- First, inject bleomycin, a nonribosomal hybrid drug, intratracheally in mice a few days before cancer cell engraftment to induce fibrosis in the lungs. Next, grow lung cancer cell lines in a tissue culture medium. Prepare cancer cell suspension by diluting cells in PBS. Inject ketamine-xylazine solution intraperitoneally to anesthetize the preconditioned mouse.
Now, place the mouse on an intubation platform by hanging its front teeth with its back against the platform. Open the mouth and gently pull the tongue out. Position a catheter into the trachea until the top reaches front teeth and dispense the desired amount of cancer cell suspension in the trachea to engraft bioluminescent cancer cells in the lungs.
Wait until the mouse inhales the entire volume. Remove the catheter gently and place the mouse on a recovery pad. After a few days, inject luciferin retro-orbitally in alternate eyes and place the mouse on an imager.
Cancer cells produce luciferase that converts luciferin into oxyluciferin in presence of oxygen and emit light. Acquire images to monitor tumor burden in the lungs by calculating luminoscore. In the following protocol, we will perform engraftment of lung adenocarcinoma cells in the mouse.
For this protocol, the mice must first be injected with bleomycin. Then after 14 days the tumor cells are injected. The procedure for both injections is very similar and described here.
Two hours prior to the injection, thaw the bleomycin stock on ice and dilute it to the working concentration. Keep it on ice. For a cell bolus, have a cell suspension prepared as described in the text protocol. The procedure time for each mouse is about 10 minutes altogether. Be certain to confirm proper anesthesia using a toe pinch and apply vet ointment to the eyes.
At this time, if the mouse has dark hair, then shave the entire rib area both ventrally and dorsally so it can be imaged correctly. Then place the mouse on the intubation platform by securing it by the front teeth with its back against the platform. If a cell bolus is to be injected, resuspend it now with gentle trituration.
Now illuminate the upper chest region, open the mouth, and gently pull out the tongue using flat forceps. Prepare an IV catheter for the trachea by removing the needle then position it over the tracheal opening. Next, insert the catheter into the trachea until the top of the catheter is near the front teeth and illuminate the interior of the catheter to confirm its placement.
Then dispense the 50 microliters of freshly made bleomycin or cell suspension directly into the catheter. Wait a few seconds for the entire volume to travel down the catheter. Then remove the catheter and dispose of it in 10% bleach.
- If the mouse is not inhaling the liquid, carefully monitor its breathing and adjust the catheter position. If the mouse stops breathing, immediately remove the catheter and allow the mouse the breath before you reinsert the catheter in.
- After removing the catheter, continue performing the procedure to have five treated mice still anesthetized and ready to image. For recovery place the mouse supine on an IACUC proved heating pad in a recovery cage and monitor it until it has fully recovered.