We describe the optimal technique for intracranial injection of GEM-derived glioblastoma cells into an immunocompetent mouse brain resulting in tumors that recapitulate key features of human glioblastoma. This technique for orthotopic injection of GEM-derived TRP cells yields highly reproducible, scalable and consistent results for tumors that originate in the cortex, yet maintain the invasiveness of GEM tumors. Mouse glioblastoma brain tumors display aggressive growth and invade the surrounding brain parenchyma.
Therefore, it can be used to evaluate therapeutics that may suppress tumor infiltration and migration in patients. This protocol can be customized for other brain tumor models, viruses, therapeutics or adapted for injection into different brain regions with the adjustments of stereotaxic coordinates. Demonstrating the procedure will be Devon Atkinson, a research associate from my laboratory.
To begin, place surface protectors under the stereotaxic apparatus and on the work surface. Connect the vinyl tube from the anesthesia machine to the in-port of the gas anesthesia platform of the stereotaxic apparatus, and another tube to its out-port. Connect the digital display to a power source and the apparatus.
Next, attach the micro pump to the manipulator arm of the apparatus after connecting the micro pump controller to the power source. Set the micro pump to 5.6 nanoliters per second. Connect the mouse heating pad to the temperature controller and to a power source.
Back-load a 30-gauge precision syringe with methyl methylcellulose cell mixture by avoiding bubbles. After inserting the plunger, attach the needle, and depress the plunger until the loaded cell mixture drops through the needle. Clean the needle with a 70%alcohol preparation pad.
Once the precision syringe is attached to the micro pump, rotate the manipulator arm away from the stage before placing the mouse to prevent syringe needle displacement. Transfer the anesthetized mouse to the stereotaxic instrument and secure it to the nose cone by placing the upper teeth on the nose cone support. After tightening the nose cone with a knob, pinch a toe to check reflexes to ensure proper anesthesia.
Then insert ear bars and tighten the knob to secure the head. Apply eye ointment to lubricate the eyes under anesthetized conditions. Inject buprenorphine SR analgesic subcutaneously, and place a rectal mouse probe to monitor internal temperature.
Sterilize the surgical field using outward circles, alternating between a surgical scrub and ethanol three times. Pull the taut skin with forceps and use a scalpel blade to make an approximately 1-centimeter-long incision between the eyes. For cell injection, return the manipulator arm with the syringe attached to the mouse.
After tightening the knobs, move the syringe using the X and Y knobs in the horizontal plane and mount it over the bregma. Lower the needle, using the Z knob, to confirm the bregma position, and set the digital readout console to zero. Then move the needle to the desired position, as described in the manuscript, using X and Y knobs and the corresponding digital readout, and use the Z knob to move the needle to the surface of the skull.
Puncture a hole in the skull using a 1-milliliter syringe with an attached 25-gauge needle. Carefully rotate the manipulator arm to the side, while placing the bevel of the needle toward the 30-gauge precision syringe. Once the manipulator arm with the loaded precision syringe needle is positioned over the hole, align the needle tip with the hole, and use the Z knob to lower the needle into the dura of the brain.
After setting the digital readout console to zero, lower the needle 1 millimeter with the Z knob, and wait for one minute. Once the 2 millimeter depth is reached, stop the micro pump, and ensure that the pump stops when 2 microliters of cell suspension has been injected. To remove the needle from the skull, raise it one millimeter, and wait one minute.
For wound closure, using the wooden end of a cotton-tipped applicator, take a piece of bone wax and shape it into a cone. After placing the wax into the opening in the skull, push it into the hole. Use the bead sterilized forceps to melt the remaining wax on the skull.
Once it becomes smooth, add two drops of anesthetic bupivacaine solution into the incision, and pull the edges of the skin together, using forceps. After pulling the skin taut, using one or two wound clips, close the skin, place the mouse in a clean recovery cage on a heating pad in a draft-free area, and closely observe the mouse. Weekly MRI scans showed increasing tumor burden within the brain, and tumor volume measurements.
The MRI tumor volume measurement revealed a lack of tumor growth suppression even after radiation treatment, and the survival of treated mice was not increased. The GBM tumors in syngeneic models appeared to be grown aggressively and breached the skull by the terminal endpoint. If extracranial growth is observed on imaging after cell implantation, this indicates cell leakage during the injection process, and more care should be taken during the withdrawal of the injection needle.
Histopathology of orthotopic TRP GBM tumors confirmed the presence of grade 4 astrocytoma, including recapitulation of the distinctive features in tumors such as pseudopalisading and necrosis. Be precise when setting the zero point at bregma to ensure correct cell injection coordinates. Lowering and raising the needle in the brain must be slow to prevent trauma and backflow.
After the injection of tumor cells, weekly MRI scans may be used to determine baseline tumor size prior to starting treatment and to visualize changing tumor burden within the brain.
