This protocol provides reproducible, immunocompetent PDAC in vivo models suitable for genotype, phenotype, and drug response studies. The main advantage of this technique is its ability to help investigate tumor progression with a syngeneic biological tumor microenvironment while maintaining the reproducibility of the experiment. Demonstrating the procedure will be Stefanie Barthel and Chiara Falcomata, both are from our laboratory.
Begin the preparation of the tumor cells by washing the pancreatic ductal adenocarcinoma, or PDAC cells, grown in a T75 culture flask with phosphate buffered saline or PBS. Then add 0.5 milliliters of trypsin for five to 10 minutes to detach from the flask. Re-suspend the detached cells in 10 milliliters of DMEM with 10%fetal bovine serum, or FBS, and 0.1%penicillin streptomycin before transferring the cell suspension to a 15 milliliter tube.
Centrifuge the cell suspension at 200G for five minutes and aspirate the supernatant. Re-suspend the cell pellet in DMEM without additives, based on the required final concentration of the cells for injection. Count the cells in 10 microliters of cell suspension, using the Neubauer Chamber and calculate the number of available cells.
Transfer one milliliter of the diluted suspension, as per the final required concentration, to a 1.5 milliliter tube. Place the tube on a rotator until implantation to prevent cell aggregation. For orthotopic implantation of PDAC cells, apply eye cream to the analgosedated mouse while the mouse is sleeping.
Check the sufficient analgosedation before shaving the left lateral abdominal flank of the analgosedated mouse. Place the mouse, lying on the right side, on a sterile heating mat and tape the legs to the surface. Wear new gloves and disinfect them.
Using surgical scissors, cut about one centimeter of the skin and subcutaneous fat tissue longitudinally on the left flank where the spleen is projected. Using scissors and forceps, separate the skin from the peritoneum to get easier access to the peritoneum. Change the pair of scissors before opening the peritoneum at the location of the spleen with a one centimeter longitudinal cut.
While mobilizing the spleen and the attached pancreas using blunt, fine tipped forceps, ensure that the organs are not attached to other tissue. Also check for the supplying blood vessels to avoid injuring the vessels or the surrounding tissue when handling the pancreas. Carefully pull the pancreas out of the incision to enable easier access to the organ's tail and observe the spleen following the pancreas.
Ensure that the organ is not folded during injection and the organ's capsule is held under tension at the needle penetration site to avoid spillage. Aim for a piece of pancreatic tissue between the visible blood vessels to minimize the risk of puncturing or injecting the vessel. Using the dominant hand, carefully penetrate the organ's capsule with a 27 gauge cannula and 50 microliter syringe following the longitudinal axis of the organ.
After injecting the 20 microliters of suspension containing 2, 500 cells into the tail area of the pancreas, keep the pancreas and inserted syringe still for approximately one minute to avoid spilling the tumor cells. Then, remove the needle from the injection site. Next, carefully rearrange the organs inside the abdomen without injuring the tissue to avoid spilling the cells.
Avoid organ adhesion by pouring one milliliter of 0.9%sodium chloride onto the organs. After suturing the peritoneum using the simple interrupted suture technique, close the skin using two to three nine millimeter stainless steel wound clips. Then antagonize the analgosedation with midazolam, medetomadine, and fentanyl, or MMF, with a subcutaneous injection of AFN according to the body weight of the mouse.
Once done, place the mouse in a 37 degree Celsius heated chamber until it is awake and completely active. Place the active mouse back into its original cage and monitor the health status of the mouse by checking the fur, skin color, and activity, and repeat it three to four hours after surgery. In MRI of implanted mouse, the engraftment of PDAC cells was abdominally palpated earliest at approximately seven days post-surgery, depending on the cell number injected and the aggressiveness and growth characteristics of the inoculated cell line.
The survival of the resulting implanted mice varied depending on the tumor cell intrinsic features of the cell lines. Successful intra-pancreatic injections led to full-blown tumors in the mouse pancreas. In contrast, unsuccessful implantations resulted in the absence of pancreatic tumors because of injection of non-viable cells, rejection of the implanted cells, or injection of an insufficient number of cells.
Remember to pay attention to not spill any cells during injections and not to injure any surrounding tissue to prevent cells from spreading inside the abdominal. Following this procedure, in vivo studies, like preclinical drug response studies, can be performed in immunocompetent mice, followed by, for example, facts, sequencing, or histological analysis to investigate tumor host interactions.
