So why is this protocol significant? Well, brain metastases is very challenging to treat and this protocol produces a clinically-relevant mouse model with minimal extracranial tumors. This technique allows objective analysis of drug distribution and response from intracranial tumors.
The main advantage of this technique is that the model is highly reproducible and the brain metastases develop in a relatively short time after injection. The disease progression is similar amongst animals, making it easier for researchers to standardize the experiments, treatment timelines, and endpoints. My advice for beginners is to practice using the forcep and syringe while looking through the dissection microscope, getting familiarized with the anatomical landmarks around the neck and practicing the protocol in the presence of a vet.
Begin by seeding BT-474 cancer cells at a seeding density of two times 10 to the six cells into a T-75 flask using 10 milliliters of complete growth media and culture till the cells reach a confluency of 70 to 80%before injection. On the day of injection, discard growth media and wash the cell monolayer with PBS twice and add five milliliters of pre-warmed cell culture dissociation reagent. Incubate at 37 degrees Celsius for five minutes or until cells have detached.
After five minutes, remove the flask from the incubator and gently tap it to aid cell detachment. Add five milliliters of complete growth media containing 10%FBS to quench the dissociation reagent activity. Gently resuspend the cells in the solution by pipetting to reduce cell clumps.
Transfer the cell suspension into a 50-milliliter tube and centrifuge at 180 G for three minutes at room temperature. Decant the supernatant and resuspend the cell pellet in 10 milliliters of HBSSS without calcium and magnesium to minimize cell clumping. Centrifuge the cell suspension at 180 G for three minutes at room temperature Decant the supernatant to remove residual serum or dissociation reagent and resuspend the cell pellet in three milliliters of HBSS.
Place a 100-micrometer cell strainer on a fresh 50-milliliter conical tube and pass the cell suspension through to remove cell clumps. Calculate the number of viable cells using trypan blue and dilute the cell suspension with HBSS to a cell concentration of 2.5 times 10 to the six cells per milliliter. Keep the tube horizontal on ice and gently rock the tube periodically to minimize clumping.
The cell suspension can be stored on ice for a maximum of six hours. Ear-punch mouse for identification and use electric clippers to shave the fur from the neck region. Clean excess hair from the exposed skin using tape.
Apply ocular lubricant to the eyes to prevent drying. Secure the mouse gently by hooking the upper incisor teeth using thread taped to the surgical board, followed by taping the front and hind legs. This step extends the body and keeps the neck straight during the procedure.
For pre-operative skin preparation, wipe the neck with a topical antiseptic such as povidone iodine to reduce skin microflora load and remove loose hair. Clean from the center of the skin, working outward to prevent recontamination of the incision site. Repeat the process using 70%ethanol and perform three alternating rounds of iodine and ethanol for disinfection.
Check for reflex via the pinch test to ensure the anesthesia death before continuing the procedure. Lay a sterile surgical drape over the animal. Under the dissection microscope, make a vertical 15-millimeter incision along the midline at the mouse's neck region using scissors, starting from five millimeters below the jaw to the thoracic inlet.
Using two pairs of angled forceps, part the skin and underlying salivary glands and apply retractors to keep the trachea exposed. Using two pairs of fine angled forceps, bluntly dissect the muscle and fat tissue adjacent to the trachea to expose the right carotid sheath. The carotid sheath is the fibrous layer covering the common carotid artery, vein, and vagus nerve, and this bundle can be visualized by the bright red common carotid artery.
Clear a segment of the common carotid artery caudal to the carotid bifurcation of the surrounding fascia and separate it from the vagus nerve and veins. Isolate and clear the carotid bifurcation from the surrounding nerves and fascia. Position fine forceps under the external carotid artery and pass a 5-0 silk suture under the artery.
Knot and tighten the suture and cut excess line. Position fine forceps under the common carotid artery and pass a 5-0 silk suture under the artery. Tie a knot and tighten the suture at a position proximal to the proposed injection site.
Cut the excess suture, leaving about 10 millimeter of the line. Cut and moisten a strip of low-lint disposable wipers about 10-by-5 millimeters. Fold the strip and place it underneath the carotid artery at the proposed site of injection.
This will support the vessel during the injection. On the common carotid artery rostral to the proposed injection site, place a third ligation with a loose knot. This is tightened only after the injection.
Gently agitate the cell suspension and draw 200 microliters of cell suspension into an insulin syringe with a 31-gauge needle. Load the syringe into the syringe driver connected to an activating foot pedal. Attach a fine cannula with a 31-gauge needle to the syringe and prime the line.
Check whether the carotid artery is well-positioned and pressurized. Using two fine-angled forceps, one gently tensioning onto the end of the first ligature and the other holding the 31-gauge needle, slowly insert the needle with the bevel up into the lumen of the blood vessel, taking care not to puncture it. Slowly inject 100 microliters of cell suspension prepared earlier into the common carotid artery at 10 microliters per second.
This will deliver 2.5 times 10 to the fifth cells into the blood vessel. Successful injection can be visualized via the clearing of blood from the carotid blood vessel. Gently lift and tighten the loose ligature immediately after withdrawing the needle to prevent backflow and bleeding.
Trim excess sutures and remove the piece of moistened low-lint disposable wipers. Using a P-200 pipette, rinse the surgical cavity twice with 150 to 200 microliters of sterile water or saline. After checking for bleeds, reposition the soft tissue, salivary glands, and skin over the carotid artery and trachea.
Close the skin layer of the incision using a suture needle-holder, forceps, and an absorbable or non-absorbable 6-0 monofilament suture in a continuous pattern. Inject 50 micrograms per kilogram of buprenorphine and one milligram per kilogram of meloxicam via subcutaneous injection as post-surgical pain relief. Move the animal to a warm and clean cage to recover from anesthesia.
Lectin was injected into the mice's common carotid artery with or without external carotid artery ligation. A reduction in lectin was observed in the cheek tissues when the external carotid artery was ligated. The results depicted that the ligation did not impact brain delivery.
Tumor progression was monitored using the luciferase-expressing, HER2-amplified breast cancer cell line. The BT474 brain metastasis model showed an increase in the bioluminescent signals from week five post-internal carotid injection and progressively grew thereafter. From weeks five to eight, heterogeneous signal intensity indicated an intracranial tumor with complex fluid perfusion.
Gadolinium concentration within the tumor region increased as it leaked from the blood circulation into tumor tissue. The accumulation of nanomedicine and brain metastases was confirmed in the BT-474 mice by higher nanomedicine levels detected in the tumor region compared to the uninvolved brain regions. As the BT-474 brain metastasis model progressed, animals began to lose up to 20%body weight, making the median survival time nine weeks post-injection.
Histology results depicted that the tumors were located unilaterally, matching the site of carotid injection where the tumors mostly appeared as solid, solitary masses. Pockets of empty spaces were frequently observed and consisted of necrotic cells, while smaller outgrowths were also present in some animals. The most important thing to remember when attempting the protocol is to prepare and position the carotid artery correctly before inserting the needle, and then to apply proper tension during insertion of the needle.
This model is useful for researchers exploring drug delivery to the brain and the efficacy of the new therapeutics for treatment of brain metastasis.
