Prostate tumor cells metabolism investigations by means of PHIP-SAH Hyperpolarized [1-13C]pyruvate (#692)
Eleonora Cavallari1, Carla Carrera1, Oksana Bondar1, Silvio Aime1, Francesca Reineri1
1 University of Torino, Dept. Molecular Biotechnology, Torino, Italy
The use of hyperpolarized (HP) pyruvate allows to detect the up-regulation of the exchange of the 13C HP label, mediated by the LDH enzyme in tumor cells. Parahydrogen Induced Polarization (PHIP) is more affordable and faster than d-DNP. In this work we used HP pyruvate achieved with the PHIP-SAH strategy (PHIP-Side Arm Hydrogenation)1 to study the difference of the metabolic phenotype of two highly aggressive and metastasizing human prostate carcinoma cell lines (PC3 and DU145). The toxicity of the solution obtained from this hyperpolarization process has also been investigated.
[1-13C]pyruvate was HP by means of PHIP-SAH and added directly in the 600 MHz spectrometer into cells suspension (9.2 ± 0.2 M cells) at the concentration of 5.0 ± 0.2 mM. A series of 13C-NMR spectra were acquired to follow the pyruvate-lactate (P-L) metabolic conversion. Three kind of samples were used: intact cells suspended in the growth medium, lysed cells and intact cells suspended medium to which extra-lactate (5.94 ± 0.14 mM) was added.
The results obtained with HP pyruvate were compared with those obtained using the conventional biochemical lactate dehydrogenase assay and 1H-NMR metabolomics.
The MTT test was applied to assess the cytotoxicity of the various components of the aqueous solutions derived from the PHIP-SAH hyperpolarization procedure.
The pyruvate-to-lactate exchange rate obtained from the 13C-hyperpolarized experiments showed that DU145 appear more glycolytic than PC3 cells. Conversely, the LDH activity was higher in the PC3 cells, according to the conventional biochemical assay.
This discrepancy might be explained considering that monocarboxylate transporters (MCTs) play an important role in determining the apparent exchange rate, but the HP experiments carried out on lysed cells show that the exchange rate is still slightly faster in the DU145 cells. Unexpectedly, the addition of lactate to the extracellular medium of intact cells made the apparent P-L conversion markedly faster in PC3 than in DU145.
This can be rationalised considering that different isoforms of LDH exist, that convert, preferentially, pyruvate into lactate or vice-versa.2 On the basis of the results obtained using HP pyruvate, we can conclude that the oxidation of lactate into pyruvate is more efficient in PC3 than in DU145 cells.
Hyperpolarized [1-13C]pyruvate, obtained through the PHIP-SAH methodology, evidenced the different metabolic phenotype of two, highly aggressive and metastasizing prostate cancer cells (PC3 and DU145). The 13C polarization level is well sufficient for these investigations and the presence of impurities due to the hyperpolarization procedure does not show toxicity effects on cells, which may alter cellular metabolism.
AcknowledgmentThis project has received funding from the Compagnia di San Paolo (Athenaeum Research 2016, n. CSTO164550) and from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 766402. The Fondazione Umberto Veronesi FUV (Post-doctoral Fellowships 2018) is gratefully aknowledged.
 Reineri, Francesca, Tommaso Boi, and Silvio Aime. 2015. 'ParaHydrogen Induced Polarization of 13C Carboxylate Resonance in Acetate and Pyruvate.' Nature Communications, 5858
 Brisson, Lucie, Piotr Ba, Martina Sboarina, Carine Michiels, Tamara Copetti, Pierre Sonveaux, Martina Sboarina, Coralie Dethier, and Pierre Danhier. 2016. “Lactate Dehydrogenase B Controls Lysosome Activity and Autophagy in Cancer” Cancer Cell 30: 418–31.
A) series of 13C-NMR spectra acquired after the perfusion of a cells suspension (PC3 cells, 10M) with the aqueous solution HP-[1-13C]pyruvate; B) expanded 13C-NMR spectrum at maximum intensity of the lactate signal; C) rate of pyruvate to lactate conversion obtained from the 13C-NMR hyperpolarized data, intact cells (DU145 and PC3) cultured in their proper culture medium; D) Pyr-Lac conversion rate, intact cells suspended in their medium with added lactate.
Keywords: hyperpolarization, cancer, metabolism
Hyperpolarized 13C-labeled tracers as NMR probes for the TCA cycle (#694)
Jaspal Singh1, Eul Hyun Suh1, Gaurav Sharma1, Jae Mo Park1, Craig Malloy1, Dean Sherry1, Zoltan Kovacs1
1 UT Southwestern Medical Center, Advanced Imaging Research Center, Dallas TX, United States of America
The TCA (citric acid) cycle is a fundamental metabolic process occurring in the mitochondria of all aerobic cells. It involves the oxidation of 2-carbon units to produce CO2 and reducing equivalents. Dysfunctional TCA cycle has been implicated in diseases such as cancer and diabetes . The cycle is primarily fed by acetyl-CoA derived from pyruvate decarboxylation and beta-oxidation. To avoid complications due to substrate competition, in this project we explore 13C labeled TCA cycle intermediates to probe TCA cycle activity by hyperpolarized 13C MRS independently from pyruvate.
Various 13C labeled oxaloacetate and 2-ketoglutarate ethyl ester derivatives were synthesized as outlined in Figure 1. The 13C tracer position was selected to have slow spin-lattice (T1) relaxation to preserve hyperpolarized (HP) magnetization. The labeled derivatives were polarized by dynamic nuclear polarization (DNP) under standard DNP conditions using the commercial HyperSense or SPINlab polarizer . The 13C polarization and T1 relaxation time values were measured by 13C NMR. For in vivo studies the HP-tracers were injected via the tail vein into normal rats and the appearance of downstream metabolites were observed by in vivo 13C MR spectroscopy in the liver using a 13C surface coil in a 3 T clinical MR scanner.
In isolated, perfused liver experiments, [4-13C]-oxaloacetic acid-1-ethyl ester disodium salt was taken up by liver cells, and was metabolized to citrate, malate and aspartate. However, in vivo experiments were inconclusive because the compound did not polarize well and the T1 value of the carboxylate was short. The metabolism of [U-13C5]-2-ketoglutarate diethyl ester was tested by 13C NMR isotopomer analysis of rat liver extracts. The 13C NMR spectra demonstrated that 2-ketoglutarate diethyl ester underwent hydrolysis and extensive metabolism to produce glutamate, succinate, glucose and other intermediates of the TCA cycle and gluconeogenesis within 3 minutes after injection. The in vivo production of hyperpolarized [13C]bicarbonate in the rat liver was successfully observed within 20 seconds after tail vein injection of hyperpolarized [1,2-13C2]-2-ketoglutarate diethyl ester.
Various 13C-labeled oxaloacetate and 2-ketoglutarate derivatives were evaluated as HP-13C MR probes of the TCA cycle. The in vivo production of HP-[13C]bicarbonate was successfully observed within seconds after injection of HP-[1,2-13C2]-2-ketoglutarate diethyl ester in rat liver indicating that this tracer could be used to monitor flux through 2-ketoglutarate dehydrogenase, an important control point in the TCA cycle.
AcknowledgmentFunding from NIH (P41-EB015908) is acknowledged.
 Brière J-J, Favier J, Gimenez-Roqueplo A-P, Rustin P 2006 'Tricarboxylic acid cycle dysfunction as a cause of human diseases and tumor formation' Am J Physiol Cell Physiol 291:C1114-C20.
 Ardenkjaer-Larsen JH, Fridlund B, Gram A, Hansson G, Hansson L, Lerche MH, Servin R, Thaning M, Golman K 2003 ‘Increase in signal-to-noise ratio of >10,000 times in liquid-state NMR’ Proc Natl Acad Sci USA 100:10158-63
Synthesis of 13C-labeled tracers
Observing the formation of HP-[13C]bicarbonate in the liver of a normal male Wistar rat after tail vein injection of HP-[1,2-13C2]-2-ketoglutaric acid diethyl ester at 3 T.
Keywords: Hyperpolarized NMR, 13C-labeled tracer, TCA cycle
Multi-compartment pH detection in healthy and tumour bearing mice using hyperpolarized deuterated [1,5-13C2]zymonic acid (#695)
Martin Grashei1, Sandra Sühnel1, Geoffrey Topping1, Elisabeth Bliemsrieder1, Stephan Düwel1, Christian Hundshammer1, Franz Schilling1
1 Technical University of Munich, Nuklearmedizinische Klinik und Poliklinik, Munich, Germany
Many pathologies such as cancer or renal failure involve alteration of the pH of the affected tissue.1 However, no fast and reliable technique in the clinic exists to non-invasively image pH.2 Previously, in vivo pH-imaging was demonstrated in rats using hyperpolarized (HP) [1,5‑13C2, 3,6,6,6‑D4]zymonic acid (ZAd)3,4. Here, we established in vivo pH-imaging in mice using HP ZAd in a 7T small animal scanner. We validated the method by non-localized and spatially resolved measurements in kidneys and show the ability to detect multiple pH-compartments per voxel in subcutaneous EL4 tumours.
Study Size: 5 C57BL/6 mice injected with 5∙106 EL4 cells and 3 healthy mice.
Hyperpolarization: 27mg ZAd and 25mg 13C-urea were co-hyperpolarized using DNP4. Dissolution was performed using D2O with 80mM TRIS, 50mM NaOD, 0.3mM Na2EDTA resulting in final concentrations of 50mM ZAd and 100mM 13C-urea.
HP MRS(I): HP MRSI was performed using FIDCSI starting 7s after end of injection with FA 15°, TR 83.1ms, matrix size 14x14, slice thickness 5mm, FOV 28x28mm2, BW 3201Hz, 256 Points. HP MRS was performed using PRESS with FAs 90°‑180°-180°, voxel size 10x5x5mm3, BW 2000Hz, 512 Points.
Data Processing: Processing of spectra, zero filling of imaging data and fitting of the difference of 13C1- and 13C5-ZAd-peaks with respect to 13C-urea peak3 to calculate pH-values was performed in MatLab.
Kidneys show three pH-compartments (Fig. 1a, b, n=3,3-6 repetitions). pH‑compartments could be grouped unambiguously and were assigned to the ureter (pH = 6.53±0.16), the medulla (pH = 7.06±0.06) and the cortex (pH = 7.38±0.03)(Fig. 1c).
pH-maps of EL4-tumours in mice (Fig. 2a) allowed detection of multiple pH-compartments (Fig. 2b,c). Tumours showed to have a physiologic (pH = 7.39±0.05,n=5) and an acidic pH-compartment (pH = 6.96±0.17,n=5) being detectable across the entire tumour (Fig. 2b,d). A second acidic pH‑compartment (pH = 6.62±0.10,n=5) is present only in parts of the tumour (Fig. 2c) which can be resolved by analysis of subregion-spectra (Fig. 2e) in several animals (n=5,Fig. 2f).
pH-compartments detected in kidneys agree with values previously reported3,5, indicating HP ZAd to be a suitable pH-sensor in mice. Further, one globally and a second, locally present, acidic pH-compartment could be detected in EL4, indicating intratumoural pH‑heterogeneity on a sub-voxel scale.
We have established an in vivo pH-imaging protocol on a 7T preclinical MRI for healthy and EL4-tumour bearing mice. We showed the ability to detect multiple pH-compartments per voxel in kidneys and EL4 tumours using hyperpolarized ZAd. Our results indicate so far unrevealed sub‑voxel pH heterogeneity in these tumours. The established method might prove useful for characterization of intratumoural heterogeneity to evaluate treatment strategies.
AcknowledgmentWe acknowledge support from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation – 391523415, SFB 824).
 Granja, S, Tavares-Valente, D 2017, 'Value of pH regulators in the diagnosis, prognosis and treatment of cancer', Seminars in Cancer Biology, 43, 17-34, Amsterdam - Netherlands: Elsevier Inc.
 Anemone, A, Consolino, L 2019, 'Imaging tumor acidosis: a survey of the available techniques for mapping in vivo tumor pH', Cancer Metastasis Rev. 38, 25-49, Cham - Switzerland: Springer Nature Switzerland AG
 Duewel, S, Hundshammer, C, 2017, 'Imaging of pH in vivo using hyperpolarized 13C‑labelled zymonic acid', Nature Commun., 8, 15126, London - UK: Nature Publishing Group
 Hundshammer, C, Duewel, S 2017, 'Deuteration of Hyperpolarized 13C‐Labeled Zymonic Acid Enables Sensitivity‐Enhanced Dynamic MRI of pH', Chemphyschem., 18, 2422-2425, Weinheim - Germany: Wiley-VCH
 Raghunand, N, Howison, C 2003, 'Renal and Systemic pH Imaging by Contrast-Enhanced MRI', Magn Reson Med., 49, 249-257, New York City - USA: Wiley
Detection of multiple pH-compartments in kidney of healthy mice
a: Mean pH-map from three kidney pH-compartments (equal weights) and anatomical T2-weighted 1H-image of a healthy mouse. Kidney ROIs are encircled with white lines.
b: PRESS-voxel spectrum of a single kidney and fit of 13C-urea-(right), parapyruvate‑hydrate‑C5(PPH5, magenta)- and ZAd-13C1- and 13C5-peaks corresponding to three pH-compartments (red, green, blue markers).
c: pH-compartments derived from multiple measurements in three animals (individual indicated by symbol type) on different days from PRESS-voxels and CSI-ROIs assigned to their anatomical kidney compartments.
Detection of multiple pH-compartments in mice bearing subcutaneous EL4-tumours
a: 1H-image with subcutaneous EL4-ROI (white) and -subregion (yellow) and [1-13C]lactate-phantom (arrow).
b: pH-map of first acidic compartment.
c: pH-map of second acidic compartment.
d: ROI-spectrum showing one physiological (pH = 7.40, red) and one acidic compartment (pH = 6.88, green).
e: Subregion-spectrum showing a physiological compartment (pH = 7.38, red) and two acidic compartments (pH = 6.88, green and pH = 6.47, blue).
f: Physiologic (pH = 7.39±0.05, n=5) and two acidic pH-compartments (pH1 = 6.96±0.14, n=4 and pH2 = 6.62±0.10, n=5), detected in EL4-subregions in several animals (n=5).
Keywords: Hyperpolarized MRSI, EL4, Zymonic Acid, pH Imaging, kidney
Fast 3D Hyperpolarised 13C Metabolic MRI at 7 T using Spectrally-Selective bSSFP (#697)
Geoffrey Topping1, Jason G. Skinner1, Irina Heid2, Maximilian Aigner1, Martin Grashei1, Christian Hundshammer1, Lukas Kritzner2, Frits van Heijster1, Rickmer Braren2, Franz Schilling1
1 Technical University of Munich, Department of Nuclear Medicine, Klinikum rechts der Isar, Munich, Germany
2 Technical University of Munich, Institute of Radiology, Klinikum rechts der Isar, Munich, Germany
Metabolic magnetic resonance spectroscopic imaging (MRSI) using hyperpolarized (HP) compounds, such as 13C-labelled pyruvate to lactate conversion, requires pulse sequences that encode spatial and spectral information quickly and efficiently. Spectrally-selective excitations exploit the sparsity of the hyperpolarized 13C spectrum, allowing spatial and temporal resolution to be improved. This work establishes a 3D balanced steady-state free precession (bSSFP) sequence using this approach, which is applied to preclinical metabolic imaging at 7 T.
A 3D bSSFP sequence was modified by disabling slice selection and rephasing gradients, and changing the excitation frequency1,2 and power after each 3D volume is acquired. Single-side-lobed sinc-shaped excitation RF pulses with narrow 400 Hz (TR 11.27 ms) or 900 Hz (TR 6.29 ms) FWHM bandwidth were used, alternating between frequencies close to each resonance, which was also in a bSSFP pass band, to produce separate images of 13C-pyruvate and lactate.
A small animal 7 T MRI (Agilent/Bruker) was used for HP 13C-pyruvate-lactate MRSI and anatomical MRI of 6 pancreatic tumour bearing (PDAC) mice3 and 12 healthy mice. 13C pyruvate was hyperpolarized (Hypersense) and injected by tail vail (80 mM) immediately before bSSFP acquisition. T2-weighted anatomical MRI of these mice were also acquired.
With 400 Hz RF pulses, images were obtained with 3 mm isotropic resolution in 950 ms per (single metabolite) 3D image (Fig. 1). With 900 Hz RF pulses, images were obtained with 1.75 mm isotropic resolution in 1212 ms per 3D image (Fig. 2).
Evidence of heterogeneity was detected in PDAC mice tumours: AUC ratios were 1.26 for ROI1 and 1.41 for ROI2 (Fig. 1).
Shortening TR by relaxing the RF pulse FWHM from 400 Hz to 900 Hz lead to a substantial increase in resolution, from (3 mm)3 to (1.75 mm)3, with only a slight increase in scantime (950 ms vs. 1212 ms). Furthermore, the passbands became broader (88.74 Hz to 158.98 Hz), simplifying interpretation of quantification, because B0 inhomogeneities are less likely to lead to banding artifacts. Placement of the excitations on the far sides of the resonances means only the target resonance is excited, despite the broader RF excitation bandwidth.
Spectrally-selective 3D bSSFP can provide high spatiotemporal resolution, banding-artefact-free images of metabolic activity in mice at 7 T. We demonstrate this in healthy mice and mice with pancreatic tumours (PDAC). Potential tumour heterogeneity was detected in PDAC mice.
AcknowledgmentWe acknowledge support from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation – 391523415, SFB 824).
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 820374.
We thank Sybille Reder and Markus Mittelhäuser for performing the PET measurements, and Sandra Sühnel for assisting with the MRSI experiments.
 Milshteyn E, von Morze C, Gordon JW, Zhu Z, Larson PE, Vigneron DBJ 2018, 'High spatiotemporal resolution bSSFP imaging of hyperpolarized [1‐13C] pyruvate and [1‐13C] lactate with spectral suppression of alanine and pyruvate‐hydrate' MRM 80(3), 1048-1060
 Shang H, Sukumar S, von Morze C, Bok RA, Marco‐Rius I, Kerr A, Reed GD, Milshteyn E, Ohliger MA, Kurhanewicz JJ 2017, 'Spectrally selective three‐dimensional dynamic balanced steady‐state free precession for hyperpolarized C‐13 metabolic imaging with spectrally selective radiofrequency pulses', MRM 78(3), 963-975
 Heid I, Steiger K, Trajkovic-Arsic M, Settles M, Eßwein MR, Erkan M, Kleeff J, Jäger C, Friess H, Haller BJ 2017, 'Co-clinical assessment of tumor cellularity in pancreatic cancer', CCR 23(6), 1461-1470.
Spectrally selective (400 Hz FWHM) 3D bSSFP in a PDAC mouse
Hyperpolarized 13C pyruvate-lactate bSSFP images of a PDAC tumour mouse. Per-metabolite scantime = 950 ms, TR = 11.27 ms, resolution = (3 mm)3, αPy = 2° (bSSFP lobe 1), αLac = 10° (bSSFP lobe 1). All 13C images are overlaid on T2w 1H anatomical images. A: 6 select lactate image timepoints marked with two ROIs (green and blue) that correspond to the metabolite signal dynamics plotted in B&C respectively. AUC ratios for ROI1 and ROI2 = 1.26 and 1.41, potentially reflecting heterogeneity within the tumour.
Spectrally selective (900 Hz FWHM) 3D bSSFP in a healthy mouse
Hyperpolarized 13C pyruvate-lactate bSSFP images of a healthy mouse. Per-metabolite scantime = 1212 ms, TR = 6.29 ms, resolution = (1.75 mm)3. 13C images are overlaid on T2w 1H anatomical images. A&B show all 12 slices of the 13C images for 14 timepoints for pyruvate (αPy = 4°, bSSFP lobe 2) and lactate (αLac = 90°, bSSFP lobe 5). C&D are select images from A&B, indicated by the white boxes. Vena cava, heart, and kidneys are visible in both metabolites. The lactate phantom is visible in D&B slice 8 and absent in A slice 8. E shows metabolite signal dynamics in the marked kidney.
Keywords: Hyperpolarized 13C, bSSFP, PDAC, MRSI