P01.11.B PATIENT-DERIVED GLIOBLASTOMA CELLS ARE CAPABLE OF FORMING TUBE-LIKE STRUCTURES IN VITRO SUGGESTIVE OF VASCULOGENIC MIMICRY

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Abstract BACKGROUND The tube formation assay (TFA), widely used to assess the angiogenic ability of endothelial cells in vitro, involves plating endothelial cells on a basement membrane matrix layer that promotes the differentiation of the cells, observed as the formation of tubule-like structures. The extent and characteristics of the tubule network are influenced by cell seeding density, addition of growth factors, and the composition of the basement membrane matrix. Tube formation by tumour cells in vitro is considered to be an indicator of the ability of these cells to undergo vasculogenic mimicry (VM), the process by which tumour cells form functional vessel-like structures. The TFA has therefore been used to assess VM in various tumours, including glioblastoma. However, aspects of the TFA, including seeding density, basement membrane matrix, media composition, and imaging timepoint, vary greatly between previous studies of VM in glioblastoma, resulting in difficulty comparing results between studies and uncertainty relating to the conditions under which glioblastoma cells may undergo tube formation. We therefore aimed to assess the tube formation ability of multiple glioblastoma cell lines assayed under the same conditions. MATERIAL AND METHODS Twelve patient-derived glioblastoma cell lines were plated for TFAs at multiple seeding densities in serum-free media on a 150µL/cm2 layer of growth factor-reduced (GFR) Matrigel. Cells were imaged 2-hourly for 24 hours using an Incucyte SX5 Live-Cell Analysis Instrument in order to determine the tube formation ability and optimal seeding density of each cell line. Human umbilical vein endothelial cells (HUVECs) were used as a positive control. The Angiogenesis Analyzer plugin for ImageJ was used to quantify number of tubules, average tubule length, tubule area, and number of branch points for HUVECs and tube forming glioblastoma cells. RESULTS Three of the 12 glioblastoma cell lines tested were considered capable of in vitro tube formation, consistently forming tubule-like networks that morphologically resembled those formed by HUVECs within 24 hours of plating for a TFA. Network characteristics differed between HUVECs and glioblastoma cells, with the glioblastoma cells generally forming a higher number of shorter tubules, and therefore having a higher number of branch points. This did not correspond to a larger total tubule area, which was comparable between the HUVECs and glioblastoma cells. CONCLUSION A subset of patient-derived glioblastoma cell lines are able to form tube-like structures in vitro when grown in serum-free media on GFR Matrigel. Further experiments assessing additional TFA conditions and subsequent analyses of gene and protein expression in tube-forming glioblastoma cell lines may provide further insight into the mechanisms by which VM may be occurring.

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