EMIM 2018 ControlCenter

Online Program Overview Session: PW-03

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Imaging Metabolism

Session chair: Uwe Himmelreich - Leuven, Belgium; Jesús Ruiz-Cabello - San Sebastián, Spain
 
Shortcut: PW-03
Date: Thursday, 22 March, 2018, 11:30 AM
Room: Banquet Hall | level -1
Session type: Poster Session

Abstract

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# 028

Monitoring the evolution of fat accumulation against Type 2 Diabetes by Computed Tomography in a high-fat diet/Streptozotocin rat model (#464)

P. C. Gutierrez1, 2, 3, R. Mares1, X. Xiao1, 3, A. Hernandez1, 3, B. Santos1, 3, C. Hernandez2, 3, J. A. Camara4, S. Aguade1, J. Castell1, 3, R. Simo2, 3, J. R. Herance1, 3

1 Vall d´Hebron Institute of Research (VHIR), Medical Molecular Imaging Research Group, Barcelona, Barcelona, Spain
2 Vall d´Hebron Institute of Research (VHIR)/CIBERDEM, Diabetis i Metabolisme, Barcelona, Barcelona, Spain
3 Universidad Autonoma de Barcelona, Medicina, Barcelona, Barcelona, Spain
4 Vall d´Hebron Institute of Research (VHIR), Preclinical Imaging Platform, Barcelona, Barcelona, Spain

Introduction

Obesity is a prevailing disease due to our social lifestyle. Accumulation of fat might be placed in different parts of the body and is a risk factor of type 2 diabetes mellitus (T2DM)1, the 7th cause of death by 20302. For this reason, the development of diagnostic tools to monitor obesity-related T2DM is required. Imaging is being investigated to show any relation between different types of fat, parameters related to T2DM and the capacity of CT to quantify fat will be an opportunity4. Thus, the aim of the current work is to determine the useful of CT to monitor T2DM and treatments.

Methods

15 male Wistar rats were used for the study, 10 were fed with high fat diet during 2 weeks. Afterward, animals were rendered diabetic by a single dose of STZ (35mg/kg). After one week, animals had blood glucose ≥ 300mg/dL. At that moment rats were placed in three groups: 2 controls (positive and negative) and chitosan treatment (300 mg/Kg). After 6 weeks, biochemical and anthropometrical parameters, density and volume of fat by CT were determined.

Image acquisition was performed using a micro CT at 160 µA and 90kV using sections of 73 mm thick for each 17 seconds.

Results/Discussion

A tendency towards different type of fat accumulation in T2DM rats against glucose, weight and cholesterol were observed. Thus, an increase of fat causes an increase of glucose, weight and cholesterol, obtaining a lineal regression with positive slopes after correlating with CT. On the other hand, the treatment with chitosan affected to the different densities of fat studied but not its volume that can explain its anti-obesity effects.

Conclusions

CT seems to be useful to determine the relation between the amounts of fat accumulation with plasmatic glucose levels. Moreover, we have observed that the distribution and the density of fat is important in this pathology, where groin fat density is more correlated than the others fat densities. In addition, this technique could be helpful for managing the evolution of fat in this disease when it is treated with chitosan.

References

  1. J Brandberg, Computed Tomography and Magnetic Resonance Imaging in Determination of Human Body Composition. Methodological and Applied Studies, 2009.

http://gupea.ub.gu.se/handle/2077/19366

  1. Yang-wei Wang et al., “Spontaneous Type 2 Diabetic Rodent Models,” Journal of Diabetes Research 2013 (2013): 1–8, doi:10.1155/2013/401723.
  2. http://www.who.int/mediacentre/factsheets/fs312/en/
  3. King, A. J. F. (2012). The use of animal models in diabetes research. British Journal of Pharmacology, 166(3), 877–89

Acknowledgement

The present work was supported by grants from Carlos III Health Institute and the European Regional Development Fund (CP13/00252 and PI16/02064) and NTRACK_H2020 NMBP 2017 from Horizon2020 EU program

# 029

Multiparametric MRI tissue characterization in experimental metal implant testing with histological validation in rats: A Holistic Approach. (#216)

M. Meier1, D. Haake2, 1, A. M. Weinberg3, R. Willumeit-Römer4

1 Hannover Medical School, ZTL-Imaging Center, Hannover, Germany
2 Hannover Medical School, Forschungslabor, OE 7720, Hannover, Germany
3 Medical University of Graz, Laura Bassi Zentrum, BRIC, Graz, Austria
4 Metallic Biomaterials, Helmholtz-Zentrum Geesthacht, Geesthacht, Germany

Introduction

We took a holistic approach to characterize the progression of the healing process after implantation and monitor the development of possible side effects. We applied enabling technology to more than our target region (bone, muscles), including brain and heart, assessing the potential impact of different materials. We looked at possible biomarkers in an effort to reduce future needs for animal testing. Magnesium as an essential element in the body makes such implants valuable. Mechanical properties resemble bone; enhancing bone formation makes it a promising material for children.

Methods

We used MRI to examine bones, joints, and soft tissues such as cartilage, muscles, and tendons. Our project customised MRI and MRS methods for determining the quality of the degradation process of implants in bone. Although mechanical properties are commonly evaluated in bigger animals, we used a rat model studying different magnesium based degradable implants and compared those with non degradable material (PEEK) and control treatment (SHAM). We used diffusion imaging, perfusion imaging, T1- and T2-mapping and UTE-methods to correlate with histology. Spectroscopy in bone marrow and surrounding muscle tissue was used to characterize the degradation process and the healing process after implantation, while in brain and liver it served also as an indicator of overall healthiness.

Results/Discussion

Changes of imaging parameters in brain and liver correlated with overall health parameters during the healing process. Quantitative MR imaging based on imaging parameters such as T1, T2, T2*and ADC could as multiparametric readout distinguish between different materials. There was no single biomarker in these parameters but as an integrative description these parameters could well describe the processes of implant integration and degradation. Early quantitation of fibrosis between the host bone and some implants was shown. We could provide a measure of the distortions in the morphological bone parameters derived from MR images due to susceptibility artefacts and partial volume effects. MRS revealed signals of tCr (3.03 ppm), Trimethyl ammonium compounds TMA (3.21 ppm), Lipid resonances (0.9–1.4 ppm), a small tCr peak, a transient Lactate signal and a short time signal of deoxymyo- globin = (de)oxygenation. (Figure 2)

Conclusions

Monitoring metal implants with MRI is challenging but feasible. 3D MRI could overcome problems of slice distortion. Corrosion of magnesium is accompanied by hydrogen evolution and local increase in pH. With additional biocompatible elements like Ca there is a way to handle such constraints. Multimodality noninvasive imaging has the potential to exhibit a suitable method in preclinical models and as a translational tool. The holistic approach, aiming at the whole animal and peeping into brain and liver gave valuable information on Host Response and Metabolism.

References

Henkelman RM, Huang X, Xiang QS, Stanisz GJ, Swanson SD, Bronskill MJ. Magn Reson Med 1993;29(6):759–766

Witte, Kalla, Meier. European Cells and Materials Vol. 23. Suppl. 2, 2012.

Xu, Yu, Zhang, Pan, Yang. Journal of Biomedical Materials Research DOI: 10.1002/jbm.a.31273.

Zhang, Yifeng et al. Nature medicine 22.10 (2016): 1160–1169.

Acknowledgement

We thank Christian Bergen for excellent technical support

Fig 1
 Holistic approch with workflow from technology to implant monitoring
Fig2
Integrative approach of imaging parameters and holistic view on anatomy and metabolism
# 030

Analyses of body fat distribution by computer tomography in a mouse model of obesity shows increased visceral fat but not subcutaneous fat associated with increased serum insulin. (#397)

O. M. Will1, J. Humbert2, T. Damm1, C. - C. Glüer1, S. Tiwari1

1 University Hospital Schleswig-Holstein, Department of Radiology & Neuroradiology / Section Molecular Imaging, Kiel, Schleswig-Holstein, Germany
2 University Hospital Schleswig-Holstein, Department of Radiology & Neuroradiology, Kiel, Schleswig-Holstein, Germany

Introduction

Obesity is a major health problem that predisposes individuals to a multitude of diseases. It is increasingly recognized that it is not the total amount of fat but the distribution of fat that is associated with risk of developing various co-morbidities. Therefore, there is a need to accurately quantify whole body fat distribution, as well sensitively quantify ectopic fat storage. We have quantified distribution of fat in an ageing mouse model that express human ceacams to determine if the distribution of fat is associated with predisposition to type 2 diabetes.

Methods

We have utilized a FVB strain of mice expressing human ceacams which develop high fat content with increasing age and compared the distribution to wild type FVB mice of similar age. Body weight was quantifed and matched with age and sex, serum insulin levels and in vivo fat distribution was quantified by micro-computer tomography. The abdominal region between the first and the fifth lumbar vertebra was scanned using a conebeam in vivo micro-CT system (vivaCT80 Scanner, Scanco Inc., Switzerland). The scan was performed using the following parameters: energy settings of the X-ray source 45kVp and 177A, voxel size 76m, integration time 300ms, 250 projections per 180. Subcutaneous and visceral adipose tissue volumes were calculated with an algorithm adapted from Lublinsky et al. (2009).

Results/Discussion

Transgenic (Tg) mice were bigger and heavier than their counterpart wildtype (WT) mice. The initial observation was made in mice of 3.5 months old and the difference in weight was observed throughout the period of study. No differences in body weight by gender was observed. Analyses of distribution of body fat indicated significantly higher levels of total adipose tissue associated with increased visceral fat (n=12, p<0.05) but no differences in subcutaneous fat was observed. The increase in visceral fat was associated with higher fasting serum levels of insulin in the transgenic mice.

Conclusions

It is well established that ceacam1, a type 1 membrane glycoprotein with a transmembrane domain, is required for insulin clearance. To what extent disruption in insulin clearance is affected requires further investigation. Increased visceral fat indicates the mouse model is a replicate of human disease. With application of CT imaging, the mouse model  offers the possibility to study the muscolskeletal phenotype associated with hyperinsulinemia.

References

Lublinsky S, Luu YK, Rubin CT, Judex S. Automated separation of visceral and subcutaneous adiposity in in vivo microcomputed tomographies of mice. J Digit Imaging 2009;22:222–231.

Lubura M, Hesse D, Neumann N, Scherneck S, Wiedmer P, Schürmann A. Non-invasive quantification of white and brown adipose tissues and liver fat content by computed tomography in mice. PLoS One. 2012;7(5):e37026. doi: 10.1371/journal.pone.0037026. Epub 2012 May 16.

Schloesser A, Campbell G, Glüer CC, Rimbach G, Huebbe P. Restriction on an energy-dense diet improves markers of metabolic health and cellular aging in mice through decreasing hepatic mTOR activity. Rejuvenation Res. 2015 Feb;18(1):30-9. doi: 10.1089/rej.2014.1630.

Keywords: visceral fat, hyperinsulinemia, ceacam 1, micro-CT
# 031

Imaging Target Engagement in Collateral Lethality, [18F]FAZA PET Imaging Reveals Pharmacodynamics of IACS-010759, a Novel Complex 1 Inhibitor (#115)

S. T. Gammon1, F. Pisaneschi1, V. G. Yennu Nanda1, Y. Sun1, M. L. Bandi1, M. Smith1, Y. Rao1, F. Muller1, E. Di Francesco1, M. Davies1, J. Marszalek1, D. Piwnica-Worms1

1 MD Anderson Cancer center, Houston, Texas, United States of America

Introduction

Tumor deletions and mutations may cause defects in glycolysis, forcing these tumors to depend on oxidative phosphorylation for growth.  Some of these deletions arise near tumor supressors.  Targeting such deletions is collateral lethality.  Tumors that rely on oxidative phosphorylation, may have simultaneous poor perfusion and highly active mitochondria, yielding hypoxic and reducing environments.  These conditions are ideal for trapping 2-nitroimidazole based reporters, such as F18-labeled fluoroazomycin arabinoside ([18F]FAZA). 

Methods

This study utilized a test retest methodology.  Mice were briefly anaesthetized (<5 min) using 1 and 2% isoflurane using O2 as a carrier.  Mice were injected iv with [18F]FAZA in  with a target of 11.1 MBq per mouse. Mice were then returned to their cages, quickly became ambulatory and could move freely, eat and drink ad libitum for 3 hr.  Mice were then re-anaesthetized using 1 and 3% isoflurane, transferred to a pre-clinical PET/SPECT/CT system (Albira PET/SPECT/CT, Bruker) and maintained at 0.5 and 2% isoflurane during the acquisition of PET and CT.  After imaging mice were treated with either vehicle or IACS-010759 po.  Mice were subsequently re-imaged the next day.   FAZA change was quantified as log fold change T/M as defined by CT.

Results/Discussion

Pre-clinical models of ENO1 deleted orthotopic GBM, niave MEK inhibitor resistant melanomas, and lung cancer were studied.  At the MTD 10 mg/kg a robust, up to 6 fold,  (2way ANOVA p<0.0001) decrease in FAZA T/B was found in  D423-Fluc orthotopic GBM, H460 NSLC, SKMEL5 melanoma, A375R melanoma.  A slight but detectable, 1.5 fold, increase in [18F]FDG was also observed at 10mg/kg in A375 and A375R cells (p=0.002), but smaller than the decrease [18F]FAZA retention.   [18F]FAZA retention could also be increased with the proton uncoupler DNP p<0.02. [18F]FAZA retention was found to correspond with IACS-010759 by IHC treatment in a dose-dependent manner, with an apparent IC50 of 1.4 mg/kg (95%CI 0.48 to 4.1 mg/kg, n=12 mice) for the A375R melanoma cell line.  This measurement was more precise than an independently scored IHC slice when stained for pimonidazole from the same animals.  

Conclusions

PET imaging of [18F]FAZA can monitor quantitatively the pharmacodynamics a novel complex-I inhibitor IACS-010759 through changes in the intracellular oxygen consumption of the tumor 24 hours post treatment with the novel complex 1 inhibitor IACS-010759.FAZA can be a powerful PD marker for the complex-I inhibitor IACS-010759 in preclinical models, and it is possible that this protocol will be useful in upcoming clinical trials.

References

Sancho P, Barneda D, Heeschen C. Hallmarks of cancer stem cell metabolism. Br J Cancer 2016;114:1305-12.

Vaupel P, Hockel M. Blood supply, oxygenation status and metabolic micromilieu of breast cancers: characterization and therapeutic relevance. Int J Oncol 2000;17:869-79.

Reischl G, Dorow DS, Cullinane C, et al. Imaging of tumor hypoxia with [124I]IAZA in comparison with [18F]FMISO and [18F]FAZA--first small animal PET results. Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques 2007;10:203-11.

Chang E, Liu H, Unterschemmann K, et al. 18F-FAZA PET imaging response tracks the reoxygenation of tumors in mice upon treatment with the mitochondrial complex I inhibitor BAY 87-2243. Clin Cancer Res 2015;21:335-46.

Acknowledgement

We would like to acknowledge the SAIF that is partially funded by the Cancer Center Support Grant (CA16672 PI – DePinho).  We would also like to acknowledge the intracranial implant core on the brain SPORE, and the MD ACC characterized cell line core for providing cells and further cell line validation.  This work was supported by the National Institutes of Health through the Washington University-MD Anderson Cancer Center Inter-Institutional Molecular Imaging Center grant (NCI P50 CA94056)

18F-FAZA retention is dramatically reduced within 24 hr of treatment with complex I inhibitor
Precise dose response curves for complex 1 inhibitor can be generated in vivo
Retention of [18F]FAZA (arrows) (a) and pimonidazole (c) in sub-cutaneous A375R tumors in mice treated with IACS-010759. Dose-response curves were calculated for each method with 95% CI.
Keywords: FAZA, Oxidative Phosphorylation, GBM, melanoma, IACS-010759, Mouse model, PET
# 033

Quantitative monitoring of brain oxygenation in vascular cognitive impairment using 19F-MRI (#125)

A. A. Khalil1, 2, 3, S. Mueller1, 4, M. Foddis1, J. Lips1, U. Dirnagl1, S. Temme5, U. Flögel5, P. Boehm-Sturm1, 4

1 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Experimental Neurology and Center for Stroke Research Berlin, Berlin, Germany
2 Humboldt-Universität zu Berlin, Berlin School of Mind & Brain, Berlin, Germany
3 Max Planck Institute for Human Cognitive & Brain Sciences, Department of Neurology, Leipzig, Germany
4 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Cluster of Excellence NeuroCure and Charité Core Facility 7T Experimental MRIs, Berlin, Germany
5 Heinrich-Heine-University of Düsseldorf, Institute of Molecular Cardiology, Düsseldorf, Germany

Introduction

Assessing tissue oxygenation plays an important role in understanding the natural history of cerebrovascular disorders and potentially for monitoring treatment. Currently, surrogate measures such as blood flow are often used for this purpose instead. These are useful, but their pathophysiological role is difficult to interpret. The longitudinal relaxation rate (R1) of perfluorocarbons increases linearly with the concentration of molecular oxygen (1). We performed T1 mapping using a 19-F cryogenic coil to assess brain oxygenation changes in a mouse model of vascular cognitive impairment.

Methods

 

C57/BL6 mice (n=17) underwent stereotactic injection of Rhodamin-labeled perfluoro-15-crown-5-ether emulsion into the striatum and corpus callosum (1 µl/deposit). Global hypoperfusion was induced by wrapping 160 µm microcoils around both common carotid arteries (2). Imaging was done under isoflurane (1.5 – 2%) on a 7T Bruker BioSpin scanner with a cryogenic 19F/1H surface coil. T1 mapping was performed with a single-slice 19F FAIR-EPI sequence with adiabatic excitation pulses (TR=5000 ms, TE=9 ms, inversion times [ms]=(25, 400,800, 1200, 1600), 20 averages, FOV [mm]=19.2 x 19.2, matrix=48 x 48, slice thickness=6 mm, acquisition time=12 min 1 s). T1 fitting was performed using a 3-parameter model accounting for inversion efficiency variations.

Results/Discussion

The signal-to-noise ratio (SNR) in the 19F FAIR-EPI images, quantified by the number of voxels exceeding an SNR threshold of 5, varied across mice but was largely stable over time (Figure 1A). Overall, the cryogenic coil has an SNR 2-3 times higher than room-temperature mouse head surface coils (depending on the distance from the coil, measured on a hexafluorobenzene phantom). T1 measured prior to surgery decreased with increasing inhaled O2 concentrations. This is shown using a voxelwise fit for one representative mouse (Figure 1B) and by fitting the mean signal across all voxels with an SNR > 5 in all mice (Figure 1C). In the mice who received all 4 scans, an increase in tissue T1 (corresponding to a decrease in pO2) was observed 24 hours after inducing hypoperfusion, followed by a gradual recovery by week 4 (Figure 2A). The longitudinal T1 changes from pre-surgery (-1W) to the 4-week post-surgery timepoint (4W) are shown spatially for a representative mouse in Figure 2B.

Conclusions

19F-MRI using water-soluble perfluorocarbon emulsions, which are more biocompatible than liquid perfluorocarbons, is a feasible and promising method for long-term monitoring of changes in tissue oxygenation over time in vivo. In the bilateral carotid stenosis model of vascular cognitive impairment, this method detected changes in tissue oxygenation that mirror known changes in blood flow (3). These changes may be related to the neovascularization and collateral blood flow recruitment seen in this model (3).

References

1.          J. Ruiz-Cabello, B. P. Barnett, P. A. Bottomley, J. W. M. Bulte, Fluorine (19F) MRS and MRI in biomedicine. NMR Biomed. 24, 114–129 (2011).

2.          M. Shibata, R. Ohtani, M. Ihara, H. Tomimoto, White matter lesions and glial activation in a novel mouse model of chronic cerebral hypoperfusion. Stroke. 35, 2598–2603 (2004).

3.          P. Boehm-Sturm et al., Neuroimaging Biomarkers Predict Brain Structural Connectivity Change in a Mouse Model of Vascular Cognitive Impairment. Stroke. 48, 468–475 (2017).

Acknowledgement

This work was supported by the Stiftung Charité (BIH_PRO_317), the Federal Ministry of Education and Research (BMBF) (grant number 01EO0801, Center for Stroke Research Berlin), and the Deutsche Forschungsgemeinschaft (DFG) (Excellence Cluster NeuroCure, Excellence Cluster BrainLinks-BrainTools).

Figure 1

1A - Number of voxels exceeding an SNR threshold of 5 for each of the mice, shown across the imaging timepoints. 

1B - Kernel density plots showing distribution of T1 values in the SNR > 5 mask for an individual mouse when inhaling different O concentrations. 

1C - Mean T1 values in the SNR > 5 mask for four mice during inhalation of different Oconcentrations. 

Figure 2

2A - Mean T1 values in the SNR > 5 mask in two mice showing T1 changes over time. "-1W" refers to baseline (1 week prior to surgery). 

2B - Spatial T1 mapping in a mouse showing changes over the imaging timepoints. 

Keywords: vascular cognitive impairment, oxygenation, 19F-MRI, global hypoperfusion, perfluorocarbon emulsions, t1 mapping
# 034

Imaging beta cells in patients with changes in glycaemic control after Roux-en-Y gastric bypass surgery by 68Ga-exendin-4 PET/CT (#15)

L. Deden1, M. Boss3, H. de Boer2, E. Aarts1, I. Janssen1, M. Brom3, F. Berends1, M. Gotthardt3

1 Rijnstate, Vitalys, Department of (bariatric) surgery, Arnhem, Netherlands
2 Rijnstate, Department of internal medicine, Arnhem, Netherlands
3 Radboud University Medical Center, Department of radiology and nuclear medicine, Nijmegen, Netherlands

Introduction

Remission of type 2 diabetes mellitus (T2DM) occurs in >60% of patients undergoing Roux-en-Y gastric bypass (RYGB) surgery. In rare cases, hyperinsulinaemic hypoglycaemia occurs. The mechanisms behind these responses are incompletely understood, but a role for beta cell activity (BCA) and beta cell mass (BCM) is hypothesized. Studying the BCM in vivo is possible using Exendin-4, a stable analogue of glucagon-like peptide-1, specifically accumulating in the beta cells. 68Ga-exendin-4 PET/CT can be used to quantify BCM and to study the role of BCM in changed glycaemic control after RYGB.

Methods

The BCA and BCM were compared between patients with various responses in glycaemic control after RYGB. Five patients with complete remission of T2DM (responders), five patients without complete remission of T2DM (non-responders) and five patients with hypoglycaemia after RYGB were included. BCA was measured by an arginine stimulation test. BCM was measured as total pancreatic uptake of 68Ga-exendin-4 by quantitative analysis of PET/CT scans.

Results/Discussion

Preoperative patient characteristics and postoperative weight loss were comparable between the groups. BCA was significantly lower in the T2DM non-responders compared to the T2DM responders, with an arginine stimulated acute C-peptide response of 0.4±0.2 and 0.9±0.3 nmol/l, respectively (p=0.02). 68Ga-exendin-4 uptake in the pancreas was 26% lower in the T2DM non-responders (83±58 kBq) compared to the responders (111± 55 kBq), although not statistically significant (p=0.088). The BCA and BCM did not correlate (p=0.40). In hypoglycaemia patients, mean BCM was significantly higher than in the T2DM responders (191±63, p=0.032).

Conclusions

The data of this study suggest that BCM after RYGB is higher in T2DM patients with complete remission compared to non-responders. Furthermore, patients with hypoglycaemia have a higher BCM than the T2DM responders. BCM did not correlate to BCA. In conclusion, there may be a role for the BCM in patient response to RYGB and 68Ga-exendin-4 PET/CT is a feasible technique to measure the BCM in vivo.

Keywords: Bariatric surgery, Type 2 diabetes mellitus, post-gastric bypass hypoglycemia, beta cell mass, GLP-1 imaging
# 035

Online MRS profiling of subnanomole quantities of metabolites in brain dialysate: application to measurements of time-varying lactate production during administration of an antitumoral drug (#80)

Y. Crémillieux1, N. Pinaud1, U. Dumont1, R. Salvati1, V. Bouchaud2, S. Sanchez2

1 Université de Bordeaux, Institut des Sciences Moléculaires, Bordeaux, France
2 Université de Bordeaux, Centre de Résonance Magnétique des Systèmes Biologiques, Bordeaux, France

Introduction

Lactate is recognized as a metabolic biomarker of tumor malignancy (1,2) associated with an increase in glycolytic flux. Unfortunately, in vivo NMR detection and quantification of lactate remains difficult. In this study, we implemented an in vivo MRI/MRS protocol for the online profiling of subnanomolar quantities of lactate sampled from the extracellular fluid using implanted microdialysis and we applied this protocol in glioma‐bearing rats for the quantification of lactate concentration and the measurement of time‐varying lactate concentration during anti-tumoral drug administration.

Methods

Wistar rats (N=10) were implanted with C6 glioblastoma cells in the striatum. At day 10, microdialysis cannulae were positioned in the periphery of the tumor and in contralateral hemisphere. At day 11, microdialysis probes (2mm long, 6kDa cutoff) were inserted in both cannulae and perfused with artificial CSF and Gd-DOTA at 1.5mM concentration during 30min. Oxamate (5mM) was then added to the perfusate. Brain and tumor dialysate was analyzed online in a 7T (Bruker Biospec) magnet using a 2 microliters NMR microsolenoid (3) positioned a few centimeters distance from the microdialysis probe (Fig.1). NMR spectra were obtained every 4 minutes using a single-pulse sequence. Dialysates were collected at the outlet of the NMR microcoil for further analysis in high resolution NMR magnet.

Results/Discussion

The limit of detection of lactate was determined as 0.37 nmol.min-1/2, corresponding to lactate concentration of 0.2 mM in the dialysate. Signal/noise ratio of lactate peak above 120 was obtained from tumor dialysate in 4 minutes. The lactate peak amplitude was 193 ± 46% higher in tumor dialysate as compared to healthy brain dialysate. Following oxamate addition in the perfusate, a monotonic decrease of the lactate peaks was observed with an average time constant of 4.6 min. Higher lactate levels observed in the tumor environment as compared to healthy brain tissue are consistent with the literature on C6 cells metabolism. Due to its inhibiting effect on lactate dehydrogenase A, oxamate is considered as a potential anti-tumoral molecule and has been investigated in several studies (4,5). Indeed, a marked decrease of lactate concentration was observed shortly after the administration of oxamate (Fig 2). High resolution NMR spectra confirmed the findings obtained using the NMR microcoil.

Conclusions

In absence of overlapping NMR peaks, robust profiling of extracellular lactate can be obtained online using dedicated NMR microcoil. MRS measurements of dynamic changes in lactate production induced by anti-tumoral drugs can be assessed accurately with temporal resolutions in the order of minute. It represents an innovative approach for the evaluation of chemiotherapeutic treatments acting on this key glycolytic pathway of tumor cells. The MRS protocol can be readily transferred to clinical environment with the use of suitable clinical microdialysis probes.

References

1. Walenta S et al. Seminars in radiation oncology 2004, 14(3): 267-274.

2. Bharadwaj S et al. Journal of Clinical Neuroscience 2015, 22:1625–1627.

3.Glöggler S et al. Sci Rep 2016;6:36080.

4.Qing-Yi L et al. Metabolomics 2015;11(1):71-78.

5.Yang Y et al. Oncotarget 2014;5(23):11886-11896.

Acknowledgement

The authors acknowledge funding by the Laboratory of Excellence TRAIL ANR‐10‐LABX‐57 (research program Oncoflux) and the ANR program
INNES. The authors are grateful to Dr Anne‐Karine Bouzier‐Sore for her helpful advice on the implementation of the microdialysis protocol.

Figure 1
Schematic drawing of the experimental protocol based on microdialysis and NMR microcoil detection. a) NMR spectra  from tumor and healthy tissue obtained using the microcoil. As a comparison in b), localized NMR spectra from tumor and healthy tissue obtained using standard surface coil.
Figure 2
Stacked plot of consecutive microcoil MR spectra obtained on tumor dialysate showing the decrease with time (from below to top) of the lactate peak amplitude attributed to the oxamate inhibitory effect on lactate dehydrogenase enzyme.
Keywords: MRI and MRS, glioblastoma, lactate, metabolite profiling, microcoil, microdialysis
# 036

PET/ME-MRI to cross-validate the simultanoues distribution of [64Cu]-Ex4 and Mn in pancreatic β-cells (#253)

F. C. Michelotti1, G. Bowden1, J. Maczewsky4, G. Drews4, A. Küppers2, V. Nischwitz2, M. Gotthardt3, A. Schmid1, B. J. Pichler1

1 Eberhard Karls University Tübingen, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Baden-Württemberg, Germany
2 Forschungszentrum Jülich GmbH, Zentralinstitut für Engineering, Elektronik und Analytik, Jülich, Germany
3 Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, Nijmegen, Netherlands
4 Eberhard Karls University Tübingen, Experimentelle Diabetologie - Pharmakologie, Pharmazeutisches Institut, Tübingen, Baden-Württemberg, Germany

Introduction

Imaging of radiolabeled exendin-derivatives targeting GLP-1R and paramagnetic manganese (Mn) gated by VDCC after glucose stimuli showed encouraging results towards the investigation of β-cell loss in patients. The main focus of this study was to quantify and compare [64Cu]NODAGA-Exendin-4 ([64Cu]-Ex4) and MnCl2 using multimodal and simultaneous PET/ME-MR imaging. Thereby, we aimed to validate the specificity of signal for β-cell mass and insulin secretion in control and transgenic RIP1-Tag2 mice using a autoradiography and elemental imaging of the pancreatic tissue.

Methods

PET and volumetric T1 mapping were performed repeatedly (1 h and 24 h) to monitor β-cell mass and VDCC activity during the development of insulinomas in the pancreas of transgenic and control mice (n=3/4). Changes in [64Cu]-Ex4 uptake and Mn concentration ([Mn]) were investigated by comparing the co-registered PET/ME-MR images of both groups. Hyperinsulinemia was monitored by weekly measuring the blood sugar levels. Autoradiography and semi-quantitative laser-ablation-inductively-coupled plasma-mass spectrometry imaging was sequentially performed to validate the co-localization of [64Cu]-Ex4 and 55Mn with the endogenous levels of 44Ca and 64Zn in the same slice tissue. Insulin response was assesed at serial glucose conditions to determine the effect of Mn on the isolated β-cell islets.

Results/Discussion

Comparison of PET/ME-MR images revealed opposing linear coefficients between 1 h and 24 h (*P<0.05), suggesting that the accumulation of Mn during the early phase was not specific for β-cells. By contrast, we observed that changes in [Mn] over time depend on the late retention of Mn within the endocrine tissue (*P<0.05, Fig. 1). The insulin response to glucose showed no differences between 1 h and 24 h after the injection of Mn.

The specific internalization of tracer has been previously demonstrated and it correlated with the tissue localization of 64Zn and 44Ca across the native β-cells and the insulinomas until 24 h. As we observed in vivo, the uptake of Mn  at 1 h was not specific for β-cells, since the levels of 55Mn were higher in the exocrine tissue and they correlated negatively with 64Zn and 44Ca and [64Cu]-Ex4 uptake. However, the spatial distribution of 55Mn was reversed after 24 h as it was lower in the exocrine tissue and correlated with the endogenous metals and [64Cu]-Ex4.

Conclusions

We validated the feasibility of a simultaneous PET/ME-MR imaging method to address β-cell mass and functionality. The quantification of [64Cu]-Ex4 was highly sensitive for β-cell islets and insulinomas during the early phases of distribution. As novel finding, we demonstrated that Mn was specific only after it was gradually released by the exocrine tissue. Furthermore, we provided evidence on the active recruitment of Mn over time within the pancreatic endocrine tissue. This mechanism can be potentially exploited in organs, whose activity depends on the intracellular influx of divalent metals.

Validation of PET/ME-MR image comparison
The analysis of PET/ME-MR images was validated by assessing the tissue co-localization of endogenous (64Zn, 44Ca and 63Cu) and exogenous metals (55Mn) with the uptake of radioactive [64Cu]-Ex4.
# 037

Metabolic phenotyping of mesenchymal stem cells (MSCs) and MSC-derived populations of adipocytes and osteoblasts using NMR spectroscopy (#457)

A. Mavousian1, 2, H. A. Lugo-Leija1, D. Constantin-Teodosiu2, H. Williams3, V. Sottile1, S. Serres2

1 University of Nottingham, Wolfson Centre for Stem CellsTissue Engineering and Modelling (STEM), School of Medicine, Nottingham, United Kingdom
2 University of Nottingham, School of Life Sciences, Nottingham, United Kingdom
3 University of Nottingham, NMR Facility, Faculty of Sciences, CBS Building, Nottingham, United Kingdom

Introduction

Mesenchymal stem cells (MSCs) are recognised as undifferentiated, self-renewing and multipotent adult progenitors with capacity to give rise to osteoblasts and adipocytes upon chemical induction. The possibility of generating new cells/tissues has made MSCs promising candidates for regenerative medicine. However, established cell phenotyping methods involve invasive and cell destructive steps, which limit their application to live cell monitoring. Metabolite footprinting has shown great promise in phenotyping of stem cell differentiation but is limited in providing quantitative information.

Methods

MSCs were differentiated into osteoblasts using MSC medium containing 50mM ascorbic acid, 1M β-glycerophosphate and 5mM dexamethasone in complete MSC medium. MSCs were differentiated into adipocytes using MSC medium containing 10μg/mL insulin, 5mM dexamethasone, 0.1M isobutylmethylxanthine and 10mM rosiglitazone. Control MSCs were maintained in the standard MSC medium. Differentiation into osteoblasts and adipocytes was confirmed using Alizarin Red and Oil Red O staining, respectively. 13C label incorporation from [3-13C]pyruvate into alanine and lactate was measured in intact cell extracts at 37°C using 1H NMR (Bruker 800Mhz) from the splitting of their respective methyl proton resonances due to 1H-13C coupling (n=1). Enzymatic and ATP measurements were performed on separate batches (n=3)

Results/Discussion

Based on the evidence that metabolic reprogramming influences stem cell differentiation, we hypothesise that metabolic pathways could be used as quantitative biomarkers of cell phenotyping. The aim of this study was, therefore, to characterise metabolic pathways during stem cell differentiation using quantitative 1H magnetic resonance spectroscopy (MRS) and biochemical assays for identifying potential non-invasive biomarkers of the fate and quality of transplantable cells/tissues. Preliminary NMR data show that MSCs, osteoblasts and adipocytes differ in their [3-13C] lactate/ [3-13C] alanine ratios and that this correlates with changes in ALT and LDH activities (Fig 1). Furthermore, control MSCs and lineage-committed cells can be discriminated based on their respective glycolytic and oxidative ATP production rates confirming a shift towards aerobic metabolism and biosynthetic pathways during stem cell differentiation (Fig 2).

Conclusions

Taken together, these findings suggest that monitoring metabolic fate of [13C] pyruvate can be used to identify metabolic signatures specific to glycolytic, oxidative and biosynthetic pathways in MSCs and MSC-derived adipocytes and osteoblasts, with implications in future live cell monitoring using sensitive hyperpolarization 13C MRS technique.

Acknowledgement

This work was funded by the Research Priority Area at the University of Nottingham

Fig1

(A) Representative images of control MSCs, Alizarin Red staining of osteoblasts and Oil Red O staining of adipocytes at day 15 after differentiation. Scale bar = 50 μm. (B) Table summarising LDH and AAT activities (mU/mL) and 13C incorporation of control MSCs, osteoblasts and adipocytes. (C) NMR spectra showing methyl proton resonances and to 1H-13C coupling methyl upon 13C labelled incorporation.

Fig2

Graphs showing maximal rates of ATP production (nmol/min) in control MSCs, adipocytes and osteoblasts for oxidative and glycolytic pathways (n=3, *p<0.05, ANOVA followed by Tukey test).

Keywords: 13C, NMR, stem cells, metabolism, biomarker
# 038

Immunometabolic profiling of arthritic joint inflammation using MRI (#256)

M. - A. Neveu1, M. A. Jarboui1, N. Bézière1, K. Fuchs1, R. Daniels2, M. Kneilling1, A. M. Schmid1, B. J. Pichler1

1 Eberhard Karls University Tuebingen, Department of Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Tuebingen, Baden-Württemberg, Germany
2 Eberhard Karls University Tuebingen, Department of Pharmaceutical Technology, Tuebingen, Baden-Württemberg, Germany

Introduction

Inflammation is involved in many disease processes. However, accurate imaging tools permitting diagnosis and characterization of inflammation are still missing. As inflamed tissues exhibit a high rate of glycolysis due to the accumulation of inflammatory cells, pyruvate metabolism may offer a unique approach to follow the inflammatory response and progression. Therefore, the aim of the study was to follow the metabolic changes occurring in arthritic ankles using Hyperpolarized 1-13C-pyruvate MRS. Additionally, the inflammatory process and immune cell recruitment was followed using 19F MRI.

Methods

Mice were injected with serum containing glucose-6-phosphate-isomerase-specific antibodies (GPI) to induce experimental arthritis. Control animals were injected with saline. We followed disease progression in inflamed ankles at day 3 and 6 after the onset of GPI-arthritis.

MRI experiments were carried out on a 7T system. To monitor pyruvate metabolism, hyperpolarized 1-13C-pyruvate was prepared using a DNP polarizer and rapidly injected intravenously. 13C spectra of the ankles were then acquired every second. To track immune cell homing, a perfluorocarbon emulsion (PFC) was injected i.v. 48h before imaging using a 19F RARE sequence.

Additionally, metabolomic profiling of mice plasma was performed using targeted LC-MS to uncover metabolic changes during systemic inflammatory response.

Results/Discussion

We observed a significant increase in ankle swelling in GPI-serum injected mice, with a maximum at day 6, while ankles of saline injected animals remained unaffected.

Lactate production in the arthritic ankles was enhanced compared to the control ankles, as measured with the lactate-to-pyruvate (Lac/Pyr) ratio. In GPI-arthritic ankles, the Lac/Pyr ratio was higher at day 3 than at day 6. Lactate production peaked before maximal joint swelling, suggesting an early onset of hypoxia presumably induced by infiltrating immune cells. The progression of joint inflammation was then followed using 19F-MRI. A strong 19F signal was identified in the GPI-arthritic ankles after PFC administration, highlighting an up-regulated inflammatory cell recruitment as early as 3 days after the onset of GPI-arthritis.

Perturbations in metabolites associated with lipids turnover, oxidative stress and hexoses balance were identified in the plasma of arthritic mice, correlating with systemic inflammatory reaction.

Conclusions

Taken together, our data suggest that the homing of activated inflammatory cells could be responsible for an early onset of hypoxia during the progression of arthritic joint inflammation, leading to increased lactate levels in the ankles. Therefore, hyperpolarized 1-13C-pyruvate represents a promising tracer to investigate the onset of arthritic joint inflammation, even in slightly inflamed ankles.

Lactate production correlates with immune cell recruitment in arthritic ankles

A: Typical 13C-MRS spectra from GPI-arthritic ankles after i.v. injection of hyperpolarized 1-13C pyruvate (171 ppm) revealing lactate (183 ppm) production.

B: In vivo 19F MRI of the ankles after i.v. injection of perfluorocarbon emulsion: representative axial images highlighting an accumulation of the 19F signal in GPI-arthritic ankles.

Keywords: Arthritis, Hyperpolarized 13C-MRS, Lactate, Perfluorocarbon, 19F-MRI
# 039

Noninvasive in vivo quantification of Brown Adipose Tissue in GC-C KO mice using chemical-shift water-fat MRI (#370)

M. Dobrivojević Radmilović1, 2, N. Habek1, 3, M. Kordić4, V. Farkaš5, R. Bagarić5, S. Škokić1, S. Gajović1, 2, A. Dugandžić1, 3

1 School of Medicine University of Zagreb, Croatian Institute for Brain Research, Zagreb, Croatia
2 School of Medicine University of Zagreb, Department of Histology and Embryology, Zagreb, Croatia
3 School of Medicine University of Zagreb, Department of Physiology and Immunology, Zagreb, Croatia
4 MKP Ltd, Zagreb, Croatia
5 Ruđer Bošković Institute,, Division of Experimental Physics, Zagreb, Croatia

Introduction

There is a renewed interest in the physiology of brown adipose tissue (BAT) consequent to the relatively recent discoveries that BAT persists long into adulthood playing a protective role in metabolic dysfunction. Recent studies showed the involvement of guanylate cyclase-C (GC-C) receptor agonists in the BAT activation. The purpose of this study was to validate a chemical-shift water-fat MRI method for quantitative BAT imaging applied to determine the effects of GC-C on BAT physiology.

Methods

To determine the effects of GC-C on BAT physiology first a spin-echo water-fat MRI, fat- and water-based images of male and female mice were acquired and ratios of fat to the combined signal from fat and water were calculated. BAT volume and fat fraction quantification were determined based on multiple segmentations. Interscapular BAT (iBAT) was characterized as having significantly higher water content than WAT. The interscapular volume quantified by MRI was compared to the iBAT activity measured after cold-induced thermogenesis by PET-CT. The body temperatures of WT and GC-C KO mice were determined by infrared thermography.

Results/Discussion

Using the fat fraction metric, the iBAT depots of wild-type and GC-C KO mice could be clearly identified and quantified. We demonstrated a significantly reduced BAT volume in GC-C KO mice vs. WT animals, which strongly correlates with the iBAT activity measured by PET-CT.

Due to BAT’s multilocular fat distribution, triglyceride content, and vascular supply, its fat content is significantly less than that of WAT, thus measuring the fat fraction is a sensitive and quantitative approach to non-invasively characterize BAT. As shown GC-C KO animals compared to WT animals have less iBAT which is less activated after cold exposure.

Conclusions

Our results demonstrate the feasibility of MR-based BAT imaging in rodents and its potential use in future humane imaging approaches.

References

  1. Rosa T. Branca, Le Zhang, Warren S. Warren, Edward Auerbach, Arjun Khanna, Simone Degan, Kamil Ugurbil, and Robert Maronpot. In Vivo Noninvasive Detection of Brown Adipose Tissue through Intermolecular Zero-Quantum MRI. PLoS One. 2013; 8(9): e74206. 
  2. Rasmussen JM, Entringer S, Nguyen A, van Erp TG, Burns J, Guijarro A, Oveisi F, Swanson JM, Piomelli D, Wadhwa PD, Buss C, Potkin SG. Brown adipose tissue quantification in human neonates using water-fat separated MRI. PLoS One. 2013 Oct 30;8(10):e77907.

Acknowledgement

We would like to thank Dr. Kris A. Steinbrecher, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA for a donation of GC-C KO animals. This study was supported by the Croatian Science Foundation project BRAIN GAIN Homing Programme National Foundation for Science, Higher Education and Technological Development of the Republic of Croatia and by Croatian Science Foundation project  IP-06-2016-1892 RepairStroke. The MR imaging was done at GlowLab Multimodal Imaging Facility, University of Zagreb School of Medicine, Croatia.

Keywords: Brown adipose tissue, MRI, PET