual information based routine.

After thresholding bone volume was measured in the slices nearest to the knee and showed a steady decline from day 20 with 23%of the bone mass lost by day 32. This set of images show co-registration of BLI tumor signal with micro ct. CT imaging not only allows direct assessment of bone lesions, but also provides anatomical reference of the tumor locations.

We have now demonstrated a non-invasive method to monitor tumor metastases and follow osteolytic lesion development using optical imaging and micro ct using the lures expressing cell line Mets can be seen in less than a week while osteolytic lesion development can be visualized at one week using micro ct. The quantum effect was used to monitor bone degradation over the course of five weeks without any adverse effects on the mice.