Analysis of three dimensional cell cultures using mass spectrometry imaging

In order to better replicate disease and response to therapeutics 3D cell culture methods have been increasingly developed for use in research and industry. Two key areas where 3D cell cultures are being used as alternative models to animals are the study of prevalent diseases such as cancer and therapeutic toxicity testing in key metabolising organs such as the liver. Matrix-assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI-MSI) is an untargeted molecular imaging technique capable of imaging multiple molecules within a single experiment. This can be utilised for the investigation of molecular mechanisms of biological function or treatment response within 3D cell cultures, however, optimised methods are required for the analysis of these models. In this thesis, a novel 3D cell culture model of osteosarcoma was developed. Sample preparation and MALDI-MSI workflows were optimised initially for small molecule analysis. Following this, doxorubicin responses in the 3D osteosarcoma model were assessed. Detection of doxorubicin-induced changes to lipids and metabolites in 3D cell culture were subsequently detected, and Principle Component Analysis (PCA) used to identify metabolite signatures associated with doxorubicin treatment. Methods were then adapted for MALDI-MSI of proteotypic peptides within the model and a novel method for peptide quantitative mass spectrometry imaging (QMSI) was developed. MALDI-MSI successfully allowed the identification and quantification of 25 proteotypic peptides, using a 120-peptide standard array, demonstrating, for the first time, that QMSI is possible for proteomic quantification in 3D cell cultures. Development of, and preliminary MSI analysis of, a novel 3D cell culture model of liver toxicity is reported. The L-pNIPAM scaffold and HepG2 cell lines used demonstrates hepatocyte differentiation and is potentially suitable for monitoring of hepatic metabolism and adverse drug reaction. Taken together, these studies represent a considerable development in simultaneous quantification of metabolites, lipids and proteins in 3D cell cultures.