(F-203) Establishing Orthotopic Murine Glioblastoma Model for Evaluating the Focused Ultrasound-Stimulated CAR-T Cell Therapy

Introduction:: Brain tumors in the pediatric population have a poor prognosis and account for the highest mortality rate of all childhood cancers. Of all the pediatric brain tumors, high-grade glioma (HGG) remains the largest challenge, with few treatment options available and an overall five-year survival rate of just 10-20 percent. Current upfront therapies include surgical resection, radiation, therapy, and chemotherapy. Despite aggressive multimodality therapy, long-term survival is poor. Chimeric antigen receptor (CAR) T cells show potential as paradigm-shifting therapeutic agents for cancer treatment by eradicating chemotherapy-resistant cancer cells. However, the phenomenon of "on-target off-tumor toxicity" presents a substantial challenge in CAR-T therapy. Recent advancements in focused ultrasound (FUS) controllable CAR-T cell technology offer the promise of controlled activation within specific tumor regions, thereby mitigating toxicity and its efficacy in treating GBM is need to be evaluated. To more accurately emulate the complex tumor microenvironment, it is crucial to employ an orthotopic model in cancer treatment investigations. Thus, in this study, we established an orthotopic glioblastoma model, which later was intergraded with the FUS system for testing FUS inducible CAR-T cells.


Materials and Methods:: To establish the orthotopic murine glioblastoma model, the mouse was put under anesthesia and positioned on the stereotaxic instrument. Subsequently, we made an incision beginning behind the right eye and ending with a 10 to 15-mm length the skull was cautiously exposed and the bregma was visualized. We targeted and marked a spot 1 mm above and 2 mm away from the bregma. Then, a syringe equipped with a 32-gauge needle was used to load 5 microliters of cells which were injected into 3 mm of the target site. Post-injection, we utilized a suture to close the skin and sterilized the wound site with iodine. The experimental process entailed the intracranial injection of U87 cells into the mouse brain, followed by continuous monitoring of tumor growth using IVIS (In Vivo Imaging System) analysis and histological analysis to validate the growth of the tumor. After tumor establishment, the FUS was applied to the mice to stimulate the mice's brains with a target temperature of 43℃ over the skull.


Results, Conclusions, and Discussions:: After 5 days of injection, mice exhibited detectable signals under the IVIS, confirming successful tumor growth in their brain hemispheres. This validates the establishment of the orthotopic glioblastoma model in mice and supports the effectiveness of the experimental approach. After 16 days of injection, both mice exhibited stronger signals compared to Day 5, indicating increased tumor growth. Using this model, we tested the FUS-mediated mild temperature raise. The stimulation process demonstrated effective performance, as the temperature was gradually increased and successfully maintained at 43℃ throughout the procedure without severe damage to the mouse brain. This successful temperature control ensures the targeted region received the intended treatment without deviation or fluctuation. The ability to sustain the desired temperature is a critical aspect of the experiment, as it allows for accurate and controlled application of the Focused Ultrasound Stimulation (FUS) system. This study has successfully established an orthotopic glioblastoma model in mice. Additionally, the successful integration of this model with the Focused Ultrasound Stimulation (FUS) system was achieved. The future research effort will be made to ensure consistent and reliable results, it is imperative to increase the sample size by including more mice in the experimental cohort. With this platform, we will test the efficacy of the ultrasound inducible CAR T cells targeting GD2, which is commonly expressed in GBM tumors. This platform should pave the way for GBM research and the design of the next generation of remotely controllable CAR-T cells.


Acknowledgements (Optional): :

References (Optional): :