17th European Molecular Imaging Meeting
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Imaging Metabolism

Session chair: Kevin Brindle (Cambridge, UK); Francesca Reineri (Torino, Italy)
 
Shortcut: PS 04
Date: Wednesday, 16 March, 2022, 11:30 a.m. - 1:00 p.m.
Session type: Parallel Session

Contents

Click at talk title to open the abstract

11:30 a.m. PS 04-01

Introductory Lecture

André Martins

Tuebingen, Germany

11:50 a.m. PS 04-02

Quantification of skin microvascular changes in patients with diabetes using Raster-Scan Optoacoustic Mesoscopy.

Hailong He1, 2, Nikolina-Alexia Fasoula1, 2, Angelos Karlas1, 2, 5, Murad Omar1, 2, Juan Aguirre1, 2, Jessica Lutz3, Michael Kallmayer5, Hans-Henning Eckstein5, 6, Annette Ziegler4, Martin Füchtenbusch3, Vasilis Ntziachristos1, 2, 7

1 Technical University of Munich, Chair of Biological Imaging, Munich, Germany
2 Helmholtz Zentrum München, Institute of Biological and Medical Imaging, Munich, Germany
3 Diabetes Center at Marienplatz, Munich, Germany
4 Helmholtz Zentrum München, Institute of Diabetes Research, Munich, Germany
5 Technical University of Munich, Clinic of Vascular and Endovascular Surgery, Klinikum Rechts der Isar, Munich, Germany
6 DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany
7 Technical University of Munich, Munich Institute of Robotics and Machine Intelligence(MIRMI), Munich, Germany

Introduction

Diabetes-related complications, such as diabetic neuropathy and atherosclerosis, are associated with degradation of the skin microvasculature, illustrating the necessity to detect microvascular changes during early stages of the disease to identify at-risk patients. Imaging skin structures and microvasculature might allow the identification of metrics sensitive to the state and progression of diabetes and related complications, which could serve as a screening tool for diabetic neuropathy and atherosclerosis in the future.

Methods

Raster-scan Optoacoustic Mesoscopy (RSOM) has demonstrated unique capabilities for non‑invasive imaging of skin microvasculature. We applied RSOM system to image skin at the lower legs of 79 patients with diabetes and 33 healthy volunteers. Imaging features afforded by RSOM in healthy and diabetic patients’ skin were investigated by a newly developed data processing pipeline, which could be used to automatically derive several metrics based on pathophysiological skin features and microvasculature in the dermis layer. For the first time, we showcase how label-free biomarkers extracted from RSOM images correlate with the effects of diabetic complications on skin microvasculature changes compared to healthy volunteer.

Results/Discussion

We present in this work, to the best of our knowledge, the first optoacoustic images of skin microvasculature and microanatomy in patients with diabetes mellitus. Our results demonstrate that changes in quantifiable metrics, such as skin epidermis thickness, blood volume, and vessel number, can significantly differentiate patients with diabetes and healthy volunteers. Furthermore, we observed statistically significant differences in these metrics between patients with different severities of diabetic neuropathy. Overall, RSOM imaging of all skin layers and their microvasculature could allow for the precise analysis of skin microanatomy and vasculature throughout all skin layers, possibly revealing novel quantitative biomarkers of disease in a non-invasive and convenient way.

Conclusions

RSOM could accurately detect skin microvascular changes in patients with diabetes and related complications, providing a novel non-invasive tool for early diabetes diagnostics and objective disease stratification.

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 687866 (INNODERM) and No 871763 (WINTHER), from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme under grant agreement No 694968 (PREMSOT) and from Helmholtz Zentrum München through Physician Scientists for Groundbreaking Projects, in part by the Helmholtz Association of German Research Center, through the Initiative and Networking Fund, i3 (ExNet-0022-Phase2-3). We thank Dr. Robert J. Wilson for his attentive reading and improvements of the manuscript and the staff at the Diabetes Center at Marienplatz in Munich and the Clinic of Vascular and Endovascular Surgery, Klinikum rechts der Isar at TUM for assisting with the patient studies.

Disclosure

Vasilis Ntziachristos is an equity owner in and consultant for iThera Medical GmbH, Munich, Germany.

Skin imaging of the lower extremities (distal pretibial region) of healthy volunteers and patients
(a) RSOM system. (b, c) RSOM images of a healthy volunteer (b) and a patient with diabetes (c);  (d) Vessel segmentation. (e) Vessel diameter and numbers of branches. Comparisons between healthy volunteers and diabetic patients; (f) the total numbers of small vessels; (g) the total numbers of large vessels; (h) the total numbers of vessels; (i) the total blood volume of the dermis vasculature; (j) the average thicknesses of the epidermis layers, and (k) the signal densities of the epidermis layers.*, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively.Scale bar = 500 µm.
Keywords: Optoacoustic imaging, skin imaging, diabetes complications
12:00 p.m. PS 04-03

Adaptation to Pregnancy at High Altitude: Lessons from a Mouse Model

Sima Stroganov1, Talia Harris2, Michal Neeman1

1 Weizmann Institute of Science, Biological Regulation, Rehovot, Israel
2 Weizmann Institute of Science, Chemical Research Support, Rehovot, Israel

Introduction

Chronic hypoxia during gestation, induced by high altitude, smoking, pollution or respiratory infections, is associated with higher incidence of IUGR (intra uterine growth restriction) and increased infant mortality. Gestational complications such as preeclampsia and gestational hypertension are more frequent at high altitudes thus increasing intrauterine mortality and negatively affecting maternal and infant health. We explored the adaptation to hypoxic environment during pregnancy to support O2 (oxygen) transport in pregnant mice and fetal survival using non-invasive in-vivo MRI technique.

Methods

MR is a powerful and versatile imaging modality with high spatial resolution that allows us to observe both structural and functional dynamic information in-vivo. Our lab has recently developed a non-invasive MRI-based method to derive Hb (Hemoglobin)-O2 affinity information1. In this study, pregnant mice were subjected to either normoxic (21% O2) or hypoxic (12.5% O2) conditions during gestation and imaged in a 15.2 Tesla ultrahigh field scanner. To further investigate the mechanism of oxygen transfer from the mother to the fetus, we looked at the placental expression of BPGM, the enzyme responsible for the synthesis of 2, 3 BPG, an allosteric modulator of Hb-O2 affinity. We also observed structural changes in the placentae following maternal hypoxia.

Results/Discussion

Our results demonstrate a higher incidence of IUGR in the embryos and the placentae of the hypoxic chamber group (Figure 1). The MR experiments showed that R2* levels were upregulated in maternal abdominal aorta of the hypoxic chamber group when subjected to hypoxic challenge, while no significant changes in R2* were observed in the placentae and embryos of the control vs hypoxic chamber group (Figure 1). We demonstrate a novel and unique polar expression of the BPGM enzyme in the spiral artery trophoblast cells (SpA TC) that come in direct contact with maternal blood, which presumably facilitates O2 transfer from the mother to the fetus (Figure 2). We also demonstrate structural changes in the hypoxic placentae as well as enlarged spiral arteries and elevation in erythrocyte levels (Figure 2). These results enabled us to explore the mechanisms of adaptation to oxygen deprivation, as well as to determine its effect on embryonic and placental development.

Conclusions

The results of our work imply on a completely novel pathway for the transfer of oxygen from the mother to the fetus. We shed light on the regulatory mechanisms controlling oxygen levels and adaptation to oxygen deprivation during gestation, revealing the ability of the placenta to compensate for systemic maternal hypoxia and further support the concept of the embryo being a “perfect parasite”.

Disclosure

 

  • a) I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.
References
[1] Avni, R. et al. MR Imaging–derived Oxygen-Hemoglobin Dissociation Curves and Fetal-Placental Oxygen-Hemoglobin Affinities. Radiology 000, 150721 (2016).
Effects of maternal hypoxia on embryos and their placentae
Effects of maternal hypoxia on the placenta
Keywords: Pregnancy, Oxygen, IUGR, MRI, Placenta
12:10 p.m. PS 04-04

[18F]FSPG PET imaging reveals elevated oxidative stress in the liver and skin of mice with argininosuccinate lyase deficiency

Oskar Vilhemsson Timmermand1, Dany P. Perocheau2, Richard Edwards1, Abigail R. Barber1, Julien Baruteau2, Timothy H. Witney1

1 King's College London, School of Biomedical Engineering and Imaging Sciences, London, United Kingdom
2 University College London, Genetics and Genomic Medicine, Great Ormond Street Institute of Child Health, London, United Kingdom

Introduction

Argininosuccinic aciduria (ASA) is an inherited metabolic disease caused by argininosuccinate lyase (ASL) deficiency. ASL enables detoxification of neurotoxic ammonia via the urea cycle and nitric oxide (NO) synthesis [1]. Consequently, ASA patients suffer from life-threatening hyperammonaemia, NO deficiency, and chronic liver disease [2].

We have previously shown that retention of the system xc- substrate [18F]FSPG is altered following oxidative stress in tumours [3,4]. Here, we assess [18F]FSPG as a sensitive marker of systemic oxidative damage in a hypomorphic mouse model of ASA.

Methods

ASL-deficient Aslneo/neo pups (Fig. 1A) were genotyped on postnatal day 1-2 and monitored for weight loss and neurological signs i.e. ataxia or stereoscopic circling. Five Aslneo/neo and 13 wild-type (WT) mice (2-4 weeks old) were injected intravenously with 1-3 MBq of [18F]FSPG and imaged dynamically over 90 min by PET/CT (nanoScan, Mediso) under terminal anaesthesia. Liver-associated radioactivity was quantified using Vivoquant (Invicro), with region of interests drawn using early timepoint PET images. Skin ROIs were drawn on the lower dorsal flank. Liver samples were collected post-imaging and homogenized on ice. In these lysates oxidative stress-regulated proteins and system xc- expression were analysed by western blot, and glutathione quantified using the GSH-Glo assay (Promega).

Results/Discussion

Aslneo/neo mice suffered from systemic disease, with high levels of ammonia and disruption of NO metabolism leading to oxidative stress, recapitulating symptoms seen in ASA patients [5]. PET imaging revealed marked differences in [18F]FSPG pharmacokinetics in Aslneo/neo pups compared to their WT littermates (Fig. 1B). Liver retention of [18F]FSPG was twice as high in Aslneo/neo pups compared to WT mice (11 ± 2 %ID/g vs. 5 ± 2 %ID/g, respectively at 30-60 min p.i.; p = 0.0006). A similar pattern was seen in the skin, reaching 12 ± 2 %ID/g at 30-60 min p.i. in Aslneo/neo pups compared to 5 ± 2 %ID/g in WT (p = 0.0006; Fig. 1C). [18F]FSPG retention was reduced in the salivary glands and pancreas of ASA mice, possibly reflecting reduced availability of [18F]FSPG due to liver and skin retention. Ex vivo analysis of liver tissue revealed upregulated levels of system xc- (Fig. 2A) and lower levels of total glutathione (Fig. 2B), signaling high levels of tissue-specific oxidative stress.

Conclusions

Herein we show that [18F]FSPG PET is a sensitive marker of systemic oxidative stress that accompanies ASA. Whilst elevated [18F]FSPG retention in the livers of ASA was predicted, large differences in [18F]FSPG skin retention was only revealed by PET imaging. Work is currently underway to determine the mechanisms that mediate [18F]FSPG tissue retention and whether [18F]FSPG can be used to monitor the efficacy of gene therapies targeting ASA.

Acknowledgement

We want to acknowledge Jana Kim and Kavitha Sunassee for their help with PET/CT calibration and maintenance, animal protocols and licensees. We also want to thank Eman Khalil for assisting with western blot procedures.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Baruteau, J, Diez-Fernandez, C, 2019, Argininosuccinic aciduria: Recent pathophysiological insights and therapeutic prospects, J Inherit Metab Dis. 42(6):1147-1161
[2] Baruteau, J, 2017, Jameson, E, Expanding the phenotype in argininosuccinic aciduria: need for new therapies, J Inherit Metab Dis 40(3):357-368
[3] McCormick, PN, Greenwood, HE2019, Assessment of Tumor Redox Status through (S)-4-(3-[F-18] fluoropropyl)-L-Glutamic Acid PET Imaging of System x(c)(-) Activity, Cancer Research 79 (4):853-863
[4] Greenwood, HE, McCormick, PN, 2019, Measurement of Tumor Antioxidant Capacity and Prediction of Chemotherapy Resistance in Preclinical Models of Ovarian Cancer by Positron Emission Tomography, Clin Cancer Res 25(8):2471-2482
[5] Baruteau, J, 2018, Perocheau, DP, Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer, Nat Commun 29;9(1):3505
Figure 1. [18F]FSPG In vivo imaging of ASL-deficient mice

A. Phenotypically, ASL-deficient pups are smaller than their wild-type (WT) litter mates and have brittle hair. This model also faithfully recapitulates the human disease and its underlying biochemistry. B. Representative [18F]FSPG PET/CT coronal slices (50-60 min p.i.) of WT and Aslneo/neo pups respectively. Pal - Palette; S – skin; P – Pancreas; K – Kidney; B – Bladder C. [18F]FSPG retention in liver and skin was significantly higher in Aslneo/neo pups compared to WT pups 30 to 80 minutes post injection. NS, not significant; *, P < 0.05; **, P < 0.01; ***, P <  0.001.

Figure 2. Oxidative stress in the liver of ASL-deficient mice

A. Western blot of the xCT subunit of system xc- in WT pups and Aslneo/neo pups show that ASL deficiency results higher expression of xCT in liver tissue. B. Analysis of total glutathione  in liver tissue show lower levels in Aslneo/neo pups compared to their WT littermates, indicating high levels of oxidative stress in ASL-deficient mice.

Keywords: [18F]FSPG, argininosuccinate lyase deficiency, Argininosuccinic aciduria, system xc-, oxidative stress
12:20 p.m. PS 04-05

Non-invasive identification of highly glycolytic, lactate-consuming tumors in pancreatic cancer

Irina Heid2, 1, Geoffrey J. Topping2, Lukas Kritzner1, Martin Grashei2, Corinna Münch3, 4, 5, Sinan Karakaya3, 4, 5, Jens T. Siveke3, 4, 5, Marcus Makowski1, Franz Schilling2, Marija Trajkovic-Arsic3, 4, 5, Rickmer F. Braren1, 6

1 Technical University of Munich, School of Medicine, Institute of Diagnostic and Interventional Radiology, Munich, Germany
2 Technical University of Munich, School of Medicine, Department of Nuclear Medicine, Munich, Germany
3 University Hospital Essen, West German Cancer Center, Bridge Institute of Experimental Tumor Therapy, Essen, Germany
4 German Cancer Consortium, Division of Solid Tumor Translational Oncology (DKTK, partner site Essen), Essen, Germany
5 German Cancer Research Center, DKFZ, Heidelberg, Germany
6 German Cancer Consortium, DKTK, partner Site Munich, Munich, Germany

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a rarely operable, highly heterogeneous tumor entity with poor prognosis. Tumor aggressiveness is associated with increased glycolytic rate and high lactate uptake to fuel the tricarboxylic acid (TCA) cycle1. In PDAC, the transcriptionally defined, aggressive quasi-mesenchymal (QM)2 subtype is related to augmented glycolysis3. Here we aimed to use magnetic resonance spectroscopy (MRS) with hyperpolarized [1-13C]pyruvate and [1-13C]lactate for metabolic phenotyping of human PDAC xenografts.

Methods

Subjects: Crl:NIH-Foxn1rnu rats were implanted subcutaneously with 107 PSN1/HPAC human PDAC cells, which were characterized using (q)RT-PCR and Seahorse Mito Fuel Flex Assays. Blood lactate concentrations were measured using Accutrend Plus Meter (Roche). Ex vivo tissue LDH enzyme activity was determined using cobas® c 701/702 system.

MRS: Metabolites were measured using multi-frame slice spectroscopy (MRS) with alternating frequency-selective excitation (FA 30°, 250 Hz, TR 2s) while injecting hyperpolarized (HP)[1-13C]pyruvate or HP[1-13C]lactate on 7T MRI (Bruker/Agilent) with a dual-tuned 1H/13C volume resonator (72 mm) and surface receiver coils (20 mm). A PRESS voxel on tumor was used for 1H frequency calibration to set the 13C center frequency for [1-13C]pyruvate4.

Results/Discussion

Gene expression analysis confirmed glycolytic orientation of the QM cells (high lactate dehydrogenase (LDH) and mono-carboxylate transporter 4 (MCT4), Fig 1A). Functional in vitro characterization revealed higher lactate consumption in the QM cells (Fig. 1B). Metabolic dependencies were preserved in vivo in the rat xenografts as measured by MRS with HP[1‑13C]pyruvate and HP[1‑13C]lactate (Fig. 2A-C). In vivo results were supported by elevated blood lactate concentrations in the QM-tumor bearing animals (Fig. 2D) and high LDH activity in tumor samples measured ex vivo (Fig. 2E).

These observations strongly support our hypothesis that QM PDAC incorporates more pyruvate and uses lactate as oxidative fuel for the TCA cycle in vitro and in vivo. Active conversion of pyruvate to lactate and re-usage of the latter by conversion to pyruvate has been suggested to correlate with tumor aggressiveness in lung and breast cancer1, 5 allowing functional metabolic subtyping.

Conclusions

We show that HP[1‑13C]pyruvate and HP[1‑13C]lactate are incorporated and utilized by human QM PDAC tumors, paving the way for functional stratification and precise treatment monitoring of PDAC. In addition, non-invasive identification of metabolic dependencies by [1-13C]pyruvate/lactate HP-MRS my lead to the development of personalized treatments targeting metabolism.

Acknowledgement

This work was supported by the grant of Wilhelm-Sander Stiftung (2019.008.1) to M.T-A and J.S and the German Research Foundation (DFG) within the SFB-Initiative 824 (collaborative research center SFB824 (391523415), projects C6 to RB and A7 to FS.

Disclosure

All authors declare no potential conflicts of interest, except:

JTS reports research funding from Bristol Myers Squibb, Celgene and Roche; consulting and personal fees from AstraZeneca, Bayer, Bristol Myers Squibb, Celgene, Immunocore, Lilly, Novartis, Roche, Shire; minor equity in iTheranostics and Pharma15 (< 3%) and member of the Board of Directors for Pharma15, all outside the submitted work.

References
[1] Faubert B, Li KY, Cai L, et al. Lactate Metabolism in Human Lung Tumors. Cell 2017;171:358-371 e9.
[2] Collisson EA, Sadanandam A, Olson P, et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nat Med 2011;17:500-3.
[3] Karasinska JM, Topham JT, Kalloger SE, et al. Altered Gene Expression along the Glycolysis-Cholesterol Synthesis Axis Is Associated with Outcome in Pancreatic Cancer. Clin Cancer Res 2020;26:135-146.
[4] Topping GJ, Heid I, Trajkovic-Arsic M, et al. Hyperpolarized (13)C Spectroscopy with Simple Slice-and-Frequency-Selective Excitation. Biomedicines 2021;9.
[5] Ros S, Wright AJ, D'Santos P, et al. Metabolic Imaging Detects Resistance to PI3Kalpha Inhibition Mediated by Persistent FOXM1 Expression in ER(+) Breast Cancer. Cancer Cell 2020;38:516-533 e9.
Figure 1: In vitro analysis of quasi-mesenchymal (QM) and classical human PDAC cell lines .

A: Quantitative (q)PCR analysis of subtype specific genes. B: Under deprived culture conditions, QM cells utilize lactate to fuel tricarboxylic acid cycle (TCA) cycle.

Figure 2: Metabolic dependencies of QM and classical human PDAC tumors in vivo.

A: T2-weighted anatomy of a rat s.c. tumor (t) covered with gel (g) showing slice and coil (c) placement of MRS measuring hyperpolarized [1-13C]pyruvate and [1-13C]lactate inter-conversions. B: Relative area under the curve (AUC) ratios in QM (PSN1, N=4) versus classical (HPAC, N=5) PDAC rat xenografts. C: Relative peak area ratios reveal high lactate consumption in QM tumors (PSN1 N=4, HPAC N=3). Elevated blood lactate levels (D) in PSN1-bearing tumor animals as well as higher LDH activity (E) measured in excised tumor samples confirm highly glycolytic phenotype of QM tumors.

Keywords: Pancreatic ductal adenocarcinoma, magnetic resonance spectroscopy, lactate, glycolysis
12:30 p.m. PS 04-06

Deuterium metabolic imaging for evaluating metabolic disorders in rodents on high fat diet

Viktoria Ehret1, Usevalad Ustsinau2, Joachim Friske3, Thomas Scherer1, Clemens Fürnsinn1, Thomas H. Helbich3, Cécile Philippe2, Martin Krššák1

1 Medical University Vienna, Division of Endocrinology and Metabolism, Department of Medicine III, Vienna, Austria
2 Medical University Vienna, Division of Nuclear Medicine, Department of Biomedical Imaging and Image-Guided Therapy, Vienna, Austria
3 Medical University Vienna, Division of Molecular and Structural Preclinical Imaging, Department of Biomedical Imaging and Image-Guided Therapy, Vienna, Austria

Introduction

Deuterium Metabolic Imaging (DMI) is a novel method to assess metabolism in vivo using 2H MR Spectroscopy (MRS) combined with the administration of deuterated substrates, giving great insight into metabolic processes of healthy and diseased brain1,2, brown adipose tissue3 and tumor tissue4. It provides metabolic maps for visualizing glucose transport and downstream metabolism2,3,5. In this pilot study, we use 2D DMI with intravenous (iv) administration of deuterated glucose (Glu) and palmitic acid (PA) to show metabolic differences in rats on high fat (HFD) and standard diet (SD) at 9.4T.

Methods

DMI measurements were performed on Biospec 94/30 (Bruker Biospin, Germany) MR with a 2H/1H surface RF coil (Ø=40mm). After an iv injection of [6,6’2H2]Glu (1.95g/kg bw) or palmitate (PA-d31) (0.0065g/kg bw), a 2D chemical shift imaging sequence (TR=350ms, FA=61.6°, matrix 12x12mm, FOV=50x36mm, avg=128 (Glu)/192 (PA)) was applied. Sprague Dawley rats on a HFD (n=3, m=525-640g, age=12 weeks) or SD (n=3, m=330-390g, age=12 weeks) were examined. Anatomic 1H MRI were acquired with an axial T1-weighted FLASH sequence (TR=30ms, avg=20, FA=30°, matrix 120x120, FOV=50x36mm).

Spectra were quantified and normalized to the 2H natural abundance water peak. Resulting maps were corrected for the hepatic baseline signal of Glu or lipids.

Results/Discussion

Chosen DMI acquisition strategy yielded sufficient SNR and spectral resolution in several 2H MRSI voxels co-registered within liver parenchyma (Fig. 1 & 2).

Quantitation yielded lower post-infusion glucose levels in rat livers on HDF (mean Glu/water=0.67±0.51) than with SD (mean Glu/water=2.21±0.90). High fat animals had higher levels of PA uptake (mean PA/water=1.01±0.27) after infusion than SD animals (mean PA/water=0.13±0.18). Higher Glu levels in livers of SD rats indicate the higher hepatic insulin sensitivity in lean animals, whereas low Glu levels following injection in livers of HFD rats point toward insulin resistance. Higher hepatic uptake of PA in HFD rats confirms their phenotype with metabolic disorder.

Future studies will focus on improved experimental setup with overnight fasting to ensure fully comparable metabolic status of animal livers during measurements. Quantification of pre-injection hepatic fat content is needed for correct quantification of metabolic fluxes.

Conclusions

DMI provides a method for visualizing metabolism in vivo making an assessment of differences in metabolically healthy and impaired rodents possible. 2H DMI spectra show a lower uptake of Glu and a higher uptake of PA in rats on HFD, confirming their metabolic syndrome.

Thus, DMI holds great potential for the detailed study of metabolic disorders associated with a wide variety of diseases, such as fatty liver disease or diabetes type 2.

Acknowledgement

This study was funded by the Vienna Science and Technology Fund (WWTF #LS19-046). We thank Robin de Graaf from Yale University for advice and support with DMIWizard.

Disclosure

I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Straathof M, Meerwald, A, De Feyter HM, et al. Deuterium Metabolic Imaging oft he Healthy and Diseased Brain. Neuroscience 2021;474:94-99.
[2] De Feyter HM, Behar KL, Corbin ZA, et al. Deuterium metabolic imaging (DMI) for MRI-based 3D mapping of metabolism in vivo. Sci Adv. 2018;4(8):eaat7314.
[3] Riis-Vestergaard M, Laustsen C, Ostergaard Mariager C, et al. Glucose metabolism in brown adipose tissue determined by deuterium metabolic imaging in rats. International Journal of Obesity 2020;44:1417-1427.
[4] Kreis F, Wright AJ, Hesse F, et al. Measuring Tumor Glytolytic Flux in Vivo by Using Fast Deuterium MRI. Radiology 2020;294:289-296.
[5] De Graaf RA, Hendriks AD, Klomp DWJ, et al. On the magnetic field dependence of deuterium metabolic imaging. NMR in Biomedicine 2020;33:e4235.
Figure 1
Liver DMI acquired after intravenous glucose administration in animals on HFD (left) and SD. The sections of the matrix maps (A) show the voxels included in the analysis, one of which is shown with spectral fit in (B) as an example. The metabolic maps in (C) clearly demonstrate the lower glucose uptake in HFD rats indicating insulin resistance and an impaired metabolism.
Figure 2
Liver DMI acquired after intravenous administration of palmitic acid in animals on HFD (left) and SD. The section of the matrix maps (A) show the voxels included in the analysis, one of which is shown with spectral fit in (B) as an example. The basal liver fat corrected metabolic map for the rats with fatty liver confirm their metabolic disorder, demonstrating the ectopic fat depots in the liver.
Keywords: Deuterium metabolic imaging, liver metabolism, preclinical MR spectroscopy
12:31 p.m. PS 04-07

Multiparametric MRI-based approaches to characterize metabolic and invasiveness landscapes in glioblastoma models

Antonella Carella1, Alessia Corrado1, Elena Botto2, Elisa Pirotta1, Daisy Villano2, Riccardo Gambino2, Edoardo Micotti3, Dario L. Longo1

1 National Research Council of Italy (CNR), Institute of Biostructures and Bioimaging (IBB), Torino, Italy
2 University of Torino, Department of Molecular Biotechnology and Health Sciences, Torino, Italy
3 Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Laboratory of Biology of Neurodegenerative Disorders, Department of Neuroscience, Torino, Italy

Introduction

Glioblastoma is the most aggressive brain tumor with very poor prognosis despite multi-modalities of treatment [1]. Despite MRI gadolinium contrast-enhanced imaging being widely utilized examination in the diagnosis and post-treatment management of patients with glioblastoma, more accurate imaging approaches are needed for identifying alterations of the tumor microenvironment. The aim of this study is to evaluate tumor pH, metabolic landscape and T1w Contrast Enhancement images for assessing tumor invasiveness and metabolic pathways in different glioblastoma models.

Methods

We compared two glioblastoma models obtained upon stereotaxic injection of 1 x 106 U87 cells and 2x105 GL261 cells (coordinates:1.5mm ML to the bregma and 3.0 mm DV to the dura) [2,3], into 8-week-old male athymic nude mice and C57BL/6 mice respectively (n=10). Tumor MRI-CEST pH imaging was obtained following Iopamidol injection and covering the whole tumor with high spatial resolution. Metabolites were assessed by single voxel spectroscopy (MRS) with a PRESS sequence with short (16ms) and long TE (135ms), VAPOR suppression, in a voxel size of 2x2x2mm3.Tumor border sharpness were assessed from T1w contrast enhanced (CE) images following Gd injection. Western blot and IHC analysis for LDHA, PDK1 and LAMP2 were performed to assess altered metabolism and acidosis.

Results/Discussion

The orthotopic model of U87 shows a strong extracellular acidification with low variability compared to the GL261 model (Fig.1a,b). This acidity is probably related to an altered metabolism. Although both orthotopic models show a metabolic alteration with a decrease in Cho and TNAA levels and an increase in mobile lipids (Fig.1c), as quantified by TARQUIN software, analyzing the spectra at 135ms TE, the U87 model shows an higher level in the lactate signal, compared to GL261(Fig.1d). This result was also confirmed in Western Blot with a slight increase of PDK1 in U87 (Fig.1e).These data confirms the hypothesis that the U87 model may has a higher glycolytic-dependent phenotype than the more OXPHOS-dependent phenotype of GL261[4]. In addition, T1w CE images of these two models(Fig.1f) showed a higher enhancement(Fig1g) and, indirectly, vascularization, of the GL261 glioblastoma model, thus confirming a more invasive pattern compared to U87 with a non-diffusive infiltrative pattern.

Conclusions

We observed in the U87 glioblastoma models a strong tumor acidosis compared to the GL261. Higher tumor acidification could be associated to the glycolytic metabolism, suggesting that MRI-CEST pH imaging can be potentially exploited for assessing the metabolic pathways involved in tumor development and potentially exploit them as therapeutic targets.

Acknowledgement

This work was supported by grant from the Associazione Italiana Ricerca Cancro (AIRC MFAG #20153)

Disclosure

 I or one of my co-authors have no financial interest or relationship to disclose regarding the subject matter of this presentation.

References
[1] Davis ME. 2016 Glioblastoma: Overview of Disease and Treatment. Clin J Oncol Nurs. Oct 1;20(5 Suppl):S2-8.
[2] Kramp TR , Camphausen K. 2012 Combination radiotherapy in an orthotopic mouse brain tumor model. J Vis Exp. Mar 6;(61):e3397
[3] Lo Dico, A et al. 2015 “Identification of imaging biomarkers for the assessment of tumour response to different treatments in a preclinical glioma model” Eur J Nucl Med Mol Imaging, June;42(7):1093-1105
[4] Zhou Y.  et al. 2011 Metabolic alterations in highly tumorigenic glioblastoma cells: preference for hypoxia and high dependency on glycolysis.J Biol Chem. Sep 16;286(37):32843-53
Fig. 1

a) Analysis of the extracellular pH in U87 and GL261 orthotopic glioblastoma models. b) Comparison of extracellular tumor pH maps on multiple slices in U87 and GL261 that allows to monitor tumor acidosis in the whole tumor. c)Quantification of the metabolites through single voxel spectroscopy (MRS) with 16ms TE. d) Evaluation of Lactate signal in U87 and GL261 orthotopic glioblastoma models, analyzed from 135ms TE. e) Western Blot analysis of PDK1 expression in U87 and GL261 cell lines. f) Comparison of axial T2 RARE and T1w CE images following Gd injection and (d) enhancement quantification.

Keywords: Glioblastoma, CEST, MRS, metabolism, T1w enhancement