The overall goal of this procedure is to establish experimental bone metastasis by delivering cancer cells directly to hind-limb tissues, including the bones, via intra-iliac artery injection. So this method can help us answer the key questions about breast cancer bone colonization, especially in the early phase of this process, how breast cancer cells interact with microenvironment cells in the bone. We did iliac artery injection today.

The main advantage of this technique is that it efficiently and selectively establish an experimental microscopic bone metastatis model for various cancer types without causing local tissue damage. Generally, individuals new to this method will struggle because finding an injection that's accurate, iliac artery injection position on the microscope may take practice to achieve higher than ninety percent injection success rate. Begin by confirming the appropriate level of sedation by toe pinch of a five to six week old twenty gram mouse.

After applying eye ointment, wipe the shaved right groin area several times with sequential, sterile, 70%isopropyl ethanol-soaked cotton swabs, followed by a betadine surgical scrub. Next, cover all of the mouse with a sterile drape. Then, use a sterile number ten carbon steel scalpel blade and a number three handle to make a one to 1.2 centimeter skin incision between the fourth and fifth nipples in the lower right abdomen.

And transfer the animal under a standard benchtop dissection microscope. Under the 4X objective, insert blunt separation forceps into the incision between the fatty tissue and the peritoneum, and push the tissues outwards on both sides to reveal the iliac vessels and nerves. Next, use straight, fine forceps to break through the connective tissue between the vessels and the nerve closest to the vessels.

Then, slide angled forceps through the connective tissue and beneath the iliac vessels, while inserting the straight forceps into the connective tissues on the other side of the vessels to guide the angled forceps through the tissue. We recommend separating and lifting the ilial rim and artery as a bundle for this step. Although the tumor cells are injected only into the artery, it saves time and effort to isolate both vessels together.

Once the angled forceps are in place, further separate the iliac vein and artery from the surrounding tissue, keeping the vessels on top of the angled forceps during the dissection. When the vessels are clear, use the straight forceps to deliver the tip of a 4-0 silk suture to the angled forceps and pull the suture slowly and gently beneath the vessels. Both of the iliac common vein and artery vessels should then be held up by the suture.

When the vessels are ready, pipette the tumor cells up and down several times to re-suspend them into a single cell solution and to break up any clumps. Then, aspirate 100 microliters of cells per mouse into a 31 gauge insulin syringe. Now, slide the angled forceps back under the vessels using the suture as a guide, and open the forceps so that the vessels rest gently on the tips.

With the other hand, insert the needle tip bevel-side up into the lumen of the section of the artery between the forceps tips. Depress the plunger slowly to inject the cells. The cell suspension will flush the blood through the vessel.

When all of the cells have been delivered, gently remove the needle, keeping the vessels raised on the forceps to prevent bleeding. Then, pull away the suture. Finally, quickly remove the forceps and press a cotton swab onto the incision for five to 10 minutes to stop the bleeding.

Here, a representative whole-animal bioluminescent image after intra-iliac artery injection of five times 10 to the fifth GFP luciferin-labeled MCF-7 breast cancer cells followed by d-luciferin administration by retro-orbital sinus injection is shown. At day 14 post injection, a strong bioluminescence signal was obtained from the intra-iliac artery injected hind bone, but not from the contralateral control bone, confirming the specific localization of the injected cells. Sectioning an H&E staining of the tumor-bearing bones also demonstrated a compact, cobblestone-like cell population with large nuclei indicative of microscopic MCF-7 metastatic lesions in the bone tissue 14 days after intra-iliac artery injection.

Further, immunoflourescent staining of the bone sections also revealed a co-mingling of the MCF-7 cells with osteoblasts and preosteoblasts. Once mastered, this technique can be completed in ten to fifteen minutes. Following these procedures, you can use other methods to answer additional questions.

For instance, we can use RNA-Seq to profile the microenvironment cells in the metastasis niche and compare them to normal cells. This technique provides one way for breast cancer researchers and prostate cancer researchers to study bone micrometastasis in animals. After watching this video, you should have a good understanding of how to establish a breast cancer bone metastasis model by intra-iliac artery injection.

Please take appropriate precautions because working with ketamine and xylazine could be extremely hazardous.