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Cancer is one of the most devastating human diseases that cause a vast number of mortalities worldwide each year. Cancer research is one of the largest fields in the life sciences and despite many astounding breakthroughs and contributions over the past few decades, there is still a considerable amount to unveil on the function of cancer that would improve diagnostics, prognostics and therapy. Since cancer is known to involve a wide range of processes, applying methods to study it from a systems perspective could reveal new properties of cancer. Systems biology is becoming an increasingly popular tool in the life sciences. The approach has been applied to many biological and biomedical analyses drawing upon recent advancements in technology that make high throughput analyses of samples and computational modelling possible. In this thesis, the effect of hypoxia inducible factor-1 (HIF-1) on cancer metabolism, the entity considered most closely related to phenotype has been investigated. This transcription factor is known to regulate a multitude of genes and proteins to promote survival in a low oxygen environment that is prevalent in solid tumours. However its effect on the metabolome is less well characterised. By revealing the effect of HIF-1 on the metabolome as a system it is hoped that phenotypic signatures, key metabolic pathways indicative of cancer function and potential targets for future cancer therapy, can be revealed.The system has been studied using two cell models: mouse hepatocellular carcinoma and human colon carcinoma, whereby metabolism has been profiled using a range of analytical platforms. In each model, wild type cells have been compared to cells deficient in HIF-1 to reveal its effect on cellular metabolism. Gas chromatography mass spectrometry (GC MS) and ultra high performance liquid chromatography - mass spectrometry (UHPLC MS) have been employed for metabolic profiling of cells exposed to a range of oxygen conditions. Additionally, time-of-flight secondary ion mass spectrometry (ToF SIMS) has been employed for imaging mass spectrometric analysis of multicellular tumour spheroids cultured from wild type cells and cells with dysfunctional HIF-1 to represent small initiating tumours. Using these techniques in metabolic profiling it has been possible to reveal metabolites associated with the effect of oxygen and HIF-1 on cancer metabolism along with key pathways and hubs that could be targeted in future therapy. Using imaging mass spectrometry it has been possible to localise metabolites in situ revealing how tumour structure relates to function. Finally, a novel approach to consider how metabolites are correlated with one another in the response to oxygen level or presence or absence of functional HIF-1 has been undertaken to better understand the systems properties of cancer metabolism. Metabolites found to be differently correlated with respect to oxygen and/or HIF-1 have been mapped onto a human metabolic network to determine their network-based origins. This allowed the simulation of sub-networks of metabolism most affected by oxygen and HIF-1, highlighting the key mechanisms in HIF 1 mediated cancer cell survival.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a sensitive technique used to analyse the molecular composition of solid samples using keV ion beam sputtering. However only a small fraction (typically < 10⁻³) of the desorbed material issecondary ions - the majority species (neutrals) cannot be extracted and detected by the mass spectrometer. To increase the sensitivity and efficiency of the SIMS technique, post-ionisation above the surface can be used. Lasers have been widely employed for molecular mass spectrometry due to the available high intensity, short pulse width, high spectral purity and spatial coherence that allow them to be highly focused. For molecular samples the challenge is to achieve efficient post-ionisation without inducing extensive fragmentation, which limits the diagnostic value of the resulting mass spectrum. An investigation was performed into the ionisation and dissociation characteristics of a series of organic molecules under the action of intense laser fields. This study is directed towards the analysis of biomolecules using laser post-ionisation. Here is reported progress towards the calibration of the experimental set-up and mass spectral data from representative biomolecules in the gas phase. In this work a Ti:Sapphire laser was used with fundamental wavelength of 800 nm and non-linear optical methods (OPAs) are used to extend the wavelength into the mid-IR region. System calibration is achieved using the ionisation of xenon atoms and comparing the results to established atomic tunnelling theory. This was followed by the analysis of representative organic and biological molecules to study ionisation-dissociation characteristics. The molecules chosen were toluene, acetone, nitroaniline and histamine. A clear transition in behavior is observed favoring molecular ion production. This behavior is discussed in the context of the underlying mechanisms, and the implications for molecular post-ionisation analysis using focused ion beams.

Introduction: Hypoxia is found in solid cancerous tumours. The presence of hypoxia within tumours inhibits anti-cancer treatment strategies such as chemotherapy from being completely effective and it is suspected that multiple mechanisms contribute to the resistance. Methods: In this project a systems biology approach was applied to determine how the toxicity of doxorubicin is affected by hypoxia at the metabolome level. A multitude of analytical techniques were applied to analyse the intracellular metabolism of a monolayer of cancer cells (MDA-MB-231). Metabolic profiling was used to determine metabolite markers related to hypoxia-induced chemoresistance. For this gas chromatography mass spectrometry (GC-MS) and ultra high performance liquid chromatography mass spectrometry (UHPLC-MS) were used. Furthermore, network-based correlation analysis was developed as a novel tool to bridge the gap between metabolomics dataset and systems biology modelling. This methodology was applied to elucidate novel metabolic pathways as potential therapeutic targets to overcome hypoxia-induced chemoresistance. This algorithm determines significant correlation differences between different physiological states, and through applying graph-theory on large genome scale models; it is possible to construct a metabolic network of the pathways connecting the pair-wise correlation. Finally, imaging mass spectrometry using time-of-flight secondary ion mass spectrometry (ToF-SIMS) was developed as a tool for in situ metabolite analysis to investigate the metabolic response to chemotherapy in multi-tumour spheroids (MTSs). Results: Metabolic fingerprinting analysis characterised a snapshot of cells exposed to various environmental perturbations. Metabolite markers associated with hypoxia-induced chemoresistance were related to metabolic pathways including gluconeogenesis, DNA synthesis and fatty acid synthesis. Furthermore, network-based correlation analysis revealed specific metabolites in the fatty acid synthesis pathways were contributing to drug resistance, which included malonyl-CoA, 3-oxoeicosanoyl-CoA, stearoyl-CoA and octadecanoic acid. To facilitate the detection of metabolites in ToF SIMS datasets, a series of metabolites standard spectra were acquired. Hypoxic metabolite markers detected in ToF-SIMS data of cell lysates included glycine, lactic acid and succinic acid, which were also shown to be metabolite markers in GC-MS metabolic data. Furthermore, MTS sections were imaged using ToF-SIMS to profile the chemical response to chemotherapy treatment within the oxygen gradient. Loadings from image PCA were explored to determine the metabolic response in the highly oxygenated outer region and hypoxic inner region of the MTS. Conclusion: A multitude of analytical techniques were able to contribute to elucidating the metabolic mechanisms associated with hypoxia-induced chemoresistance. Metabolic profiling combined with a systems biology approach was further able to identify potential underlying metabolic regulation of resistance. Finally ToF-SIMS was developed as a tool for metabolite analysis in complex biological systems in situ.

Imaging Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) has been developed to perform 2D imaging and depth profiling of biological systems with micron or submicron scale lateral resolution, which can be attributed to the advent of polyatomic ion beam particularly C60+ and new concept of ToF-SIMS instrument, the J105 3D Chemical Imager (J105). These recent advances in ToF-SIMS have opened a new dimension for biological analysis. In this study, 2D and 3D imaging have been performed on two biological systems, Xenopus laevis (X. laevis) zygote/embryo and murine embryonic fibroblasts NIH 3T3 BXB-ER cells to explore the capability of ToF-SIMS to handle the biological samples with extreme topography and high resolution depth profiling of microdomains, which still represent major challenges for the ToF-SIMS. The study on X. laevis embryo explored the capability of ToF-SIMS to handle spherical samples (approx. 1-1.2 mm in diameter), identify lipid species in mixtures of lipid extraction from the zygotes and image of an intact embryo in 2D/3D during dynamic biological events, e.g., fertilisation and early embryo development. For the first time the J105 and conventional BioToF-SIMS instrument were employed for the study of developmental biology. The major classes of lipid were identified through multiple lipid assay in a single analytical run using ToF-SIMS. Topography effects of the embryo were assessed through imaging a single intact zygote/embryo that revealed secondary ions loss at the edge of the single cell. However, the topography effects on the mass resolution could be minimised using the J105. Moreover, in situ lipid profiling of the zygote revealed different lipid compositions and intensities on the membrane of the animal and vegetal hemispheres. Furthermore, high resolution imaging and depth profiling that performed on a single intact cell in a time course study visualised the egg-sperm fusion sites on the membrane of the zygote 10 min post-insemination and lipids arrangement on the membrane of the embryo through the early development stages. Subcellular signalling upon the fertilisation was also spatially located on the serial cryosections of a single zygote. With the NIH 3T3 BXB-ER cells, the study firstly adopted a finely focused C60+ beam to track morphological changes and rearrangement of subcellular organelle mitochondria (0.5-2 µm) in response to the activation of Raf/ERK (extracellular signal regulated kinase) pathway using the J105. The SIMS images of the unlabelled cells showed the shifting of membrane distribution and nuclei shrinking following Raf/ERK activation. The mitochondria fluorescence probe within the cells were located 3-dimensionally using confocal microscopy and ToF-SIMS, which revealed the distribution pattern of condensing in the two sides of the nuclei following the Raf/ERK activation. Coupled with scanning electron microscopy (SEM), the three imaging modes showed good agreement in cellular morphological changes and subcellular mitochondrial rearrangement without or following Raf/ERK activation, demonstrating an integrated approaching to study the biological processes at subcellular dimension.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) in principle has the capability to detect and localise complex chemistry on the sub-cellular scale. Robust sample preparation protocols are therefore of great importance when attempting to preserve the natural biochemistry and architecture of biological specimens and extract meaningful information. It is likely that the optimum methodology will depend on the nature of the sample, and the goal of the analysis. The following study focussed on the preparation of cancer cells for ToF-SIMS analysis. Air drying, freeze fracturing, freeze drying and cytospinning-based preparation techniques and variations thereof were evaluated. Using image contrast, assessing spectra for the gain, or loss of native and non-native cellular species and the ratio of inorganic to organic ion yields, the investigation provides a systematic evaluation of the features of each preparation technique. This study then employed the optimum methods to determine the influence of the various phases of the cell cycle on classification of ToF-SIMS spectral data. A number of cell types, both cancerous and non-cancerous in G1, S and G2/M growth phases are separated by anticancer drug treatment and FACS (fluorescence activated cell sorting) and subjected to ToF-SIMS analysis. The results indicate that ToF-SIMS can detect surface biochemical changes induced by the cell cycle. Importantly it is also capable of detecting subtle alterations in cellular chemistry caused by anticancer drugs. Furthermore, the ToF-SIMS data was compared to results obtained from synchrotron sourced FTIR microspectroscopy to provide a more robust complementary analysis and support chemical evidence gathered.

Time-of-flight the secondary ion mass spectrometry (ToF-SIMS) is becoming a promising technique in analysis of a biological system due to its chemical specificity and the ability to perform high spatial resolution imaging. The combination of novel cluster and polyatomic beams has allowed generating images to map the distribution of biological components in tissue sections and cell surfaces. However, under these conditions the cluster beams have to be operated in the static regime, which limits sensitivity and confines the molecular imaging to 2 μm spatial resolution. The polyatomic beams offer the benefits of high yields and low sub-surface damage, allowing the analysis to be performed at high ion fluence and abandoning the static limit. This presents a new approach to molecular imaging in which 'voxels' are used rather than pixels, thus increasing sensitivity. As a result, the current SIMS instrumentation then becomes a limitation and has to be modified. A novel SIMS instrument, the J105 3D Chemical Imager has been developed with Ionoptika, which allows taking full advantage of the polyatomic primary beam, particularly the C60 by using it in a dc mode with buncher-ToF configuration. The aim of this project was to prove the concept and the potential of this new instrument. Initially standard organic samples have been used to show the current performance of the J105 for the analysis of organic samples with respect to a conventional ToF-SIMS instrument, the BioToF. The tandem MS capability of the instrument has been tested and proved using standard samples and a lipid mixture of brain extract. HeLa cells, an immortalised cell line were analysed using this instrument in 2D and 3D imaging mode. The images generated show molecular localisation on a sub-cellular scale, over a practical timeframe, whilst sustaining high mass resolution at 4000. The cells were imaged using a 40 keV C60+ dc beam and a clear differentiation between the material within the nuclei and lipid membrane can be made. Investigation of cell preparation suggested that the frozen-hydrated approach may be beneficial. The data presented in this thesis validates the new instrument concept offering the advantages of higher mass detection, increase in sensitivity, and an increase in the duty cycle while at the same time allowing imaging at sub-cellular scale with higher mass resolution.