The overall goal of this procedure is to deliver tumor cells directly to the murine liver for the experimental modeling of breast cancer liver metastasis. This method can be used to study the later stages of the metastatic cascade in the murine liver. Including breast cancer cell extravasation, seeding, death, proliferation, and dormancy decisions, as well as outgrowth.
The main advantage of this technique is that it delivers tumor cells directly to the liver without the removal of the spleen or concomitant multi organ metastasis. Visual demonstration of this method is critical, as the portal vein injection requires precise manipulations that rely on direct anatomical surveillance. Assisting me with this procedure will be Alexandra Quackenbush, a graduate student in Pepershadine's laboratory.
Before beginning the procedure, wipe down all the surfaces of the surgical area with 10%bleach. One hour before the injections, treat eight 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 mice 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 one inch incision into the skin between the median and sagittal planes on the left side of the mouse, 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 re-suspend the cell solution, and load a 25 microliter 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 a sterile cotton swab and insert the needle three to five 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, remove 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 one centimeter square piece of hemostatic gauze over the injection site. After five minutes, carefully lift the gauze to asses 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 the sterile four zero 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. The accurate anatomical identification of the portal vein and successful intra-portal injection can be confirmed by practicing the injection protocol with india ink or a similar dye.
With a correct injection via the portal vein, resulting in the ink being delivered immediately and specifically to the liver without spreading to the lung. Using mouse memory tumor cells tagged with GFP, tumor cell dispersal throughout the liver is apparent at 90 minutes post injection, confirming a successful portal vein delivery to the liver. The tumor cells are found within the sinusoids as well as within the liver parenchyma in close proximity to portal triads, where the portal vein blood enters the liver, suggesting that active tumor cell extravasation is taking place at this time.
As demonstrated by these survival curves, the adoptive portal vein transfer of aggressive mammary tumor cell lines results in shorter metastasis-free survival rates compared to mice injected with a less aggressive tumor cell line. Metastases from these animals can be confirmed by sectioning through the liver in 250 micron levels and subsequent H&E analysis of the samples. Further, immunofluorescence analysis is useful for micro and macro metastatic lesion analyses of untagged syngenic lines such as the D2A1 tumor line, which are CD45 negative, Heppar-1 negative, and CK18 positive as demonstrated in these representative images.
Once mastered, this technique can be completed in 12-14 minutes per mouse, if it is performed properly. While attempting this procedure, it's important to remember to take the necessary time to properly visualize and access the portal vein prior to attempting the injection. Following this procedure, other methods like intravital imaging following tumor cell injection can be performed to answer additional questions about how tumor cells interact with the lubramicro environment.
With its development, this technique permits researchers in the field of metastasis to explore later stages of the metastatic cascade of various cancers in mice. After watching this video you should have a good understanding of where to make the surgical incision, how to gently move the internal organs to access the portal vein, and how to carry out the intra-portal injection. Don't forget, working with syringes loaded with tumor cells can be hazardous, and that precautions must be taken to avoid needle sticks while performing this procedure.
