Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE credits
Gliomas are brain tumors of glial origin and the most aggressive form is called glioblastoma multiforme (GBM). In Sweden, 300 new GBM cases are reported each year. Although advance treatments including radiotherapy, chemotherapy and surgery, are available, the survival of GBM is less than two years from diagnosis. Cell lines derived from GBM and cultured as neurospheres are known to be very heterogenic, that is they comprise many different cells, therefore it is difficult to study what factors may influence their proliferation, differentiation, apoptosis etc.
Several recent studies have suggested Notch signalling as a potential therapeutic target . Notch signalling occurs via direct cell-cell interactions and influence many different cell fate decisions including, proliferation, differentiation, apoptosis and migration. Abnormal function in this pathway is related to many different diseases including cancer. Furthermore, Notch play a crucial role in normal development of central nervous system and is involved in regulation response to hypoxia and angiogenesis which are associated with the development of GBM [2, 3]. Notch signalling is far from fully understood. Moreover antibodies that specifically block signalling through individual Botch receptors can provide tools to investigate this in detail, potentially leading to new cancer treatments.
The aim of this study was to utilize antibodies to specifically target Notch1, Notch2 and Notch3 in glioma cell lines, derived from glioma neural stem (GNS) cells, grown in both proliferating and differentiating conditions. The blocking antibodies were previously generated from a scFv phage display library and shown to interfere with Notch in a receptor-specific way . After adherent culturing of glioma cell lines, the anti-Notch antibodies were employed to inhibit Notch signalling. Moreover, the gene expression of different markers related to Notch signalling pathway were analysed using RT-PCR and a gel-based semi-quantitative method.
We produced antibodies and verified their full activity after being incubated at 37°C during four weeks and also after long term storage at 4°C. Durign the project, a protocol for adherent culturing of three different GNS cell lines, in both proliferating and differentiating conditions, were established. Further, gene expression of several Notch related proteins, including Notch1-3 and the ligands Jag1, Jag2, D111, D113 were verified using RT-PCR suggesting Notch to be highly active in GNS cell lines. On the contrary, Notch4 receptor and D114 ligand were not expressed in these cell linces. Hence these systems provide a good platform addressing the role of Notch signalling in initiation and progression of GBM. The involvement of Notch in these cells i further indicated by the fact that eve after several days in differentiating conditions, Notch target genes such as Hes5 and Hey1 were not fully down regulated. In addition, inhibition of single receptors with blocking antibodies does not seem to be enough and therefore additional approaches are required for a significant down regulation of Notch signalling GNS cells.