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Three-Dimensional (3D) Tumor Spheroid Invasion Assay
Published on: May 1, 2015
Effect of brain- and tumor-derived connective tissue growth factor on glioma invasion
Lincoln A Edwards1, Kevin Woolard, Myung Jin Son
1Neuro-Oncology Branch, National Cancer Institute/National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-1002, USA.
Background:
Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets.
Methods:
Highly infiltrative patient-derived glioma tumor-initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin β1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided.
Results:
Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1-TrkA receptor complexes and nuclear factor kappa B (NF-κB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF(200 ng/mL): 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2, P < .001). NF-κB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF(200 ng/mL) + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most infiltrative glioma cells in situ and that the surrounding reactive astrocytes secrete CTGF.
Conclusion:
A CTGF-rich microenvironment facilitates CTGF-ITGB1-TrkA complex activation in TIC/TSCs, thereby increasing the invasiveness of malignant gliomas.
Insights
Connective tissue growth factor (CTGF) drives malignant glioma invasion by activating integrin β1 (ITGB1) and TrkA receptors. Targeting this pathway offers a potential therapeutic strategy for glioblastoma. Keywords: CTGF, glioma invasion, ITGB1, TrkA, therapeutic target.
Area of Science:
- Neuro-oncology
- Cancer Biology
- Molecular Mechanisms of Cancer
Background:
- Malignant glioma cell invasion is a primary driver of treatment failure and mortality.
- Connective tissue growth factor (CTGF) is implicated in the metastasis and invasion of various cancers.
- Understanding CTGF-mediated glioma infiltration is crucial for developing effective therapies.
Purpose of the Study:
- To elucidate the mechanism of CTGF-induced glioma cell infiltration.
- To identify and evaluate potential therapeutic targets within the CTGF signaling pathway.
Main Methods:
- Utilized patient-derived glioma tumor-initiating/stem cells (TIC/TSCs) to investigate the CTGF signaling pathway.
- Employed luciferase reporter assays, ChIP, qPCR, and immunoblotting to study signal transduction.
- Tested small-molecule inhibitors of integrin β1 (ITGB1) and TrkA, and CTGF-targeting shRNAs.
- Assessed TIC/TSC infiltration in vitro and in vivo using orthotopic xenograft mouse models.
Main Results:
- CTGF treatment induced CTGF binding to ITGB1-TrkA receptor complexes and activated nuclear factor kappa B (NF-κB).
- NF-κB activation led to ZEB-1 binding to the E-cadherin promoter, suppressing E-cadherin expression.
- TrkA was selectively expressed in highly infiltrative glioma cells, and surrounding astrocytes secreted CTGF.
Conclusions:
- A CTGF-rich microenvironment promotes malignant glioma invasiveness via CTGF-ITGB1-TrkA complex activation in TIC/TSCs.
- This pathway represents a significant mechanism contributing to glioma cell infiltration.
- Targeting the CTGF-ITGB1-TrkA axis may offer a novel therapeutic approach for malignant gliomas.
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