Online Program Overview Session: PO 03

Introduction

Recently, optogenetic cardiac stimulation including structured illumination has been described as a promising experimental tool for cardiac arrhythmia research [1,2].  However, for optogenetic modulation light scattering, distribution, and attenuation through cardiac tissues and blood, is considered to affect the efficiency of illumination. To optimize photostimulation, we underlined differences among whole optogenetic murine hearts, ventricles and septum. Moreover, optical mapping of membrane potential is used to shed light on mechanisms underlying arrhythmia.

Methods

Isolated optogenetic murine hearts were transferred to a Langendorff perfusion system. Hearts were perfused with DI-4-ANBDQPQ (50 µM as bolus, Biozol) and Blebbistatin (45 µM, Sigma-Aldrich) for at least 20 min before pacing. The experimental system for determination of light penetrance and absorption through whole heart, left ventricle (LV), right ventricle (RV) and septum, was designed to globally illuminate with a LED source (Thorlabs). Applied light intensities were modulated using a function generator (Agilent Technologies). A photometer  (Fa. Thorlabs) detected the power of light fully penetrating the tissue. Furthermore, the system used for pacing the ventricles was designed to locally illuminate either ventricle with optical fibers of divers core diameters.

Results/Discussion

Light penetrance in blood filled hearts is significantly lower than in tyrode perfused hearts. This also applies to attenuation in perfused and non-perfused LV and septum. Non-perfused LV showed significantly higher penetrance than a non-perfused heart at 470 nm illumination (p≤0.01). No significant change was found in perfused and non-perfused RV. Testing for light penetrance efficiency between 470 nm and 630 nm illumination showed that 630 nm exhibited a significantly higher penetrance (p≤0.0001). Comparing the 470 nm light attenuation between LV and RV released that the illumination of RV exhibited a significantly lower light attenuation. Pacing the RV with an optical fiber led to a significant decrease (p≤0.05) in intensity unequal to LV. Measurements of different pacing intensities with and without voltage dye depicted that LV perfused without voltage dye show a significant decrease (p≤0.01). However, pacing through RV with and without voltage dye, showed non-significant changes.

Conclusions

Here we investigated optogenetic properties among divers cardiac tissues for a better understanding of tissue structure as well as experimental condition relevance in optogenetic modulation. In consequence, we managed to reveal the importance of specific experimental parameter ubiquitary used in cardiac research, which in turn contributes to an improvement in the efficiency of illumination and optimization of cardiac optogenetic approaches.  

References

Bruegmann, T. et al., Nature Methods 7, 897-900 (2010)

Quinonez Uribe, R.A. et al., Front. Physiol. 9, 1651 (2018)

Acknowledgement

The research leading to these results has received funding from the DZHK e.V. (partner site Göttingen), the German Federal Ministry of Education and Research (BMBF, Go-Bio),  the German Research Foundation (DFG, SFB 1002 (B05 and C03), SFB 937 (A18)), the European Union’s Horizon 2020 Programme (BiomEdical OPTICAL, grant number 675512) and the Max Planck Society.

Introduction

Inflammation is the key driver in cardiovascular atherosclerosis disease progression and plays an instrumental role in causing life-threatening clinical complications by predisposing vulnerable risky lesions to rupture¹ . This study proposes a novel approach that may allow for quantitative early detection of high-risk patients. Using SPECT molecular imaging, a novel dual-tracer protocol was tested to target all inflammation leukocyte cells [Novel tracer:¹¹¹In-DANBIRT] and the subset of activated macrophage cells [rtracer:^99mTc-DEMOTATE] simultaneously in human diseased atherosclerotic samples.

Methods

Human symptomatic carotid plaque samples were incubated in radioactive solution of (¹¹¹In-DANBIRT+^99mTc-DEMOTATE - radioactivity=100MBq). Dual-SPECT coupled with micro-CT scanning was performed using ultrahigh-resolution focused imaging. A high-energy VECTor collimator was used which has a <500μm reconstructed resolution and sensitivity >2800cps/MBq (Vector 5, MiLabs Utrecht). After scanning, plaque samples were further cryo-sectioned into 5µm slices and were immunohistochemically stained to assess co-localization of inflammation cells and macrophages positive cells in the plaque segments with respect to the dual-SPECT tracer’s signal uptake.

Results/Discussion

Novel radiotracer DANBIRT detected global inflammation levels in the plaque samples. Inflammatory leukocyte uptake signal was significantly higher compared to the subset of activated macrophages. The average total uptake was [Leukocyte; 16.92±5.13MBq/g] vs [Macrophage; 7.07±1.98MBq/g] (P<0.001). Dual-SPECT analysis further identified the colocalization in radiotracer uptake between the inflammation leukocytes and activated macrophages, and importantly in high risk shoulder regions with evidence of collagen fiber degradation. Local leukocyte detection in the vulnerable fibrous cap and shoulder regions revealed a significant positive correlation between SPECT leukocyte signal and immunohistochemistry cell area fraction [r=0.561; P<0.001] and a weak positive trend was identified for the macrophages alone [r=0.123; P=0.494]. A quantitative inflammation map was developed to indicate local regions of high inflammation heterogeneously distributed throughout the whole plaque lesion length.

Conclusions

This study demonstrates a quantitative approach to measure global inflammation levels locally in high-risk regions of vulnerable plaques prone to rupture and additionally identification of the intricate interplay between the key cellular interactions that drive the disease progression. This approach may offer the opportunity for the early diagnosis of highly inflamed vasculature and subsequent intervention to eliminate cardiovascular event.

References

[1] Libby, P et al. 2016 Leukocytes Link Local and Systemic Inflammation in Ischemic Cardiovascular Disease, VOL 67, NO.9.

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No.707404.

Introduction

Preclinical studies rely on imaging under anaesthesia, which has several limitations: 1) Not a true physiological representation of the disease, potentially modifying therapeutic-response and measurements likely confounded by effects of anaesthesia; 2) Numerous studies documenting cardio-depressive effects of anaesthesia, hence it is especially not suited for studies of the heart; 3) Clinically, patients are imaged in the ‘awake’ state, hence difference in method does not allow horizontal-comparison of results and often leads to promising preclinical research not translated into the clinic.

Methods

Here, we have developed a clinically-relevant preclinical method of measuring cardiac function in ‘awake’ and ‘asleep’ mice using real-time cardiac ultrasound. We aim to inform researchers on the various differences in cardiac function between animals imaged under anaesthesia and in the ‘awake’ state, and hope to propose a new regime for studying cardiovascular function via echocardiography in the preclinical setting.

Ten female BALB/c mice were shaved one-day prior (-1d) to echocardiography and were consecutively imaged on a daily basis (1d to 5d), first in the ‘awake’ state (securely- but gently-scruffed) and then in the ‘slept’ state (anaesthetised, 2% isoflurane in 100% oxygen). Parasternal long- and short-axis views in B- and M-modes were obtained.

Results/Discussion

Ejection fraction (EF) was significantly higher in awake mice and showed less variability at all time-points compared to anaesthetised (96.0±5.3 vs 47.9±9.6%), as expected without the cardio-depressive effects of anaesthesia, thereby allowing greater-sensitivity in detecting changes after therapy/treatment. Variability was also reduced, possibly due to the absence of sinus rhythm-disturbance caused by anaesthesia thus increasing the chances of observing a true effect.

Stroke volume (SV) initially-similar, but reduced overtime in the conscious mice (22.8±6.7 vs 27.7±4.4μl at 1d; 9.5±4.7 vs 25.1±6.6μl at 5d), possibly reflecting the series of adaptation of the animals to the procedure.

Cardiac output (CO) was comparable between the two states when taking an average over the 5-day period. (12.2±5.5 vs 11.6±2.5ml/min). However, there is a downward-trend in the awake animals due to decreasing end-diastolic volume (EDV) over time.

Conclusions

In conclusion, we have developed a clinically-relevant preclinical method of measuring cardiac function and showed the feasibility of imaging conscious mice using real-time cardiac ultrasound. Taken all together, we propose this new method of imaging to better-align preclinical to clinical ultrasound imaging of cardiac function.

References

Reference 1: Cardiovascular diseases (CVDs) fact sheet. World Health Organization. 2015.

Reference 2: Eisele JH, Trenchard D, Stubbs J, Guz A. The immediate cardiac depression by anaesthetics in conscious dogs. Br J Anaesth. 1969;41(2):86-93.

Reference 3: Murray DJ, Forbes RB, Mahoney LT. Comparative hemodynamic depression of halothane versus isoflurane in neonates and infants: an echocardiographic study. Anesth Analg. 1992;74(3):329-37.

Introduction

Macrophages cause atherosclerotic plaque rupture often leading to thrombosis and myocardial infarction. Their detection in coronary artery plaques would enable stratification of patients for better treatment of “vulnerable” phenotypes. Optical Coherence Tomography (OCT) enables visualisation of vulnerable plaque morphology and has the potential to detect the signal variance created by increased numbers of macrophages. Our aim was to enhance this effect using a clinically approved solution of Ultra-Small Paramagnetic Iron Oxide particles (USPIO) which are readily uptaken by plaque macrophages.

Methods

A “phantom” artery model was designed and 3D printed for standardised in-vitro imaging with an intra-coronary OCT system (St Jude OPTIS^TM). USPIO-laden macrophages were tested in this system to determine OCT contrast enhancement. Aortic atherosclerotic plaques of Apo-lipoprotein E knockout mice, treated with USPIO or saline were OCT imaged with the same system and sectioned for histological analysis. Patients presenting with acute coronary artery disease were randomised to receive USPIO or saline treatment, followed by OCT imaging of the culprit plaque and non-culprit control. The local pixel Standard Deviation (SD) of OCT images were processed in Matlab as a metric of contrast enhancement across all the experiments (in-vitro, ex-vivo and in-vivo imaging).

Results/Discussion

The OCT slices of the phantom gels in figure 1 appear more grainy when macrophages are present and this effect is enhanced with USPIO-laden cells. This is confirmed by the significantly higher mean SD in USPIO-laden cell gels compared to untreated cells in all 7 phantoms (p<0.001). USPIO treated mouse plaques (n=3) showed significantly higher mean SD than control plaques (n=3) (p<0.05). USPIO treated patients (n=8) demonstrated a significantly increased mean SD compared to control patients (n=10) in culprit and non-culprit plaques (p<0.05). Within patients the mean SD was significantly higher for culprit compared to non-culprit plaques in both USPIO and control groups which concurs with previous findings of increased macrophage presence in the caps of culprit plaques compared to stable ones. These results show that USPIO presence is associated with a significant increase in OCT contrast in a novel in-vitro phantom model, an atherosclerotic mouse study and in a human clinical trial.

Conclusions

USPIO was shown here to enhance the detection of macrophages in-vitro, ex-vivo and in-vivo using an intra-coronary OCT system. These results suggest that the use of USPIO, in conjunction with an automated SD colour-map tool could assist cardiologists in identifying vulnerable plaques during coronary interventions therefore enabling better stratification and treatment of patients post intervention.

Acknowledgement

Clara acknowledges the support of the EPSRC and the MRC through the OPTIMA (Optical Medical Imaging) CDT programme based at the Universities of Edinburgh and Strathclyde.

Introduction

Photoacoustic (PA) imaging is an emerging modality which uses pulsed laser light-induced acoustic waves to obtain optical contrast. Because oxy- and deoxyhemoglobin absorb light at different wavelengths, multispectral imaging can be used to quantify oxygen saturation within tissue. A limitation, however, include reduced frame rate due to the laser pulse repetition frequency. To image mouse hearts, faster frame rates are needed to analyze PA signals throughout the cardiac cycle. We have developed a method to post-process multispectral PA imaging data to account for limitations in frame rate.

Methods

A high-frequency small animal ultrasound (US) and PA imaging system (Vevo LAZR-X) was used to image the anterior myocardium of mice before and after myocardial infarction (MI) and phenylephrine infusion. The left coronary artery was permanently ligated to induce an MI, and a tail vein catheter was inserted for phenylephrine infusion. Mice were anesthetized, and ECG and respiration signals were collected. PA and radio frequency data were simultaneously acquired at 750nm and 850nm wavelengths at 20Hz. Images were retrospectively analyzed to remove aberrant frames and rearranged according to their cardiac phase to create a parametric map of myocardium O₂ saturation. 4DUS images were acquired and processed to study how oxygen saturation levels contribute to myocardial strain changes.

Results/Discussion

We have developed a retrospectively gated PA technique to measure oxygen saturation throughout the cardiac cycle. Our method allows us to acquire US and PA images at 20Hz, which can be post-processed to reorganize the acquired images based on the cardiac cycle and estimate sO₂% (Figure 1). Moreover, our results showed that the majority of oxygenated blood perfuses into the myocardium during diastole and phenylephrine leads to vasoconstriction. 3D Green-Lagrange maximum principal strain of the left ventricle shows decreased strain after MI in regions where blood supply to the myocardium is occluded (Healthy E_i = 0.40±0.05; Infarcted E_i = 0.05±0.03; Figure 2).

Conclusions

The results of these experiments show promising advancement in PA imaging to study oxygen saturation in various murine cardiac disease models. Future work will focus on developing this technique into a four-dimensional retrospectively gated PA technique to better understand myocardial oxygen demand throughout the cardiac cycle. Taken together, this work opens a new door for 3D spatial mapping of oxygen perfusion of the myocardium.

Introduction

Non-invasive imaging of atherosclerotic plaque development remains a major challenge. Imaging plaque inflammation targeting Somatostatin Subtype Receptor 2 (SSTR2) expressing macrophages has proven successful in studies using the receptor agonist [DOTA,Tyr³]-octreotate (DOTATATE). DOTA-JR11 is a receptor antagonist, with a reported 5-10 times higher uptake than DOTATATE in SSTR2 imaging in oncology, and therefore may also improve imaging of plaque inflammation. The aim of this study was to assess the feasibility of nuclear imaging and monitoring of plaque using [¹¹¹In]-DOTA-JR11.

Methods

Atherosclerotic mice (ApoE ^-/-) fed an atherogenic diet for 10 or 20 weeks (n=16) were imaged by SPECT/CT 2 hours post injection of ¹¹¹In-JR11 (~200 pmol, ~50 MBq). In vivo imaging was validated by ex vivo imaging and ex vivo autoradiography (ARG) of excised arteries. Oil Red O (ORO) staining of lipids was used to validate plaque presence in the arteries. Presence of macrophages and expression of SSTR2 in plaque was evaluated by immunohistochemical analysis of tissue sections. Specific binding was assessed by competition binding experiments with an excess of unlabelled JR11 in 3 mice and in vitro ARG on human Carotid Endarterectomy (CEA) sections. Levels of ¹¹¹In-JR11 uptake were compared between mice on atherogenic diet for 10 and 20 weeks to assess the potential for plaque monitoring.

Results/Discussion

In vivo SPECT/CT (fig. 1), ex vivo SPECT/CT (fig. 2), and ex vivo ARG showed high specific tracer uptake in the aortic arch and brachiocephalic artery, and ORO staining confirmed plaque build-up in corresponding locations inside the aortic arch of all mice. Mice which were fed an atherogenic diet for 20 weeks showed a 2.2 fold higher target to background ratio than mice on atherogenic diet for 10 weeks.

Conclusions

Our study shows the feasibility of plaque detection and monitoring with [¹¹¹In]In-DOTA-JR11. Monitoring of plaque inflammation with JR11 could be used to assess plaque progression over time or regression during treatment.

Introduction

Abdominal aortic aneurysms (AAA) are diagnosed and monitored via ultrasound screening to measure the aortic size. Molecular imaging may help stratify patients with early-stage AAA to reduced surveillance. AAA uptake of [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) has been demonstrated previously; however, its glucose-dependent uptake may overlook other key mechanisms. [¹⁸F]fluorothymidine ([¹⁸F]FLT), a cell proliferation marker, is primarily used in tumour imaging. We aimed to explore the feasibility of [¹⁸F]FLT positron emission tomography/computed tomography (PET/CT) to study AAA in murine models.

Methods

Fourteen-week-old apolipoprotein E-knockout mice received 28-day saline (n=5) or angiotensin II (n=5) infusions via subcutaneously implanted osmotic mini-pumps. Ninety-minute dynamic PET/CT under 2% isoflurane anaesthesia was performed after [¹⁸F]FDG (day 28, 9.79±2.10 MBq) or [¹⁸F]FLT (day 14, 7.63±3.02 MBq; day 28, 9.61±1.34 MBq) injections in the lateral tail vein. Images were reconstructed using a three-dimensional (3D) ordered subsets expectation maximisation algorithm (2 iterations, 16 subsets). 3D isocontour regions of interest were manually drawn in all aortic regions at 80–90 minutes to obtain maximum standardised uptake values. Ex vivo gamma counting, autoradiography, and Western blotting for thymidine kinase-1 (TK-1) in different cohorts corroborated the in vivo PET/CT findings.

Results/Discussion

[¹⁸F]FDG and [¹⁸F]FLT uptake in the inferior vena cava were observed at 0–15 s of each scan, reflecting intravenous delivery, and in the myocardium and spleen, respectively, consistent with expected uptake in all mice. There was significantly greater abdominal aortic [¹⁸F]FLT uptake at 80–90 minutes in AAA mice than in control mice (percentage differences (PDs): day 14, 4496.80, p<0.001; day 28, 430.35). This enhanced uptake was more consistently demonstrated across different mice with [¹⁸F]FLT than with [¹⁸F]FDG (standard deviations: [¹⁸F]FDG, day 28, 0.14; [¹⁸F]FLT, day 14, 0.04, day 28, 0.02). There were significantly more [¹⁸F]FLT gamma counts in the abdominal aortae relative to the hearts from AAA mice than those from control mice (PD: 671.33, p<0.05). Autoradiography revealed greater [¹⁸F]FLT uptake in AAA than in control aortae. Greater TK-1 expression was found in 14-day AAA than in 28-day AAA and control aortae (PDs: 28-day AAA, 128.14, p<0.001; control, 604.72, p<0.001).

Conclusions

To our best knowledge, this is the first study to demonstrate the potential utility of [¹⁸F]FLT PET/CT to visualise AAA. The increased uptake of [¹⁸F]FLT in AAA, and lack thereof in control aortae, was consistent across all 5 AAA and 5 control murine models, respectively. Quantitative and biological analyses to validate these preliminary findings and to investigate the time course and molecular mechanisms of AAA are ongoing.

Introduction

Currently, there are no available Positron Emission Tomography (PET) tracers for the clinical imaging of atherosclerosis. LO9 is an antibody that targets adherent low-density lipoprotein(LDL), a component of atherosclerotic plaque. Pretargeted labelling of antibodies decouples their slow pharmacokinetics from the half-life of the radioisotope, facilitating their use as tracers. This methodology involves the conjugation of a specific antibody, such as LO9, to a trans-cyclooctene (TCO), followed by the selective in vivo reaction with a radiolabelled tetrazine (Tz) as shown in figure.

Methods

Two different tetrazines were synthesised; Tz-PEG-vivotag (VT) a near-infrared emitting one and Tz-PEG-NODA that was subsequently radiolabelled via chelation of Al-18F to give Tz-PEG-NODA-Al-18F. LO9 was conjugated to TCO via lysine residue, to yield LO9-TCO. The affinity of LO9-TCO towards adherent LDL was tested in an indirect-enzyme-linked immunosorbent assay. LO9-TCO was reacted with the NIRF tetrazine in PBS and the product of the reaction was analysed by sodium dodecyl sulphate–polyacrylamide electrophoresis (SDS-PAGE). Aortic root sections from LDL-R-/- mice fed a high-fat diet for 14 weeks were coated with LO9-TCO and stained with Tz-vivotag, and high magnification studies were performed.

Results/Discussion

The development of an automated synthesis for the labelling of the Tz allowed for fast and reproducible labelling. Tz-NODA-Al-18F was obtained in good yield with a molar activity of 38GBq/nmol. The newly conjugated LO9-TCO was found to have slightly reduced function towards adherent LDL when compared to native LO9, but still retained significant function compared to control antibody. The product of the reaction between LO9-TCO and Tz-PEG-VT was found to exhibit fluorescence signal corresponding to the NIRF emitting tetrazine, therefore suggesting that the reaction was successful. The stained tissue sections showed areas of positive staining along plaque edges. The positive results on tissue simultaneously tested for the two different aspects: the ability of LO9-TCO to bind to the target and the capacity for the reaction between LO9-TCO and NIRF-tetrazine to happen in a complex environment such as fixed tissue with a sufficiently high yield to allow for the detection of atherosclerosis.

Conclusions

Our work so far has been focused on the preparation, characterization, and optimization of the two constructs that are needed in the pretargeted detection of atherosclerosis with PET. Two tetrazines were prepared and the antibody successfully conjugated to TCO. The in vitro results obtained so far suggested that the pretargeted PET imaging of a plaque-specific antibody such as LO9 is a promising strategy for the detection of atherosclerosis.

References

[1] Botnar, R. M. et al. Molecular Imaging in Cardiovascular Diseases TT  - Molekulare kardiovaskuläre MRT-Bildgebung. Fortschr Röntgenstr 36, 92–101 (2015)

[2] R.Y, K. Abstract. Heart 98–103 (2012) (Paper submitted)

[3] Rossin, R. et al. In vivo Chemistry for Pretargeted Tumor Imaging in Live Mice. Angew. Chemie Int. Ed. 49, 3375–3378 (2010).

[4] Meyer, J. P. et al. 18F-Based Pretargeted PET Imaging Based on Bioorthogonal Diels-Alder Click Chemistry. Bioconjug. Chem. 27, 298–301 (2016)

Acknowledgement

L. Vázquez

Introduction

Cardiovascular disease (CVD) is one of the leading causes of death world-wide. CVDs are a class of diseases that relates to conditions that involve narrowing of blood vessels and can lead to myocardial infarction (MI). MI is caused by a complete blockage of one of the coronary artery that supply the heart with oxygen and the lack of oxygen causes severe damage to the heart tissue. Elucidating the biochemical changes occurring in the heart after an infarction leads to better understanding of breakdown and repair of heart tissue, hence, better treatment methods can be developed.

Methods

In this study, multimodal mass spectrometry imaging, using MALDI and ToF-SIMS, has been applied to mouse heart tissue 6, 24 and 48- hours following an infarction to elucidate changes in the lipid and protein distribution in the tissue over time. Myocardial infarction was surgically induced by permanently ligating the left anterior descending coronary artery of anaesthetised mice. Extracted hearts were snap frozen and sectioned using a cryo-microtome and mounted on to silicon wafer and ITO glass slides, consecutive sections were mounted on to glass microscope slides for histological staining. Imaging MS analysis was performed using a J105 ToF-SIMS instruments (Ionoptika Ltd) equipped with a 40 keV GCIB and an Ultraflextreme (Bruker) imaging MALDI instrument.

Results/Discussion

Results show that differences in the lipid distribution between the infarcted region and the normal heart tissue region can be detected, as well as accumulation of acyl-carnitine species at the boundary of the damaged region which indicated impaired mitochondrial function. The ToF-SIMS imaging shows differences in salt adduct formation between different regions within the heart due to changes in salt concentration during and after infarction and a general decrease in phosphatidylinositol in the infarcted region can be seen. The complementarity of SIMS and MALDI is clearly demonstrated in the ability of each technique to deliver information regarding different lipid classes while there is enough overlap of chemical coverage for simple comparison to be made of the images. MALDI analysis also provides information of specific protein distribution in the border region. The time course experiments allow for tracking of the progression of the infarction across the heart.

Conclusions

Multimodal mass spectrometry imaging studies provides a tool to studythe progression of the infarction chemically which offers insights into how the tissue responds to the hypoxic environment. The border region is the tissue in the frontline of the progressing infarction, hence it can reveal the changes occurring in the acute phase of the infarction while the tissue in the infarcted region reveals more long-term changes.

Introduction

We recently implemented a spatially resolved navigator (PolyGate) enabling to extract the cardiac and respiratory motions of up to three separate animals at the same time and to retrospectively reconstruct cardiac gated PET data of multiple animals. Although the implementation of PolyGate was primarily focus on the possibility to scan multiple animals simultaneously (1), it has many advantages and potential application. In this work we are reporting 2 main cases for which the PolyGate method has proved to be outperforming the conventional FID-based navigator (2).

Methods

All data were acquired on a BioSpec 70/30 MRI system equipped with a SiPM based PET insert (Bruker Biospin), using a quadrature volume coil (inner diameter of 40 mm). PolyGate-FLASH (TE/TR: 4.2/14.5ms, 20 degrees flip angle, FOV: 30×30 mm, matrix 192×192, single slice package of 7 slices of 1 mm thickness, 250 oversampling, flow compensated, total acquisition time: 54 min 16 s) was acquired on a mouse subject to permanent ligation of the left anterior descending artery (2 weeks post-ligation). Navigator was positioned at the level of the atria and aortic arch (fig.1). In this second case, the navigator was placed in the neck region of a naive mouse in order to investigate the potential use of such method for any other application than cardiac imaging.

Results/Discussion

PolyGate method provided a more robust way to extract cardiac and respiratory motions compared to conventional IntraGate method (98.3% of motion retrieval versus 82.8% using the full navigator signal) in animal subject to myocardial infarction where motion and ECG are altered. In our case since we used 250 oversampling, only little effect was seen in the image quality (Fig.1). However in situation where the oversampling was kept minimal in order to reduce the acquisition time, this may lead to partially sampled k-space for certain cardiac frames. In the second case, with the navigator placed remotely in the neck region, motion was far more difficult to be identified using the full profile of the navigator, however a small fraction of the profile corresponding to pulsatile flow and tracheal movement enables us to reconstruct appropriate cardiac cine frames (Fig.2).

Conclusions

PolyGate and more specifically the introduction of spatially resolved navigators showed many benefits such as the possibility to scan multiple animals (increase of throughput) and increase of the robustness of the self-gated MRI sequence. In some cases, motion from pulsatile flow of main arteries may be identified providing a way to improve vascular imaging or motion correction of remote tissues such as tumors.

References

1. Retrospectively gated cardiac PET-MR imaging in rodents using MRI-based cardiac motion information. (#549). W. Gsell, A. Nauerth, C. Molinos, C. Correcher, A. J. Gonzalez, S. Sven, T. Greeb, R. Polo, B. Holvoet, C. M. Deroose, U. Himmelreich, M. Heidenreich. 13th annual meeting of the European Society for Molecular Imaging, ESMI. 20-23 March 2018.

2. Evaluation of infarcted murine heart function: comparison of prospectively triggered with self-gated MRI. Bovens SM, te Boekhorst BC, den Ouden K, van de Kolk KW, Nauerth A, Nederhoff MG, Pasterkamp G, ten Hove M, van Echteld CJ. NMR Biomed. 2011 Apr;24(3):307-15.

Introduction

Manganese is a calcium analogue and potent T1-contrast agent for MRI that enters cardiomyocytes through active calcium channels, thus reducing T1 in viable myocardium (1). We hypothesized that T1-mapping-MRI could measure Mn levels in the myocardium acutely after myocardial infarction (MI) allowing quantification of intracellular Ca response to ischemic injury

Methods

Mice received intraperitoneal injections of 0.10mmol/kg MnCl₂ 40min before MI. T1-mapping-MRI was performed at 1, 2 and 3 hours and 2 days after MI. R1 values (1/T1=the relaxivity of the tissue) were analysed from the area-at-risk of infarction segments (AAR-MI, n=12) and viable segments (Viable-MI, n=12) of infarcted hearts, and naïve control heart (Viable-Naïve, n=12). Imaging was performed using a 9.4T Agilent MRI system and a multi inversion time Look-Locker sequence in the short-axis orientation (TE/TR =  3.04/1.11ms, 18 inversion times at consecutive R waves, 10⁰ excitation pulse, slice thickness = 1.0mm, FOV = 25.6 x 25.6 mm, matrix size = 128 x 128) as described (2)

Results/Discussion

As soon as 1 hour after MI, R1 values increased in Viable-MI tissue compared with AAR-MI (p<0.0001) and naïve controls. R1 values continued to rise in the Viable-MI at 2 and 3 hours (p=0.02, p=0.01. Fig1 & 2). When the same animals were imaged 2 days post-MI, R1 values were still significantly higher in the Viable-MI tissue compared with AAR-MI tissue (p=0.03). However, Viable-MI tissue had similar R1 to naïve hearts, while R1 in the infarcted AAR-MI was lower than of the naïve myocardium (p=0.03. Fig1 & 2).

Acutely after ischemic injury a large increase in R1 (reflecting increased Mn²⁺ uptake) occurred in Viable-MI myocytes, likely due to elevated catecholamine levels acutely post-MI; increased cardiac work and thus increased Ca²⁺/Mn²⁺ uptake. By 2 days the catecholamine storm has passed and R1 levels in the surviving myocardium normalise, while Mn uptake in the dead infarct region was reduced due to lack of functional myocytes.

Conclusions

T1-Mapping Manganese-enhanced-MRI offers a valuable in vivo tool for optimisation of the many emerging pharmacological and biological interventions which aim to modulate Ca²⁺ homeostasis acuity after MI.

References

(1) Waghorn, B., Edwards, T., Yang, Y., Chuang, K.-H., Yanasak, N., & Hu, T. C.-C. (2008). Monitoring dynamic alterations in calcium homeostasis by T1-weighted and T1-mapping cardiac manganese-enhanced MRI in a murine myocardial infarction model. NMR in Biomedicine, 1102–1111.

(2) Stuckey, D. J., McSweeney, S. J., Thin, M. Z., Habib, J., Price, a. N., Fiedler, L. R., … Schneider, M. D. (2014). T1 Mapping Detects Pharmacological Retardation of Diffuse Cardiac Fibrosis in Mouse Pressure-Overload Hypertrophy. Circulation: Cardiovascular Imaging, 7, 240–249.

Acknowledgement

Daniel J Stuckey would like to acknowledge the support of his BHF Fellowship FS/15/33/31608 and MRC Project Grant MR/R026416/1

Introduction

Multimodal imaging using PET or SPECT with optical imaging (OI) presents a useful tool for the evaluation of new drugs or intraoperative imaging. First, the biodistribution of the drug or tumour marker is monitored by a radiotracer. A fluorophore then visualizes the uptake on the cellular level or the exact tumour margins. Ideally, this two-step procedure is performed by using only one molecule, where the multimodal label is comparatively small to minimize effects on pharmacokinetics. Cyanine dyes present versatile structural and functional platforms for the build-up of such multimodal labels.

Methods

A new bifunctional asymmetric indocarbocyanine dye (ICC) was synthesized and linked to a DOTA-based chelator. The resulting multimodal DOTA-ICC label was attached to the octapeptide TOC as a model compound, which binds to the somatostatin receptor SSTR2. DOTA-TOC – without the dye – is already used in clinics for diagnostics and treatment. Toxicity tests and cellular uptake studies were performed with the cold DOTA-ICC-TOC conjugate. The conjugate was furthermore subjected to radiolabelling with ⁶⁸Ga and then injected into unhatched eggs and into mice, all carrying a tumour overexpressing the SSTR2-receptor, to perform a PET/MRI scan and fluorescence microscopy afterwards.

Results/Discussion

In comparison to other multimodal labels, the new ICC dye allows the attachment of the chelator as close as possible without any spacer groups which generally increase the size of multimodal labels and risk to influence the pharmacokinetic properties of linked bioactive molecules. Using TOC as a model compound, no toxicity up to 10 μM was detectable for the cold DOTA-ICC-TOC conjugate, which additionally showed uptake at low concentrations via endocytosis and retention of high SSTR2-receptor affinity in cellular uptake studies. Successful radiolabelling with ⁶⁸Ga allowed the injection of ⁶⁸Ga-DOTA-ICC-TOC into unhatched eggs and mice bearing an SSTR2-positive tumour. The in vivo studies indicate specific uptake in the tumour tissue, making this new type of small multimodal imaging agent DOTA-ICC usable for the labelling of new drug candidates and disease markers.

Conclusions

A new bifunctional indocarbocyanine dye was synthesized and attached to a DOTA-chelator via the shortest possible linkage to reduce effects on pharmacokinetics of linked bioactive molecules. The resulting small multimodal label is applicable for PET/OI or SPECT/OI in the course of drug evaluation or intraoperative imaging. Its usage is exemplified in cellular uptake and in vivo studies by labelling of the somatostatin analogue TOC.

References

[1] K. Licha, U. Resch-Genger, Comprehensive Biomedical Physics, Elsevier, Oxford, 2014, 85 – 109.
[2] S. C Ghosh, A. Azhdarinia et al. J. Med. Chem. 2013, 56, 406 – 416.
[3] S. Exner, V. Prasad, B. Wiedenmann, C. Grötzinger, Front. Endocrinol. 2018, 9, 146.

Acknowledgement

The Dahlem Research School and the Studienstiftung des deutschen Volkes are acknowledged for funding.

Introduction

Pancreatic cancer is the 4^th leading cause of death by cancer in Europe. This cancer has a poor prognosis, which is due to late clinical presentation, rapid progression and limited effect of chemotherapy. Moreover, 30% to 50% of patients, who have had surgery, relapse after a few years due to difficulties to accurately delineating tumor margins. An interesting target involved in tumor proliferation and progression of pancreatic cancer is the neurotensin receptor 1 (NTSR1). In this study, we describe the development and in vivo evaluation of PET/fluorescent imaging agents targeting the NTSR1.

Methods

Five bimodal imaging agents were synthesized, based on the structure of the peptide agonist NT20.3.¹ Several spacers (no spacer, a PEG₂ or PEG₄ chain) were introduced between proline⁷ and N-terminal lysine⁶ residue which serve as a trivalent platform. The radiometal chelator, (R)-NODAGA, and three cyanine 5 derivatives (modified with 0, 2 or 4 sulfonate groups) were conjugated to the lysine. The affinity of ligands for NTSR1 was determined in vitro after ^natGa labeling. The compounds were radiolabeled with ⁶⁸Ga and their biodistribution was evaluated on nude mice bearing a subcutaneous xenograft of AsPC1 cells. PET images were acquired 1 h post injection. Mice were sacrificed and the uptake of tracers into different organs was measured by gamma-counting and fluorescence imaging.

Results/Discussion

Bimodal imaging agents were obtained with an overall yield of 10% to 33%. In vitro studies confirmed the high affinity of all compounds for NTSR1 with Ki values in the nanomolar range. Spacers did not have a significant impact on affinity for the receptor. In contrast, the addition of sulfonate groups resulted in a decrease in affinity from 1.39 nM to 8.25 nM. The radiolabeling with ⁶⁸Ga (37°C, 10 min) was achieved with good radiochemical yields (95%) and purity (>99%). PET imaging studies allowed us to identify the compound [⁶⁸Ga]-NODAGA-Lys(Cy5**)-PEG₄-[Me-Arg⁸, Tle¹²]-NT(7-13), as the one showing the most promising pharmacokinetic profile in a mice model of xenografted AsPC1 (human pancreatic adenocarcinoma) cells. Biodistribution data showed a high tumor uptake (14.34%ID/g 1.5 h p.i.) with a high tumor-to-normal tissues ratio. Similar results were obtained by fluorescence imaging, in vivo and ex vivo on isolated organs.

Conclusions

A novel peptide-based bimodal imaging agent for NTSR1-positive tumors was developed. This compound showed high tumor uptake and fast clearance from non-targeted organs, leading to high contrast in PET and fluorescence imaging. These results highlight the potential of this agent for the diagnosis and the fluorescence-guided surgery of pancreatic cancer.

References

(1)       Alshoukr, F.; Prignon, A.; Brans, L.; Jallane, A.; Mendes, S.; Talbot, J.-N.; Tourwé, D.; Barbet, J.; Gruaz-Guyon, A. Novel DOTA-Neurotensin Analogues for 111In Scintigraphy and 68Ga PET Imaging of Neurotensin Receptor-Positive Tumors. Bioconjugate Chem. 2011, 22, 1374-1385.

Acknowledgement

Financial support was provided by France Life Imaging (project FluoNTEP), the French National Research Agency (ANR) under the programs AAP Générique 2017 (project ZINELABEL), the CNRS and the Université de Bourgogne. This work is also part of the project Pharmaco-imagerie et agents théranostiques supported by the Conseil Régional de Bourgogne Franche-Comté through the Plan d’Action Régional pour l'Innovation (PARI) and by the European Union through the PO FEDER-FSE 2014/2020 Bourgogne program. The authors thank the "Plateforme d'Analyse Chimique et de Synthèse Moléculaire de l'Université de Bourgogne” (http://www.wpcm.fr) for access to analytical instrumentation.

Introduction

Surgery is usually the main treatment for primary brain tumors. With the ultimate goal of assuring complete resection of the cancerous tissue while avoiding neurological damage, fluorescence-guided surgery is gaining a lot of interest as a tool to guide the surgeon in real-time during the surgery itself. Within this study, we aim to validate a near-infrared fluorescent nanobody (Nb) targeting the human Epidermal Growth Factor Receptor (EGFR) as tracer for brain tumors in a preclinical setting.

Methods

The anti-EGFR Nb 7C12, labeled site-specifically with IRDye800CW-maleimide was prepared and characterized in vitro. Next, 2 nmol dye was injected I.v. in nude mice (n≥3 / group) bearing an intracranial EGFR-transfected F98-tumor (Luc⁺and GFP⁺).Control groups included a non-specific Nb and sham-operated animals. 1 or 3 hours after tracer injection, animals were killed and craniotomy was performed. Tumor targeting was assessed through 2D fluorescence imaging (FluoBeam800, Fluoptics). Signal was co-localized with bioluminescence and GFP signal emitted by the tumor (Optima, Biospace). Finally, 5 µm-thick cryosections of the brain were prepared and location of IRDye800CW-labeled anti-EGFR Nb was correlated with histology and GFP⁺signal of tumor cells (Odyssey Reader and Evos FL microscope).

Results/Discussion

IRdye800CW was successfully conjugated to Nb 7C12, with a dye-to-protein ratio of 0.7. Cell binding studies demonstrated that affinity of the Nb was not affected by the conjugation.

Uptake of NIR-Nb 7C12 was observed in brain tumor lesions by intra-operative imaging and ex vivo analysis, while no uptake was seen in sham-operated animals. This signal is co-localized with BLI and GFP signal emitted by the tumor (Figure 1, 2)). However, at 1h the control Nb showed comparable tumor uptake as Nb 7C12, while at 3h significantly less uptake was noted in localized lesions for the control Nb.

Conclusions

This study demonstrates the potential of Nb-based fluorescent tracer for use in image-guided surgery of brain tumors. 

Acknowledgement

The authors would like to thank the Wetenschappelijk Fonds Willy Gepts for funding the stereotaxic frame, used for tumor inoculations.

Introduction

Dual-labeled biovectors, developed for bimodal imaging consisting of an initial PET or SPECT scan to localize tumors and a subsequent intraoperative optical imaging (OI) to identify tumor margins, are promising.^1-3 However, the sequential conjugation classically used to produce such imaging agents does not allow a precise control of modifications and can deeply impact the pharmacokinetic properties. To overcome these limitations, we have developed a monomolecular bimodal imaging probe (PET/OI) conjugated in one step to an antibody to detect and optimally resect glioblastoma.

Methods

Glioblastoma are very heterogeneous and contain aggressive subpopulations referred as glioblastoma cancer stem cells (hGSC), thought to be responsible for tumor relapse. To detect these cells, high affinity antibody named Rendomab A63 (RA63), directed against human endothelin 1 receptors (ET1R) – overexpressed in hGSC – has been developed⁴ and functionalized with a monomolecular bimodal imaging probe based on a lysine trivalent platform (fig 1, 2) bearing both a fluorophore (Cy5) and a ⁶⁴Cu chelator (NODAGA). The immunoconjugate was purified by affinity chromatography (HiTrap Mabselect Protein A) and the degree of labeling was determined using both MALDI-ToF and UV spectroscopy. Fluorescence binding assays using CHO cells expressing ET1R were used to assess the affinity of the construct.

Results/Discussion

The monomolecular multimodal probe – ensuring a better control of the radionuclide/fluorophore ratio as well as a single attachment point to the antibody – was obtained in 7 steps with an overall yield of 10%. Briefly, an amine-bearing derivative of NODAGA was coupled to the Fmoc-L-Lys(Dde)-NHS, followed by the addition of an NHS activated ester-bearing Cy5 on the deprotected a-amino group. Finally, after deprotection of the side-chain amine, the p-phenylene diisiothiocyanate was introduced. Next, the random bioconjugation to the lysines of RA63 and the affinity chromatography purification were optimized to obtain a concordant degree of labeling (1 probe/antibody) with two different techniques: MALDI-ToF and UV spectroscopy. In vitro assay (fluorescence binding assay with CHO cells expressing ET1R) confirmed that the conjugate retained its affinity for ET1R (Kd = 0.1nM).

Conclusions

We successfully developed a high affinity single bifunctional probe able to generate both fluorescent and nuclear signals. Ex vivo fluorescence imaging on patient’s biopsies and in vivo fluorescence imaging on relevant mouse model will be performed, before proceeding with PET imaging. The influence of ⁶⁴Cu on the fluorescence properties will also be investigated. Alternatively, DFO (⁸⁹Zr) variant of the probe could be synthesized and evaluated.

References

1. Thorp-Greenwood FL. et al., Dalton Trans. (2011), 40, 6129-6143.
2. Azhdarinia A. et al., Mol. Imaging Biol. (2012), 14, 261–276.
3. Seibold U. et al., Biomed. Res. Int. (2014), 153741.
4. Boquet D. et al., French patent 07/02/2018 FR 18 51026.

Acknowledgement

This work was financially supported by France Life Imaging (grant ANR-11-INBS-0006 from the French “Investissements d’Avenir” program), the “Université de Bourgogne”, the “Conseil Régional de Bourgogne Franche-Comté”, the CNRS, the CEA, and the European Union (PO FEDER/FSE 2014/2020).

Introduction

Targeted fluorescence-guided surgery is a technique that provides surgeons with real-time cancer detection and margin delineation. SGM-101 is a fluorescent antibody conjugate that targets CEA, making it highly suitable for image-guided surgery in colorectal cancer[1]. Labeling the antibody conjugate with the radionuclide ¹¹¹In creates a powerful multimodal tracer that allows for preoperative SPECT imaging, intraoperative gamma detection, fluorescence imaging, and straightforward quantification of the tracer[2] Here, we show that SGM-101 can be radiolabeled for multimodal image-guided surgery.

Methods

SGM-101 was conjugated with different amounts of the chelator diethylenetriaminepentaacetic acid (ITC-DTPA). Substitution ratios were determined after ¹¹¹In-labeling and fluorescence was measured to determine the effect of chelator conjugation on the fluorescent properties of the multimodal tracer. Subsequently, the conjugates were labelled with different amounts of ¹¹¹In. ITLC was performed to determine labelling efficiency. HPLC was performed to evaluate the integrity of the conjugate and possible formation of micro-aggregates. Immunoreactivity was investigated with a Lindmo assay using the colorectal cancer cell line LS174T.

Results/Discussion

The conjugate with a antibody/chelator substitution ratio of 1 was selected for further experiments. Fluorescence measurements corrected for concentration showed no significant decrease of the fluorescent signal after conjugation to DTPA. HPLC analysis of the conjugate revealed no signs of micro-aggregate formation and a purity of 98.6%. Labelling of 1 ug of the conjugate with 1 Mbq/ug and 3 Mbq/ug ¹¹¹In resulted in labelling efficiencies of 98.0% and 95.7% respectively. HPLC analysis of the ¹¹¹In-labeled product showed no signs of micro-aggregate formation and a radiochemical purity  of 100% of PD10-purified conjugates. The  immunoreactive fraction was >0.8. Currently, the first preclinical in vivo studies are performed on biodistribution and multimodal image-guided surgery in a colorectal cancer xenograft model. These results will be presented at the EMIM 2019.

Conclusions

The multimodal tracer ¹¹¹In-DTPA-SGM-101 can easily be made from SGM-101 without loss of integrity or fluorescence. The specific activity and  high immunoreactivity allow the use of this tracer for in vivo multimodal image-guided surgery studies.

References

1.            Boogerd, L.S.F., et al., Safety and effectiveness of SGM-101, a fluorescent antibody targeting carcinoembryonic antigen, for intraoperative detection of colorectal cancer: a dose-escalation pilot study. Lancet Gastroenterol Hepatol, 2018. 3(3): p. 181-191.

2.            Hekman, M.C., et al., Tumor-targeted Dual-modality Imaging to Improve Intraoperative Visualization of Clear Cell Renal Cell Carcinoma: A First in Man Study. Theranostics, 2018. 8(8): p. 2161-2170.

Introduction

Currently, no technology is available during surgery which reliably supports intra-operative margin assessment during tumor excision. Molecular fluorescence-guided surgery (MFGS) using tumor specific fluorescent optical tracers can improve intra-operative tumor detection. Our aim was to determine the safety and the optimal dose of Cetuximab-IRDye800CW for in- and ex-vivo tumor detection using MFGS and spectroscopy to be used in a subsequent phase 2 study to establish the positive predictive value of Cetuximab-IRDye800CW as marker for tumor positive resection margins.

Methods

Cetuximab-IRDye800CW targeting endothelial growth factor receptor (EGFR) was prepared at the UMCG and administered 4 days prior to surgery in a dose escalation scheme (3x10mg, 3x25mg and 3x50mg). Intra-operative images using a flexible endoscopic fluorescence camera device were collected of the tumor before and after excision and of the wound bed. After excision ex vivo fluorescence images were obtained from tissue slices and formalin fixated paraffin embedded tissue blocks. Fluorescence images were correlated with histology on Hematoxylin and Eosin (H/E) and EGFR stained sections. Additionally, MFGS was augmented using multi-diameter single fiber spectroscopy (MDSFR) to quantify the intrinsic intensity of fluorescence (SFF), both in-vivo, during surgery and in ex-vivo samples.     

Results/Discussion

In this ongoing clinical trial, nine patients with pathological proven HNSCC were enrolled between December 2017 and May 2018. No tracer related (serious) adverse events higher than CTCAE grade I were observed. Tumor tissue shows strong fluorescence signals in all nine patients and good localization of the conjugate in tumor tissue, both in- and ex-vivo. Analysis of SFF spectroscopy showed significantly higher fluorescence in tumor over normal tissue with a Tumor-to-Background Ratio (respective median and IQR: 10mg: 2.7±0.34, 25mg: 2.0±0.67 and 50mg: 1.7±0.22), which proves the intrinsic accumulation of the tracer in the tumor compared to the background. Interestingly, in one patient (25mg cohort), an intra-operatively clinically unnoticed tumor positive margin was detected using fluorescence imaging and SFF spectroscopy, which emphasizes the potential role and clinical benefit of MFGS.

Conclusions

First results show that Cetuximab-IRDye800CW is safe and allows tumor visualization both in- and ex-vivo in HNSCC patients already at a 10mg dose. Additionally, we show that MFGS could be used as a tool for intra-operative margin assessment. The optimal dosing scheme will be determined during the rest of this trial (N=12-15), after which our phase 2 trial will commence that might lead to new insights for intra-operative margin assessment.

Introduction

Photodynamic therapy (PDT) relies on the combination of a photosensitizer and light of a specific wavelength, to create a toxic environment by formation of radical (oxygen) species. In oncology, tumor-targeted PDT might increase the efficacy of this technique by specific accumulation of the photosensitizer in tumor tissue. CEA may serve as a target for colorectal tumor-targeted PDT [1]. The aim of the current work is to evaluate the efficacy of tumor-targeted PDT in colorectal tumor xenografts, using the humanized monoclonal antibody MN-14 conjugated with the photosensitizer IRDye700DX.

Methods

CEA-expressing cell lines LoVo, LS174T, SW48, HT29, SW620, WiDr, DLD-1, SW480, SW1222 were evaluated. First, cells were plated into transparent 24 well-plates and subsequently incubated with different concentrations 0.5 and 3.0 µg/mL and labetuzumab:IRDye700DX substitution ratios. Then, cells were washed and irradiated with different intensities (5-300 W/cm²) of NIR light using a LED light source at 690nm. Post-therapeutic cell viability was analyzed with the ATP dependent Celltiter Glo® Luminescent Cell Viability Assay. For comparison of cell lines, cells were irradiated with 96 W/cm².Optimal MN-14:IRDye700DX substitution ratio and irradiation time and intensity were tested in a nude mouse model with s.c. LoVo Tumors. Tumor growth was monitored for 8 weeks post irradiation.

Results/Discussion

The CEA-expressing cell lines were evaluated for susceptibility for tumor-targeted PDT in vitro. Post-therapeutic cell viability levels ranged from 20-90% and were dependent on the MN-14:IRDye700DX substitution ratio, conjugate concentration, irradiation time and intensity, and cell type. In vitro, LoVo, LS174T and SW48 were more susceptible to tumor-targeted PDT than the other cell lines. As expected, more light and higher conjugate concentrations resulted in larger effects. When plotted against the relative binding (corrected for amount of cells), only cells with high binding show a significant PDT effect (Figure 1). This suggests that levels of CEA expression seems to be partly related to the effect of PDT in identical conditions. Our in vivo results show a trend towards reduced tumor growth in the PDT treated group (Figure 2).

Conclusions

CEA-targeted PDT is feasible and leads to cell death in vitro. CEA availability on the cellular membrane surface plays a role in the efficacy of tumor-targeted PDT using MN-14-700DX, however differences in cellular regulation of protective mechanisms remain unclear and should be investigated in the future.

References

1. Shirasu N., et al. Potent and specific antitumor effect of CEA-targeted photoimmunotherapy. Int J Cancer, 2014. 135, 2697-2710.
2. Sato, K., Hanaoka, H., Watanabe, R., Nakajima, T., Choyke, P. L., & Kobayashi, H. (2015). Near infrared photoimmunotherapy in the treatment of disseminated peritoneal ovarian cancer. Mol Cancer Ther, 14(1), 141-150.

Acknowledgement

The authors thank the biotechnicians from the Preclinical imaging centre Nijmegen (PRIME) for their technical assistance with the animals.

Introduction

Complete cytoreductive surgery is essential in the treatment of peritoneal metastases of colorectal origin. Intraoperative fluorescence imaging of colorectal peritoneal metastasis using tumor-targeting tracers can aid surgeons to achieve complete cytoreduction [1,2]. Since fluorescence has a limited penetration depth, radiotracers might be of added value during image-guided surgery [3]. The current study investigates the potential of the dual-labeled anti-CEA humanized antibody ¹¹¹In-DOTA-hMN-14-800CW for intraoperative multimodal imaging in tumor specimens from colorectal cancer patients.

Methods

Colorectal peritoneal tumor specimens were obtained from patients and placed in cold DMEM supplemented with 0.1% bovine serum albumin and 1% penicillin-streptomycin. Within 1.5h specimens were cut in half (max 5x5x2 mm). Tumors were placed in DMEM with ¹¹¹In-DOTA-hMN-14-800CW (0.04 MBq/µg, 4 µg/ml) and kept on a shaker in an incubator at 37° C with 5% CO₂ for 16-20h. Tumors were repeatedly washed in PBS + 0.1% BSA for >6 hours. Washed tumors were imaged by fluorescence flatbed scanning and autoradiography. Next, tumors were fixed in formalin and embedded in paraffin, and slides were stained (H&E, CEA, CK20, CDx2). Lastly, we analyzed correlations between fluorescence, radioactivity and cancer-related CEA-expression.

Results/Discussion

Twenty-nine peritoneal tumors were resected in nine patients. After manual sharp division of several tumors, this resulted in 42 tumor pieces. The first 8 tumors resulted in 12 pieces and were used for determination of antibody penetration dept, optimization of (preincubation) cutting procedures and the washing time. Radiolabeling efficiency ranged between 88% and 98.2% for all procedures. After incubation, tumors were cut at 100 µm from the surface, as the labeled antibody did not penetrate much deeper into the tissue by diffusion. In all cases, the fluorescence signal and radiotracer signals showed overlapping patterns (Figure 1). We found a good visual correlation with CEA expression and presence of cancer cells, as confirmed with H&E and immunohistochemical staining. Specific uptake is a prerequisite for successful image-guided surgery. Fluorescence intensity was high in CEA-expressing tumor tissue, whereas non-tumor tissue did not show tracer uptake.

Conclusions

Ex vivo analyses of patient-derived colorectal peritoneal tumors showed specific tumor targeting using ¹¹¹In-DOTA-hMN-14-800CW, with overlapping patterns of fluorescence and radiosignals in CEA-expressing tumor tissue. These promising results serve as a translational step for the upcoming clinical trial on multimodal image-guided surgery in colorectal cancer patients.

References

1.           Harlaar, N.J., et al., Molecular fluorescence-guided surgery of peritoneal carcinomatosis of colorectal origin: a single-centre feasibility study. The Lancet Gastroenterology & Hepatology, 2016. 1(4): p. 283-290.

2.            Hekman, M.C., et al., Tumor-targeted Dual-modality Imaging to Improve Intraoperative Visualization of Clear Cell Renal Cell Carcinoma: A First in Man Study. Theranostics, 2018. 8(8): p. 2161-2170.

3.            Hoogstins, C.E.S., et al., Image-Guided Surgery in Patients with Pancreatic Cancer: First Results of a Clinical Trial Using SGM-101, a Novel Carcinoembryonic Antigen-Targeting, Near-Infrared Fluorescent Agent. Ann Surg Oncol, 2018. 25(11): p. 3350-3357.

Acknowledgement

The authours would like to thank D.L. Bos and C. Frielink for their assistance with the labeling and preparation of the tracer prior to each patient.

Introduction

Ovarian cancer is the leading cause of death among gynecological malignancies with a 5 years survival rate of 17 % for disseminated disease. The current treatment is based on extensive cytoreductive surgery associated with chemotherapy [1]. However, to improve overall survival of patients the most important prognostic factor is the absence of post-operative microscopic residue [2]. The present study evaluated the inputs of intraoperative fluorescence to improve the quality of surgical resection in mice with peritoneal carcinomatosis of ovarian origin.

Methods

A mice model of human ovarian carcinomatosis was induced by intraperitoneal implantation of human ovarian carcinoma cells (SKOV3) stably expressing luciferase thus allowing to monitor tumor growth by bioluminescence. At the time of established carcinomatosis, mice were divided into 2 groups (n=8). Sixteen hours before surgery, mice were injected intravenously with either ICG or Angiostamp™800. Then surgical laparotomy was performed in 2 steps: first, a conventional surgical procedure under normal light and then, cytoreduction was completed by fluorescence-guided surgery. At the end, all resected nodules as well as potential tumor residue in the mouse were assessed by bioluminescence imaging to determine total tumor load and the respective resection rates of each experimental step.

Results/Discussion

Peritoneal carcinomatosis was well established 35 days after tumor cells implantation. Tumor resection by conventional surgical procedure removed 84±8% of total tumor load. This percentage rose to 84% and 94% when completed by ICG and Angiostamp™800 fluorescence-guided steps. All resected nodules were evaluated by bioluminescence to check their tumor status and interestingly, while false positive rate was high with ICG (28%) it was only 1% with Angiostamp™800.

Conclusions

NIR fluorescence-guided surgery significantly improved tumor resection of peritoneal carcinomatosis of ovarian origin and in particular, Angiostamp800™ displayed very high specificity and sensitivity compared to ICG. This very promising fluorescent tracer should therefore move towards clinical trials.

References

1.         Siegel, R., D. Naishadham, and A. Jemal, Cancer statistics, 2013. CA Cancer J Clin, 2013. 63(1): p. 11-30.

2.         Bristow, R.E., et al., Survival effect of maximal cytoreductive surgery for advanced ovarian carcinoma during the platinum era: a meta-analysis. J Clin Oncol, 2002. 20(5): p. 1248-59.

Introduction

Presently, the cornerstone of breast cancer treatment is Breast-Conserving Surgery (BCS), in which, both the tumour and a margin of surrounding tissue are resected, while the healthy breast tissue is spared. Local cancer recurrence has been shown to positively correlate with resection margins of insufficient width, raising the following challenge: How can the surgeons decide the optimal width of the surrounding tissue to resect (1)? This problem could be palliated by the use of fluorescence visualisation of fluorescent drugs, accumulated preferentially in the tumour (2-5).

Methods

Here, a prototype real-time imaging device for surgical navigation is presented, which overlays fluorescence signals and colour images. Excitation of the contrast agent, indocyanine green (ICG), is achieved with a customised light source of digitally controlled spectral band light output or a filtered broadband light source. Detection of both fluorescence, which indicates where the tumour is, and colour image, which serves as a localisation reference map, is achieved with a two-camera head, shown in figure 1. Live control of illumination and detection equipment, as well as, Fluorescence signal segmentation and colour coding is implemented in LabVIEW^TM software. Full device characterisation, including sensitivity to ICG and resolution (explained below) has been investigated.

Results/Discussion

The lowest ICG concentration in aqueous solution that can be detected is 30 [nM] while the greatest feasible resolution is 12.7 [lp/mm]. The system can also accommodate a range of different working distances, from 6 [cm] to 48 [cm] and corresponding fields of view, from 3.0×4.5 [cm] up to 8.41×12.6 [cm]. The control and display interface is shown in figure 2. The user is able to track on-line the raw fluorescence, the colour and the fluorescence/colour final overlay images. The above indicate that the system has potential to be applied in a real clinical scenario since it is sensitive to clinically relevant in-vivo ICG concentrations and yields high quality images. Further validation of the device during BCS (10 different clinical cases) is planned, where our prototype will be compared with a commercial analogue.

Conclusions

Image guided resection during BCS is currently an unmet clinical challenge, since none of the existed intraoperative imaging modalities has been established in the surgical routine (1). The herein system fulfils the requirements for clinical adaptation since it is user-friendly and ergonomic. Provided that our technology will be able to detect accurately the tumour during the clinical trials, it could be the answer to the above clinical problem.

References

1. Morrow M et al., JCO, 2016, 23(12), p.3801-3810; 2. Blacker TS, Free Radic Biol Med, 2016, 100, p.53-65; 3. Carmeliet P, Nat Rev,2000, 407(6801), p.249-257; 4. Hagen A, Grosenick, Opt Express, 2009, 17(19), p.17016-17033; 5. Zhang RR, Nat Rev Clin Oncol, 2017, 14(6), p.347-364;

Acknowledgement

This research was funded by the NIHR i4i programme (grant II-LB-0214-20009) with infrastructure support from the Cancer Research UK Imperial Centre. Maria Leiloglou was supported by the NIHR Imperial BRC (Biomedical Research Centre ) and the Foundation for Education and European Culture for her studies.

Introduction

Intra-operative optical imaging can provide real-time detection of tumour lesions and thereby contribute to optimal surgical procedures. Insulinomas and focal lesions in congenital hyperinsulinism (CHI) are GLP-1-receptor (GLP-1R) positive pancreatic lesions (1, 2). Surgery is challenging because of the small size of these lesions and the need to conserve healthy pancreatic tissue (3). The peptide exendin binds the GLP-1R on pancreatic beta cells. We hypothesize that exendin-4 labelled with the near-infrared (NIR) dye IRDye800CW enables intra-operative visualization of GLP-1R positive lesions.

Methods

Exendin-IRDye800CW was characterized in vitro and in vivo. A competitive binding assay was performed using CHL-cells transfected with the human GLP-1R. In mice bearing subcutaneous GLP-1R positive tumours, biodistribution studies were performed, as well as in vivo fluorescence imaging using an IVIS optical imaging system. The applied fluorescent biodistribution method was validated by combined radioactive and fluorescent biodistribution studies using the dual-labelled tracer [¹¹¹In]In-DTPA-exendin-4-Cy5.5. In healthy mini pigs, in vivo fluorescence imaging of the pancreas was performed using a laparoscopic NIR imaging system. Ex vivo fluorescence imaging of pancreatic tissue was performed with a flatbed scanner and fluorescence microscopy.

Results/Discussion

Exendin-IRDye800CW has a high affinity for the GLP-1R with an IC₅₀ of 4 nM, which is comparable to unlabeled exendin. Injection of 3 µg exendin-IRDye800CW in BALB/c nude mice with subcutaneous GLP-1R positive tumours resulted in a tumour uptake of 3.2 ± 1.4 %ID/g, which decreased with increasing peptide doses to 2.5 ± 0.2 %ID/g, 1.4 ± 0.1 %ID/g and 0.6 ± 0.3 %ID/g for 10, 30 and 100 µg, respectively. Tumours were successfully detected by fluorescence imaging. In mini pigs after injection of 1.4 µg/kg exendin-IRDye-800CW, a clear fluorescent signal was detected in the pancreas. Specific uptake in the pancreatic islets was shown by detection of individual islets in pancreas tissue by flatbed fluorescence scanning and fluorescence microscopy.

Conclusions

These data indicate that exendin-IRDye800CW is a promising tracer for intra-operative optical imaging of GLP-1R positive lesions.

References

 1. Lord K, Dzata E, Snider KE, Gallagher PR, De Leon DD. Clinical presentation and management of children with diffuse and focal hyperinsulinism: a review of 223 cases. The Journal of clinical endocrinology and metabolism. 2013;98(11):E1786-9.

2. Okabayashi T, Shima Y, Sumiyoshi T, Kozuki A, Ito S, Ogawa Y, et al. Diagnosis and management of insulinoma. World journal of gastroenterology. 2013;19(6):829-37.

3. Richards ML, Gauger PG, Thompson NW, Kloos RG, Giordano TJ. Pitfalls in the surgical treatment of insulinoma. Surgery. 2002;132(6):1040-9

Introduction

A prosthetic joint infection (PJI) is a serious complication following a total joint replacement. Early and accurate diagnosis is vital to increase the chance of successful treatment and optimise antibiotic use¹. Current diagnostic techniques to detect PJIs are, however, time consuming, often non-specific and unable to detect bacterial biofilm². This proof-of-principle study explores the use of fluorescently labelled vancomycin (vancomycin-IRDye800CW; vanco-800CW)³ as a bacteria-targeting imaging modality to detect Gram-positive bacterial biofilms.

Methods

In this study, we have explored real-time optical imaging and visualised infected knee prostheses using a near-infrared (NIR) fluorescent fiber during arthroscopy in a human post-mortem model. Femoral components of two knee prostheses were coated on the lateral sides with Staphylococcus epidermidis biofilm. The biofilm-coated prostheses were surgically implanted on the distal femurs of a human cadaver, followed by standard watertight suturing. Vanco-800CW was injected inside each knee cavity and after incubation, flushed with NaCl 0.9%. Imaging was performed with both, white and NIR light before and after administration of the tracer. The post-mortem experiments were conducted according to institutional guidelines.

Results/Discussion

The ex vivo knee prosthesis, after incubation with vanco-800CW, shows a strong fluorescent signal from the lateral part containing biofilm, whereas no fluorescent signal was detected of the medial site. Images of the post-mortem experiment, prior to administration of the tracer, showed no fluorescence compared to the background. After injection of vanco-800CW, the biofilm-coated part of the prosthesis emitted a strong fluorescent signal, while the uncoated part showed no fluorescence compared to the background.

Conclusions

The promising results of the post-mortem experiment indicate that bacteria-targeted arthroscopic imaging of bacterial biofilms is possible and can potentially be achieved in clinical setting in the very near future. Aspects of this approach, like high resolution, the possibility to image in real-time and bacteria-specific tracers, make this imaging modality superior to current diagnostic modalities³.  

References

1. Steward PS. Antimicrobial Tolerance in Biofilms. Microbiol Spectr 2015; 3(3).
2. Tande AJ, Patel R. Prosthetic joint infection. Clin Microbiol Rev 2014; 27(2):302-345.
3. van Oosten M, Schafer T, Gazendam JA, Ohlsen K, Tsompanidou E, de Goffau MC, et al. Real time in vivo imaging of invasive- and biomaterial-associated bacterial infections using fluorescently labelled vacomycin. Nat Commun 2013; 4:2584.

Introduction

Bacterial communication is of key importance in the regulation of bacterial symbiotic processes and pathogenesis of infections.[1] It is mainly based on quorum sensing (QS); a process in which bacteria produce and excrete QS autoinducers responsible for controlling cellular organization, virulence and biofilm formation, amongst others.[2] External control over the activity of QS autoinducers with light, coupled with optical imaging methods, would enable theranostic regulation of biofilm formation, which is becoming a major consideration in the treatment of bacterial infections.[3,4]

Methods

A small library of light-switchable QS inducers and inhibitors was prepared by introducing an azobenzene molecular photoswitch into the structure of known QS inducer molecules. The photochemical properties of the products were fully characterized using UV-Vis and NMR spectroscopy methods. Biological evaluation focused on the Las network of Pseudomonas aeruginosa, which can be quantified by the induction of LasQS as measured by a functional readout of bioluminescence in a QS reporter strain (E.coli JM109 pSB1075). In this strain, the compounds potentially bind to the transcriptional activator LasR to form a stable dimer, which can bind to the responsive promoter region of the LasQS system proceeding the luxCDABE-lasR promoter fusion reporter genes.

Results/Discussion

At the thermally stable states, several compounds from the library proved to be agonists of quorum sensing with 15-18% induction, compared to the native autoinducer. However, irradiation (λ = 365 nm for 5 min) and subsequent evaluation as quorum sensing agonists revealed a dramatic increase in activity. Especially the compound presented in Figure A stood out in this respect. Subsequent investigation to the dose response of its irradiated and non-irradiated forms (Figure B) revealed a stunning >700-times difference in activity between the irradiated and non-irradiated forms. This represents an unprecedented selectivity in photopharmacology.

Several compounds from the library showed good inhibitory activity against a native autoinducer, albeit with a small difference in activity between the irradiated and non-irradiated states. Notably another compound reported before by our group[3] proved to be the most potent photoswitchable inhibitor in our library with 57% inhibition of QS activity.

Conclusions

We present here a library of photoswitchable QS agonists and antagonists. Up to 71% QS induction was obtained, whereas the best inhibitor showed almost 60% inhibition. Our lead compound, showed privileged properties for further development in optical imaging-coupled photopharmacology, due to an unprecedented difference in activity between the inactive and light-activated state, which could be interconverted without apparent fatigue.

References

[1] Greenberg, E. P. Nature 2003, 424, 134–134.

[2] Welsh, M. A.; Blackwell, H. E. FEMS Microbiol. Rev. 2016, 40, 774–794.

[3] Van der Berg, J. P.; Velema, W. A.; Szymanski, W.; Driessen, A. J. M.; Feringa, B. L. Chem. Sci. 2015, 6, 3593–3598

[4] Hansen, M. J.; Hille, J. I. C.; Szymanski, W.; Driessen, A M.; Feringa, B.L. 2018 submitted

Acknowledgement

This work was financially supported by the Netherlands Organization for Scientific Research (NWO-CW, Top grant to B.L.F. and NWO VIDI grant no. 723.014.001 for W.S.), the Royal Netherlands Academy of Arts and Sciences (KNAW), the Ministry of Education, Culture and Science (Gravitation program 024.001.035), and the European Research Council (Advanced Investigator Grant no. 694345 to B.L.F.).

Introduction

Osteomyelitis and implant-associated infections are severe complications after bone fracture treatment¹. Clinical suspicion might result in removal of infected bone or biomaterials. However, there is no adequate tool to directly distinguish infection from sterile inflammation. Accurate visualization and diagnosis of an infection during revision surgery will improve clinical decision-making and the chance of a successful treatment. Here, we evaluated the specificity and sensitivity of vancomycin-IDRye800CW (vanco-800CW)² in presumably infected patient materials obtained during revision surgery.

Methods

In this ex vivo study, we aim to establish a bacteria-targeted fluorescent imaging approach to discriminate between infected materials (such as tissue, bone marrow and infected plates and screws) and non-infected materials. Tissue and implants extracted during revision surgery were incubated with vanco-800CW for 15 minutes at 37^oC and subsequently washed with phosphate-buffered saline. Fluorescent macroscopic imaging was performed with the IVIS Lumina II and an intraoperative camera system (SurgVision Explorer Air). Imaging results were confirmed using standard culture techniques, including diagnostic culture by sonication. Institutional review board permission for this study was obtained.

Results/Discussion

Our results with explant material from 8 patients demonstrate that sites infected with Gram-positive bacteria show a fluorescent vanco-800CW signal, whereas no fluorescent signal is detectable on non-infected materials. Gram-negative bacterial infections did not result in a fluorescent signal as expected.

Conclusions

Vanco-800CW is a specific and effective tracer for ex vivo detection of Gram-positive bacteria on implants and osteomyelitis. Exact sensitivity and specificity have still to be determined using a larger sample size. Fluorescent targeted imaging with vanco-800CW is a promising technique that can provide fast, accurate and real-time information, potentially allowing faster diagnosis and treatment of fracture-related infections.

References

1. Engelsman, A. F. et al. The risk of biomaterial-associated infection after revision surgery due to an experimental primary implant infection. Biofouling (2010).
2. van Oosten, M. et al. Real-time in vivo imaging of invasive- and biomaterial-associated bacterial infections using fluorescently labelled vancomycin. Nat. Commun. (2013).

Introduction

Pseudomonas aeruginosa (P.a.) infections have become a serious problem, especially in immunocompromised patients (1). The development of novel therapeutic and diagnostic strategies is intensively pursued. One of the promising diagnostic strategies for the detection of P.a. could be utilization of its siderophores. Siderophores are small iron-chelating molecules produced by almost all microorganisms (2). Replacing iron in siderophores by radiometal, such as gallium-68, opens approaches for targeted imaging of P.a. by means of PET. Here we report on ⁶⁸Ga-siderophores for P.a. infection imaging.

Methods

Radiolabelling of selected siderophores (three different types of pyoverdines and deferoxamine) with Ga-68 was performed using acetate buffer. Stability in human serum, towards competitive chelator and iron challenge as well as protein binding and partition coefficient values were determined. In vitro uptake of ⁶⁸Ga-siderophores was tested in various microbial cultures including different P.a. strains. In vivo behaviour of ⁶⁸Ga-siderophores was studied in normal Balb/c mice by ex vivo biodistribution study and PET/CT imaging. Mouse and rat P.a. infection models were developed and used for the evaluation of ⁶⁸Ga-siderophores under study by means of PET/CT.

Results/Discussion

All studied siderophores were labelled with ⁶⁸Ga with high (>95%) radiochemical purity. The resulting complexes showed hydrophilic properties (log P = -3), low protein binding (<5%) and high stability in human serum (>95%) up to 120 min incubation time. In vitro uptake of ⁶⁸Ga-siderophores was highly dependent on type of microbial culture. ⁶⁸Ga-pyoverdines displayed different levels of uptake in tested P.a. strains, while ⁶⁸Ga-deferoxamine was taken up by all P.a. strains under study. In normal mice, all studied ⁶⁸Ga labelled siderophores showed similar results manifested by rapid renal excretion and low blood values even at a short time period (90 min) after the application. PET/CT imaging of selected ⁶⁸Ga-siderophores in P.a. infected animals showed focal accumulation of ⁶⁸Ga-pyoverdine I and ⁶⁸Ga-deferoxamine in infected tissues.

Conclusions

Studied siderophores can be labelled with Ga-68 with high affinity and radiochemical purity. ⁶⁸Ga-siderophores showed very similar and satisfactory in vitro characteristics. In vitro uptake of ⁶⁸Ga-siderophores in tested microbial cultures was diverse even within the microbial species. ⁶⁸Ga-siderophores revealed excellent pharmacokinetics and can be used for imaging of P.a. infections.

References

1. de Bentzmann S, Plésiat P. The Pseudomonas aeruginosa opportunistic pathogen and human infections. Environ. Microbiol. 2011;13:1655-1665.

2. Hider RC, Kong X. Chemistry and biology of siderophores. Nat. Prod. Rep. 2010;27:637-657.

Acknowledgement

We gratefully acknowledge the financial support of Technology Agency of the Czech Republic (Project No. TE01020028) and Ministry of Education Youth and Sports of the Czech Republic (Project No. LO1304).

Introduction

Pneumonia remains a leading cause of death^1,2. Newly designed rapid diagnostic tools enable greater accuracy in clinical decision making and optimise antimicrobial management thus leading to better clinical outcomes. Visualisation of the distal respiratory tract evolved following the development of probe-based confocal laser endomicroscopy and molecular imaging³. Additional fluorescence techniques and microbe specific agents can provide unique insights into processes in the alveolar space and can rapidly identify structural change and microbial presence during infection⁴.

Methods

From April 2018 we received explanted lungs or lobes from patients undergoing lung transplant or resection for malignancy at the University Medical Center Groningen (UMCG). The protocol was ethically approved and initially trialled with a porcine, slaughterhouse lung model.

We establish mechanical ventilation, with varying pressures, through direct intubation of the available airway and perform bronchoscopy examination of the specimens. Cellvizio and the alveoflex, fiber-based bundle (Mauna Kea Technologies, Paris) were used with optical molecular imaging agents e.g. BAC 1⁵ BAC 2⁶, capable of identifying specific bacterial and fungal targets. Thereafter, image algorithm analysis was performed on the recorded datasets, using previously described techniques in MATLAB (The Mathworks Inc.)⁷.

Results/Discussion

Preliminary laboratory and animal lung models were used to optimise our protocol and image bacteria in the distal lung. Subsequent to this, we performed 17 procedures on human lung explants at the UMCG. Thus far, we have obtained specimens from 7 lung cancer patients, 1 rejected donor-lung following ex vivo lung perfusion (EVLP) and 8 double explanted lungs (aetiologies: chronic obstructive airways disease – COPD, cystic fibrosis – CF, pulmonary hypertension and interstitial lung diseases). With the use of antimicrobial specific fluorescence imaging agents, Gram-positive and Gram-negative bacteria in the alveolar space were visualised. This correlates with established microbiology culturing and identification methods following recognised sampling techniques (lavage and endobronchial brushing).

Conclusions

We have established a working model to ventilate end-stage, diseased, human lung explants and image the distal respiratory tract using optical molecular imaging techniques. New insights regarding the presence of bacteria in the alveolar space of explants in varying diseases are provided. Further clinical human studies are required to validate and assess the translatability of these novel techniques for real time infection imaging in patients.

References

1 Mizgerd, Joseph P. "Acute lower respiratory tract infection." New England Journal of Medicine 358.7 (2008): 716-727.

2 World Health Organization (WHO). The global burden of disease: 2004 update. Geneva: WHO; 2008 [accessed 2016 Jun 29]. Available from: http://www.who.int/healthinfo/global_burden_disease/GBD_report_2004update_full.pdf

3 Thiberville, Luc, et al. "In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy." American journal of respiratory and critical care medicine 175.1 (2007): 22-31.

4 Mills, Bethany, Mark Bradley, and Kevin Dhaliwal. "Optical imaging of bacterial infections." Clinical and translational imaging 4.3 (2016): 163-174.

5 Akram, A. R., et al. "A Labelled-Ubiquicidin Antimicrobial Peptide for Immediate In Situ Optical Detection of Live Bacteria in Human Alveolar Lung Tissue."

6 Akram, Ahsan R., et al. "In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A." Science Translational Medicine 10.464 (2018): eaal0033.

7 Seth, Sohan, et al. "Estimating Bacterial Load in FCFM Imaging." Annual Conference on Medical Image Understanding and Analysis. Springer, Cham, 2017.

Acknowledgement

Proteus, Edinburgh, UK, have provided intellectual insight, discussion and the molecular agents for this work. We would like to acknowledge the assistance of the Cardiology and Thorax Surgery and Pathology departments of the UMCG in providing specimens according to an ethically approved protocol. Furthermore, the abundance of services provided by the clinical skills department of the UMCG.

Introduction

Cryptococcal brain lesions are often difficult to distinguish from other pathologies (e.g. tumors and abscesses) on anatomical MRI. By using quantitative, multiparametric MRI, we studied the typical MR features of C. neoformans H99 (CN) and C. gattii R265 (CG) brain lesions to identify potential markers for differential diagnosis. To explain differences in the MR properties between both strains, we assessed in vivo fungal cell density and size using fibered confocal fluorescence microscopy (FCFM) and ex vivo microscopy.

Methods

Cryptococcal brain lesions were induced by stereotactic injection of 10⁴ GFP-expressing CN H99 or CG R265 cells in the striatum of female Balb/c mice. Using a 9.4T preclinical MRI scanner (94/20 Biospec, Bruker Biospin), we acquired T₂-weighted anatomical brain scans (2D, axial and coronal), diffusion-weighted images (b-values 31 and 1561 s/mm³), T₁ maps (RARE-VTR with 5 TRs) and T₂ maps (MSME with 16 TEs). For FCFM (Cellvizio, Mauna Kea), the fiberoptic probe was mounted on a stereotactic frame and inserted into the lesion. After sacrificing the animals, colony-forming unit (CFU) counting on brain homogenates was performed and the size of the fungal cells and their surrounding capsule was measured after india ink staining using a microscope with digital camera (Leica DM LS2 with ICC50W)¹.

Results/Discussion

Lesions were characterized by higher apparent diffusion coefficients (ADC), T₁ and T₂ relaxation times than in the contralateral (normal) hemisphere. The ADC and T₂ relaxation times were significantly higher in CG lesions than in CN lesions, without differences in the T₁ relaxation time. FCFM showed that the cryptococcal cell density was higher in lesions caused by CN H99 than by CG R265. The number of cells (CFU) per mm³ of lesion material (MRI) was significantly higher in CN lesions, confirming more densely packed cells in CN lesions. India ink staining of the homogenized brain tissue showed that CG R265 cells are in general larger than CN H99 cells, with a thicker capsule around the cell and a larger cell body size, leading to a lower cell density compared to CN. The larger fluid-filled intercellular spaces and higher amount of the highly hydrated capsular material in CG lesions can explain the higher ADC and T₂ relaxation time.

Conclusions

Increased ADCs can potentially be used to differentiate cryptococcomas from e.g. bacterial abscesses that show restricted diffusion². T₂ relaxation times and ADCs differed between CG R265 and CN H99 lesions, which was related to the capsule size and associated fungal density in the respective lesions. This shows that pathogen characteristics can impact MR read-outs and that in vivo MRI can potentially provide indirect measures for cell density.

References

1. Dragotakes, Q. & Casadevall, A. Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body. J. Vis. Exp. e56957–e56957 (2018). doi:10.3791/56957
2. Nadal Desbarats, L. et al. Differential MRI diagnosis between brain abscesses and necrotic or cystic brain tumors using the apparent diffusion coefficient and normalized diffusion-weighted images. Magn. Reson. Imaging 21, 645–50 (2003).

Acknowledgement

We are thankful for financial support from the European Commission for the Infect-ERA project CryptoVIEW. LV is an SB PhD fellow at Research Foundation Flanders (FWO).

Introduction

Tuberculosis (TB) remains the number one killer of infectious diseases. Worldwide, a person dies of TB about every 20 seconds and antibiotic resistance poses a growing global health challenge also in TB. The interaction between the pathogen and the host is complex, and many aspects underlying disease susceptibility and protective host defence remain elusive still. A better understanding of disease dynamics and immune mechanisms is key to the development of improved therapies. PET-CT ¹⁸F-FDG based imaging has opened new opportunities, especially in non-human primates (NHPs), to clarify this.

Methods

PET-CT was performed at week 0, 4, 6, 8 and 10 post-infection mycobacterium tuberculosis (Mtb). Images were obtained using a preclinical MultiScan LFER 150 PET-CT (Mediso Medical Imaging Systems) on anesthetized rhesus macaques (RM; n=24) as NHP model of TB. All CTs were acquired with a breath-hold with and without CT contrast (Omnipaque 300, 2 ml/kg). To detect the metabolically, inflammatory, activity of TB lesions, 45min after intravenous injection of 100 MBq ¹⁸F-FDG a 20min static PET scan was acquired. Image were analysed using Osirix MD viewer. ROIs were drawn on every lesion and data were exported as standardized uptake value (SUV) within these granulomas.

Results/Discussion

Due to progressive TB some RM reached a humane endpoint before wk10 which were euthanized immediately without performing an endpoint PET-CT. In total 80 scans were recorded for analyses (24, 23, 20, and 13 scans on week 4, 6, 8 and 10, respectively).  We determined on CT the lesion size and on PET the uptake of ¹⁸F-FDG in those granulomas, after which we performed a correlation analysis. We found a clear correlation between the lung pathology score (at endpoint) vs SUV of 0.72, 0.75, 0.91 and 0.88 at the four different timepoints, week 4,6, 8 and 10 post-infection, respectively, with p-value <0.0001 for all. A similar trend was observed for bacterial tissue load at endpoint vs the lung pathology score. The first is measured via the amount of colony forming units (CFUs)) from the lung.  The lung pathology score is semi quantative and based on the number and size of granulomas per lung lobe with a maximum score of 10 per lobe times 7 lobes.

Conclusions

Our results demonstrate the utility of PET-CT to monitor TB infection dynamics over time and implicitly the ability to monitor treatment response in a non-invasive manner over time. Using imaging in combination with conventional pathology assessment and microbiological measurements, we shall gain a deeper understanding of both the development of active TB and the underlying pathogen-host interaction in this clinically relevant NHP model of TB.

Introduction

Antimicrobial peptides as infection imaging agents are of increased interest in Nuclear Medicine. Infection imaging provides a solution for often misdiagnosed, complicated infections and monitoring of therapy. The aim of this study is to validate the radiosynthesis of [68Ga]Ga-NODAGA-/DOTA-CDP1, to investigate bacterial binding and mammalian cell toxocity, and to establish the pharmacokinetic tracer profile in healthy mice using micro PET-CT imaging.

Methods

Sytheses of [68Ga]Ga-NODAGA-/DOTA-CDP including optimisation for pH, molarity, incubation time and temperature, and product purification was conducted (1). [68Ga]Ga-DOTA-CDP1 integrity and protein binding was investigated at ambient temperature and 37ºC, [68Ga]Ga-chloride and [68Ga]Ga-DOTATATE were internal refernces. DOTA-CDP1 cytotoxicity was investigated against HepG2 and Vero cells (2).Bacterial cell binding of [68Ga]Ga-DOTA-CDP1 with/without ecxess DOTA-CDP1 was determined in the following microbes: Escherichia coli (EC), Staphylococcus aureus (SA) and Mycobacterium smegmatis (MS). The pharmacokinetic profiling protocol for [68Ga]Ga-DOTA-CDP1 in healthy BALB/c mice employed  45 min dynamic PET/CT imaging followed by 1 h and 2 h static PET-CT and subsequent ex vivo biodistribution (3).

Results/Discussion

[68Ga]Ga-DOTA-CDP1 showed >90% integrity after 2h incubation in PBS or serum whereas [68Ga]Ga-NODAGA-CDP1 was lacking stability (p<0.01). LC₅₀ analysis indicated DOTA-CDP1 as non-toxic in both mammalian cell-lines with 86-89% [68Ga]Ga-DOTA-CDP1 unbound. Rapid 0h tracer binding in EC occurred  (40+/- 6.8%)  and MS (18+/-2.6%; p<0.01), at 1h uptake increased slowly  to 45+/-5.3 and 20+/-3.3%, respectively (p<0.02), EC uptake was higher than SA (29+/-8;p<0.003). PET imaging showed moderate blood pool clearance over 1h. Static [68Ga]Ga-DOTA-CDP1 PET showed significantly decreased heart SUV comparing images at 1h (2.53+/-0.5)  and 2h (1.5+/-0.3; p<0.01). Liver / spleen uptake increased with time. Ex vivo biodistribution showed lower liver and lung activity [68Ga]Ga-DOTA-CDP1 (15.7+/-7.6 and 11.3+/-4) compared to [68Ga]Ga-chloride (18.2+/-10.5 and 18.3+/-11.8) and significantly higher uptake in heart (7.5+/-1.7; p<0.003), blood pool (18.3+/-10.6; p<0.004) and brain (0.66+/-0.2; p<0.01).

Conclusions

[68Ga]Ga-DOTA-CDP1 exhibits good bench and serum stability. The concentration used in bacterial uptake is non-toxic due to low binding of [68Ga]Ga-DOTA-CDP1 to mammalian cells, thereby increasing bioavailability to host infection sites.Accumulation observed in the bacterial stratins at different sensitivities gives promise in distinguishing between infections. Biodistribution in healthy mice show delayed clearance from blood and blood rich organs

References

1. Ebenhan, T., et al. (2014). Nucl Med Biol 41(5):390-400
2. Mosmann, T. (1983).J Immunol Methods 65(1-2):55-63
3. Mokaleng, B.B., et al. (2015). Biomed Res Int 2015:284354

Introduction

Sepsis is a systemic inflammatory response to a suspected infection that often leads to multi-organ dysfunction. Among the different clinical signs, metabolic acidosis is mainly a consequence of renal failure that impairs the homeostasis of the systemic acid-base regulation [1]. Recently, several MRI approaches were proposed to monitor sepsis-induced changes in renal physiology [2,3]. In this work, MRI-pH mapping was applied to investigate whether variations of pH balance in kidneys might be a predictive and specific biomarker for early diagnosis of sepsis-induced shock.

Methods

Male C57BL/6J mice (n=12) were i.p. administered with 10 mg/kg body weight (b.w).of LPS from E. coli to induce sepsis condition or with 1 ml of 4% thioglycollate (TG) broth as a control model of local inflammation. After 18 hours, MR images were acquired on kidney coronal sections on a 7T Bruker MRI scanner (FOV: 3 cm, in-plane resolution: 234 µm, slice thickness: 1.5 mm). MRI-CEST pH mapping [4] was performed using a fast spin-echo sequence with a RF saturation pulse of 3 μT x 5s and Z-spectra acquired before and after the injection of 1.5 g I/kg b.w. iopamidol into the tail vein. After MRI acquisition, mice were sacrificed, several organs collected and stained with H&E. Concentration of cyto/chemochines and creatinine were detected in mouse sera by cytometric beads and ELISA kits.

Results/Discussion

T_2w MR-images of kidneys failed to report macroscopic lesions in both LPS and TG mice. Conversely, CEST-pH mapping allowed detecting renal dysfunctional changes. Extracellular pH values significantly raised upon LPS, whereas any pH changes were reported in TG and vehicle (mean pH: 6.48 and 6.83, p<0.001 for LPS;6.65 and 6.70 for TG; 6.55 and 6.64 for vehicle, before and after treatment.Fig.1A-B). Besides common sepsis-induced systemic signs (dishevelled hair, reduced mobility, diarrhoea), LPS-endotoxic shock stimulated a marked recruitment of proinflammatory mediators, as IL-6 and MCP-1, whereas a marginal increase was detected in TG mice (Fig.2A). In addition, only LPS mice reported a strong body weight loss (Δweight= -2.2 g), which variation significantly correlated with ΔpH changes (r=-0.77, P<0.05.Fig.2B). Increased concentration of serum creatinine (5.52 ng/µL) confirmed LPS-induced renal functional impairment in comparison to TG (0.46 ng/µL) and vehicle (1.2 ng/µL, Fig.2C)

Conclusions

These results showed that renal pH variations occur in LPS-treated mice and can be non-invasively visualized by the proposed CEST-MRI approach. In particular, alterations of renal pH balance are specific for systemic sepsis and are not detected in a local inflammation model induced by TG. Therefore, this study suggests that further evaluation of renal pH mapping might be considered as a potential tool for diagnosis of sepsis.

References

1. Kellum JA., Critical care and resuscitation 2004, 6:197-203
2. Tran M. et al., Clin Invest. 2011,121 :4003–4014
3. Liu J. et al., NMR in Biomed 2018, 31:e3942
4. Longo DL et al., NMR in Biomedicine. 2017;30:e3720

Acknowledgement

The authors acknowledge the framework of the EU COST Action CA16103 - PARENCHIMA “Magnetic Resonance Imaging Biomarkers for Chronic Kidney Disease”.

Introduction

Imaging of inflammation and infection is widely done with the positron emission tomography (PET) tracer fluorine-18-fluorodeoxyglucose (18F-FDG). However, 18F-FDG cannot reliably distinguish bacterial infection from a tumour or sterile inflammation¹. Therefore, there is a need for bacterial targeted tracers². Sorbitol is a sugar analogue only metabolized by bacteria of the Enterobacteriaceae family and not by mammalian cells, which makes it an ideal candidate for targeted bacterial PET imaging³.

Methods

In this study, a sorbitol based PET tracer 2-[18F]-fluorodeoxysorbitol (¹⁸F-FDS) is synthesised from ¹⁸F-FDG through a single reduction step according to established methods⁴. Purity analysis is done by thin liquid chromatography. ¹⁸F-FDS is tested in vitro on a wide variety of 12 clinically relevant isolates, 11 infected clinical blood samples and 2 infected clinical ascites samples. Samples were collected from the department of Medical Microbiology at the University Medical Centre Groningen, the Netherlands. Heatkilled cultures and sterile patient materials were used as controls. The uptake of ¹⁸F-FDS was assessed by measuring activity (Bq) with a gamma-counter and correlating this to the number of colony forming units (CFU).

Results/Discussion

Clinical isolates from the family Enterobacteriaceae accumulated ¹⁸F-FDS substantially in comparison to the heatkilled controls. Additionally, Pseudomonas aeruginosa and Corynebacterium jeikeium showed relevant uptake, which has not been described before. The uptake in Enterobacteriaceae was more than a two-fold increase compared to the tested Gram-positive bacteria. In both blood and ascites samples, a high¹⁸F-FDS uptake correlated with infection. The highest uptake of ¹⁸F-FDS was seen in Klebsiella pneumonia, both in clinical strains as well as in infected blood samples.

Conclusions

¹⁸F-FDS was successful in detecting infected patient materials which makes it a promising bacterial-targeted PET tracer for clinical translation.

References

1. Love C, Palestro CJ. Radionuclide imaging of infection. J Nucl Med Technol. 2004;32(2):47-57; quiz 58-59.
2. Glaudemans AWJM, Slart RHJA, van Dijl JM, van Oosten M, van Dam GM. Molecular imaging of infectious and inflammatory diseases: a terra incognita. J Nucl Med. 2015 May 1;56(5):659–61.
3. Weinstein EA, Ordonez AA, Demarco VP, Murawski AM, Pokkali S, Macdonald EM, et al. Imaging Enterobacteriaceae infection in vivo with 18 F-fluorodeoxysorbitol positron emission tomography.
4. Li Z-B, Wu Z, Cao Q, Dick DW, Tseng JR, Gambhir SS, et al. The Synthesis of 18 F-FDS and Its Potential Application in Molecular Imaging. B Acad Mol Imaging Mol Imaging Biol. 2007;10:92–8.

Introduction

Although non-human primates (NHP) provide a good model for infectious diseases, their exploration for viral transmission and dissemination by in vivo imaging has not been used extensively. This can be explained by the limited access to adequate structures for imaging these large animals with a high resolution while having specific regulations according to the level of confinement for the human pathogens. Our main objectives are to develop minimally invasive technologies for the longitudinal monitoring of infections, host response and treatments in NHP.

Methods

IDMIT (Infectious disease models for innovative therapies) is a national infrastructure (www.idmitcenter.fr ) founded by five major academic institutions in France (the CEA, the INSERM, ANRS, the Université Paris Sud and the Institut Pasteur) which is located at Fontenay-aux-Roses (France). The infrastructure develops NHP models for immune related disorders and human infectious diseases for preclinical evaluation of human vaccines, immunotherapies and anti-microbial treatments. IDMIT has implemented a unique set of facilities for in vivo imaging in BSL2 and BSL3 constrains (near infrared fluorescence, probe based confocal endomicroscopy, echography, two-photon microscopy and PET-CT).

Results/Discussion

So far, we have developed non-invasive and longitudinal fluorescence imaging approaches using probe based confocal endomicroscopy to study the behavior of skin antigen presenting cells following intradermal immunization with different vaccine vectors in order to better understand the mechanisms leading to the induction of cellular and humoral immune responses after vaccination^1-2. Furthermore, non-invasive in vivo imaging procedures were developed using probe based confocal endomicroscopy coupled with bronchoscopy to track bacterial localization and cellular interactions with host cells in the lower respiratory tract of challenged and naturally infected animals in a model of fluorescent bordetella pertussis in baboons³. Development of PET-CT approaches to track pathogens and inflammation related to infection is ongoing.

Conclusions

This type of approaches could now be used for extended characterization of diverse cell types in vivo and their interactions with other vaccine antigens and/or pathogens. Furthermore the implementation of the PET-CT system will allow us to study whole body dissemination of pathogens with a high sensitivity. 

References

1.           Todorova, B., Salabert, N., Tricot, S., Boisgard, R., Rathaux, M., Le Grand, R. et al. Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques. Contrast Media Mol Imaging. 2017, 3127908  (2017).

2.           Todorova, B., Adam, L., Culina, S., Boisgard, R., Martinon, F., Cosma, A. et al. Electroporation as a vaccine delivery system and a natural adjuvant to intradermal administration of plasmid DNA in macaques. Sci Rep. 7, 4122  (2017).

3.           Naninck, T., Coutte, L., Mayet, C., Contreras, V., Locht, C., Le Grand, R. et al. In vivo imaging of bacterial colonization of the lower respiratory tract in a baboon model of Bordetella pertussis infection and transmission. Sci Rep. 8, 12297  (2018).

Acknowledgement

We warmly thank the IDMIT infrastructure staff for excellent technical assistance, morte specifically the Animal Science and Welfare core facility. 

Introduction

Staphylococcus aureus-induced infective endocarditis (IE) is a life threatening disease with high mortality rates up to 30% [1]. Due to the currently prevalent long diagnostic latency (29 ± 35 days [2]) and unspecific diagnostic methods of IE, there is an urgent need for improved detection tools. Here, we assess the binding affinity of peptide fragments from specific bacteriophages and antimicrobial peptides to S. aureus and design a reference fluorescence imaging probe for use in diagnosis of infective endocarditis.

Methods

Binding efficiency of peptide candidates (­~20 aa) to the S. aureus´ surface was assessed using ELISA (n=8) and fluorescence measurements (n=6) with GFP-expressing bacteria.

Peptide candidate ubiquicidin (UBI29-41) was synthetized with a polyhistidin-tag and conjugated to a nickel-containing fluorophore (NTA-Atto 488, Sigma Aldrich).

Antimicrobial capacity, binding affinity, specificity and detection thresholds were assessed in incubation experiments with naïve S. aureus. A bacterial pellet (10⁸ CFU) was incubated with different concentrations of the fluorescent probe and observed in fluorescence microscopy and fluorescence reflectance imaging (FRI). Binding specificity was assessed in competition experiments with unlabelled UBI 29-41.

Results/Discussion

Among the investigated peptide fragments the peptide UBI showed the best pathogen affinity. UBI29-41 was therefore used for design of a reference imaging probe. In growth experiments with different concentrations of S. aureus bacteria (10⁴-10⁸CFU) in presence of different amounts (0,125 – 2mmol/l) of UBI29-41, the peptide fragment did not exert antimicrobial effects.

In bacterial labeling assays the imaging probe showed pronounced fluorescence intensity as observed in fluorescence microscopy and FRI. In contrast, the bacteria did not emit a fluorescent signal after incubation with the uncoupled fluorophore. After incubation with 0.1 mmol/l of the probe, bacteria showed well detectable signal. In competition experiments a signal difference of 62% between the preincubated bacteria (100 fold excess unlabeled UBI29-41) and the pellet only incubated with the dye was observed, indicating a specific binding on the bacterial surface.

Conclusions

Our results show that our imaging marker binds specifically to S. aureus. The coordinative binding of the His-tagged UBI 29-41 to the Ni-containing fluorophore is stable under the tested conditions, and suggests that, Ni-coated iron oxide nanoparticles (IONP) can be coupled to UBI29-41 to detect bacterial infections via MRI imaging. An UBI-based MRI marker appears likely to reproduce previous results obtained with an UBI-based SPECT-tracer [3].

References

[1] Baddour LM, et al. (2015): Infective Endocarditis in Adults: Diagnosis, Antimicrobial Therapy, and Management of Complication. Circulation, 132 (15): 1435-1486

[2] Habib, Gilbert, et al. (2015): 2015 ESC Guidelines for the management of infective endocarditis. European heart journal, 36 (44): 3075–3128

[3] Sathekge, Mike, et al. (2018): Molecular imaging in musculoskeletal infections with 99mTc-UBI 29-41 SPECT/CT. Ann Nucl Med (2018) 32: 54.

Introduction

Nanoparticles (NPs) in biomedicine hold great promise in improving a range of therapies. Vital in the evolution of nanoparticle-based approaches is tracking biodistribution and degradation, as they impact delivery efficacy. In vivoimaging of both biodistribution and degradation of NPs still faces several challenges, but it is necessary to advance the performance of NPs ^[1]. We developed a dual perfluorocarbon (PFC) loaded NP which acts as a biodistribution and degradation sensor simultaneously, using the magnetic resonance properties of both PFCs, each with a single, distinct resonance.

Methods

For synthesis of NPs loaded with PERFECTA and Perfluoro-15-crown-5-ether (PFCE) a miniemulsion and nanoprecipitation method was used ^[2,3]. Hexafluoroisopropanol was used to dissolve both PFCs and PLGA. Particle size was measured using dynamic light scattering (DLS) and cryoSEM (Scanning electron microscope). PERFECTA and PFCE content, relaxation times and ¹⁹F-¹⁹F Nuclear Overhauser Enhancement (NOE) were measured on a Bruker Advance III 400 or 500MHz. NPs were incubated with dendritic cells for 3 days, subsequently an MMT assay was used to investigate toxicity of the particles. In vitro imaging was done on a 11.7T Biospec using a RARE sequence with selective excitation of either PERFECTA or PFCE. Internal structure of the NPs was measured using Small Angle Neutron Scattering (SANS)^[4].

Results/Discussion

Synthesis yielded NPs measuring a radius of 164±7nm in DLS and 96±23nm in cryoSEM. Magnetic resonance spectroscopy showed a PFC encapsulation of 8.7wt% PERFECTA and 10wt% PFCE (Fig 1a). An MTT assay demonstrated no toxicity of the NPs on cells, showing safety for applications such a cell therapy (Fig.1b). Selective imaging is shown in Fig. 1c. SANS and ¹⁹F NMR showed that these particles have a fractal structure, opposed to the conventional core-shell structure in PFC-emulsions. Whether the nanoparticles are intact in vivo,can be established using Nuclear Overhauser Effect (NOE) and relaxation times. NOE is only observed when two, in this case fluorinated, compounds are within a few Ångströms from each other (Fig. 2a). When the PERFECTA-PFCE NPs were hydrolyzed with NaOH the NOE strongly decreased and relaxation times changed, suggesting dissociation of the 2 fluorinated compounds (Fig. 2b). Location of degradation can be established, as PFCs stay visible but becomes immobile.

Conclusions

Simultaneous imaging of biodistribution and degradation in vivocan help to optimize nanoparticle-based therapeutics. Here we present nanoparticles containing two different PFCs that can be imaged to give information on biodistribution. Furthermore, between these two PFCs we observed NOE. This effect strongly decreased after hydrolysis of the particles, giving information on degradation, which is supported by changing relaxation times.

References

1. D. Rosenblum, N. Joshi,W. Tao et al., Progress and challenges towards targeted delivery of cancer therapeutics. Nat. Commun. 2018; 9(12).
2. E. Swider, A.H.J. Staal, N.K. van Riessen, et al., Design of triphasic poly(lactic-co-glycolic acid) nanoparticles containing a perfluorocarbon phase for biomedical applications. Rsc Advances, 2018; 8(12): 6460-70.
3. I. Tirotta, A. Mastropietro, C. Cordiglieri, et al., A Superfluorinated Molecular Probe for Highly Sensitive in Vivo 19F-MRI. J. Am. Chem. Soc., 2014; 36(24): 8524–8527
4. In preparation: O. Koshkina, P.B. White. A.H.J. Staal, et al.,Two-Color Perfluorocarbon-Loaded Fractal Nanoparticles for Imaging their Biodistribution and Degradation by ¹⁹F Magnetic Resonance Imaging.

Introduction

Nanoscaffolds combining paramagnetic and radioactive metal-ions offer the benefit of the excellent soft-tissue resolution of MRI together with the high sensitivity of PET. Nanozeolites, with their unique crystalline framework forming channels and cavities, are especially interesting due to their ability for stable accommodation of metal-ions with different physical properties. Here, we demonstrate the potential of Gd³⁺-loaded nanozeolite-LTL system with very high and pH-responsive r₁- and r₂-relaxivities^[1] for the additional radiolabeling with ⁸⁹Zr²⁺ for simultaneous PET imaging.

Methods

⁸⁹Zr-labeling was done by incubation of Gd³⁺-LTL with aqueous ⁸⁹ZrCl₃(100 MBq) obtained by ion-exchange from originally produced ⁸⁹Zr-oxalate, followed by purification and PEGylation of the surface. The final ⁸⁹Zr/Gd-LTL-PEG probe (5 MBq, 3.7 wt% Gd) suspended in saline was injected into healthy nude mice and the biodistribution was followed by PET/CT imaging. The images were acquired at 10 min, 2h, 18h and 4 days post-injection. The kinetic behavior was also studied by in vivoMRI following the fate of Gd-LTL-PEG during the first hour after injection. The biodistribution of the probe was investigated by the ex-vivoICP-analysis of the organs and the amounts of Al (representing the framework of LTL) and Gd were correlated to assess the integrity of the probe.

Results/Discussion

The straightforward synthesis of ⁸⁹Zr/Gd-LTL-PEG probe is attractive for application in multimodal imaging: it can be done in a facile manner with 74% radiolabeling yield. The used method differs from the common ⁸⁹Zr radiolabeling, done typically in the presence of oxalic acid. The extra ion-exchange purification step was included to avoid the formation of highly stable ⁸⁹Zr-oxalate complexes that prevent ⁸⁹Zr²⁺ from entering the inner cavities. Our method reinsured encapsulation of the radiolabel inside the LTL, which resulted in an increased stability of the final probe. The relaxivity of the cold probe after addition of Zr²⁺ remained practically unchanged (r₁=34 s⁻¹mM⁻¹) compared to its Gd-loaded analogue.^[2] The in vivoMRI study showed no particular organ accumulation within 1h post-injection. PET images revealed initial lung accumulation of the probe and further elimination through the liver (Fig. 1). Ex vivo ICP and g-counting biodistribution confirmed the integrity of the probe.

Conclusions

The PET/MRI ⁸⁹Zr/Gd-LTL-PEG probe can be easily prepared with a good radiolabeling yield. The additional loading of ⁸⁹Zr²⁺into the inner cavities next to the already present Gd³⁺and the subsequent PEGylation of the surface do not change the relaxivity of the system. The biodistribution profiles confirm the stability of the probe in vivo, which along with absence of adverse effects render the probe as promising for theranostic applications.

References

^[1]W. Zhang, J.A. Peters, F. Mayer, L. Helm, K. Djanashvili, J. Phys. Chem. C, 2015, 119, 5080
^[2]W. Zhang, J. Martinelli, J.A. Peters, J.M.A. van Hengst, H. Bouwmeester, E. Kramer, C.S. Bonnet, F. Szeremeta, É. Tóth, K. Djanashvili. Appl. Mater. Interfaces2017, 9, 23458−23465.

Acknowledgement

The work was supported by Le Studium, Loire Valley Institute for Advanced Studies, co-funded by the EU’s Horizon 2020 under the Marie Skłodowska-Curie programme (agreement No. 665790). Dr. Sandra Même is acknowledged for the MRI measurements.

Introduction

Iron oxide nanoparticles (IONPs) have been developed for several applications including magnetic hyperthermia for cancer, and cell tracking. MRI gives sensitive non-invasive detection of IONPs in vivo, but can be ambiguous due to endogenous contrast sources and saturation. Radio-labelling avoids this problem, allowing quantification with nuclear imaging – yet requires IONP modification with chelators. A new method achieves labelling without chelators via heating with radiometals^[1,2] thus simplifying the labelling process. However the resulting radiometal interaction is yet to be understood.

Methods

Heat-induced, chelate-free labelling of magnetite (50nm) and maghemite (40nm) nanoparticles was done with cold InCl₃ and ZrCl₄ according to Boros et al^[1]. To determine the physical basis of metal incorporation, labelled and control IONPs were analysed with Time-of-flight surface ionisation mass spectroscopy (ToF-SIMS), X-ray Photoelectron Spectroscopy (XPS) Mössbauer Spectroscopy, and X-ray Diffraction (XRD). Magnetic properties were measured with SQUID. Radio-chemical yield (RCY) was measured after labelling with ¹¹¹In and ⁸⁹Zr for SPECT and PET respectively, for a panel of 8 commercial IONPs, using both magnetic separation and thin-layer chromatography. PET and SPECT CT imaging (Mediso), and MRI (Bruker 1T Icon) was done following subcutaneous particle injection into C57 Blk/6 mice.

Results/Discussion

We showed that heat-induced labelling of maghemite / magnetite nanoparticles with InCl₃ and ZrCl₄ operates via amorphous metal oxide deposition (In₂O₃ or ZrO₂) using XPS and XRD analysis, while ToF-SIMS showed localization to the particle surface. The original magnetite and maghemite cores were shown to be unaltered using Mössbauer Spectroscopy and XRD, with no doping of In or Zr into the lattice structure. SQUID magnetometry showed retention of magnetic properties. Eight commercially-available IONPs of varying size/coating were labelled with RCYs of 65 to 95% using both ¹¹¹In and ⁸⁹Zr.

To show biodistribution tracking we labelled a new clinical magnetic hyperthermia iron oxide agent RCL-01 (Resonant Circuits Ltd)^[3] using ⁸⁹ZrCl₄ (95% RCY). ⁸⁹Zr-RCL-01 (5mg) was injected subcutaneously in mice to model dosing for melanoma, and PET-CT and MRI tracked biodistribution for 8 days (Fig2A). MRI, PET, and CT contrast co-localised, consistent with particle retention of radiolabel (Fig2B-E).

Conclusions

We show that heat-induced radio-labelling works by mineralization of radio-metals onto the surface of the particle in trace amounts, without changing the original iron oxide material or magnetic properties. This method promises rapid radio-labelling and whole-body quantification of IONPs across a range of bio-medical applications, which we illustrate using a new particle formulation (RCL-01) developed for clinical hyperthermia therapy.

References

[1] Boros et al. Chem Sci. 2015   [2] Huan et al. Nat Protocols 2018. [3] www.resonantcircuits.com

Introduction

Magnetic “nanoflower”-shaped iron oxide cores have been of increased interest for application to Magnetic Particle Imaging (MPI) and Magnetic Hyperthermia Therapy (MHT).^1-3 These nanoflowers contain densely packed iron oxide cores with a larger average particle diameter then current particles used for MPI.  In this study we compared the MPI signal properties and in vivo biodistribution of a commercial iron oxide nanoflower nanoparticle with two different surface coatings.   

Methods

Plain dextran coated and polyethylene glycol (PEG) coated synomag^®-D nanoparticles (micromod Partikeltechnologie GmbH, Germany) were evaluated. Particle stability was also measured under conditions with particles stored at room temperature, at 4°C, and at -20°C.

For in vivo blood half-life measurements, C57Bl/6 mice were injected IV with 50 μL tracer (5.5 mg/mL). Two cohorts (n=2) were evaluated comparing the dextran coated and PEG nanoparticles. Imaging was performed at Stanford University with the MOMENTUM MPI system (Magnetic Insight Inc., CA, USA). Projection imaging was performed serially over 2 hours after which a tomographic image was acquired prior to animal sacrifice. An additional synomag^®-D + PEG cohort (n=2) was imaged at 1, 2, 4, 8, 12 and 24 hours after injection.      

Results/Discussion

The synomag^®-D particles produced ~3.5x more signal per mass of Fe than ferucarbotran with a measured resolution of 900µm (6 T/m selection field gradient). No significant differences were observed in the peak signal amplitude and resolution between the plain dextran coated and PEG coated synomag^®-D particles.  Following intravenous tail vein injection, dextran-coated synomag^®-D particles were rapidly taken up by the liver and spleen (t_1/2 < 4 minutes, see Fig 1B). In contrast, the PEG-coated synomag^®-D particles were retained within the blood pool for a longer period of time (Fig 1C). After 90 minutes, nanoparticles were observed in the cranial vasculature, pulmonary vasculature, bone marrow, and liver. We believe that the blood pool signal stabilized after 45 minutes due to saturation of the liver at the injected dosage. Surprisingly, the blood pool signal did not continue to decrease as rapidly, which we believe is the result of saturating the Kupffer cells in the liver.

Conclusions

As shown here, synomag^®-D particles, with and without PEG coatings, are promising tracers for MPI.  By changing the coating, it is possible to both have short and long tracer blood half-life.  Importantly, the change of coating does not change the magnetic behavior of the tracer. We believe that these tracers are ideal for measurements of tissue perfusion, functional tracking, and in situ inflammation detection.

References

[1]  Gavilan et al. Colloidal flower-shaped iron oxide nanoparticles: synthesis strategies and coats. Part. Part. Syst. Charact., 34:1700094. 2017 doi: 10.1002/ppsc.201700094

[2]   P. Bender et al. Relating magnetic properties and hyperthermia performance of iron oxide nanoflowers. J. Phys. Chem. 122:3068-3077. 2018. Doi: 10.1021/acs.jpcc.7b11255

[3]   Kratz et al. Novel magnetic multicore nanoparticles designed for MPI and other biomedical applications: From synthesis to first in vivo studies. PLOS ONE  2018 doi: 10.1371/journal.pone.0190214

Acknowledgement

Research funding: Research reported in this publication was supported by NIBIB of the NIH under award number R43EB020463. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Introduction

The use of molecular MRI probes gives information at cellular and molecular level allowing to assess biomarkers such as pH, enzymes or ions. The main drawback of such MRI probes is that even if they are responsive to the presence of a biomarker, it is not possible to quantify them since the relaxivity depends both on the local concentration and on its environement. To overcome this issue, we propose a cocktail of ¹⁶⁵Er³⁺ (active in SPECT, quantificative technique) and Gd³⁺ complexes of a new ligand designed for the quantification of zinc, deregulated in diseases such as cancer or diabetes. 

Methods

¹⁶⁵Er obtained by proton beam irradiation of ^natHo at the CEMTHI cyclotron was efficiently purified by ion-exchange chromatography and its radionuclidic purity assessed by γ-spectrometry. The new ligand was synthesised and successfully radiolabeled with ¹⁶⁵Er³⁺ and complexed with Gd³⁺. A cocktail with a known GdL/¹⁶⁵ErL mole ratio, in 0.6mM HSA, was diluted to give 5 samples to which unknown concentrations of Zn²⁺ were added. The activities were measured by γ-spectrometry and imaged in a γ-camera, enabling the calculation of ¹⁶⁵Er³⁺ concentration and thus the Gd³⁺ ones. Upon decay, the T1-weighted MRI was recorded on a 1.5T scanner. Using a calibration curve, the experimental Zn²⁺ concentrations were calculated from the measured relaxivities. All concentrations were confirmed by ICP/BMS.

Results/Discussion

The proposed ligand contains 2 units: one for Gd³⁺ complexation and another one for Zn²⁺ (fig 1) yielding its Gd³⁺ complexes responsive to Zn²⁺, in the presence of HSA. Importantly, the relaxivity changes linearly with Zn²⁺ concentration. The sussessful production of ¹⁶⁵Er³⁺ allowed radiolabeling of the same ligand. The choice of a radiolanthanide enables a similar biodistribution to GdL. The use of a fixed mole ratio of these complexes (as a single cocktail) allows to respect the different sensitivity of each imaging techniques, MRI and SPECT. The unknown test samples prepared were assessed by γ-spectrometry which allowed to determine their ¹⁶⁵Er concentration, and thus calculate the Gd ones, with 6% error as confirmed by BMS. T1-weighted MR images of the same samples where then measured and converted in relaxivity accordingly. Based on a relaxivity calibration curve obtained in the same conditions, the Zn concentrations were calculated with an overall accuracy of ca. 20%.

Conclusions

The use of a Gd/¹⁶⁵Er³⁺ cocktail of the same ligand combines MRI and SPECT detection, providing an elegant approach for in vitro proof-of-concept of quantitative assessment of Zn²⁺ concentration. ¹⁶⁵Er³⁺ (t_1/2=10.4h) is easily produced and possible to be separated from the parent ¹⁶⁵Ho³⁺. The use of ¹⁶⁵Er³⁺ on quantitative responsive imaging of a biomarker, acting as surrogate to Gd³⁺ for in vivo biodistribution studies, is highly interesting.

References

K.P. Malikidogo, I. Da Silva, J-F. Morfin, S. Lacerda, L. Barantin, T. Sauvage, J. Sobilo, S. Lerondel, É. Tóth and C.S. Bonnet Chem. Commun., 2018, 54, 7597-7600.

Acknowledgement

We thank Agnès Pallier for ICP measurements. CSB thanks the French National Research Agency (grant ANR-13-JS07-0007).

Introduction

Fluorine-19 Magnetic Resonance Imaging (¹⁹F MRI) is an upcoming imaging modality, using¹ the 100% naturally abundant and stable ¹⁹F isotope, that can be imaged as a result of its spin magnetic moment.² MRI has low sensitivity, therefore most of the used imaging agents are composed of heavily fluorinated molecules. Most used is perfluorocrown-ether, because its 20 identical fluorine bonds give it a high single resonance signal. Unfortunately, these perfluorocarbons have clearance half-lives of 200 days, leading to regulatory and safety concerns, which in turn hamper clinical adaptation.³

Methods

¹⁹F Nanoparticles (NP), consisting of poly(D,L-lactide-co-glyolide) polymer and PFCE were prepared using a miniemulsion technique⁴. PFCE-nanoemulsions NE) were made by microfluidization using egg-lecithin⁵. Equivalent amounts of PFCE (20mg NP dissolved in 400ul 0,9% NaCl and 3,3% PFCE NE) were injected into the tail vein of wild type mice. At 6 timepoints spanning 2 weeks biodistribution was measured using an optimized ¹⁹F 3D RARE sequence (12min scan time). With the aid of a reference tube with a known concentration of imaging agent, biodistribution is shown as % of injected dose per organ. NP were dissolved in PBS and agitated at 37°C for 2 weeks to study in vitro degradation through hydrolysis. Dynamic light scatering and atomic force microscopy were used to measure NP size over time.

Results/Discussion

Whereas PLGA-NPs had a diameter of about 200nm, PFCE-NEs were about 130nm in size.  Fluoride concentration and MRI signal are linear (R² 0,99 for linear fit) allowing quantification based on a reference. 2 hours after injection, the liver contains 40% of the injected dose for both NE and NP, spleen contains 2,5% and 11% of injected dose for NE and NP respectively (Fig. 1). Kidneys and lungs did not show any signal above the detection limit. Over the time course of 2 weeks the liver signal halved and the spleen signal was reduced to 1/5 of the initial signal for NPs, for NEs the liver concentration stayed stable and the spleen concentration dropped below the detection threshold. Degradation experiments show a unique degradation profile when compared to conventional PLGA particles. NPs degrade into smaller domains, averaging around 50nm diameter, while retaining PFCE (Fig 2).

Conclusions

PFC-based ¹⁹F imaging agents are cleared by exhalation. Single resonance PFCs, such as PFCE and the commercially available perfluoropolyether are excreted slowly via this process. Here we show a poly(D,L-lactide-co-glyolide) nanoparticle, containing PFCE with a half-life of only 2 weeks. A unique degradation profile is hypothesized to facilitate faster clearance.

References

1. Ruiz-Cabello J, Barnett BP, Bottomley PA, Bulte JW. Fluorine (19F) MRS and MRI in biomedicine. NMR Biomed. 2011 Feb;24(2):114-29

2. G.N. Holland, P.A. Bottomley, W.S. Hinshaw. 19F magnetic resonance imaging. J. Magn Res. 1977; 28(1):133-136

3. C. Jacoby, S. Temme, F. Mayenfels et al., Probing different perfluorocarbons for in vivo inflammation imaging by 19F MRI: image reconstruction, biological half‐lives and sensitivity. NMR in Biomed. 2014; 27(3):261-271

4. E. Swider, A. H. J. Staal, N. K. van Riessen, et al., Design of triphasic poly(lactic-co-glycolic acid) nanoparticles containing a perfluorocarbon phase for biomedical applications. RCS Adv. 2018;12

5. U. Flögel, Z. Ding, H. Hardung, et al., In Vivo Monitoring of Inflammation After Cardiac and Cerebral Ischemia by Fluorine Magnetic Resonance Imaging. Circ. 2008; 118:140–148

Introduction

A major advantage of fluorine MRI is the signal specificity. Micelles were developed to carry a branched fluorinated probe (PERFECTA) bearing 36 equivalent ¹⁹F atoms which resonate at a single NMR frequency¹. Micelles are made from the assembly of amphiphilic units and have a diameter of approximately 15 nm. The micelles can be loaded with PERFECTA and a drug, and be used as a theranostic agent to target tumors through the enhanced permeability and retention (EPR) effect². The goal of this study is to characterize the detection of PERFECTA micelles (PM) in vitro and in vivo by fluorine MRI.

Methods

PERFECTA micelles in PBS were formulated at different concentrations in fluorine which were determined by ¹⁹F-NMR analysis. Samples were then imaged using a 7 T preclinical scanner (Bruker) and a dual channel (¹H/¹⁹F) coil (Rapid Biomed) with MSME sequence to assess the detection sensitivity.

First in vivo studies were performed on non-implanted C57BL/6 mice (Group 1, N = 3) and 150 µL of PM were injected by intravenous route (IV). In a second experiment, we used mice bearing subcutaneous tumors from murine colon MC-38 cells (Group 2, N = 2) and 30 µL of PM were injected by intra-tumoral route (IT).

MRI sequences were acquired 20 min after injection, and then after 24, 48, 72 and 96 h using FcFLASH and RARE sequences for proton and fluorine acquisition.

Results/Discussion

According to the in vitro study, the detection sensitivity of MP with MSME sequence (35 min) was 0.118 M (tube C in Fig. 1), meaning about 7.10¹⁶ of ¹⁹F atoms per mm³.

The in vivo study showed fluorine signal in the heart 24 h after IV injection (Fig. 2/A) and suggested that PM were circulating in the blood 24 h after injection. In vivo biodistribution was found to be repeatable: for the 3 non-implanted mice, the PM target the liver, as previously described in another study.² For IT injections, the fluorine signal remained for 4 days in the tumor area (Fig. 2/B&C) which suggests that PM were trapped in the tumor.

Conclusions

In this study, we characterized the sensitivity detection of PM in vitro and the biodistribution of these nano-objects in vivo using a preclinical MRI scanner at 7 T. Moreover, in vivo images showed retention of the PM in the tumor 4 days after IT injection. This result suggests that the fluorinated nanocarrier (PM) can be used to target tumors through the EPR effect which also opens the way to its use as theranostic agent.

References

1. Tirotta, I. et al. A Superfluorinated Molecular Probe for Highly Sensitive in Vivo19F-MRI. J. Am. Chem. Soc. 136, 8524–8527 (2014).

2. Mackiewicz, N. et al. Tumor-targeted polydiacetylene micelles for in vivo imaging and drug delivery. Small 7, 2786–2792 (2011).

Introduction

The synthesis and in vitro validation of a multifunctional theranostic nanoparticle, which combines photodynamic therapy (PDT)/magnetic resonance imaging (MRI), is described. The combination of diagnostic and therapeutic modalities in a single construct represents an important step towards optimising and personalising diagnosis and treatment of solid tumours.

Methods

In this project, a hydrophilic polyacrylamide nanoparticle capable of orthogonal surface functionalisation through “click” chemistry and peptide-coupling chemistry, was synthesised and subsequently surface functionalised with a porphyrin (PDT agent) and a gadolinium complex (MRI contrast agent). The compounds where tested for hydrodynamic diameter, relaxometry, PDT efficacy in vitro and in vivo.

Results/Discussion

Upon incubation of this theransostic agent with human adenocarcinoma (HT-29) cells, and subsequent irradiation with visible light, > 90% cell death was achieved at a porphyrin concentration of 0.62 µM. Minimal toxicity was observed without light irradiation (95% cell survival at 99 µM). T1 measurements and phantom MRI experiments were carried out to evaluate the MRI contrast efficiency of the construct. Relaxivity (r₁) of the nanoparticle, measured at 60 MHz, was 14.7 mM^-1 s^-1, and compares favourably with the clinical relevant contrast agent [Gd(DTPA)]^2- which gave an r₁ of 3.3 mM^-1 s^-1 under the same conditions.

Conclusions

We have shown the synthesis and validation in vitro and in vivo of a novel theranostic based on MRI and PDT. Using hydrophilic polyacrylamide nanoparticles as vector for both modalities. 

References

1. “Functionalized polyacrylamide nanoparticles as theranostic PDT/MRI agents” S.Y. Yap, L. Kenning, H. L. Perry, J. D. E. T. Wilton Ely, H. Savoie, G. J. Stasiuk*, and Ross W. Boyle*. Chem. Sci. (under revision 2018).

Introduction

Vesicular acetylcholine transporter (VAChT) protein plays a crucial role in cholinergic neurotransmission, alterations of VAChT expression occur in different diseases such as Alzheimer disease, sepsis, hypertension, and Huntington disease[1]. VAChT is a reliable biomarker for cholinergic neuron loss that relates to cognitive dysfunction. We had reported that [¹⁸F]VAT is a promising VAChT PET radiotracer[2]. Herein, we report in vitro characterization of a tritium version [³H]VAT binding properties for human VAChT cell and nonhuman primate brain tissues.

Methods

Post-nuclear supernatant from PC12^A123.7 cell stably transfected with human VAChT (hVAChT) was prepared and used to characterize the binding affinity of [³H]VAT through saturation and competition binding assay. Five VAChT inhibitors, vesamicol, TZ6-59, FBBV, TZ45-091B, and TZ21-44, as well as four other central nervous system (CNS) ligands, MP-10 for PDE10A enzyme, Yun-122 for sigma-1 receptor, eticlopride and quinpirole for D₂ receptor were used for competition studies to check [³H]VAT binding specificity. In vitro autoradiography quantification using [³H]VAT was also performed in normal monkey brain slices.

Results/Discussion

A saturation binding curve was obtained with K_d = 6.5 ± 1.7 nM and B_max = 22893 ± 1934 fmol/mg protein (Figure 1A, B). As control, a PC12A123.7 cell line without transfected hVAChT had no binding, suggesting [³H]VAT is highly specific for VAChT protein. For competition studies, [³H]VAT binding to VAChT was inhibited by different VAChT inhibitors (K_i = 5.4-33.3 nM), and a panel of other common used CNS ligands did not interfere [³H]VAT binding to VAChT (Figure 2). Further, in vitro autoradiography study of [³H]VAT using monkey brain slice confirmed that the striatum had the highest ligand uptake compared to other brain regions (Figure 3).

Conclusions

[³H]VAT has high potent and specific binding to VAChT. The promise data of [³H]VAT combining with our previous PET study data of [¹⁸F]VAT in the brains of animals suggested that [¹⁸F]VAT could be a promising PET tracer for quantifying the VAChT expression in the brain for CNS disorders.

References

[1] V.F. Prado, A. Roy, B. Kolisnyk, R. Gros and M.A. Prado. Regulation of cholinergic activity by the vesicular acetylcholine transporter. Biochem J. 2013 Mar 1;450(2):265-74.

[2] Z. Tu, X. Zhang, H. Jin, X. Yue, P.K. Padakanti, L. Yu, H. Liu, H.P. Flores, K. Kaneshige, S.M. Parsons, and J.S. Perlmutter. Synthesis and biological characterization of a promising F-18 PET tracer for vesicular acetylcholine transporter. Bioorg Med Chem. 2015 Aug 1;23(15):4699-709.

Acknowledgement

This study was sponsored by the USA the National Institute of Neurological Disorders and Stroke, and the National Institute on Aging of National Institutes of Health NS075527 and NS103988.

Introduction

Fenretinide (4-HPR) is a synthetic retinoid which has been investigated for decades as an anticancer agent and more recently as a potential drug to treat metabolic syndrome.^1-3 Here we synthesised a library of 4-HPR analogues - using a SAR approach - with the final goal to improve both anti-cancer activity and anti-obesity/diabetes properties of 4-HPR. Among the designed 4-HPR analogues, we selected the click-type analogue 3b, which is amenable to radiofluorination, to develop a PET tracer to further investigate the pharmacological profile of 4-HPR and analogues in vivo.

Methods

After developing a methodology for the synthesis and purification of 4-HPR analogues, their anticancer activity and selectivity were evaluated using MTT assays in MCF-7 cells and HEK 293 cells to detect the amount of viable cells after treatment. The potential anti-obesity properties were tested by measuring the inhibition of adipogenesis using Oil Red O stain in differentiated 3T3-L1 adipocytes after a chronic treatment. We then proceeded with the [¹⁸F]labelling of compound 3b – which gave positive in vitro results - by producing a [¹⁸F]tosylazide that was subjected to a click reaction with the 4-HPR propargylamide derivative.

Results/Discussion

Our results demonstrate that introduction of a fluorine in meta position (2b) onto the 4-HPR aryl amide group increases the anti-proliferative activity in MCF-7 cells, reducing the IC₅₀ from 12 µM to 46 nM, also inducing a moderate selectivity for cancer cells. On the other hand, replacement of the OH in para position with a phenyl ring (1d) increases the cytotoxicity (IC₅₀ 81 nM) but reduces the selectivity. The replacement of the phenol with a triazol ring (3b) increases both anti-proliferative activity (IC₅₀ 1.2 µM) and selectivity. The same modifications however do not improve the anti-adipogenic activity, in particular 2b and 3b are able to reduce lipid accumulation in 3T3-L1 adipocytes but to a lesser degree in comparison with 4-HPR. The [¹⁸F]Fluorobutyl azide was labelled and distilled (40-60% DCY) and the click reaction occurred with a satisfactory rate. The next step will be using [¹⁸F]3b to perform pre-clinical imaging to investigate the pharmacokinetic properties in vivo.

Conclusions

In this study we demonstrated that specific substitutions on the aromatic ring of the 4-HPR scaffold (1d, 2b, 3b), can lead to an increase of cytotoxicity or selectivity towards MCF-7 cancer cells. Importantly we also radio-synthesised [¹⁸F]3b as a novel PET  tracer to support the development of 4-HPR and analogues as drug candidates.

References

1. Mcilroy, G. D. et al. Fenretinide mediated retinoic acid receptor signalling and inhibition of ceramide biosynthesis regulates adipogenesis, lipid accumulation, mitochondrial function and nutrient stress signalling in adipocytes and adipose tissue. Biochem. Pharmacol. 100, 86–97 (2016).

2. Veronesi, U. Fifteen-year results of a randomized phase III trial of fenretinide to prevent second breast cancer. Ann. Oncol. 17, 1065–1071 (2006).

3. Mody, N. & Mcilroy, G. D. The mechanisms of Fenretinide-mediated anti-cancer activity and prevention of obesity and type-2 diabetes. Biochem. Pharmacol. 91, 277–286 (2014).

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska–Curie grant agreement No 675417.

Introduction

Chemokine receptors are implicated in various diseases, such as asthma, rheumatoid arthritis, AIDS and cancer, making them interesting targets for the development of antagonists and imaging agents. Among these receptors, CXCR4 and ACKR3 (CXCR7) have been shown to be over-expressed in different types of cancers [1] and are usually associated with poor prognosis [2]. Successfully targeting and imaging the expression density of these receptors could allow earlier diagnosis of some cancers, inform treatment selection and lead to better outcome for the patients.

Methods

We developed a library of configurationally restricted multimacrocyclic compounds and their corresponding copper(II), zinc(II) and nickel(II) metal complexes that target both CXCR4 [3] and ACKR3. In vitro affinity for CXCR4 was determined by Ca²⁺ signalling assays and affinity for ACKR3 was determined by competition binding with fluorescent CXCL12, using U87 glioblastoma cell lines transfected to stably express each receptor. Affinity for CXCR4 was assessed in vivo through a series of imaging blocking studies of the known CXCR4-specific PET tracer [⁶⁸Ga]Pentixafor, in SCID/Beige mice implanted with CXCR4 over-expressing xenografts.

Results/Discussion

We have produced a library of these multi-macrocyclic compounds and their corresponding Cu(II), Zn(II) and Ni(II) metal complexes specific to CXCR4 with affinities up to 4 nM [3]. We extended our work to the in vitro validation of a related set of novel compounds that also bind to ACKR3. The structure-activity relationship of these compounds is currently under investigation and preliminary results have already shown affinity for ACKR3 below 100 nM. In vivo blocking studies with this set of compounds confirmed high specificity for CXCR4 in tumour-bearing animals, successfully blocking [⁶⁸Ga]Pentixafor with doses as low as 4 mg/kg, showing a 6-fold decrease in tumour uptake.

Conclusions

We successfully synthesised a set of novel configurationally restricted multi-macrocyclic metal complexes and demonstrated their affinity for both CXCR4 and ACKR3 in vitro. We also confirmed the affinity of these derivatives for CXCR4 in vivo through a series of blocking studies of the known CXCR4-specific PET imaging Pentixafor. Further ACKR3 in vivo validation is currently under investigation.

References

[1] N. A. Wani et al. Breast Cancer Research, 2014, 16, R54.

[2] A. Zlotnik et al. Nat. Rev. Immunol., 2011, 11, 597.

[3] A. Khan et al. J. Am. Chem. Soc., 2009, 131, 3416.

Acknowledgement

The authors would like to thank Dr Assem Allam for fellowship funding and his generous contribution to the University of Hull PET Research Centre.

Introduction

Chemokine receptors are involved in cell trafficking and implicated in diseases including atherosclerosis, asthma, rheumatoid arthritis, HIV infection and cancer.¹ Imaging CXCR4 chemokine receptor expression levels in cancers as a prognostic marker or to inform treatment selection is of interest.² We have targeted the CXCR4 chemokine receptor using configurationally restricted tetraazamacrocyclic transition metal complexes to give nanomolar level receptor affinities and long receptor residence times,^3,4 offering an alternative approach to the gallium-68 pentixafor in clinical trials.⁵

Methods

This new class of probes can be directly labelled with copper-64 with no structural change or modified to incorporate gallium-68 in a second chelating component or prosthetic. The influence of structural variation was investigated using a series of in vitro assays to select compounds for in vivo imaging and optimised radiolabelling protocols. Probes were synthesised and assessed in a series of in vitro assays. Radiochemistry has been carried out with copper-64 and gallium-68. In vitro cell uptake experiments were carried out on U87-CXCR4 and U87 glioblastoma cells. In vivo 90 minute dynamic PET/CT imaging was carried out on CD1 mice bearing U87-CXCR4 and U87 glioblastoma xenografts with the copper-64 and gallium-68 derivatives.

Results/Discussion

Copper-64 was used to directly label the compounds to form a complex with an affinity of 60 nM (IC₅₀, determined by a calcium flux assay) and shown to be stable in serum and urine. In vitro uptake experiments show selective accumulation in cells overexpressing the CXCR4 receptor. The tumour-to-muscle ratio at 90 min post-injection in U87-CXCR4 tumours (23.6 ± 2.7) was 8-fold higher than that found for U87 tumours (3.0 ± 0.5). Blocking with 10 mg/kg of a higher affinity antagonist (IC₅₀ = 4 nM) shows a > 92% reduction of radioactivity in the U87.CXCR4 tumour.

Conclusions

To target CXCR4, a new configurationally restricted tetraazamacrocyclic CXCR4 antagonist was developed and radiolabelled with copper-64 and gallium-68. In vitro and in vivo data showed high specificity for the CXCR4 receptor. The significant reduction in liver uptake is consistent with high in vivo stability. Detailed assessment of a library of tracers is underway to identify the lead tracer for translational studies.

References

[1] F. Balkwill, Semin. Cancer Biol., 2004, 14, 171.

[2] J. Kuil et al. Chem. Soc. Rev., 2012, 41, 5239.

[3] R. Smith, et al., Dalton Trans., 2012, 41, 11369.

[4] A. Khan, et al., JACS, 2009[SA1] , 131, 3416.

[5] C. Lapa et al., Theranostics., 2017, 7(1), 205-212.

Acknowledgement

We gratefully acknowledge Yorkshire Cancer Research (Grant: HEND376), Dr Assem Allam and the Daisy Appeal Charity (Grant: DAhul0211) for funding.

Introduction

Management of IBD patients is often difficult due to poor correlation between clinical symptoms and diagnostic techniques. Several new biological therapies have been recently introduced, but not all patients seem to respond. Therefore, the prediction of the most appropriate therapy is still an open challenge. Molecular nuclear medicine can help in therapy decision-making by highlighting the presence or absence of specific therapeutic targets. To this aim we developed three radiopharmaceuticals to evaluate the expression of specific biomarkers and tested them in a DSS mouse model of IBD.

Methods

We labelled, with ^99mTc, three monoclonal antibodies (mAbs) that specifically bind TNFα, α₄β₇ integrin and F4/80. In vitro quality controls and binding assays were performed to assess labelling efficiency, stability and affinity for their targets. In vivo studies were performed in normal and DSS-treated mice to evaluate the uptake of these radiopharmaceuticals in inflamed intestine by planar and SPECT/CT imaging. After each scan, mice were sacrificed to perform ex-vivo studies and histology on excised organs. Ex vivo imaging of each colon was performed prior to cut them in 20 small rings and performing immunohistochemistry to evaluate biomarkers’ distribution.

Results/Discussion

After several titrations, all mAbs were radiolabelled with high labelling efficiency, stability and affinity for their target. In vivo, higher uptake of each radiopharmaceutical was observed in the colon of DSS mice as compared to controls. We also observed variable uptake, thus confirming the patchy nature of IBD as also observed by ex-vivo planar imaging of the whole colon and immunohistochemistry studies. Increased expression of TNFα and α₄β₇ integrin was observed from the mid-distal colon and proximal-mid colon respectively. Expression of F4/80 was evenly distributed among the different colon segments.

Conclusions

We were able to develop a panel of radiopharmaceuticals and perform an in vivo non-invasive evaluation of therapeutic biomarkers involved in IBD. This approach is feasible and studies are ongoing to evaluate drug efficacy in preclinical models. In the future, this strategy may be translated into humans for therapy decision-making and follow up of patients undergoing biological therapies, providing the use of humanized mAbs or Fab fragments.

References

Auletta S, Bonfiglio R, Schonberger T, Varani M, Galli F, Borri F, Scimeca M, Wunder A, Niessen HG, Signore A, Bonanno E. Animal models for studying inflammatory bowel diseases (IBD): a meta-analysis on modalities for imaging inflammatory lesions. Q J Nucl Med Mol Imaging 2018;62:78-100.

Acknowledgement

We acknowledge “Sapienza” University grants for funding.

Introduction

We recently reported [⁵²Mn]Mn(oxinate)₂ (t_1/2 = 5.6 d) as a cell/liposome labelling agent [1]. The neutral, lipophilic metal complex can passively cross lipid bilayers and release the radionuclide which is trapped by binding to intracellular macromolecules, or intraliposomal drugs [2]. Due to its promising liposome labelling properties, and the half-life of manganese-52 potentially allowing longer term tracking, we explored the cell-labelling ability of the metastable [⁵²Mn]Mn(oxinate)₂ complex in comparison with a stable lipophilic ⁵²Mn porphyrin complex, [⁵²Mn]Mn-PPh1.

Methods

[⁵²Mn]Mn(oxinate)₂ was synthesised as recently reported [1] and used to radiolabel various cell lines - including MDA-MB 231 breast cancer cells and gamma-delta T-cells - and the cell viability and cellular retention of the radiometal tested. [⁵²Mn]Mn-PPh1 was synthesized using a microwave reactor after 1 h at 165^oC with a radiolabeling yield of >98% (Figure 1A) and a preliminary comparison of its cell-labelling properties in comparison with  [⁵²Mn]Mn(oxinate)₂ was carried out using the MDA-MB 231 cell line. Additionally, the clinically-available nanomedicine DOXIL^® was radiolabelled using [⁵²Mn]Mn(oxinate)₂ and tracked in vivo. Healthy B6CBAF1 mice (n = 3) were injected with [⁵²Mn]Mn-DOXIL and imaged over 3 days, with ex vivo biodistribution performed 3 d post-injection.

Results/Discussion

[⁵²Mn]Mn(oxinate)₂ uptake in MDA-MB 231 cells was higher than with [⁵²Mn]Mn-PPh1 (42% and 12 %, respectively, Figure 1C). However, cellular retention of ⁵²Mn using oxine was three-fold lower than with the stable ⁵²Mn porphyrin complex (14% and 45% respectively, Figure 1D). PET images of [⁵²Mn]Mn-DOXIL at 1 h and 24 h post-injection showed a distribution consistent with previously imaged liposomal nanomedicines (Figure 2A). PET imaging and ex vivo biodistribution at 3 d post-injection showed a profile consistent with release of free manganese-52 (Figure 2A & B), which may be indicative of drug release after destruction of the liposomes.

Conclusions

Whilst the cell tracking ability of [⁵²Mn]Mn(oxinate)₂ is limited by its cellular retention, it may be used as a tool for studying intracellular manganese trafficking. [⁵²Mn]Mn-PPh1, shows promise as cell labelling agent. We also propose that [⁵²Mn]Mn(oxinate)₂ is an effective means to image liposomal drug delivery and release.^[2]

References

[1] Gawne P. et al., Dalton Trans. 2018, 47, 9283-9293

[2] Edmonds S. et al., ACS Nano. 2016, 10, 10294−10307

Introduction

Nanobodies are desirable probes for use in PET imaging, to overcome disadvantages of directly fluorinating proteins, ¹⁸F-labelled prosthetic groups are initially radiolabelled and utilized but have bioconjugation limitations [1,3]. [¹⁸F]FDR is a prosthetic group that has been successfully coupled to different peptides via oxime-bond formations [2]. It can be synthesised in automated PET synthesisers while linking to biomolecules requires shorter reaction times under milder conditions in comparison to [¹⁸F]FDG. In this project,  [¹⁸F]FDR-nanobodies are synthesiszed and characterised.

Methods

Nanobodies were functionalised with an optimal PEG linker that can exploit the aminooxy-carbonyl coupling. [¹⁸F]FDR was produced in an automated E&Z PET synthesiser according to a literature procedure at the John Mallard Scottish PET centre [2]. For radiolabelling of nanobodies, various reaction conditions (time, temperature, catalyst) were tested to obtain a suitable protocol. Radiolabelled nanobody was purified using a NAP5 cartridge and a 0.22 µm filter. This purified fluorinated nanobody, [¹⁸F]FDR-nanobody, was analysed by RP-HPLC, SEC and iTLC.

Results/Discussion

A preliminary [¹⁸F]FDR labelling protocol for nanobodies was successfully developed. Influence of the oxime catalyst (aniline) was evident since the conversion measured by RP-HPLC and SEC was ~85%, which is higher in comparison to conditions that lacked this. Our findings demonstrate the influence of temperature and reaction time [VC1] was marginal with conversion; increasing temperature and extending reaction time over 30 minutes showed little or no improvements. Radiochemical purity of the [¹⁸F]FDR-nanobody conjugate following purification was >98%.

Conclusions

Here we report the first example of radiolabelling of nanobodies with [¹⁸F]FDR. These preliminary results will be used to prepare [¹⁸F]FDR-nanobodies and evaluate their in vivo properties during studies that will comprise of stability tests, biodistribution evaluation in various animal models along with molecular imaging.

References

[1] Xavier, C. et al., Nucl Med Biol. 2016 Apr; 43(4): 247-52.

[2] Li, X.-G.; Dall'Angelo S. et al.,  Chem. Commun. 2012; 48(43): 5247–5249.

[3] Devoogdt, N et al., Methods Mol Biol. 2012; 911: 559-67

Acknowledgement

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska–Curie grant agreement No 675417.

Introduction

Nuclear Imaging techniques are particularly well-suited for in vivo tracking of intravenously injected cells, due to their high sensitivity, quantitative nature and whole-body imaging capability^1-3. Previously, we have developed PLGA-based nanoaprticles (PLGA-NPs), containing ¹⁹F for high resolution MR imaging and different fluorescent dyes for optical imaging. These nanoaprticles are suitable for clinical use⁴. Here we describe the modification of PLGA-NPs for labelling with ¹¹¹In to include SPECT in the multimodal tracking of cells in vivo.

Methods

PLGA-NP modified with a diamine cap (PLGA-AA) were conjugated with an excess DTPA. After washing, the NPs were labelled with 3 MBq/mg of [¹¹¹In]InCl₃ in 0.5 M NH₄Ac buffer. Radiochemical yield was measured using a dose calibrator, radiochemical purity with instant Thin Layer Chromatography (iTLC), and stability was assessed in PBS or 100% human serum (HS). A range of 0.1-50 mM EDTA was used for an overtime challange experiment at 37 C. Lymphocytes and monocytes were incubated with 1.6-1.7 MBq/mg [¹¹¹In]In-PLGA-AA overnight at culture conditions and labelling efficiency was measured in a dose calibrator. Cell viability after radiolabelling was measured using CellTiterGlo assay.

Results/Discussion

Three types of particles were made: PLGA-NPs (i.e. non-modified), PLGA-AA, and DTPA-PLGA-AA (PLGA-AA conjugated with DTPA). The results showed specific radiolabelling of PLGA-AA with ¹¹¹In (Fig. 1A). Labelling efficiency was 87%, 42%, and <10% for PLGA-AA, DTPA-PLGA-AA, and PLGA-NP respectively. ¹¹¹In retention was >90% up to 3 days in PBS and 100% serum (Fig. 1B). ¹¹¹In retention remained stable during EDTA challenge at low concentrations, but was released at higher concentrations (Fig. 1C). Labelling of lymphocytes and monocytes with a range of [¹¹¹In]In-PLGA-AA showed a labelling efficiency of 1.4-3.1%, with the monocytes having a higher labelling efficiency than lymphocytes (Fig. 2A). The specific activity increased with increasing concentration of particles: 10 mg PLGA-AA resulted in a specific activity of >0.8 MBq/10^6 cells and >3 MBq/10^6 cells for lymphocytes and monocytes, respectively (Fig. 2B). No variation in cell viability was seen between the groups (Fig. 2C).

Conclusions

We have shown efficient and specific labelling of PLGA-AA-NP with ¹¹¹In compared to normal PLGA-NP. Preliminary results using lymphocytes and monocytes indicate sufficient labelling efficiency for in vivo imaging. Importantly, overnight incubation of cells with high concentration of ¹¹¹In did not result in decreased cell viability. Therefore, we conclude that modified PLGA particles are suitable for cell labelling, in vivo work is in process.

References

1.        Srinivas, M., Heerschap, A., Ahrens, E. T., Figdor, C. G. & de Vries, I. J. M. 19F MRI for quantitative in vivo cell tracking. Trends Biotechnol. 28, 363–370 (2010).

2.        Srinivas, M. et al. Imaging of cellular therapies. Adv. Drug Deliv. Rev. 62, 1080–1093 (2010).

3.        Aarntzen, E. H. J. G. et al. In vivo imaging of therapy-induced anti-cancer immune responses in humans. Cell. Mol. Life Sci. 70, 2237–2257 (2013).

4.        Srinivas, M. et al. PLGA-encapsulated perfluorocarbon nanoparticles for simultaneous visualization of distinct cell populations by ¹⁹ F MRI. Nanomedicine 10, 2339–2348 (2015).

Acknowledgement

This work is supported by a Radboudumc PhD grant and the European Research Council (ERC) Starting Grant (CoNQUeST Grant no.336454) to MS.

Introduction

Gallium-68 (⁶⁸Ga) has been the subject of increasing interest for its potential in the production of radio-tracers for diagnosis of diseases. In this work we report the complexation of ⁶⁸Ga by acyclic chelates under a range of conditions with particular emphasis on the rapid complexation of ⁶⁸Ga at pH 7.4.

Typical gallium-68 radiolabelling conditions are harsh – with acidic pHs and high temperatures required for efficient radiolabelling of macrocyclic chelators such as DOTA.¹ This may be unsuitable for use with targeting motifs that are unstable under these conditions.

Methods

With a view to performing radiolabelling under milder conditions, at neutral pH and without heating, we have studied a number of acyclic chelate designs for gallium-68.

The tripodal chelate H₃Dpaa and derivatives, are made up of two picolinate arms coupled to an amino acid. This allows for bifunctional derivatives to be prepared by substitution of the central amino acid, yielding H₄Dpaa.ga, H₃Dpaa.dab, H₃Dpaa.lys.^2,3

Modifying this system to incorporate three picolinate arms around a central amine increased the number of potential coordinating atoms, and yielded the chelate H₃Tpaa.

A more flexible system, L₁, was also investigated. The effects of pH, time, temperature, and ligand concentration have been investigated. The biodistribution of this system has been investigated in vivo.

Results/Discussion

The Dpaa chelates could be radiolabelled across a wide pH range (Figure 1); with RCYs of 99% achieved at pH 4 with no heating and  95% achieved at pH 7.4 at 37 ^oC. Whilst this chelate design achieved efficient radiolabelling under mild conditions, the resulting ⁶⁸Ga complex was not stable when subjected to a serum challenge. Further investigation highlighted the presence of a bound water molecule, with only 5 of the coordination sites of Ga³⁺ being fulfilled by the ligands (Figure 1).

The crystal structure of [Ga(Tpaa)] confirmed the ligand saturated the coordination sphere of Ga³⁺ in a hexadentate manner (Figure 1). Radiolabelling of Tpaa with ⁶⁸Ga was achieved at pH 7 with a 99% RCY. The resulting radiolabelled complex was more stable to serum with 32% of the activity being retained after 30 minutes.⁴

The L₁ system has a pH dependent speciation (Figure 2), and the radiolabelled complex formed at pH 7.4 was shown to be stable in serum over 2 hours.

Conclusions

The acyclic chelate designs investigated here show the feasibility of radiolabelling with gallium-68 at high pH. Further development of these chelates will enable radiolabelling of pH sensitive targeting motifs, broadening the scope of gallium-68 imaging.

References

¹ T. W. Price, J. Greenman and G. J. Stasiuk, Dalton Trans., 2016, 45, 15702-15724

² T. W. Price, J. Gallo, V. Kubíček, Z. Böhmová, T. J. Prior, J. Greenman, P. Hermann and G. J. Stasiuk, Dalton Trans., 2017, 46, 16973-16982

³ S. Y. Yap, T. W. Price, H. Savoie, R. W. Boyle and G. J. Stasiuk, Chem. Comm., 2018, 54, 7952-7954

⁴ T. W. Price, T. J. Prior and G. J. Stasiuk, Dalton Trans., submitted

Acknowledgement

Funding for this work was obtained from the Royal Society (grant RG160156).Thanks to the EPSRC UK National Crystallography Service at the University of Southampton for the collection of crystallographic data.

Introduction

N-triphos derivatives (NP₃^R, R = alkyl, aryl), and asymmetric variants (NP₂^RX^R’, R’ = alkyl, aryl, X  = OH, SH, SR’’) represent an underexplored class of tuneable, tripodal ligands in the coordination chemistry of Re and Tc for biomedical applications.¹ Importantly, these ligands have the potential to form well-defined and isomer-free coordination spheres.^2,3 Our aim is to prepare a series of model complexes containing these tripodal phosphine ligands, and to assess their structure and reactivity prior to attempted translation to ^99mTc and ^186/188Re radiopharmaceutical formulation.  

Methods

NP₃^Ph and NP₂^PhOH^Ar are readily synthesised via phosphorus-based Mannich reactions.⁴ This reaction facilitates the introduction of functionalised secondary phosphines into an appropriate ligand scaffold. The reactivity between these ligands and a number of translationally relevant non-radioactive Re(I) and Re(V) precursors, including [NEt₄][ReBr₃(CO)₃], ReOCl₃(PPh₃)₂ and [NBu₄][ReOCl₄], has been investigated. The resultant complexes have been fully characterised using standard techniques (NMR, IR, mass spectrometry, and in many cases, X-ray crystallography) and present themselves as suitable model compounds for further investigation of ^99mTc and ^186/188Re complexes.

Results/Discussion

NP₃^Ph has been shown to form kinetically stable complexes exhibiting both bidentate and tridentate coordination to the fac-[Re(CO)₃]⁺ fragment, depending upon the synthetic conditions. However, these symmetric ligands exhibit only bidentate coordination in Re(V) complexes. The use of the asymmetric NP₂^PhOH^Ar ligand has been shown to facilitate tridentate coordination towards both fac-[Re(CO)₃]⁺ and [ReO]³⁺ units. The latter case provides the basis for a novel, reactive metal fragment for stabilisation of the [ReO]³⁺ core; [ReOCl₂(κ³-NP₂^PhO^Ar)] (Figure 1). The reactivity of this species towards further functionalisation with π-donors has also been explored. Furthermore, these chelators have the potential to be chemically modified to incorporate additional functionality that would enable them to act as bifunctional chelators.

Conclusions

Non-radioactive rhenium studies have illustrated the potential of these tripodal, N-centred phosphine ligands towards the stabilisation of Re cores in both +1 and +5 oxidation states in a series of model complexes. We are currently investigating these ligands for the synthesis and characterisation of isostructural ⁹⁹Tc, and ^99mTc, complexes and we are anticipating presenting this data in due course.

References

1           S. Liu, Adv. Drug Deliv. Rev., 2008, 60, 1347–1370.

2           A. Phanopoulos, A. J. P. White, N. J. Long and P. W. Miller, Dalton Trans., 2016, 45, 5536–5548.

3           S. L. Apps, A. J. P. White, P. W. Miller and N. J. Long, Dalton Trans., 2018, 47, 11386–11396.

4           B. Cao, M. R. J. Elsegood, N. Lastra-Calvo and M. B. Smith, J. Organomet. Chem., 2017, 853, 159–167.

Acknowledgement

This project is funded as part of the EPSRC Centre for Doctoral Training in Medical Imaging at Imperial College London and King’s College London.

Introduction

Nowadays, the combined use of photoacoustic/ultrasonographic techniques is increasing ^(1,2). Photoacoustics provides complementary information capable to identify a molecular behavior, otherwise unpredictable by US methods. This potential can be used in different bioimaging applications⁽³⁾, like the tracking, distribution or signal enhancing of nanoparticles. Here, we present a new kind of bioinspired nanoparticles, characterized to be made of a novel conjugation between melanin and silver nanoparticles, capable to improve the quality of PA images in terms of signal and contrast to noise ratio

Methods

This new design of photoacoustic (PA) nanoprobes, denoted MelaSil_Ag-NPs, is based on a tricomponent hybrid nanoplatform constructed by redox interaction of melanin silica (MelaSil), hybrid nanoparticles (NPs) and Ag+ ions. MelaSil_Ag-NPs are featured by embedded silver nanoclusters, appearing as punctate small vesicles distributed in the cytoplasm. We firstly characterized the MelaSil_Ag-NPs in test-object phantoms, in terms of spectral trend, photo-stability (PHSt) and linearity; after that, to obtain a similar human environment, we tested the MelaSil_Ag-NPs in biological samples of tissue got from chicken breast (ex-vivo tests).

Results/Discussion

MelaSil_Ag-NPs proved to be stable towards aggregation and efficiently endocytosed by human pancreatic cancer cells. The PA results obtained during test-object acquisitions are showed in Fig.1: we reported the nanoparticles PA spectral trend (Fig.1A), characterized to the presence of melanin, the PHSt exhibited under prolonged laser illumination (Fig.1B) and the linear matching between PA signal and concentrations (Fig.1C). The ex-vivo tests are shown in Fig.2, where we resumed the high quality of PA results obtained by the PA imaging of samples injected with a bolus of MelaSil_Ag-NPs. The spectral trend shows the correlation with test-object test (Fig.2A) and an impressive photo-stability of nanoparticles, when injected into the tissue. The trend during time of contrast-to-noise ratio was reported in Fig.2C and the related PA sample images (Fig. 2D,E). PA imaging matches finely the internal distribution of nanoparticles bolus visible in the section of the biological sample (Fig.2F).

Conclusions

The hybrid nature of the material allowed a fine control of morphology and stability to aggregation of the final systems ensuring cellular uptake. The results also suggest how the synergism between the silver-nanocluster and melanin is capable to generate a strong PA signal. The test-object and ex-vivo tests showed the NPs photo-stability, by which we obtained high value of CNR (up to 95), and the related fine pattern of internal distribution.

References

1. “Advanced Photoacoustic Imaging Applications of Near‐Infrared Absorbing Organic Nanoparticles”, Yuyan Jiang, Kanyi Pu, Small (13) (30), 2018, doi:10.1002/smll.201700710
2. “Strategies for non-invasive imaging of polymeric biomaterial in vascular tissue engineering and regenerative medicine using ultrasound and photoacoustic techniques“, C. Avigo, A. Flori, P. Armanetti, N. Di Lascio, C. Kusmic, J. Jose, P. Losi, G. Soldani, F. Faita, L. Menichetti, Polymer International , 2016
3. “Pattern of distribution and kinetics of accumulation of gold nanorods in mouse spleen”, N. Di Lascio; C. Avigo; P. Armanetti; F. Stea; L. Cavigli; F. Ratto; R. Pini; C. Kusmic; L. Menichetti; F. Faita, SPIE 9323, Photons Plus Ultrasound: Imaging and Sensing 2015, doi:10.1117/12.2077947

Acknowledgement

This work was supported by the University of Naples Federico II Research Grant (Finanziamento per la Ricerca di Ateneo, BioDressMel, DR/2017/408−07/02/2017)

Introduction

Indocyanine Green (ICG) has been extensively used in optical and photoacoustic (PAI) studies. ICG loaded mesoporous silica nanoparticles (ICG-MSNs) have shown enhanced PAI properties and increased stability.¹ Unfortunately, ICG is sequestered by human serum albumin (HSA), strongly hampering its in vivo application. Herein, ICG was covalently bound to MSNs surface to avoid its removal by HSA. Silica pores were loaded with the chemotherapeutic drug mitoxantrone (MTX). Chemical and PAI properties of this theranostic nanosystem were analysed and a preliminary in vivo study was carried out

Methods

MSNs were prepared through a sol-gel procedure and functionalized in the surface with aminopropyl groups and PEG molecules. In a second step, Indocyanine green NHS active ester was covalently attached to NH₂ groups of MSNs. Finally, mitoxantrone was confined in the pores by wet-impregnation. MSNs were fully characterized by UV-Visible and photoluminescence spectroscopy. PAI analysis was carried out by using a VisualSonics Vevo 2100 LAZR Instrument. For in vitro PAI, ICG-MTX-MSNs suspensions were loaded onto plastic capillaries surrounded by agarose gel. For in vivo PAI, balb/c mice bearing intramuscolar TS/A breast cancer cells were injected with 3 mg ICG-MTX-MSNs and PA images of tumour region were acquired.

Results/Discussion

ICG-MTX-MSNs have a radius of 120nm and pores of 2.0nm. 0.3µmol/g of ICG are conjugated on the surface and 22.6µmol/g of MTX are loaded into pores. The samples show UV-vis absorptions (π→π*) of both ICG and MTX in the 550-800 nm range and luminescence emissions at 690 and 810 nm.  PA analysis of ICG-MTX-MSNs reveals the presence of signals at 700nm (MTX) and 810nm (ICG)(Fig.1A). The drug release was measured by spectrophotometry and PAI. The PAI peak of MTX is strongly reduced 24h after MSNs dissolution (Fig.1B). Then, ICG-MTX-MSNs were i.v. administrated to cancer-bearing mice. In pre-contrast PA image, only endogenous haemoglobin signal is present (Fig. 2B). In post contrast PA image, the presence of ICG-MTX-MSNs is detectable in the tumour. This can be visualizable in PA spectra of a selected ROI, where both PA signals of MTX(700 nm) and of ICG(810 nm) are present (Fig. 2B).After 30min, the signal of MTX is significantly reduced, whereas ICG signal in only slightly reduced (Fig. 2B)

Conclusions

ICG-MTX-MSNs are a truly promising theranostic PAI probe. The covalent binding of ICG onto MSNs surface prevents sequestering from albumin thus increasing bioavailability of the probe.  The release of the drug MTX can be monitored by PAI. This system can be employed in vivo, and PA modality can report on the distribution of the nanosystem and, simultaneously, on the release of the drug.

References

Ferrauto G. et al. Nanoscale 2017 Jan 7;9(1):99-103.

Introduction

The cytotoxic activity of CD8+ T cells is a critical component of the human adaptive immune system, which is dysregulated in most tumours. There is a lack of functional probes to measure CD8+ T cell activity in tumours and to identify combinations of therapies that can effectively reprogram their cytotoxic potential. We have created novel small molecule fluorescent probes targeting granzyme B to measure CD8+ T cell activity in cancer as biomarkers to monitor the effect of pharmaceutical compounds on stimulating the immune response.

Methods

Probe synthesis: We generated a library of activity-based probes through a combination of solid-phase and solution-phase coupling reactions. Kinetic Assays: Fluorescence amplification was monitored by incubating the probes with granzyme B, granzyme A and caspases to identify JIS-2 as a new granzyme B-specific fluorescent marker. Cancer/T cell co-cultures: Murine CD8+ T cells were isolated and cultured for 2 days before co-culture with E0771-LG2 cancer cells for 2 additional days. CD8+ T cells were then either activated or not and treated with JIS-2 before imaging by confocal microscopy under live-cell conditions (Figure 1). We also used JIS-2 in high-throughput phenotypic screenings as well as in vivo in mouse cancer models and tumour biopsies.

Results/Discussion

We assessed the cytotoxicity of CD8+ T cells against cancer cells through direct imaging of the activity of granzyme B, a serine protease that acts as a key mediator of their cytotoxic potential. From a small library of activatable fluorescent probes, we identified JIS-2 as a specific activity-based probe for granzyme B with 150-fold fluoresence fold increase upon rapid reaction with the enzyme. We have employed JIS-2 in a cancer killing assay where CD8+ T cells were incubated with E0771-LG2 cancer cells and activated by addition of IL-2. Images revealed that JIS-2 was able to detect active granzyme B inside cancer cells upon CD8+ T cell stimulation. The selectivity of the probe was confirmed by co-incubation with granzyme B inhibitors which led to reduced fluorescence signals. JIS-2 can be employed as a fluorescent biomarker of T cell activity in high-throughput screenings for drug libraries, in vivo imaging and ex vivo analysis of tumour biopsies.

Conclusions

We have developed a new smart functional probe for monitoring the activity of granzyme B in tumours. JIS-2 rapidly and selectively reacts with granzyme B to produce a remarkable 150-fold fluorescence increase in emission. The utility of JIS-2 for in vitro cancer-T cell co-cultures as well as for in vivo imaging will be discussed. 

Acknowledgement

J.I.S acknowledges funding from the EPSRC and MRC Centre for Doctoral Training in Optical Medical Imaging and the Scottish Funding Council (H14052).

Introduction

Ultrasound imaging (US) is one of the most widespread imaging modalities. Microbubbles (MB) with a size of 1-10 µm are routinely used contrast agents for US with a gas core-stabilizing shell based on lipids or polymers¹. The thick shell of polymeric MB enables utilization as a platform technology for functional and molecular US imaging, as well as for direct and indirect drug delivery via sonopermeation (Figure 1). Here we demonstrate the suitability of poly n-butylcyanoacrylate (PBCA) MB for multiple biomedical applications, including US imaging and US-mediated drug delivery.

Methods

PBCA MB are prepared by vigorous stirring of an aqueous solution of the monomer n-butyl cyanoacrylate (BCA) with 1% Triton-X at pH 2.5. After in vivo TNF-α stimulation, the murine carotid artery was explanted for ex vivo 3D two-photon laser scanning microscopy (3D-2PM) of endothelial cells and rhodamine-labeled VCAM-1-targeted MB. Tumors were induced by s.c. inoculation of A431 and CT26 cells in mice. US of tumor microvasculature was performed with antibody-labeled VEGFR-2-targeted and peptide-labeled E-selectin-targeted MB. Sonopermeation-enhanced drug delivery across the blood brain-barrier and the tumor vasculature was evaluated by 3D-2PM and 2D fluorescence microscopy.

Results/Discussion

We could demonstrate retention of VCAM-1-targeted MB binding to stimulated murine carotid arteries and for E-selectin-targeted MB retention to the tumor vasculature in A431 epidermoid carcinomas (Figure 1). Via sonopermeation, MB are useful for both indirect drug delivery (in which the (model) drug is co-injected with MB) and for direct drug delivery (in which the (model) drug is incorporated within the MB shell). We could show the accumulation and penetration of FITC-dextran across the murine blood-brain barrier upon transcranial US-induced MB destruction (Figure 1). In CT26 colon carcinoma tumors, US-mediated MB destruction of VEGFR-2-targeted and coumarin 6-loaded MB enhanced model drug (coumarin 6) delivery to tumor tissue². These findings exemplify the suitability of PBCA-based polymeric MB for molecular imaging in oncology and angiology, and for US-enhanced drug delivery to tumors and to the brain.

Conclusions

Model drug molecules can be efficiently loaded into the shell of PBCA MB. In addition, targeting of inflamed and angiogenic blood vessels is demonstrated. Drug delivery across the vascular wall into the brain and tumor tissue is confirmed. These results are in line with recently published proof-of-principle studies showing the usefulness of phospholipid-based MB for molecular US imaging and US-enhanced drug delivery in patients³.

References

1          Appold L, et al. Macromol Biosci. 2017;17(10).  

2          Koczera P, et al. J Control Release. 2017;259:128-135.

3          Dimcevski G, et al. J Control Release. 2016;243:172-181.

Acknowledgement

This work was financially supported by the ERC (starting grant 309495: NeoNaNo), the DFG (La2937/1-2) and by the i³tm Seed Fund Program (SF_14-4-09-Sonoporation). 

Introduction

Abdominal aortic aneurysm (AAA) is a major health concern, carrying an astonishingly high acute mortality rate. Current condition monitoring for high-risk patients is ultrasound imaging, without secondary prevention therapies available. Limited high-risk surgical interventions are available for severe cases.

Methods

Targeted microbubbles for the delivery of microRNA-126 (miR126) mimics downregulate vascular cell adhesion molecule-1 (VCAM-1) expression, rectify endothelial inflammation and ameliorate the development of abdominal aortic aneurysm.

Results/Discussion

Using the angiotensin II induced model of murine AAA, we observed that targeted delivery of mimic-miR126 hinder the dilation of the abdominal aorta both before (1.12 ± 0.11 vs. 1.36 ± 0.12; mm mean ± SD; p>0.05; N=5-9) and after (0.88 ± 0.06 vs. 1.05 ± 0.05; p>0.001; N=5-9) the renal arteries. Further confirmation using 3D ultrasound vessel reconstructions, immunohistological examination, and gene expression analyses (p>0.01; N=5-9) confer an overall decrease in VCAM-1 expression and aneurysm severity.

Conclusions

We describe a novel theranostic approach towards effective reestablishment of intravascular endothelial homeostasis under pro-inflammatory conditions. This technology holds immense potential for a safe, non-invasive, targeted treatment towards conditions driven by inflammation of the endothelial, including AAA.

Introduction

Low optical absorption and rapid clearance of small molecule dyes impose considerable limitations for contrast enhanced in vivo optoacoustic imaging (OAI). Using DNA nanotechnology it is possible to precisely position individual dye molecules to enhance the optoacoustic (OA) signal generation capabilities and also prolong the blood retention time of the contrast agent in vivo. Here, we report the synthesis, characterization and in vivo imaging of DNA nanotechnology-derived nano-carriers (NCs) that exhibit superior blood retention time, tumor accumulation and OA signal generation capabilities.

Methods

DNA NCs having different structural designs, sizes and inter dye (IRDye 800CW) separation distances were synthesized and characterized.  Structural and optical characterizations were performed to evaluate the stability and optical response of the NCs. Cell viability of the NCs was studied using the MTT assay. Multispectral optoacoustic tomography (MSOT) studies were performed using phantoms to evaluate the OA response of NCs and free dye. In-vivo fluorescence imaging and MSOT studies were performed in healthy and tumor bearing mice to evaluate the bio-distribution, clearance and tumor uptake. investigated These parameters were investigated on two controls: the free dye and NC2Q+, which is a single double stranded DNA helix 21 nucleotides-long bearing two molecules of dye.

Results/Discussion

The nano-carrier (NC6Q+) comprised of closely positioned IRDye 800CW molecules offers good serum stability and biocompatibility. Absorption and emission measurements show that NC6Q+ exhibits a blue shifted absorption peak at 705nm due to exciton coupling and offers more than 80% fluorescence quenching efficiency. As a result, OAI data from NC6Q+ in tissue mimicking phantoms show a 72% enhancement in OA signal when compared to free IRDye 800CW. In vivo bio-distribution (n=2) and kinetics studies of the nano-carriers indicate 87 % increase in blood retention time for NC6Q+  when compared to free dye, while maintaining renal clearance. Multispectral optoacoustic imaging of tumor bearing mice (n=3) showed 88% enhancement in tumor PA signal for NC6Q+ when compared to free IRDye 800CW. These results suggest that DNA nano-carriers hold promise to create PAI contrast agents with enhanced tumor uptake and PA signal generation capabilities.

Conclusions

We demonstrate that DNA nanotechnology-derived nanocarriers can be used to generate NCs that successfully enhance the OA signal by liaising pairs of the IRDye 800CW, thereby presenting a way of straight-forward fabrication of biocompatible contrast agents for in vivo OAI. Size dependent bio-distribution and clearance of contrast agents was studeid using MSOT. Our studies also show enhancements in tumor uptake of contrast agents.

Acknowledgement

This work was funded by a Cancer Research UK Cambridge Centre Pump Prime Research Grant. K. N. B.  acknowledge the ERASMUS placement organization for ERASMUS+ funding.  K. N. B.  acknowledges the DAAD for a PROMOS scholarship. JJ and SEB are funded by the EPSRC-CRUK Cancer Imaging Centre in Cambridge and Manchester (C197/A16465); CRUK (C14303/A17197, C47594/A16267) and the EU-FP7-agreement FP7-PEOPLE-2013-CIG-630729. S.H.A. acknowledges funding by the University of Zaragoza (UZ2018-CIE-04) and the support of ARAID.

Introduction

Optical biosensing based on affinity or activity of fluorescent reporters¹ offers a powerful methodology for the real-time molecular interrogation of disease.² Here we report on the recent progress in fluorescent probes for sensing Thrombin and MMP activities with a number of fluorophores and quenchers.³ The design, synthesis, in vitro and ex vivo validation of the sensors are herein reported as a step towards their application in the direct analysis of lung fibrosis using fibre-based, dual colour optical microendoscopy technology.⁴

Methods

FRET-peptides were synthesised by Fmoc solid-phase synthesis with multiple fluorophores and quenchers incorporated into the peptides. Enzyme specificity was measured as a fluorescence-fold increase with the probes and analysed with target/off-targeted enzymes and inhibitors. Ex vivo validation was carried out using diseased human lung tissue.

Results/Discussion

The probes were designed to monitor, at two distant wavelengths, extracellular MMPs and thrombin in the lung providing an OFF/ON fluorescent signal. Probes were tuned for optimal signal outputs, solubility and specificity. When human tissue samples were treated with these probes a significant increase in fluorescence was observed, with this rise suppressed by known inhibitors.

Conclusions

A series of activatable fluorescent proteases reporters were synthesised and evaluated. They allowed the rapid, specific and sensitive analysis for key enzymes involved in fibrosis and are suitable for the detection of enzymatic activity in lung tissue.

References

1. H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke and Y. Urano, Chem Rev, 2010, 110, 2620.

2. A. R. Akram, S. V. Chankeshwara, E. Scholefield, T. Aslam, N. McDonald, A. Megia-Fernandez, A. Marshall, B. Mills, N. Avlonitis, T. H. Craven, A. M. Smyth, D. S. Collie, C. Gray, N. Hirani, A. T. Hill, J. R. Govan, T. Walsh, C. Haslett, M. Bradley and K. Dhaliwal, Sci Transl Med, 2018, 10, eaal0033.

3. a) A. Megia-Fernandez, B. Mills, C. Michels, S. V. Chankeshwara, K. Dhaliwal and M. Bradley, Org Biomol Chem, 2017, 15, 4344.

b) A. Megia-Fernandez, B. Mills, C. Michels, S. V. Chankeshwara, N. Krstajić, C. Haslett, K. Dhaliwal and M. Bradley, Org Biomol Chem, 2018, 16, 8056.

4. N. Krstajić, B. Mills, I. Murray, A. Marshall, D. Norberg, T. H. Craven, P. Emanuel, T. R. Choudhary, G. O. S. Williams, E. Scholefield, A. R. Akram, A. Davie, N. Hirani, A. Bruce, A. Moore, M. Bradley and K. Dhaliwal, J Biomed Opt, 2018, 23, 076005.

Acknowledgement

EPSRC, Interdisciplinary Research Collaboration grant EP/K03197X/1 and Medical Research Council (under the Developmental Pathway Funding Scheme grant number MR/J014702/1) for funding this work.

Introduction

Among the various molecular imaging approaches, optical imaging has recently gained interest, especially with the rise of fluorescence guided surgery. However, because of its low tissue penetration it is necessary to use NIR fluorophores, which present superior resolution, tissue penetration, and lower background signal. Currently, the only clinically approved NIR probe is ICG, and many of the emerging fluorophores suffer from high hydrophobicity or poor fluorescence. There is a crucial need for the development of NIR probes with better biocompatibility and high quantum yield.   

Methods

Aza-BODIPY dyes were synthetized and different functional groups were introduced on the boron atom, such as hydrosolubilizing groups and bioconjugatable handles. One water soluble aza-BODIPY (Wazaby) was bioconjugated to anti PD-L1 antibody. The purity and stability of the resulting bioconjugate was evaluated by electrophoresis. After checking that Wazaby derivatives did not display significant cytotoxicity on cells, the ability of this molecule to target PD-L1 expressing tumors was investigated in vivo in a model of colon cancer based on the subcutaneous xenograft of CT26 tumor cells in syngenic BALB/c mice. Whole-body fluorescence images were recorded with an IVIS Lumina III at different time points (1, 6, 24 and 48 h). The organs biodistribution was evaluated 48 h post-injection. 

Results/Discussion

In this multidisciplinary project, we designed, synthetized, and characterized new water-soluble aza BODIPY dyes, obtained by functionalization of the boron atom with various hydrosolubilizing groups.

The photophysical properties were evaluated, revealing high fluorescence of the probes. One of these dyes was chosen for further imaging studies. The azaBODIPY was bioconjugated to an anti-PD-L1 antibody, and the polyacrylamine gel electrophoresis studies showed high purity and stability of the resulting bioconjugate. The high affinity of the bioconjugate for PD-L1 was assessed by flux cytometry.

The biodistribution was studied using non-invasive optical imaging on CT26 bearing tumor mice. The fluorescent aza-BODIPY probe emitted an intense signal in vivo, therefore validating its use as optical imaging probe. Additionally, an uptake of the bioconjugate anti PD-L1 at the tumor site could be observed, with a maximum at 24 h post injection.

Conclusions

In conclusion, a new family of highly efficient water-soluble aza-BODIPY dyes was developed, by boron functionalization of the aza-BODIPY. They have been proved to be very promising imaging probes for optical molecular imaging applications, and represent a robust alternative to the classically used cyanine dyes for molecular imaging but also for fluorescence image-guided surgery.

References

[1] Daly, H.C.; Sampedro,G.; Bon, C.; Wu, D.; Ismail ,G.; Cahill, R.A.;  O’Shea, D.F. Eur. J. Med. Chem, 2017, 135, 392-400.

[2] James, M.L.; Gambhir, S.S. Physiol. Rev. 2012, 92, 897-965.

[3] Ge, Y.; O Shea, D.F.; Chem.Soc.Rev., 2016, 45, 3846-3864.

[4] Kamkaew, A.; Burgess, K. Chemical Commun. 2015, 51, 10664-10667.

[5] Bodio, E.; Goze, C.; Dyes Pigm. 2019, 160, 700-710.

Acknowledgement

Support was provided by the Conseil Régional de Bourgogne (PhD JCE grant # 2015-9205AAO033S04139 / BG0003203), and the Conseil Régional de Bourgogne Franche-Comté. The Ministère de l’Enseignement Supérieur et de la Recherche, the Centre National de la Recherche Scientifique (CNRS), and the French Research National Agency (ANR) via project JCJC ‘‘SPID” ANR-16-CE07-0020 and project JCJC “WazaBY” ANR-18-CE18-0012-01 are gratefully acknowledged. Pr. Anthony Romieu and Iris Biotech are warmly thanked for the TOTA and the Cy5 synthesis, and Dr. Victor Goncalves for the diethylsquarate. This work is part of the projects ‘‘Pharmacoimagerie et agents théranostiques” et ‘‘Chimie durable, environnement et agroalimentaire” supported by the Université de Bourgogne and the Conseil Régional de Bourgogne through the Plan d’Actions Régional pour l’Innovation (PARI) and the European Union through the PO FEDER-FSE Bourgogne 2014/2020 programs. FrenchBIC and GDR AIM are acknowledged for fruitful discussion. Oncodesign® is acknowledged for fruitful discussion and for their support.

Introduction

Albumin is playing an important role as a drug carrier in clinical settings. To improve target-to-background (TBR) ratios of small molecular imaging probes in target tissue we intended to prolong the blood half-life by exploiting albumin-based transport mechanisms. The endothelin-(ET-)axis plays a crucial role in cancer progression. Especially, signalling of ET-1 via the endothelin-A-receptor (ET_AR) has been related to adverse effects. We modified a small molecule fluorescent probe directed towards ET_AR by albumin-binding moieties to evaluate receptor status in a murine xenograft model.

Methods

Different hydrophobic albumin-binding moieties (tags) were attached to an existing fluorescent probe targeted to ET_AR [1]. Evaluation of albumin-binding was first carried out in vitro by agarose gel electrophoresis and photometry. Target binding was proved by cell binding assays. In nude mice, HT-1080 fibrosarcoma xenografts were established by subcutaneous injection of 1-2 x10⁶ cells into breast tissue. Imaging experiments were started when tumors reached 4-5mm diameter. The probe was injected via the tail vein (2nmol/animal) and fluorescence reflectance imaging (FRI) was used to detect probe accumulation in tumor tissue.

Results/Discussion

In vitro, the modified probes retain their high target binding on cells. Specificity could be proven by predosing with atrasentan. In solution, the probe showed a significant shift of the absorbtion and emission maxima in the presence of albumin and electrophoretic evaluation showed fluorescence signal in the protein band. Longitudinal studies in murine xenograft models revealed distinct accumulation profiles of the different tags. In comparison to the established ET_AR probe we observed a reduced elimination and a prolonged circulation of the probes resulting in higher signal intensity and a higher TBR between 6h and 96h after injection [2].

Conclusions

The reversible binding to albumin enhances the biological half-life of the probes substantially and enables near infrared optical imaging of subcutaneous tumors for several days. This approach of reversibly attaching probes to serum albumin may serve as a tool to optimize tracer distribution for more precise target characterization and an improved TBR in molecular imaging experiments.

References

[1] Höltke C. et al A fluorescent photoprobe for the imaging of endothelin receptors, Bioconjug. Chem. 2007 18(3):685-694.

[2] Hahnenkamp A. et al. Optimizing the bioavailability of small molecular optical imaging probes by conjugation to an albumin affinity tag, J Control Release 186 (2014) 32–40.

Introduction

Near-infrared (NIR) fluorescence imaging has been applied in oncology to aid in tumor detection, delineation and resection. Visible light imaging is used in a majority of diagnosis, disease staging and treatment procedures in the field of gastroenterology. Therefore, the application of NIR imaging in inflammatory bowel disease (IBD) could be of benefit to expand the current visual imaging procedures and get insight in drug distribution and therapy efficacy. In order to achieve this, an IBD-specific tracer was developed.

Methods

Near-infrared fluorescent dye IRDye 800CW was conjugated to the monoclonal antibody vedolizumab, which is licenced for use in IBD patients. Critical process parameters to obtain a stable labelling of high purity were investigated, and possibilities for production of a drug product of sufficient purity for use in phase I clinical trials were investigated. Stability of the drug product designs were tested using SE-HPLC to assess protein stability and indirect ELISA assay for binding capacity.

Results/Discussion

IRDye 800CW was conjugated to vedolizumab. Purification by gel-filtraton resulted in very low levels of impurities in the resulting vedolizumab-800CW. Dye-to-protein ratio (D/P) was optimal when below a molar ratio of 4:1, as higher ratio’s resulted in inefficient conjugations and quenching of the fluorescent signal. A shift in conditions for optimal stability was seen between the original product and the conjugated product. The protein component of vedolizumab-800CW appeared stable in all labelling experiments. When increasing the D/P, a linear relationship between D/P and both absorbance and fluorescence could be seen for D/P of no more than 4. When D/P exceeded 8:1, both absorbance and fluorescence signals showed plateauing and subsequent decrease of the signal. This indicates a limit for feasible tracer production. In a panel of buffers to investigate stability, a shift in stability could be seen from the original formulation to a formulation with higher pH and salt levels.

Conclusions

Initial development of vedolizumab-800CW shows favourable results for further development of this tracer to a “for human use” product. The purified product showed low levels of impurities, excellent integrity, and initial stability data suggest compatibility with a formulation fit for human application. Additional stability studies and scale-up to GMP processes will be required to finalize product development for phase 1 trials.

Introduction

Tissue hypoxia occurs when oxygen supply in insufficient to meet physiological demands, leading to changes in gene expression. Due to the irregular vasculature of tumors, hypoxia is a characteristic of the tumor microenvironment.¹ Because it has been corelated with tumor stage and treatment resistance, detecting hypoxia has implications in both treatment planning and predicting patient prognosis.² We have developed a series of small-molecule Hypoxia Probes (HyP) for photoacoustic (PA) imaging. These probes feature a novel N-oxide molecular trigger that responds selectively to hypoxia in vivo.

Methods

The HyP series design utilizes the near-infrared aza-BODIPY dye platform. The probes are equipped with an N-oxide that is bioreduced in the absence of oxygen by heme proteins such as CYP450 enzymes. Reduction produces a spectrally distinct product, facilitating detection via photoacoustic imaging. HyP-1, the first probe in the series, is also compatible with ratiometric fluorescence imaging. HyP-1 was fully characterized in vitro, in hypoxic cell culture, and in two in vivo models of hypoxia to determine the capabilities of the N-oxide trigger. To improve the photophysical properties and enable ratiometric PA imaging, chemical tuning was employed to shift the absorbance further into the NIR. Additionally, a variety of N-oxide analogs were synthesized and evaluated in vitro and in vivo. 

Results/Discussion

Through a series of selectivity experiments in vitro and in cell culture, the N-oxide trigger of HyP-1 was found to be highly specific for activation by heme proteins under hypoxic conditions. The trigger was stable to a variety of common biological reductants and metal ions, and inhibition experiments in cell culture verified that activation occurs via heme proteins. The probe produced a PA response to tumor hypoxia in 4T1 mammary carcinoma allografts following systemic injection, and was able to respond to rapidly-developing hypoxia in a surgically-induced hind limb ischemia model. These results were confirmed using NIR ratiometric fluorescence imaging. Modifications to HyP-1 produced red-shifted, ratiometric probes (rHyPs) with enhanced PA signals, and enabled evaluation of N-oxide probes with various chemical substitutions. Ratiometric PA imaging with the optimal probe in this series enabled visualization of specific hypoxic regions within tumor tissue.

Conclusions

HyP-1 represents the first hypoxia-responsive, small-molecule probe designed for PA imaging. The combination of PA imaging with activatable probes enables high-resolution molecular imaging in deep tissue using non-ionizing light. This work outlines key design principles for the development of small-molecule PA probes, and future studies will be aimed at the application of these probes for tumor staging and predicting treatment responses.

References

(1)           McKeown, S. R. Defining Normoxia, Physoxia and Hypoxia in Tumours—Implications for Treatment Response. Br. J. Radiol. 2014, 87, 20130676.

(2)           Rankin, E. B.; Giaccia, A. J. Hypoxic Control of Metastasis. Science (80-. ). 2016, 352, 175–180.

Acknowledgement

This work was supported by the Alfred P. Sloan Fellowship (FG-2017-8964 to J.C.) and the National Science Foundation (CHE 1752879 to J.C.). Funding for the 500 MHz Bruker CryoProbe was provided to the School of Chemical Sciences NMR Lab by the Roy J. Carver Charitable trust (Muscatine, Iowa; Grant #15-4521). Purchase of the Q-Tof Ultima mass spectrometer was made possible by a grant from the National Science Foundation, Division of Biological Infrastructure (DBI-0100085). H.J.K and J.H. were supported by fellowships from the Beckman Institute (UIUC) and the Tissue Microenvironment Training Grant (T32 EB019944).

Introduction

Optoacoustic imaging offers high spatial resolution and penetration depth well beyond the conventional optical techniques, advantages that could be favorable for a variety of use. However, similar to PET and optical fluorescence imaging, exogenous contrasts agents (sonophores), need to be developed for molecular targeting. Despite some contrast agents having been reported, more rational development of sonophores needs to be performed (1). Here, we systematically evaluated twelve commercially available near infrared and high absorbing dyes for multi-spectral optoacoustic tomography (MSOT).

Methods

We have formulated the near-infrared dyes in nanoemulsions (NE) adapting a previously described method (2). Size, zeta potential, stability, dye encapsulation, loading efficiency and photophysical properties were evaluated. Optical and optoacoustic intensities as a function of concentration using standardized approaches were measured. Using the optoacoustic signal intensities directly measured from the phantom experiments as our reference, a direct classical least-square (DCLS) approach was implemented (3). The mouse breast cancer cell line 4T1 was used for in-vivo mouse experiments. In-vivo MSOT imaging was performed before and after (24 h) intravenous injection of sonophores.

Results/Discussion

The NEs had a mean effective diameter of 130-160 nm that form a more stable, biocompatible optoacoustic agent as compared to its native dye form. Comparisons made between absorbers reveal compelling evidence for higher optoacoustic signals with dark quenchers as opposed to weakly fluorescent dyes. Using the most promising near-infrared NE, we have confirmed in in vivo 4T1 tumor model experiments using MSOT that the dark quencher IRDyeQC-1 COOH is a highly potent tumor targeting candidate when encapsulated in a biocompatible (O/W) NE system. IRDye QC-1 carboxylic acid is non-fluorescent but exhibits a high extinction coefficient and broad near infrared UV/Vis and optoacoustic spectrum, all favorable characteristics of an optoacoustic agent.

Conclusions

MSOT in combination with our NE system enables non-invasive detection of tumors. Specifically, NE IRDye QC1 offers a superior optoacoustic performance and detection compared to related near-infrared NEs. Using a library-screening approach, we have demonstrated that NEs when loaded with dark quenchers represent a flexible and new class of exogenous sonophores suitable for non-invasive pre-clinical optoacoustic imaging.

References

(1)Weber, J. et al., Nature Methods, 2016 (2)Pérez-Medina, C. et al., Nat. Commun., 2016. (3) Tzoumas, S. et al., Nat. Commun., 2016

Acknowledgement

The authors thank the support of MSKCC’s Animal Imaging Core, Radiochemistry & Molecular Imaging Probes Core and Molecular Cytology Facilities. This work was supported by NIH grants R01 HL125703 (T.R.), R01 CA204441 (T.R.), R01 CA214820 (T.R.), R21CA191679 (T.R.), P30 CA008748, NIH R01 EB017748 (M.F.K.) and K08 CA16396 (M.F.K.); M.F.K. is a Damon Runyon-Rachleff Innovator supported (in part) by the Damon Runyon Cancer Research Foundation (DRR-29-14). 

Introduction

Gold nanoclusters (Au NCs) are atomically precise nanoparticles, composed of few to hundred atoms.¹ Due to their ultra-small core size (below 2 nm), surface easy to functionalize and fluorescence in the near-infrared region, they have been recently developed for tumor imaging.² One of the main drawback is related to their low uptake efficiency.³ We therefore developed a set of Au NCs functionalized with glutathione modified arginine peptides, often used as cell-penetrating peptides,⁴ to favour their tumor internalization.

Methods

Au NCs stabilized by glutathione and arginine modified-glutathione peptides were prepared by wet chemistry. First, the optical and physio-chemical properties of these Au NCs were characterized by agarose gel, zeta potential and fluorescence spectrometry. Then, particle uptake (30 min at 37°C with 10, 25 and 50 μgAu/mL) in human melanoma cell line (Colo 829) was evaluated by flow cytometry and by confocal microscopy. Finally, pharmaco-kinetic and biodistribution of these Au NCs were investigated on healthy nude mice after intravenously injection (200 μL, 2.5 μgAu/mL).

Results/Discussion

Characterizations confirmed the ultra-small size and fluorescence properties of these particles with a maximum of emission around 680 nm (λ_exc~450 nm). In vitro studies showed a stronger cell accumulation of Au NCs containing arginine than with only glutathione. The first in vivo experiment has confirmed the optical detection of these Au NCs in the near-infrared and their fast renal elimination without major toxicity.

Conclusions

At this stage, we believe Arginine stabilized Au NCs could be an interesting nanosystems for cancer diagnostics and therapy.

References

1- J. Chemical Reviews, Atomically Precise Colloidal Metal Nanoclusters and Nanoparticles: Fundamentals and Opportunities 116, 10346-10413 (2016).

2- D. S. APL Materials, Zwitterion functionalized gold nanoclusters for multimodal near infrared fluorescence and photoacoustic imaging 5, 053404 (2017).

3- L. Z. Nano Today, Metal nanoclusters: New fluorescent probes for sensors and bioimaging 9, 132–157 (2014).

4- Nanoscale, Promoting siRNA delivery via enhanced cellular uptake using an arginine-decorated amphiphilic dendrimer 9, 3867-3875 (2015).

Acknowledgement

I acknowledge the Auvergne-Rhône-Alpes region for the financial support of EP (ARC1 Santé fellowship 2016) and CLARA-Oncostarter (CVPPRCAN000162).

Introduction

Firefly luciferases (Flucs), both wild-type and engineered - are widely utilised for BLI in biomedical research, to track cells, molecules or proteins in living moving animals. Fluc genetic labels are more sensitive and less invasive than fluorescent proteins, which often require imaging of tissues ex vivo. However, immunogenicity of bioluminescent insect enzymes leads to variability and limitations in the scope and duration of experiments that can be performed. Thus, in BLI cohort sizes must be large partly to counteract such statistical uncertainties, which raises ethical concerns.

Methods

Enzymes were tested as pure proteins prior to human codon optimisation and cloning of gblocks gene fragments into pCCL lentiviral vectors co-expressing EGFP via internal ribosome entry sites (IRES). Human embryonic kidney (HEK) 293 cells were transfected with transfer vectors using Genejuice and 48h later BLI in PhotonIMAGER Optima with addition of 1mM D-LH2 potassium (K+) salt in phosphate buffered saline (PBS). Flow cytometric analyses and sorting were carried out using Fortessa and FACS Aria Fusion instruments, respectively. Retrovirus was produced by co-transfection of HEK 293 cells with transfer vectors and packaging vectors purchased from Addgene to make virus, then harvested, filtered and used for transduction of HEK cells using 2uL/mL of media and 10mg/mL hexadimethrine bromide.

Results/Discussion

The PhD is on-going but we have generated results that demonstrate the efficacy of our approach by creating human codon optimised insect-mammal protein hybrid reporters (Fluc and Mus musculus “unnamed protein” enzyme hybrids) which have reached ~20% mouse protein and allow high sensitivity in BLI in vivo but are better expressed in human cells than firefly luciferase. We display pilot in vivo data in Figure 2. Remarkably, even after normalisation of the expression levels of the new reporters and Photinus pyralis (Ppy) Fluc by flow cytometric cell sorting, in HEK 293 xenograft models in NOD/SCID/gamma (NSG) mice our brightest variant MusLuc (n = 6) gave 76% of the in vivo light yield of Ppy Fluc (n = 3) and was c.20nm redshifted, advantageous for bioimaging due to less scatter, or absorption by haemoglobin (Hb) in mammalian tissues. We are currently testing the effect on the immune response in BALB/c mice using a mouse 4T1 cell line model and testing effects on silencing in stem cells.

Conclusions

This work represents a new approach to reporter engineering: replacing redundant segments of ectopically expressed reporters with homologous functional sequences from target organism as a novel initiative for biocompatibility. Any sensitive reporter that is highly homologous to a native cellular mammalian (particularly mouse) protein is likely to be of great benefit to research using small animals.

References

1. Mol. Cell Biol. 7: 725-37. 2. Bioorg. Chem. 1: 92-122. 3. Biochem. J. 397: 305-12. 4. Anal. Biochem. 361: 253–62. 5. In Biolumin.—Recent Adv. Ocean. Meas. Lab. Appl. (D. Lapota). InTech: DOI: 10.5772/37170. 6. Annu. Rev Biomed. Eng. 4: 235-60. 7. ILAR J. 49: 103-15. 8. Science 359: 935-9. 9. Cancer Biol. Ther. 6: 781-6. 10. Gene Ther. 16: 441-7. 11. Hum. Gene Ther. 25: 955-65. 12. Sci Rep. 7: 7715.

Acknowledgement

This PhD project is wholly funded by the National Centre for the Replacement Refinement & Reduction of Animals and carried out at the Murray Lab at Cardiff University under the supervision of Prof Jim Murray and the mentorship of Dr Amit Jathoul

Introduction

 A very popular microscopy technique is Light Sheet Fluorescence Microscopy (LSFM SPIM) where a light-sheet is used to illuminate a sample in sequential slices vertically to a camera system. The resulting stacks of slices render a tomographic representation of the sample.  Usually, to remove regions affected by occlusion or scattering  scans from different viewing angles (0^o, 90^o, 180^o, 270^o), are acquired offering complementary information and image registration is necessary  to resolve any misalignment issues by transforming different sets of data into one common coordinate system.

Methods

We propose a method for the reduction of the dimensionality of the 3D problem of aligning the various viewpoints by sequential registration on 2D max projections created from the initial volumes following the steps described in the graph 1 for the case of 4 viewpoints. We should note that any 2D image registration algorithm can be used for the registration of the projections (e.g.  image based methods). Finally, for difficult datasets, where not much common information exists among views an easy semi-automatic selection of few 2D landmarks (usually 4-5) can be used.

Results/Discussion

We used the dataset http://fly.mpi-cbg.de/preibisch/nm/HisYFP-SPIM.zip and compare it with the fluorescent beads method described in [1]. Four views of the  Drosophila melanogaster embryo were used 0^o (red), 90^o (yellow), 180^o (green), 270^o (blue), and registration was performed using both the   projection method figure a) and figure b) and the method described in  [1] figure c) and figure d). For the max projection method the beads were excluded to show the possibility to register without the artificial inclusion of landmarks.   Which can be difficult or even impossible, as in the case where beads visible in a multispectral acquisition, don’t exist.

Conclusions

With projections registration we managed to create volumetric images of high information for multiangle LSFM, and overcame the problems arising from the high complexity of 3D algorithms. We achieved fast and accurate results without the need for the introduction of artificial fluorescence beads. Our implementation is easy to be used by an experimentalist, and it has shown to work even on samples with minimal common information between angles

References

1. S. Preibisch,   Nature Methods, 7, (2010)
2. M. Rieckher   PLoS ONE 10, (2015)

Acknowledgement

We acknowledge support of this work by the project “BIOIMAGING-GR” (MIS5002755) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund).

Introduction

The physiological and pathological roles of both endogenous and exogenous elements are of major interest for the medical community. Elemental imaging of biological tissues is a technological challenge, generally requiring complex instruments with restricted accesses. We recently developed an all-optical method, fully compatible with standard microscopy systems, for multi-elemental imaging of biological tissues. In this work, we used LIBS to image the elemental composition of peri-prosthetic tissues to identify metallic wear and corrosion products from hip arthroplasty revision cases.

Methods

We upgraded our Laser Induced Breakdown Spectroscopy (LIBS) instrument to work faster and to image the elements contained in paraffin-embedded samples¹. Our LIBS system allows the in situ imaging and quantification of the elements of the periodic table within biological/human tissues, with ppm-scale sensitivity and a pixel size of 10x10 µm². A laser is focused on the sample surface, which generates a plasma. Elemental images (maps) are obtained by scanning the surface of the sample. Different spectrometers are used to collect the signal of various metallic elements, including Cr, Co, Ti and Al, which are the main components of implants. Peri-prosthetic tissues blocks represent a spectrum of wear debris types, based on implant retrieval findings and background knowledge of the case.

Results/Discussion

Particles were observed in all cases except the control tissue but their appearance and distribution were highly variable. By contrast, LIBS demonstrated the distribution of the various elements of the tissues, including areas where visible particles were not obvious histologically. Generally, there was good agreement with the known background sources of debris. Notably, Co was very low or absent the tissues with CoCr components while Cr was abundant (Fig. 1). Areas with high Cr density typically contained reddish-brown colored macrophages both with and without visible particles. High peaks for Ti and Cr were identified in the tissues from a worn CoCr stem and a worn Ti cup (Fig.2).  Areas with large green-gold solid corrosion products were associated with a high count for Cr, while darker and more granular corrosion products were associated with high Ti and Fe counts. Fe distribution was variable and only sometimes seemed to correspond to areas with red blood cells or fibrin deposits.

Conclusions

Arthoplasty-derived metal particles and corrosion products cannot be accurately characterized based on light microscopy alone. LIBS overcomes these limitations and allows a more direct and quantitative elemental identification², used with direct reference to the H&E slide features. LIBS can detect submicron, nanometer and ion-protein complex forms of biomaterials as well as endogenous particles that are undetectable at the light microscope level.

References

[1]. Busser et al, Modern Pathology, 2018

[2]. Busser et al, Coord Chem Rev, 2018

Acknowledgement

This work was supported by the ITMO Cancer et ITMO Technologies pour la santé de l'alliance nationale pour les sciences de la vie et de la santé (AVIESAN), the Institut National du Cancer (INCa) and INSERM within the project LAST (#PC201513). This work was also supported by the French national grant ANR-17-CE18-0028 “MEDI-LIBS”.

Introduction

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Early sepsis diagnosis using biomolecular information can accelerate clinical decision-making. In the past decade, biomolecular information obtained from non-linear multimodal imaging, the combination of coherent anti-Stokes Raman scattering (CARS), two-photon excited autofluorescence (TPEF) and second-harmonic generation (SHG), was used for characterizing various inflammatory diseases. In this study, we present non-linear multimodal imaging as a competent technique for early sepsis diagnosis.

Methods

To investigate the molecular consequences of septic liver injury, six mouse liver sections derived 24 hours after the induction of abdominal sepsis (n_PCI = 5) and peritoneal injection of NaCl in the control group (n_sham = 5) were studied using non-linear multimodal microscopy. The multimodal images of the liver sections were visualized as an RGB image (red: CARS, green: TPEF, blue: SHG) and a 20 µm region around the periportal and pericentral veins in the multimodal image was used for extracting texture features. A Mann-Whitney U test was used to prove the significance of each feature for discriminating the sepsis and control group. Furthermore, a binary classification of the two groups based on the texture features was performed and the receiver-operating characteristic curve was studied.

Results/Discussion

Statistical analysis showed a significant difference between the PCI and the sham group which was confirmed by seven statistically significant features (p<0.05). A significant difference between the two veins in the PCI group was found by one feature (skewness TPEF), whereas no statistically significant feature was observed for these veins in the sham group. This different behaviour between the PCI and the sham group can be associated with different functions and biomolecular activities of these two veins. The multivariate analysis showed that the classifier based on the features from all modalities discriminate the two groups effectively (AUC = 0.78). The classification performance was improved using only CARS features (AUC = 0.94) and only TPEF features (AUC = 0.81). The classifier based only on the SHG features achieved a poor performance (AUC =0.50), possibly due to poor collagen metabolism in early sepsis.

Conclusions

Non-linear multimodal imaging along with machine learning reveals significant differences between PCI and sham group, and between the periportal and pericentral veins of the PCI group resulting from inflammation. The binary classification based on CARS and TPEF features showed a good diagnostic ability for early sepsis detection. Therefore it can serve as a complementary label-free technique to study an organ damage associated with sepsis.

Acknowledgement

The funding of the DFG for the project BO4700/1-1, PO563/30-1 and STA295/11-1 is highly acknowledged.

Introduction

In vivo optical imaging is nowadays widely used for cancer diagnosis and image-guided therapy with various devices that have already entered into the clinic. However, current fluorescence imaging is worth improving in spatial and temporal resolution in deep tissues. A novel approach relies on collecting optical signal from the second near-infrared window between 850 to 1700 nm called ShortWave InfraRed (SWIR). The use of this wavelength range should considerably reduce light scattering and absorption by blood and tissues in vivo improving sensitivity and spatial resolution in depth [1-3].

Methods

To address a clinical need for non-invasive high-resolution imaging of vascular and lymphatic networks at cellular level, we propose the development of a new SWIR imaging platform coupled with advanced imaging processing. This development comprises i) the imaging platform set up and characterization, 2) in silico image processing tools, 3) nanoscale contrast agents development, and 4) in vivo experiments to investigate lymphatic and vascular disorders in pathological animal models.

Results/Discussion

We show how a large panel of ultra-small gold particles (<5nm) called gold nanoclusters could be used as new SWIR contrast agents to visualize the vascularization in mice with high spatial and temporal resolution thanks to a careful design of these nanocompounds.

Conclusions

Contrast agents based on ultra-small gold particles exhibiting intense photoluminescence in the SWIR region combine with low toxicity represent an useful tool for in vivooptical imaging notably in the field of angiogenesis. 

References

[1]G. Hong, A. Antaris, H. Dai, Nature biom. Eng. 2017, 1, 0010 (0011-0022)

[2]O. Bruns, … M. Bawendi, Nature biom. Eng. 2017, 1, 0056(0051-0011)

[3] Y. Chen, D. Montana, H. Wei, J. Cordero, M. Schneider, X. Le Guevel, O. Chen, O. Bruns, M. Bawendi, Nanoletters 2017, 17, 6330-6334.

Acknowledgement

Xavier Le Guevel would like to thank l’agence pour la recherche contre le cancer ARC (RAC17012CCA) and Plan cancer (C18038CS) fundings.

Introduction

Visualizing radiolabeled compound using optical systems can be a powerful tool for preclinical studies. However, the usefulness of optical systems to detect radioactivity emitted by ²²⁵Ac was not demonstrated so far. The aim of our work was to standardize measurement of Cerenkov radiation produced by α- and β-emitting radiotracers using OptiImager device with cooled and highly sensitive CCD camera. We examined how the geometry and radioactivity of the tested system affects the record of Cerenkov luminescence imaging (CLI).

Methods

The standardization tests were carried out using ²²⁵Ac (JRC, Karlsruhe, Germany) and ⁶⁸Ga (E&Z generator) solutions as α- and β-emitting sources in n-well plates. In the present study, we attempted to show how the size, shape and internal structure of samples influence the Cerenkov luminescence counting by the PhotonIMAGER™ (BioSpace Lab) optical imaging system.

Results/Discussion

The standardization studies were performed considering Cerenkov sources of the same activity but different volumes and thickness. We compared also an influence of the medium (water and gelatin) on efficiency of Cerenkov radiation production by ionizing α and β particles. Results shows that both the geometry and dielectric medium used for standardization are of great importance for efficient counting of the emitted Cerenkov luminescence. We observed the linearity of the Cerenkov counting for ²²⁵Ac up to 200 kBq (r²=0.992) and for ⁶⁸Ga up to 6 MBq (r²=0.993) when water was as a medium, and 12 MBq (r²=0.994) when gelatin was as a medium. Based on this results, optical imaging of ²²⁵Ac- and ⁶⁸Ga-DOTATATE distribution in mice were performed.

Conclusions

In our research we showed that the use of Cerenkov emissions for α- and β-emitting radiotracers has a potential for optical imaging of their biodistribution.

Introduction

The Beatson Institute’s High Content Image Analysis Resource (HiCAR) group design and implement image analysis solutions to many model experimental systems. Images are typically collected using the Perkin Elmer (PE) Opera Phenix high-throughput microscope and analysed primarily in PE’s proprietary Harmony 4.8 Image Analysis software. Many different experimental systems are able to be analysed, though here we will be showing the creation of a 3 D experimental system using a panel of pancreatic cancer patient derived cell lines.

Methods

A panel of patient derived cell lines, some with established knockdown of specific epigenetic regulators, were imaged and analysed in 2 and 3 dimensions to create a databank of morphological and intensity measurements. This was analysed using machine learning as well as other methods to establish if there are any differences between the cell lines.

Results/Discussion

Currently this project is ongoing and there is much work to be done. However from the machine learning analysis and clustering that has been carried out so far is very promising. From our preliminary analysis it is clear that there is a possibility to cluster the different cell lines into different subgroups based upon their morphological data. The various genetic subgroups determined by Bailey et al 2016 also appear to cluster in a similar fashion in their morphological properties. The cells lines have also been shown to behave in a variable manner when exposed to a wounding assay. The cell lines described as pancreatic progenitor appear to invade into the wound in a much slower fashion compared to those described as squamous. We’re also currently in the process of examining the effects of SWI/SNF component knockdown and the changes in the invasive capabilities of cell lines.

Conclusions

This work is currently ongoing however the establishment of protocols and analysis sequences suggests this is a viable avenue of enquiry. Initial data seems to suggest that pancreatic cancer patient derived cell lines can be sub grouped into distinct groups based upon their morphology and other associated staining. Knockdown of epigenetic components is another line of enquiry and and their effects on invasiveness is promising.

References

1. Bailey, P. et al. Genomic analyses identify molecular subtypes of pancreatic cancer . Nature. 531, 47 (2016).
2. Buchser W, Collins M, Garyantes T, et al. Assay Development Guidelines for Image-Based High Content Screening, High Content Analysis and High Content Imaging. 2012 Oct 1 [Updated 2014 Sep 22]. In: Sittampalam GS, Coussens NP, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-.Available from: https://www.ncbi.nlm.nih.gov/books/NBK100913/

Acknowledgement

Thanks go to Emma Shanks and Peter Bailey for their help and guidance on this project and the provision of cell lines. Special thanks goes to Lynn McGarry, David Bryant, Eva Freckman and John Halpin for their image analysis and machine learning help.

Introduction

Studying spatio-temporal characteristics of oxygen perfusion in solid tumors is essential for assessing their long term responses to therapy [1,2]. However, efficient visualization of multi-scale oxygenation dynamics in heterogeneous tumors remains difficult with the commonly employed imaging modalities due to inefficient trade-offs between image acquisition time and effective field of view.

Methods

Here we used a multi-scale imaging approach based on the spiral volumetric optoacoustic tomography (SVOT) technique [3] (Fig. 1). This truly 3D imaging system offers high quality visualization of the intricate anatomy at the whole-body scale along with the ability for fast tracking of kinetics and functional changes at the whole tumor level. In our study, female hairless NOD.SCID mice bearing orthotopic MDA-MB-231 breast cancer tumors were imaged during rest and oxygen stress challenge [4]. Time sequences of three-dimensional maps of blood oxygen saturation (sO₂), total hemoglobin (THb) and deoxy-hemoglobin (Hb) were built with an unprecedented isotropic 3D resolution of ~100 μm.

Results/Discussion

Segmentation of the tumor into sub-regions was achieved from the sO₂ distribution (Fig. 2a), which enabled volumetric quantification of volumetric hypoxic fractions (vHF) and visualization of distinct hypoxic centers in the core. The oxygen stress challenge further revealed distinctive dynamic sO₂ responses in the sub-regions of the tumors. The dynamic analysis clearly revealed the presence of cyclic hypoxia. We have further observed discrepancies between volumetric cyclic fractions (vCF) values manifested by fluctuations in sO₂ versus Hb in the hypoxic core (Fig. 2b).

Conclusions

The excellent spatio-temporal resolution performance of SVOT system combined with its capacity for rapid volumetric quantification of the sO₂ gradients offers new powerful capabilities for assessment of heterogeneity and perfusion dynamics in solid tumors. The multiple functional readouts provided by SVOT may enable better quantification of vCF as compared to conventional measurements with BOLD-MRI.

References

[1] Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer and Metastasis Reviews. 2007;26(2):225-39.

[2] Alizadeh AA, Aranda V, Bardelli A, Blanpain C, Bock C, Borowski C, et al. Toward understanding and exploiting tumor heterogeneity. Nature medicine. 2015;21(8):846.

[3] Deán-Ben XL, Fehm TF, Ford SJ, Gottschalk S, Razansky D. Spiral volumetric optoacoustic tomography visualizes multi-scale dynamics in mice. Light: Science & Applications. 2017;6(4):e16247.

[4] Tomaszewski MR, Gonzalez IQ, O'Connor JP, Abeyakoon O, Parker GJ, Williams KJ, et al. Oxygen enhanced optoacoustic tomography (OE-OT) reveals vascular dynamics in murine models of prostate cancer. Theranostics. 2017;7(11):2900.

Acknowledgement

The authors would like to acknowledge grant support from the Human Frontier Science Program (RGY0070/2016) and Deutsche Forschungsgemeinschaft (RA1848/5-1).

Introduction

Traditional cancer imaging devices are limited in their ability to screen for multiple contrast agents simultaneously in real time. Multispectral Optoacoustic Tomography (MSOT) is an emerging imaging modality capable of real-time imaging of numerous contrast agents with enhanced spatial resolution. As the simultaneous detection of multiple exogenous contrast agents in MSOT remains largely unexplored, we developed two species of spectrally distinct tumor targeted gold nanorods to serve as contrast agents detectable using MSOT. 

Methods

Two gold nanorod species were created using hydrogen peroxide (GNR-H2O2) or ascorbic (GNR-ASC) acid as reducing agents to modify the length of each species to create nanorods with individual light absorbance spectra in the IR range (680-900 nm). The particles were then coated with mesoporous silica to improve stability and targeted with Trastuzumab.  Characterization of particles included identification of absorbance spectra, zeta potential, electron microscopy, and HER2+ specificity.  HER2+ and HER2- breast cancer cells were treated with each particle alone and in combination with uptake evaluated using electron microscopy and within tissue mimicking phantoms detected using MSOT.  Multispectral processing was conducted using both linear regression.

Results/Discussion

The initial GNR-H202 particle was 18 nm in length and the GNR-ASC particle was 53 nm in length with similar aspect ratios of 2:1 (l:w). Both TRA-CMG-ASC and TRA-CMG-H2O2 resulted in optoacoustic spectrally distinct signals when imaged in tissue phantoms both individually as well as mixed within the same well after multispectral processing using linear regression. Treatment of HER2+ and Her 2- breast cancer cells with a combination of TRA-CMG-H2O2 and TRA-CMG-ASC particles evaluated using TEM (Fig 1). In DY36T2 and SKBR3 cells treated with TRA-CMG-H2O2 resulted in 2.5x and 3.1x enhanced signal, respectively, as compared to HER2- MDA-MD-231 cells in tissue mimicking phantoms detected using MSOT p=0.003. Treatment of DY36T2Q and SKBR3 cells with TRA-CMG-ASC demonstrated 3.7x and 6.9x, respectively, compared to MDA-MD-231p=0.001. Combination treatment of TRA-CMG-H2O2’s and TRA-CMG-ASC’s clear and distinct signals were observed using MSOT for each particle individually and together.

Conclusions

Both particles have demonstrated that they can be simultaneously administered and targeted at HER2+ cell while also maintaining distinct photoacoustic signals in MSOT upon consolidation. Each particle species, targeted to the same cells, were capable of being monitored individually in the presence of the other gold nanorod contrast agent.

Acknowledgement

This work was supported by NIH grant R01EB020125, R01CA212350, R01CA205941.

Introduction

The targeting of tumor cells often relies on the availability of cancer-associated biomarkers at the tumor site. In general, however, expression levels of such biomarkers are unknown at initial diagnosis. To enable accumulation of theranostic agents independent of biomarker availability, star nanoparticles were developed, consisting of center-crosslinked arms functionalized with imaging components (Gd³⁺ for MRI) and therapeutic components (¹⁷⁷Lu for endoradiotherapy), that may utilize the enhanced permeability and retention (EPR) effect for passive tumor targeting.

Methods

A star copolymer was synthesized, consisting of oligoethylene glycol methyl ether acrylate (OEGA) units, 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) units modified with [Gd³⁺]1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid)-10-(aminoethylacetamide) ([Gd³⁺]DO3A) and aminoethyl acrylate (AEA) units modified trans-cyclooctene (TCO). Tetrazine-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Tz-DOTA) was radiolabeled with lutetium-177 (¹⁷⁷Lu) and conjugated to the star copolymer. Biodistribution and dosimetry were assessed in BALB/c mice carrying subcutaneous CT26 allografts. ¹H longitudinal relaxivities were determined over a continuum of magnetic field strengths ranging from 0.24 mT – 0.94 T at 37 °C (nuclear magnetic relaxation dispersion (NMRD) profile).

Results/Discussion

The star polymer (84 kDa, PDI=1.16, D_h= 11 nm) consisted of 7-8 polymer arms, each containing 19×OEGA units, 5×VDM-[Gd³⁺]DO3A units and 4×AEA-DFO units. Radiolabeling of Tz-DOTA with ¹⁷⁷Lu was achieved with high radiochemical yield (93.9%), with good conjugation efficacy to the star copolymer (70.9%) and high radiochemical purity (>99%). Accumulation of the star copolymers in CT26 allografts after three days was 14.8±4 %ID/g. Dosimetry data will be discussed. The NMRD profile indicated maximal relaxivity (16.9 mM^-1s^-1) at ~30 MHz.

Conclusions

Theranostic star nanoparticles were developed that passively accumulated at the tumor site with high tissue contrast. Such nanoparticles would allow for simultaneous tumor imaging and therapy without prior assessment of biomarker expression.

Acknowledgement

Funding from Australian Research Council (ARC) Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036) and NIH Cancer Center Support Grant P30 CA008748-48, supporting MSK Small Animal Imaging Core, Radiation Core and Research Animal Resource Facilities are gratefully acknowledged.

Introduction

Intratumor heterogeneity has been recognized as cause for failing cancer therapies and the occurrence of resistance (1). Besides the heterogeneity of tumor cells, different compartments can be identified within the tumor such as the invasive margin, superficial tumor rim and tumor core (1). The core differs from other tumor regions since it shows metabolic reprogramming caused by nutrient deficiency, hypoxia and immature vasculature; however, direct analysis of this compartment by intravital two-photon microscopy is hampered by the limited tissue penetration of near-infrared light (2, 3).

Methods

To overcome the depth limitations of conventional two-photon microscopy, we developed a rotational side-view endomicroscope, which enables optical access to the tumor core. The microobjective consists of a GRIN lens (NA = 0.28) embedded in a smooth polyimide tubing (diameter: 420µm), allowing nearly friction-free gliding into the tumor. Endomicroscopy was performed with subcellular resolution (1.2µm lateral, 25µm axial) reaching up to 2mm deep inside the tissue, including multicolor fluorescence and second harmonic generation of fibrillar collagen. Microendoscopic penetration into the tumor caused only minimal damage, with intact perfused micro- and macrovessels near the imaging interface, minimal bleeding after retraction, and lack of circulating tumor cell (CTC) release during imaging.

Results/Discussion

We applied endomicroscopy in a murine melanoma model located in the deep dermis to elucidate intratumor heterogeneity. Using 3D and time-lapse detection in the tumor core, we identified a pool of intravascular resident tumor cells (IRTC) that are located inside tumor vessels in focalized microniches (“hotspots”). Analysis of arrival and departure kinetics of IRTC revealed long residence times (days) and slow turn-over, indicating a persistent pool of intravascular non-circulating tumor cells. IRTC as intratumor compartment were confirmed in 75% of clinical samples from melanoma patients. Moreover, their occurrence was positively correlated with the tumor stage. To test whether IRTC contribute to the CTC pool, we increased  the intratumor blood flow by systemic administration of prazosin, an α-adrenergic receptor antagonist, and detected 4-fold increased CTC counts in the periphery within 10 min, as well as mildly decreased IRTC counts in the tumor core.

Conclusions

We developed rotational side-view multiphoton endomicroscopy as strategy for subcellular-resolved intravital deep-tissue imaging. Using intratumor endomicroscopy, we identified IRTC as a previously unappreciated tumor compartment, in which tumor cells reside long-term in blood vessels and can be released in response to varying blood flow, suggesting that they might serve as reservoir for CTCs and, possibly, contribute to metastatic seeding.

References

1.  Pribluda, A. et al. Intratumoral Heterogeneity: From Diversity Comes Resistance. Clin Cancer Res. 21:2916-2923 (2015).
2. Andresen, V. et al.  Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging. Curr Opin Biotechnol. 20:54-62 (2009).
3. Ouzounov, D.G. et al. In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain. Nat. Methods 14, 388–390 (2017)

Acknowledgement

We would like to thank Anke Kraeplin (Grintech, Jena, Germany), Esther Wagena, Manon Vullings and Lenny Droesen (RIMLS, Nijmegen, NL) for technical support. The work was funded by an ERC Consolidator grant.

Introduction

For solid tumors, such as breast cancer, complete tumor resection is of utmost importance, since tumor-positive resection margins may result in impaired overall survival.¹ Current optical imaging techniques using endogenous contrast are limited to point measurements or long acquisition times. Broadband hyperspectral cameras, which provide a complete spectral fingerprint of the object at pixel level, are needed for two-dimensional imaging of the operative region.² An ex vivo study was conducted to evaluate the feasibility of hyperspectral imaging for breast cancer detection. 

Methods

Fresh (<3 hours after surgery) resected breast cancer slices were imaged with a snapshot hyperspectral camera (Quest Medical Imaging, Middenmeer, The Netherlands), which has 41 spectral bands, equally distributed in the visible and near-infrared (VIS-NIR) range (450 – 950 nm). Supervised analysis of the hyperspectral images was performed by using the pathology annotations and unsupervised analysis was performed by using the hierarchical stochastic neighbour embedding (h-SNE) algorithm.³  

Results/Discussion

So far, eight resected specimens, of which five invasive carcinomas and three mucinous adenocarcinomas, were imaged. Significant spectral differences were found between the non-tumor, malign and benign regions on the resected specimens, as illustrated in Figure 1. The most characterising wavelengths were located in the NIR range, however a relatively high variety between the individual tumors was found. Furthermore, automatic feature classification, i.e. selection of tumor tissue, using the h-SNE algorithm was possible in selected cases (Figure 2).

Conclusions

Hyperspectral imaging showed potential for discrimination of benign and malign breast tissue by using specific wavelengths bands in an ex vivo setting. Further analysis will be performed to determine whether it is possible to select tumor-specific wavelengths.

References

1. Chagpar AB, Martin RC, 2nd, Hagendoorn LJ, Chao C, & McMasters KM (2004) Lumpectomy margins are affected by tumor size and histologic subtype but not by biopsy technique. American journal of surgery 188(4):399-402.
2. Lu G & Fei B (2014) Medical hyperspectral imaging: a review. Journal of biomedical optics 19(1):10901.
3. van Unen V, Höllt T, Pezzotti N, Li N, Reinders MJT, Eisemann E, Koning F, Vilanova A, Lelieveldt BPF.Visual analysis of mass cytometry data by hierarchical stochastic neighbour embedding reveals rare cell types. Nat Commun. 2017 Nov 23;8(1):1740.

Acknowledgement

This work was supported by the European Union Horizon 2020 Program under grant agreement number 692470 (ASTONISH project).

Introduction

Hepatocellular carcinoma (HCC) is a type of highly vascular tumor, and therefore linked with major vascular changes. The HCC tumors show a typical contrast enhancement pattern where, compared to liver parenchyma: they are first hyperdense during the arterial phase (wash-in) and then hypodense in the portal venous phase (wash-out) [1-3].

In this work we develop a contrast-enhanced ultrasound (CEUS) method to detect hepatic focal lesions. Quantitative parameters from the CEUS time intensity curve (TIC) of HCC were obtained in order to describe pathological changes in blood flow.

Methods

An inducible HCC mouse model (SV40-Tag Oncogene) was used. 22 developed HCC lesions with diameter >2mm and were considered suitable the study. Tumor dimensions were assessed using 7T-MRI.

CEUS studies were performed using the VEVO 2100 system using a 21 MHz probe, after tail vein injection of a 100 μl-bolus of PolySon L. Scanning was performed over a period of 70 sec to allow assessment of the arterial and portal venous phases. Region of interest (ROI), drawn in the interior of HCCs and in the surrounding liver parenchyma (reference ROI) were analyzed off-line using the VEVO 2100 Software for the quantification of peak-enhancement (PE) and time to peak (TTP). In order to find the slope of the arterial and portal venous phase two gamma variate functions were used to fit the TIC.

Results/Discussion

The TIC of the HCCs lesions, shows two distinct peaks: the main one with a PE=15,19±11,94 a.u. and a TTP=3,235±1,328 sec and a second one with a PE=13,27±10,88 a.u. and a TTP=17,35±10,59 sec. The surrounding liver parenchyma shows two peaks: the first one with a PE=21,83±16,23 a.u. and a TTP=5,968±1,030 sec and a second one with a PE= 28,44±18,99 a.u. and a TTP=17,77±7,905 sec. Comparing the TIC of the HCCs and the surrounding liver parenchyma we observed a statistical significant difference between the TTP of the first peaks (p<0,0001). We also observed a statistical significant difference between the PE and the derivative of the wash-in (p=0,0099) relative to the peaks observed in the second phase (p=0,0041). The value of the difference between the PE of the first and second peak, shows a significant difference (p<0,0001) between the HCCs and the surrounding liver parenchyma.

Conclusions

The data presented in this work describe the decrease of portal blood supply in HCC lesions replaced by an abnormal supply from arterial tumor vessels in genetically engineered mouse models (GEM).

More precisely we showed that CEUS imaging provide further information about tumor characterization on functional (perfusion analyses) levels, useful in the evaluation of efficacy of new therapies.

References

1. Jang HJ, Kim TK, Wilson SR. Imaging of malignant liver masses: characterization and detection. Ultrasound Q. 2006 Mar;22(1):19-29. Review.
2. Pei XQ, Liu LZ, Liu M, Zheng W, Han F, Li AH, Cai MY. Contrast-enhanced ultrasonography of hepatocellular carcinoma: correlation between quantitative parameters and histological grading. Br J Radiol. 2012 Sep;85(1017):e740-7. doi: 10.1259/bjr/20402927. Epub 2011 Nov 17.
3. Liao AH, Cheng YC, Weng CH, Tsai TF, Lin WH, Yeh SH, Yeh WC, Li PC. Characterization of malignant focal liver lesions with contrast-enhanced 40 MHz ultrasound imaging in hepatitis B virus X transgenic mice: a feasibility study. Ultrason Imaging. 2008 Oct;30(4):203-16.

Introduction

DWI MRI provides an early marker for therapy response in tumor treatment, being sensitive to changes in tissue density, cellularity, or vascularization. How exactly structural and physiological changes influence diffusion parameters, is not fully clear. DWI using oscillating instead of constant pulsed gradients may provide more detailed insight into the microstructure of tumors. We have used oscillating gradient DWI and the IMPULSED¹ approach to calculate diameters of cells in spheroids of three cancer cell lines, and one in vivo model, and compared those to laser scanning microscopy data.

Methods

Tumor spheroids of SK-Mel-30, MG63 and A375 cells were grown to a diameter of 500-700 µm. For the in vivo model 10⁶ 4T1 cells were injected into the mammary fat pad of Balb-c mice and grown for three to six days (n=3). DWI with a spin echo sequence using oscillating gradients was performed at 9.4 T on a Bruker BioSpec using a CryoProbe. For spheroids in PCR tubes: TR/TE:2000/89.4 ms; resolution: 63 µm; modulation: 200, 150, 100, 50Hz; nine b-values per frequency: 0-1000 s/mm². For in vivo scans: TR/TE:3000/21.5 ms; matrix 108x96, FOV: 18x15 mm².

For IMPULSED approach analysis, tumors were manually segmented in the images and intensities fitted¹, yielding five parameters including average cell radius.

Laser scanning microscopy data (LSM 800, Zeiss) were analyzed manually using ImageJ.

Results/Discussion

SNR values in DWI in vitro ranged between 30 and 70, and allowed for delineating the spheroids. For A375, SK-Mel-30, and MG-63 cells average radii of 11.1±0.9 µm (n=6 spheroids), 11.7±1.3 µm (n=9), and 13.9±1.4 µm (n=7) were calculated. These agreed well with the values obtained for the different cell lines by microscopy of 8.7±2.4 µm (n=41 cells), 11.5±3.0 µm (n=91), and 13.6±2.1 µm (n=50) (Fig. 1). Eccentricity values were determined as 0.34, 0.42, and 0.34, respectively.

For in vivo experiments, MR analysis of the 4T1 cells yielded an average radius of 9.9±1.9 µm, exceeding the radius obtained from laser scanning microscopy of 6.4±1.0 µm. The eccentricity was determined as 0.72 (Fig. 2).

Determining cell radii from oscillating gradient DWI is based on assumptions about geometry and exchange between compartments. Deviation between DWI and microscopy observed in vivo may be explained by the larger eccentricity of the 4T1 cells and the presence of vessels and small areas of necrosis.

Conclusions

The IMPULSED approach provides highly accurate radii for spherical cells with no other disturbing structures, as observed in tumor spheroids. For in vivo tumors however, more complex geometrical assumptions may be required to provide correct estimates of the cellular microstructure, a potentially reliable marker for treatment response in cancer.

References

Jiang X, Li H, Xie J, et al. Quantification of cell size using temporal diffusion spectroscopy. Magn Reson Med. 2016; 75:1076-1085.

Introduction

Since electron paramagnetic resonance (EPR) phenomenon is used to study biological systems to obtain unique functional information such as oxygen concentration[1], tissue redox state[2], thiol concentration[3], pH[4] etc. the possibility of imaging live animals have been highly desirable. Distributions of oxygen concentration (pO₂) are a critical determinant of normal tissue health as well as tumour aggressiveness and response to therapy [5,6]. EPR tomography is recognized as the gold standard in the measurement of the absolute level of oxygen in tissue.

Methods

Imaging was performed on TM 600 ERI tomograph (Novilet, Poland) capable to analysing 1000 EPR signal harmonics and to performing continues wave (CW) with multiharmonic (MHA) or rapid scan (RS) techniques with a 1kHz field modulation. The loop-gap resonator with a 3.4 cm diameter dedicated to imaging whole mice. 3D and 4D functional imaging were performed on conducted on C57bl/6J mice aged 3-8 weeks. As a spin probe, 90mM OX063 was used.

Results/Discussion

Imaging procedures and obtained images reflecting the oxygen level in healthy and tumour tissues were presented as results of this work. This is the first report describing in vivo imaging of oxygen concentration with sub-millimetre resolution and obtained in sub-minutes time scale, performed in-vivo on mice using advanced EPR imaging technology such as multiharmonic analysis, rapid scan detection and fast imaging protocol. Fast and continues acquisition of 4D images combined with advanced reconstruction algorithm allowed for tracking oxygen changes in time intervals of several seconds.

Conclusions

Presented results confirm the effectiveness of application EPR tomography for in vivo imaging of oxygen concentration in murine tumour models. Spatial and temporal resolutions of obtained images allow to monitor the temporary changes of the oxygen concentration and introduce new standards in vivo oxygen imaging

References

1. Epel B, Kotecha M, Halpern HJ. In vivo preclinical cancer and tissue engineering applications of absolute oxygen imaging using pulse EPR. J Magn Reson. 2017 Jul;280:149-157.

2. Fujii H, Sato-Akaba H, Kawanishi K, Hirata H. Mapping of redox status in a brain-disease mouse model by three-dimensional EPR imaging. Magn Reson Med. 2011 Jan;65(1):295-303.

3. Epel B, Sundramoorthy SV, Krzykawska-Serda M, Maggio MC, Tseytlin M, Eaton GR, Eaton SS, Rosen GM, Kao JPY, Halpern HJ. Imaging thiol redox status in murine tumours in vivo with rapid-scan electron paramagnetic resonance. J Magn Reson. 2017 Mar;276:31-36.

4. Komarov DA, Ichikawa Y, Yamamoto K, Stewart NJ, Matsumoto S, Yasui H, Kirilyuk IA, Khramtsov VV, Inanami O, Hirata H. In Vivo Extracellular pH Mapping of Tumors Using Electron Paramagnetic Resonance. Anal Chem. 2018 Nov 8

5. Richmond KN, Burnite S, Lynch RM. Oxygen sensitivity of mitochondrial metabolic state in isolated skeletal and cardiac myocytes. Am J Physiol. 1997 Nov;273(5 Pt 1):C1613-22.

6. Chapman, J.D., Franko, A.J., & Koch, C.J. (1983). The fraction of hypoxic clonogenic cells in tumour populations. United States: Masson Publishing USA, Inc.

Acknowledgement

This work was supported by the Polish National Science Centre project UMO-2014/15/B/NZ5/01488

Introduction

The Gastrin Releasing Peptide Receptor (GRPr) is overexpressed on a variety of human tumours including prostate cancer. Radiolabelled peptides for this receptor are of potential use for diagnosis and therapy of GRPr positive tumours. Given the mitogenic properties of GRPr agonists, GRPr antagonists have been developed recently.

Here we compared the properties of two GRPr antagonists, RM2 and NeoBOMB in a PC-3 xenograft mouse model. Beside tumour uptake, retention in healthy tissue was evaluated to understand potential limitations for GRPr targeting radiotherapy.

Methods

[⁶⁸Ga]RM2 and [⁶⁸Ga]NeoBOMB have been evaluated by small animal Positron-Emission-Tomography (PET) imaging using NMRI mice bearing PC-3 xenografts (n= 3 mice per compound). PET acquisition was started at 60 min p.i. for 10 min. Uptake in tumour, liver, kidney and pancreas was quantified using ROI analysis.

Biodistribution of [¹⁷⁷Lu]RM2 and [¹⁷⁷Lu]NeoBOMB was studied in the same model. 3 mice were sacrificed at 1 h, 4 h and 24 h p.i. . In 3 additional mice 100 µg of unlabelled RM2 or NeoBOMB were injected shortly before animals were sacrificed at the 4 h time point. For the biodistribution, all organs were collected, weighted and activity determined in a gamma counter.

Results/Discussion

[⁶⁸Ga]RM2 and [⁶⁸Ga]NeoBOMB showed comparably uptake in the PC-3 xenografts at 1h p.i. (RM2 3.5±0.8 %ID/g, NeoBOMB 2.75±0.4 % ID/g). In contrast to [⁶⁸Ga]RM2, [⁶⁸Ga]NeoBOMB PET images showed significantly increased background with high uptake in liver and kidney.

Tumour uptake of [¹⁷⁷Lu]RM2 and [¹⁷⁷Lu]NeoBOMB at 1h p.i. was comparable for both compounds (6.6±0.9 %ID/g for [¹⁷⁷Lu]RM2, and 5.3±0.6 % ID/g for [¹⁷⁷Lu]NeoBOMB). Pancreas uptake was significantly higher for [¹⁷⁷Lu]NeoBOMB at all time points. Tumour to pancreas ratios of 0.05 at 1 h, 0.09 at 4 h and 0.4 at 24 h were found for [¹⁷⁷Lu]NeoBOMB. Higher tumour to pancreas ratios were observed with [¹⁷⁷Lu]RM2 (0.17 at 1 h, 0.6 at 4 h and 6.3 at 24 h).

Addition of unlabelled peptide at 4h did not reduce tumour uptake for both compounds. However, [¹⁷⁷Lu]RM2 uptake in pancreas was further reduced which was not observed for [¹⁷⁷Lu]NeoBOMB (tumour to pancreas ratio at 4 h for [¹⁷⁷Lu]RM2: 4.8, for [¹⁷⁷Lu]NeoBOMB: 0.1).

Conclusions

Both compounds show high specific and rapid localization of PC-3 tumour xenografts. [⁶⁸Ga/¹⁷⁷Lu]RM2 is characterized by a rapid clearance from normal tissues whereas [⁶⁸Ga/¹⁷⁷Lu]NeoBOMB1 showed significantly increased background signal with high activity in liver and kidney. Pancreas uptake was found to be significantly higher for [¹⁷⁷Lu]NeoBOMB which favors [¹⁷⁷Lu]RM2 for use in radiotherapeutic applications.

Introduction

Glioblastoma (GB) adjuvant chemotherapy includes Temozolomide (TMZ); yet, not all GB patients are responsive [1]. The latest trends in GB (pre-)clinical trials [2-5] usually refer to Doxorubicin (DOX); yet, it is unable to adequately overpass the blood brain barrier [5] with no adverse reaction [6]. However, DOX’s effective concentration against GB cells is reported to be very low [7]. A range of TMZ and DOX doses were used to treat primary GB spheroids. We used Light Sheet Fluorescence Microscopy (LSFM) in order to discriminate growth inhibition in cell division arrest from cell death.

Methods

An in-house primary GB cell culture was used. 3D spheroids were generated using the hanging drop technique growing for up to 23 days. The spheroids were treated with a range of concentrations of the anticancer agents DOX and TMZ from day 4 to day 7 (as in [8]), based on the IC₅₀ values previously estimated in 2D. Optical Microscopy was used to monitor the growth pattern and LSFM was used to visualize the drug penetration (DOX autofluorescence) and necrosis (Draq7) in multiple time points. Multiple projections LSFM scans were performed to obtain for multispectral high resolution 3D imaging and optical sectioning. Diametrical projections were stacked and registered for all the emission data and finally, combined together in order to achieve the isotropic resolution in all directions.

Results/Discussion

Interestingly, we observed that unlike DOX, which shows a similar dose-response pattern in both 2D and 3D experiments, TMZ has a noisy unidentified response pattern in 3D. Comparing the effective dose regime of the two drugs in the primary GB spheroids, we observed that DOX appeared to be very effective in less concentrated doses; even in three orders of magnitude less than TMZ. On the other hand, in TMZ-treated spheroids growth-inhibiting effects were observed in a non-consistent dose-response relationship.

Given the growth inhibition effects observed in vitro for both drugs, as well as the reported mechanisms of action [9, 10], we investigated whether this result can be further discriminated in either cell proliferation arrest and/or cell death using LSFM imaging. According to the fluorescent images, DOX had a low elimination rate over time and was able to accumulatively cause necrosis. There was no differential pattern of the TMZ-induced cell death compared to the control spheroids.

Conclusions

Our results demonstrate a preclinical drug screening tool to assess the distribution, penetration and cytotoxic potency of the anti-neoplastic agents TMZ and DOX in primary 3D cell cultures. Alternative therapeutic schemes and time-efficient drug tests are expected to serve the ambitious goal of approaching cancer simulations based on patient-specific data in order to optimize individualized therapy decisions and GB pathophysiology understanding.

References

1.            Lee, S.Y., Temozolomide resistance in glioblastoma multiforme. Genes & Diseases, 2016. 3(3): p. 198-210.

2.            Villodre, E.S., et al., Low Dose of Doxorubicin Potentiates the Effect of Temozolomide in Glioblastoma Cells. Mol Neurobiol, 2018. 55(5): p. 4185-4194.

3.            Jiang, P., et al., Novel anti-glioblastoma agents and therapeutic combinations identified from a collection of FDA approved drugs. J Transl Med, 2014. 12: p. 13.

4.            MacDiarmid, J.A., et al., Targeted Doxorubicin Delivery to Brain Tumors via Minicells: Proof of Principle Using Dogs with Spontaneously Occurring Tumors as a Model. PLoS ONE, 2016. 11(4): p. e0151832.

5.            Lesniak, M.S., et al., Local Delivery of Doxorubicin for the Treatment of Malignant Brain Tumors in Rats. Anticancer research, 2005. 25(6B): p. 3825-3831.

6.            Edwardson, D.W., et al., Role of Drug Metabolism in the Cytotoxicity and Clinical Efficacy of Anthracyclines. Current Drug Metabolism, 2015. 16(6): p. 412-426.

7.            LLC, N.B., 3D Tumor Spheroid Analysis Method for HTS Drug Discovery using Celigo Imaging Cytometer.

8.            Tzedakis, G., et al. A hybrid discrete-continuous model of in vitro spheroid tumor growth and drug response. in Proceedings of the Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS. 2016.

9.            Thomas, R.P., L. Recht, and S. Nagpal, Advances in the management of glioblastoma: the role of temozolomide and MGMT testing. Clinical Pharmacology : Advances and Applications, 2013. 5: p. 1-9.

10.          Thorn, C.F., et al., Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenetics and Genomics, 2011. 21(7): p. 440-446.

Acknowledgement

Authors would like to thank Evangelos Liapis for all the help he provided, Elias Drakos for his collaboration, and Katerina Manolitsi for her advisory comments, as well as Despina Tsoukatou and Venediktos Makatounakis for the expert technical assistance. This work was supported by the project “BIOIMAGING-GR” (MIS5002755) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund). Mariam-Eleni Oraiopoulou acknowledges support from the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), under the HFRI PhD Fellowship grant (GA. no. 130178/I2/31-7-2017).

Introduction

Among the large variety of anticancer metallic complexes, titanocene dichloride Cp₂TiCl₂ reached Phase 2 clinical trial. However, its poor solubility and stability in water severely hampered its development as anticancer agent. Besides, the mechanism of action of such complexes is still unclear since the usual biodistribution studies by ICP-MS cannot be performed due to the “natural” occurrence of titanium. Scintigraphy and optical imaging appear then as promising tools to visualize in vivo and in vitro metal biodistribution and clarify the mechanism of action of titanocene derivatives.

Methods

To deal with the previously mentioned issues, Gansäuer developed the synthesis of water-soluble and stable cationic titanium complexes [1]. Seeking to synthesize new trackable titanium-based drugs, we decided to use this strategy to introduce a SPECT probe (DOTA-¹¹¹In) and a fluorophore (BODIPY dye) on the titanocene moiety [2]. The complexes were characterized and their antiproliferative properties were evaluated in vitro on 3 cancer cell lines (A2780, B16F1, and PC3). As a proof of concept, preliminary studies in healthy mice were performed with the Ti/¹¹¹In derivative to obtain information about its uptake, its biodistribution, and its excretion. Confocal microscopy experiments were performed with the fluorescent complex to track it in vitro in cancer cells.

Results/Discussion

A novel Ti/¹¹¹In radiotheranostic was reported thanks to the challenging synthesis a titanocene-DOTA ligand. This radiolabeled complex is stable until 24 h in biological medium. The antiproliferative activity of the complex was evaluated on 3 cancer cell lines, but no significant cytotoxicity was observed. A cellular uptake study was performed and indicated that the complex weakly enters the cell because of its high hydrophilicity. Comparisons between the antiproliferative activities of our complexes lead to the conclusion that, for this family of titanocene complexes, the lipophilicity balance is a key parameter for the cytotoxicity. However, a biodistribution study on healthy mice gave the proof of concept of the radiotrackability of such complexes. The fluorescent analogue bearing a BODIPY probe was developed for in vitro tracking by optical imaging, highlighting a good cellular uptake, an accumulation in the cytoplasm and giving insights on the biological target of the complex.

Conclusions

A radiolabeled Ti-DOTA and a fluorescent Ti-BODIPY were described for the first time. Imaging studies proved the feasibility of tracking these complexes by scintigraphy or optical imaging. This study highlighted the crucial role of the lipophilicity of the complexes for the cytotoxicity. For future investigations, vectorisation of the complexes will be performed to increase the cell uptake, the cytotoxicity, and the selectivity of the complexes.

References

[1] Gansäuer, A. et al., J. Am. Chem. Soc., 2005, 127, 11622

[2] Florès, O. et al., Dalton Trans., 2017, 46, 14548

Acknowledgement

Support was provided by the Ministère de l’Enseignement Supérieur et de la Recherche, the Centre National de la Recherche Scientifique (CNRS), and the French Research National Agency (ANR) via project JCJC “SPID” ANR-16-CE07-0020. This work is part of the projects ‘‘Pharmacoimagerie et agents théranostiques” et ‘‘Chimie durable, environnement et agroalimentaire” supported by the Université de Bourgogne and the Conseil Régional de Bourgogne through the Plan d’Actions Régional pour l’Innovation (PARI) and the European Union through the PO FEDER-FSE Bourgogne 2014/2020 programs.

Introduction

Diagnosis of neuroblastoma (NB) historically relies upon scintigraphy with the noradrenaline analogue ¹²³I-mIBG (meta-iodobenzylguanadine), yet there is discordance between patients being identified as potential responders, and the actual response rate to targeted radiotherapy (¹³¹I-mIBG). We postulate that positron emission tomography (PET) with a fluorinated analogue, ¹⁸F-mFBG, can guide ¹³¹I-mIBG delivery based on the actual expression level of noradrenaline transporters (NET-1), and thus have a greater potential in stratifying patients that may benefit from this treatment regimen.

Methods

Cell-associated ¹⁸F-mFBG tracer uptake was evaluated in vitro in a panel of NB cells lines treated with dual mTORC1/2 inhibitor AZD2014 (0-500nM), measure by γ-counter. Tracer pharmacokinetics was studied using Kelly and SK-N-BE(2)C xenografts. Additionally, ¹⁸F-mFBG imaging was performed to evaluate NET-1 expression level in response to AZD2014 (20 or 25 mg/kg/day; 3 doses) in SK-N-BE(2)C tumour bearing mice. Ex vivo biodistribution of major organs, and Western blot, autoradiography and IHC analyses of the tumour were correlated with PET data. For dosimetry estimations, mice received ~8 MBq ¹⁸F-mFBG and were scanned up to 6 h post injection. The activity concentrations in the tumours [%ID/g] were used for image-guided delivery of ¹³¹I-mIBG.

Results/Discussion

Uptake of ¹⁸F-mFBG correlated with NET-1 protein expression in NB cell lines. Following AZD2014 treatment, there was a concentration-dependent increase in NET-1 protein expression, and equivalent radiotracer uptake, in both Kelly and SK-N-BE(2)C cells in vitro. PET imaging allowed for accurate delineation of NET-1 expressing SK-N-BE(2)C xenografts. Quantitative analysis of PET images revealed an increased ¹⁸F-mFBG uptake following 3 days treatment with AZD2014, paralleled by upregulation in tumour NET-1 protein expression confirmed by Western blot. The response to ¹³¹I-mIBG, in vivo, was in line with NET-1 expression level calculated from the dosimetry estimations obtained from ¹⁸F-mFBG acquisitions.

Conclusions

¹⁸F-mFBG can quantitatively assess different levels of NET-1 in vitro and in vivo, and therefore provide guidance for ¹³¹I-mIBG dose optimisation and monitoring changes in transporter level in response to therapeutic intervention.

Acknowledgement

We would like to thank Dr. Orli Yogev and Professor Louis Chesler for providing AZD2014. 

Introduction

Nanoparticles (NPs) with rapidly eliminated from the body offer great potential in clinical translation. However, preparation of an ultra-small nanotheranostic agent with renal clearance behaviors is still challenging. In this work, we report a facile method to prepare nanoparticles with small size that showed renal clearable behavior for imaging-guided photodynamic therapy (PDT).

Methods

A carboxyl group on pyropheoporbide-a (Pa) was first activated with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC), then an excess amount of a single terminated NH₂ polyethylene glycol (1:1.5, Pa:PEG) was added to ensure the entire carboxylic group of Pa was occupied with PEG. Finally, PEGylated-Pa (Pa-PEG) was obtained after dialysis against DI water with the size of ~2 nm.

Results/Discussion

Pa-PEG nanodots exhibited high singlet oxygen generation upon irradiation with 660 nm lamp. In vitro studies revealed good PDT effect of the Pa-PEG nanodots. When 4T1 cells incubated with the Pa-PEG nanodots at concentration as low as 0.25 μM, most of the cells were destroyed after being exposed to the irradiation for 30 min. Utilizing the good optical properties of the Pa-PEG nanodots, in vivo photoacoustic (PA) and fluorescence (FL) imaging techniques were used to assess the proper time for PDT after intravenous injection. Guided by the PA/FL imaging, the nanodots could accumulate in the tumor site and reached up to maximum concentration after 8 h post injection. The tumor bearing balb/c mice were devastated by PDT and the tumor sizes were obviously smaller than the control groups. Moreover, Pa-PEG nanodots showed high PA/FL signals in kidneys implying the ultra-small nanodots could be physically excreted out of the body via renal clearance.

Conclusions

We demonstrated excellent properties of Pa-PEG that can be an in vivo imaging-guide PDT agent with renal clearable function, which could be a good candidate for future clinical translation materials.

References

1. M. Ethirajan, Y. Chen, P. Joshi and R. K. Pandey, Chem. Soc. Rev., 2011, 40, 340–362.
2. L. Cheng, D. Jiang, A. Kamkaew, H. F. Valdovinos, H. J. Im, L. Feng, C. G. England, S. Goel, T. E. Barnhart and Z. Liu, Adv Funct Mater, 2017, 27, 1702928.

Introduction

The introduction of tailored radiation techniques such as image-guided radiotherapy has contributed to improve the therapeutic ratio of radical radiotherapy (RT) for prostate cancer (PCa).

A fraction of irradiated patients experience RT-induced side effects affecting their quality-of-life. A promising approach to minimizing urinary toxicity (UT) is reducing the detrimental RT-induced tissue remodeling using androgen deprivation therapy (ADT).

The main goal of this work is to test whether the anti-inflammatory and anti-fibrotic effect of ADT could lead to diminished anti-tumoral activity of RT.

Methods

An established orthotopic bioluminescent syngeneic mouse model of urothelial carcinoma was used. The model was obtained by instilling 5x10⁴ luciferase expressing MB49 cells into the bladder of 7-weeks old C57BL/6 female mice.

Nine animals were included in the experiment as follow: 3 animals were exposed to 25Gy 10 days after cell inoculation, 3 animals had been administered with Degarelix 10 days before irradiation then exposed to 25 Gy, 3 animals were treated with Degarelix only for 10 days.

RT delivery were performed using the SmART irradiator system, the dose was calculated using a Monte Carlo based planning system and delivered using a box (4 beams) technique. Bioluminescence imaging (BLI) was performed using the IVIS SpectrumCT system within 1-2 days after inoculating the cells.

Results/Discussion

Figure 1 shows a comparisons between the normalized flux obtained using BLI of mice treated with RT+Degarelix, RT only, or Degarelix only.

The normalized flux was calculated by dividing the bioluminescence signal (total flux=photons/sec) after and before RT.

No differences were observed in the normalized flux between RT+Degarelix and RT only; the normalized flux increases when treating the mice only with Degarelix.

These preliminary data show no impact of Degarelix on tumor control and thus no significant difference in terms of antitumoral activity between mices receiving RT only or RT +Degarelix.

Conclusions

In this preliminary work we found using an orthotopic bladder cancer mouse model that ADT does not affect nor reduce the antitumoral activity of RT.

Introduction

Contrast-enhanced ultrasound (US) is an established imaging technique in preclinical routine for the characterization of tumor vascularization and expression of molecular markers on the tumor endothelium. It is known that US contrast agents can be used to permeabilize vessels during sonoporation¹, but no data are available to exclude an influence of diagnostic contrast enhanced US on tumor biology. Therefore, this study should reveal possible influences of diagnostic contrast-enhanced US on tumor biology and therapy outcome after antiangiogenic and antitumoral treatment.

Methods

Female orthotopic 4T1-tumor bearing Balb/c mice (n=40) were allocated randomly to the following groups: (i) no imaging, (ii) isoflurane anesthesia alone, (iii) non-destructive ultrasound (US) or (iiii) destructive US. Animals of each group were subdivided to either receive 10 mg/kg regorafenib or vehicle solution daily. US measurements were performed on day 7, 10 and 14 using phospholipid MB for non-destructive US and VEGFR2-targeting phospholipid MB for destructive US. Impact of anesthesia and US imaging on animal welfare (heartrate, motor coordination, fecal corticosterone) and tumor size were evaluated daily. Before sacrifice lectin was injected intravenously and a histological characterization of tumors concerning vascularization, apoptosis and immune cell infiltration was performed.

Results/Discussion

Longitudinal contrast enhanced ultrasound examinations had no impact on heartrate, motor coordination or fecal corticosterone levels of the animals. Furthermore, no influence of isoflurane anesthesia on tumor growth could be detected in vehicle treated animals, whereas non-destructive and destructive US in vehicle treated animals tended to reduce the tumor growth rate (fig. 1). Histological characterization of tumors showed a reduction in CD31 positive area in regorafenib treated animals, whereas no impact of US imaging on the amount of CD31 positive area could be detected. Surprisingly, the application of diagnostic non-destructive as well as destructive US strongly increased the amount of perfused vessels in vehicle and regorafenib treated animals (fig. 2). Furthermore, initial results on immune cell infiltration indicate a reduced amount of leukocytes in tumors after US imaging in vehicle treated animals.

Conclusions

Although longitudinal US imaging did not increase the burden of the animals during the experiment, diagnostic contrast enhanced US imaging influenced tumor growth, perfusion and immune cell infiltration. Experiments are ongoing to unravel whether these effects are related to the ultrasound, the contrast agent injection or the combination of both.

References

¹Theek B, Baues M, Ojha T et. al. Sonoporation enhances liposome accumulation and penetration in tumors with low EPR J Control Release. 2016 Jun 10; 231: 77–85.

Acknowledgement

This work is supported by Deutsche Forschungsgemeinschaft (DFG) project number 321137804.

Introduction

Amyotrophic lateral sclerosis (ALS) is an adult onset fatal neurodegenerative disease The immune mechanisms in the disease are prevalent. Immunoglobulins G from ALS patients (ALS IgGs) increase intracellular Ca²⁺ in motor neurons, enhance release from synapses onto motor neurons and at the neuromuscular junction [1-3]. We have also shown that ALS IgGs induce excitotoxic synaptic activity in vitro in non- motor neurons [4], and may also affect glial cells (Ca²⁺ homeostasis, vesicle trafficking, and intracellular oxidative status;  5-7).

Methods

The cell cultures of rat astrocytes and microglia treated with ALS and control IgGs, and ATP (as physiological control) were grown on CaF₂ slides. After fixing  cells were and scanned for high resolution synchrotron radiation-based Fourier Transform Infrared (FTIR) micro-spectroscopy with the aperture 10x10μm to achieve single cell resolution. Prinicipal Componenat Analysis (PCA) was used to investigate macromolecular expression and biochemical characteristics of glial cells treated with ALS IgGs from 2 patients (ALS 1 & 2) compared to healthy and disease (polydiscopathy) control IgGs and 0.2 mM ATP action. All the treatments were performed for 20 min or 24 h.

Results/Discussion

The PCA revealed the following prominent observations: 1) in astrocytes only the 20 min treatment gave apparent differences for the lipids (3100 - 2800 cm^-1) and protein and esters (1800-1480 cm^-1) bands between the two ALS patients, and between ALS1 and healthy control and disease control as well; 2) the difference between the two ALS patients was also retained for the carbohydrate and nucleic acids band (1480-900 cm^-1); 3) in microglia after 20 min treatment again more apparent effects were observed as compared to 24 h, thus, most apparent differences were seen in the lipids band with ALS1 IgG compared to the healthy control while 4) microglia from ALS1 and 2 patients differed in the protein and ester band.

Conclusions

Basic changes in glial cell metabolism were revealed as induced by immune humoral factors, that may also serve for better stratification of patients for personalized medicine.

References

1) Appel et al. 1991. Proc. Natl. Acad. Sci. 88: 647; 2) Uchitel et al.1992. Neurology 42: 2175; 3) Mosier et al. 1995. Ann. Neurol. 37, 102; 4) Andjus et al. 1997. J Physiol. 504 ( Pt 1):103; 5) Milošević et al. 2013. Cell Calcium. 54:17; 6) Stenovec  et al. 2011. Acta Physiol (Oxf). 203:457; 7) Milošević et al. 2017. Front Immunol. 8:1619.

Acknowledgement

Supported by EC H2020 MSCA-RISE project No 778405. The experiments were performed at BL01-MIRAS beamline at ALBA AV-2018022751, CALIPSOplus Grant 730872.

Introduction

Ischemic brain injury evokes in situ inflammatory response mediated by Toll-like receptors (TLR) located on the microglia. Different studies of Tlr2-deficient mice assessed Tlr2 function in ischemic brain injury, but showed different, even opposing results, suggesting a dual effect consisting of both increasing the lesion but as well its repair. Therefore, the aim of the current study was to clarify this issue by performing a longitudinal in vivo MRI assessment of the evolution of the ischemic injury in Tlr2-deficient mice.

Methods

Transient middle cerebral artery occlusion (MCAO) was induced in male 2-4 month old Tlr2-deficient (Tlr2-/-) and C57Bl6 (WT) mice for 60 minutes followed by reperfusion. To compare functional consequences between groups, the animals were scored for neurological deficit 7 days prior to MCAO and 1, 2, 3, 7, 14 and 28 days after surgery. At the same time points the animals were imaged by a 7T BioSpec 70/20 USR MRI system. The scans included a high-resolution T2-weighted anatomical scan and a T2-map scan protocol. Segmentation was performed by manual delineation of ipsilateral and contralateral hemisphere and ischemic lesion in ImageJ. At day 28 after MCAO the animals were sacrificed, and the brains were processed for histological analysis.

Results/Discussion

The survival analysis showed lower mortality for Tlr2-/- group compared to WT group. Most of the non-surviving animals were lost during the first 7 days after surgery. The WT non-survivors had significantly higher neurological score than the animals which reached day 28 after surgery, which was not the case for Tlr2-/- mice. The Tlr2-/- group showed higher neurological deficit in the acute phase, bigger lesion volume in the first week, and higher edema index in the first 3 days reaching similar levels as WT at day 7 after surgery. Both groups showed a marked reduction of the ipsilateral cortical and striatal tissue at 14 days after MCAO, progressing over time, more prominently for Tlr2-/-. Longitudinal MRI monitoring revealed that modulated neuroinflammation due to Tlr2 deficiency increases the ischemic lesion, but, interestingly, at the same time enhances the survival rate.

Conclusions

Tlr2 deficiency can be correlated with better survival after ischemic brain injury, regardless of the higher neurological deficit and lesion volume in the acute phase shown by longitudinal in vivo MRI volumetric assessment.

References

1. Bohacek I et al. J Neuroinflammation. 2012, 9:19
2. Ziegler G et al. Biochem Biophys Res Commun 2007, 359:574–579.
3. Tang SC et al. Proc Natl Acad Sci U S A 2007, 104:13798–1380

Acknowledgement

EU European Regional Development Fund, Operational Programme Competitiveness and Cohesion, grant agreement No.KK.01.1.1.01.0007, CoRE – Neuro; Croatian Science Foundation projects RepairStroke (IP-06-2016-1892), and BRADISCHEMIA (UIP-2017-05-8082). The work of doctoral students Anja Barić and Paula Josić has been fully supported by the “Young researchers' career development project – training of doctoral students” of the Croatian Science Foundation funded by the European Union from the European Social Fund. Multimodal imaging was done at Laboratory for Regenerative Neuroscience - GlowLab, University of Zagreb School of Medicine.

Introduction

Cerebral small vessel disease (SVD) is a leading cause of vascular cognitive impairment (VCI) and dementia. Despite characterisation of disease features (micro haemorrhage, infarcts and white matter pathology) the underlying molecular basis of these processes remains poorly understood. Central to SVD pathophysiology is the disruption of the finely tuned interplay between the cells of the neuro-glial-vascular unit. Mutations in the genes COL 4 A1/A2 (collagen IV alpha chains 1 and 2) are known to cause familial, SVD and polymorphisms in these genes are also associated with sporadic SVD risk.

Methods

By utilising mouse models relevant to SVD (COL4a1^SVD mutant), we aim to investigate how mutations in the COL4 gene lead to neuro-glial-vascular alterations, SVD pathology and VCI. Male and female COL4a1^SVD and wild type litter mates (n=10) aged 3-8 months underwent magnetic resonance imaging (MRI) scanning (T2, T2*, SWI, DTI, ASL) to assess structural, white matter and perfusion changes. Subsequently, neurovascular function (NVC) was assessed by measuring blood flow responses to whisker stimulation by laser speckle contrast imaging in the same mice. Following in vivo imaging, mice were sacrificed and brains were extracted for pathological analysis. Histology analysis was used to further assess structural alterations investigated with MRI and characterise SVD-like pathology in the model.

Results/Discussion

Preliminary data from neuroimaging showed white matter alterations and subcortical micro haemorrhages. These changes were most evident on susceptibility-weighted MRI (Figure 1). Pathological investigations showed increases in vascular collagen deposition and fibrinoid necrosis. A fuller characterisation of the model will be presented at the conference.

Conclusions

This work will provide information on the neurovascular unit, structural, white matter and perfusion changes relevant to familial and sporadic SVD.

Introduction

Neuroimaging studies in patients with spinal cord injury (SCI) and animal models of the disease have demonstrated neurodegenerative changes, both rostral and caudal, from the site of the lesion. However, the spatio-temporal evolution of these changes and its relation to the underlying cellular pathology are not well characterized. We aimed to use high resolution anatomical MRI to assess degenerative changes above the level of the lesion after chronic SCI in ex vivo rat specimens.

Methods

Sprague-Dawley rats underwent thoracic contusion (T10) of mild, moderate, severe, or sham severity. Locomotor performance was assessed using the Basso, Bresnahan, and Beattie scoring scale. SCI rats were euthanized 90 days after injury. SCs were dissected above the injury, rostral at cervical (C1-C2) level and caudal at thoracic (T6-T8) level. High resolution images were acquired at a 9.4T small animal MRI scanner (Bruker Co.) using a cryogenic RF coil. Region-of-interest-based analysis of the T₂ transverse relaxation time constant was performed on axial and coronal T₂ maps with ParaVision 6.0.1 Software (Bruker Co.). Left-right and anterior-posterior width as well as tissue surface area were measured on coronal T₁-weighted SC images at cervical (C1, C5) and thoracic (T2, T6) levels.

Results/Discussion

Region-of-interest-based analysis of cervical SC samples revealed significant increase (p<0.05) in T₂ relaxation time constant in the grey matter at T2 level in animals with severe SCI compared to naïve tissue (Figure 1A). No changes occurred in T₂ relaxation time constants at C1 and T6-T8 levels. Severe SCI did not result in width and tissue surface area changes at all levels of the spinal cord (Figure 1B).

Conclusions

Longer T₂ relaxation times may be associated with chronic gliosis in severely injured SCs. The severity of SCI might also influence the course of changes in tissue relaxation times. Thus, we are currently analyzing samples from rats after mild or moderate injury. The tissue composition and cellular changes in normal and pathological tissue with gold standard histology, immunohistochemistry and chemical element imaging will be investigated.

Introduction

Despite progress in acute stroke therapy, patients are at high risk of mortality and disability. Stroke sequelae include drastic post-ischemic neuroimmune responses. Therapeutic approaches targeting α7 nicotinic acetylcholine receptors (α7 nAChRs), involved in the modulation of neuroinflammatory processes, have already been evaluated in small animal models of stroke [1]. However, the understanding of the role of α7 nAChR in stroke is still incomplete.  We therefore aimed to assess α7 nAChRs in stroke-associated neuroinflammation by long-term PET studies in a large animal model of stroke [2].

Methods

The left middle cerebral artery of male merino sheep was permanently occluded (pMCAO, n=10). Cerebral blood flow was determined with [¹⁵O]H₂O and standard stroke MRI sequences were employed to segment the stroke compartments, prior to PET imaging of α7 nAChR using [¹⁸F]DBT10 [3]. Before and on selected time points after pMCAO (3 hours, 7 and 14 days) simultaneous dynamic brain PET/MRI scans (Siemens Biograph mMR, 300 MBq [¹⁸F]DBT10, 120 min) were conducted. Arterial blood sampling and metabolite analysis via RP-HPLC or micellar chromatography was performed at selected time points. Immediately after PET/MRI on day 14, ex vivo brain autoradiography and histopathology were conducted. Triple fluorescence labeling identified endothelial cells, astrocytes, and microglia/macrophages.

Results/Discussion

Baseline scans exhibited a high initial uptake of [¹⁸F]DBT10 in all brain regions (SUV_max: 4-8) followed by a fast washout. Time-to-SUVR_Cb curves reach a plateau at 60-80 min p.i. depending on the brain region. In the pMCAO-sheep, MR and [¹⁵O]H₂O PET imaging at ~3 hrs after surgery revealed findings typically observed in acute ischemic stroke, which was in agreement with a reduction in the uptake of [¹⁸F]DBT10 at days 1 and 7. In contrast, 14 days after surgery the uptake of [¹⁸F]DBT10 in the stroke border was significantly higher in comparison to the contralateral control region (+82%, p<0.01, Fig.). Histopathological investigations confirmed the presence of ischemic necrosis in the infarct core along with a substantial microglia activation and macrophage infiltration.  

Conclusions

The combined PET/MR imaging and histopathological findings in the sheep stroke model support our hypothesis that PET imaging of α7 nAChR using [¹⁸F]DBT10 reflects stroke-related neuroinflammation. To further assess the suitability of α7 nAChR as stroke biomarker and of [¹⁸F]DBT10 PET to noninvasively quantify pathology- and therapy-related changes of this target, additional in-depth evaluation applying this unique stroke model will be performed.

References

[1] Ren, C. et al.  The Protective Effect of Alpha 7 Nicotinic Acetylcholine Receptor Activation on Critical Illness and Its Mechanism. Int. J. Biol. Sci. 2017, 13 (1), 46-56.

[2] Nitzsche, B. et al.  Focal Cerebral Ischemia by Permanent Middle Cerebral Artery Occlusion in Sheep: Surgical Technique, Clinical Imaging, and Histopathological Results. In: Experimental Neurosurgery in Animal Models, Janowski, M., Ed. Springer New York: New York, NY, 2016, 195-225.

[3] Teodoro, R. et al. A promising PET tracer for imaging of α7 nicotinic acetylcholine receptors in the brain: design, synthesis, and in vivo evaluation of a dibenzothiophene-based Radioligand. Molecules 2015, 20 (10), 18387-18421.

Acknowledgement

The authors acknowledge Deutsche Forschungsgemeinschaft (DFG, 1165-2/3) for the financial support.

Introduction

Crohn’s disease (CD), a subtype of inflammatory bowel disease, is caused by immune-mediated inflammation in the gastrointestinal tract. It is known to cause functional [1] and morphological brain changes. In this study, morphological parameters such as volume, cortical thickness and mean intensity among others of each white and grey matter region are used as features for classification. Parcellation of structural MRI of CD patients and controls was done with Freesurfer [2]. Different feature selection approaches were applied to identify features serving as bio-markers to identify CD patients.

Methods

MRI was acquired using a SIEMENS TRIO 3T clinical scanner. 17 CD patients (all responders) underwent 3 different MRI measurements, before treatment (M1), 24 hours after (M2), as well as 27 days (M3) after treatment. 33 controls with matching age and BMI were included.

R packages (Boruta, RandomForest and mixOmics) were used for feature selection and classification. Boruta, a feature selection approach, filters out all features that contribute to the classification between groups. A Random forest model was built on 14 CED patients and 25 controls M1 datasets and the remaining were used as test data. The model was cross-validated 100 times. For comparison, Sparse Partial Least Squares for Discriminant Analysis (sPLS-DA) was alternatively used for combined feature selection and classification.

Results/Discussion

Structures from all Freesurfer atlases were included and the top 50 features from boruta and sPLS-DA were taken into account. The random forest model built on boruta features showed a balance accuracy of 79.1%, 78.3% and 75.6% while the sPLS-DA model read 77.5%, 73.7%, 73.6% on the test dataset for M 1, 2 and 3 respectively.

An extended analysis of the top 50 selected features yielded two major findings.

First, features from the wmparc atlas were highly important (cf. Fig. 1). The intensity of white matter proximal to the cortical structures showed considerable changes indicating neuropil reduction in these structures.

Secondly, the volume of cuneus, precuneus, superior parietal and others were substantially reduced for CD patients (cf. Fig. 2). These volume changes improved after the successful treatment already within 27 days (M3) approaching that of controls.

These changes constitute a biomarker for separating CD patients from controls.

Conclusions

The biomarker to classify between CD patients and controls is mainly comprised of the structures shown in Fig. 2 and the parameters volume and grey value intensity. The linear reduction in accuracy over consecutive measurements show that the CD patients respond to treatment and the anatomy of the brain structures moves back i.e. closer to the control group. A more detailed analysis provides deeper insights into brain changes in CD patients.

References

[1] Hess et al., 2015. Functional Brain Imaging Reveals Rapid Blockade of Abdominal Pain Response Upon Anti-TNF Therapy in Crohn's Disease., Gastroenterology 2015, 149(4):864-6, doi: 10.1053/j.gastro.2015.05.063

[2] Reuter, M., Schmansky, N.J., Rosas, H.D., Fischl, B. 2012. Within-Subject Template Estimation for Unbiased Longitudinal Image Analysis. Neuroimage 61 (4), 1402-1418. http://surfer.nmr.mgh.harvard.edu

Acknowledgement

We thank the Dr. Pfleger Stiftung and the IOIBD for financial support of this study.

Introduction

Fluoroacetate (FACE) has been known to be preferentially incorporated into astrocyte because of its high expression of monocarboxylic transporter and acts as a metabolic toxin by blocking the tricarboxylic acid cycle. Recently, astrocyte has been reported to play an important role in neuronal cell death after ischemic injury in response to intracellular acidosis driven by its emergent lactate production. To investigate changes in glial metabolism during ischemia, we performed PET imaging with [¹⁸F]FACE in rat model of ischemic/reperfusion injury.

Methods

For the experimental model of temporary focal cerebral ischemia, rats were anesthetized with 1.5% isoflurane. The right middle cerebral artery (MCA) was occluded by the insertion of 18-mm silicon-coated nylon suture (4-0) through the proximal external carotid artery into the internal carotid artery and up to the MCA. After 60 min of occlusion, reperfusion of blood was performed by removing the suture. For visualization of FACE uptake, we synthesized FACE labeled with fluorine-18, [¹⁸F]FACE. [¹⁸F]FACE-PET scan was conducted for 60 min under isoflurane anesthesia during occlusion in the right middle cerebral artery. We also analyzed metabolite and subcellular localization of [¹⁸F]FACE in the ischemic region and histological assessment for astrocytic activation.

Results/Discussion

PET imaging clearly showed that [¹⁸F]FACE uptake in the ipsilateral side, where blood flow was reduced to approximately half of pre-occlusion flow, was significantly elevated (approximately 1.5 times) compared with the contralateral side. The kinetic analysis revealed that cellular influx (K1) of [¹⁸F]FACE was similar between the ipsilateral and contralateral sides, while the efflux constant (k2) was significantly slower in the ipsilateral side than in the contralateral side. [¹⁸F]FACE was distributed into cytosolic but not into mitochondrial fraction and almost no [¹⁸F]FACE metabolite was observed. Histological analyses also revealed that [¹⁸F]FACE accumulation was not associated with appearance of reactive astrocyte and breakdown of blood brain barrier. These finding indicate that ischemia may cause changes in function of waste clearance pathway in brain, and suggest that accumulation of [¹⁸F]FACE is caused by efflux transporter dysfunction in the ischemic regions.

Conclusions

We have revealed that [¹⁸F]FACE accumulated in the ischemic brain at early stage of reperfusion injury in PET study with rat transient MCA occlusion model. [¹⁸F]FACE can be a promising PET probe for monitoring the function of waste clearance pathway which is useable for the early detection of cerebral ischemia.

References

Yamauchi H, Kagawa S, Kishibe Y, Takahashi M, Nishii R, Mizuma H, Takahashi K, Onoe H, Higashi T. Increase in [¹⁸F]-fluoroacetate uptake in patients with chronic hemodynamic cerebral ischemia. Stroke 46(9):2669-72, 2015.

Introduction

Diffusion Kurtosis Imaging (DKI) has shown promise in assessing microstructural heterogeneity of gliomas and its degree of diffusion restriction (1-3) by measuring the deviation of diffusivity from a Gaussian distribution. DKI has been used to grade human gliomas (4), assess cellularity (5), structural heterogeneity in human diseases and rodent models (6-8). In this study, we assessed the tumor growth by examining the longitudinal changes in diffusion and kurtosis properties of the intracranial F98 tumor.

Methods

Six F344 female (100-120 g) rats were injected with 50,000 F98 cells 3 mm right and 3 mm posterior from the bregma, and 2.5 mm into the right cortex.

In vivo DKI MRI was carried out on day 8, 11 and 14 post-tumor cell implantation at 9.4T using a respiratory-gated spin echo EPI-DTI sequence (TE/TR=23/2500ms, 5 averages, 4 EPI segments, 38 slices, matrix=128x64, FOV=40x20mm, voxel resolution: 0.3x0.3x0.3mm, b-values=0,1000,2000s/mm², δ/Δ=4/11ms, 15 directions).

Diffusion and kurtosis parametric maps were processed in DKE (Medical University of South Carolina, USA). Whole tumor, peritumoral edema and the contralateral cortex VOIs were manually segmented and the masks were applied to the parametric maps. The median values were extracted from each region and used for statistical analysis.

Results/Discussion

Fig.1 shows a greater mean diffusivity (MD) in the tumor compared to the contralateral cortex. The mean kurtosis (MK) was also higher in the tumor compared to the contralateral cortex. The radial diffusivity followed the same trend (Fig.2). High median diffusivity in the tumor might originate from the high cellular density of the tumoral rim and the heterogeneity of the necrotic core. An increased MK was also observed in higher grade human glioma with higher cellularity (9) and in a traumatic brain injury rat model (10).

Lower fractional anisotropy values measured in the tumor indicate decreased diffusion anisotropy due to a more chaotic tumor cell organization. Kurtosis fractional anisotropy (KFA), representing the anisotropy of the kurtosis tensor, has been described as useful microstructural contrast (11,12). Lower KFA observed in the tumor indicates a much lower degree of tissue organization.

The diffusion kurtosis did not show any significant change with tumor growth.

Conclusions

The observed diffusional kurtosis profiles did separate the tumor from edema and the healthy brain tissue but did not evolve over time and might require the use of complementary imaging methods (e.g. conductivity measurements or time-dependent DTI) allowing for the separation of the intracellular and extracellular compartments, thus providing more insight on the tumoral microenvironment.

References

1.            Fieremans E, Jensen JH, Helpern JA. White matter characterization with diffusional kurtosis imaging. NeuroImage 2011;58(1):177-188.

2.            Jensen JH, Helpern JA, Ramani A, Lu H, Kaczynski K. Diffusional kurtosis imaging: The quantification of non-gaussian water diffusion by means of magnetic resonance imaging. Magnetic Resonance in Medicine 2005;53(6):1432-1440.

3.            Jensen JH, Helpern JA. MRI quantification of non-Gaussian water diffusion by kurtosis analysis. NMR in Biomedicine 2010;23(7):698-710.

4.            Hempel JM, Schittenhelm J, Bisdas S, Brendle C, Bender B, Bier G, Skardelly M, Tabatabai G, Castaneda Vega S, Ernemann U, Klose U. In vivo assessment of tumor heterogeneity in WHO 2016 glioma grades using diffusion kurtosis imaging: Diagnostic performance and improvement of feasibility in routine clinical practice. Journal of Neuroradiology 2018;45(1):32-40.

5.            Liu H, Shen W, Zhang C, Cui Y, Li J, Zhang T, Chen W, Wang D. Diffusion kurtosis imaging evaluating epithelial–mesenchymal transition in colorectal carcinoma xenografts model: a preliminary study. Scientific Reports 2017;7(1):11424.

6.            Hansen B, Shemesh N, Jespersen SN. Fast imaging of mean, axial and radial diffusion kurtosis. NeuroImage 2016;142:381-393.

7.            Ding G, Chen J, Chopp M, Li L, Yan T, Davoodi-Bojd E, Li Q, Davarani SPN, Jiang Q. White matter changes after stroke in type 2 diabetic rats measured by diffusion magnetic resonance imaging. Journal of Cerebral Blood Flow & Metabolism 2015;37(1):241-251.

8.            Chen X-r, Zeng J-y, Shen Z-W, Kong L-m, Zheng W-b. Diffusion Kurtosis Imaging Detects Microstructural Changes in the Brain after Acute Alcohol Intoxication in Rats. BioMed Research International 2017;2017:6.

9.            Van Cauter S, Veraart J, Sijbers J, Peeters RR, Himmelreich U, De Keyzer F, Van Gool SW, Van Calenbergh F, De Vleeschouwer S, Van Hecke W, Sunaert S. Gliomas: Diffusion Kurtosis MR Imaging in Grading. Radiology 2012;263(2):492-501.

10.          Zhuo J, Xu S, Proctor JL, Mullins RJ, Simon JZ, Fiskum G, Gullapalli RP. Diffusion kurtosis as an in vivo imaging marker for reactive astrogliosis in traumatic brain injury. NeuroImage 2012;59(1):467-477.

11.          Hansen B, Jespersen SN. Kurtosis fractional anisotropy, its contrast and estimation by proxy. Scientific Reports 2016;6:23999.

12.          Glenn GR, Helpern JA, Tabesh A, Jensen JH. Quantitative assessment of diffusional kurtosis anisotropy. NMR in Biomedicine 2015;28(4):448-459.

Acknowledgement

Imaging data were obtained at the Centre for Preclinical Imaging (CPI) of the University of Liverpool.

Introduction

The cuprizone model for multiple sclerosis (MS) is increasingly used, since it allows to study both demyelination (after the toxic insult) and the spontaneous remyelination that follows, with no abrupt immune response. However, the integrity of the BBB is still under discussion in this model. While Gd-enhanced MRI has shown no visible alterations of the BBB, [1] others claim to observe minor disruptions, revealed by Evans blue. [2] We have used Gadofluorine-P, an MRI contrast agent especially sensitive to BBB alterations, [3] to further assess the integrity of the BBB by MRI, in this model.

Methods

C57BL/6 mice were fed for 5 weeks with a diet containing 0.2% of Cuprizone, switching to standard (Cuprizone-free) matching diet for additional 5 weeks. Animals were scanned weekly at 11.7 T (Bruker Biospec USR117/16) to obtain T1w-MRI images prior and post (up to 1h) injection of 0.1 mmol/kg of Gadofluorine-P (InvivoContrast GmbH) in the tail vein. A capillary filled with contrast agent solution was included in the field-of-view to normalize signal intensity of temporal series at image analysis stage. Up to 75 µm resollution T2w and DTI imaging was used to correlate areas of de- and remyelination with areas of potential BBB alterations, in this model. All images where analyzed using FSL and Image-J platforms, with self-developed routines.

Results/Discussion

T2 and DTI images revealed areas of intensive demyelination during poisoning with cuprizone, specially at areas containing large white matter tracks, such as the central section corpus callosum (cc) and cerebellum. Increasing remyelination was observed  after discontinuing cuprizone (Fig. 1). The high spatial resolution used helped us to build precise maps of de- and remyelination. Contrarily to what it has been published with conventional MRI contrast agents, we could permeability of the BBB to gadolufluorine-P at different regions in the brain (Fig. 2) , showing 2 peaks of higher % of signal change, one at week 1, after starting the intoxication with Cuprizone, and a second at week 8, 3 weeks after discontinuing Cuprizone. Some regions like the cc showed a 3rd peak at week 5 (maximal demyelination). On-going histological analysis of excised brain tissues should bring some insights on the physiological processes associated with these transient disruptions of the BBB.  

Conclusions

Gadofluorine-P (InvivoContrast GmbH) seems to be a sensitive MRI contrast agent to detect the mild disruptions of the BBB associated to the Cuprizone model of MS.

References

[1] Boretius S, et al. Neuroimage 2012;59:2678-88

[2] Berghoff SA, et al. Nature Commun 2017;8:14241-56

[3] Bendszus M, et al. Brain 2008;131:2341-52

Acknowledgement

Financial support: Spanish State Research Agency, grant SAF2017-87670-R

Introduction

Diffusion fMRI (DfMRI) has been proposed as an alternative to BOLD fMRI. Observed earlier onsets for DfMRI signals suggest a signal mechanism more directly related to neuronal changes [1,2]. However, also vascular contributions to the DfMRI signal have been observed [3] and the DfMRI signal suffers from low signal to noise ratio due to long echo times and strong dephasing gradients. We performed DfMRI and BOLD fMRI at high (250 ms) temporal resolution to assess temporal characteristics upon sensory stimulation. We performed measurements with a CO₂ challenge to assess vascular contributions.

Methods

We acquired GE-BOLD (n=18) and SE diffusion measurements with b=500 and b=1000 (each n=20) in 7 naïve female Fischer rats with electric forepaw stimulation (5 s stimulation, 25 s rest, 9 Hz, 1.5 mA, 1 ms-pulses). All measurements were performed using a 9.4 T small animal MRI and a 1 cm surface coil as receiver coil under medetomidine sedation and ventilation. MR measurements were performed with a TR of 250 ms and resolution of 325 µm x 350 µm x 1.2 mm in 2-3 slices. Parameters for SE diffusion: TE=35.85 ms, δ=2 ms, Δ=15 ms, b-values=500 and 1000, FA=150°, single SE preparation.

Additionally in 3 animals measurements with a CO₂ challenge (5 % CO₂ in inspiratory air for 3 min, 0 % CO₂ for 3 min) and TR of 1 s (b-values=1000, 1500 and 1800) were performed.

Results/Discussion

We observed positive signal changes upon electric stimulation in DfMRI measurements with b=500 (n=14/20) and b=1000 (n=6/20). Mean DfMRI signal amplitudes were larger (b=500 2.7 % and b=1000 2.8%) than mean BOLD fMRI amplitudes (1.1 %) (Fig. 1a). Notably, larger areas were activated in BOLD fMRI. Summed amplitudes weighted by the number of activated voxels were almost four times larger for BOLD fMRI than for DfMRI with b=500 (Fig. 1b).

The mean onset (defined by the first value above baseline after start of the stimulation) for DfMRI with b=500 was 500 ms and for BOLD fMRI 1250 ms after stimulation (Fig. 1), suggesting a non-venous contribution to the DfMRI signal.

BOLD fMRI and DfMRI showed positive signal changes upon inspiration of 5 % CO₂, suggesting also a vascular signal contribution. Amplitudes were 1 ± 0.5 % (n=3) for GE-BOLD, 1.8 ± 0.9 % (n=4) for b=1000 and 1.6 ± 0.7 % (n=5) for b=1500 or 1800 (Fig. 2).

Conclusions

Both, our observed earlier onsets for DfMRI with b=500 and the signal increase upon CO₂ are in line with DfMRI observations in humans [1,3].

Generating sufficient SNR in DfMRI measurements with high b-values remains challenging even at 9.4 T with very sensitive coils. For measurements at high temporal resolution BOLD fMRI remains superior.

References

[1] Darquié A, Poline JB, Poupon C, et al. Transient decrease in water diffusion observed in human occipital cortex during visual stimulation. PNAS 2001;98(16):9391-9395.

[2] Tsurugizawa T, Ciobanu L and Le Bihan D. Water diffusion in brain cortex closely tracks underlying neuronal activity. PNAS 2013;110(28):11636-11641.

[3] Miller KL, Bulte DP, Devlin H, et al. Evidence for a vascular contribution to diffusion FMRI at high b value. PNAS 2007;104(52):20967-20972.

Introduction

It has been described previously that arterial vasodilation can create damped oscillations of blood flow¹. This can result in oscillations in T2*-weighted fMRI signal. Together with other physiological oscillations and signal fluctuations caused by aliasing², these may superimpose the BOLD response. Models for analysis of BOLD data usually ignore such oscillations, and may therefore be prone to producing false results. To characterize the occurrence and shape of these oscillations, we have performed fMRI measurements upon sensory stimulation, with high temporal resolution of 100 ms.

Methods

BOLD fMRI was performed on 12 medetomedine sedated Fischer rats at 9.4 T with single-shot GE-EPI upon electrical paw stimulation (9 Hz, 1ms-pulses, 1.5 mA) using 3 paradigms (ON/OFF: 4/26s, 5/25s, 10/30s). For some measurements we added delays of random length (0–5 s) between repetitions of the basic paradigm.
Using MATLAB, a U-test determined activated voxels, and signal was summed up and averaged over stimulation trials, resulting in time courses of the BOLD responses. A damped oscillation was fitted to these BOLD time courses, according to:
-A•exp(-ct)cos(2πft)+b
Additionally, all time courses were filtered and cropped from start of stimulation until five seconds after stimulation end. Resulting BOLD peaks with and without oscillations were compared using a customized functional t-test³.  

Results/Discussion

52 % of the measurements (n=62) showed periodic signal oscillations (Figure 1). 25 time courses with oscillations could be fitted successfully. The mean frequency of oscillations was highly reproducible (0.20 ± 0.02) Hz and did not differ for the used stimulation lengths.
Measurements (n=21) with random delays between the stimulation periods were analyzed separately. 57 % of these measurements showed oscillations, confirming that oscillations were induced by stimulation.
A simultaneous onset of signal oscillations with the BOLD response may affect signal analysis, if neglecting the contribution of signal oscillations. We therefore compared BOLD profiles for measurements with and without oscillation. No significant differences were observed for all stimulation lengths (Figure 2), indicating that oscillations start only after end of the stimulation period. The observation that oscillation frequency was independent of stimulation length corroborates this result.

Conclusions

In 52 % of our data sensory stimulation gave rise to signal oscillations with a defined frequency of 0.2 Hz. BOLD responses of data with oscillation showed no significant difference from those without oscillation and the frequency was independent from stimulation lengths. Both indicate that the oscillations start after end of the stimulation period. Further investigation into starting point and mechanism of the signal oscillations is warranted.

References

1. Kim JH, Ress D. Arterial impulse model for the BOLD response to brief neural activation. NeuroImage 2016;124:394-408
2. Pais-Roldán P, Biswal B, Scheffler K, et al. Identifying Respiration-Related Aliasing Artifacts in the Rodent Resting-State fMRI. Front. Neurosci.  2018; doi:10.3389/fnins.2018.00788.
3. Ramsay, J., Hooker, G., Spencer, G., 2009. Functional Data Analysis with R and MATLAB, 1st ed. Springer, Dordrecht, Heidelberg, London, New York.

Introduction

The abnormal deposition of fibrillar beta-amyloid (Aβ) deposits in the brain is one of the major hallmarks of Alzheimer’s disease (AD). Here we describe novel 3D high-resolution whole brain imaging methods to quantify brain Aβ fibrils by using non-invasive photoacoustic tomography (PAT) and light-sheet microscopy in aged arcAβ mice, a transgenic mouse model of AD amyloidosis.

Methods

Transgenic arcAβ mice, and non-transgenic littermates of 18-24 months-of-age underwent whole brain imaging of Aβ deposits by using in vivo PAT with CRANAD-2. T2 magnetic resonance imaging were performed on a 7/16 small animal MR Pharmascan for structrual reference. Light-sheet microscopy in cleared brain with luminescent conjugated oligothiophenes that bind with high-affinity to β-sheet structures and histrochemical staining were performed ex vivo.

Results/Discussion

We demonstrate that the probe CRANAD-2 binds in vitro to recombinant Aβ₄₂ fibrils in a quantitative way. We show that in vivo PAT (resolution 100 mm) registered with magnetic resonance imaging successfully detect increased accumulation of fibrillar Aβ deposits in the cortical brain regions of arcAβ mice compared to non-transgenic littermates. Fluorescent microscopy showed co-localization of Thioflavin-S and CRANAD-2 signals in brain tissue slices. Ex vivo selective plane illumination microscopy (resolution 3 mm) using luminescent conjugated oligothiophenes validated the distribution of Aβ deposits in the brain parenchyma and cerebral amyloid angiopathy inside the vessels in cleared whole brain from arcAβ mice.

Conclusions

In conclusion, we demonstrate new high-resolution in vivo/ex vivo imaging platforms for detecting Aβ deposits at whole brain scale in animal model, thereby facilitating mechanistic studies and monitoring treatment targeting at Aβ deposits.

Acknowledgement

This work was funded by the University of Zurich and the ETH Zurich Foundation through a Seed Grant of "University Medicine Zurich/Hochschulmedizin Zürich", from the Olga Mayenfisch Stiftung, Hartmann Muller Stiftung to JK; and University of Zurich Forschungskredit (Nr. FK-17-052), and Synapsis foundation career development award (2017 CDA-03) to RN.

Introduction

Vascular dysfunction such as cerebral blood flow (CBF) decrease is predicted an early event in Alzheimer’s disease (AD) onset¹. Amyloid-beta (Aβ) accumulation in the brain is a hallmark of AD², but whether and how this leads to CBF decrease is unknown. Decreased vessel density could be an underlying link, but the microvasculature is too small for in vivo imaging, and conventional histology does not allow volumetric imaging. Here, we use tissue clearing to image a large 3D volume at microscopic resolution and compare vessel densities in mouse brains with and without Aβ accumulation.

Methods

Shortly before sacrifice, 18 months old APP^swe/PS1^dE9 (TG; n=4) and wild type (WT) littermates (n=5) were injected with methoxy-XO4 and alexa594-lectin to visualize Aβ and vessels respectively. The brains were isolated, 4% PFA-fixed, cut in two and cleared with CUBIC³ (left hemisphere only). A Zeiss laser scanning microscope (LSM7) with a 20x lens (NA=1.0; WD=5.6mm) and a motorized stage allowed full-hemisphere imaging at 3.32um² (9.96um step size). Fluorophores were 2-photon excited (800nm) and their emission separated with filters. The resulting Z-stacks were stitched, shading corrected and vessels were segmented using an Otsu threshold. Vessel density (VD) was calculated in patches of 15x15x5 voxels and compared in different brain regions after registration to the Allen Brain template.

Results/Discussion

Figure 1 shows a maximum intensity projection (MIP) of 5 slices from the LSM data for representative WT (a) and TG (b) examples, together with the resulting vessel segmentation and vessel density images. Methoxy-XO4 bleeding and autofluorescence signal in the red channel were removed by subtracting the green from the red channel for both WT and TG mice. Alexa594 fluorescence signal dropped after around 1mm of depth and was lost after 3mm (not shown), resulting in dropping VD values from 1mm onwards. Quantification was therefore limited to the most superficial 0.75mm. Voxels with a VD higher than 20%, probably representing large vessels, were excluded. Figure 2 shows the result of the VD per genotype for 6 different brain regions. A mixed anova showed that VDs were significantly different per brain region (p=0.01), but not per genotype, despite a trend towards lower VD in the TG group. This is probably due to the low group size.

Conclusions

Here, we show that it is feasible to quantify vessel densities in a large volume and reproducibly in mouse brains using the CUBIC clearing protocol and laser scanning microscopy. The group size is however low, potentially explaining the absence of significant differences between the genotypes. It remains to be elucidated whether a low vessel density could be an underlying link between Aβ accumulation and CBF decrease in Alzheimer's disease.

References

1. Iturria-Medina Y et al., Early role of vascular dysregulation on late-onset Alzheimer's disease based on multifactorial data-driven analysis. Nat Commun. 2016 Jun 21;7:11934.
2. Alzheimer, A. Über eine eigenartige Erkrankung der Hirnrinde. Allgemeine Zeitschrift für Psychiatrie und Psychisch-gerichtliche Medizin 64, 146–148 (1907).
3. Susaki et al., Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc. 2015 Nov;10(11):1709-27.

Acknowledgement

This study was funded by the Dutch National Science Organization (NWO) – Innovational Research Incentives Scheme Vidi (‘Amyloid and vessels’, number 864.13.014).

Introduction

Using MRE¹, we found a reduced brain stiffness in both MS patients² and the EAE mouse model of the disease³, indicating that brain tissue becomes softer during inflammation. However, the mechanisms behind these alterations remain unclear. Remodeling of the extracellular matrix (ECM)⁴ during pathological processes may influence brain stiffness. In both, MS and EAE, a disease-related deposition of the ECM component fibronectin (FN) has been well-documented⁵. Here we have investigated whether FN expression during the peak of EAE may be associated with stiffness alterations of the brain tissue.

Methods

6-8 weeks old, female SJL/J mice were divided into two groups, EAE (n=25, clinical disability recorded everyday) and healthy controls (n=7). In both groups, MRE was performed at a 7-T small-animal scanner (Bruker PharmaScan, Ettlingen, Germany) at the peak of disease. The vibration was generated by an air-cooled electromagnetic Lorentz coil and transferred to the head via a head holder. Acquisition time for the 1mm-midsagittal slices of 128×128 matrix, 25mm² FOV, two averages, eight dynamic scans was 12min. MRE data were processed by 2D-Helmholtz inversion, yielding parameter of the magnitude modulus [Pa] surrogating tissue stiffness. After MRE measurement, mice were sacrificed and gene expression of FN, as well as FN distribution were analyzed by qPCR and immunostaining, respectively.

Results/Discussion

MRE with tomoelastography data processing provided a magnitude image with definitions of the cerebrum/anterior and cerebellum/posterior as regions of interest, a wave image and a magnitude image superimposed on the magnitude image of the mouse brain (Fig. 1 A). During relapsing-remitting EAE, Reduction of brain stiffness correlated with clinical disability (Spearman correlation, p=0.0117, r=-0.6306, Fig. 1 C). Brain magnitude modulus was significantly reduced at peak EAE in comparison to heathy mice (Fig. 1 D), and was more pronounced in the cerebellum, a region with predominant inflammation in this model (Fig. 1 E). Furthermore, FN deposits were found principally in perivascular areas (Fig. 2 A), and the reduction of brain magnitude modulus was associated with an enhanced gene expression of the matrix protein FN (Pearson correlation, p=0.0041, r=-0.9473, Fig. 2 B).

Conclusions

Reduction of cerebellar stiffness during EAE coincides with an increased expression of FN and with aggregation of FN molecules at sites of inflammation. Thus, MRE may represent an excellent non-invasive method to visualize ECM pathological alterations and lesion formation in vivo.

References

1.  Di Ieva, A., et al. Magnetic resonance elastography: a general overview of its current and future applications in brain imaging. Neurosurg Rev 33, 137-145; discussion 145 (2010).

2.  Streitberger, K.J., et al. Brain viscoelasticity alteration in chronic-progressive multiple sclerosis. PLoS One 7, e29888 (2012).

3.  Riek, K., et al. Magnetic resonance elastography reveals altered brain viscoelasticity in experimental autoimmune encephalomyelitis. Neuroimage Clin 1, 81-90 (2012).

4.  Schregel, K., et al. Demyelination reduces brain parenchymal stiffness quantified in vivo by magnetic resonance elastography. Proc Natl Acad Sci U S A 109, 6650-6655 (2012).

5.  Stoffels, J.M., et al. Fibronectin aggregation in multiple sclerosis lesions impairs remyelination. Brain 136, 116-131 (2013).

Acknowledgement

We thank Natascha Asselborn for expert technical assistance. This work was supported by grants of the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) to CID and IS (IN 156/4-1 and SA 901/16-1), and the Federal Ministry of Education and Research to PBS (BMBF; 01EO0801, Center for Stroke Research Berlin). Shuangqing Wang acknowledges financial support from China Scholarship Council.

Introduction

The combination of MR (rather than CT) with PET imaging provides a range of potential advantages, including much better soft tissue contrast and reduced radiation dose. However, quantitative PET requires accurate attenuation correction and that is not directly available from MR images. Ultrashort or zero echo time (ZTE) MRI methods have been introduced since they are expected to give a better respresentation of bone. The objective of this study is to compare the effect of attenuation correction (MRAC) strategies, especially considering the distance from bone as determined by CT.

Methods

Nine patients with cognitive impairment, referred for FDG PETCT (Siemens Biograph-40) were imaged by PETMR (GE 3T Signa, MP24) approximately 30 min later. Default MR attenuation used an atlas method and ZTE MR images were also obtained. The images were processed and compared through an automated protocol:
1. Registration of CT to MR images with Elastix
2. Segmentation of MRI with a combination of BEaST, SPM12 and Elastix for whole brain, brain tissue types (white matter, grey matter and CSF) and specific brain regions
3. PET attenuation correction used CT (Siemens PETCT); atlas-based MRAC (default on GE PET-MR); CT correction of PETMR; ZTE correction of PETMR
4. Analysis of FDG SUV in whole brain and gross tissue types as a function of distance from bone in CT

Results/Discussion

Bland-Altman analysis of PETCT and PETMR (atlas MRAC) data shows a higher FDG SUV in PETMR than PETCT with whole brain mean = 9.7 +/- 6.2% (s.d.). Using CTAC for the PETMR reconstruction gave similar results for whole brain mean = 13.7 +/- 6.5% implying the AC difference is not responsible for the systematic increase in FDG SUV.

Linear regression of whole brain difference against delay between PETCT and PETMR scans for each patient, a positive correlation was found, indicating that this delay contributed to the effect. The largest differences (positive and negative) were found within 5mm of bone. There is also a peak at 60mm due to CSF (since that disappears on analysis of white or grey matter only). The near to bone effects were less with PETMR reconstruction using CT or ZTE-MR for AC.

Conclusions

Higher brain FDG uptake was measured by PETMR than by PETCT. Part of the effect was due to a delay between PETCT and PETMR imaging (correlation with delay time; no whole-brain improvement from co-registered CT data for PETMR AC). In addition, FDG SUV showed substantial differences close to bone, suggesting that the atlas-based MRAC method was inadequate. Quantitative improvements near to bone were observed with co-registered CT or ZTE MR for AC.

References

Sekine-T. et al., Radiology 286: 249 (2018)
Wiesinger-F. et al., Magn. Reson. Med. 80: 1440 (2018)
Khalife-M. Phys. Med. Biol. 62: 7814 (2017)

Introduction

Fibrillar aggregation of misshaped endogenous proteins is the pathological hallmark of dementias. This work intends to characterize these fibrils with Magnetic Resonance Elastography (MRE), a technique already used to study the spatial distribution of sub-voxel obstacles.^1,2 Stiffness changes in transgenic mice have already been reported;^3-5 but the molecular substrate of these changes is not well understood. The purpose of this work is to study the changes induced by fibrils on the brain mechanical properties.

Methods

Four Sprague-Dawley rats were injected with 10µL of pre-formed α-synuclein fibrils at 200µM in the striatum area. The opposite hemisphere received a control injection of PBS. Animals were sacrificed 2 to 4 weeks after injections and the brains were imaged using a Bruker 4.7T scanner with a home-designed volume coil. RARE sequence was used for anatomical imaging. Real part k_r of the complex wave number was extracted using 3D direct curl inversion⁶ from MRE images acquired at 4 excitation frequencies between 800 and 1200 Hz in ROIs delimiting the whole brain, the fibrils injection site and the contralateral site. k_r was fitted using Origin software on a power law, in which exponent y characterizes microstructure of the sample.

Results/Discussion

No significant difference was found between k_r estimated from the three ROIs whatever the frequency investigated (Fig. 1). y was non-significantly superior in average in the α-synuclein ROI (0.80±0.09) compared to the whole brain (0.65±0.07) and the contralateral injection (0.58±0.04) (Fig.2). In two cases, y in the injection ROI was close to y in the whole brain. For two rats, y in PBS seemed close to y in the whole brain, but less for two others. Histological analysis is ongoing to confirm the localization of fibrils. Replication in another batch of animals is planned.

Conclusions

α-synuclein fibrils, without over toxicity and inflammation at the sub-acute stage, could hardly be detected using multi-frequency MRE, which resulting parameter y is sensitive to microarchitecture alteration, as indicated by Schregel et al.⁷  These preliminary results are in line with recent findings in Alzheimer’s transgenic mice suggesting that fibrils alone are not responsible for viscoelasticity changes at 1000Hz.³

References

1. Jugé L, Petiet A, Lambert SA, et al.: Microvasculature alters the dispersion properties of shear waves--a multi-frequency MR elastography study. NMR Biomed 2015; 28:1763–1771.

2. Lambert SA, Näsholm SP, Nordsletten D, et al.: Bridging Three Orders of Magnitude: Multiple Scattered Waves Sense Fractal Microscopic Structures via Dispersion. Phys Rev Lett 2015; 115:094301.

3. Majumdar S, Mishra R, Lazarov O, Klatt D: Early-stage analysis of murine models of Familial Alzheimer’s disease: Preliminary results. 1^st MRE Workshop Berlin, Germany; 2017:25.

4. Murphy MC, Curran GL, Glaser KJ, et al.: Magnetic resonance elastography of the brain in a mouse model of Alzheimer’s disease: initial results. Magn Reson Imaging 2012; 30:535–539.

5. Munder T, Pfeffer A, Schreyer S, et al.: MR elastography detection of early viscoelastic response of the murine hippocampus to amyloid β accumulation and neuronal cell loss due to Alzheimer’s disease. J Magn Reson Imaging 2017; 47:234–245.

6. Sinkus R, Daire J-L, Beers BEV, Vilgrain V: Elasticity reconstruction: Beyond the assumption of local homogeneity. C R Mécanique 2010; 338.

7. Schregel K, Wuerfel E, Garteiser P, et al.: Demyelination reduces brain parenchymal stiffness quantified in vivo by magnetic resonance elastography. Proc Natl Acad Sci U S A 2012; 109:6650–6655.

Acknowledgement

Performed on the PILoT platform, member of France Life Imaging network (ANR-11-INBS-0006).

LABEX PRIMES (ANR-11-LABX-0063) of Univ. Lyon; IDEX (ANR-11-IDEX-0007). PEPS CNRS “Balanced”.

Introduction

The involvement of the innate immune system in regulating inflammatory processes in the CNS is heavily discussed, with potentially detrimental and disease-promoting effects on CNS disorders like Alzheimer Disease (AD). Neutrophils are involved in inflammation and were shown to contribute to AD pathology, however, with ex vivo methods only (Zenaro et al. 2015). The aim of this study was to evaluate an in vivo dual label probe for PET imaging and flow cytometric analysis (FC) to track neutrophil infiltration into the brain of AD-diseased mice without the need for adoptive cell transfer.

Methods

The Ly6G-specific monoclonal antibody (mAb) 1A8 was dually labeled with [⁶⁴Cu]NODAGA for in vivo PET and AF700 for ex vivo FC. Whole body in vivo static PET and anatomic MRI measurements were conducted in 20-months old male transgenic APPPS1 mice and C57BL/6 (WT, n=5) littermates at 3h, 24h and 48h postinjection (pi) of [⁶⁴Cu]NODAGA-AF700-anti-Ly6G (10MBq, 20µg) to investigate neutrophil migration. At 24h pi mice were injected with granulocyte colony stimulating factor (G-CSF, 0.1µg/g bodyweight) to induce neutrophil migration. FC of spleen, bone marrow and brain was performed at 48h pi. For control experiments, Ly6G-specific mAb 1A8 (100µg/kg) was injected to deplete neutrophils in APPPS1 and WT (n=4). Two-tailed Student`s t-test was used, p<0.05 was considered statistically significant.

Results/Discussion

At 48h pi, PET imaging showed a significantly elevated uptake of [⁶⁴Cu]NODAGA-AF700-anti-Ly6G in APPPS1 brains (WT: 0.61±0.07 %ID/cc; APPPS1: 0.75±0.06 %ID/cc, n=5; p=0.01) and APPPS1 spleens (WT: 2.22±0.53 %ID/cc; APPPS1: 3.79± 0.73 %ID/cc; p=0.004) compared to control mice. Ex vivo FC, however, could not confirm in vivo PET results for brain, showing no significant difference in AF700⁺ populations (WT: 5.62±8.54 % of CD45.2; APPPS1: 1.95±1.39 % of CD45.2; p=0.37). No significant differences were detected for spleen and bone marrow. Neutrophil depletion resulted in a robust uptake decrease in PET in spleen and bone marrow compared to non-depleted mice, but no differences in brains between those conditions. Likewise, in FC, neutrophil depletion resulted in cell number reduction in spleen and bone marrow, however, brain values were not affected, even resulting in a higher AF700⁺ population in APPPS1 brains than in WT (WT: 0.20±0.08 % of CD45.2; APPPS1: 1.23±0.56 % of CD45.2; p=0.01).

Conclusions

Using the dual-labeled mAb, neutrophil migration could be tracked in vivo in the CNS and in peripheral organs in AD mice. Correlation with ex vivo FC analysis, however, revealed certain mismatches in the brain while data in the periphery fit. Although a promising first approach to correlate in vivo cell migration with ex vivo analysis was made, this probe needs further optimization such as the use of a different fluorochrome for ex vivo FC.

References

Zenaro, E. et al. Neutrophils promote Alzheimer’s disease–like pathology and cognitive decline via LFA-1 integrin. Nat. Med. 21, 880–886 (2015).