Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

The technique focuses on modeling patient mutation profiles of glioblastoma in the immunocompetent mouse model and how these different mutations are influencing the tumor microenvironment and response to treatment. Genomic and single-cell sequencing identify that the genetic driver mutations of glioblastoma are associated with the aggressiveness of cancer and components of the tumor microenvironment such as the immune cell population. Glioblastoma mouse models in preclinical trials show great success with immunotherapy, leading to curative results and no sign of tumor growth after treatment.

However, these effects are not reflected in patients for outcome or survival. This protocol recapitulates patient tumor mutation profiles of glioblastoma in the immunocompetent mouse model, allowing gradual autochthonous tumor growth and better prediction of treatment efficacy, especially with immunotherapy. This modeling system integrates DNA plasmids into the genome through electroporation of immunocompetent mice.

It avoids the use of viruses and transplanting thousands of tumor cells, both of which can have their own immune effects, confounding downstream results. Utilizing an immunocompetent mouse model that recapitulates patient mutation profiles allows for a more accurate assessment of treatment outcomes, preventing the laborious task of taking false positive results to the clinic. To begin, place the anesthetized mouse pup on the table in the ventral position.

Pull the back of the pup's head slightly to visualize the cranial skull sutures through the skin. Find lambda on the skull. The ventricle or injection site will be visible midway between lambda and the eye.

At a perpendicular angle, pierce the skin with a glass pipette tip. An initial resistance and slight concave indentation will be observed. Once pierced, hold the tip in place and press the microinjector foot pedal once to inject one microliter of the plasmid mix.

Begin electroporation-based DNA plasmid delivery by placing a square piece of paraffin film on the table and squeezing the electrode gel on top of the film. Then apply a generous amount of the electrode gel on each paddle to cover the electrode paddles completely. Next, hold the pup microinjected with the plasmid DNA mix in the non-dominant hand, keeping the fingers behind the head.

To position the pup's head for electroporation, gently slide a finger on the next dorsal side posterior to the head, and position the positive electrode outside the head where the ventricle is targeted. Place the electrode over the pup's eye and lightly squeeze the electrode paddles on the rostral part of the pup's head. Press the electrode foot pedal to start the electroporation.

With each pulse, rotate the electrode paddles slightly in a counterclockwise direction so the positive paddle moves towards the top of the head. After the final pulse, remove the paddles and place them aside. Mice were moribund by five months post-electroporation with full tumor growth detected through the reporter protein and histological analysis.