Isolation and Characterization of Exosome from Cancer Stem Cells-Derived from Glioblastoma Cell Line

Background: Cancer stem cells (CSCs) are a stubborn subpopulation that fuel therapy resistance, tumor recurrence, and metastasis. In glioblastoma multiforme (GBM) — one of the deadliest brain tumors — CSCs make treatment especially challenging. Here, we set out to isolate, characterize, and compare exosomes released by CSCs and non-CSCs in the U87 glioblastoma cell line. Methods: We grew U87 cells in serum-free, growth-factor-rich conditions to encourage tumor sphere formation, isolating CSCs. Flow cytometry confirmed CSC identity with strong CD133 positivity. To test chemo-resistance, we measured temozolomide (TMZ) IC50 values using an MTT assay. We isolated exosomes from CSCs and regular cancer cells (CCs) by ultracentrifugation, analyzed their size with NanoSight, checked their shape via TEM, and confirmed CD81 expression by flow cytometry. Their biochemical makeup was profiled using FTIR and Raman spectroscopy. Results: The isolated CSCs showed strong sphere formation and 96.7% CD133 positivity. They were notably more resistant to TMZ, with a higher IC50 (12.56 ± 0.065 μg/mL) compared to CCs (6.032 ± 0.277 μg/mL). Exosomes from CSCs were slightly smaller (157 ± 12 nm) than those from CCs (193 ± 5 nm) but maintained the classic spherical shape (50–200 nm) under TEM. FTIR and Raman spectroscopy revealed distinct molecular fingerprints between the two exosome types — especially in protein, lipid, and nucleic acid signatures. Conclusion: We successfully isolated CSCs and their exosomes from U87 cells, uncovering clear biochemical and morphological differences between CSC- and CC-derived exosomes. These findings pave the way for targeting CSC exosomes in future glioblastoma therapies.