This study allows for flexibility in creating primary bone tumors or bone metastasis by placing solid tumor grafts into bone. This technique is a departure from injecting tumor cell suspensions in the bone. This method overcomes the limitation of tumor cell suspension injections lung emboli.
Another advantage is creating a more uniform model when compared to spontaneous bone metastasis or after intravascular cell injection. Based on solid tumor graft implantation, possibilities exist beyond human or mouse models, but the use of patient-derived xenografts which can lead to therapies tailored to specific patients. The primary focus is for use at cancer research such as tumor microenvironment, metastasis, drug discovery, cancer genetics, and precision medicine.
Visual demonstration is key to showing the key steps of creating the subcutaneous tumor, dissection and creation of the tumor graft, and surgical implantation into the tibia. For subcutaneous tumor induction, harvest the cell line of interest using an appropriate cell dissociation solution and resuspend the cells at a one to two times 10 to the fifth cells to 100 to 150 microliter sterile PBS concentration. Place the cells on ice.
Clean the exposed skin with 70%ethanol, and load the cells into a Tuberculin syringe equipped with a 27 gauge needle. Then inject the cell subcutaneously into a region of disinfected skin that will not be impacted by the movement of the shoulder blades. Use a caliper to monitor the size of the subcutaneous tumor overlying the dorsal thorax or abdomen, and measure the animal's body weight weekly to ensure that the tumor does not ulcerate or that the mouse does not meet early removal criteria as established by the Institutional Animal Care and Use Committee.
When the tumor reaches 15 millimeters in any dimension, use 70%ethanol to sterilize the skin and use a 15 scalpel to incise the skin overlying the tumor. Use sterile surgical scissors to dissect the tumor from the surrounding attached soft tissues sharply and place the tumor in one well of a six-well plate containing complete growth medium. Use the scalpel to mince the tumor into appropriately sized fragments and place the fragments into a new container of sterile complete growth medium at room temperature.
For cryopreservation, pool multiple fragments in a single cryovial in complete growth medium supplemented with 20%fetal bovine serum and 10%dimethyl sulfoxide and use a commercial cryopreservation system at minus 80 degrees Celsius to gradually freeze the tissue before long-term storage in liquid nitrogen. To preserve tumor fragments for subsequent analysis but not future implantation, snap freeze the samples by liquid nitrogen immersion and store the frozen tumor fragments long term at minus 80 degrees Celsius. For subcutaneous tumor fragment implantation, first bring the tumor fragments suspension to room temperature.
Next, confirm a lack of response to pedal reflex in an anesthetized recipient mouse and subcutaneously administer analgesia. To minimize the potential trauma to the skin, use depilating cream to remove the hair on the right knee joint and proximal tibia of the connected hindlimb. Scrub the exposed skin with 70%ethanol, followed by alternating chlorhexidine and saline scrubs.
While flexing and extending the joint, visualize the proximal tibia as the region just distal to the knee joint and use a 15 scalpel blade to create a three to four millimeter incision at the level of the proximal tibia on the medial aspect of the limb through the skin and subcutaneous tissue to expose the medial cortex of the proximal tibia. To create a small hole in the medial cortex of the proximal tibia, use the tip of a 25 gauge needle to apply gentle pressure while rotating the tip approximately two millimeters distal to the knee joint and equidistant between the cranial and caudal tibial cortexes. When the hole has been created, use sterile forceps or a 27 to 30 gauge needle to insert a minimum of 0.5 cubed millimeter tumor fragments into the medullary cavity of the proximal tibia and use a 27 to 30 gauge needle to manipulate the tumor fragments into the medullary canal.
After placing all the fragments, oppose the skin edges with the appropriate material and return the animal to its cage with monitoring until ambulatory. Then evaluate the tibial tumor growth noninvasively by weekly digital radiography, bioluminescence, or fluorescence imaging, and use calipers to measure the limb at the implantation site. Successful engraftment can be documented with advanced imaging.
In this representative analysis, the cortical defect created for tumor graft implantation was visible in the proximal tibia one week after implantation. By week two, visible osteolysis and bone remodeling adjacent to the cortical defect could be observed. From weeks two to five, additional progressive bone destruction and the formation of new bone associated with tumor engraftment and growth occurred.
Care should be taken while creating a small hole in the proximal tibia. Rely on rotating the tip with gentle to minimal pressure allowing the needle tip to do the work. Following this procedure, tumor graft placement in other bone sites, placement of organoids or tumor cells in the extracellular matrix into the bone defect or molecular and histologic analysis in addition to imaging can be performed.
