The overall goal of this procedure is to create a preclinical mirroring pancreatic cancer model of hepatic metastases via Hemi spleen technique. This is accomplished by first creating a single cell suspension of cultured pan O2 tumor cells. The second step of the procedure is to express and divide the spleen of a fully anesthetized mouse.
The next steps are to inject tumor cells into a single Hemi spleen and remove the injected hemi spleen. The final step is to close the abdomen and recover the mouse from anesthesia. Ultimately, results can be obtained that show differences in metastatic disease progression by monitoring survival, followed by liver necropsy, and additional non-invasive imaging.
These techniques can help answer key questions in the cancer research field, such as identify key factors in the metastatic process, testing novel therapeutic agents, and conducting tumor immunology research. The implications of this technique extend toward therapy of pancreatic cancer because immunocompetent mice undergo identical tumor inoculation, consistently develop tumor and atic consistent time point Prior to preparing for the surgery. Make ready panco two cells in PBS at 20 million cells per milliliter stored on ice.
Next anesthetize an eight to 12 week old female C 57 black six mouse. Using divided doses then ensure that the mouse is anesthetized by testing its reflexes. Administer additional doses as needed, tend to the mouse's eyes with ointment, and then proceed with shaving the abdomen.
Once shaved, clean the skin off with alternate scrubs of 70%ethanol and iodine. Then drape the mouse. Begin with the left subcostal incision in line with the left ear through the skin and peritoneum using a scalpel.
Then express the spleen through the incision by applying simultaneous digital pressure along the cranial and coddle aspects of the incision. With the spleen visible, locate the splenic blood vessels at its inferior end. Next, divide the spleen.
Place two horizon medium-sized ligating clips in its center. Be careful of the splenic blood vessels at the splenic poles. They are easily damaged.
Now, place the upper pole of the spleen back into the peritoneum to avoid contamination. Prepare for the tumor injection by first drawing up 150 microliters of PBS into a 26 gauge five eight inch syringe. Then draw up 100 microliters of the prepared Panko two cells on ice into the same syringe.
Keep the syringe upright so that the layers do not mix too much before making the injection dip the needle in a 70%ethanol solution. Then slowly inject the cells into the exposed hemi spleen with the syringe upright. Observe the bevel of the syringe through the splenic capsule to be sure cells under the spleen are not injured.
The spleen should whiten with the injection. Now elevate the spleen and apply one medium horizon clip under the spleen to occlude the splenic blood vessels. Then apply a small horizon clip to the most distal aspect of the pancreas and splenic vessels.
With the clips in place, ligate the pancreas and splenic vessels from the hemi spleen by cutting above the clips. After the ligation, place the mouse on its side and irrigate the incision with distilled water. Finish the surgery by closing the peritoneum with four oh running sutures.
Then apply two to three skin clips to close the skin incision immediately administer carprofen at four milligrams per kilogram subcutaneously to alleviate post-surgical pain. Transfer the mouse to a heating pad until it is mobile and breathes regularly. Do not return the mouse to the company of other animals until it shows regular breathing and is moving about normally 30 to 60 days After the surgery, the mouse will begin to show clinical symptoms of metastasis and require euthanasia.
Once the animal is non-responsive, use scissors to make an incision through the peritoneum to expose the abdomen. Then with forceps and scissors, carefully isolate the liver from the abdomen. Transfer the liver to OCT compound flash freeze using liquid nitrogen and store the liver at minus 80 degrees Celsius.
Alternatively, fix the liver in 16%Neutral buffer Formin at room temperature for 24 hours, followed by paraffin. Embedding for processing. Presented is the technique for creating pancreatic hepatic metastases.
With panco two tumor cells in 100%of over 100 properly processed mice panco two tumor cells injected into the hemi spleen formed liver metastases. In addition, lung and peritoneal metastases were never observed in all experiments conducted thus far. Panko two injected mice that were left untreated died with liver metastases between 30 and 60 days.
In the necropsies, multiple liver metastases were always observed injecting other cells resulted in similar metastases, such as with KPC tumor cells. KPC tumor cells are a syngeneic pancreatic tumor cell line derived from mice with tissue specific KRAS and P 53 knockin mutations. With known therapeutic treatment, an inhibition or delay in formation of metastases can occur.
Thus, this can be an effective model for testing new therapeutic agents. Another tool to use is small animal ultrasound, which allows in vivo analysis of the liver with enough resolution to view developing metastases After this development. This techniques has paved the way for pate cancer research to explore the metastatic process as well as testing novel therapy agents in pancreatic cancer.
After watching this video, you should have a good understanding of how to establish a preclinical mirroring pancreatic cancer model of hepatic metastases using a Hemi spleen technique.
