Nuclear magnetic resonance spectroscopy based metabolomics of breast cancer in hypoxia

Hypoxia has emerged as a crucial part of the aetiology of tumours. It is a negative prognostic factor which is associated to chemoresistance, invasiveness and metastasis. There is a strong association between hypoxia and metabolic transformation in breast cancer due to the alterations of multiple metabolic pathways. However, the current understanding of the nature of metabolic alterations in hypoxia is insufficient. This thesis uses NMR as a tool to investigate both the static metabolome by measuring metabolite concentrations, as well as to determine \(^1\)\(^3\)C metabolic fluxes using stable isotope tracers to reveal metabolic pathway alterations by hypoxia \(in\) \(vitro\) and by tumour growth \(in\) \(vivo\). Firstly, we developed the \(^1\)\(^3\)C isotopomer distribution (CID) analysis to quantify metabolic fluxes by following the evolution of specific isotopomers of specific pathways of interests. MCF7 breast cancer cells were analysed in hypoxia using an integrated approach using gene expression, steady-state metabolite levels and \(^1\)\(^3\)C metabolic flux analysis to pinpoint hypoxia induced metabolic alterations. These most significant alterations were an up-regulation of the pentose phosphate pathway and a down-regulation of mitochondrial oxidative metabolism by lowering the PDH flux. The latter was partially compensated by carbon entry into the mitochondria by increasing flux through pyruvate carboxylase (PC). Further attention was focused towards identifying the shifts in metabolic activity in PC altered cells using [1,2-\(^1\)\(^3\)C]glucose and [3-\(^1\)\(^3\)C]glutamine as precursor nutrients correlated to cellular transformation potential accessed by cell viability. Finally, the \(^1\)\(^3\)C labelled glucose strategy was applied to a cancer model in mice model by infusing mice with [1,2-\(^1\)\(^3\)C]glucose. \(^1\)\(^3\)C glucose administration protocol was optimised in order to enable an investigation of \(^1\)\(^3\)C metabolic fluxes in tumour tissue to identify metabolic pathway differences between earlier stage and advanced stage of mammary gland tumours. In conclusion, an NMR based metabolomics analysis is suitable for discovering metabolic pathway alterations using both \(in\) \(vitro\) and \(in\) \(vivo\) models