Description of tibial tumor implantation
Six-eight-week-old mice are anesthetized with isoflurane. Murine tumor cells (e.g. K7M2 (ATCC)) are implanted intratibially into female BALB/C mice and human tumor cells (e.g. HOS 143B (ATCC)) are implanted into female NOD SCID mice. Aseptic techniques will be followed as per the APLAC Guidelines for Rodent Survival Surgery. In brief, the surgical area is cleaned and disinfected with soap and water followed by a disinfectant (Virkon S). Surgical instruments are autoclaved followed by hot bead sterilization between surgeries of different mice. The surgeon wears a clean lab coat, and hands are cleaned with soap and water. Sterile gloves are donned and cleaned between animals with Virkon S. Under anesthesia (1-3% Isoflurane), surgery will be performed under aseptic conditions. Ophthalmic ointment is applied to the eyes to prevent drying of the cornea. The surgical site is shaved and cleaned with Betadine. The surgical site is prepped with three alternating wipes of betadine and alcohol. The right knee will be flexed, and a 30G1/2 needle will be inserted into the proximal end of tibia followed by injection of 10 μL of cell suspension. The cell suspension contains 1 × 106 tumor cells in RPMI medium. The contralateral tibia can be injected with PBS alone to serve as a control. The animals will be maintained on a heating pad during and after the operation (37°C), and monitored until they ambulate. Please note that during recovery from anesthesia, the cage will be half-on and half-off the heating pad to maintain the desired temperature. To prevent pain from the procedure, mice are treated with buprenorphine SR (0.5-1 mg/kg SC, lasts 48-72 hours) pre-op and Carprofen post-op and every 24 hours for 3 days. Developing tumors can be palpated under the skin of the proximal tibia. The tumor size is measured every day with a caliper. Once tumors are approximately 5 mm in size (approximately after two weeks of tumor cell implantation), they can be referred to imaging studies and/or therapy. Note that proper tumor implantation should not lead to any impairment in ambulation of the affected leg.
MRI scans of osteosarcomas in mouse models
Animal Preparation: The investigator wears a clean lab coat and sterile gloves. Hands are cleaned with soap and water before and after every procedure. Sterile gloves are donned, and hard surfaces are cleaned between different animals using Virkon S. The whole procedure is done under inhalational anesthesia with 1-3% isoflurane anesthetic. Parameters monitored during anesthesia include skin color of the ears or toes as an indicator for oxygenation, respiratory rate, and depth of anesthesia. Ophthalmic ointment is applied to the eyes to prevent drying of the cornea. The tail of the animal is scrubbed 2 X with betadine/70%ethanol scrubs, then placed on a heating pad to dilate the tail veins for easier injection. After successful dilation of the tail veins, an intravenous needle is placed. MR contrast agent is injected slowly with simultaneous monitoring of the respiratory rate. For imaging of tumor associated macrophages, we administer a dose of iron oxide nanoparticles (Ferumoxytol) of 30mg Fe/kg. After the procedure, the injection area is cleaned with 70% ethanol scrubs.
Procedural Steps for MRI:
- Anesthetize mouse per regimen above; fill anesthesia humidifier vessel with deionized water; place ophthalmic ointment in both eyes.
- Position mouse for imaging on an MRI-compatible temperature probe. Place an appropriate surface coil around the anatomical area of interest, in our case the leg with osteosarcoma.
- Turn on warm air blower; monitor proper anesthetic depth by regular respiratory rate.
- Apply appropriate pulse sequences before and/or after contrast media injection. Check acquired images for quality and completeness. Carefully check animal vital signs. Length of scan should not exceed 180 min; monitor respiration throughout procedure.
Post-Procedural Monitoring:
After scanning, place mouse in a warm recovery cage (e.g., paper-lined cage on top of heating blanket), check and record vital signs every 15 min, and observe until fully conscious and ambulatory.
During recovery from the anesthesia, the mouse is placed on a clean paper towel (and not directly on the bedding) to avoid aspiration of the bedding.