Interrogating Cholinergic Chemical Transmission of U-87 MG Glioblastoma Cells with Nanoelectrochemistry

Acetylcholine was identified over a century ago with early studies focusing on its release from neuronal systems; while a growing interest in non-neuronal systems has emerged, little is known about acetylcholine release from glioblastoma cells, and its mechanism remains unexplored. Here, we quantified the real-time acetylcholine release from live, single U-87 MG glioblastoma cells and investigated its mechanistic framework through the combination of nanoelectrodes with an interface between two immiscible electrolyte solutions and nano-resolved positioning using scanning electrochemical microscopy. Complementary to conventional solid electrodes, this measuring technique enabled quantitative and selective detection of redox-inactive acetylcholine. We hypothesized that U-87 MG glioblastoma cells may release acetylcholine, considering they express cholinergic markers and exhibited a ∼3-fold increase in intracellular calcium activity following KCl stimulation. We achieved the highly sensitive local measurement of the acetylcholine release by positioning the electrode ∼200 nm away from the glioblastoma cell surface. The release of acetylcholine from U-87 MG glioblastoma cells in the substantially reduced calcium environment or with the treatment of vesamicol, an inhibitor of vesicular acetylcholine transporter, only decreased slightly from the control condition. This suggests the presence of a possible nonvesicular mechanism of acetylcholine release in cancer cells, differing from neurons. The unprecedented findings on the acetylcholine release mechanism in U-87 MG glioblastoma cells presented here provide new insights in understanding the role of acetylcholine in glioblastoma disease progression.