Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

This protocol allows the study of pancreatic cancer in the native tissue site, while minimizing inflammation during tumor cell injection and provides a high throughput method for generating large cohorts of mice. By using ultrasound guidance, the need for abdominal surgery is eliminated. In our hands, UG-OTIM has a much lower rate of peritoneal wall seeding compared to traditional orthotopic implantation.

UG-OTIM produces tumors that recapitulate histological and immunobiological features characteristic of the pancreatic tumor micro environment and can therefore be used to investigate novel therapeutic combinations in a clinically relevant setting. We developed this protocol for immuno-oncology and cancer biology therapies, but it could be used in other research areas, including investigations of normal pancreatic function or disease states such as diabetes. The real-time ultrasound imaging of the needle insertion and injection is an integral part of this method.

So visual demonstration of the step is critical for the proper technique. Before beginning the procedure, we suspend the pancreatic ductile adenocarcinoma cell line of interest in an appropriate volume of sterile, cold PBS on ice. Place cages on a 37 degree Celsius warming plate, and thoroughly clean 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 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 one minute, use a dry gauze pad to gently remove the hair before removing the excess depilatory cream with a 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 gage 1/2 inch insulin syringe with 25 micro-liters 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 the 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 tripamboo injections and confirm fluid bolus by both ultrasound and necropsy.

Monitoring of the tumor implantation and growth rate by weekly ultrasound imaging reveals tumors that are contained within the borders of the pancreas throughout the entire experimental period. High titer tumor injections result in a higher proportion of tumor bearing animals three weeks after injection compared to the low titer cohort. Despite the delay in tumor onset, the overall tumor growth rate is not significantly different between the two doses.

Similarly, while the survival rate between the two cohorts is not significantly different, they tend to trend toward slightly improved survival in the low titer cohort. The high titer cohort also produces a greater proportion of mice with tumors that are inrollable in pre-clinical studies, by four weeks post-injection. Upon sacrifice, the gross anatomy of ultrasound guided orthotopic tumor implantation model tumors is similar to that of spontaneous KPC tumors and histologic analysis demonstrates a pattern of abnormal ductal structures that is similar in both models and that recapitulates the morphology of the human disease.

In both models, the tumors are poorly infiltrated by T-Cells, but highly infiltrated by macrophages. Remember to hold the syringe at the appropriate angle to allow smooth entry into the pancreas, and to confirm that the needle's in the pancreas before proceeding with the injection. Tumor growth can be monitored and tumors can be harvested for analysis by pho cytometry or immunohistochemistry to determine the impact of an intervention.