不可逆エレクトロポレーションの効果を研究する同系膵癌マウス モデル

The overall goal of this model is to study the effects of irreversible electroporation or IRE, on pancreatic cancer in immunocompetent mice. This model can help answer many key questions regarding the role of IRE in pancreatic cancer. Such as, how does IRE affect the local tumor microenvironment?

The main advantage of this model is that it facilitate accurate tumor growth assessment in an immunocompetent system, without requiring knockout or mutant mouse models. This method can provide insight into the effects of IRE on the immune system, in an immunocompetent mouse model of pancreatic cancer. For subcutaneous tumor induction, first, shave the injection site on an anesthetized six-week old, male, C57 black six mouse, and use an alcohol swab to clean the site two times.

Next, load a cold one-milliliter syringe with one to two times ten to the seven cells per milliliter and 250 microliters of basement membrane matrix. Attach a 25-gauge needle to the syringe, and load the needle with the tumor cell suspension. Press and retract the plunger a few times to remove any air bubbles.

Carefully insert the needle under the skin of the injection site, at a 30 degree angle. With the needle parallel to the body, slowly inject 50 microliters of the tumor cells. After 10 seconds, slowly withdraw the needle, keeping it parallel without any sideways movement, and gently clean the injection site with an alcohol swab, before returning the mouse to its cage.

When the tumor reaches five to seven millimeters, use sterile forceps and micro scissors to excise the subcutaneous tumor, under sterile conditions in a biosafety level two hood, taking care not to puncture the peritoneal cavity. Immediately wash the excised tumor, two times, in sterile PBS. And use sterile scalpels to mince the tumor into one to two cubed millimeter pieces.

Then, transfer the tumor pieces into a Petri dish, containing cold, sterile DMEM basal medium on ice. To implant the tumor fragments, after confirming a lack of response to toe pinch, apply ointment to the anesthetized recipient animal's eyes, and place the mouse on its right side, so that the left side of the abdomen is exposed. Remove the abdominal hair with depilatory cream, and remove the cream with gauze.

After disinfecting the exposed skin with three sequential 10%povidone plus iodine and alcohol wipes, use a sterile scalpel to make a 1.5 centimeter incision in the skin, one centimeter to the left of the midline, below the rib cage, and slightly medial to the spleen. Gently externalize the spleen with flat-tipped forceps and a sterile cotton applicator, and use the forceps to laterally retract the tail of the pancreas, at the bottom of the spleen. Using sterile forceps, grasp one finely-minced tumor piece, and pass a fine 6-O sterile suturing needle through the piece.

Insert the needle containing tumor fragment into the tail of the pancreas, passing the suture slowly through the tissue. Take care to avoid contact between the tumor and the abdominal wall musculature and peritoneum, to prevent tumor seeding. When the tumor fragment has been secured, completely remove the needle from the tissue, and keep the organs completely externalized, for an additional 60 seconds, while inspecting for any signs of hemorrhage.

If no bleeding is observed, use blunt forceps, or a cotton tip applicator, to gently return the spleen, pancreas, and tumor to the abdominal cavity. Before the tumor reaches six millimeters in diameter, place the anesthetized animal on its right side to allow access to the tumor, and use clippers and an alcohol swab to clean the tumor site. Using forceps, elevate the skin directly under the tumor, and insert two needle array electrodes through the skin, parallel to the body, to bracket the tumor, taking care that the electrodes do not penetrate the peritoneal cavity.

Set the electroporator to deliver 100 microsecond pulses at a one hertz frequency, at 1500 volts per centimeter, for a total of 150 pulses, and use the foot pedal to deliver the pulses, separating each set of 10 pulses by 10 seconds, to allow heat dissipation between each set of pulses. As muscle twitching occurs during the IRE delivery, and is more pronounced during the initial set of pulses, ensure that the electrodes are still in the desired location, and that the animal is still sedated after each set of pulses. After no more than 200 seconds of dosing, remove the electrodes and record the actual voltage delivered, as displayed on the electroporator.

Then, return the mouse to its cage. For IRE of an orthotopic tumor, eight to 10 days post tumor implantation, use blunt-nosed forceps to externalize the tumor with the forceps, and tightly capture the tumor with the platinum electrodes of the Tweezertrode, deliver the electroporation pulses, as just demonstrated, keeping the tumor externalized for the last set of pulses, for at least 60 seconds, to monitor for any signs of hemorrhage. Then, return the tumor to the abdominal cavity and close the incision, as demonstrated, monitoring the animal until it has fully recovered.

In this representative experiment, subcutaneous tumors, in nearly 50%of the mice, regressed completely after 150 IRE pulses, with only minor regression after 75 pulses. Histological analysis of the tumor tissue, one week post IRE, revealed large regions of central tumor necrosis, that were not observed in control, untreated tumors, and that were flanked by live tumor tissue, in cases of incomplete tumor regression. Monitoring implanted orthotopic tumors growth rates revealed a consistent tumor growth over time in recipient animals, that could also be disrupted by IRE, demonstrating the ability of this model to simulate the effects of IRE, in an immunocompetent mouse model.

Once mastered, this technique can be completed in 15 minutes per mouse, if it is performed properly. While attempting this procedure, it is important to remember to keep the tumor size within the working distance of the IRE electrodes available, as larger tumors will produce inconsistent responses due to incomplete ablation. Other treatment approaches, such as radiation or chemotherapies, can also be used to answer additional questions about whether combining treatments can improve tumor responses to IRE.

After watching this video, you should have a good understanding of how to establish subcutaneous and orthotopic pancreatic cancers in mice, and how to perform IRE on these tumors.