P01.19.A FUNCTIONAL INVESTIGATION OF SIALYLTRANSFERASES IN GLIOBLASTOMA

Abstract BACKROUND Glioblastoma (GBM) is the most prevalent and aggressive brain tumour, and is associated with an unfavourable prognosis. Consequently, there is an imperative for the identification of novel therapeutic targets. The attachment of sialic acids (Sia) to the cell surface represents a potential target for therapeutic intervention, as this process is already employed clinically and prognostically in other tumour entities. The sialyltransferases (ST) ST8SIA2 and ST8SIA4, which influence the interaction of cells via long sugar chains, are mainly responsible for polysialylation (PolySia) in tumours. The project investigates the expression of STs in different GBMs, and functionally characterises the influence of PolySia on malignancy. MATERIALS AND METHODS For the expression analysis of STs in primary tumours (PT) and primary cultures (PC), quantitative PCR (qPCR) and bulk RNAseq were utilised. The knockout of ST8SIA2 and ST8SIA4 is performed using CRISPR/Cas9. Following the transfection of cell lines and primary cultures, the influence of the knockout of both STs alone and the double knockout will be characterised using Real-Time Cell Analysis (RTCA). The effect on cellular properties will be measured by changes in proliferation, invasion, resistance to therapy and co-culture experiments. RESULTS The qPCR revealed a heterogeneous ST profile, with the ST3 group showing notable expressions in PCs. The data indicate changes in ST expression between PTs and corresponding PCs. The presence of poly-STs has been detected, while the predominance of either ST8SIA2 or ST8SIA4 in PTs is maintained in 2D culture. ST8SIA2 was stronger expressed in some PCs and ST8SIA4 in others. However, higher expressions were observed in 2D cultures than in PTs. With regard to gene editing, sgRNA was cloned into a plasmid, and the transfection protocol could be tested. In addition, the formation of spheroids from cell lines and PCs was achieved. CONCLUSION GBM is distinguished by intertumoural heterogeneity, a property that extends to the ST expression. The elevated expression of ST8SIA2 or ST8SIA4 in 2D cultures when compared to PTs may be attributed to the selection of cells, suggesting that these cells possess a growth advantage. Consequently, 3D GBM models have been developed to achieve maximum comparability with PTs. Given the divergent expression of ST8SIA2 to ST8SIA4 between PCs, the double knockout of both STs represents a promising prospect, with the potential to be used therapeutically in future.