Portal Vein Injection: A Method to Study Cancer Metastasis to the Liver

The liver is a secondary metastatic site for most cancers. To study metastasis, we can inject cancer cells into the portal vein, which carries blood from other parts of the body to the liver.

Start by placing an anesthetized mouse in a supine position. Sterilize the bare abdominal area using chlorhexidine and alcohol. Using microscissors, make an incision between ribs and the fourth mammary gland on the animal's left side without damaging internal organs.

Place a gauze pad soaked in sterile saline near the incision. Gently pull the intestine out using a sterile cotton swab to reveal the portal vein, and place it on the gauze. Cover the organs with the gauze pad to prevent drying. Insert the syringe needle containing fluorescently labeled cancer cells into the portal vein and slowly inject the cells. Wait for a few seconds to prevent the backflow of cells and carefully remove the needle.

After injection, cancer cells usually travel in the sinusoid and invade into the hepatocytes near the portal triad to metastasize. Place organs back into the abdominal cavity and suture the incision. In the example protocol, we will perform portal vein injection to study breast cancer metastasis to the liver.

Before beginning the procedure, wipe down all the surfaces of the surgical area with 10% bleach. One hour before the injections, treat 8 to 15-week-old BALB/c female mice with 100 microliters of analgesia subcutaneously for pain management. Apply ointment to the animal's eyes. Then, place the mouse in the supine position and confirm the appropriate level of sedation by toe pinch.

Next, disinfect the surgical and surrounding areas of the animal, including the tail, with three alternating sterile gauze-soaked 2% chlorhexidine wipes and two alcohol prep pad wipes. After the last chlorhexidine wipe, use a sterile scalpel to make a 1 inch incision into the skin between the median and sagittal plains on the left side of the mouth, starting just below the ribs and ending above the fourth inguinal mammary gland teat, followed by a similar one inch incision through the peritoneum.

Now, pipette the tumor cells up and down several times to resuspend the cell solution, and load a 25 microliters removable needle syringe, equipped with a 32 gauge needle with 10 microliters of the cells. Depress the plunger until the cells are at the tip of the needle and the plunger is at the appropriate volume for injection. Wipe the outside of the loaded needle with a sterile alcohol pad to remove any external tumor cells, taking care to avoid needle sticks.

Then, holding the skin and peritoneal lining of the median side of the incision with the forceps, use a sterile cotton swab to carefully move the large and small intestines onto a sterile gauze soaked in sterile saline. Cover the internal organs in the saline-soaked gauze to maintain the internal moisture and sterility. When the portal vein is visible, have an assistant gently hold the intestines out of the way to expose the portal vein with the sterile cotton swab and insert the needle 3 to 5 millimeters into the portal vein, approximately 10 millimeters below the liver at a less than 5 degree angle, bevel side up.

Slowly inject the full volume of tumor cells, allowing the blood to flow past the needle head for several seconds to avoid a backflow of the tumor cells out of the vein.

As the proper visualization, access, and injection into the portal vein is essential, take the time to ensure that the positioning of the portal vein in relation to the needle is satisfactory prior to attempting the injection.

When all of the cells have been injected, removed the needle while simultaneously applying gentle pressure to the vein with a sterile cotton tip applicator. Then, use a new sterile cotton tip applicator to hold a 0.5 to 1 centimeter square piece of hemostatic gauze over the injection site.

After five minutes, carefully lift the gauze to assess the vein closure. When the blood flow has ceased completely, remove the gauze and return the intestines to the abdominal cavity. Using a simple, continuous, or interrupted suture pattern, close the peritoneal lining with a sterile 4-0 vicryl suture and taper.

After closing the skin in the same manner, inject 100 microliters of local anesthetic along the incision site, followed by subcutaneous delivery of 0.5 milliliters of sterile saline.