Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

This protocol replicates an intraventricular catheter system used in ongoing clinical trials to test local regional delivery of CAR T-cell therapy directed against central nervous system tumors. The main advantage of this technique is the ability to provide multiple repeated doses of local regional CAR T-cell therapy without performing multiple invasive surgical procedures. This system could be used to investigate different therapeutics in different delivery sites, and thus may provide insight into a myriad of research areas in neuroscience.

To begin, place the anesthetized mouse with shaved head on the operation table and gently open the bottom of the stereotactic arm using a spatula. Insert the cannula with forceps and secure it by tightening the screw on the arm until half to two-thirds of the white plastic portion and five millimeters of the cannula protrude from the bottom of the opening. Insert and secure the mouse's top teeth in the bite bar of the stereotaxic apparatus.

Pull the nose cone forward and tighten it, ensuring the mouse inhales isoflurane. Then, mount the mouse on the warmed stereotaxic apparatus using ear cuffs or ear bars, avoiding excessive pressure. Disinfect the surgical site and make a cut parallel to the skull as described in the manuscript.

Use cotton tip swabs to push the fascia away. Identify the landmarks, bregma and lambda on the skull corresponding to anterior and posterior marks where the cranial plates meet. To create a surface for attaching the acrylic, gently make several slits across the skull using a scalpel.

Using the stereotaxic arm, localize the cannula to the landmark of interest. Raise the cannula tip one to two millimeters above the skull surface and move to the desired coordinates. Using an 18 gauge needle or surgical drill, make two screw holes on the exposed skull away from where the cannula enters, ensuring enough space for the cannula.

Twist the drill through the screw hole until it attaches to the skull. With a flat tip screwdriver, insert and fasten two screws into the holes. Then, gently pull the screw up to ensure it is secured.

For inserting a cannula, create a hole in the skull at the identified coordinates using an 18 gauge needle or surgical drill. Using the stereotactic arm, lower the cannula to the desired DV coordinate. At approximately 0.3 grams of acrylic resin powder and 10 to 15 drops of liquid in a porcelain 12-well plate, load the prepared viscous white-colored material into a one-milliliter syringe.

Coat the skull and fill the spaces around the cannula and screw. Loosen the screw on the stereotactic arm while the cement is flexible. Gently use a spatula to release the cannula from the holder at the bottom opening and retract the stereotactic arm away from the mouse.

Once the cement is dried, insert the dummy cannula into the guide cannula and rotate clockwise to secure it tightly. Once the procedure is complete, return the mouse to its warmed home cage for recovery. To prepare the treatment cannula, insert its top into PKG tubing.

Then, fill the treatment syringe with CAR T-cell suspension and insert it through the other end of the PKG tube, covering the top of the treatment cannula. After anesthetizing the mouse, fix the guide cannula at the base using forceps. Unscrew and remove the dummy cannula, allowing access to the guide cannula.

Infuse the CAR T-cells for one minute and hold the treatment cannula in place for an additional minute. After removing the treatment cannula, screw back a dummy cannula tightly. Then, administer meloxicam subcutaneously for pain control.

The GPC2-directed CAR T-cell therapy induced significant tumor regression in the medulloblastoma and significantly prolonged survival in the thalamic diffuse midline glioma model. When inserting the guide cannula into an intratumoral location, it's important to understand that the dorsal-ventral coordinates may be more superficial than tumor injection in order to account for the dummy and treatment cannula projection lengths.