Glioblastoma (GB) is the most aggressive brain tumor known. GB stem cells (GSC) are resistant to ordinary therapy and contribute to the development of recurrences. Toll-like receptors (TLR) are expressed in immunity cells to recognize multiple ligands from multiple infectious agents to unleash the immune response. TLRs are also expressed in neural cells, such as GB and neural stem cells, where its activation may imply cell differentiation. The aim of this study was to prove whether the stimulation of GSC TLRs induces a phenotype more sensitive to therapy and less aggressive.
Two GB cell lines were used: U-87 and U-118 (ATCC), cultured in the presence of Neurobasal® medium and in the absence of fetal bovine serum. The culture obtained was formed by neurospheres, which include a high proportion of GSC. On the one hand, the enrichment in GSC was confirmed by flow cytometry by the expression of stem cell markers CD133 and CD44. On the other hand, the analysis of TLR expression was performed by RT-PCR. According to its TLR expression, neurosphere cells were exposed to the pertinent TLR ligands for 24 hours in vitro and cultured in the presence or absence of temozolomide (TMZ). After stimulation, the expression of CD133 and CD44 was measured by flow cytometry.
Flow cytometry results showed a higher proportion of GSC in the culture with Neurobasal®. RT-PCR results demonstrated expression in stem cells of the genes corresponding to TLR2, TLR3, TLR4 and TLR6 receptors. Flow cytometry post-stimulation proved a decrease of stem cell markers in the ligands of the TLR2 and TLR4 in both lines. Cultures with TMZ did not show significantly altered expression of GSC, although survival was lower than cultures without TMZ.
These results show a relation between the activation of the TLR and the increase of the differentiation rate in GSCs, especially through TLR2 and TLR4. Based on the results obtained, a new therapy for GB treatment, might be possible, which would include the differentiation of GSC by the exposition to TLR2 and TLR4 ligands, previous or concomitant to chemotherapy.
Clinical trial identification
Legal entity responsible for the study
University of Valencia.
Has not received any funding.
All authors have declared no conflicts of interest.