31P-CEST: A new diagnostic tool for MRI based Molecular Imaging

For the last decades, preclinical and clinical oncology diagnostics have made extensive use of exogenous contrast agents. Despite these substances having proven their efficiency for imaging tumors and diseased tissues, the issue of their environmental disposal has arisen. Additionally, using these compounds, for instance, heavy metal-based shift reagents may cause major health problems for patients suffering from renal failure making it impossible for them to perform MRI exams. This work aims to find valid alternatives to avoid the high concentration of injected contrast agents and to reduce the polluting impact derived from their employment. The focus of this research has been based on how to implement an established magnetic resonance technique such as the Chemical Exchange Saturation Transfer, by exploiting the heteronuclear application of the method and reporting a new way of evaluating metabolism through the shift of the bulk signal of exchange from water to inorganic phosphate (Pi). In the first part of this thesis, we reported for the very first-time system in vitro for the performance of CEST on a bulk of Pi using a paramagnetic complex, found to have a high affinity for the phosphate group and an effective role as a shift reagent. The effectiveness of the system resulted in the decrease of the detection threshold of three orders of magnitude (from mM to M range) and the use of a lower concentration of contrast agents with a proof of concept for cell labelling. In the second part of this project, we pursued an even more challenging goal: to eliminate the need for contrast agents or exogenous tracers to monitor glycolysis deregulation, a hallmark of many pathologies, using endogenous 31P CEST. In vitro experiments were run on murine mammary carcinoma cell lines, leading to the acquisition of fingerprint Z-spectra at 14 T by comparing the chemical exchange efficiency, allowing for the discrimination in the exchange outcome of the phosphate group of the glycolytic molecules with the bulk signal of intracellular Pi. The different 5 shape of the spectra was discovered to be linked to the intrinsic aggressiveness of the various cell lines, linking the spectral features with tumor malignancy. Inhibition experiments, along with different temperature settings, were also carried out to confirm the involvement of the glycolysis exchange pathway. The promising results obtained in vitro led to the translation of the method in vivo on a preclinical 7T MRI scanner. The exchange of the phosphate group was reported for -ATP and Phosphocreatine (PCr) bulk, due to the low intensity of the Pi on the 31P spectrum in vivo. We began with the acquisition of the 31P signal in a “whole mouse” setting, obtaining a fingerprint on healthy subjects using -ATP resonance as exchange bulk. Inhibition experiments were carried out and led to positive outcomes on the differences in glycolytic exchange trend. We moved forward to a CEST-localized asset. The implementation of the localization protocol led to the acquisition of CEST spectra using PCr as the bulk of exchange, to detect glycolysis alterations in mammary carcinoma compared to healthy tissues. Overall, this thesis reports the discovery and advancement of a heteronuclear CEST methodology that employs the ³¹P nucleus to diminish the concentration of the bulk pool of exchange. This development allowed for the use of considerably reduced doses of contrast agents, proving that inorganic phosphate (Pi) is an efficient exchange pool at a concentration of three orders of magnitude less than that of water, and drove the application of the technique for the endogenous metabolic characterization of cancer cells in vitro, ultimately leading to its translation in vivo.