This methodology can be used to reproducibly generate bladder tumors using the BBN carcinogen and to image the size and extension of the bladder tumor burden through MRI. This technique uses an operator independent rapid imaging approach with short acquisition times at a high diagnostic quality to conduct high-throughput assays of mice at intermediate stages of tumor development. To induce bladder cancer tumor formation, add 0.05%BBN to an opaque drinking water container and provide the water to 6-week old C57 black six mice ad libitum.
Then monitor the animals two times a week for sign of distress associated with bladder tumors in accordance with the local IACUC guidelines. 16 to 24 weeks after exposure, load a one milliliter syringe equipped with a 25 to 27 gauge needle with 100 to 200 microliters of sterile saline and inject the saline subcutaneously into a BBN-treated animal. After 10 minutes, transfer the mouse into the imaging holder of a magnetic resonance imager and insert a rectal temperature probe connected to the physiological recording computer to monitor the animal's body temperature.
Next, place a 4-channel quadrature receiver coil over the lower abdomen of the mouse and initiate automatic adjustments in the integrated imaging software to acquire a triaxial set of images of the whole mouse body. From this reference set of images, identify the region of interest and acquire three sets of orthogonal sliced images along the axial, coronal, and sagittal planes. Using the true fast imaging with steady-state progression imaging sequence, set the TR to 900 milliseconds, the TE to two milliseconds, and the FA to 70, 14 averages.
Then select the appropriate geometric parameters within the imaging platform software to obtain a series of slices across the whole bladder of 0.5 milliliter thickness and an in-plane resolution of 0.148 millimeters. When all of the images have been obtained, export the set of slices covering the whole bladder to an appropriate medical image analysis software program and scroll through the generated images to locate a slice at the midpoint of the bladder that allows visualization of the bladder wall and lumen to select the representative axial view at the center of the bladder for quantitative analysis. Then trace the boundaries around the outer edge and inner lumen of the bladder to carefully delineate the region of interest within the selected representative axial view and subtract the inner lumen from the outer edge to calculate the surface area of the bladder wall.
20 weeks after BBN exposure, clean the area of incision with 70%ethanol and use forceps to lift the abdominal wall skin. Make a midline incision from the pubic symphysis to the xiphoid process and grasp the peritoneum to allow a sharp incision of the peritoneal cavity. Identify the bladder in the midline lower abdomen, using forceps to grasp the dome of the bladder.
Dissect the bladder away from the surrounding structures including the seminal vesicles, rectum, and fat. Cut the median umbilical ligament connecting the dome of the bladder to the umbilicus and abdominal wall. Cut the ureters close to the bladder and lifting the bladder cephalad, cut the urethra to remove the bladder.
Then rinse the excised bladder with PBS and immediately weigh the tissue on a balance. Here, representative true fast imaging with steady-state progression magnetic resonance images of bladder wall 3-D reconstructions and pathologic images of a mouse with a large tumor and of a control mouse without a tumor are shown. The bladder wall thickness as derived from the magnetic resonance imaging correlates weekly with the ex-vivo obtained bladder weight measurements.
Examination of the MRI-derived bladder wall thickness and ex-vivo obtained bladder weight data also demonstrate an association with the tumor stage as well as an association when stratifying the pathology by non-muscle invasive bladder cancer and muscle invasive bladder cancer. Keep in mind that it is best to acquire the images in mice when they have a full bladder. Following MR imaging of the bladder tumors, mice can be split into cohorts with similar tumor characteristics, as appropriate.
This technique allows researchers to explore new immunotherapies and other treatments to improve outcomes for our patients. It is important to remember that BBN is a carcinogen and that the appropriate precaution should always be taken when handling this compound.
