Hyperpolarized 13C-pyruvate Research Articles

Multi-Modal Investigation of Metabolism in Murine Breast Cancer Cell Lines Using Fluorescence Lifetime Microscopy and Hyperpolarized 13C-Pyruvate Magnetic Resonance Spectroscopy.

Background/Objectives: Despite the role of metabolism in breast cancer metastasis, we still cannot predict which breast tumors will progress to distal metastatic lesions or remain dormant. This work uses metabolic imaging to study breast cancer cell lines (4T1, 4T07, and 67NR) with differing metastatic potential in a 3D collagen gel bioreactor system. Methods: Within the bioreactor, hyperpolarized magnetic resonance spectroscopy (HP-MRS) is used to image lactate/pyruvate ratios, while fluorescence lifetime imaging microscopy (FLIM) of endogenous metabolites measures metabolism at the cellular scale. Results: HP-MRS results showed no lactate peak for 67NR and a comparatively large lactate/pyruvate ratio for both 4T1 and 4T07 cell lines, suggestive of greater pyruvate utilization with greater metastatic potential. Similar patterns were observed using FLIM with significant increases in FAD intensity, redox ratio, and NAD(P)H lifetime. The lactate/pyruvate ratio was strongly correlated to NAD(P)H lifetime, consistent with the role of NADH as an electron donor for the glycolytic pathway, suggestive of an overall upregulation of metabolism (both glycolytic and oxidative), for the 4T07 and 4T1 cell lines compared to the non-metastatic 67NR cell line. Conclusions: These findings support a complementary role for HP-MRS and FLIM enabled by a novel collagen gel bioreactor system to investigate metastatic potential and cancer metabolism.

Targeting NAD+ Metabolism Vulnerability in FH-Deficient Hereditary Leiomyomatosis and Renal Cell Carcinoma with the novel NAMPT Inhibitor OT-82.

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an inherited cancer syndrome caused by germline pathogenic variants in the fumarate hydratase (FH) gene. Affected individuals are at risk for developing cutaneous and uterine leiomyomas and aggressive FH-deficient renal cell carcinoma (RCC) with a papillary histology. Due to a disrupted TCA cycle, FH-deficient kidney cancers rely on aerobic glycolysis for energy production, potentially creating compensatory metabolic vulnerabilities. This study conducted a high-throughput drug screen in HLRCC cell lines, which identified a critical dependency on nicotinamide adenine dinucleotide (NAD), a redox cofactor produced by the biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). Human HLRCC tumors and HLRCC-derived cell lines exhibited elevated NAMPT expression compared to controls. FH-deficient HLRCC cells, but not FH-restored HLRCC or normal kidney cells, were sensitive to NAMPT inhibition. HLRCC cell line viability was significantly decreased in both 2D and 3D in vitro cultures in response to the clinically relevant NAMPT inhibitor OT-82. NAMPT inhibition in vitro significantly decreased the total amount of NAD+, NADH, NADP, NADPH, and PAR levels and the effects of NAMPT inhibition could be rescued by the downstream NAD precursor nicotinamide mononucleotide, confirming the on-target activity of OT-82. Moreover, NAMPT inhibition by OT-82 in two HLRCC xenograft models resulted in severely reduced tumor growth. OT-82 treatment of HLRCC xenograft tumors in vivo inhibited glycolytic flux as demonstrated by reduced lactate/pyruvate ratio in hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging experiments. Overall, our data define NAMPT inhibition as a potential therapeutic approach for FH-deficient HLRCC-associated renal cell carcinoma.

Dual hyperpolarized [1-13C]pyruvate and [13C]urea magnetic resonance imaging of prostate cancer

BackgroundAlthough multiparametric (mp) 1H magnetic resonance imaging (MRI) is increasingly used to detect and localize prostate cancer (PC), its correlation with tumor grade is limited. Hyperpolarized (HP) carbon-13 (13C) MR is an emerging imaging technique, which can be used to interrogate key biologic processes through in vivo detection of various HP probes. A distinct attribute of HP 13C MRI is the ability to detect multiple HP probes within a single acquisition. Here we report on the first simultaneous dual HP [1-13C]pyruvate and [13C]urea MRI with correlations to histopathologic findings in a patient with localized PC scheduled for radical prostatectomy. Material and methodsPaired HP 13C and standard mp 1H MRI were performed in a patient with biopsy-proven Gleason score 4 + 3 = 7 adenocarcinoma of the prostate scheduled for radical prostatectomy through a first-in-human pilot study of dual-agent HP MRI (NCT02526368). HP 13C MRI was performed using a clinical 3T scanner with 13C transmit-and-receive capabilities. Dynamic series of HP 13C pyruvate, lactate and urea imaging were acquired following intravenous (IV) injection of co-hyperpolarized [13C]urea (25 mM) and [1–13C]pyruvate (125 mM). The [1-13C]pyruvate-to-[1-13C]lactate conversion rate (kPL) was calculated using an inputless two-site exchange model; AUCurea was the [13C]urea signal summed over time. Following radical prostatectomy, whole-mount prostate histopathological slides were prepared and reviewed by an experienced genitourinary pathologist. ResultsFollowing informed consent, the patient underwent paired mp 1H MRI and dual-agent HP MRI. mp 1H MRI revealed a 1.2 cm lesion in the left apical posterior zone. Dual-agent HP MRI identified a focus of increased [1-13C]pyruvate-to-[1-13C]lactate conversion rate (kPL) extending from the left apical posterior peripheral zone to the right gland. A corresponding area of abnormal tissue perfusion (AUCurea) was seen in the left gland. Metabolism-perfusion mismatch (with several foci of increased kPL/AUCurea) was observed throughout the tumor. Tumor extension to the right midgland was confirmed at the time of radical prostatectomy and staining for lactate dehydrogenase-A was increased throughout the tumor relative to surrounding benign prostatic tissue. ConclusionThis first-in-human radiopathologic study demonstrates the feasibility of dual-agent HP MRI in PC patients. Simultaneous assessment of tumor metabolism and perfusion was able to detect occult disease as well as to show a significant mismatch between intra-tumoral metabolism and tissue perfusion in high-grade PC. Prospective validation of these findings is warranted.

Detection of early-stage NASH using non-invasive hyperpolarized 13C metabolic imaging

Non-alcoholic steatohepatitis (NASH) is characterized from its early stages by a profound remodeling of the liver microenvironment, encompassing changes in the composition and activities of multiple cell types and associated gene expression patterns. Hyperpolarized (HP) 13C MRI provides a unique view of the metabolic microenvironment, with potential relevance for early diagnosis of liver disease. Previous studies have detected changes in HP 13C pyruvate to lactate conversion, catalyzed by lactate dehydrogenase (LDH), with experimental liver injury. HP ∝\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\propto $$\\end{document}-ketobutyrate (∝\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\propto $$\\end{document} KB) is a close molecular analog of pyruvate with modified specificity for LDH isoforms, specifically attenuated activity with their LDHA-expressed subunits that dominate liver parenchyma. Building on recent results with pyruvate, we investigated HP ∝\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\propto $$\\end{document} KB in methionine-choline deficient (MCD) diet as a model of early-stage NASH. Similarity of results between this new agent and pyruvate (~ 50% drop in cytoplasmic reducing capacity), interpreted together with gene expression data from the model, suggests that changes are mediated through broad effects on intermediary metabolism. Plausible mechanisms are depletion of the lactate pool by upregulation of gluconeogenesis (GNG) and pentose phosphate pathway (PPP) flux, and a possible shift toward increased lactate oxidation. These changes may reflect high levels of oxidative stress and/or shifting macrophage populations in NASH.

Abstract 4156: Monitoring early chemotherapy response in murine pancreatic ductal adenocarcinoma using non-invasive hyperpolarized magnetic resonance spectroscopic imaging with 13C-pyruvate

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a rarely operable cancer with a very poor prognosis. Nearly 100% of affected patients receive one of two alternative first-line chemotherapies during the course of their disease: 1.) Gemcitabine-Abraxane (G/A) or 2.) FOLFIRINOX. Treatment success varies widely between the patients due to individual molecular, structural tumor and metabolic heterogeneity. In this project, we use hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging (HP-MRSI) to non-invasively characterize early metabolic changes in murine endogenous PDAC in response to the mentioned chemotherapies. Methods: 18 KPC (Ptf1awt/cre(C)Kraswt/G12D(K)Trp53fl/fl(P)) mice with 74 distinct tumour nodules, visually identified based on T2-weighted anatomical imaging, were used in this study. The conversion of pyruvate to lactate was detected by HP-MRSI, performed on 7 T preclinical system using modified 3D balanced steady-state free precession (bSSFP) sequence on day 0 pre-therapy and on day 3 post-therapy. 100μg/g G (intraperitoneally) and 30μg/g A (intravenously) were applied twice on day 0 post-scan and day 2 (n=6). FOLFIRINOX (60μg/g 5-Fluoruracil, 27μg/g Irinotecan, 12.8μg/g Oxaliplatin, 60μg/g Leucovorin) was injected intravenously once on day 0 post-scan (n=6). Vehicle mice (n=6) were left untreated. Area under the curves (AUC) of lactate to pyruvate time-courses (AUCl/AUCp) were calculated. Primary murine PDAC cell lines were established from biopsies of 58 imaging region, sequenced using 3’RNA-Seq (Poly-ASeq) method and analysed using Dseq2 R package. Results: G/A and FOLFIRINOX-treated tumors exhibited heterogeneous metabolic responses in distinct nodules, while non-treated group tumors remained stable. A significant difference in metabolic changes (p = 0.01, ANOVA) from day 0 to day 3 was observed between FOLFIRINOX, G/A, and non-treated groups. Low glycolytic tumors showed a positive trend, more pronounced with FOLFIRINOX than G/A treatment. Metabolically active nodules with high AUCl/AUCp on day 0 reduced lactate turnover under therapy. These findings suggest that metabolic HP-MRSI can detect treatment-induced changes in tumor composition and subtype-specific epithelial/mesenchymal plasticity, correlating with the metabolic phenotype. Further investigation will include histology, subtype-specific biomarkers, transcriptional signatures, and regional transcriptome analysis to explain molecular changes in metabolism. Conclusions: We have applied a non-invasive approach for detection of metabolic changes under chemotherapy in murine PDAC. These findings will contribute in the long term to the establishment of predictive imaging biomarkers for PDAC. Citation Format: Irina Heid, Anna-Maria Schmidmüller, Simon Baller, Jason G. Skinner, Geoffrey J. Topping, Wolfgang Gottwald, Hussein Trabulssi, Rupert Öllinger, Katja Steiger, Roland Rad, Franz Schilling, Rickmer F. Braren. Monitoring early chemotherapy response in murine pancreatic ductal adenocarcinoma using non-invasive hyperpolarized magnetic resonance spectroscopic imaging with 13C-pyruvate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4156.

Current methods for hyperpolarized [1-13C]pyruvate MRI human studies.

MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of HP agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate-by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation; (2) MRI system setup and calibrations; (3) data acquisition and image reconstruction; and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the "HP 13C MRI Consensus Group" as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods and Equipment study groups. It further aims to provide a comprehensive reference for future consensus, building as the field continues to advance human studies with this metabolic imaging modality.

Characterizing Metabolic Heterogeneity of Hepatocellular Carcinoma with Hyperpolarized 13C Pyruvate MRI and Mass Spectrometry.

Purpose To characterize the metabolomic profiles of two hepatocellular carcinoma (HCC) rat models, track evolution of these profiles to a stimulated tumor state, and assess their effect on lactate flux with hyperpolarized (HP) carbon 13 (13C) MRI. Materials and Methods Forty-three female adult Fischer rats were implanted with N1S1 or McA-RH7777 HCC tumors. In vivo lactate-to-pyruvate ratio (LPR) was measured with HP 13C MRI at 9.4 T. Ex vivo mass spectrometry was used to measure intratumoral metabolites, and Ki67 labeling was used to quantify proliferation. Tumors were first compared with three normal liver controls. The tumors were then compared with stimulated variants via off-target hepatic thermal ablation treatment. All comparisons were made using the Mann-Whitney test. Results HP 13C pyruvate MRI showed greater LPR in N1S1 tumors compared with normal liver (mean [SD], 0.564 ± 0.194 vs 0.311 ± 0.057; P < .001 [n = 9]), but not for McA-RH7777 (P = .44 [n = 8]). Mass spectrometry confirmed that the glycolysis pathway was increased in N1S1 tumors and decreased in McA-RH7777 tumors. The pentose phosphate pathway was also decreased only in McA-RH7777 tumors. Increased proliferation in stimulated N1S1 tumors corresponded to a net increase in LPR (six stimulated vs six nonstimulated, 0.269 ± 0.148 vs 0.027 ± 0.08; P = .009), but not in McA-RH7777 (eight stimulated vs six nonstimulated, P = .13), despite increased proliferation and metastases. Mass spectrometry demonstrated relatively increased lactate production with stimulation in N1S1 tumors only. Conclusion Two HCC subtypes showed divergent glycolytic dependency at baseline and during transformation to a high proliferation state. This metabolic heterogeneity in HCC should be considered with use of HP 13C MRI for diagnosis and tracking. Keywords: Molecular Imaging-Probe Development, Liver, Abdomen/GI, Oncology, Hepatocellular Carcinoma © RSNA, 2024 See also commentary by Ohliger in this issue.

Hyperpolarized 13C Pyruvate MRI: An Important Window into Tumor Metabolism.

Hyperpolarized 13C Pyruvate MRI: An Important Window into Tumor Metabolism.

O099 A relevant metabolic model for monitoring human metabolism in vivo using dynamic nuclear polarization with 13C magnetic resonance spectroscopy

Abstract Introduction DNP increases the signal of carbon-13 molecules on MR scans (&amp;gt;10,000-fold), enabling real-time, dynamic quantitation of tissue metabolism in vivo with significant clinical potential. 13C quantification of metabolism is affected by confounding factors (T1 signal decay, flip angle, and metabolite escape), which necessitates metabolic modelling. Here, we have assessed a mathematical model that requires minimal information on confounding factors to measure metabolic rates with reliability. Methods Four healthy male volunteers (age; 24 ± 1.4 years; BMI; 23 ± 0.4 kg/m2) were recruited. Sterile hyperpolarized 13C pyruvate was safely produced using a clinical DNP polariser (SPINlab GE-Healthcare) and administered intravenously to volunteers performing in-bore plantar-flexion exercise to increase muscle pyruvate flux. 13C MR scans of the calf muscle were collected during exercise and used to test our model against 5000 computer simulations using randomised kinetic constants and confounding factors. Results The signal of metabolites was used to extract the kinetic constants of hyperpolarised-13C pyruvate conversion to lactate (KpL), alanine (KpA), and bicarbonate (KpB) with reproducibility and satisfactory signal-to-noise ratio (8.2 ± 1.8×102 s−1, 2.3 ± 2.1×102 s−1 to 2.1 ± 1.3 x103 s−1, respectively; mean ± SD). Model predictions were in statistical agreement with the kinetic constants used in the simulations. Conclusion Our metabolic model could be used to measure metabolic conversion of hyperpolarised-13C metabolites in the context of surgery, where postoperative insulin resistance is well-known to be linked to stress-induced upregulation of pyruvate dehydrogenase inhibitor.

Abstract 3585: Probing the relationship between renal carcinoma perfusion, hypoxia, and metabolism during response to VEGFR inhibition and at resistance

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common primary kidney cancer. VEGF-receptor tyrosine kinase inhibitors remain a second-line or concurrent (with immunotherapy) treatment option for advanced RCC, but resistance inevitably occurs. There remains a need for biomarkers predictive of response to VEGFR targeting. Anti-VEGFR therapy-induced hypoxia can result in changes to both tumor metabolism and vascularization that contribute to the aggressiveness of treatment-resistant tumors. In this study we used in vivo hyperpolarized 13C-tert-butanol, a freely-diffusible novel MRI tracer that provides high-SNR tissue perfusion mapping, and hyperpolarized 13C pyruvate MRI, which enables real-time investigation of glycolytic lactate production, to understand the evolution of RCC perfusion and metabolism in response to VEGFR targeting, while correlating to histologic measures of tumor proliferation (Ki67) and vascularization (CD34). A498, a human ccRCC cell line, was implanted subcutaneously into 12-week-old nude mice. When tumors reached 15mm, the mice were placed into three arms, untreated (UT), sunitinib sensitive (SS), and sunitinib resistant (SR). SS mice were treated with sunitinib (25 mg/kg via gavage) then the mice were imaged with MRI at 48 hours. SR mice were treated with sunitinib daily until 2mm of re-growth was detected, then MRI was performed. UT mice were imaged when tumors reached 20mm. Pimonidazole infusion immediately after MRI, then the tumors were harvested. Histology included Ki67, CD34, and pimonidazole (hypoxia) staining. Ki67 expression was greater in the UT group compared to SS (37.56±5.21 vs 26.23±3.78, p=0.004), but not compared to SR (37.56±5.21 vs 34.32±1.12, p=0.93). The UT group demonstrated greater vascularization with CD-34 vessel counting than SS (UT 6.70±1.81 vs. SS 2.39±1.16, p=0.002), while vascularization was increased in SR compared to SS (SR 3.82±1.81 vs SS 2.39±1.16, p=0.05). Ki67 expression was positively correlated with CD34 expression. MR-measured tumor perfusion of the SS group was greater than UT (0.0086±0.0056 vs 0.0035±0.0026, p=0.0001) and SR (0.0086±0.0056 vs 0.0043±0.0033, p=0.0003). Tumor lactate production in the SS group was lower than UT (0.752±0.268 vs 0.609±0.188, p=0.04) and SR (0.752±0.268 vs 0.593±0.246, p=0.006). However, tumor perfusion was negatively correlated with lactate production ratio only in the SR group. Untreated and sunitinib-resistent RCC tumors are associated with increased perfusion and vascularity, and decreased lactate production. An inverse relationship between lactate production and perfusion in SR may be attributable to vascular normalization. Analysis of the spatial relationship between tumor metabolism and perfusion will further yield additional detail on the interplay between hypoxia and metabolism. Citation Format: Qianhui Dou, Patricia Coutinho de Souza, Xiaoen Wang, Aaron K. Grant, Leo L. Tsai. Probing the relationship between renal carcinoma perfusion, hypoxia, and metabolism during response to VEGFR inhibition and at resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3585.

Metabolite-Specific Echo Planar Imaging for Preclinical Studies with Hyperpolarized 13C-Pyruvate MRI.

Metabolite-specific echo-planar imaging (EPI) sequences with spectral-spatial (spsp) excitation are commonly used in clinical hyperpolarized [1-13C]pyruvate studies because of their speed, efficiency, and flexibility. In contrast, preclinical systems typically rely on slower spectroscopic methods, such as chemical shift imaging (CSI). In this study, a 2D spspEPI sequence was developed for use on a preclinical 3T Bruker system and tested on in vivo mice experiments with patient-derived xenograft renal cell carcinoma (RCC) or prostate cancer tissues implanted in the kidney or liver. Compared to spspEPI sequences, CSI were found to have a broader point spread function via simulations and exhibited signal bleeding between vasculature and tumors in vivo. Parameters for the spspEPI sequence were optimized using simulations and verified with in vivo data. The expected lactate SNR and pharmacokinetic modeling accuracy increased with lower pyruvate flip angles (less than 15°), intermediate lactate flip angles (25° to 40°), and temporal resolution of 3 s. Overall SNR was also higher with coarser spatial resolution (4 mm isotropic vs. 2 mm isotropic). Pharmacokinetic modelling used to fit kPL maps showed results consistent with the previous literature and across different sequences and tumor xenografts. This work describes and justifies the pulse design and parameter choices for preclinical spspEPI hyperpolarized 13C-pyruvate studies and shows superior image quality to CSI.

Regional quantification of cardiac metabolism with hyperpolarized [1-13C]-pyruvate CMR evaluated in an oral glucose challenge

BackgroundThe heart has metabolic flexibility, which is influenced by fed/fasting states, and pathologies such as myocardial ischemia and hypertrophic cardiomyopathy (HCM). Hyperpolarized (HP) 13C-pyruvate MRI is a promising new tool for non-invasive quantification of myocardial glycolytic and Krebs cycle flux. However, human studies of HP 13C-MRI have yet to demonstrate regional quantification of metabolism, which is important in regional ischemia and HCM patients with asymmetric septal/apical hypertrophy. MethodsWe developed and applied methods for whole-heart imaging of 13C-pyruvate, 13C-lactate and 13C-bicarbonate, following intravenous administration of [1-13C]-pyruvate. The image acquisition used an autonomous scanning method including bolus tracking, real-time magnetic field calibrations and metabolite-specific imaging. For quantification of metabolism, we evaluated 13C metabolite images, ratio metrics, and pharmacokinetic modeling to provide measurements of myocardial lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) mediated metabolic conversion in 5 healthy volunteers (fasting & 30 min following oral glucose load). ResultsWe demonstrate whole heart coverage for dynamic measurement of pyruvate-to-lactate conversion via LDH and pyruvate-to-bicarbonate conversion via PDH at a resolution of 6 × 6 × 21 mm3 (13C-pyruvate) and 12 × 12 × 21 mm3 (13C-lactate, 13C-bicarbonate). 13C-pyruvate and 13C-lactate were detected simultaneously in the RV blood pool, immediately after intravenous injection, reflecting LDH activity in blood. In healthy volunteers, myocardial 13C-pyruvate-SNR, 13C-lactate-SNR, 13C-bicarbonate-SNR, 13C-lactate/pyruvate ratio, 13C-pyruvate-to-lactate conversion rate, kPL, and 13C-pyruvate-to-bicarbonate conversion rate, kPB, all had statistically significant increases following oral glucose challenge. kPB, reflecting PDH activity and pyruvate entering the Krebs Cycle, had the highest correlation with blood glucose levels and was statistically significant. ConclusionsWe demonstrate first-in-human regional quantifications of cardiac metabolism by HP 13C-pyruvate MRI that aims to reflect LDH and PDH activity.

Hyperpolarized 13C-Pyruvate to Assess Response to Anti-PD1 Immune Checkpoint Inhibition in YUMMER 1.7 Melanoma Xenografts.

There is currently no consensus to determine which advanced melanoma patients will benefit from immunotherapy, highlighting the critical need to identify early-response biomarkers to immune checkpoint inhibitors. The aim of this work was to evaluate in vivo metabolic spectroscopy using hyperpolarized (HP) 13C-pyruvate and 13C-glucose to assess early response to anti-PD1 therapy in the YUMMER1.7 syngeneic melanoma model. The xenografts showed a significant tumor growth delay when treated with two cycles of an anti-PD1 antibody compared to an isotype control antibody. 13C-MRS was performed in vivo after the injection of hyperpolarized 13C-pyruvate, at baseline and after one cycle of immunotherapy, to evaluate early dynamic changes in 13C-pyruvate-13C-lactate exchange. Furthermore, ex vivo 13C-MRS metabolic tracing experiments were performed after U-13C-glucose injection following one cycle of immunotherapy. A significant decrease in the ratio of HP 13C-lactate to 13C-pyruvate was observed in vivo in comparison with the isotype control group, while there was a lack of change in the levels of 13C lactate and 13C alanine issued from 13C glucose infusion, following ex vivo assessment on resected tumors. Thus, these results suggest that hyperpolarized 13C-pyruvate could be used to assess early response to immune checkpoint inhibitors in melanoma patients.

Early Monitoring of Redox Status Based on Reduced Molecules Using In Vivo DNP-MRI for Cancer Treatment

<h3>Purpose/Objective(s)</h3> Radiation treatment effectiveness is assessed through the observation of morphological changes with CT or MRI images after treatment. The treatment efficacy of radiotherapy depends on redox modulation in tumor tissue, such as hydroxyl radicals and of reactive oxygen species generated by irradiation. Therefore, redox status may be a prognostic factor response to radiotherapy. However, spatiotemporal response of the redox metabolism in tumor tissue post-irradiation remains unknown. The purpose of this study was to detect early changes in the redox status in tumor tissue after irradiation using <i>in vivo</i> dynamic nuclear polarization (DNP)-MRI, to clarify the key molecules that cause these changes, and to compare these changes with early changes in lactate metabolism using hyperpolarized ¹³C magnetic resonance spectroscopy (MRS). <h3>Materials/Methods</h3> To monitor the response of tumor redox status after irradiation, redox imaging using in vivo DNP MRI/ Carbamoyl PROXYL (CmP) as redox sensitive DNP probe was performed after 5 Gy irradiation. To clarify the relationship between redox status alteration and treatment efficacy, in vivo DNP MRI was performed before and after irradiation by 2 and 5 Gy. In addition, tissue GSH and AsA levels on day 1 post irradiation were determined by HPLC. To observe the Warburg effect after radiation treatment, dissolution DNP study using hyperpolarized ¹³C pyruvate was performed before and after irradiation. <h3>Results</h3> The DNP image intensity in the tumor demonstrated a maximum value approximately four minutes after CmP administration, thereafter the image intensity was decreased by the redox reaction. The tumor GSH and AsA level on day 1 post irradiation were decreased compared to the control group. Production of hyperpolarized ¹³C lactate in tumor tissue did not change before and after irradiation. <h3>Conclusion</h3> Our study highlights that redox status in tumor tissue is already significantly decreased on day 1 before the detection of any morphological changes, even after irradiation with a dose as low as 2 Gy. Early changes in this redox status may be related decreases in glutathione and ascorbic acid levels in the tumor tissue after irradiation, which may alter earlier than ¹³C pyruvate hyperpolarized MRS, based on lactate metabolism. Therefore, early assessment of change in decay rate using in vivo DNP-MRI/CmP probing may predict the subsequent radiotherapy effect.

A 13C/31P surface coil to visualize metabolism and energetics in the rodent brain at 3 Tesla

PurposeWe constructed a 13C/31P surface coil at 3 T for studying cancer metabolism and bioenergetics. In a single scan session, hyperpolarized 13C-pyruvate MRS and 31P MRS was carried out for a healthy rat brain. MethodsAll experiments were carried out at 3 Tesla. The multinuclear surface coil was designed as two coplanar loops each tuned to either the 13C or 31P operating frequency with an LCC trap on the 13C loop. A commercial volume proton coil was used for anatomical localization and B0 shimming. Single tuned coils operating at either the 13C or 31P frequency were built to evaluate the relative performance of the multinuclear coil. Coil performance metrics consisted of measuring Q factor ratio, calculating system input power using a single-pulse acquisition, and acquiring SNR and flip angle maps using 2D CSI sequences. To observe in vivo spectra, a bolus of hyperpolarized [1-13C] pyruvate was administered via tail vein. In vivo13C and endogenous 31P spectra were obtained in a single scan session using 1D slice selective acquisitions. ResultsWhen compared with single tuned surface coils, the multinuclear coil performance showed a decrease in Q factor ratio, SNR, and transmit efficiency. Flip angle maps showed adequate flip angles within the phantom when the transmit voltage was set using an external phantom. Results show good detection of 13C labeled lactate, alanine, and bicarbonate in addition to ATP from 31P MRS. ConclusionsThe coil enables obtaining complementary information within a scan session, thus reducing the number of trials and minimizing biological variability for studies of metabolism and bioenergetics.

Improved total sensitivity estimation for multiple receive coils in MRI using ratios of first-order statistics.

Spatial variation in the sensitivity profiles of receive coils in MRI leads to spatially dependent scaling of the signal amplitude across an image. In practice, total sensitivity of the coil array is either calibrated or corrected directly by comparison to a uniform sensitivity image, fitting of coil profiles, or indirectly by constraining the reconstructed image or coil profiles. In the absence of these corrections, popular coil summation strategies are often designed to maximize the signal-to-noise ratio or optimize under-sampled encoding but not necessarily estimate the value of the signal unscaled by the coil spatial sensitivity. We use ratios of first-order statistics to approach the unscaled value of the signal at any position. Motivated by the assumption that the coil array is a sample from much larger number of possible coils, we present two approaches to scale the mean signal in all coils: (1) an argument for use of the mode of the normalized signals, and (2) using a one-dimensional analog derive an approximate expression for scaling with the ratio of the square-of-the-mean to the mean-of-the-squares. We test these approaches with simulation where idealized coil elements are arrayed around an object, and on directly acquired data with an 8-channel coil array on a uniform 13C phantom, and on Hyperpolarized 13C pyruvate brain MRI. We show improved image uniformity using the ratios of first order statistics compared to a simple homomorphic filter, noting that these approaches are more sensitive to noise. We present simple methods for correcting the spatial variation in sensitivity profiles in the context of a coil array. These methods can be used as an initial or adjunct step in data post-processing.

Abstract 6222: Comprehensive monitoring of pyruvate metabolism in cancer cells and tumors reveals vulnerability to metabolic inhibition therapy with small molecules

Abstract Introduction: Pyruvate(Pyr) metabolism is a keystone in cancer metabolism, as well as normal cells, and has two major metabolic fluxes; Glycolysis and Oxidative Phosphorylation (OXPHOS). Especially in cancer cells, increased Pyr metabolism meets the demand of higher energy consumption during rapid cell growing state. Therefore, monitoring tumor Pyr metabolic flux in vitro and in vivo should be necessary to develop a therapeutic strategy using small molecules which can inhibit the fluxes.Aim: We performed in vitro metabolic analysis on pancreatic cancer cells (MiaPaCa2) and applied a novel in vivo imaging technology “hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging (HP-MRSI)” to dynamic monitoring of the impact of Glycolysis and OXPHOS inhibitors on the MiaPaCa2 xenografts. Methods: We used a novel LDH inhibitor “NCI-006” for Glycolysis inhibition and Mitochondrial Complex 1(MC1) Inhibitor "Metformin(Met)", which is already clinically available, and “IACS-010759(IACS)” for OXPHOS inhibition, in vitro and in vivo. Extracellular flux (EXF) analysis and HP-MRSI were performed before and after administration of each or both to assess inhibitor impact on metabolic flux in vitro and in vivo, respectively. Results: HP-MRSI confirmed that LDH activity in the tumor was suppressed by NCI-006 (83.3±4.4% decrease) and accelerated by Met (69.9±6.6% increase) and IACS (88.6±25.1% increase). We confirmed these in vivo observations are fully consistent with the effect of those inhibitors in vitro on the energy profile of MiaPaCa2, and also the close correlation of these data with the results of the ex vivo LDH activity assay, suggesting HP-MRSI can reliably monitor in vivo on target effects of the inhibitors without need for tissue sampling. In addition, combined treatment with the NCI-006 and MC1 inhibitor significantly suppressed in vitro cell growth and tumor growth in an efficacy study of MiaPaCa2 xenografts, compared to each single administration. Apoptosis assay showed the combined treatment induced apoptosis in MIA Paca2 cells. Conclusion: Using EXF analyzer and HP-MRSI revealed that Glycolysis inhibition redirects tumor Pyr toward OXPHOS and also MC1 inhibition redirects tumor Pyr toward Glycolysis. Combination therapy suppresses metabolic plasticity, causing metabolic quiescence in vitro and tumor growth inhibition in vivo. HP-MRSI is thought to be useful for pancreatic cancer patients to establish the current treatment model because of no need to obtain clinical samples, such as patients-derived cancer cells or spheroid from the patients. The current proof of concept can be of great value in developing new therapeutic strategies using metabolic inhibitors to treat cancers. Citation Format: Nobu Oshima, Yuki Aisu, Shigeo Hisamori, Shigeru Tsunoda, Hiroki Hashida, Kenji Uryuhara, Hiroyuki Kobayashi, Masato Kondo, koji Kitamura, Satoshi Kaihara, Kazutaka Obama, Krishna Murali, Len Neckers. Comprehensive monitoring of pyruvate metabolism in cancer cells and tumors reveals vulnerability to metabolic inhibition therapy with small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6222.

Deuterium metabolic imaging and hyperpolarized 13C-MRI of the normal human brain at clinical field strength reveals differential cerebral metabolism

Deuterium metabolic imaging (DMI) and hyperpolarized 13C-pyruvate MRI (13C-HPMRI) are two emerging methods for non-invasive and non-ionizing imaging of tissue metabolism. Imaging cerebral metabolism has potential applications in cancer, neurodegeneration, multiple sclerosis, traumatic brain injury, stroke, and inborn errors of metabolism. Here we directly compare these two non-invasive methods at 3 T for the first time in humans and show how they simultaneously probe both oxidative and non-oxidative metabolism. DMI was undertaken 1–2 h after oral administration of [6,6′-2H2]glucose, and 13C-MRI was performed immediately following intravenous injection of hyperpolarized [1–13C]pyruvate in ten and nine normal volunteers within each arm respectively. DMI was used to generate maps of deuterium-labelled water, glucose, lactate, and glutamate/glutamine (Glx) and the spectral separation demonstrated that DMI is feasible at 3 T. 13C-HPMRI generated maps of hyperpolarized carbon-13 labelled pyruvate, lactate, and bicarbonate. The ratio of 13C-lactate/13C-bicarbonate (mean 3.7 ± 1.2) acquired with 13C-HPMRI was higher than the equivalent 2H-lactate/2H-Glx ratio (mean 0.18 ± 0.09) acquired using DMI. These differences can be explained by the route of administering each probe, the timing of imaging after ingestion or injection, as well as the biological differences in cerebral uptake and cellular physiology between the two molecules. The results demonstrate these two metabolic imaging methods provide different yet complementary readouts of oxidative and reductive metabolism within a clinically feasible timescale. Furthermore, as DMI was undertaken at a clinical field strength within a ten-minute scan time, it demonstrates its potential as a routine clinical tool in the future.

Combined HP 13C Pyruvate and 13C-Glucose Fluxomic as a Potential Marker of Response to Targeted Therapies in YUMM1.7 Melanoma Xenografts.

A vast majority of BRAF V600E mutated melanoma patients will develop resistance to combined BRAF/MEK inhibition after initial clinical response. Resistance to targeted therapy is described to be accompanied by specific metabolic changes in melanoma. The aim of this work was to evaluate metabolic imaging using 13C-MRS (Magnetic Resonance Spectroscopy) as a marker of response to BRAF/MEK inhibition in a syngeneic melanoma model. Tumor growth was significantly delayed in mice bearing YUMM1.7 melanoma xenografts treated with the BRAF inhibitor vemurafenib, and/or with the MEK inhibitor trametinib, in comparison with the control group. 13C-MRS was performed in vivo after injection of hyperpolarized (HP) 13C-pyruvate, at baseline and 24 h after treatment, to evaluate dynamic changes in pyruvate-lactate exchange. Furthermore, ex vivo 13C-MRS steady state metabolic tracing experiments were performed after U-13C-glucose or 5-13C-glutamine injection, 24 h after treatment. The HP 13C-lactate-to-pyruvate ratio was not modified in response to BRAF/MEK inhibition, whereas the production of 13C-lactate from 13C-glucose was significantly reduced 24 h after treatment with vemurafenib, trametinib, or with the combined inhibitors. Conversely, 13C-glutamine metabolism was not modified in response to BRAF/MEK inhibition. In conclusion, we identified 13C-glucose fluxomic as a potential marker of response to BRAF/MEK inhibition in YUMM1.7 melanoma xenografts.

Spatiotemporal imaging of redox status using in vivo dynamic nuclear polarization magnetic resonance imaging system for early monitoring of response to radiation treatment of tumor

In general, the effectiveness of radiation treatment is evaluated through the observation of morphological changes with computed tomography (CT) or magnetic resonance imaging (MRI) images after treatment. However, the evaluation of the treatment effects can be very time consuming, and thus can delay the verification of patient cases where treatment has not been fully effective. It is known that the treatment efficacy depends on redox modulation in tumor tissues, which is an indirect effect of oxidizing redox molecules such as hydroxyl radicals and of reactive oxygen species generated by radiation treatment. In vivo dynamic nuclear polarization-MRI (DNP-MRI) using carbamoyl-PROXYL (CmP) as a redox sensitive DNP probe enables the accurate monitoring of the anatomical distribution of free radicals based on interactions of electrons and nuclear spin, known as Overhauser effect. However, spatiotemporal response of the redox status in tumor tissues post-irradiation remains unknown. In this study, we demonstrate the usefulness of spatiotemporal redox status as an early imaging biomarker of tumor response after irradiation using in vivo DNP-MRI. Our results highlight that in vivo DNP-MRI/CmP allowed us to visualize the tumor redox status responses significantly faster and earlier compared to the verification of morphological changes observed with 1.5 T MRI and cancer metabolism (Warburg effect) obtained by hyperpolarized 13C pyruvate MRS. Our findings suggest that the early assessment of redox status alterations with in vivo DNP-MRI/CmP probe may provide very efficient information regarding the effectiveness of the subsequent radiation treatment.