BACKGROUND: Glioblastoma is an incurable primary brain tumors with one of the worst 5-year survival rates among all cancers. Despite multimodal treatment involving neurosurgery, irradiation and temozolomide chemotherapy, the median overall survival is in the range of 15-16 months even in selected clinical trial populations. Thymosin beta 4 (TB4) is a pleiotropic actin-sequestering polypeptide that is involved in wound healing and developmental processes. TB4 gene silencing promotes differentiation of neural progenitor cells whereas TB4 overexpression initiates cortical folding of developing brain hemispheres. Moreover, TB4 is involved in migration, invasion and epithelial to mesenchymal transition in other cancer entities, e.g. colon cancer. METHODS: We analyzed TB4 expression on tissue microarrays and performed REMBRANDT and TCGA database interrogations. We analyzed TB4 expression in 8 long-term glioma cell lines and 7 glioma-initiating cell lines. Using lentiviral transduction, we modulated TB4 expression in the long-term glioma cell lines LNT-229 and U87MG, and in the glioma-initiating cell line GS-2. We studied clonogenic survival, migration, invasion, sphere formation anddifferentiation capacity of TB4-depleted or TB4-overexpressing glioma cells in vitro, and tumorigenicity upon orthotopic implantation in vivo. Finally, we performed a genome wide mRNA expression analysis to unravel the molecular network of TB4 signaling effects. RESULTS: TB4 expression increased with the grade of malignancy in gliomas and correlated with patient survival in glioblastoma. In vitro, TB4 gene silencing by lentiviral transduction decreased migration, invasion, growth and self-renewal, and promoted differentiation and the susceptibility to undergo apoptotic cell death upon nutrient depletion in LNT-229, U87MG and the glioma stem-cell line GS-2, respectively. In vivo, survival of nude mice bearing tumors derived from TB4-depleted glioma cells was improved and the tumorigenicity of the GS-2 glioma stem-cell line was decreased. The gene expression pattern was shifted from the mesenchymal towards the pro-neural gene signature upon TB4 gene silencing, thereby modulating TGF-beta and p53 signaling networks. CONCLUSIONS: TB4 is involved in the regulation of key malignancy features in glioblastoma and may therefore be a novel candidate therapeutic target. SECONDARY CATEGORY: n/a.