Electrophysiology of autocrine and paracrine NMDA receptor signalling in invasive mouse pancreatic neuroendocrine tumour cells
Session type: Poster / e-Poster / Silent Theatre session
N-methyl-D-aspartate receptor (NMDAR) activation is implicated in the malignant progression of many cancer types, including pancreatic neuroendocrine tumours (PanNETs). (Li and Hanahan, 2013, Cell 153:86). In previous studies, NMDAR expression in cancer cells has been demonstrated at RNA and/or protein levels, and functional validation has mostly been achieved by using NMDAR antagonists. However, NMDAR-mediated calcium influx and its downstream signalling depend critically on the dynamics of membrane potential and of ambient glutamate concentration; notably, however, these factors have been little studied in the context of cancer. We therefore set out to unravel glutamate-driven electrical signalling in a mouse model of PanNET.
The RIP1-Tag2 mouse model of PanNET (Hanahan, 1985, Nature 314:115) was studied in 2D cell cultures, using patch-clamp recording, optical measurement of calcium, and computational models of glutamate transport and voltage-dependent ion channel gating.
Activation of NMDARs by extracellular perfusion of agonist caused depolarization, action potential firing and intracellular calcium elevation in PanNET cells. Intracellular perfusion with physiological (1-10 mM) levels of glutamate caused autocrine NMDAR activation, observable as single-channel transitions in low-noise whole-cell currents. Autocrine NMDAR activation could be blocked by inhibitors of VGLUT transporters, suggesting their involvement in glutamate efflux. Lower-level paracrine NMDAR activation could also be resolved when glutamate was omitted from the intracellular solution. Paracrine activation was stimulated by mechanically-induced necrosis of nearby cells, which caused intense NMDAR activation, resulting from release of endogenous cytoplasmic glutamate and aspartate.
Computational modelling based on these findings supported the idea that NMDARs in cancer cells are strongly activated by glutamate arising from autocrine and paracrine release, and that necrosis is a novel potential source of invasiveness-promoting extracellular glutamate in the tumour microenvironment. Collectively, this study presents unprecedented, comprehensive electrophysiological evidence of how cancer cells can hijack the neuronal NMDAR receptor signalling circuit.