Heterogenous Model of Temozolomide Resistance in Glioblastoma Reveals Phenotypic Shifts in Drug Response and Migratory Potential

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, with limited survival outcomes due to tumor recurrence, mainly driven by GBM cell invasion and therapy resistance. Although temozolomide (TMZ) remains the standard‐of‐care chemotherapeutic, its long‐term efficacy is often compromised by rapid emergence of acquired resistance, largely mediated by the DNA repair enzyme, methylguanine methyltransferase (MGMT). To investigate the interplay between tumor heterogeneity, drug resistance, and the extracellular matrix (ECM) microenvironment, we adapted a 3D methacrylamide‐functionalized gelatin (GelMA) hydrogel model to study the behavior of mixed populations of TMZ‐sensitive and TMZ‐resistant GBM cells. Using both single‐cell distributions and multicellular spheroids, we report the impact of heterogeneous cell populations and TMZ dosing regimens, including physiological, supraphysiological, and metronomic TMZ schedules, on drug response and migration. We show that the combination therapy of TMZ with an MGMT inhibitor, lomeguatrib, can modulate TMZ resistance in vitro. This hydrogel model enables systematic investigation of GBM heterogeneity, “go‐or‐grow” phenotypic plasticity, and therapeutic resistance in an ECM‐rich microenvironment, offering a valuable platform for future translational research.