The overall goal of this procedure is to create orthotopic bladder cancer xenografts in a rapid, precise, and minimally invasive fashion. This is accomplished by first expanding the particular tumor cell line and suspension of the tumor cells in Matrigel. Next, the animal is mounted on the imaging platform and the bladder is visualized with ultrasound.
Then the mucosal and muscular layers in the bladder are separated with a saline injection. Finally, the tumor cell matrigel suspension is injected into this artificially created space. Ultimately, results can be obtained that show tumor growth through ultrasound imaging and bioluminescence.
The main advantage of this technique over other existing methods, such as use of laparotomy for inoculation of primary xenografts, is that this approach is rapid, precise, and minimally invasive. The implications of these techniques extend to the diagnosis and therapy of bladder cancer because auto xenografts are the gold standard for preclinical study of novel treatments. Visual demonstration of this method is critical because injection steps are quite difficult to learn, and water was is very thin, Demonstrating this procedure will be.
Igor Mosca have a research assistant and Wolfgang Yeager, a postdoctoral fellow. Both are urologists After confirming the identity of the respective human bladder cancer cell lines. For this study through DNA fingerprinting, use a lentiviral construct carrying the firefly luciferase gene for bioluminescence growth analysis of xenograft tumors to transfect the cell lines.
When ready to begin the procedure, thaw and use DMEM with 10%FBS to expand the existing cell lines, incubate at 37 degrees Celsius in a humidified 5%CO2 atmosphere. To prepare a cell suspension for injection after thawing, matrigel, and keeping on ice trypsin eyes, 70%confluence cells and use normal growth medium to suspend, then count the cells and spin the suspension at 200 times G for five minutes. After removing the supernatant, add the appropriate amount of equal volumes of D-M-E-M-F-B-S and MATRIGEL to reach the desired cell concentration for injecting a volume of 40 microliters per animal.
Mix well and avoid creating air bubbles from any kind of flat, rigid plastic material. Cut a bladder stabilizer carefully inspect the strap and remove any sharp edges before application to the mice. After anesthetizing female mice with 3%isoflurane in oxygen and performing a toe pinch.
To ensure adequate sedation, mount the animal on the heating imaging table of the small animal imaging platform, continuously monitoring its vital signs with a rubber band. Fix the lower limbs and use 0.5%chlorhexidine gluconate to disinfect the abdomen before using a sterile cotton tip to wipe the skin. Next, using the bladder stabilization strap, immobilize the bladder to avoid an evasion of it during intramural injection.
Then apply sterile ultrasound gel to the lower abdomen. Slowly advance the ultrasound scan, head to the skin and visualize the bladder on the ultrasound screen. If the bladder is empty, use 50 microliters of warm PBS in a transurethral 24 gauge angio catheter to fill it to separate the bladder wall layers.
Begin by attaching a 1.0 milliliter syringe filled with PBS and connected to a 30 gauge threequarter inch needle to the syringe clamp. Position the needle towards the skin just above the pubic bone. At a 30 to 45 degree angle, detect the needle on the ultrasound screen before slowly perforating the skin and abdominal wall muscles.
Next, rotate the bevel of the needle 180 degrees so that it is facing posteriorly, and insert the tip of the needle into the bladder wall without penetrating the mucosa. Then to create an artificial space, slowly inject 50 microliters of PBS between the muscle layer and the mucosa before withdrawing the needle. To inject bladder cancer cells, attach a second 1.0 milliliter syringe filled with the cancer cell matrigel suspension, and a 30 gauge three-quarter inch needle to the syringe clamp.
Guide the tip of the needle to the PBS filled space. Just created and inject 40 microliters of the suspension into the space. Then withdraw the needle after dismounting the mouse from the imaging platform.
Keep it in a warm and comfortable environment under continuous monitoring. After regaining consciousness and normal ambulation, place the animal back in its home cage. Intramural injection of three different tumor cell lines was performed in 50 animals under ultrasound guidance on three consecutive days.
The inoculation was performed efficiently and was not associated with any intra or post interventional complications. Monitoring of tumor growth was performed by ultrasound imaging and bioluminescence. On day three, a tumor could be detected by ultrasound in the anterior bladder of all 50 animals, and 98%of the mice exhibited constant tumor growth during the follow-up period following the inoculation of uc.Three.
Luke one mouse developed intraperitoneal tumor dissemination and the tumor involuted after day seven in a second animal. This was the first group of mice inoculated with this new technique after inoculation of uc.Three. Luke uc, one Luke, and uc.
13 Luke. The mice were sacrificed on days 24, 28 and 37 respectively. Xenograft tumors were harvested and examined via h and d sections.
All tumors were muscle invasive and some infiltrated into the per visceral fat, but no invasion into adjacent organs was observed. 60%of the uc, 13 Luke, and 20%of the uc. Three Luke tumor bearing mice developed retroperitoneal lymph node metastases, which were confirmed by h and e staining.
Once mastery, this technique can be done in three minutes. Familiarity with ultrasound imaging is a prerequisite for performing this procedure After tumors have been inoculated in this fashion, the ultrasound allows us not only to monitor tumor growth over time and also to look at tumor perfusion with microbubbles. It allows us also to perform intratumoral injection with novel experimental agents.
