Image Denoising of Low-Dose PET Mouse Scans with Deep Learning: Validation Study for Preclinical Imaging Applicability.

PURPOSE: Positron emission tomography (PET) image quality can be improved by higher injected activity and/or longer acquisition time, but both may often not be practical in preclinical imaging. Common preclinical radioactive doses (10 MBq) have been shown to cause deterministic changes in biological pathways. Reducing the injected tracer activity and/or shortening the scan time inevitably results in low-count acquisitions which poses a challenge because of the inherent noise introduction. We present an image-based deep learning (DL) framework for denoising lower count micro-PET images. PROCEDURES: For 36 mice, a 15-min [18F]FDG (8.15 ± 1.34 MBq) PET scan was acquired at 40 min post-injection on the Molecubes ß-CUBE (in list mode). The 15-min acquisition (high-count) was parsed into smaller time fractions of 7.50, 3.75, 1.50, and 0.75 min to emulate images reconstructed at 50, 25, 10, and 5% of the full counts, respectively. A 2D U-Net was trained with mean-squared-error loss on 28 high-low count image pairs. RESULTS: The DL algorithms were visually and quantitatively compared to spatial and edge-preserving denoising filters; the DL-based methods effectively removed image noise and recovered image details much better while keeping quantitative (SUV) accuracy. The largest improvement in image quality was seen in the images reconstructed with 10 and 5% of the counts (equivalent to sub-1 MBq or sub-1 min mouse imaging). The DL-based denoising framework was also successfully applied on the NEMA-NU4 phantom and different tracer studies ([18F]PSMA, [18F]FAPI, and [68 Ga]FAPI). CONCLUSION: Visual and quantitative results support the superior performance and robustness in image denoising of the implemented DL models for low statistics micro-PET. This offers much more flexibility in optimizing preclinical, longitudinal imaging protocols with reduced tracer doses or shorter durations.

Post Mortem Study on the Effects of Routine Handling and Manipulation of Laboratory Mice.

Routine handling and manipulation of laboratory mice are integral components of most preclinical studies. Any type of handling and manipulation may cause stress and result in physical harm to mice, potentially leading to unintended consequences of experimental outcomes. Nevertheless, the pathological effects of these interventions are poorly documented and assumed to have a negligible effect on experimental variables. In that context, we provide a comprehensive post mortem overview of the main pathological changes associated with routine interventions (i.e., restraint, blood drawing, and intraperitoneal injections) of laboratory mice with an emphasis on presumed traumatic osteoarticular lesions. A total of 1000 mice from various studies were included, with 864 animals being heavily manipulated and 136 being handled for routine husbandry procedures only. The most common lesions observed were associated with blood collection or intraperitoneal injections, as well as a series of traumatic osteoarticular lesions likely resulting from restraint. Osteoarticular lesions were found in 62 animals (61 heavily manipulated; 1 unmanipulated) with rib fractures and avulsion of the dens of the axis being over-represented. Histopathology and micro-CT confirmed the traumatic nature of the rib fractures. While these lesions might be unavoidable if mice are manipulated according to the current standards, intentional training of research personnel on appropriate mouse handling and restraint techniques could help reduce their frequency and the impact on animal wellbeing as well as study reproducibility.

5-[18F]Fluoroalkyl pyrimidine nucleosides: probes for positron emission tomography imaging of herpes simplex virus type 1 thymidine kinase gene expression.

INTRODUCTION: The preliminary in vivo evaluation of novel 5-[(18)F]fluoroalkyl-2'-deoxyuridines ([(18)F]FPrDU, [(18)F]FBuDU, [(18)F]FPeDU; [(18)F]1a-c, respectively) and 2'-fluoro-2'-deoxy-5-[(18)F]fluoroalkyl-1-beta-d-arabinofuranosyl uracils ([(18)F]FFPrAU, [(18)F]FFBuAU, [(18)F]FFPeAU; [(18)F]1d-f, respectively) as probes for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression is described. METHODS: [(18)F]1a-f were successfully synthesized by a rapid and efficient two-step one-pot nucleophilic fluorination reaction using 5-O-mesylate precursors and [(18)F]F(-). For in vivo studies, tumor xenografts were grown in nude mice by implanting RG2 cells stably expressing HSV1-tk (RG2TK+) and wild-type cells (RG2). RESULTS: Biodistribution studies at 2 h pi revealed that the uptake of [(18)F]1a-b and [(18)F]1d-e in RG2TK+ tumors was not significantly different from control tumors. However, [(18)F]1c and [(18)F]1f had an average 1.6- and 1.7-fold higher uptake in RG2TK+ tumors than control RG2 tumors. Blood activity curves for [(18)F]1c and [(18)F]1f highlight rapid clearance of radioactivity in the blood. Dynamic small animal PET (A-PET) imaging studies of tumor-bearing mice with [(18)F]1c and [(18)F]1f showed higher initial uptake (3.5- and 1.4-fold, respectively) in RG2TK+ tumors than in control tumors, with continued washout of activity from both tumors over time. CONCLUSIONS: Biological evaluations suggest that [(18)F]1c and [(18)F]1f may have limited potential for imaging HSV1-tk gene expression due to fast washout of activity from the blood, thus significantly decreasing sensitivity and specificity of tracer accumulation in HSV1-tk-expressing tumors.

In vivo imaging of beta-cell mass in rats using 18F-FP-(+)-DTBZ: a potential PET ligand for studying diabetes mellitus.

UNLABELLED: Recent studies on gene expression of beta-cell mass (BCM) in the pancreas showed that vesicular monoamine transporter 2 (VMAT2) is highly expressed in the BCM (mainly in the islets of Langerhans). Imaging pancreatic BCM may provide an important tool for understanding the relationship between loss of insulin-secreting beta-cells and onset of diabetes mellitus. In this article, 9-fluoropropyl-(+)-dihydrotetrabenazine (FP-(+)-DTBZ), which is a VMAT2 imaging agent, was evaluated as a PET agent for estimating BCM in vivo. METHODS: Organ biodistribution after an intravenous injection of (18)F-FP-(+)-DTBZ (active isomer) and (18)F-FP-(-)-DTBZ (inactive isomer) was evaluated in normal rats. The specificity of uptake of (18)F-FP-(+)-DTBZ was assessed by a pretreatment (3.8 mg of (+)-DTBZ per kilogram and 3.5 mg of FP-(+)-DTBZ per kilogram, intravenously, 5 min prior) or coadministration (2 mg of (+)-DTBZ per kilogram). PET studies were performed in normal rats. RESULTS: The in vivo biodistribution of (18)F-FP-(+)-DTBZ in rats showed the highest uptake in the pancreas (5% dose/g at 30 min after injection), whereas (18)F-FP-(-)-DTBZ showed a very low pancreas uptake. Rats pretreated with FP-(+)-DTBZ displayed a 78% blockade of pancreas uptake. PET studies in normal rats demonstrated an avid pancreas uptake of (18)F-FP-(+)-DTBZ. CONCLUSION: The preliminary data obtained with (18)F-FP-(+)-DTBZ suggest that this fluorinated derivative of DTBZ shows good pancreas specificity and has the potential to be useful for quantitative measurement of VMAT2 binding sites reflecting BCM in the pancreas.

In vivo imaging of vesicular monoamine transporter 2 in pancreas using an (18)F epoxide derivative of tetrabenazine.

OBJECTIVES: Development of imaging agents for pancreatic beta cell mass may provide tools for studying insulin-secreting beta cells and their relationship with diabetes mellitus. In this paper, a new imaging agent, [(18)F](+)-2-oxiranyl-3-isobutyl-9-(3-fluoropropoxy)-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline [(18)F](+)4, which displays properties targeting vesicular monoamine transporter 2 (VMAT2) binding sites of beta cells in the pancreas, was evaluated as a positron emission tomography (PET) agent for estimating beta cell mass in vivo. The hydrolyzable epoxide group of (+)4 may provide a mechanism for shifting biodistribution from liver to kidney, thus reducing the background signal. METHODS: Both (18)F- and (19)F-labeled (+) and (-) isomers of 4 were synthesized and evaluated. Organ distribution was carried out in normal rats. Uptake of [(18)F](+)4 in pancreas of normal rats was measured and correlated with blocking studies using competing drugs, (+)dihydrotetrabenazine [(+)-DTBZ] or 9-fluoropropyl-(+)dihydro tetrabenazine [FP-(+)-DTBZ, (+)2]. RESULTS: In vitro binding study of VMAT2 using rat brain striatum showed a K(i) value of 0.08 and 0.15 nM for the (+)4 and (+/-)4, respectively. The in vivo biodistribution of [(18)F](+)4 in rats showed the highest uptake in the pancreas (2.68 %ID/g at 60 min postinjection). In vivo competition experiments with cold FP-(+)-DTBZ, (+)2, (3.5 mg/kg, 5 min iv pretreatment) led to a significant reduction of pancreas uptake (85% blockade at 60 min). The inactive isomer [(18)F](-)4 showed significantly lower pancreas uptake (0.22 %ID/g at 30 min postinjection). Animal PET imaging studies of [(18)F](+)4 in normal rats demonstrated an avid pancreatic uptake in rats. CONCLUSION: The preliminary results suggest that the epoxide, [(18)F](+)4, is highly selective in binding to VMAT2 and it has an excellent uptake in the pancreas of rats. The liver uptake was significantly reduced through the use of the epoxide group. Therefore, it may be potentially useful for imaging beta cell mass in the pancreas.

Performance evaluation of the MOLECUBES ß-CUBE-a high spatial resolution and high sensitivity small animal PET scanner utilizing monolithic LYSO scintillation detectors.

The MOLECUBES ß-CUBE scanner is the newest amongst commercially available preclinical PET scanners for dedicated small animal imaging. The scanner is compact, lightweight and utilizes a small footprint to facilitate bench-top imaging. It can be used individually, or in combination with the X-CUBE CT scanner, which provides the ability to perform all necessary PET data corrections and provide fully quantitative PET images. The PET detector comprises of an 8 mm thick monolithic LYSO scintillator read-out by an array of 3 mm × 3 mm Hamamatsu silicon photomultipliers. The monolithic scintillator provides the ability to measure depth-of-interaction which aids in the development of such a compact scanner. With a scanner diameter of 7.6 cm and axial length of 13 cm it is suitable for imaging both whole-body mice and rats. This paper presents the design and imaging performance of the ß-CUBE scanner. NEMA NU4-2008 characterization and a variety of phantom and animal imaging studies to demonstrate the quantitative imaging performance of the PET scanner are presented. Spatial resolution of 1 mm is measured with a filtered-back projection reconstruction algorithm at the center of the scanner and DOI measurement helps maintain the excellent spatial resolution over the entire imaging FOV. An absolute peak sensitivity of 12.4% is measured with a 255-765 keV energy window. The scanner demonstrates good count-rate performance, with a peak NEC of 300 kcps and 160 kcps measured with ~900 µCi in the NEMA mouse and rat phantoms, respectively. Imaging data with the NEMA image quality phantom and Micro Derenzo phantoms demonstrate the ability to achieve good image quality and accurate quantitative data. Image uniformity of 7.4% and spill-over ratio of 8% were measured. The superior spatial resolution, excellent energy resolution and sensitivity also provide superior contrast recovery, with ~70% recovery for the 2 mm rods. While current commercial preclinical PET scanners have spatial resolution in the 1-2 mm range, the 1 mm3 volumetric resolution presents significant improvement over current commercially available preclinical PET scanners. In combination with the X-CUBE scanner it provides the ability to perform fully quantitative imaging with spatially co-registered high-resolution 3D PET-CT images.

[18F](2S,4R)4-Fluoroglutamine PET Detects Glutamine Pool Size Changes in Triple-Negative Breast Cancer in Response to Glutaminase Inhibition.

Glutaminolysis is a metabolic pathway adapted by many aggressive cancers, including triple-negative breast cancers (TNBC), to utilize glutamine for survival and growth. In this study, we examined the utility of [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) PET to measure tumor cellular glutamine pool size, whose change might reveal the pharmacodynamic (PD) effect of drugs targeting this cancer-specific metabolic pathway. High glutaminase (GLS) activity in TNBC tumors resulted in low cellular glutamine pool size assayed via high-resolution 1H magnetic resonance spectroscopy (MRS). GLS inhibition significantly increased glutamine pool size in TNBC tumors. MCF-7 tumors, with inherently low GLS activity compared with TNBC, displayed a larger baseline glutamine pool size that did not change as much in response to GLS inhibition. The tumor-to-blood-activity ratios (T/B) obtained from [18F]4F-Gln PET images matched the distinct glutamine pool sizes of both tumor models at baseline. After a short course of GLS inhibitor treatment, the T/B values increased significantly in TNBC, but did not change in MCF-7 tumors. Across both tumor types and after GLS inhibitor or vehicle treatment, we observed a strong positive correlation between T/B values and tumor glutamine pool size measured using MRS (r2 = 0.71). In conclusion, [18F]4F-Gln PET tracked cellular glutamine pool size in breast cancers with differential GLS activity and detected increases in cellular glutamine pool size induced by GLS inhibitors. This study accomplished the first necessary step toward validating [18F]4F-Gln PET as a PD marker for GLS-targeting drugs. Cancer Res; 77(6); 1476-84. ©2017 AACR.

Cerenkov-specific contrast agents for detection of pH in vivo.

UNLABELLED: We report the design, testing, and in vivo application of pH-sensitive contrast agents designed specifically for Cerenkov imaging. Radioisotopes used for PET emit photons via Cerenkov radiation. The multispectral emission of Cerenkov radiation allows for selective bandwidth quenching, in which a band of photons is quenched by absorption by a functional dye. Under acidic conditions, (18)F-labeled derivatives emit the full spectrum of Cerenkov light. Under basic conditions, the dyes change color and a wavelength-dependent quenching of Cerenkov emission is observed. METHODS: Mono- and di-(18)F-labeled derivatives of phenolsulfonphthalein (phenol red) and meta-cresolsulfonphthalein (cresol purple) were synthesized by electrophilic fluorination. Cerenkov emission was measured at different wavelengths as a function of pH in vitro. Intramolecular response was measured in fluorinated probes and intermolecular quenching by mixing phenolphthalein with (18)F-FDG. Monofluorocresol purple (MFCP) was tested in mice treated with acetazolamide to cause urinary alkalinization, and Cerenkov images were compared with PET images. RESULTS: Fluorinated pH indicators were produced with radiochemical yields of 4%-11% at greater than 90% purity. Selective Cerenkov quenching was observed intramolecularly with difluorophenol red or monofluorocresol purple and intermolecularly in phenolphthalein (18)F-FDG mixtures. The probes were selectively quenched in the bandwidth closest to the indicator's absorption maximum (λmax) at pHs above the indicator pKa (the negative logarithm of the acid dissociation constant). Addition of acid or base to the probes resulted in reversible switching from unquenched to quenched emission. In vivo, the bladders of acetazolamide-treated mice exhibited a wavelength-dependent quenching in Cerenkov emission, with the greatest reduction occurring near the λmax. Ratiometric imaging at 2 wavelengths showed significant decreases in Cerenkov emission at basic pH and allowed the estimation of absolute pH in vivo. CONCLUSION: We have created contrast agents that selectively quench photons emitted during Cerenkov radiation within a given bandwidth. In the presence of a functional dye, such as a pH indicator, this selective quenching allows for a functional determination of pH in vitro and in vivo. This method can be used to obtain functional information from radiolabeled probes using multimodal imaging. This approach allows for the imaging of nonfluorescent chromophores and is generalizable to any functional dye that absorbs at suitable wavelengths.

Oncogene pathway activation in mammary tumors dictates FDG-PET uptake.

Increased glucose utilization is a hallmark of human cancer that is used to image tumors clinically. In this widely used application, glucose uptake by tumors is monitored by positron emission tomography of the labeled glucose analogue 2[(18)F]fluoro-2-deoxy-D-glucose (FDG). Despite its widespread clinical use, the cellular and molecular mechanisms that determine FDG uptake--and that underlie the heterogeneity observed across cancers-remain poorly understood. In this study, we compared FDG uptake in mammary tumors driven by the Akt1, c-MYC, HER2/neu, Wnt1, or H-Ras oncogenes in genetically engineered mice, correlating it to tumor growth, cell proliferation, and expression levels of gene involved in key steps of glycolytic metabolism. We found that FDG uptake by tumors was dictated principally by the driver oncogene and was not independently associated with tumor growth or cellular proliferation. Oncogene downregulation resulted in a rapid decrease in FDG uptake, preceding effects on tumor regression, irrespective of the baseline level of uptake. FDG uptake correlated positively with expression of hexokinase-2 (HK2) and hypoxia-inducible factor-1α (HIF1α) and associated negatively with PFK-2b expression and p-AMPK. The correlation between HK2 and FDG uptake was independent of all variables tested, including the initiating oncogene, suggesting that HK2 is an independent predictor of FDG uptake. In contrast, expression of Glut1 was correlated with FDG uptake only in tumors driven by Akt or HER2/neu. Together, these results demonstrate that the oncogenic pathway activated within a tumor is a primary determinant of its FDG uptake, mediated by key glycolytic enzymes, and provide a framework to interpret effects on this key parameter in clinical imaging.

Dextran coated bismuth-iron oxide nanohybrid contrast agents for computed tomography and magnetic resonance imaging.

Bismuth nanoparticles have been proposed as a novel CT contrast agent, however few syntheses of biocompatible bismuth nanoparticles have been achieved. We herein report the synthesis of composite bismuth-iron oxide nanoparticles (BION) that are based on a clinically approved, dextran-coated iron oxide formulation; the particles have the advantage of acting as contrast agents for both CT and MRI. BION were synthesized and characterized using various analytical methods. BION CT phantom images revealed that the X-ray attenuation of the different formulations was dependent upon the amount of bismuth present in the nanoparticle, while T2-weighted MRI contrast decreased with increasing bismuth content. No cytotoxicity was observed in Hep G2 and BJ5ta cells after 24 hours incubation with BION. The above properties, as well as the yield of synthesis and bismuth inclusion efficiency, led us to select the Bi-30 formulation for in vivo experiments, performed in mice using a micro-CT and a 9.4 T MRI system. X-ray contrast was observed in the heart and blood vessels over a 2 hour period, indicating that Bi-30 has a prolonged circulation half-life. Considerable signal loss in T2-weighted MR images was observed in the liver compared to pre-injection scans. Evaluation of the biodistribution of Bi-30 revealed that bismuth is excreted via the urine, with significant concentrations found in the kidneys and urine. In vitro experiments confirmed the degradability of Bi-30. In summary, dextran coated BION are biocompatible, biodegradable, possess strong X-ray attenuation properties and also can be used as T2-weighted MR contrast agents.

Reduction of nanoparticle avidity enhances the selectivity of vascular targeting and PET detection of pulmonary inflammation.

Targeting nanoparticles (NPs) loaded with drugs and probes to precise locations in the body may improve the treatment and detection of many diseases. Generally, to achieve targeting, affinity ligands are introduced on the surface of NPs that can bind to molecules present on the cell of interest. Optimization of ligand density is a critical parameter in controlling NP binding to target cells, and a higher ligand density is not always the most effective. In this study, we investigated how NP avidity affects targeting to the pulmonary vasculature, using NPs targeted to ICAM-1. This cell adhesion molecule is expressed by quiescent endothelium at modest levels and is upregulated in a variety of pathological settings. NP avidity was controlled by ligand density, with the expected result that higher avidity NPs demonstrated greater pulmonary uptake than lower avidity NPs in both naive and pathological mice. However, in comparison with high-avidity NPs, low-avidity NPs exhibited several-fold higher selectivity of targeting to pathological endothelium. This finding was translated into a PET imaging platform that was more effective in detecting pulmonary vascular inflammation using low-avidity NPs. Furthermore, computational modeling revealed that elevated expression of ICAM-1 on the endothelium is critical for multivalent anchoring of NPs with low avidity, while high-avidity NPs anchor effectively to both quiescent and activated endothelium. These results provide a paradigm that can be used to optimize NP targeting by manipulating ligand density and may find biomedical utility for increasing detection of pathological vasculature.

Endothelial targeting of polymeric nanoparticles stably labeled with the PET imaging radioisotope iodine-124.

Targeting of therapeutics or imaging agents to the endothelium has the potential to improve specificity and effectiveness of treatment for many diseases. One strategy to achieve this goal is the use of nanoparticles (NPs) targeted to the endothelium by ligands of protein determinants present on this tissue, including cell adhesion molecules, peptidases, and cell receptors. However, detachment of the radiolabel probes from NPs poses a significant problem. In this study, we devised polymeric NPs directly labeled with radioiodine isotopes including the positron emission tomography (PET) isotope (124)I, and characterized their targeting to specific endothelial determinants. This approach provided sizable, targetable probes for specific detection of endothelial surface determinants non-invasively in live animals. Direct conjugation of radiolabel to NPs allowed for stable longitudinal tracking of tissue distribution without label detachment even in an aggressive proteolytic environment. Further, this approach permits tracking of NP pharmacokinetics in real-time and non-invasive imaging of the lung in mice using micro-PET imaging. The use of this strategy will considerably improve investigation of NP interactions with target cells and PET imaging in small animals, which ultimately can aid in the optimization of targeted drug delivery.

NEMA NU 4-2008 comparison of preclinical PET imaging systems.

UNLABELLED: The National Electrical Manufacturers Association (NEMA) standard NU 4-2008 for performance measurements of small-animal tomographs was recently published. Before this standard, there were no standard testing procedures for preclinical PET systems, and manufacturers could not provide clear specifications similar to those available for clinical systems under NEMA NU 2-1994 and 2-2001. Consequently, performance evaluation papers used methods that were modified ad hoc from the clinical PET NEMA standard, thus making comparisons between systems difficult. METHODS: We acquired NEMA NU 4-2008 performance data for a collection of commercial animal PET systems manufactured since 2000: microPET P4, microPET R4, microPET Focus 120, microPET Focus 220, Inveon, ClearPET, Mosaic HP, Argus (formerly eXplore Vista), VrPET, LabPET 8, and LabPET 12. The data included spatial resolution, counting-rate performance, scatter fraction, sensitivity, and image quality and were acquired using settings for routine PET. RESULTS: The data showed a steady improvement in system performance for newer systems as compared with first-generation systems, with notable improvements in spatial resolution and sensitivity. CONCLUSION: Variation in system design makes direct comparisons between systems from different vendors difficult. When considering the results from NEMA testing, one must also consider the suitability of the PET system for the specific imaging task at hand.