An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

This VX2 endometrial cancer model with retroperitoneal lymph node metastasis can be used to study technology for image-guided surgery. The main advantages of this technique are that it is reliable, simple, and that it create consistent metastatic spread, which mimics the pattern of human endometrial cancer. Overall, this is a simple technique.

However, practicing the suturing prior to attempting to create the model will help to make the process easier. Visual demonstration of this technique is important, as it helps to demonstrate the surgical steps involved in model establishment. Demonstrating the procedure with Harley Chan and I will be Lili Ding, a technician from our laboratory.

To prepare in vitro-propagated VX2 cells for injection, begin by warming frozen vials of VX2 cells in a 37 degrees Celsius bead or water bath for one minute. When the cells have thawed, pull the cell suspensions in a 15-milliliter tube containing 10 milliliters of culture medium. Sediment the cells by centrifugation.

Resuspend the pellet in nine milliliters of medium. Transfer the cells to a large culture flask for incubation at 37 degrees Celsius without shaking, checking the cells daily for confluence by light microscopy. When the cells have reached 80%confluency, add three milliliters of 0.2%trypsin to the flask and return the cells to the cell culture incubator for five minutes.

When the cells have detached, transfer the cell suspension to a conical tube to collect the cells by centrifugation. Wash the cell pellet three times with nine milliliters of fresh PBS per wash. After the last wash, resuspend the cells in fresh PBS for counting before diluting to a four times 10 to the seven VX2 cells per milliliter of PBS concentration in a new conical tube on ice.

To prepare in vivo-propagated VX2 cells for injection, thaw frozen one by one-centimeter VX2 tumor blocks and use a scalpel to mince the tissue samples in a culture plate. Transfer the tissue fragments to a 70-micrometer filter and add small aliquots of up to one milliliter of Hank's balanced salt solution, or HBSS, to ensure that all of the cells are passed through the strainer. Then count the cells before diluting the cell suspension to a one times 10 to the seven VX2 cells per milliliter of HBSS concentration in a sterile tube on ice.

After confirming a lack of response to stimulus, place the anesthetized female white New Zealand rabbit in the dorsal position on the operating table. Clip the hair over the pelvis and abdomen, and use a three step surgical skin prep to clean the exposed skin. Wearing a surgical cap and face mask, scrub the hands with disinfectant before putting on a sterile gown and sterile surgical gloves.

Landmark the top of the pubic bone and drape the surgical field with laparotomy drapes, leaving a five by five-centimeter area of the lower abdomen exposed. Next, use a number 11 scalpel blade to make a 2.5-centimeter incision one centimeter cranial to the symphysis pubis through skin and subcutaneous tissue. Incise the rectus fascia and dissect the rectus muscles laterally to expose the underlying peritoneum.

Confirm that the undersurface is clear of bowel or other abdominal organs. Sharply enter the peritoneum to identify the urinary bladder and sweep a gloved finger superiorly, posteriorly, and laterally over the apex of the bladder to locate the uterine horns. Extract the uterine horns through the abdominal incision to rest on the abdominal wall.

Use a 3-0 braided absorbable suture to perform a single suture ligation of each uterine horn, approximately 1.5 to two centimeters distal to the cervices and just medial to the uterine arteries. Then tie the sutures snugly to include the distal uterine horns. To inoculate the myometrium with the previously prepared VX2 cell suspension, load a one-milliliter syringe equipped with a 27-gauge needle with one milliliter of cells.

Slowly inject 0.5 milliliters of the cell suspension over one minute into each uterine horn proximal to the suture site between the suture and the cervix. Then apply pressure to the myometrial injection site for 30 seconds to minimize cell leakage. Inspect the injection and suture sites for hemostasis before placing the uterine horns back into the abdomen.

For deep abdominal wall closure, identify the apex of the peritoneal incision and use a surgical clamp to grasp the peritoneum rectus muscle and fascia. Anchor the suture by placing a 3-0 absorbable polyfilament suture, superficial to deep on one apex of the incision, and deep to superficial on the other. Using the attached suture, make running stitches perpendicular to the incision through the layers of the abdominal wall, working step-wise along the incision from side to side before tying off the suture and cutting the loose ends.

For superficial abdominal wall closure, use a buried running subcuticular 3-0 absorbable polyfilament suture to suture deep to superficial on one side of the incision and superficial to deep on the other. After knotting the sutures, use the anchored sutures to make running stitches in the dermal layer parallel to the incision, working step-wise along the incision from side to side. Tie the sutures and remove the loose ends.

Apply surgical glue to the closed incision to appose the skin edges. Then, place the animal on a warm blanket with oxygen and monitoring, until the animal is alert and able to sit independently. When establishing this model, a dose of two times 10 to the seven cells per uterine horn was successful in four rabbits, in an average time of 45 days from inoculation to experiment and was thus determined as the optimal injection dose for the cultured VX2 model.

For the in vivo-propagated VX2 model, which was successfully created in 16 rabbits with an average time from inoculation to experiment of 29 days, five times 10 to the six cells per uterine horn was determined as the optimal injection dose. All of the models resulted in a successful metastatic transformation of the retroperitoneal lymph nodes. Further, tumors and metastatic lymph nodes from cultured VX2 cells appeared similar to tumors from in vivo-propagated VX2 cells by histological analysis, with densymetoxolin-stained cells observed in the invading muscle and forming glandular like structures with many pathological mytotic figures.

Place your suture medial to the uterine artery to avoid bleeding from a vessel puncture and take care to infiltrate the cells into the myometrium and not the endometrial cavity. Take care to use proper sterile technique during the model creation to prevent post-operative infection and to protect yourself with the barium material during the surgery.