Flow cytometry protocol for cell death analysis in glioblastoma organoids: A technical note

Area of Science:

• * Cancer Research
• * Molecular Biology
• * Drug Discovery

Background:

• * Tumor organoids preserve patient-specific heterogeneity for drug screening.
• * Flow cytometry is crucial for cell death analysis but lacks validated methods for complex organoids.
• * Glioblastoma organoids (GBOs) are valuable models for studying brain tumors.

Purpose of the Study:

• * To develop and validate a flow cytometry protocol for quantifying cell death in glioblastoma organoids (GBOs).
• * To assess the efficacy of common glioblastoma chemotherapies (Temozolomide and Lomustine) in GBOs.
• * To establish a reliable method for evaluating drug responses in complex tumor models.

Main Methods:

• * Generated human GBOs from patient tumor samples.
• * Treated GBOs with Temozolomide (TMZ) and Lomustine (CCNU).
• * Developed a single-stain flow cytometry protocol using propidium iodide (PI) for cell death analysis after enzymatic and mechanical dissociation.
• * Validated results with Hoechst 33258 staining, LDH release assays, and GBO diameter measurements.

Main Results:

• * The developed flow cytometry protocol accurately quantified cell death in GBOs.
• * Chemotherapy treatment (TMZ and CCNU) induced significant cell death, reaching up to 63% after 288 hours.
• * Lomustine (CCNU) showed a more pronounced effect than Temozolomide (TMZ) at tested concentrations.
• * The protocol demonstrated low variability across replicates and validated findings from other assays.

Conclusions:

• * A robust and scalable flow cytometry protocol for cell death analysis in large, dense glioblastoma organoids was established.
• * This method provides a reliable readout for drug response in physiologically relevant tumor models.
• * The protocol supports the development and evaluation of subtype-specific glioblastoma therapies.