C3PO: complex 3D patient organoids for preclinical drug testing in brain tumors

Background . Glioblastoma (GBM) remains one of the most aggressive and therapeutically challenging brain tumors, with current treatments offering limited survival benefits. One of the major obstacles in developing effective therapies is the lack of physiologically relevant preclinical models that can accurately recapitulate the structural, molecular, and cellular complexity of the human tumor microenvironment. Conventional 2D cultures and animal models often fail to predict clinical efficacy and are associated with high variability and limited translational value. Methods . To overcome these limitations, we have developed advanced ex vivo 3D glioblastoma models, termed Complex 3D Patient Organoids (C3POs), designed to reproduce the architecture, multicellular composition, and microenvironmental gradients of GBM. Each C3PO integrates patient-derived glioma stem-like cells (PD-GSCs) with astrocytes and microglia in defined ratios, generating self-assembled spheroids that mimic the tumor-stroma interactions observed in vivo . To ensure biological relevance, all patient-derived GSCs were also profiled for key genomic alterations commonly associated with GBM. Distinct models representing different molecular GBM subtypes were established and characterized using high-content imaging and fluorescent tracking of individual cell populations. These models have been used to evaluate both proprietary compounds and clinically used agents such as temozolomide, under conditions that more faithfully reproduce the pathophysiological features of GBM. Results . Preliminary findings revealed subtype-specific responses to treatment, with C3POs of mesenchymal background displaying higher drug resistance and C3POs of proneural background showing higher sensitivity, consistent with a subtype-specific clinical outcome. Furthermore, 3D co-culture systems exhibited reduced drug sensitivity compared to monocultures, underscoring their higher biological fidelity. Current studies are focused on correlating in vitro efficacy and pharmacokinetic (PK) data from C3PO models with results previously obtained in orthotopic GBM mouse models, to quantitatively assess their predictive potential. Conclusions . The C3PO platform provides a robust and scalable tool for ex vivo pharmacological testing of anti-GBM agents. By improving translational predictivity and reducing reliance on animal models, these complex 3D systems represent a promising advancement toward more ethical, cost-effective, and clinically relevant preclinical strategies for the development of new therapies targeting brain tumors.