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Updated: Jan 7, 2026
Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion
Published on: August 29, 2018
NO/iNOS axis impact on glioma stem cells and temozolomide resistance
Elsa Hincapié-Arias1,2, Carolina Moughty-Cueto3, Juan Manuel Zaloff-Dakoff3
1Universidad de Buenos Aires, Facultad de Medicina, Instituto de Oncología Ángel H. Roffo, Área de Investigación, Buenos Aires, Argentina.
Abstract:
Glioblastoma (GB), the most aggressive brain tumor invariably recurs despite conventional treatment (surgery, chemotherapy with temozolomide (TMZ), radiotherapy), a phenomenon linked to glioma stem cells (GSC). This study demonstrates that inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) sustains the GSC niche and contributes to TMZ resistance. In silico analysis of 695 glioma samples from The Cancer Genome Atlas showed that higher iNOS expression correlates with poor patient survival. In vitro, GSC-enriched cultures (spheres) from GB cell lines (LN229, U87, U251, GL26) exhibited elevated pluripotency (Nestin gene expression), iNOS expression and NO levels threefold higher than more differentiated cell cultures (2D and 3D-spheroids). The iNOS inhibitor S-methylisothiourea (SMT) effectively reduced NO production. Sequential treatment with TMZ followed by TMZ + SMT more effectively reduced viability in 2D and 3D-spheroids growth compared to TMZ alone. In GSC enriched spheres, SMT decreased GSC frequency and growth, and combined TMZ + SMT showed enhanced reduction. Primary human glioma cultures (n = 11) expressed iNOS and their GSC spheres formation efficiency was reduced by SMT. These findings suggest that iNOS/NO axis is critical for GSC maintenance and TMZ resistance. Sequential TMZ followed by iNOS inhibition shows promise as a therapeutic strategy against GB recurrence by targeting GSCs. KEY MESSAGES: Elevated iNOS expression correlates with poor glioma prognosis markers. Temozolomide acts on more differentiated glioma cells and enriches stem cells. iNOS inhibition reduces the stem cell niche, enhancing TMZ sensitization.
Insights
Inducible nitric oxide synthase (iNOS) fuels glioma stem cells (GSCs) and temozolomide (TMZ) resistance in glioblastoma. Inhibiting iNOS alongside TMZ treatment offers a promising strategy to target GSCs and prevent tumor recurrence.
Area of Science:
- Neuro-oncology
- Cancer Stem Cell Biology
- Molecular Oncology
Background:
- Glioblastoma (GB) invariably recurs due to glioma stem cells (GSCs).
- Temozolomide (TMZ) resistance is a major challenge in glioblastoma treatment.
- The role of inducible nitric oxide synthase (iNOS) in GSC maintenance and drug resistance is not fully understood.
Purpose of the Study:
- To investigate the role of the iNOS/nitric oxide (NO) axis in sustaining the GSC niche.
- To determine the impact of iNOS inhibition on GSC frequency and TMZ resistance.
- To evaluate a sequential treatment strategy combining TMZ and iNOS inhibition for glioblastoma.
Main Methods:
- In silico analysis of The Cancer Genome Atlas (TCGA) data for iNOS expression and patient survival.
- In vitro studies using glioblastoma cell lines and primary human glioma cultures to assess GSC characteristics.
- Treatment with the iNOS inhibitor S-methylisothiourea (SMT) alone and in combination with TMZ.
- Assessment of cell viability, GSC frequency, and sphere formation efficiency.
Main Results:
- Higher iNOS expression in glioma samples correlated with poorer patient survival.
- GSC-enriched cultures showed significantly higher iNOS expression and NO levels compared to differentiated cells.
- iNOS inhibition with SMT reduced GSC frequency and growth.
- Sequential treatment with TMZ followed by TMZ + SMT demonstrated enhanced efficacy in reducing glioblastoma cell viability and GSC growth compared to TMZ alone.
Conclusions:
- The iNOS/NO axis is crucial for maintaining the GSC niche and contributes to TMZ resistance in glioblastoma.
- Targeting iNOS can disrupt the GSC niche and sensitize glioblastoma cells to TMZ.
- Sequential TMZ and iNOS inhibition presents a potential therapeutic strategy to overcome glioblastoma recurrence by targeting GSCs.
