The overall goal of this procedure is to directly test the effect of tumor hypoxia on metastasis using an animal model. This method can help to identify the mechanism underlying the prometastatic effects of hypoxia and potential novel therapeutic targets for preventing tumor dissemination. Two advantages of this technique are the ability to test the effects of tumor hypoxia in vivo, and to recapitulate the steps of metastasis and cell interactions that occur in cancer patients.

In our model, tumor hypoxia is created by ligation of the femoral artery supplying blood to the tumor-bearing hilum of the mouse. Subsequent hind limb amputation prevents the mobility associated with repeat primary tumor growth, allowing for metastasis to form. Demonstrating the procedure will be Jason Tilan, Sung-Kyeok Hung, and Dr.Olga Rodriguez, co-director of the Preclinical Imaging Research Laboratory here at Georgetown.

To inject the Ewing sarcoma cells, gently restrain a four to six week old female SCID Beige mouse, stabilizing the left leg between the fourth and fifth fingers to expose the medial side of the lower hind limb. Insert a 28 gauge 1/2"inch needle at an approximately 30-45 degree angle anteriorly into the gastrocnemius muscle toward the tibial crest. When the needle tip touches the crest, withdraw the needle slightly and slowly inject 0.1 milliliters of the Ewing sarcoma cells while continuing to withdraw the needle.

Then use digital calipers to measure the calf size daily via the medial/lateral and anterior/posterior lengths until the tumor size reaches the desired volume. Before beginning the ligation, subcutaneously inject an analgesic agent followed by the administration of Hypoxyprobe-1 for hypoxia confirmation. Next, place the anesthetized mouse on a warming pad on the operating surface and apply ointment to the animal's eyes.

Then, extend and secure the hind limb with a piece of tape approximately 45 degrees from the midline. Depilate the surgical area with hair-removal cream removing the cream with an ethanol prep pad after 10 seconds and disinfect the exposed skin with three consecutive 10%povidone-iodine and ethanol wipes. Transfer the mouse under a stereo microscope and make a 1 centimeter incision in the skin from the mid-thigh towards the inguinal region.

Using saline-moistened fine cotton swabs, gently brush away the subcutaneous fat tissue surrounding the thigh muscle. Then carefully blunt dissect the subcutaneous fat tissue to reveal the underlying femoral artery. Stabilize the wound and surgical field to expose the vasculature of the mid-upper adductor muscle and use fine forceps to gently pierce through the membranous femoral sheath to expose the neurovascular bundle.

Using a clean set of fine forceps, dissect and separate the femoral artery from the femoral vein and nerve at the proximal location near the groin, distal to the inguinal ligament. Then, pass a 6-0 silk suture under the femoral artery distal to the brach of the lateral circumflex femoral artery, and occlude the femoral artery with double knots. Close the incision with 6-0 polypropylene sutures and subcutaneously inject 0.5 milliliters of warm saline for fluid balance therapy.

Then place the animal on top of a draped warm pad in the recovery cage with monitoring until fully recovered. At the appropriate experimental time point, gently restrain the animal and use clippers to shave the hair from the tumor-bearing limb from the distal tibia to the pelvic region. Inject the analgesic agent and place the anesthetized animal in the right lateral recumbent position on a sterile drape placed on a warming pad.

Apply ointment to the eyes and disinfect the surgical site as just demonstrated. Then, place a sterile drape over the animal and make a middle femoral circumferential skin incision followed by blunt dissection and proximal retraction of the skin. Expose the medial femoral neurovascular pedicle on the median side of the leg.

Then use a 4-0 polyglactin 910 coated absorbable suture to ligate near the inguinal ligament. Using scissors, perform a mid-femoral transection of the muscle followed by blunt dissection of the soft tissue to the coxofemoral joint. Using a bone-cutter, perform a mid-femoral osteotomy followed by gentle pressure at the site of osteotomy with a absorbable gelatin sponge to minimize the bleeding.

Then use surgical wound clips to close the overlying skin and subcutaneously inject 0.5 milliliters of warm saline for fluid balance therapy. At least twice a week, carefully palpate the head, neck, and axillary regions and contralateral hind limb of the tumor-injected animals. Check for internal organ metastases via abdominal distension and for the presence of lung metastases via gentle xiphoid process pressure with the index finger.

To evaluate the internal organ metastasis by MRI at the appropriate experimental time points, turn on the pump connected to a warm water circulating system to maintain the mouse at a physiological body temperature. Then, place the respiratory sensor in contact with the upper abdomen of the animal and turn on the BIOPAC Systems respiratory monitoring equipment. Place the anesthetized mouse onto a custom-designed mouse stereotaxic holder and apply ophthalmic ointment to the animal's eyes.

To reduce the motion artifacts and noise, select the gating option in the MRI protocol to synchronize the data collection with the respiratory cycle. Then, run a two dimensional T2-weighted anatomical imaging sequence to image the metastases. For primary tumor cell culture after amputation, incision, or necropsy, harvest two to three 2-3 millimeter segments of wet lustrous pink-red viable tumor tissue, from each tissue sample of interest and place the isolated segments in 6 centimeter cell culture plates containing fresh primary culture medium.

Then, transfer the tissues to a cell culture incubator for the appropriate cellular outgrowth period. Following injection of the Ewing sarcoma cells into the gastrocnemius muscle, the primary tumors are allowed to grow to a calf size of 250 cubic millimeters. Control tumors at this volume exhibit a relatively low level of endogenous hypoxia solely in the cells distant from the vasculature as determined by Hypoxyprobe-1 staining.

In contrast, femoral artery ligation induces profound hypoxia in the vast majority of the tumor cells, even those proximal to the blood vessels. The effectiveness of the femoral artery ligation is supported by the complete inhibition of the primary tumor growth over the three-day period between the femoral artery ligation and the amputation. Histopathological analysis reveals extensive areas of necrosis in femoral artery ligation treated primary tumors harvested three days post-surgery.

Nevertheless, there are groups of viable tumor cells present within the tumor tissue located at the edges of the tumor close to the vasculature. These remaining viable tumor cells are highly invasive as evidenced by their massive intravasation. Many cells within the vessel lumen are Hypoxyprobe-1 positive, indicating that cells that are hypoxic post femoral artery ligation may have initiated angioinvasion.

In these experiments, hypoxia significantly increased the frequency and multiplicity of the metastases, often observed in locations not affected in mice bearing control tumors. Once mastered, each surgical procedure, including preparation, anesthesia, and recovery can be completed in 30 minutes per animal. The length of the entire experiment depends on the cell line used and its rates of growth and metastasis.

The MRI and euthanasia timelines vary according to the latency of the metastases, and must be established for each individual cell line in a pilot experiment. While attempting this procedure, it's important to remember to maintain the appropriate pain management and animal monitoring post-procedure. If the procedure is performed perfectly, no severe adverse effect or death related to the surgery should be observed.

Following this procedure, tissues and cells from control and hypoxic primary tumors as well as from their corresponding metastasis can be subjected to further analysis, including global gene expression profiling for identifying the mechanisms of hypoxia-induced metastasis. This technique can be used for the researchers in the field of cancer metastasis for directly testing the effects of tumor hypoxia on disease dissemination. Although here we focused on Ewing sarcoma, a similar approach can be applied to other tumors.

After watching this video, you should have a very good understanding of how to create an animal model of tumor hypoxia and how to monitor the resulting metastases both in vivo and ex vivo.