real-time-metabolic-detection-living-cells-using-hyperpolarized-13c

Real-Time Metabolic Detection in Living Cells Using Hyperpolarized <sup>13</sup>C NMR

The protocol presents a method to integrate an in-house-built bioreactor system with hyperpolarized 13C nuclear magnetic resonance (NMR) spectroscopy to ...

National Institutes for Quantum Science and Technology

Reoxygenation has a significant impact on the tumor response to radiotherapy. With developments in radiotherapy technology, the relevance of the reoxygenation phenomenon in treatment efficacy has been a topic of interest. Evaluating the reoxygenation in the tumor microenvironment throughout the course of radiation therapy is important in developing effective treatment strategies. In the current study, we used electron paramagnetic resonance imaging (EPRI) to directly map and quantify the partial oxygen pressure (pO ) in tumor tissues. Human colorectal cancer cell lines, HT29 and HCT116, were used to induce tumor growth in female athymic nude mice. Tumors were irradiated with 3, 10, or 20 Gy using an x-ray irradiator. Prior to each EPRI scan, magnetic resonance imaging (MRI) was performed to obtain T2-weighted anatomical images for reference. The differences in the mean pO were determined through two-tailed Student's t-test and one-way analysis of variance. The median pO 60 min after irradiation was found to be lower in HCT116 than in HT29 (9.1 ± 1.5 vs. 14.0 ± 1.0 mmHg, p = 0.045). There was a tendency for delayed and incomplete recovery of pO in the HT29 tumor when a higher dose of irradiation (10 and 20 Gy) was applied. Moreover, there was a dose-dependent increase in the hypoxic areas (pO  < 10 mmHg) 2 and 24 h after irradiation in all groups. In addition, an area that showed pO fluctuation between hypoxia and normoxia (pO  > 10 mmHg) was also identified surrounding the region with stable hypoxia, and it slightly enlarged after recovery from acute hypoxia. In conclusion, we demonstrated the reoxygenation phenomenon in an in vivo xenograft model study using EPRI. These findings may lead to new knowledge regarding the reoxygenation process and possibilities of a new radiation therapy concept, namely, reoxygenation-based radiation therapy.

Continuous monitoring of postirradiation reoxygenation and cycling hypoxia using electron paramagnetic resonance imaging.

Imaging tumor microenvironments such as hypoxia, oxygenation, redox status, and/or glycolytic metabolism in tissues/cells is useful for diagnostic and prognostic purposes. New imaging modalities are under development for imaging various aspects of tumor microenvironments. Electron Paramagnetic Resonance Imaging (EPRI) though similar to NMR/MRI is unique in its ability to provide quantitative images of pO in vivo. The short electron spin relaxation times have been posing formidable challenge to the technology development for clinical application. With the availability of the narrow line width trityl compounds, pulsed EPR imaging techniques were developed for pO imaging. EPRI visualizes the exogenously administered spin probes/contrast agents and hence lacks the complementary morphological information. Dynamic nuclear polarization (DNP), a phenomenon that transfers the high electron spin polarization to the surrounding nuclear spins (H and C) opened new capabilities in molecular imaging. DNP of C nuclei is utilized in metabolic imaging of 13C-labeled compounds by imaging specific enzyme kinetics. In this article, imaging strategies mapping physiologic and metabolic aspects in vivo are reviewed within the framework of their application in cancer research, highlighting the potential and challenges of each of them.

EPR and Related Magnetic Resonance Imaging Techniques in Cancer Research.

Peptides play essential roles in biological phenomena, and, thus, there is a growing interest in detecting in vivo dynamics of peptide metabolisms. Dissolution-dynamic nuclear polarization (d-DNP) is a state-of-the-art technology that can markedly enhance the sensitivity of nuclear magnetic resonance (NMR), providing metabolic and physiological information in vivo. However, the hyperpolarized state exponentially decays back to the thermal equilibrium, depending on the spin-lattice relaxation time (). Because of the limitation in , peptide-based DNP NMR molecular probes applicable in vivo have been limited to amino acids or dipeptides. Here, we report the direct detection of in vivo metabolic conversions of hyperpolarized C-oligopeptides. Structure-based  relaxation analysis suggests that the C-terminal [1-C]Gly- residue affords sufficient  for biological uses, even in relatively large oligopeptides, and allowed us to develop C-β-casomorphin-5 and C-glutathione. It was found that the metabolic response and perfusion of the hyperpolarized C-glutathione in the mouse kidney were significantly altered in a model of cisplatin-induced acute kidney injury.

Directly monitoring the dynamic in vivo metabolisms of hyperpolarized C-oligopeptides.

Abnormalities and alterations in the glycolytic pathway in the pathology of neurodegenerative diseases and brain aging have received much attention, as clinical applications of proton-based magnetic resonance spectroscopy (MRS) have recently illuminated the elevation of lactate concentrations in the brains of patients with neurodegenerative diseases, including Alzheimer's disease. Hyperpolarized [1-C]pyruvate MRS has shown promise for neurological applications because it enables the real-time  detection of glycolysis and oxidative phosphorylation flux. In studies of the mouse brain using hyperpolarized [1-C]pyruvate, there are few reports that the signal of [C]bicarbonate, a product of oxidative phosphorylation metabolized from [1-C]pyruvate, was detected using MR spectroscopic imaging (MRSI) that allows spatial mapping of metabolism, although there have been reports of [C]bicarbonate signals being detected by pulse-acquire sequences in the entire brain. In the present study, we compared hyperpolarized [1-C]pyruvate metabolism between the brains of awake and isoflurane-anesthetized mice using a custom-made awake mouse restraint device with MRSI. Although the signal for [1-C]lactate, a product of glycolysis metabolized from [1-C]pyruvate, was detectable in multiple brain regions that include the orbitofrontal cortex and hippocampus in both awake and anesthetized mice, the signal for [C]bicarbonate metabolized from [1-C]pyruvate was only detectable in the brains of awake mice. Moreover, a comparison of hyperpolarized [1-C]pyruvate metabolism in young and aged mouse brains using awake MRSI detected age-related decreases in oxidative phosphorylation flux in brain regions that include the hippocampus with variations in the extent of these changes across different brain regions. These results demonstrate that hyperpolarized [1-C]pyruvate MRSI under awake conditions is useful for the spatial detection of abnormalities and alterations in glycolysis and oxidative phosphorylation flux in the brains of mice. Thus, the use of hyperpolarized [1-C]pyruvate MRSI has potential in pathological and mechanistic studies of brain diseases and brain aging.

Awake brain MRSI reveals anesthetic sensitivity and regional aging effects on [C]bicarbonate metabolism in mice.

Hyperpolarized (HP) [1-C]pyruvate nuclear magnetic resonance (NMR) spectroscopy was employed to investigate tumor energy metabolism in microscale multicellular spheroids of a few hundred micrometers in diameter, serving as a model of early-phase tumorigenesis in vivo. A three-dimensional static culture of murine squamous cell carcinoma (SCCVII) cells formed uniform smaller multicellular spheroids (~ 150 μm in diameter), without hypoxic or necrotic cores, yet these spheroids exhibited resistance to anti-tumor drugs. HP [1-C]pyruvate NMR spectroscopy of SCCVII spheroids revealed an increased conversion of pyruvate to lactate compared to monolayer cultures, indicating enhanced aerobic glycolysis in the aggregated cells. Additionally, HP spectroscopy differentiated the degree of aerobic glycolysis in human prostate tumor spheroids-DU145 (~ 120 μm) and PC-3 (~ 230 μm)-as evidenced by the upregulation of genes associated with lactate production and cellular transport. The Lac/Pyr ratio among spheroids correlated with those observed in homogenate samples of corresponding tumors grown in mice. These findings suggest that HP [1-C]pyruvate NMR spectroscopy may serve as a metabolic biomarker for early-phase tumorigenesis in vivo.

Article	Year
Continuous monitoring of postirradiation reoxygenation and cycling hypoxia using electron paramagnetic resonance imaging. NMR in biomedicine\| PubMed ID: 35661282	2022
EPR and Related Magnetic Resonance Imaging Techniques in Cancer Research. Metabolites\| PubMed ID: 36676994	2023
Directly monitoring the dynamic in vivo metabolisms of hyperpolarized C-oligopeptides. Science advances\| PubMed ID: 39413185	2024
Awake brain MRSI reveals anesthetic sensitivity and regional aging effects on [C]bicarbonate metabolism in mice. Frontiers in neuroimaging\| PubMed ID: 40017822	2025
Hyperpolarized [1-C]pyruvate NMR spectroscopy reveals transition of tumor energy metabolism in microscale multicellular spheroids. Scientific reports\| PubMed ID: 40456829	2025

Keita Saito

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