Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

The protocol describes a method to generate an orthotopic mouse model of pancreatic cancer using a system that combines the injection of cells and the evaluation of engraftment and development of tumors. This technique has great reproducibility, is minimally invasive, allows to overcome many drawbacks caused by the surgical procedure, and complies with the 3Rs principles. The echo-guided injection could be used for other orthotopic models, such as injections in the biliary tree or breast cancer models.

The use of such a technique could be coupled with the use of small molecules. Start preparing an 8-week-old 20 Athymic Nude-Foxn-1nu female mice for ultrasound-guided injection by administering tramadol intraperitoneally at a dose of 5 milligrams per kilogram using a tuberculin syringe with a 27-gauge needle. Next, turn on the imager and select Mouse, Small, Abdominal"on the application menu from the transducer panel.

Ensure that the B-Mode, or Brightness Mode, imaging window appears and the system is ready to acquire B-Mode data. Go to the Study Browser to select New Study"and type the study name and information, such as the date of the study. Then, fill in all the necessary information in the Series Name, like animal strain, ID, date of birth.

After the information is entered, tap Done"to prepare the program for B-Mode imaging. Place the anesthetized mouse on its right flank onto the handling table heated at 37 degrees Celsius with its snout in a nose cone and apply a drop of vet ointment on the mouse's eyes. Then, use an adhesive gauze to tape the right hand, right foot, and tail of the mouse onto the electrode pads on the animal platform.

The mouse's respiration rate and electrocardiogram are recorded through the electrode pads. Before injecting the mouse with PANC1 cells, sanitize the mouse skin with 70%ethanol and keep the skin of the left flank stretched using adhesive gauze. Use a 20 milliliter syringe without the needle to apply ultrasound gel on the abdomen and left flank of the mouse.

With the help of the height control knob of the ultrasound transducer, lower the transducer to touch the left flank of the mouse and place the transducer transversely to the body of the animal. Then, move the transducer to visualize the pancreas on the transducer display using B-Mode imaging. Prepare a 50 microliter Hamilton syringe, containing 10 million PANC1 cells suspended in 20 microliters of PBS with a sanitized 30-millimeter 28-gauge needle and place the syringe on the appropriate holder.

Using the holder micromanipulator, lower the syringe to the mouse skin, with the needle bevel face up and in the same plane as the ultrasound transducer, forming an angle of 45 degrees with the transducer. Perforate the skin with the micromanipulator and insert the syringe needle into the pancreas while observing the ultrasound image on display to follow the trajectory. Once the needle is inserted into the pancreas, inject a 20 microliters bolus containing the cells by applying constant pressure on the syringe's plunger.

The correct injection procedure can be checked by the presence of a small bubble in the pancreas and the flow of hypoechogenic fluid. After 5 to 10 seconds of the injection, retract the needle. Then, remove the ultrasound transducer and clean the gel from the flank.

When done, place the mouse alone in a new cage under observation until it has regained sufficient consciousness to maintain sternal recumbency. The representative image shows the presence of a tumor mass in the tail of the pancreas after echo-guided cell injection, confirming the reproducibility of the protocol. The tumor volume of orthotopic pancreatic tumors derived from PANC1 cells was evaluated by ultrasound imaging once a week, starting from Day 8 after cell injection.

At the end point of the experiment on Day 43, ultrasound images of the tumor masses were acquired. In the necropsy examination, tumor mass, a small portion of a healthy pancreas, spleen, stomach, and liver was observed. Additionally, the tumor masses were explanted for histologic analysis.

It is important to position the mouse on its right side onto the table, and the skin must be properly stretched to avoid the risk of the cells spilling out of the pancreas. Future applications of the pancreatic cancer model obtained by the ultrasound-guided injection include the studies aimed at the development of appropriate therapeutic treatments or treatment combinations.