| Summary: | The molecule neuron-glia 2 (NG2) is a transmembrane chondroitin sulphate proteoglycan that is present on a distinct population of glial cells in the central nervous system (eNS). NG2 has been used to identify this subgroup of cells as an additional major glial cell population, in addition to, oligodendrocytes, astrocytes, ependymal cells and microglia. In the adult brain, these cells retain the ability to differentiate into oligodendrocytes and astrocytes. NG2 is often re-expressed by brain tumours and has been found to have increased expression levels in the high grade glioma and glioma-derived cell lines. Within the scope of this thesis it has been demonstrated that culture conditions (different media and serum supplementation) influence expression of NG2 and other antigens on glioma cell cultures. Seven cell lines covering a range of histopathological diagnoses have been fully characterised with regard to their antigenic expression profiles. With one cell line in particular, UP AB, the influence of different serum supplementation conditions on the expression of a wide range of gliomarelated antigens was elucidated. NG2 expression was also demonstrated for each of these cell lines, with results confiming higher expression in the more malignant gliomas. A panel of NG2 antibodies were considered and the most specific one was taken through for the immunotoxin studies. Proof of principle was then demonstrated that NG2 positive gliomas may be selectively and effectively ablated using a secondary imunnotoxin approach. This secondary immunotoxin approach comprised of an antibody directed against NG2 being conjugated to a secondary antibody with the immunotoxin Saporin attached. This immunotoxin system proved to be adaptable in its ability to target different antigens with the sequential treatment of ablating NG2 positive cells and then the ganglioside markers GD3 and GD3A positive cells respectively. The expression of NG2 is associated with proliferative activity in gliomas, whereas invasiveness is associated with the expression of GD3. Spatial 1 distribution of NG2 and GD3 in tumour biopsies has been reported, indicating that NG2 expression is confined to the main tumour mass and is reduced towards the tumour periphery, and GD3 expression is evident both in the main tumour mass and at the brain/tumour interface. Our studies suggest differential functions ofNG2 and GD3 on the same glioma cell populations, the former facilitating proliferation and the latter invasion. Thus NG2 expression may confer increased proliferation, while a "switch" to GD3A facilitates tumour invasion. NG2 is also deposited between substrate and migrating glioma cells, and interacts with extracellular matrix proteins (ECM), synthesised by neoplastic cells during invasion and migration, indicating that NG2 may modulate glioma cell adhesion and angiogenesis. NG2 may also negatively regulate glioma adhesion by interacting with integrins. Thereby, this dual approach of ablating NG2 and GD3A positive glioma cells was adopted to attempt to kill 100% of the tumour cell population. With the highest concentration of Mab-Zap (5J.lglml) combining both NG2 and GD3A resulted in approximately 94% of cell death in three cell lines UPAB, UPMC and UPPP. It is with the identification of specific tumour-associated antigens present in tumour tissue such as NG2 and GD3A and the production of high affinity MAbs to such antigens and the use of compartmental administration (intrathecal or intracystic), the promise of passive immunotherapy of primary and metastatic central nervous system neoplasms has been recognized. This involvement of NG2 in multiple aspects of tumour biology makes this proteoglycan an attractive candidate for future therapies against cancer. NG2 may provide a suitable target for cytotoxic therapy, particularly when harnessed with approaches which aim to target the shared glioma cell antigen GD3A •
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