膵臓癌の正交性切除マウスモデル

This protocol describes a robust and reproducible surgical resection model of pancreatic cancer in mice, which may be used for testing adjuvant and neoadjuvant therapy. This technique combines a co-infection orthotopic model, which is effective at recapitulating human pancreatic cancer with a safe and effective pancreatic resection technique. While an immunodeficient model is described in this protocol, an alternative immunocompetent model, using KPC tumor cells implanted into C57 black six mice may be performed.

This alternative would be useful for testing immune therapies. To begin, place the anesthetized mouse on a sterile field in a supine position and apply povidone iodine, followed by 70%ethanol for skin preparation. Dissect the skin by making a longitudinal incision on the left cranial quadrant of the abdomen and incise the muscular layers between forceps.

After mounting the syringe with the cell suspension on the injection device, place the purse string swab over the laparotomy incision and exteriorize the spleen and pancreatic tail through the swab’s opening. Tighten the purse strings swab gently and expose the pancreatic tail for injection, making sure that the gauze contacts the pancreas circumferentially, but does not constrict it. Grasp the tail of the pancreas, using forceps and gently place lateral tension on it.

Puncture the pancreas with the needle, bevel up and advance it while the needle tip is lifted slightly upward until the bevel has completely entered the peritoneum. Inject the parenchyma of the pancreas with the cell suspension in a slow and controlled fashion. After injection, hold the needle in place for a few seconds before withdrawing to minimize leakage.

Use a povidone iodine soaked swab to carefully dab the site to absorb any inadvertently leaked cell suspension. Replace the spleen and pancreas back to its orthotopic position and close the abdominal wall with 5-0 polyglycolic acid suture. Close the skin with clips.

Make a longitudinal incision in the skin of the left cranial quadrant of the abdomen. Preferably through the previous incision site. Bluntly dissect the skin off the underlying, muscular abdominal wall and place an all self retaining retractor to hold the wound open.

Incise the muscular layer between forceps, just to one side of the suture line of the previous operation. And then extend the incision to excise the entire previous suture line. Exteriorize the spleen and pancreas by retracting it cranially.

In this case, some adhesions were found attached to the inferior aspect of the tumor. This was divided by cautery. The colon may be found attached to the caudal aspect of the pancreas by filmy adhesions.

If this is found, bluntly dissect the colon off. Expose the segment of pancreas for ligation by carefully passing a pair of forceps dorsal to the splenic vessels at the body of the pancreas. Ligate the pancreas proximal to the tumor with a titanium legation clip and then transect the pancreas distal to this with cautery.

Alternatively, ligate the pancreas in continuity with 5-0 polyglycolic acid suture before transection, in order to control the pancreatic stump. Carefully retract the pancreas caudally and cauterize the gastro splenic vessels between the cranial pole of the spleen and the stomach. Keeping in mind that the vessels are small and may easily bleed if avulse or inadequately cauterized.

Such vessels may retract into the abdominal cavity and cause bleeding, which may be difficult to control. Remove the specimen and examine the surgical site to confirm hemostasis. Close the abdominal wall with a 5-0 polyglycolic acid suture in a continuous fashion and close the skin with clips.

Out of 59 consecutive mice who underwent implantation surgery, gross leakage occurred in eight mice. After three weeks of tumor growth, the pre-resection bioluminescence imaging allowed the exclusion of mice with gross metastatic disease from surgical resection. This left 45 mice that proceeded to pancreatectomy.

The procedure was successfully carried out in 100%of mice. A macroscopic proximal pancreatic margin, greater than five millimeters was achieved in 43 of these mice. At the time of resection, local metastasis was found in nine out of 45 mice.

In addition to metastasis to the suture line, isolated metastatic nodules on the greater curve of the stomach were observed in three mice and a subcapsular nodule on the liver in one mouse. Local extra pancreatic invasion occurred in six mice with direct invasion into the suture line in five mice and into the liver in one mouse. One week post-resection, 32 mice had a maximum radiance ratio of less than 10, indicating minimal or no residual disease.

The mean surgery time from induction of anesthesia to closure was 22 minutes. The key step in the resection procedure is recognizing the bloodless embryological plane dorsal to the splenic vessels and pancreas. Dissecting this plane frees up a segment of the pancreas for subsequent ligation.

After resection, the mice may be treated with adjuvant therapies. Progression of disease may then be assessed, using bioluminescence imaging in combination with necropsy findings and animal survival, depending on the local animal ethics protocols.