Ultrasound-Guided Implantation: A Technique to Implant Pancreatic Ductal Adenocarcinoma Cells into an Adult Mouse

- Ultrasound provides high-resolution images that help implant cancer cells in a specific tissue site with minimal inflammation during tumor cell injection. Primarily, we use orthotopic models to graft tumor cells into a relevant organ of tumor origin. The organ mimics the original tumor microenvironment and facilitates metastasis.

Begin by suspending the tumor cells in cold PBS to maintain pH and place the tube on ice. Next, place the anesthetic mouse on the ultrasound stage and secure the limbs with lab tape. Apply the required amount of ultrasound gel on the shaved abdomen and adjust the ultrasound probe position until the pancreas is clearly visible. Load the syringe with tumor cell suspension and carefully inject the cells into the pancreas.

A fluid bubble appears in the ultrasound image, confirming successful implantation. Wait for 5 seconds to prevent the backflow of cells and gently remove the needle. Return the animal to a clean cage and monitor until it recovers from anesthesia. Within a few weeks of injection, the injected cancer cells in the pancreas divide rapidly and form a tumor. In the following protocol, we will perform ultrasound-guided implantation of murine-derived pancreatic ductal adenocarcinoma cells into the pancreas of the orthotopic model.

- Before beginning the procedure, resuspend the pancreatic ductal adenocarcinoma cell line of interest in an appropriate volume of sterile cold PBS on ice. Place cages on a 37 degrees Celsius warming plate and thoroughly cleaned the biological safety cabinet, induction chamber, and ultrasound stage with an appropriate sterilant. Set the warming function of the ultrasound stage to 37 degrees Celsius and after induction of anesthesia, apply ointment to the first animal's eyes.

Place the mouse in dorsal recumbency on the ultrasound stage and gently secure the upper and lower extremities to the stage with the tape. Use a sterile cotton-tip applicator to apply a generous layer of depilatory cream to the upper-left quadrant of the abdomen over the region of the spleen to the midline. After 1 minute, use a dry gauze pad to gently remove the hair before removing the excess depilatory cream with the saline wet gauze. Then, place the mouse in one of the new clean cages on the warmer.

For ultrasound-guided tumor cell implantation, adjust the ultrasound platform so that the surface is parallel to the floor and stand to the left side of the animal with the head of the mouse facing to the right. Adjust the transducer position so that a transverse abdominal image is obtained and secure the mouse limbs to the platform with tape. Gently lower the transducer to contact the mouse abdomen and adjust the transducer until the pancreas is clearly visible. Locate the left kidney and spleen to provide an accurate orientation in the abdominal cavity.

When the injection site has been located, load a 29 gauge, half-inch insulin syringe with 25 microliters of the tumor cell suspension and wipe the needle tip with a sterile alcohol prep pad. Use blunt edge forceps to grasp the mouse skin and peritoneal wall to increase tension at the injection site. Holding the syringe at an approximately 25 to 45-degree angle to the ultrasound platform surface, slowly advance the needle through the skin and the peritoneal wall. Confirm the needle has punctured through the peritoneal wall before utilizing ultrasound visualization to guide the needle directly into the pancreas.

When the needle is in place, slowly inject the tumor cells. The formation of a fluid bolus within the pancreas will be evident in a correctly injected pancreas by ultrasound. Once the full volume of suspension has been injected, and a fluid bolus can be observed by ultrasound, hold the syringe very still for several seconds before slowly retracting the needle from the mouse abdomen, taking care not to disturb the injected cells. Then, place the mouse into a new clean cage on the warmer with monitoring until full recovery.

- Proper needle placement in the pancreas, including needle depth and angle, is a critical step in this protocol. Visualization of the fluid bolus helps confirm a successful tumor injection. Controlling the depth of injection is the most difficult aspect. My advice is to practice with trypan-blue injections and confirm fluid bolus by both ultrasound and necropsy.