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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - ASSIMILES (Advanced Spectroscopy and Spectrometry for Imaging Metabolism using Isotopically-Labeled Endogenous Substrates)

Teaser

Summary

A technological revolution is currently taking place making it possible to noninvasively study 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 and spectroscopy becomes possible. The first clinical translations of hyperpolarization MR technology has recently been demonstrated in cancer patients.

We have played an active role in these exciting developments, through design and construction of hyperpolarization MR setups that are defining the cutting-edge for in vivo preclinical metabolic studies. However, important obstacles still exist for the technology to fulfill its enormous potential.

With this highly interdisciplinary project, we aim at overcoming the principal drawbacks of current hyperpolarization technology, namely: 1) A limited time window for hyperpolarized MR detection; 2) the conventional use of potentially toxic polarizing agents; 3) the necessity to use supra-physiological doses of metabolic substrates to reach detectable MR signal.

We will develop a novel hyperpolarization instrument making use of photo-excited compounds as polarizing agents to produce hyperpolarized solutions containing exclusively endogenous compounds. It will become possible to deliver hyperpolarized solutions in a quasi-continuous manner, permitting infusion of physiological doses and greatly increasing sensitivity. I will also use a complementary isotope imaging technique, the so-called CryoNanoSIMS, which can image isotopic distributions in frozen tissue sections and reveal the localization of injected substrates and their metabolites with subcellular spatial resolution. Case studies will include liver and brain cancer models. This work will create a new frontier in molecular imaging, with a high potential for clinical translation.

Work performed

We have implemented a new set of instruments to prepare hyperpolarized 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. We demonstrated that when operating at 7 T and a temperature of 1.4 K, this instrument allows us to polarize 13C-molecules to more than 30%, which is higher than in the commercial preclinical hyperpolarizer. This hyperpolarizer, which is now fully operational can be used for in vitro and in vivo experiments.

We have demonstrated that most keto acids, in particular pyruvic acid, alpha-ketoisocaproic acid, and phenylglyoxylic acid, which are all 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 hyperpolarized molecules are brought to room-temperature, forming a small amount of endogenous byproduct. We showed that the absence of radicals in the solution containing the hyperpolarized 13C-substrates may simplify the translation to clinical use, as no radical filtration is required prior to injection.

We have investigated the potential of glucose and lactate as hyperpolarized 13C-substrates for in vivo metabolic studies at physiological level. We have demonstrated that it is possible to measure cerebral glucose metabolism in vivo with sub-second time resolution. In particular, the dynamic 13C-labeling of pyruvate and lactate formed from 13C-glucose was observed in real time. This unique method allows direct detection of glycolysis in vivo in the healthy brain in a noninvasive manner. We have also studied lactate uptake and intracellular metabolism in the mouse brain using hyperpolarized 13C MR. Following the intravenous injection of hyperpolarized 13C-lactate, we observed that the distribution of the 13C label between lactate and pyruvate can be linked to a higher level of cerebral lactate dehydrogenase A (Ldha) expression.

We have also imaged, with a sub-cellular resolution, the 13C-enrichment in macromolecules following the injection of 13C-glucose in a brain cancer model exhibiting increased lactate level. NanoSIMS analyses showed a nearly two-fold higher 13C enrichment in the tumor region as compared with the contralateral hemisphere demonstrating that not only glycolysis was upregulated in the brain tumor region but that glucose was also used for biosynthesis. Since cell nuclei were more 13C-enriched in the glioma cells than in healthy neurons and astrocytes, we conclude that the flux through the pentose phosphate pathway was significantly higher in the tumor.

Final results

We have demonstrated that it is possible to polarize and quench the radicals that have been photo-induced in pyruvic acid by thermalizing the sample above 200K without melting it using helium gas. This method can be extended to all keto acids that have been photo-irradiated. We have also shown that the polarized solid can be subsequently extracted from the hyperpolarizer and melted ex situ without substantial losses in 13C polarization. This rapid radical quench provides a great potential for storage and transport of hyperpolarized substrates due to an extended solid-state nuclear spin relaxation time constant, reaching times on the order of many hours. By coupling this rapid thermalization method and low-temperature DNP with photo-induced radicals, it becomes possible to produce highly polarized solid consisting of exclusively endogenous substrates for metabolic imaging. This opens the way to the development of methods to prepare hyperpolarized substrates for biomedical applications without using exogenous substances. This technology will be further developed to produce nearly continuous flow of hyperpolarized solutions that could eventually be used for metabolic imaging in humans.

We have developed a light-weight system for transferring vitrified biological samples among imaging modalities, even over significant (i.e. 200 km) distances. More specifically, this transport allows transferring frozen vitrified samples from a cryo-electron microscope to our newly developed cryo-NanoSIMS. This system allows contamination-free sample transportation between different imaging modalities. We aim at using these technical advances to perform ex vivo metabolic imaging at sub-cellular resolution in intact frozen mammalian tissue.