We utilized the tail vein method to generate brain metastasis in mouse models using cell lines derived from patients with inflammatory breast cancer, or IBC, a highly aggressive form of primary breast cancer. The generation of brain metastasis in mouse models typically involves intracardiac or intracarotid arterial injections. However, the tail vein method offers advantage over these techniques as it better mimics the steps of brain metastasis colonization, and is relatively straightforward to perform.
Doctors Xiaoding Hu and Emilly Villodre, both of whom are instructors in my laboratory, will demonstrate the procedure. To detect brain metastases by GFP imaging eight to 10 weeks after tumor cell injection, disinfect the mouse with 70%ethanol and remove the fur from the head and ears. Cut the cervical area of the neck.
Excise and remove the skull to allow the brain to be harvested. Place the brain into a tissue cassette and place the cassette into a container of cold DPBS for no more than one hour. For imaging by stereo microscopy, transfer the brain into a 100-centimeter tissue dish lid under a stereo microscope equipped with a UV light and select the 0.5x objective to allow visualization of the entire brain.
Press live view and focus the view while the brain is in the ventral position. Select the appropriate filter in the software and microscope for the fluorescent marker in the cells, and take a picture. Without moving the sample, switch to brightfield imaging and select the brightfield filter.
Take a picture. Then image the brain in the dorsal position as demonstrated earlier. To analyze the whole brain images, first convert the image files from TIFF to JPEG so they can be opened with any computer that does not have the associated software.
Next, open a pair of brightfield and fluorescent images and select file and merge channels. Select the proper components, click okay, and save the merged image. To quantify the brain tumor area in the fluorescent image, select automatic selection.
Move the arrow to the GFP position area and click to create a selection automatically. Then click again to confirm the selection. All the measurements will be shown.
If more than one GFP positive area can be observed, repeat the measurement with the subsequent area. Brain metastatic lesions can be detected as early as eight weeks after injection by luciferase imaging and stereo fluorescent microscopy. After imaging, portions of the brain metastases can be formalin fixed and processed for hematoxylin and eosin staining to validate the presence of brain metastasis lesions, and for immunohistochemical staining to detect specific protein markers of interest.
When attempting This protocol, it is important to maintain cell viability by keeping the cells on ice for no more than one hour, and to avoid air bubbles in the cell suspension to prevent emboli formation and mouse mortality. Following tail vein injection, we monitor brain metastasis growths and progression with luciferase imaging. After euthanasia, stereo fluorescent microscopy and histology confirmed metastasis lesions.
