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Updated: Jun 22, 2026
Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion
Published on: August 29, 2018
Glioblastoma stem cells resistant to temozolomide-induced autophagy
Jun Fu1, Zhi-gang Liu, Xiao-mei Liu
1State Key Laboratory for Cancer Research in Southern China and Department of Neurosurgery/Neuro-oncology, Cancer Center, Sun Yat-sen University, Guangzhou, Guangdong 510060, China.
Background:
Recent studies have demonstrated the existence of a small fraction of cells with features of primitive neural progenitor cells and tumor-initiating function in brain tumors. These cells might represent primary therapeutic target for complete eradication of the tumors. This study aimed to determine the resistant phenotype of glioblastoma stem cells (GSCs) to temozolomide (TMZ) and to explore the possible molecular mechanisms underlying TMZ resistance.
Methods:
Freshly resected glioblastoma specimen was collected and magnetic isolation of GSCs was carried out using the Miltenyi Biotec CD133 Cell Isolation kit. The cytotoxic effect of TMZ on CD133(+) and CD133(-) glioblastoma cells was determined by using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Autophagy-related proteins (Beclin-1, LC3 and Atg5) and cleaved caspase-3 (p17) were analyzed by Western blotting. Immunofluorescent staining was used to detect Atg5, glial fibrillary acidic protein (GFAP) and CD133 expression in glioblastoma cells. Statistical analysis was carried out using SPSS 10.0 software. For all tests, the level of statistical significance was set at P < 0.05.
Results:
CD133(+) glioblastoma cells exhibited neurosphere-like growth in vitro and high expression of CD133 stem cell marker. The growth-inhibiting rate in CD133(-) glioblastoma cells treated with 5 or 50 micromol/L TMZ was significantly higher than that in CD133(+) glioblastoma cells ((14.36 +/- 3.75)% vs (2.54 +/- 1.36)% or (25.95 +/- 5.25)% vs (2.72 +/- 1.84)%, respectively, P < 0.05). Atg5, LC3-II and Beclin-1 levels were significantly lower in CD133(+) glioblastoma cells than those in autologous CD133(-) cells after TMZ treatment (P < 0.05). Caspase-3 was mildly activated only in CD133(-) glioblastoma cells after exposure to TMZ (P < 0.05). Immunofluorescent staining revealed elevated expression of Atg5 in GFAP(+) cells following TMZ treatment.
Conclusions:
The GSCs display strong capability of tumor's resistance to TMZ. This resistance is probably contributed by the CD133(+) cells with down-regulation of autophagy-related proteins. Future treatment should target this small population of cancer stem cells in tumors to improve survival of patients.
Insights
Glioblastoma stem cells (GSCs) expressing CD133 show resistance to temozolomide (TMZ) due to down-regulated autophagy. Targeting these CD133(+) GSCs may improve patient survival in brain tumors.
Area of Science:
- Neuro-oncology
- Cancer Stem Cell Biology
- Chemotherapy Resistance Mechanisms
Background:
- Brain tumors contain a small fraction of cells with neural progenitor features and tumor-initiating potential.
- These cancer stem cells are potential therapeutic targets for complete tumor eradication.
- Glioblastoma stem cells (GSCs) are implicated in tumor recurrence and treatment resistance.
Purpose of the Study:
- To determine the temozolomide (TMZ)-resistant phenotype of glioblastoma stem cells (GSCs).
- To investigate the molecular mechanisms underlying TMZ resistance in GSCs.
- To compare the sensitivity of CD133(+) and CD133(-) glioblastoma cells to TMZ.
Main Methods:
- Isolation of CD133(+) and CD133(-) GSCs from glioblastoma specimens.
- Assessment of TMZ cytotoxicity using MTT assay.
- Analysis of autophagy-related proteins (Beclin-1, LC3, Atg5) and cleaved caspase-3 via Western blotting.
- Immunofluorescent staining for Atg5, GFAP, and CD133 expression.
Main Results:
- CD133(+) GSCs exhibited neurosphere-like growth and high CD133 expression.
- CD133(+) GSCs showed significantly higher resistance to TMZ compared to CD133(-) cells.
- TMZ treatment led to down-regulation of autophagy proteins (Atg5, LC3-II, Beclin-1) in CD133(+) GSCs.
- Caspase-3 activation was observed primarily in CD133(-) cells, indicating apoptosis resistance in CD133(+) cells.
Conclusions:
- GSCs possess a strong resistance phenotype to TMZ.
- Down-regulation of autophagy-related proteins in CD133(+) GSCs likely contributes to TMZ resistance.
- Targeting CD133(+) cancer stem cells is a promising strategy to enhance glioblastoma treatment outcomes and improve patient survival.
