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Summary: Development of spatial-integrative pre-clinical models is needed for glioblastoma, which are heterogenous tumors with poor prognosis. Here, we present an optimized protocol to generate three-dimensional ex vivo explant slice glioma model from orthotopic tumors, genetically engineered mouse models, and fresh patient-derived specimens. We describe a step-by-step workflow for tissue acquisition, dissection, and sectioning of 300-μm tumor slices maintaining cell viability. The explant slice model allows the integration of confocal time-lapse imaging with spatial analysis for studying migration, invasion, and tumor microenvironment, making it a valuable platform for testing effective treatment modalities.For complete details on the use and execution of this protocol, please refer to Comba et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a mass cytometry protocol optimized to examine the phenotype of immune cells within the mouse glioma microenvironment, using a Sleeping Beauty transposon-mediated mouse glioma model. We describe antibody conjugation and titrations for analysis of immune cells. We then detail mouse brain tumor tissue collection and processing, staining, followed by data acquisition, analysis, and gating strategy. This protocol can be applied to any brain tumor-harboring mouse model.For complete details on the use and execution of this protocol, please refer to Alghamri et al. (2021).

Summary: Brainstem gliomas are aggressive tumors that are more prevalent in pediatric patients. The location of these tumors makes them inoperable, and currently there is no effective treatment. Recent genomic data revealed the unique biology of these tumors. The following protocol provides a method to incorporate these specific genetic lesions in a mouse glioma model. Using this model, the effects of these mutations in tumor progression and response to treatments can be studied within a relevant in vivo context.For complete details on the use and execution of this protocol, please refer to Mendez et al. (2020).

Summary: Malignant gliomas are the most common and aggressive primary brain tumor in adults, and high mitotic rates are associated with their malignancy. Gliomas were modeled in mice using the Sleeping Beauty system to encode genetic lesions recapitulating the human disease. The presented workflow allows the study of the proliferation of glioma cells in vivo, enabling the identification of different phases of the cell cycle, with the advantage that 5-ethynyl-2′-deoxyuridine staining does not involve denaturation steps and samples do not require histological processing.For complete details on the use and execution of this protocol, please refer to Núñez et al. (2019).