Triple-Survival Stereotactic Brain Surgeries for the Intracranial Injections of Glioblastoma Stem-like Cells and Oncolytic Herpes Simplex Viruses

Glioblastoma (GBM) is an aggressive primary brain tumor associated with poor prognosis and resistance to therapy, underscoring the need for reliable preclinical models to evaluate emerging treatments. The orthotopic implantation of GBM stem-like cells (GSCs), combined with the intratumoral delivery of therapeutic agents, represents a widely used approach for modeling GBM tumor growth and studying treatment response. In particular, oncolytic herpes simplex viruses (oHSVs) have emerged as a promising strategy to selectively target malignant cells while inducing antitumor immune responses with minimal systemic toxicity. However, performing repeated survival stereotactic neurosurgeries in the same animal poses significant technical challenges. Here, we describe a comprehensive and reproducible protocol for triple survival stereotactic neurosurgery in mice. This approach involves (i) the intracranial implantation of GSCs to establish orthotopic tumors, (ii) the intratumoral delivery of oHSV using the same stereotactic coordinates, and (iii) contralateral intracranial rechallenge with GSCs to evaluate therapeutic efficacy and resistance to tumor rechallenge as a measure of immune memory. Using this protocol, consistent tumor establishment was achieved, and mice tolerated repeated neurosurgical procedures with stable postoperative recovery. Successful intracranial rechallenge in the same animal demonstrates the technical feasibility of multiple survival surgeries while minimizing procedure-related variability and complications. This method enables longitudinal assessment of tumor progression, therapeutic response, and durable memory protection within a single subject. Furthermore, this protocol provides a versatile platform for evaluating oncolytic virotherapy and other localized treatment strategies for GBM.