Abstract 2670: Spatial potassium buffering among clustered glioblastoma cells is proposed to enable survival scavenging of nutrients released by interior necrotic cells

Abstract

Abstract Glioblastoma cells are known for surviving harsh environments and for forming secondary structures of Scherer (cell clusters around neurons/axons, blood vessels, regions of coagulative necrosis, & in the subpia). The astrocytic lineage of glioblastomas suggests recapitulation of astrocytic-type syncytia that buffer potassium released from neurons/axons. Here, multiple glioblastoma cell lines were stressed by nutrient withdrawal to observe cell behavior in view of their astrocytic lineage & known genome sequence of 1 cell line. Methods: Single cell suspensions of 4 glioblastoma cell lines (U87, LN229, T98G & A172) were added to a settling chamber (Neuro Probe, Gaithersburg, MD). After 0.5 hr of gradual nutrient withdrawal, slides (Diff Quik stain) were analyzed, 2 replicate wells per cell line. U87 cells with cytoplasmic vacuoles were quantified to indicate the degeneration in cell clusters. When vacuoles collectively had a diameter at least 1/3 of an ave. nuclear diameter in the same 100X microscopic field, the vacuolated cell was deemed +. Potassium ion channel genes potentially used by glioblastoma cells per the Online Mendelian Inheritance in Man (OMIM) database were checked in genome sequence results for U87 cells (Clark MJ, et al. PLoS Genet 2010 6(1): e10000832). Results: All glioblastoma cell lines displayed cell adherence & wrapping around single swollen, often heavily vacuolated degenerating cells of the same lineage after nutrient withdrawal. As clustered U87 cells (putative syncytia) increased in cell number, the number of constituent cells with vacuoles decreased (Pearson regressions, p=.04, p=.006) in 2 replicate wells. Whereas single cells were +, 72% and 51%, vacuolated cells fell to 18% and 11% in putative syncytia of over 20 cells in 100X microscopic fields across the width (7 mm) of each well. Thus, within increasing-sized putative syncytia, which offer increasing potassium buffering capacity, significantly fewer constituent cells displayed vacuoles. Presumably this was due to available nutrients from one to few necrotic cells in their midst during nutrient withdrawal. Potassium ion channeling within astrocytic-type syncytia, termed "spatial buffering" explains how viable glioblastoma cells adhere to degenerating cells despite high potassium levels. Genome sequencing of U87 cells revealed that only KCNMB2 & KCNJ16 of 47 potassium channeling genes pertinent to brain, were negatively affected by genetic defects. Functional losses of KCNMB2 as an enhancer for KCNMA1 (intact), and KCNJ16 as a co-assembler with other family members (intact), are unlikely to prevent potassium buffering. Preservation of nearly all potassium channeling genes in U87 suggests that maintenance of spatial buffering in astrocytic-type syncytia permits viable cells to surround necrotic cells and salvage released nutrients as a survival strategy. Citation Format: Marie Beckner. Spatial potassium buffering among clustered glioblastoma cells is proposed to enable survival scavenging of nutrients released by interior necrotic cells [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 2670.