Abstract/Summary

Glioblastoma cancer stem cells (CSCs) are a subpopulation of cells that play important roles in tumour metastasis, tumour recurrence and anticancer therapy resistance. Toll-like receptors (TLRs) are type I integral membrane proteins found on glioblastoma CSCs, which are also essential pathogen recognition receptors within the innate immune system. MyD88-dependent pathway and MyD88-independent pathway are two major signalling pathways triggered by TLRs, and Toll-like receptor 4 (TLR4) is the only member of TLRs that can trigger both in a biased manner. Depending on the ligand, signalling via TLR4 can either activate the pro-inflammatory transcription factor ‘Kappa-light-chain-enhancer’ of activated B cells (NF-κB) or the anti-viral and anti-inflammatory interferon regulatory factor 3 (IRF3). Currently, TLR4 in cancer was widely studied. However, the TLR4 and inflammation in glioblastoma, especially how the biased signalling is impacting the glioblastoma CSCs behaviours is a gap of the field. In this study, using 2D and 3D cell cultivation, I demonstrated that TLR4 mediated NF-κB and IRF3 have opposing effects on U251 cells. The activation of TLR4- NF-κB pathway is able to increase the tumourigenicity, migration, and the overall percentage of CSCs. Activation of TLR4-IRF3 pathway is able to increase the differentiation of CSCs, decrease the stemness and suppress tumourigenicity of glioblastoma. In addition, I demonstrated that in 3D, U251 cells showed significant increase of tumourigenesis and stemness. Also, 3D cultivated U251 cells proliferated quicker than 2D cultivated cells, and had higher resistance to Temozolomide. I have also demonstrated that curcumin is an efficient anti-inflammatory compound and showed that a novel curcumin microemulsion increases its anti-inflammatory potential. Lastly, I have developed an IRF3 reporter system that can be used to generate a quad-reporter cell line, which in the future could be a very useful tool to study the biased signalling downstream of TLR4. In general, my research has clinical Impact of Toll-like receptor 4 biased signaling on the behaviours of glioma cancer stem cells application potential. By developing drugs to regulate TLR4 downstream pathways, it could offer new treatment options for glioblastoma either alone or in combination with conventional treatment options. In addition, 3D cell culture and curcumin also have great prospects in anti-cancer drug development.