An Orthotopic Xenograft Model for Preclinical Evaluation of CAR T Cell Therapy in Glioblastoma

Glioblastoma (GBM) is the most lethal primary brain tumor in adults, and despite intensive research efforts, effective therapeutic breakthroughs remain elusive. Consequently, the standard of care has remained largely unchanged for nearly 30 years, underscoring the need for robust preclinical models to evaluate experimental therapies. One particularly promising approach is chimeric antigen receptor (CAR) T cell therapy, which has demonstrated remarkable success in several hematologic malignancies but has not yet achieved comparable efficacy in solid tumors. This article describes the generation of orthotopic GBM xenografts in NSG mice, including anesthesia, surgical procedures, and postoperative care, followed by locoregional delivery of human CAR T cells into the lateral ventricle and subsequent assessment of therapeutic efficacy. Together, these protocols provide a reliable platform for evaluating GBM-directed CAR T cell therapies in both basic and translational research. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Intracranial tumor implantation Basic Protocol 2: Intraventricular CAR T cell administration Support Protocol 1: Bioluminescence imaging of xenografts Support Protocol 2: Endpoint analysis of residual tumor burden by flow cytometry.