Monitoring apoptosis in intact cells by high-resolution magic angle spinning 1H NMR spectroscopy

Apoptosis is a cellular process that maintains an equilibrium between cell proliferation and cell death. Induction of apoptosis is a well-known strategy in developing cancer treatments, therefore non-invasive monitoring of apoptosis in intact cells could contribute towards the characterisation of drug efficacy and hence drug discovery. A known metabolic marker of apoptosis is a notable increase in proton NMR resonances associated with lipids stored in lipid droplets (LDs). MRI-based studies of lipid accumulation have been used to monitor apoptosis-based cancer treatments. However, the cellular processes which lead to the accumulation of lipid droplets remain poorly understood, causing a bottleneck for targeting lipid metabolism in cancer cells. This thesis investigates the application of High-Resolution Magic Angle Spinning (HRMAS) 1H NMR spectroscopy in monitoring metabolic changes in intact cells during apoptosis. The technique is used to analyse metabolic profiles of cells treated with cisplatin and etoposide after 3, 8, 24 and 48 h. The results are compared to the analysis of organic cell extracts by solution-state 1H NMR spectroscopy to demonstrate lipid compartmentalisation and highlight the advantages of HRMAS 1H NMR spectroscopy in monitoring lipid metabolism during apoptosis. The lipid compartmentalisation is also confirmed by differential centrifugation and mass spectrometry-based lipidomics further validating the importance of LDs. Previous work linked NMR-visible lipid resonances to increased LD size. In this thesis, an NMR-based diffusion method is described for differentiating between control, apoptotic and necrotic cells. I demonstrate that as apoptosis-induced LDs become larger, the diffusion coefficient of NMR-visible lipids decreases. Therefore, diffusion measurements in conjunction with HRMAS 1H NMR-derived lipid signals provide a novel means of following apoptosis in intact cells. Mass spectrometry and transcriptomic analysis was used to elucidate the origin of LD during cisplatin and etoposide treatments. This work identifies two treatment-dependent mechanisms of lipid particle organisation, contributing to our understanding of LD formation during apoptosis. It may help validate magnetic resonance spectroscopy as a non-invasive tool for following the efficacy of apoptosis-inducing drugs.AstraZeneca, EPSR