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Functional tumour genomics using metabolomic profiling

Final Report Summary - FUTUGEMET (Functional tumour genomics using metabolomic profiling)

The metabolic characterisation of tumour cells before and after treatment with agents that induce cell death was the main objective of the project. The results obtained will provide a better understanding of the relationship between tumour cell metabolism and putative molecular imaging tracers, allowing a better evaluation and characterization of a given tumour. The work was divided in two parts. On one side, the effect of different pathways on tumour cell metabolism was analysed. For that, different genes were knocked down using a lentiviral system in several cancer cell lines. Evaluation of the knock-downs was performed by quantitative PCR and, in some cases, also by Western blot. To be able to compare the metabolic profiles of the different knocked-down cell lines, proliferation rates were measured by assessing the percentage of confluence in a well over time.

After proving that the knock-down was not affecting cell proliferation, metabolite extracts were obtained and analysed by nuclear magnetic resonance spectroscopy and by gas chromatography coupled to mass spectrometry. Cells transduced with lentivirus expressing non-targeting shRNA were used as controls. The metabolic profile obtained was analysed by multivariate statistical analysis. A clear separation between the different knocked-down cell lines and the different controls was obtained, indicating that tumour cell metabolism (the cell's phenotype) correlates with changes in gene expression. This method, if used in different tumour samples or in blood from cancer patients, could provide a readout to evaluate which are the signalling pathways altered in a given patient by comparing the patient's metabolic profile and a set of metabolic profiles from different pathway alterations. The results obtained can also provide insight into new metabolites, which can be used as tracers for imaging different cell characteristics (for instance, the effect of a drug on a given oncogene or on a cell signalling pathway) or to check the response to a treatment.

The second part of the project consisted in the evaluation of proposed imaging methods to detect tumours. It has been proposed that detection of high-resolution nuclear magnetic resonance signals originating from lipid droplets in apoptotic cells could be used to image a tumour's response to treatment. On the other hand, 11C-acetate has been proposed for PET imaging of cancer as a readout of the increased lipid synthesis occurring in tumours due to increased fatty acid synthase levels. These increases in lipid synthesis during cell death and also during tumour growth prompted us to characterize the mechanism by which there is an increase in lipid droplets after induction of cell death. Studies of acetate uptake by apoptotic cells were performed to understand the mechanism of this incorporation.

It was observed that the increase in lipid accumulation was due to increased de novo lipid synthesis, although the levels and activities of the majority of enzymes involved in lipogenesis were decreased. This increase resulted from the inhibition of mitochondrial fatty acid beta-oxidation, which coupled with an increase in acyl-CoA synthetase activity, diverted fatty acids away from oxidation and into lipid synthesis. These results are of significant importance to understand 11C acetate results in in vivo imaging and need to be taken into account when measuring tumour growth. A preclinical evaluation of acetate incorporation into tumours was also performed to understand acetate metabolism and to improve the protocol for its utilization by PET. For that, C4-2b cells were transplanted into NSG mice and acetate was administered for different times. It was observed that acetate was taken up by all the tissues, although it was cleared from the body one hour after injection except in those tissues where acetate had been converted into lipids. The results obtained will contribute to a better understanding of tumour PET imaging with 11C acetate.