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051. Three-dimensional organoid culture unveils resistance to clinical therapies in adult and pediatric glioblastoma

Authors: Swetha Sundar, MD

Introduction:
Glioblastoma (GBM) is the most common primary brain tumor with a dismal prognosis. The inherent cellular diversity and interactions within tumor microenvironments represent significant challenges to effective treatment. Traditional culture methods such as adherent or sphere cultures may mask such complexities whereas three-dimensional (3D) organoid culture systems derived from patient cancer stem cells (CSCs) can preserve cellular complexity and microenvironments. The objective of this study was to determine whether GBM organoid cultures show increased patterns of resistance to potential clinical therapies compared to traditional sphere cultures.Methods: Adult and pediatric surgical specimens were collected and established as organoids. We created organoid microarrays and visualized bulk and spatial differences in cell proliferation using immunohistochemistry (IHC) staining, and cell cycle analysis by flow cytometry paired with 3D regional labeling. We tested the response of CSCs grown in each culture method to temozolomide, ibrutinib, lomustine, ruxolitinib, and radiotherapy.
Results:
Compared to patient-matched sphere cultures, GBM organoids showed diverse proliferative cell populations and broad resistance to all therapies tested, albeit with both intraspecimen and interspecimen variability in the extent of resistance. Organoids demonstrated a blunt response to current GBM standard-of-care therapy (combination temozolomide and radiotherapy) and maintained both cellular proliferation in their outer rim and overall structure and viability compared to matched sphere specimens.
Conclusion:
Our results suggest that GBM organoids offer a valuable complement to standard GBM sphere culture and may better reflect tumor-like proliferative diversity and therapeutic responses. Therapeutic resistance in organoids appears to be driven by altered biological mechanisms in 3D rather than physical limitations of therapeutic access.