A “Patient-Like” Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a “patient-like” orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The overall goal of this procedure is to establish a patient like Orthotopic Syngeneic mouse model system for studying the mechanisms of metastasis in hepatocellular carcinoma. This is accomplished by first preparing mouse hepatocellular carcinoma cells for implantation. In the second step, the abdomen of a recipient syngeneic animal is opened and the cells are carefully injected into the right lobe of the liver.

Ultimately, the success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of hepatocellular carcinoma metastasis. The main advantage of this technique over existing techniques like mouse xenograft tumor models, is that our syn genetic tumor model system allows the investigation of the role of the immune response in tumor metastasis. Demonstrating this procedure with me will be Michelle Nadu, an undergraduate student from our lab To prepare the cells for implantation begin by washing a 60 to 70%confluent culture of hepatocellular carcinoma cells two times with five milliliters of PBS.

Incubate the cell culture with two milliliters of trypsin at 37 degrees Celsius. After five minutes, count the cells and dilute them at five times 10 to the fifth cells per milliliter of PBS. Next, transfer 100 microliter aliquots of the cells into one sterile 1.5 milliliter micro centrifuge tube per mouse, and place the cells on ice.

Before beginning the surgical procedure, confirm the proper level of anesthesia by toe. Pinch, apply ointment to the eyes of a 16 to 20 gram healthy adult bsy mouse and shave in approximately one inch area around the midline of the animal's abdomen. Then dawn a pair of latex gloves and wash the exposed skin several times with a soapy disinfectant scrub, followed by a sterile PBS rinse for approximately five minutes.

When the skin has been prepared, discard the gloves and put on the appropriate personal protective equipment. Provide an external heat source to prevent anesthetic hypothermia during the procedure, and then place surgical tape on the animal's, limbs and tail to fix the mouse in a supine position. Next, make a straight incision immediately inferior to the xiphos sternum, and use a sterile self retaining surgical retractor to open the abdomen.

When the liver is visible, aspirate one aliquot of cells into a one milliliter syringe equipped with a 20 gauge needle, and slowly and carefully inject all 100 microliters of the hepatocellular carcinoma cells into the right lobe of the liver. After removing the needle, apply a sterile cotton swab for at least one minute to stop any bleeding. Then use an absorbable suture to close the subcutaneous tissue, followed by closure of the skin.

With surgical clips, clean the wound with another disinfectant scrub and subcutaneously, administer 100 microliters of normal saline. Then immediately after the surgery, transfer the mouse to a clean cage without companions and with paper bedding for at least 24 hours. These BSY mouse livers were implanted with five times 10 to the six one ME.A mouse hepatocellular carcinoma cells as just demonstrated.

Clinic clinical evidence of hepatocellular carcinoma development was observable 63 days later as indicated by the arrow in one mouse. A superficial tumor was observed on the surface of the liver with some attachment to the abdominal wall. The lungs from this mouse exhibited an abnormal morphology as well displaying areas of visible hemorrhaging necrosis.

In a second mouse, the liver appeared grossly abnormal with the abnormality very strongly resembling a superficial tumor as expected control. Mice that were not implanted did not develop tumors in either the liver or the lungs. Once mastered, this orthotopic syn genetic mouse model can provide a good platform for elucidating the biological mechanisms of metastasis and hepatocellular carcinoma.