<em>In Vivo</em> Glioblastoma Tumor Modeling via Stereotaxic Injection in Mice for Tumor Progression Studies

Glioblastoma (GB) is a highly aggressive brain cancer with a median survival of under 2 years despite conventional therapy. Research efforts frequently use murine in vivo models to study cancer progression and tumor growth, as these models recapitulate critical aspects of the tumor microenvironment, immune responses, and the appearance of classical GB pathology. Here, we describe a detailed protocol for performing stereotaxic injections to establish GB tumor models in immunocompetent C57BL/6 mice. Stereotaxic surgery enables rapid and precise delivery of GB cells grown as 2D cultures or spheroids into the brain using anatomical landmarks without the need for image guidance. Specifically, GL261 murine cell lines are injected into the striatum, allowing subsequent assessment of behavior and physiology. On day 21 post surgery, animals are euthanized following perfusion and their brains extracted and fixed. Hematoxylin-eosin staining on brain slices enables measurement of tumor area and volume, while immunohistochemistry enables imaging of tumor proliferation markers. This paper provides detailed protocols for a reliable approach to generate in vivo GB models for studying tumor growth and cancer progression. Additionally, this method is adaptable for other GB cell lines, transgenic mice, or patient-derived xenografts broadening its applicability for GB research.