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Magnetic resonance spectroscopy (MRS) methods offer a potentially valuable window into cellular metabolism. Measurement of flux between inorganic phosphate (Pi) and ATP using (31)P MRS magnetization transfer has been used in resting muscle to assess what is claimed to be mitochondrial ATP synthesis and has been particularly popular in the study of insulin effects and insulin resistance. However, the measured Pi→ATP flux in resting skeletal muscle is far higher than the true rate of oxidative ATP synthesis, being dominated by a glycolytically mediated Pi↔ATP exchange reaction that is unrelated to mitochondrial function. Furthermore, even if measured accurately, the ATP production rate in resting muscle has no simple relationship to mitochondrial capacity as measured either ex vivo or in vivo. We summarize the published measurements of Pi→ATP flux, concentrating on work relevant to diabetes and insulin, relate it to current understanding of the physiology of mitochondrial ATP synthesis and glycolytic Pi↔ATP exchange, and discuss some possible implications of recently reported correlations between Pi→ATP flux and other physiological measures. [ABSTRACT FROM AUTHOR]

Purpose: In humans, 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) tumour-to-background contrast continues to increase long after a typical uptake period of 45 - 60 min. Similar studies have not been performed in mice and the static imaging time point for most studies is arbitrarily set at 30 - 60 min post-injection of [ 18 F]FDG. Ideally, static PET imaging should be performed after the initial period of rapid uptake but this period has not been defined in mice, with previous dynamic studies in mice being limited to 60 min. This study aimed to define the kinetics of [ 18 F]FDG biodistribution over periods of 3 - 4 h in different murine tumour models, both subcutaneous and autochthonous, and to further refine fasting and warming protocols used prior to imaging., Procedures: Dynamic [ 18 F]FDG PET-CT scans lasting 3 or 4 h were performed with C57BL/6 J and Balb/c nude mice bearing subcutaneous EL4 murine T-cell lymphoma and Colo205 human colorectal tumours, respectively, and with transgenic Eμ-Myc lymphoma mice. Prior to [ 18 F]FDG injection, four combinations of different animal handling conditions were used: warming for 1 h at 31 °C; maintenance at room temperature (20 - 24 °C), fasting for 6 - 10 h and a fed state., Results: Tumour mean standardised uptake value (SUV mean ) peaked at 147 ± 48 min post injection in subcutaneous tumours and 74 ± 31 min in autochthonous Eμ-Myc lymphomas. The tumour-to-blood ratio (TBR) peaked at 171 ± 57 and 83 ± 33 min in subcutaneous and autochthonous Eμ-Myc tumours, respectively. Fasting increased tumour [ 18 F]FDG uptake and suppressed myocardial uptake in EL4 tumour-bearing mice. There was a good correlation between tumour SUV mean and K i calculated using an input function (IDIF) derived from the inferior vena cava., Conclusions: Delayed static [ 18 F]FDG-PET imaging (> 60 min) in both autochthonous and subcutaneous tumours in improved tumour-to-background contrast and increased reproducibility., Competing Interests: Declarations Ethical Approval All applicable institutional and/or national guidelines for the care and use of animals were followed. Conflict of Interest The authors declare that they have no conflict of interest., (© 2024. Crown.)

Purpose: Serine is a major source of one-carbon units needed for the synthesis of nucleotides and the production of intramitochondrial nicotinamide adenine dinucleotide phosphate (NADPH), and it plays an important role in cancer cell proliferation. The aim of this study was to develop a deuterium ( 2 H) MRS imaging method for imaging tumor serine metabolism., Methods: Sequential ( 2 H) spectra and spectroscopic images were used to monitor the metabolism of [2,3,3- 2 H 3 ]serine in patient-derived glioblastoma cells in vitro and in tumors obtained by their orthotopic implantation in mouse brain., Results: [14,14- 2 H 2 ] 5,10-methylene-tetrahydrofolate, [ 2 H]glycine, [ 2 H]formate, and labeled water were detected in cell suspensions and water labeling in spectroscopic images of tumors. Studies in cells and tumors with variable mitochondrial content and inhibitor studies in cells demonstrated that most of the labeled serine was metabolized in the mitochondria. Water labeling in the cell suspensions was correlated with formate labeling; therefore, water labeling observed in tumors could be used to provide a surrogate measure of flux in the pathway of one-carbon metabolism in vivo., Conclusion: The method has the potential to be used clinically to select patients for treatment with inhibitors of one-carbon metabolism and subsequently to detect their early responses to such treatment., (© 2024 The Author(s). Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Metabolic subtypes of glioblastoma (GBM) have different prognoses and responses to treatment. Deuterium metabolic imaging with 2H-labeled substrates is a potential approach to stratify patients into metabolic subtypes for targeted treatment. In this study, we used 2H magnetic resonance spectroscopy and magnetic resonance spectroscopic imaging (MRSI) measurements of [6,6'-2H2]glucose metabolism to identify metabolic subtypes and their responses to chemoradiotherapy in patient-derived GBM xenografts in vivo. The metabolism of patient-derived cells was first characterized in vitro by measuring the oxygen consumption rate, a marker of mitochondrial tricarboxylic acid cycle activity, as well as the extracellular acidification rate and 2H-labeled lactate production from [6,6'-2H2]glucose, which are markers of glycolytic activity. Two cell lines representative of a glycolytic subtype and two representative of a mitochondrial subtype were identified. 2H magnetic resonance spectroscopy and MRSI measurements showed similar concentrations of 2H-labeled glucose from [6,6'-2H2]glucose in all four tumor models when implanted orthotopically in mice. The glycolytic subtypes showed higher concentrations of 2H-labeled lactate than the mitochondrial subtypes and normal-appearing brain tissue, whereas the mitochondrial subtypes showed more glutamate/glutamine labeling, a surrogate for tricarboxylic acid cycle activity, than the glycolytic subtypes and normal-appearing brain tissue. The response of the tumors to chemoradiation could be detected within 24 hours of treatment completion, with the mitochondrial subtypes showing a decrease in both 2H-labeled glutamate/glutamine and lactate concentrations and glycolytic tumors showing a decrease in 2H-labeled lactate concentration. This technique has the potential to be used clinically for treatment selection and early detection of treatment response., Significance: Deuterium magnetic resonance spectroscopic imaging of glucose metabolism has the potential to differentiate between glycolytic and mitochondrial metabolic subtypes in glioblastoma and to evaluate early treatment responses, which could guide patient treatment., (©2024 The Authors; Published by the American Association for Cancer Research.)

Hyperpolarization techniques significantly enhance the sensitivity of magnetic resonance (MR) and thus present fascinating new directions for research and applications with in vivo MR imaging and spectroscopy (MRI/S). Hyperpolarized 13 C MRI/S, in particular, enables real-time non-invasive assessment of metabolic processes and holds great promise for a diverse range of clinical applications spanning fields like oncology, neurology, and cardiology, with a potential for improving early diagnosis of disease, patient stratification, and therapy response assessment. Despite its potential, technical challenges remain for achieving clinical translation. This paper provides an overview of the discussions that took place at the international workshop "New Horizons in Hyperpolarized 13 C MRI," in March 2023 at the Bavarian Academy of Sciences and Humanities, Munich, Germany. The workshop covered new developments, as well as future directions, in topics including polarization techniques (particularly focusing on parahydrogen-based methods), novel probes, considerations related to data acquisition and analysis, and emerging clinical applications in oncology and other fields., (© 2023. The Author(s).)

Imaging agents capable of detecting the extent, timing, and distribution of tumor cell death following treatment could be used in clinical trials of novel cancer therapies to get an early indication of efficacy and subsequently in the clinic to guide treatment in individual patients. We have shown how the C2A domain of synaptotagmin I, which binds the phosphatidylserine exposed by apoptotic and necrotic cells, can be used to image cell death (Bulat et al., EJNMMI Res 10(1):151, 2020; Neves et al. J Nucl Med 58(6):881-887, 2017). We describe here the semi-automated 18 F labeling of the single cysteine residue in the protein (C2Am) that had been introduced by site-directed mutagenesis., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Imaging the metabolism of [2,3- 2 H 2 ]fumarate to produce malate can be used to detect tumor cell death post-treatment. Here, we assess the sensitivity of the technique for detecting cell death by lowering the concentration of injected [2,3- 2 H 2 ]fumarate and by varying the extent of tumor cell death through changes in drug concentration. Mice were implanted subcutaneously with human triple negative breast cancer cells (MDA-MB-231) and injected with 0.1, 0.3, and 0.5 g/kg [2,3- 2 H 2 ]fumarate before and after treatment with a multivalent TRAlL-R2 agonist (MEDI3039) at 0.1, 0.4, and 0.8 mg/kg. Tumor conversion of [2,3- 2 H 2 ]fumarate to [2,3- 2 H 2 ]malate was assessed from a series of 13 spatially localized 2 H MR spectra acquired over 65 min using a pulse-acquire sequence with a 2-ms BIR4 adiabatic excitation pulse. Tumors were then excised and stained for histopathological markers of cell death: cleaved caspase 3 (CC3) and DNA damage (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]). The rate of malate production and the malate/fumarate ratio plateaued at tumor fumarate concentrations of 2 mM, which were obtained with injected [2,3- 2 H 2 ]fumarate concentrations of 0.3 g/kg and above. Tumor malate concentration and the malate/fumarate ratio increased linearly with the extent of cell death determined histologically. At an injected [2,3- 2 H 2 ]fumarate concentration of 0.3 g/kg, 20% CC3 staining corresponded to a malate concentration of 0.62 mM and a malate/fumarate ratio of 0.21. Extrapolation indicated that there would be no detectable malate at 0% CC3 staining. The use of low and nontoxic fumarate concentrations and the production of [2,3- 2 H 2 ]malate at concentrations that are within the range that can be detected clinically suggest this technique could translate to the clinic., (© 2023 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Background: Branched-chain aminotransferase 1 (BCAT1) has been proposed to drive proliferation and invasion of isocitrate dehydrogenase ( IDH ) wild-type glioblastoma cells. However, the Cancer Genome Atlas (TCGA) dataset shows considerable variation in the expression of this enzyme in glioblastoma. The aim of this study was to determine the role of BCAT1 in driving the proliferation and invasion of glioblastoma cells and xenografts that have widely differing levels of BCAT1 expression and the mechanism responsible., Methods: The activity of BCAT1 was modulated in IDH wild-type patient-derived glioblastoma cell lines, and in orthotopically implanted tumors derived from these cells, to examine the effects of BCAT1 expression on tumor phenotype., Results: In cells with constitutively high BCAT1 expression and a glycolytic metabolic phenotype, inducible shRNA knockdown of the enzyme resulted in reduced proliferation and invasion by increasing the concentration of α-ketoglutarate, leading to reduced DNA methylation, HIF-1α destabilization, and reduced expression of the transcription factor Forkhead box protein M1 (FOXM1). Conversely, overexpression of the enzyme increased HIF-1α expression and promoted proliferation and invasion. However, in cells with an oxidative phenotype and very low constitutive expression of BCAT1 increased expression of the enzyme had no effect on invasion and reduced cell proliferation. This occurred despite an increase in HIF-1α levels and could be explained by decreased TCA cycle flux., Conclusions: There is a wide variation in BCAT1 expression in glioblastoma and its role in proliferation and invasion is dependent on tumor subtype., Competing Interests: The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

One of the hallmarks of cancer is metabolic reprogramming, including high levels of aerobic glycolysis (the Warburg effect). Pyruvate is a product of glucose metabolism, and 13 C-MR imaging of the metabolism of hyperpolarized (HP) [1- 13 C]pyruvate (HP 13 C-MRI) has been shown to be a potentially versatile tool for the clinical evaluation of tumor metabolism. Hyperpolarization of the 13 C nuclear spin can increase the sensitivity of detection by 4-5 orders of magnitude. Therefore, following intravenous injection, the location of hyperpolarized 13 C-labeled pyruvate in the body and its subsequent metabolism can be tracked using 13 C-MRI. Hyperpolarized [ 13 C]urea and [1,4- 13 C 2 ]fumarate are also likely to translate to the clinic in the near future as tools for imaging tissue perfusion and post-treatment tumor cell death, respectively. For clinical breast imaging, HP 13 C-MRI can be combined with 1 H-MRI to address the need for detailed anatomical imaging combined with improved functional tumor phenotyping and very early identification of patients not responding to standard and novel neoadjuvant treatments. If the technical complexity of the hyperpolarization process and the relatively high associated costs can be reduced, then hyperpolarized 13 C-MRI has the potential to become more widely available for large-scale clinical trials.

Deuterium metabolic imaging (DMI) is an emerging clinically-applicable technique for the non-invasive investigation of tissue metabolism. The generally short T 1 values of 2 H-labeled metabolites in vivo can compensate for the relatively low sensitivity of detection by allowing rapid signal acquisition in the absence of significant signal saturation. Studies with deuterated substrates, including [6,6'- 2 H 2 ]glucose, [ 2 H 3 ]acetate, [ 2 H 9 ]choline and [2,3- 2 H 2 ]fumarate have demonstrated the considerable potential of DMI for imaging tissue metabolism and cell death in vivo. The technique is evaluated here in comparison with established metabolic imaging techniques, including PET measurements of 2-deoxy-2-[ 18 F]fluoro-d-glucose (FDG) uptake and 13 C MR imaging of the metabolism of hyperpolarized 13 C-labeled substrates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

There is an unmet clinical need for imaging agents capable of detecting early evidence of tumor cell death, since the timing, extent, and distribution of cell death in tumors following treatment can give an indication of treatment outcome. We describe here 68 Ga-labeled C2Am, which is a phosphatidylserine-binding protein, for imaging tumor cell death in vivo using positron emission tomography (PET). A one-pot synthesis of 68 Ga-C2Am (20 min, 25 °C, >95% radiochemical purity) has been developed, using a NODAGA-maleimide chelator. The binding of 68 Ga-C2Am to apoptotic and necrotic tumor cells was assessed in vitro using human breast and colorectal cancer cell lines, and in vivo, using dynamic PET measurements in mice implanted subcutaneously with the colorectal tumor cells and treated with a TRAIL-R2 agonist. 68 Ga-C2Am showed predominantly renal clearance and low retention in the liver, spleen, small intestine, and bone and generated a tumor-to-muscle (T/m) ratio of 2.3 ± 0.4, at 2 h post probe administration and at 24 h following treatment. 68 Ga-C2Am has the potential to be used in the clinic as a PET tracer for assessing early treatment response in tumors.

Mutations in the TP53 gene are common in cancer with the R248Q missense mutation conferring an increased propensity to aggregate. Previous p53 aggregation studies showed that, at micromolar concentrations, protein unfolding to produce aggregation-prone species is the rate-determining step. Here we show that, at physiological concentrations, aggregation kinetics of insect cell-derived full-length wild-type p53 and p53R248Q are determined by a nucleation-growth model, rather than formation of aggregation-prone monomeric species. Self-seeding, but not cross-seeding, increases aggregation rate, confirming the aggregation process as rate determining. p53R248Q displays enhanced aggregation propensity due to decreased solubility and increased aggregation rate, forming greater numbers of larger amorphous aggregates that disrupt lipid bilayers and invokes an inflammatory response. These results suggest that p53 aggregation can occur under physiological conditions, a rate enhanced by R248Q mutation, and that aggregates formed can cause membrane damage and inflammation that may influence tumorigenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Purpose: There is an unmet clinical need for direct and sensitive methods to detect cell death in vivo, especially with regard to monitoring tumor treatment response. We have shown previously that tumor cell death can be detected in vivo from 2 H MRS and MRSI measurements of increased [2,3- 2 H 2 ]malate production following intravenous injection of [2,3- 2 H 2 ]fumarate. We show here that cell death can be detected with similar sensitivity following oral administration of the 2 H-labeled fumarate., Methods: Mice with subcutaneously implanted EL4 tumors were fasted for 1 h before administration (200 μl) of [2,3- 2 H 2 ]fumarate (2 g/kg bodyweight) via oral gavage without anesthesia. The animals were then anesthetized, and after 30 min, tumor conversion of [2,3- 2 H 2 ]fumarate to [2,3- 2 H 2 ]malate was assessed from a series of 13 2 H spectra acquired over a period of 65 min. The 2 H spectra and 2 H spectroscopic images were acquired using a surface coil before and at 48 h after treatment with a chemotherapeutic drug (etoposide, 67 mg/kg)., Results: The malate/fumarate signal ratio increased from 0.022 ± 0.03 before drug treatment to 0.12 ± 0.04 following treatment (p = 0.023, n = 4). Labeled malate was undetectable in spectroscopic images acquired before treatment and increased in the tumor area following treatment. The increase in the malate/fumarate signal ratio was similar to that observed previously following intravenous administration of labeled fumarate., Conclusion: Orally administered [2,3- 2 H 2 ]fumarate can be used to detect tumor cell death noninvasively following treatment with a sensitivity that is similar to that obtained with intravenous administration., (© 2022 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Early detection of tumor cell death in glioblastoma following treatment with chemoradiation has the potential to distinguish between true disease progression and pseudoprogression. Tumor cell death can be detected noninvasively in vivo by imaging the production of [2,3-2H2]malate from [2,3-2H2]fumarate using 2H magnetic resonance (MR) spectroscopic imaging. We show here that 2H MR spectroscopy and spectroscopic imaging measurements of [2,3-2H2]fumarate metabolism can detect tumor cell death in orthotopically implanted glioblastoma models within 48 hours following the completion of chemoradiation. Following the injection of [2,3-2H2]fumarate into tumor-bearing mice, production of [2,3-2H2]malate was measured in a human cell line-derived model and in radiosensitive and radioresistant patient-derived models of glioblastoma that were treated with temozolomide followed by targeted fractionated irradiation. The increase in the [2,3-2H2]malate/[2,3-2H2]fumarate signal ratio posttreatment, which correlated with histologic assessment of cell death, was a more sensitive indicator of treatment response than diffusion-weighted and contrast agent-enhanced 1H MRI measurements, which have been used clinically to detect responses of glioblastoma to chemoradiation. Overall, early detection of glioblastoma cell death using 2H MRI of malate production from fumarate could help improve the clinical evaluation of response to chemoradiation., Significance: 2H magnetic resonance imaging of labeled fumarate metabolism can detect early evidence of tumor cell death following chemoradiation, meeting a clinical need to reliably detect treatment response in glioblastoma., (©2022 The Authors; Published by the American Association for Cancer Research.)

MRI-based gene reporters allow imaging of gene expression at depth (tens of centimetres) and at relatively high resolution (~10-100 μm) and have the potential to be translated to the clinic. The reporters exploit either endogenous contrast mechanisms or they modulate the response to an introduced exogenous contrast agent., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Untargeted Nuclear Magnetic Resonance (NMR) metabolomics of polar extracts from the pancreata of a caerulin-induced mouse model of pancreatitis (Pt) and of a transgenic mouse model of pancreatic cancer (PCa) were used to find metabolic markers of Pt and to characterize the metabolic changes accompanying PCa progression. Using multivariate analysis a 10-metabolite metabolic signature specific to Pt tissue was found to distinguish the benign condition from both normal tissue and precancerous tissue (low grade pancreatic intraepithelial neoplasia, PanIN, lesions). The mice pancreata showed significant changes in the progression from normal tissue, through low-grade and high-grade PanIN lesions to pancreatic ductal adenocarcinoma (PDA). These included increased lactate production, amino acid changes consistent with enhanced anaplerosis, decreased concentrations of intermediates in membrane biosynthesis (phosphocholine and phosphoethanolamine) and decreased glycosylated uridine phosphates, reflecting activation of the hexosamine biosynthesis pathway and protein glycosylation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Carneiro, Pinto, Serrao, Barros, Brindle and Gil.)

Methylcyclopropene (Cyoc)-tagged tetra-acetylated monosaccharides, and in particular mannosamine derivatives, are promising tools for medical imaging of cancer using metabolic oligosaccharide engineering and the extremely fast inverse electron-demand Diels-Alder bioorthogonal reaction. However, the in vivo potential of these monosaccharide derivatives has yet to be fully explored due to their low aqueous solubility. To address this issue, we sought to vary the extent of acetylation of Cyoc-tagged monosaccharides and probe its effect on the extent of glycan labeling in various cancer cell lines. We demonstrate that, in the case of Ac x ManNCyoc, tri- and diacetylated derivatives generated significantly enhanced cell labeling compared to the tetra-acetylated monosaccharide. In contrast, for the more readily soluble azide-tagged sugars, a decrease in acetylation led to decreased glycan labeling. Ac 3 ManNCyoc gave better labeling than the azido-tagged Ac 4 ManNAz and has significant potential for in vitro and in vivo imaging of glycosylated cancer biomarkers.

Purpose To evaluate glioblastoma (GBM) metabolism by using hyperpolarized carbon 13 ( 13 C) MRI to monitor the exchange of the hyperpolarized 13 C label between injected [1- 13 C]pyruvate and tumor lactate and bicarbonate. Materials and Methods In this prospective study, seven treatment-naive patients (age [mean ± SD], 60 years ± 11; five men) with GBM were imaged at 3 T by using a dual-tuned 13 C-hydrogen 1 head coil. Hyperpolarized [1- 13 C]pyruvate was injected, and signal was acquired by using a dynamic MRI spiral sequence. Metabolism was assessed within the tumor, in the normal-appearing brain parenchyma (NABP), and in healthy volunteers by using paired or unpaired t tests and a Wilcoxon signed rank test. The Spearman ρ correlation coefficient was used to correlate metabolite labeling with lactate dehydrogenase A (LDH-A) expression and some immunohistochemical markers. The Benjamini-Hochberg procedure was used to correct for multiple comparisons. Results The bicarbonate-to-pyruvate (BP) ratio was lower in the tumor than in the contralateral NABP ( P < .01). The tumor lactate-to-pyruvate (LP) ratio was not different from that in the NABP ( P = .38). The LP and BP ratios in the NABP were higher than those observed previously in healthy volunteers ( P < .05). Tumor lactate and bicarbonate signal intensities were strongly correlated with the pyruvate signal intensity (ρ = 0.92, P < .001, and ρ = 0.66, P < .001, respectively), and the LP ratio was weakly correlated with LDH-A expression in biopsy samples (ρ = 0.43, P = .04). Conclusion Hyperpolarized 13 C MRI demonstrated variation in lactate labeling in GBM, both within and between tumors. In contrast, bicarbonate labeling was consistently lower in tumors than in the surrounding NABP. Keywords: Hyperpolarized 13 C MRI, Glioblastoma, Metabolism, Cancer, MRI, Neuro-oncology Supplemental material is available for this article. Published under a CC BY 4.0 license.

Purpose: The performance of pulse sequences in vivo can be limited by fast relaxation rates, magnetic field inhomogeneity, and nonuniform spin excitation. We describe here a method for pulse sequence optimization that uses a stochastic numerical solver that in principle is capable of finding a global optimum. The method provides a simple framework for incorporating any constraint and implementing arbitrarily complex cost functions. Efficient methods for simulating spin dynamics and incorporating frequency selectivity are also described., Methods: Optimized pulse sequences for polarization transfer between protons and X-nuclei and excitation pulses that eliminate J-coupling modulation were evaluated experimentally using a surface coil on phantoms, and also the detection of hyperpolarized [2- 13 C]lactate in vivo in the case of J-coupling modulation-free excitation., Results: The optimized polarization transfer pulses improved the SNR by ~50% with a more than twofold reduction in the B 1 field, and J-coupling modulation-free excitation was achieved with a more than threefold reduction in pulse length., Conclusion: This process could be used to optimize any pulse when there is a need to improve the uniformity and frequency selectivity of excitation as well as to design new pulses to steer the spin system to any desired achievable state., (© 2021 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Survival improves when cancer is detected early. However, ~50% of cancers are at an advanced stage when diagnosed. Early detection of cancer or precancerous change allows early intervention to try to slow or prevent cancer development and lethality. To achieve early detection of all cancers, numerous challenges must be overcome. It is vital to better understand who is at greatest risk of developing cancer. We also need to elucidate the biology and trajectory of precancer and early cancer to identify consequential disease that requires intervention. Insights must be translated into sensitive and specific early detection technologies and be appropriately evaluated to support practical clinical implementation. Interdisciplinary collaboration is key; advances in technology and biological understanding highlight that it is time to accelerate early detection research and transform cancer survival.

Hyperpolarised magnetic resonance imaging (HP  13 C-MRI) is an emerging clinical technique to detect [1- 13 C]lactate production in prostate cancer (PCa) following intravenous injection of hyperpolarised [1- 13 C]pyruvate. Here we differentiate clinically significant PCa from indolent disease in a low/intermediate-risk population by correlating [1- 13 C]lactate labelling on MRI with the percentage of Gleason pattern 4 (%GP4) disease. Using immunohistochemistry and spatial transcriptomics, we show that HP  13 C-MRI predominantly measures metabolism in the epithelial compartment of the tumour, rather than the stroma. MRI-derived tumour [1- 13 C]lactate labelling correlated with epithelial mRNA expression of the enzyme lactate dehydrogenase (LDHA and LDHB combined), and the ratio of lactate transporter expression between the epithelial and stromal compartments (epithelium-to-stroma MCT4). We observe similar changes in MCT4, LDHA, and LDHB between tumours with primary Gleason patterns 3 and 4 in an independent TCGA cohort. Therefore, HP  13 C-MRI can metabolically phenotype clinically significant disease based on underlying metabolic differences in the epithelial and stromal tumour compartments., (© 2022. The Author(s).)

Differentiating aggressive clear cell renal cell carcinoma (ccRCC) from indolent lesions is challenging using conventional imaging. This work prospectively compared the metabolic imaging phenotype of renal tumors using carbon-13 MRI following injection of hyperpolarized [1- 13 C]pyruvate (HP- 13 C-MRI) and validated these findings with histopathology. Nine patients with treatment-naïve renal tumors (6 ccRCCs, 1 liposarcoma, 1 pheochromocytoma, 1 oncocytoma) underwent pre-operative HP- 13 C-MRI and conventional proton ( 1 H) MRI. Multi-regional tissue samples were collected using patient-specific 3D-printed tumor molds for spatial registration between imaging and molecular analysis. The apparent exchange rate constant ( k PL ) between 13 C-pyruvate and 13 C-lactate was calculated. Immunohistochemistry for the pyruvate transporter (MCT1) from 44 multi-regional samples, as well as associations between MCT1 expression and outcome in the TCGA-KIRC dataset, were investigated. Increasing k PL in ccRCC was correlated with increasing overall tumor grade (ρ = 0.92, p = 0.009) and MCT1 expression (r = 0.89, p = 0.016), with similar results acquired from the multi-regional analysis. Conventional 1 H-MRI parameters did not discriminate tumor grades. The correlation between MCT1 and ccRCC grade was confirmed within a TCGA dataset ( p < 0.001), where MCT1 expression was a predictor of overall and disease-free survival. In conclusion, metabolic imaging using HP- 13 C-MRI differentiates tumor aggressiveness in ccRCC and correlates with the expression of MCT1, a predictor of survival. HP- 13 C-MRI may non-invasively characterize metabolic phenotypes within renal cancer.

Hyperpolarized (HP) 13 C magnetic resonance enables non-invasive probing of metabolism in vivo. To date, only 13 C-molecules hyperpolarized with persistent trityl radicals have been injected in humans. We show here that the free radical photo-induced in alpha-ketoglutaric acid (α-KG) can be used to hyperpolarize photo-inactive 13 C-molecules such as [1- 13 C]lactate. α-KG is an endogenous molecule with an exceptionally high radical yield under photo-irradiation, up to 50 %, and its breakdown product, succinic acid, is also endogenous. This radical precursor therefore exhibits an excellent safety profile for translation to human studies. The labile nature of the radical means that no filtration is required prior to injection while also offering the opportunity to extend the 13 C relaxation time in frozen HP 13 C-molecules for storage and transport. The potential for in vivo metabolic studies is demonstrated in the rat liver following the injection of a physiological dose of HP [1- 13 C]lactate., Competing Interests: Arnaud Comment was employed by General Electric Medical Systems Inc. at the time of manuscript preparation and submission., (© 2021 The Authors. Angewandte Chemie published by Wiley-VCH GmbH.)

Radioligand theranostics (RT) in oncology use cancer-type specific biomarkers and molecular imaging (MI), including positron emission tomography (PET), single-photon emission computed tomography (SPECT) and planar scintigraphy, for patient diagnosis, therapy, and personalized management. While the definition of theranostics was initially restricted to a single compound allowing visualization and therapy simultaneously, the concept has been widened with the development of theranostic pairs and the combination of nuclear medicine with different types of cancer therapies. Here, we review the clinical applications of different theranostic radiopharmaceuticals in managing different tumor types (differentiated thyroid, neuroendocrine prostate, and breast cancer) that support the combination of innovative oncological therapies such as gene and cell-based therapies with RT.

Hyperpolarized 13 C-MRI is an emerging tool for probing tissue metabolism by measuring 13 C-label exchange between intravenously injected hyperpolarized [1- 13 C]pyruvate and endogenous tissue lactate. Here, we demonstrate that hyperpolarized 13 C-MRI can be used to detect early response to neoadjuvant therapy in breast cancer. Seven patients underwent multiparametric 1 H-MRI and hyperpolarized 13 C-MRI before and 7-11 days after commencing treatment. An increase in the lactate-to-pyruvate ratio of approximately 20% identified three patients who, following 5-6 cycles of treatment, showed pathological complete response. This ratio correlated with gene expression of the pyruvate transporter MCT1 and lactate dehydrogenase A ( LDHA ), the enzyme catalyzing label exchange between pyruvate and lactate. Analysis of approximately 2,000 breast tumors showed that overexpression of LDHA and the hypoxia marker CAIX was associated with reduced relapse-free and overall survival. Hyperpolarized 13 C-MRI represents a promising method for monitoring very early treatment response in breast cancer and has demonstrated prognostic potential. SIGNIFICANCE: Hyperpolarized carbon-13 MRI allows response assessment in patients with breast cancer after 7-11 days of neoadjuvant chemotherapy and outperformed state-of-the-art and research quantitative proton MRI techniques., (©2021 The Authors; Published by the American Association for Cancer Research.)

Background: Immune checkpoint inhibitors are now standard of care treatment for many cancers. Treatment failure in metastatic melanoma is often due to tumor heterogeneity, which is not easily captured by conventional CT or tumor biopsy. The aim of this prospective study was to investigate early microstructural and functional changes within melanoma metastases following immune checkpoint blockade using multiparametric MRI., Methods: Fifteen treatment-naïve metastatic melanoma patients (total 27 measurable target lesions) were imaged at baseline and following 3 and 12 weeks of treatment on immune checkpoint inhibitors using: T 2 -weighted imaging, diffusion kurtosis imaging, and dynamic contrast-enhanced MRI. Treatment timepoint changes in tumor cellularity, vascularity, and heterogeneity within individual metastases were evaluated and correlated to the clinical outcome in each patient based on Response Evaluation Criteria in Solid Tumors V.1.1 at 1 year., Results: Differential tumor growth kinetics in response to immune checkpoint blockade were measured in individual metastases within the same patient, demonstrating significant intertumoral heterogeneity in some patients. Early detection of tumor cell death or cell loss measured by a significant increase in the apparent diffusivity (D app ) (p<0.05) was observed in both responding and pseudoprogressive lesions after 3 weeks of treatment. Tumor heterogeneity, as measured by apparent diffusional kurtosis (K app ), was consistently higher in the pseudoprogressive and true progressive lesions, compared with the responding lesions throughout the first 12 weeks of treatment. These preceded tumor regression and significant tumor vascularity changes (K trans , v e , and v p ) detected after 12 weeks of immunotherapy (p<0.05)., Conclusions: Multiparametric MRI demonstrated potential for early detection of successful response to immune checkpoint inhibitors in metastatic melanoma., Competing Interests: Competing interests: No conflict of interest to declare with respect to the content of this work. AL, MS, LB, and J-ML are employees of AstraZeneca UK., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

13 C nuclear spin hyperpolarization can increase the sensitivity of detection in an MRI experiment by more than 10,000-fold. 13 C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized 13 C label exchange between injected [1- 13 C]pyruvate and the endogenous tumor lactate pool can be used clinically to assess tumor grade and response to treatment. We describe here an experimental protocol for using this technique in patient-derived and established cell line xenograft models of breast cancer in the mouse. For complete details on the use and execution of this protocol, please refer to Ros et al. (2020)., Competing Interests: The hyperpolarizer is on loan from GE Healthcare (GEH) and is the subject of a research agreement between the University of Cambridge, Cancer Research UK, and GEH. K.M.B. holds patents with GEH on some aspects of dissolution dynamic nuclear polarization (DNP) technology. C.C. reports receiving speakers' bureau honoraria from Illumina, is a member of the AstraZeneca (AZ) External Science Panel, and reports receiving research grants from Roche, Genentech, AZ, and Servier that are administered by the University of Cambridge., (© 2021 The Authors.)

Purpose: To compare carbon-13 ( 13 C) MRSI of hyperpolarized [1- 13 C]pyruvate metabolism in a murine tumor model with mass spectrometric (MS) imaging of the corresponding tumor sections in order to cross validate these metabolic imaging techniques and to investigate the effects of pyruvate delivery and tumor lactate concentration on lactate labeling., Methods: [1- 13 C]lactate images were obtained from tumor-bearing mice, following injection of hyperpolarized [1- 13 C]pyruvate, using a single-shot 3D 13 C spectroscopic imaging sequence in vivo and using desorption electrospray ionization MS imaging of the corresponding rapidly frozen tumor sections ex vivo. The images were coregistered, and levels of association were determined by means of Spearman rank correlation and Cohen kappa coefficients as well as linear mixed models. The correlation between [1- 13 C]pyruvate and [1- 13 C]lactate in the MRS images and between [ 12 C] and [1- 13 C]lactate in the MS images were determined by means of Pearson correlation coefficients., Results: [1- 13 C]lactate images generated by MS imaging were significantly correlated with the corresponding MRS images. The correlation coefficient between [1- 13 C]lactate and [1- 13 C]pyruvate in the MRS images was higher than between [1- 13 C]lactate and [ 12 C]lactate in the MS images., Conclusion: The inhomogeneous distribution of labeled lactate observed in the MRS images was confirmed by MS imaging of the corresponding tumor sections. The images acquired using both techniques show that the rate of 13 C label exchange between the injected pyruvate and endogenous tumor lactate pool is more correlated with the rate of pyruvate delivery to the tumor cells and is less affected by the endogenous lactate concentration., (© 2021 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

2 H magnetic resonance spectroscopic imaging has been shown recently to be a viable technique for metabolic imaging in the clinic. We show here that 2 H MR spectroscopy and spectroscopic imaging measurements of [2,3- 2 H 2 ]malate production from [2,3- 2 H 2 ]fumarate can be used to detect tumor cell death in vivo via the production of labeled malate. Production of [2,3- 2 H 2 ]malate, following injection of [2,3- 2 H 2 ]fumarate (1 g/kg) into tumor-bearing mice, was measured in a murine lymphoma (EL4) treated with etoposide, and in human breast (MDA-MB-231) and colorectal (Colo205) xenografts treated with a TRAILR2 agonist, using surface-coil localized 2 H MR spectroscopy at 7 T. Malate production was also imaged in EL4 tumors using a fast 2 H chemical shift imaging sequence. The malate/fumarate ratio increased from 0.016 ± 0.02 to 0.16 ± 0.14 in EL4 tumors 48 h after drug treatment ( P = 0.0024, n = 3), and from 0.019 ± 0.03 to 0.25 ± 0.23 in MDA-MB-231 tumors ( P = 0.0001, n = 5) and from 0.016 ± 0.04 to 0.28 ± 0.26 in Colo205 tumors ( P = 0.0002, n = 5) 24 h after drug treatment. These increases were correlated with increased levels of cell death measured in excised tumor sections obtained immediately after imaging. 2 H MR measurements of [2,3- 2 H 2 ]malate production from [2,3- 2 H 2 ]fumarate provide a potentially less expensive and more sensitive method for detecting cell death in vivo than 13 C MR measurements of hyperpolarized [1,4- 13 C 2 ]fumarate metabolism, which have been used previously for this purpose., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Ductal carcinoma in situ (DCIS) is a non-invasive form of early breast cancer, with a poorly understood natural history of invasive transformation. Necrosis is a well-recognized adverse prognostic feature of DCIS, and non-invasive detection of its presence and spatial extent could provide information not obtainable by biopsy. We describe here imaging of the distribution and extent of comedo-type necrosis in a model of human DCIS using C2Am, an imaging agent that binds to the phosphatidylserine exposed by necrotic cells., Methods: We used an established xenograft model of human DCIS that mimics the histopathological features of the disease. Planar near-infrared and optoacoustic imaging, using fluorescently labeled C2Am, were used to image non-invasively the presence and extent of lesion necrosis., Results: C2Am showed specific and sensitive binding to necrotic areas in DCIS tissue, detectable both in vivo and ex vivo. The imaging signal generated in vivo using near-infrared (NIR) fluorescence imaging was up to 6-fold higher in DCIS lesions than in surrounding fat pad or skin tissue. There was a correlation between the C2Am NIR fluorescence (Pearson R = 0.783, P = 0.0125) and optoacoustic signals (R > 0.875, P < 0.022) in the DCIS lesions in vivo and the corresponding levels of cell death detected histologically., Conclusions: C2Am is a targeted multi-modal imaging agent that could complement current anatomical imaging methods for detecting DCIS. Imaging the presence and spatial extent of necrosis may give better prognostic information than that obtained by biopsy alone.

Objectives: To enhance detection of the products of hyperpolarized [2- 13 C]dihydroxyacetone metabolism for assessment of three metabolic pathways in the liver in vivo. Hyperpolarized [2- 13 C]DHAc emerged as a promising substrate to follow gluconeogenesis, glycolysis and the glycerol pathways. However, the use of [2- 13 C]DHAc in vivo has not taken off because (i) the chemical shift range of [2- 13 C]DHAc and its metabolic products span over 144 ppm, and (ii) 1 H decoupling is required to increase spectral resolution and sensitivity. While these issues are trivial for high-field vertical-bore NMR spectrometers, horizontal-bore small-animal MR scanners are seldom equipped for such experiments., Methods: Real-time hepatic metabolism of three fed mice was probed by 1 H-decoupled 13 C-MR following injection of hyperpolarized [2- 13 C]DHAc. The spectra of [2- 13 C]DHAc and its metabolic products were acquired in a 7 T small-animal MR scanner using three purpose-designed spectral-spatial radiofrequency pulses that excited a spatial bandwidth of 8 mm with varying spectral bandwidths and central frequencies (chemical shifts)., Results: The metabolic products detected in vivo include glycerol 3-phosphate, glycerol, phosphoenolpyruvate, lactate, alanine, glyceraldehyde 3-phosphate and glucose 6-phosphate. The metabolite-to-substrate ratios were comparable to those reported previously in perfused liver., Discussion: Three metabolic pathways can be probed simultaneously in the mouse liver in vivo, in real time,  using hyperpolarized DHAc.

Introduction: Trialing novel cancer therapies in the clinic would benefit from imaging agents that can detect early evidence of treatment response. The timing, extent and distribution of cell death in tumors following treatment can give an indication of outcome. We describe here an 18 F-labeled derivative of a phosphatidylserine-binding protein, the C2A domain of Synaptotagmin-I (C2Am), for imaging tumor cell death in vivo using PET., Methods: A one-pot, two-step automated synthesis of N-(5-[ 18 F]fluoropentyl)maleimide (60 min synthesis time, > 98% radiochemical purity) has been developed, which was used to label the single cysteine residue in C2Am within 30 min at room temperature. Binding of 18 F-C2Am to apoptotic and necrotic tumor cells was assessed in vitro, and also in vivo, by dynamic PET and biodistribution measurements in mice bearing human tumor xenografts treated with a TRAILR2 agonist or with conventional chemotherapy. C2Am detection of tumor cell death was validated by correlation of probe binding with histological markers of cell death in tumor sections obtained immediately after imaging., Results: 18 F-C2Am showed a favorable biodistribution profile, with predominantly renal clearance and minimal retention in spleen, liver, small intestine, bone and kidney, at 2 h following probe administration. 18 F-C2Am generated tumor-to-muscle (T/m) ratios of 6.1 ± 2.1 and 10.7 ± 2.4 within 2 h of probe administration in colorectal and breast tumor models, respectively, following treatment with the TRAILR2 agonist. The levels of cell death (CC3 positivity) following treatment were 12.9-58.8% and 11.3-79.7% in the breast and colorectal xenografts, respectively. Overall, a 20% increase in CC3 positivity generated a one unit increase in the post/pre-treatment tumor contrast. Significant correlations were found between tracer uptake post-treatment, at 2 h post-probe administration, and histological markers of cell death (CC3: Pearson R = 0.733, P = 0.0005; TUNEL: Pearson R = 0.532, P = 0.023)., Conclusion: The rapid clearance of 18 F-C2Am from the blood pool and low kidney retention allowed the spatial distribution of cell death in a tumor to be imaged during the course of therapy, providing a rapid assessment of tumor treatment response. 18 F-C2Am has the potential to be used in the clinic to assess early treatment response in tumors.