Three-dimensional in vitro models for assessing temozolomide sensitivity in glioblastoma

IDH-wildtype glioblastoma, or glioblastoma multiforme (GBM), is the most aggressive primary malignancy of the central nervous system. It is characterized by marked invasiveness, intratumoral heterogeneity, and high recurrence rates, largely driven by intrinsic and acquired resistance to standard temozolomide (TMZ) therapy. TMZ is an oral alkylating agent that methylates nitrogenous bases, mainly forming O⁶-methylguanine (O⁶-meG) lesions, the main cytotoxic and mutagenic events responsible for its antitumor effects. This review first characterizes gliomas and the mechanism of action of the main GBM treatment drug (TMZ), then critically examines in vitro cell culture models investigating these mechanisms. This review emphasizes the development and application of three-dimensional (3D) culture systems, including tumor spheroids and organoids, which more effectively recapitulate tumor architecture, cell-cell interactions, and drug penetration gradients than conventional two-dimensional models. This review discusses the strengths and limitations of these models and their translational relevance to modeling TMZ sensitivity and resistance in GBM.