Abstract 241: An efficient cell-based assay for quantification of cellular polysialic acid in neuroblastoma

Abstract

Abstract Introduction Polysialic acid (polySia) decorates the surface of NCAM (neuronal cell adhesion molecule) on neuroendocrine tumors, notably neuroblastoma and small cell lung cancer, and is strongly associated with poor prognosis and aggressive disease in patients in the clinic [1]. PolySia modulates tumor cell-cell and cell-matrix adhesion, migration, invasion and metastasis. SiRNA knockdown of polysialyltransferase (polyST) ST8SiaII, the enzyme primarily responsible for polySia synthesis in tumors, abrogates tumor cell migration and invasion. PolyST is a selective and largely unexplored therapeutic target for neuroblastoma dissemination [1]. Methods and Results We describe the development of a cell-based assay that quantifies the formation of polySia on the cell surface of neuroblastoma cells. This is a sensitive assay that detects the polySia that is released after pH adjustment, organic precipitation and acid hydrolysis. PolySia was first separated from cell lysate by mild acid hydrolysis and acetone precipitation. It was then purified by ethanol precipitation. We further analysed the role of lactonisation of polySia during organic precipitation. The purified polySia is further hydrolysed into sialic acid monomers, followed by DMB labelling and RPLC-fluorescence analysis. This method was validated by analysis of polySia in C6-ST8SiaII cells, following treatment with Endo-NF in a various concentrations. This method proved to be useful to assess polySia changes after treatment of cells with novel polyST inhibitors. The effect of lactonisation of polySia during organic precipitation was also investigated. It was found that lactonised polySia was mainly soluble in 90% acetone/90% ethanol, while natural polySia (non-lactonised) can be precipitated by ethanol in the presence salts. Based on the different solubility of polySia, a cost-efficient assay was designed to purify polySia released from cells with a low level of non-polySia-derived sialic acid contamination before further acid hydrolysis. With this method we quantified the different levels of cell-surface polySia-derived sialic acid between polySia-positive and polySia-negative cell lines. Furthermore, we successfully validated this method by Endo-NF treatment in C6-ST8SiaII cells: the EC50 of Endo-NF digestion of polySia in C6-ST8SiaII cells was approximately 5.4 pM. The assay has been successfully utilised to evaluate novel polyST inhibitors in vitro. [1] Falconer, R.A. et al., Curr. Cancer Drug Targets, 2012, 12, 925-939; [2] Al-Saraireh YMJ et al., PLoS ONE, 2013, 8:e73366. Citation Format: Xiaoxiao Guo, Francis Mprah Barnieh, Jodie Malcolm, Amanda D Race, Goreti Ribeiro Morais, Steven D Shnyder, Paul M Loadman, Laurence H Patterson, Robert A. Falconer. An efficient cell-based assay for quantification of cellular polysialic acid in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 241.