Periodic Reporting for period 4 - ASSIMILES (Advanced Spectroscopy and Spectrometry for Imaging Metabolism using Isotopically-Labeled Endogenous Substrates)
Periodo di rendicontazione: 2021-03-01 al 2022-04-30
A technological revolution is currently taking place making it possible to noninvasively probe metabolism in mammals (incl. humans) in vivo with unprecedented temporal and spatial resolution. Central to these developments is the phenomenon of hyperpolarization, which transiently enhances the magnetic resonance (MR) signals so much that real-time metabolic imaging becomes possible. The first clinical translations of hyperpolarized (HP) MR technology has recently been demonstrated in patients with cancer. However, important obstacles still exist for the technology to fulfill its enormous potential and become an integral part of patient care in radiology departments.
With this highly interdisciplinary project, we aimed at overcoming the principal drawbacks of current hyperpolarization technology, namely: 1) a very short time window between the polarization and injection of HP probes; 2) the conventional use of synthetic polarizing agents that need to be filtered out prior to injection; 3) the necessity to use supra-physiological doses of metabolic substrates to reach detectable MR signal.
We have developed a novel methodology based on the use of photo-excited molecules as polarizing agents, including custom-designed instruments, to produce HP probes containing exclusively endogenous compounds. We have demonstrated that, based on this novel technology, metabolic probes more sensitive than the ones currently used in patients can be prepared without the use of exogenous synthetic molecules. The preparation of these exclusively endogenous probes does not require any filtration prior to injection into humans and they can also be stored and transported, reducing the burden and costs for clinical applications of metabolic imaging by MR imaging (MRI). A complementary isotope imaging method based on mass spectrometry was used to reveal, in tissue sections, the localization of metabolic products formed from the injected probes with subcellular spatial resolution. This method was used to highlight the upregulated metabolic pathways in cancer models, aiming at better understanding the contrast measured in vivo with HP MRI.
With this highly interdisciplinary project, we aimed at overcoming the principal drawbacks of current hyperpolarization technology, namely: 1) a very short time window between the polarization and injection of HP probes; 2) the conventional use of synthetic polarizing agents that need to be filtered out prior to injection; 3) the necessity to use supra-physiological doses of metabolic substrates to reach detectable MR signal.
We have developed a novel methodology based on the use of photo-excited molecules as polarizing agents, including custom-designed instruments, to produce HP probes containing exclusively endogenous compounds. We have demonstrated that, based on this novel technology, metabolic probes more sensitive than the ones currently used in patients can be prepared without the use of exogenous synthetic molecules. The preparation of these exclusively endogenous probes does not require any filtration prior to injection into humans and they can also be stored and transported, reducing the burden and costs for clinical applications of metabolic imaging by MR imaging (MRI). A complementary isotope imaging method based on mass spectrometry was used to reveal, in tissue sections, the localization of metabolic products formed from the injected probes with subcellular spatial resolution. This method was used to highlight the upregulated metabolic pathways in cancer models, aiming at better understanding the contrast measured in vivo with HP MRI.
We implemented a new set of instruments to prepare HP molecules by dynamic nuclear polarization (DNP) for metabolic imaging, in particular a stand-alone hyperpolarizer containing a cryogen-free magnet that can be operated at any field up to 7 T and does not require the use of liquid helium, an expensive consumable. This novel system is by far the most performant preclinical hyperpolarizer that has been developed to date, providing up to three doses of HP molecules with more than 50% carbon-13 (13C) polarization. It will be a model for the future commercial systems. It was used for both in vitro and in vivo HP MR experiments.
We demonstrated that most keto acids, in particular pyruvic acid and phenylglyoxylic acid, which are endogenous molecules, have the propensity to yield radicals under photo-excitation in the ultraviolet-visible spectrum. These non-persistent radicals play the role of polarizing agent and they disappear when the HP molecules are brought to room-temperature, forming a small amount of endogenous byproduct. While investigating these keto acids, we also unravelled the key photochemical mechanism enabling the creation of non-persistent radicals in pyruvic acid. This had remained elusive despite more than 85 years of research.
We investigated the feasibility of reducing the pyruvate dose used for in vivo HP MR experiments. In particular, we demonstrated that that liver metabolism can be measured in vivo with HP pyruvate administered at near basal plasma concentration. We showed that hepatic gluconeogenesis can be directly probed in vivo with HP pyruvate. We also investigated the potential of glucose and lactate as HP substrates for in vivo metabolic studies at physiological level. More specifically, we demonstrated that it is possible to measure cerebral glucose metabolism in vivo with sub-second time resolution. This unique method allows direct detection of glycolysis in vivo in the healthy brain in a noninvasive manner. We also studied lactate uptake and intracellular metabolism in the brain. We demonstrated that HP lactate can be used to detect a variation in cerebral lactate uptake of <40 nmol in a healthy brain during an in vivo experiment lasting only 75 s.
We proposed a new ex vivo application of HP MR to detect the metabolic changes induced by the activation of immune cells. We demonstrated that the metabolic adaptation triggered by human T lymphocytes stimulation can be rapidly and noninvasively detected by HP MR. We imaged, with a sub-cellular resolution, the 13C enrichment in macromolecules following the injection of 13C-glucose in tumors, highlighting the higher 13C enrichment in the tumor region as compared with healthy tissue.
In terms of dissemination, these results led to the publication of 13 articles in scientific journals, 4 book chapters, 21 conference abstracts, and I gave 13 invited talks at international conferences. We also submitted two patent applications and are aiming at exploiting the technology developed in this project to make HP MR a clinically viable modality.
We demonstrated that most keto acids, in particular pyruvic acid and phenylglyoxylic acid, which are endogenous molecules, have the propensity to yield radicals under photo-excitation in the ultraviolet-visible spectrum. These non-persistent radicals play the role of polarizing agent and they disappear when the HP molecules are brought to room-temperature, forming a small amount of endogenous byproduct. While investigating these keto acids, we also unravelled the key photochemical mechanism enabling the creation of non-persistent radicals in pyruvic acid. This had remained elusive despite more than 85 years of research.
We investigated the feasibility of reducing the pyruvate dose used for in vivo HP MR experiments. In particular, we demonstrated that that liver metabolism can be measured in vivo with HP pyruvate administered at near basal plasma concentration. We showed that hepatic gluconeogenesis can be directly probed in vivo with HP pyruvate. We also investigated the potential of glucose and lactate as HP substrates for in vivo metabolic studies at physiological level. More specifically, we demonstrated that it is possible to measure cerebral glucose metabolism in vivo with sub-second time resolution. This unique method allows direct detection of glycolysis in vivo in the healthy brain in a noninvasive manner. We also studied lactate uptake and intracellular metabolism in the brain. We demonstrated that HP lactate can be used to detect a variation in cerebral lactate uptake of <40 nmol in a healthy brain during an in vivo experiment lasting only 75 s.
We proposed a new ex vivo application of HP MR to detect the metabolic changes induced by the activation of immune cells. We demonstrated that the metabolic adaptation triggered by human T lymphocytes stimulation can be rapidly and noninvasively detected by HP MR. We imaged, with a sub-cellular resolution, the 13C enrichment in macromolecules following the injection of 13C-glucose in tumors, highlighting the higher 13C enrichment in the tumor region as compared with healthy tissue.
In terms of dissemination, these results led to the publication of 13 articles in scientific journals, 4 book chapters, 21 conference abstracts, and I gave 13 invited talks at international conferences. We also submitted two patent applications and are aiming at exploiting the technology developed in this project to make HP MR a clinically viable modality.
We developed a novel methodology to hyperpolarize molecules using photoinduced radicals, including a complete workflow for in vivo HP MR experiments. A central part of this methodology is based on the efficient generation of photoinduced radicals inside frozen solutions containing alpha-keto acids. These radicals are required for hyperpolarization. We showed that besides pyruvic acid, two alpha-keto acids are particularly interesting for HP MR experiments: phenylglyoxylic acid and alpha-keto glutaric acid. They are both endogenous molecules with an excellent safety profile for in vivo experiments and they can be used as radical precursor to hyperpolarize any molecule of interest. Non-persistent radicals are formed upon photoirradiation of these precursors at 77K and they disappear when the HP molecules are brought to room-temperature, forming a small amount of endogenous byproduct. We demonstrated that at high magnetic field (7T), the polarization obtained with these photoinduced radicals is comparable to that achieved in samples doped with synthetic radical molecules. We also showed that the absence of radicals in the solution containing the HP molecules will simplify the translation to clinical use, as no radical filtration is required prior to injection.
We also demonstrated that it is possible to polarize and quench the radicals that are photoinduced in alpha-keto acids by thermalizing the frozen sample containing the alpha-keto acids above 200K without melting it. We showed that the polarized solid can be subsequently extracted from the hyperpolarizer and melted ex situ without substantial losses in carbon-13 polarization. We implemented a new insert to optimize the thermalization and extraction in a newly developed cryogen-free polarizer. We showed that the rapid radical quench provides a great potential for storage and transport of HP molecules due to an extended life time of the polarization, many hours at low temperature.
We also demonstrated that it is possible to polarize and quench the radicals that are photoinduced in alpha-keto acids by thermalizing the frozen sample containing the alpha-keto acids above 200K without melting it. We showed that the polarized solid can be subsequently extracted from the hyperpolarizer and melted ex situ without substantial losses in carbon-13 polarization. We implemented a new insert to optimize the thermalization and extraction in a newly developed cryogen-free polarizer. We showed that the rapid radical quench provides a great potential for storage and transport of HP molecules due to an extended life time of the polarization, many hours at low temperature.