The Use of a Non-Conventional Long-Lived Gallium Radioisotope 66Ga Improves Imaging Contrast of EGFR Expression in Malignant Tumours Using DFO-ZEGFR:2377 Affibody Molecule

Maryam Oroujeni¹, Tianqi Xu¹, Katherine Gagnon^2,3, Sara S. Rinne³, Jan Weis⁴, Javad Garousi¹, Ken G. Andersson⁵, John Löfblom⁵, Anna Orlova^3,6, Vladimir Tolmachev^1,6

Effects of administration route on uptake kinetics of ¹⁸F‑sodium fluoride positron emission tomography in mice

 Zaniah N. Gonzalez‑Galofre^1,2, Carlos J. Alcaide‑Corral^1,2 & Adriana A. S. Tavares^1,2

¹British Heart Foundation/University of Edinburgh Centre for Cardiovascular Science

²Edinburgh Imaging, University of Edinburgh, Little France Campus, Edinburgh

³British Heart Foundation/University of Edinburgh Centre for Cardiovascular Science

 https://doi.org/10.1038/s41598-021-85073-0

Summary

¹⁸F-sodium fluoride (¹⁸F-NaF) is a positron emission tomography (PET) radiotracer widely used in skeletal imaging and has also been proposed as a biomarker of active calcification in atherosclerosis. Like most PET radiotracers, ¹⁸F-NaF is typically administered intravenously. 

However, in small animal research intravenous administrations can be challenging, because partial paravenous injection is common due to the small calibre of the superficial tail veins and repeat administrations via tail veins can lead to tissue injury therefore limiting the total number of longitudinal scanning points. In this paper, the feasibility of using intra-peritoneal route of injection of 18F-NaF to study calcification in mice was studied by looking at the kinetic and uptake profiles of normal soft tissues and bones versus intravascular injections.

In soft tissue, the ¹⁸F-NaF perfusion phase changes depending on the type on injection route, whereas the uptake phase is similar regardless of the administration route. In bone tissue standardised uptake value (SUV), standardised uptake value ratio (SUVr) and uptake constant (Ki) measures were not significantly different between the three administration routes.

 Authors have concluded that, intra-peritoneal injection is a valid and practical alternative to the intra-vascular injections in small-animal ¹⁸F-NaF PET imaging and provides equivalent pharmacokinetic data. Furthermore, their data show that CT outcomes report on sites of stablished calcification whereas PET measures sites of higher complexity and active calcification.

Results from nanoScan PET/CT

Mice received an intra-vascular or intra-peritoneal injection of ¹⁸F-NaF as follows: intra-arterial administration (8.68 ± 3.94 MBq, mean ± SD, n = 7) via femoral artery; intra-venous administration via femoral vein (7.43 ± 4.69 MBq, mean ± SD, n = 6); and intra-peritoneal administration (7.85 ± 2.18 MBq, mean ± SD, n = 6). Immediately post-radiotracer administration, a 60 min whole-body emission scan was obtained. PET data was reconstructed into 6 × 30 s, 3 × 60 s, 2 × 120 s, 10 × 300 s frames using Mediso’s iterative Tera-Tomo 3D reconstruction algorithm and the following settings: 4 iterations, 6 subsets, full detector model, low regularisation, spike filter on, voxel size 0.4 mm and 400–600 keV energy window.

Figure 1.  Representative examples of biodistribution of ¹⁸F-sodium fluoride (NaF) after femoral artery, femoral vein and intra peritoneal injections presented as average sagittal section images between 45 and 60 min post-injection (A). Mean time-activity curves of the vena cava (B), heart (C), lungs (D), liver (E) and kidneys (F) after femoral, artery, femoral vein or intraperitoneal (IP) injection. Error bars represent one SEM. SUV standardised uptake value.

An In Vivo Study of a Rat Fluid-Percussion-Induced Traumatic Brain Injury Model with ¹¹C-PBR28 and ¹⁸F-flumazenil PET Imaging

Krishna Kanta Ghosh¹, Parasuraman Padmanabhan^1,2, Chang-Tong Yang^1,3,4, Zhimin Wang¹, Mathangi Palanivel¹ , Kian Chye Ng⁵, Jia Lu⁵, Jan Carlstedt-Duke⁶, Christer Halldin^1,7 and Balázs Gulyás^1,2,7

1 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore

2 Cognitive Neuroimaging Centre, Nanyang Technological University, Singapore

3 Department of Nuclear Medicine and Molecular Imaging, Radiological Sciences Division, Singapore General Hospital, Singapore

4 Duke-NUS Medical School, Singapore

5 DSO National Laboratories (Kent Ridge), Singapore;

6 President’s Office, Nanyang Technological University, Singapore;

7 Department of Clinical Neuroscience, Karolinska Institute, Sweden

https://doi.org/10.3390/ijms22020951

Summary

Traumatic brain injury (TBI) modelled by lateral fluid percussion-induction (LFPI) in rats is a widely used experimental rodent model to explore and understand the underlying cellular and molecular alterations in the brain caused by TBI in humans. The present study aims to investigate whether the two adioligands, ¹¹C-PBR28 and ¹⁸F-flumazenil, are able to accurately quantify in vivo molecular-cellular changes in a rodent TBI-model for two different biochemical targets of the processes. As ¹¹C-PBR28 is a radioligand of the 18 kD translocator protein (TSPO), the up-regulation of which is coupled to the level of neuroinflammation in the brain, and ¹⁸F-flumazenil is a radioligand for GABAA-benzodiazepine receptors, whose level capable of indicating at a high precision the neuronal loss that ensues in various brain disorders and injuries, the use of the two radioligands may reveal two critical features of TBI. An up-regulation in the ¹¹C-PBR28 uptake triggered by the LFP in the injured (right) hemisphere was noted on day 14, while the uptake of ¹⁸F-flumazenil was down-regulated on day 14. When comparing the left (contralateral) and right (LFPI) hemispheres, the differences between the two in neuroinflammation were obvious. In vitro immunohistochemical analyses on the corpus callosum and hippocampal sections of the cerebrum were done to validate the results obtained in the PET imaging Results demonstrate a potential way to measure the molecular alterations in a rodent-based TBI model using PET imaging with ¹¹C-PBR28 and ¹⁸F-flumazenil. These radioligands are promising options that can be eventually used in exploring the complex in vivo pharmacokinetics and delivery mechanisms of nanoparticles in TBI treatment.

Results from nanoScan PET/MRI

According to a standard LFP procedure, a combination of focal and diffuse injury was inflicted on the cerebral cortex and hippocampus of the right hemisphere of rats to create a TBI model for the study. The left hemisphere served as an internal control for the study. Following the LFP procedure on the brain’s right hemisphere of 12 Sprague Dawley rats, day 2 post operation 3D dynamic PET scans were performed using the nanoScan PET/MRI scanner. After injecting approximately 30±4MBq ¹¹C-PBR28 or 18±4MBq ¹⁸F-flumazenil to the tail vein, dynamic 63min PET scan was performed with ¹¹C-PBR28. The second PET radioligand ¹⁸F-flumazenil was injected 80min later, and the animals were scanned for a duration of 90min. The detailed time frames for the respective scan protocols were as follows: 8x15s, 4x30s, 2x1min, 2x2min, 4x5min, 3x10min for ¹¹C-PBR28; 8x15s, 4x30s, 2x1min, 2x2min, 4x5min and 6x10min for ¹⁸F-flumazenil.

Results show:

• Compared to day 2 post-op, there is an increase in the uptake of ¹¹C-PBR28 on day 14 due to the LFP in the right hemisphere (injured)
• ¹⁸F-flumazenil uptake was down-regulated on day 14, compared to day 2
• The time activity curves (TACs) of the whole brain also clearly demonstrate that there was a higher ¹¹C-PBR28 and lower ¹⁸F-flumazenil uptake in day 14 as compared to day 2

• When juxtaposing the right and left hemispheres using an area-under-the-curve (AUC) measure, the discrepancies between the two hemispheres for the ¹¹C-PBR28 radiotracer were apparent. This is an indication that local increases in neuroinflammation due to the physical impact can be observed in the LFPI TBI rodent model. On the other hand, while ¹⁸F-flumazenil uptake is slightly higher in the left hemisphere, the lack of marked changes between the two hemispheres may reflect either the lack of neuron density alterations or the inappropriateness of the radioligand in indicating neuron density changes. This is the first LFPI TBI rat study to evaluate neuroinflammation and loss of neuronal density using two radioligands subsequently on the same day.

Myocardial perfusion recovery induced by an a-calcitonin gene-related peptide analogue

Simon Bentsen, MD¹, Anette Sams, PhD², Philip Hasbak, MD, DMSc¹, Lars Edvinsson, MD, PhD, DMSc², Andreas Kjaer, MD, PhD, DMSc¹, Rasmus S. Ripa, DMSc¹

¹Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Rigshospitalet and University of Copenhagen, Copenhagen, Denmark
²Department of Clinical Experimental Research, Glostrup Research Institute, Glostrup University Hospital, Glostrup, Denmark

https://doi.org/10.1007/s12350-021-02678-8

Summary
Myocardial infarction (MI) is the leading cause of heart failure, despite the introduction of primary percutaneous coronary intervention (PCI). PCI has improved survival after an MI, but the recovering myocardium is still impaired after PCI. Therefore, it is crucial to develop new treatments to protect and improve myocardial recovery after an MI.
After the onset of an MI, there is insufficient oxygen supply to the myocytes. To protect the myocytes prior to PCI, it is crucial to reduce cardiac workload. Vasodilation of the coronary arteries, the peripheral arterial bed, and venous capacitance vessels is a known and used method of reducing afterload and the myocardial workload. However, peripheral vasodilation can increase the workload of the heart because of compensatory elevated heart rate, which could lead to increased ischemic damage. Calcitonin gene-related peptide (CGRP) is one of the most potent vasodilators known.
Exogenous CGRP has been shown to induce cardioprotective effects in isolated hearts and CGRP antagonism reduces the cardioprotective effects. Also, exogenous CGRP induces positive chronotropic and inotropic effects on isolated guinea pig hearts and isolated human myocardial trabeculae. In three clinical studies with CGRP infusion for the treatment of cerebral vasospasm, adverse effects of increased heart rate and peripheral hypotension were observed, and the studies were stopped before completion.
Recently, a metabolically stable CGRP analogue, SAX, with a lipophilic tail has been synthesized. This analogue reverses hypertension and complications of hypertension in experimental animal studies. Data show that SAX and CGRP display similar pharmacological actions, but the potency of peripheral vasodilation induced by SAX is approximately 10 times lower than native CGRP.
The aim of this study was to examine a potential cardioprotective effect of SAX in rats undergoing experimental acute myocardial infarction by permanent left anterior descending (LAD) occlusion. The authors hypothesized that SAX will increase perfusion of the acutely hypoperfused area without promoting increased workload on the heart and that this will cause improved recovery of the myocardial perfusion.

Results from the nanoScan SPECT/CT
The rats underwent acute SPECT/CT scan one hour after surgery (to determine baseline non-perfused area). In Sprague-Dawley rats, [99mTc]Tc-sestamibi clears from the blood rapidly and is taken up in the heart with very little redistribution. Therefore, the injection of [99mTc]Tc-sestamibi 20 min before injection of SAX or placebo enables the detection of the non-perfused area before any potential impact of the first SAX injection. Three weeks after surgery, the rats were follow-up scanned with SPECT/CT. This enabled a determination of final infarct size. All imaging was performed on the Mediso nanoScan SPECT/CT scanner.
The rats were anesthetized with sevoflurane 4% for the SPECT/CT scan. On the day of the surgery, the rats were perioperatively injected with [99mTc]Tc-sestamibi (median 106 mBq[99; 116]), as described above. Twenty-one days after surgery, the rats were anesthetized with sevoflurane 4%. A 24G intravenous catheter was placed in the tail vein (Vasofix Safety, Braun, Denmark). The rats were then injected with [99mTc]Tc-sestamibi (median 122 mBq[106; 129]). During the SPECT/CT scan, the rats were placed on MultiCell Rat bed, in a prone position. The rats were monitored by way of electrocardiography (ECG), respiratory rate, and core temperature. A computed tomography (CT) scout image was obtained to ensure correct positioning of the SPECT detector’s field of view over the heart. One hour after injection of [99mTc]Tc-sestamibi, the SPECT acquisition was initiated. Scan time was between 20 and 40 min, depending on injected activity, adjusted to ensure a number of counts above 100,000 counts/frame/detector. For attenuation correction and anatomical co-registration, a CT scan was acquired after the SPECT. All images were reconstructed using vendor software (Mediso Nanoscan, Hungary) and vendor-recommended parameters.
Figure 1. shows the aboved mentioned workflow of the experiment:

Figure 2. shows two representative examples of rats with chronic LAD occlusion from the SPECT/CT scans. 4DM software was used to analyze [^99mTc]Tc-sestamibi SPECT/CT. Top panels (A and B) show a SAX-treated rat. Bottom panels (C and D) show a placebo-treated rat.(A) Large perfusion defect in the anterior and apical wall in the acute scan, with a smaller perfusion defect at follow-up scan after SAX treatment (white arrow).(B) The perfusion defects from panel A in a 17-segment polar map.(C) Medium perfusion defect in the anterior wall at the acute scan, and a more severe perfusion defect at follow-up after placebo treatment (red arrow).(D) The perfusion defects from panel C in a 17-segment polar map. HLA horizontal long axis, VLA vertical long axis, SRS summed rest score.

• The results show that an analogue of CGRP that induces both coronary and peripheral vasodilation significantly improves myocardial perfusion recovery after experimental myocardial infarction in rats. There was no significant difference in overall survival between the two groups suggesting that SAX does not have a damaging effect on the animals or the myocardium.
• In conclusion, the CGRP analogue, SAX, seems to have a cardioprotective effect on a rat model of myocardial infarction, by improving the perfusion recovery after a chronic occlusion of the coronary artery.

One Size Fits All? Not in In Vivo Modeling of Tuberculosis Chemotherapeutics

 Hee-Jeong Yang¹, Decheng Wang^{2 , 3} , Xin Wen^{2 , 3} ,Danielle M. Weiner^{1 , 4} and Laura E. Via^{1 , 4 , 5 , *}

¹ Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research (DIR), National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States,

² Medical College, China Three Gorges University, Yichang, China,

³ Institute of Infection and Inflammation, China Three Gorges University, Yichang, China,

⁴ Tuberculosis Imaging Program, DIR, NIAID, NIH, Bethesda, MD, United States,

⁵ Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa

 doi: 10.3389/fcimb.2021.613149

Abstract

Tuberculosis (TB) remains a global health problem despite almost universal efforts to provide patients with highly effective chemotherapy, in part, because many infected individuals are not diagnosed and treated, others do not complete treatment, and a small proportion harbor Mycobacterium tuberculosis (Mtb) strains that have become resistant to drugs in the standard regimen. Development and approval of new drugs for TB have accelerated in the last 10 years, but more drugs are needed due to both Mtb’s development of resistance and the desire to shorten therapy to 4 months or less. The drug development process needs predictive animal models that recapitulate the complex pathology and bacterial burden distribution of human disease. The human host response to pulmonary infection with Mtb is granulomatous inflammation usually resulting in contained lesions and limited bacterial replication. In those who develop progressive or active disease, regions of necrosis and cavitation can develop leading to lasting lung damage and possible death. This review describes the major vertebrate animal models used in evaluating compound activity against Mtb and the disease presentation that develops. Each of the models, including the zebrafish, various mice, guinea pigs, rabbits, and non-human primates provides data on number of Mtb bacteria and pathology resolution. The models where individual lesions can be dissected from the tissue or sampled can also provide data on lesion-specific bacterial loads and lesion-specific drug concentrations. With the inclusion of medical imaging, a compound’s effect on resolution of pathology within individual lesions and animals can also be determined over time. Incorporation of measurement of drug exposure and drug distribution within animals and their tissues is important for choosing the best compounds to push toward the clinic and to the development of better regimens. We review the practical aspects of each model and the advantages and limitations of each in order to promote choosing a rational combination of them for a compound’s development. 

Results from MultiScan LFER PET/CT

Tuberculosis Imaging group under the direction of Laura E. Via is using 2 MultiScan LFER PET/CT scanner for studying tuberculosis on Rabbit, Common Marmoset and Rhesus Macaque at National Institute of Allergy and Infectious Diseases.

In this substantial comprehensive review, the researchers also compared these 3 animal models in respect of PET/CT imaging:

• In longitudinal studies that combined PET/CT imaging, using FDG injected IV as a probe for metabolic activity, FDG concentrated in metabolically active regions within the Mtb HN878 lesions as they developed, but as necrosis progressed, the acellular centers of granulomas and cavities accumulated less FDG due to the lack of live cells (Figure 3A)
• NHP models are amenable to most diagnostic and therapeutic methods used in clinical studies, such as medical imaging (Figures 3B, C), serial blood sampling, and bronchoalveolar lavage (BAL) sampling for pharmacokinetic monitoring within an individual (Lewinsohn et al., 2006; Lin et al., 2013; Via et al., 2013). As serial imaging is feasible, methods to follow both individual lesion development and regression PI have been developed

Figure 3. FDG PET/CTs of a rabbit (A), marmoset (B), and rhesus macaque (C) with cavitary disease. The animals were infected for 69 to 90 days with M. tuberculosis at the time of imaging. Cavities (blue arrows) have been partially emptied of their necrotic contents and filled with air indicated by a darker central region in the lesions (lower density) surrounded by lighter walls (higher density). The scales show the range of CT Hounsfield units from higher to low density in shades of gray (+400 to −1000) on the left and FDG uptake in PET standard uptake units/body weight from high to low uptake in bright yellow to red to black (14 to 0) on the right. The width of the animal’s midsection is indicated with a bar and label to highlight the difference in size of the three animals.

Para-chloro-2-[18F]fluoroethyl-etomidate: A promising new PET radiotracer for adrenocortical imaging

Isabella Silins¹, Anders Sundin¹, Patrik Nordeman², Mahabuba Jahan², Sergio Estrada², Azita Monazzam³, Mark Lubberink¹, Franklin Aigbirhio⁴, Per Hellman¹, Gunnar Antoni²

¹ Department of Surgical Sciences, Uppsala University

² Medicinal Chemistry and Uppsala University

³ Medical Sciences at Uppsala University

⁴ Wolfson Brain Imaging Centre, University of Cambridge

https://www.medsci.org/v18p2187.htm

Summary

¹¹C-Metomidate (¹¹C-MTO) was developed as a PET radiotracer for adrenocortical tumours and has also been suggested for imaging in primary aldosteronism (PA). However, the use of ¹¹C-MTO is somewhat hampered by considerable accumulation in the liver, which, because of its close proximity to the right adrenal gland, may obscure adrenal pathology and make PET measurements unreliable. Moreover, increased ¹¹C-MTO uptake has been found in various liver lesions, such as adenoma, hepatocellular cancer and focal nodular hyperplasia, with the risk of false positive imaging results. Another disadventage of the tracer is that the clinical availability is restricted because of the short half-life of carbon-11 (T1/2= 20.4 min), which limits its use to PET centres with an in-house cyclotron and radiopharmacy.

The aim of this study was to evaluate the binding properties and in vivo behaviour of the previously published 18F-labeled (halflife 109.5min) etomidate analogue, para-chloro-2-¹⁸F-fluoroethyl etomidate; (¹⁸F-CETO), as an adrenal PET tracer. Comparative studies were also performed with ¹¹C-MTO and with ¹⁸F-FETO, another adrenocortical imaging agent, which has not reached widespread clinical use, partial due to its two-stage radiosynthesis.

Autoradiography on human and cynomolgus monkey tissues show specific, high ¹⁸F-CETO uptake in normal adrenal cortex, as well as in human adrenocortical adenomas and adrenocortical carcinomas.

Following in vitro binding kinetic analysis and the evaluation of ex vivo biodistribution, in vivo imaging studies revealed high specificity of ¹⁸F-CETO accumulation in the adrenal cortex qualitatively surpassing those of ¹¹C-MTO. Non-specific binding to the liver was significantly lower than that of ¹¹C-MTO. ¹⁸F-CETO is a promising new PET tracer for imaging of adrenocortical disease and should be evaluated further in humans.

Results from nanoScan PET/MRI

¹⁸F-CETO imaging in rats and mice:

Eight female C57BL/6 mice and two male Sprague Dawley rats were were injected with of ¹⁸F-CETO (1.6±1MBq or 4-5MBq, respectively) and the 1h long dynamic PET imaging was started immediately. Four of the mice and one of the rats were co-injected with metomidate (1μmol/kg). The PET examination was followed by an MRI acquisition.

• ¹⁸F-CETO accumulated predominantly in the liver and in the adrenal glands, thus obfuscating the view of the adrenal glands in mice (Figure 1A: baseline; B: after blockage with metomidate)
• in contrast in rats the uptake was concentrated mainly in the adrenal glands with 120min p.i. peak adrenal uptake (Figure 2A: baseline; B: after blockage with metomidate)

¹⁸F-FETO imaging in rats:

Rats were were iv. injected with 4.0-4.8 MBq of ¹⁸F-FETO, one of the rats was given metomidate (1μmol/kg) and 1h long PET scan was started immediately.

• ¹⁸F-FETO in rats was also concentrated mainly in the adrenal glands. However, it was unable to block the uptake with metomidate, thus, re-evaluation of ¹⁸F-FETO has to be discontinued (Figure 2C: baseline; D: after blockage with metomidate)

Bispecific GRPR-Antagonistic Anti-PSMA/GRPR Heterodimer for PET and SPECT Diagnostic Imaging of Prostate Cancer

Bogdan Mitran¹, Zohreh Varasteh^1,2, Ayman Abouzayed¹, Sara S. Rinne¹, Emmi Puuvuori¹, Maria De Rosa^1,3, Mats Larhed^1,4, Vladimir Tolmachev⁵, Anna Orlova^1,4, Ulrika Rosenström¹

¹Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden
²Department of Nuclear Medicine, Klinikum rechts der Isar der TUM, 81675 Munich, German
³Drug Discovery Unit, Ri.MED Foundation, 90133 Palermo, Italy
⁴Science for Life Laboratory, Department of Medicinal Chemistry, Uppsala University, 751 23 Uppsala, Sweden
⁵Department of Immunology, Genetics and Pathology, Uppsala University, 751 23 Uppsala, Sweden

https://doi.org/10.3390/cancers11091371

Summary
Until recently, the diagnosis of prostate cancer (PCa) was based on measuring the concentration of prostate-specific antigen (PSA) in blood, a pathological examination of biopsy material, positron emission tomography (PET) imaging of metabolic activity using ¹⁸F-flourodeoxyglucose (FDG) or ¹¹C-acetate, and proliferation activity using radiolabeled choline. Bombesin agonistic analogues for the imaging of gastrin-releasing peptide receptor (GRPR) expression were also proposed, but their clinical implementation was limited due to their strong physiological action.
The development of small-molecular-weight prostate-specific membrane antigen (PSMA)-targeting derivatives of urea was a big breakthrough in the diagnosis of PCa, however despite the apparent success, the imaging of PCa lesions requires further improvement because of the sensitivity of the imaging.
Another relevant target for the diagnostic imaging of PCa is GRPR, which is expressed in earlier stages of PCa. The gallium-68-labeled GRPR antagonist RM2 was used in several clinical studies, with promising results.
In this study, Mitran et al. report the synthesis of a new PSMA/GRPR-targeting heterodimer that includes a Glu-Ureido PSMA-binding moiety and GRPR antagonist RM26 coupled via a glutamic acid bearing NOTA chelator. The aim of this study was to preclinically evaluate the gallium- and indium-labeled heterodimer in terms of the binding properties to PSMA and GRPR, cellular processing, in vivo targeting, and biodistribution, as well as the imaging properties.

Results from the nanoScan PET/MRI 3T and nanoScan SPECT/CT
For the small animal imaging, the authors have used a nanoScan PET/MRI 3T and a nanoScan SPECT/CT, to follow the biodistribution of the the bispecific heterodimeric molecule Glu-Urea-Glu-Aoc-Lys(NOTA)-(PEG)₆-RM26, which was labeled with indium-111 and gallium-68.
Whole body SPECT/CT scans of the mice bearing PC3-PIP xenografts injected with ¹¹¹In-6 (400 kBq, 100 pmol/mouse) were performed using nanoScan SPECT/CT at 1, 3, and 24 h pi. Additionally, three mice were co-injected with either non-labeled PSMA-617 (1.5 nmol), non-labeled NOTA-PEG₆-RM26 (1.5 nmol), or a combination of both, and imaged 1 h pi. CT scans were acquired at the following parameters: 50 keV, 670 μA, 480 projections, and 5 min acquisition time. SPECT scans were carried out using ¹¹¹In energy windows (154 keV–188 keV; 221 keV–270 keV), a 256  ×  256 matrix, and a 30 min acquisition time. The CT raw data were reconstructed using Nucline 2.03 Software. SPECT raw data were reconstructed using Tera-Tomo™ 3D SPECT.
PET/CT scans of the mice injected with ⁶⁸Ga-6 (1.8 MBq, 100 pmol/mouse) were performed using nanoScan PET/MRI 3T at 1 h pi. To evaluate the in vivo specificity, one mouse was co-injected with a combination of non-labeled PSMA-617 (1.5 nmol) and non-labeled NOTA-PEG₆-RM26 (1.5 nmol). CT acquisitions were performed as previously described using nanoScan SPECT/CT immediately after PET acquisition employing the same bed position. PET scans were performed for 30 min. PET data were reconstructed into a static image using the Tera-Tomo™ 3D reconstruction engine. CT data were reconstructed using Filter Back Projection. PET and CT files were fused and analyzed using Nucline 2.03 Software.
Figure 4. shows the main results from the in vivo studies. (A) micro positron emission tomography (microPET)/CT and (B) microSPECT/CT MIP images of PC3-PIP-xenografted mice (PSMA positive/GRPR positive) after an iv injection of ⁶⁸Ga-6 and ¹¹¹In-6. Blocked animals were co-injected with PSMA-617 for the blocking of PSMA and RM26 for the blocking of GRPR, or both.

• The imaging of PC3-PIP tumors using microPET/CT for ⁶⁸Ga-6 (1 h pi) and microSPECT/CT for ¹¹¹In-6 (1, 3, and 24 h pi) confirmed the ex vivo biodistribution data. Tumors were clearly visualized at all time-points, while weak activity accumulation was only observed in the kidneys among healthy organs. In agreement with ex vivo measurements, imaging contrast improved with time up to 3 h pi, despite the rapid washout of activity from tumors. The superior imaging contrast obtained for ¹¹¹In-6 compared to ⁶⁸Ga-6 was also in agreement with the biodistribution data. The imaging of mice after the co-injection of excess PSMA- and GRPR-targeting monomers corroborated with the ex vivo biodistribution pattern: partially decreased activity uptake in tumors, but increased activity uptake in kidneys, were exhibited in the case of PSMA blocking.
• MicroPET/CT and microSPECT/CT scans confirmed biodistribution data, suggesting that ⁶⁸Ga-NOTA-DUPA-RM26 and ¹¹¹In-NOTA-DUPA-RM26 are suitable candidates for the imaging of GRPR and PSMA expression in PCa shortly after administration.

Sigma-1 Receptor Positron Emission Tomography: A New Molecular Imaging Approach Using (S)-(−)-[18F]Fluspidine in Glioblastoma

Magali Toussaint ¹, Winnie Deuther-Conrad ¹, Mathias Kranz ^{1 2 3}, Steffen Fischer ¹, Friedrich-Alexander Ludwig ¹, Tareq A Juratli ⁴, Marianne Patt ⁵, Bernhard Wünsch ⁶, Gabriele Schackert ⁴, Osama Sabri ⁵, Peter Brust ¹

¹ Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Institute of Radiopharmaceutical Cancer Research, Department of Neuroradiopharmaceuticals, Research site Leipzig, 04318 Leipzig, Germany.

² PET Imaging Center, University Hospital of North Norway (UNN), 9009 Tromsø, Norway.

³ Nuclear Medicine and Radiation Biology Research Group, The Arctic University of Norway, 9009 Tromsø, Norway.

⁴ Department of Neurosurgery, Technische Universität Dresden (TUD), University Hospital Carl Gustav Carus, 01307 Dresden, Germany.

⁵ Department of Nuclear Medicine, University Hospital Leipzig, 04318 Leipzig, Germany.

⁶ Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, 48149 Münster, Germany.

https://doi.org/10.3390/molecules25092170

Summary

Glioblastoma multiforme (GBM) is the most common primary tumors of the central nervous system. The survival rate for patients with GBM is dramatically low compared to patients with other brain tumor types. An important aspect contributing to this poor outcome is the genetic heterogeneity of GBM, which translates into heterogeneous expression patterns of potentially druggable targets. Hence the understanding of how spatiotemporal patterns evolve and change during pathogenesis would help to develop new targeted therapies, and biomarkers for treatment response.

The sigma-1 receptor (sig1R), an endoplasmic reticulum chaperone protein, is involved in signaling pathways assumed to control the proliferation of cancer cells and thus could serve as candidate for molecular characterization of GBM. The authors have used a selective sig1R ligand (S)-(−)-[18F]fluspidine to test this hypothesis with PET noninvasive molecular imaging.

In conclusion, the data obtained in the U87-MG mouse model of GBM along with the detection of sig1R in human GBM tissue for the first time by a PET radioligand, indicate not only the relevance of this target but also the suitability of (S)-(−)-[18F]fluspidine for sig1R-targeted cancer research and drug development.

Results from nanoScan PET/MRI

• Dynamic PET imaging showed that the uptake of (S)-(−)-[18F]fluspidine has higher retention in the tumor region compared to the CL at 60 min p.i., with SUVs of 0.38 and 0.28, respectively (Figure 4.)

Figure 4. PET/MR imaging of sig1R in mice with orthotopic xenograft of human GBM cells (U87-MG). Average time-activity curves after i.v. administration of (S)-(−)-[18F]fluspidine of the tumor (red dots) and the contralateral (black squares) regions of interest (n = 3). Statistical test: Student t-test, * p < 0.05.

• The early dynamic PET images between 2 and 9 min after injection show a heterogeneous uptake of (S)-(−)-[18F]fluspidine into the tumor (Figure 5D, upper panel), which may be caused by reduced blood supply to the tumor center. The PET image at later time points (45 to -60 min p.i.; Figure 5D, lower panel) pictures a more homogenous uptake of the tracer, along with a low slope, reflecting an accumulation.

Figure 5. (D) Representatives coronal PET/MR images of U87-MG tumor-bearing mouse after i.v. administration of (S)-(−)-[18F]fluspidine. The upper panel exhibits the distribution of (S)-(−)-[18F]fluspidine at early times p.i. (averaged time frames from 2 to 9 min), and the lower panel exhibits the distribution of (S)-(−)-[18F]fluspidine at later times (averaged time frames from 45 to 60 min). The regions-of-interest (ROIs) were delineated on the T2-weighted MR images and then applied on the PET data to generate the regional TACs.

Simultaneous Visualization of ¹⁶¹Tb- and ¹⁷⁷Lu-Labeled Somatostatin Analogues Using Dual-Isotope SPECT Imaging

Francesca Borgna¹, Patrick Barritt¹, Pascal V. Grundler¹, Zeynep Talip¹, Susan Cohrs¹, Jan Rijn Zeevaart², Ulli Köster³, Roger Schibli^1,4, Nicholas P. van der Meulen^1,5 and Cristina Müller^1,4

¹ Center for Radiopharmaceutical Sciences, Paul Scherrer Institute, 5232 Villigen-PSI, Switzerland

² Radiochemistry, South African Nuclear Energy Corporation (Necsa), Brits 0240, South Africa

³ Institut Laue-Langevin, 38042 Grenoble, France

⁴ Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland

⁵ Laboratory of Radiochemistry, Paul Scherrer Institute, 5232 Villigen-PSI, Switzerland

https://doi.org/10.3390/pharmaceutics13040536

Summary

¹⁷⁷Lu (half-life 6.7d) is currently the most often applied radiometal for therapeutic purposes, as it has a particulate emission (β− or Auger electron) for effecting therapy and emits several accompanying γ-photons of 208 keV (11%) and 113 keV (6.4%), which are used for diagnostic evaluation and dosimetry.

¹⁶¹Tb is a more recently introduced radiolanthanide for therapeutic applications. ¹⁶¹Tb decays with a half-life of 6.89 days to stable ¹⁶¹Dy, while emitting β¯-particles (Eβ͞av = 154 keV) suitable for therapeutic purposes and γ-radiation (Eγ = 49 keV, I = 17.0%; Eγ = 75 keV, I = 10.2%) useful for SPECT imaging. ¹⁶¹Tb also emits a substantial number of low-energy conversion and Auger electrons, which makes this radionuclide exceptionally interesting for the treatment of disseminated cancers with multiple metastases ranging from a single cell (diameter: ~10μm) to micro cell clusters (diameter: < 1mm). Monte Carlo simulations assessed the dose delivered to 10μm spheres revealed a 3.5-fold increased value when using ¹⁶¹Tb as compared to ¹⁷⁷Lu. In larger tumors (diameter > 10mm), the emitted electron energy from ¹⁶¹Tb and ¹⁷⁷Lu respectively is almost entirely absorbed, resulting in a 1.3-fold higher absorbed electron energy fraction per decay for ¹⁶¹Tb compared to ¹⁷⁷Lu, making ¹⁶¹Tb more potent than ¹⁷⁷Lu.

The aim of the present study was to use dual-isotope SPECT imaging in order to demonstrate that ¹⁶¹Tb and ¹⁷⁷Lu are interchangeable without compromising the pharmacokinetic profile of the radiopharmaceutical.

After in vitro characterization, ¹⁶¹Tb- and ¹⁷⁷Lu-labeled somatostatin (SST) analogues DOTATOC (agonist) and DOTA-LM3 (antagonist) were injected to AR42J tumor-bearing nude mice. In vivo disptribution profiles were investigatd by dual-isotope SPECT/CT imaging. Results revealed identical pharmacokinetic profiles of the two peptides, irrespective of whether it was labeled with ¹⁶¹Tb or ¹⁷⁷Lu. Moreover, the visualization of the sub-organ distribution confirmed similar behavior of ¹⁶¹Tb- and ¹⁷⁷Lu-labeled SST analogues. These and previous findings suggest that any future (pre)clinical studies with ¹⁶¹Tb can be based on preclinical data obtained with its ¹⁷⁷Lu-labeled counterpart. This will allow the focusing of future investigations directly on the therapeutic efficacy of ¹⁶¹Tb, which is likely to be superior to the effect obtained with ¹⁷⁷Lu.

Results from nanoSPECT/CT

Five-week-old female CD1 nude mice were subcutaneously inoculated with AR42J tumor cells (5x106 cells in 100µl PBS). The scans were performed 10–14 days after tumor cell inoculation when the tumor size reached a volume of ~250mm3.

Mice were i.v. injected with a mixture of ¹⁶¹Tb-DOTATOC (~15MBq) and ¹⁷⁷Lu-DOTATOC (~15MBq) or a mixture of ¹⁶¹Tb-DOTA-LM3 and ¹⁷⁷Lu-DOTA-LM3 (~30MBq) at a ¹⁶¹Tb/¹⁷⁷Lu activity ratio of 1:1. For specificity test, blocking studies were performed under the same experimental conditions; however, in this case, an excess of unlabeled DOTATOC or DOTA-LM3 was added to the injection solution. SPECT/CT scans were acquired 2h, 4h, and 24h after injection of the radiopeptides using the dual-isotope SPECT acquisition protocol with a frame time of 60s resulting in a scan time of 45min.

For the SPECT/CT scan, simultaneous acquisition of counts stemming from ¹⁶¹Tb and ¹⁷⁷Lu, respectively, was performed by the selection of distinct energy windows for the two radionuclides. The two energy windows chosen for ¹⁶¹Tb were set at 47.7keV±10%, which enabled the detection of X-rays and γ-rays (46.0keV, 48.9keV and 52.0keV), and at 74.6keV±10%, enabling the detection of the γ-rays at 74.6keV. For ¹⁷⁷Lu, the windows were set at 112.9keV±10% and 208.4±10% to detect the γ-rays. Prior to animal studies, phantom scans were carried out in order to verify of the dual-isotope SPECT imaging protocol using Eppendorf vials filled with ¹⁶¹Tb or ¹⁷⁷Lu or both: analysis revealed no interference between ¹⁶¹Tb and ¹⁷⁷Lu in the acquired scans; each radionuclide was visualized independently of the other with high accuracy.

Analysis of the SPECT/CT images revealed:

• Equal in vivo distribution of simultaneously injected ¹⁶¹Tb-DOTATOC and ¹⁷⁷Lu-DOTATOC. The same observation was made for ¹⁶¹Tb-DOTA-LM3 and ¹⁷⁷Lu-DOTA-LM3. Images reconstructed using the energies of either radiolanthanide (red-to-yellow scale and green-to-yellow scale for ¹⁶¹Tb and ¹⁷⁷Lu, respectively), provided the distribution for each radiopeptide separately in the same mouse.

• Experiments performed by co-injection of excess unlabeled peptide resulted in SSTR blockade and, hence, accumulation of the radiopeptides in AR42J tumors was not observed. These additional studies proved that the uptake of the SST analogues in AR42J tumor xenografts was SSTR-specific.
• Quantification of the accumulated activity in AR42J tumors and kidneys confirmed equal distribution of the ¹⁶¹Tb- and ¹⁷⁷Lu-labeled counterparts. This was the ultimate proof that the chosen radiolanthanide did not have an impact on the tissue distribution profile of the radiopeptides.
• The SPECT/CT images showed activity accumulation in the AR42J xenografts, which was higher for the antagonist than for the agonist. In agreement with quantitative data from biodistribution studies, the activity was efficiently cleared through the kidneys over time and almost entirely excreted after 24h. Due to the favorable uptake of radiolabeled DOTA-LM3 in the tumor tissue, the tumor-to-kidney ratio was higher as compared to the ratio obtained after injection of radiolabeled DOTATOC.
• Dual-isotope SPECT image sections enabled, for the first time, visualization of the ¹⁶¹Tb- and ¹⁷⁷Lu-labeled peptide distribution at a sub-organ level in the same animal. Most important to note is that the pattern of activity distribution in tumors and kidneys was the same, irrespective of whether ¹⁶¹Tb or ¹⁷⁷Lu was used. The uptake in the tumor was quite homogenous, which can be ascribed to the well-vascularized AR42J xenograft. Accumulation of activity in the kidneys was more prominent in the cortex where the megalin-mediated reabsorption of radiopeptides occurs, and where various SSTR subtypes are known to be expressed

Inhibition of HIF-1α by Atorvastatin During ¹³¹I-RTX Therapy in Burkitt’s Lymphoma Model

Eun-Ho Kim^1,2, Hae Young Ko^3,4, A Ram Yu⁵, Hyeongi Kim³, Javeria Zaheer^3,6, Hyun Ji Kang^3,6, Young-Cheol Lim³, Kyung Deuk Cho³, Hyun-Yoo Joo¹, Min Kyoung Kang⁵, Jae Jun Lee⁵, Seung-Sook Lee⁷, Hye Jin Kang⁸, Sang Moo Lim^3,9, Jin Su Kim^3,6

A non-human primate model for stable chronic Parkinson’s disease induced by MPTP administration based on individual behavioral quantification

https://doi.org/10.1016/j.jneumeth.2018.10.037

Summary

Parkinson’s disease (PD) is a neurodegenerative disease that affects about 3% of the population over 65 years of age. PD is characterized by a selective loss of dopaminergic neurons accompanied by movement disorders, including rigidity, bradykinesia, akinesia, tremors, and postural instability.

The authors aimed to develop a MPTP-induced chronic NHP model with stable symptoms by adjusting MPTP treatment based on individual behavioral quantification using a video-based analysis system. To validate this strategy, they assessed a parkinsonian behavior score, an immunohistochemistry Western blot, and PET imaging of dopamine transporter (DAT) with [¹⁸F] N-(3-fluoropropyl)-2ß-carboxymethoxy-3β-(4-iodophenyl) nortropane (¹⁸F-FP-CIT).

The authors stated that the novel strategy of MPTP administration based on global activity evaluations using a video-based analysis system provides an important conceptual advance of this method for the development of chronic NHP PD models.

Results from nanoScan PET/CT

Mediso’s nanoScan PET/CT with 16cm bore size, 12 cm transaxial and 10 cm axial Field of View (FOV) were suitable to execute PET/CT scans in cynomolgus monkeys (Macaca fascicularis). Furthermore high spatial and temporal resolution of PET allowed to determine the dopamine transporter biding potential (BP) in different striatal sub-regions.

Following a CT scan for attenuation correction, 185 MBq 18 F-FP-CIT in 1.5 ml saline was injected intravenously via the saphenous vein. To monitor presynaptic dopamine transporter activity in vivo, serial 18F-FP-CIT PET imaging was performed after MPTP administration.

• The biding potential analysis revealed that the 18F-FP-CIT BP were significantly reduced in all sub-regions of the striatum at 8, 16, 24, 32,40 and 48 weeks following the first MPTP administration (Fig. 4A and 4B).
• Monkeys with fewer striatal DAT tended to have lower global activity (GA), indicating that a decrease in GA reflects damage in dopaminergic neurons.
• The severity of Parkinsonian symptoms and the loss rates of DAT in each striatal sub-region did not correlate with the total dose of MPTP, indicating that a fixed MPTP dose is not a good strategy for development of chronic NHP PD models

Fig. 4. (A) Representative 18F-FP-CIT PET images fused with individual MRI. (B) Histogram representing 18F-FP-CIT binding potential (BP) in the MPTP-injection group. (#P < 0.05, *P<0.01 vs. baseline). (C) Histogram representing time activity curve. The radiotracer uptake in each region of interest was estimated as the standardized uptake value (SUV), which was calculated as decay-corrected activity per milliliter of tissue volume per injected radiotracer activity per body mass ([kBq/mL]/[kBq/g]). Ant, anterior; binding potential, BP; Ext, external; pallidum, globus pallidus; Int, internal; Post, posterior.

Iodine‑124 PET quantification of organ‑specific delivery and expression of NIS‑encoding RNA

Matthias Miederer¹, Stefanie Pektor¹, Isabelle Miederer¹, Nicole Bausbacher¹, Isabell Sofia Keil²,

Hossam Hefesha³, Heinrich Haas³, Ugur Sahin^2,3 and Mustafa Diken^2,3

¹ Department of Nuclear Medicine, University Medical Center of Johannes Gutenberg University, Mainz, Germany

² TRON - Translational Oncology at the University Medical Center, Johannes Gutenberg University Mainz GmbH, Mainz, Germany

³ Biopharmaceutical New Technologies (BioNTech) SE, Mainz, Germany

https://doi.org/10.1186/s13550-021-00753-2

Summary

There has been increased interest in the development of mRNA-based vaccines for protection against various infectious diseases and also for cancer immunotherapies since lipid-based nanoparticles opened the possibility to deliver RNA to specific sites within the body, overcoming the limitation of rapid degradation in the bloodstream. In the present study, RNA-lipoplex nanoparticles were assembled by complexing sodium-iodide-symporter (NIS) coding mRNA with liposomes at different charge ratios. Two kinds of RNA-lipoplex systems were used: one system with net anionic charge mediating translation primarily within the spleen, and the other with net positive charge yielding translation primarily within the lungs. After in vitro analysis of the expression kinetics, mice were iv. injected with the mRNA-lipoplexes then 6h later with ¹²⁴Iodine. Functional NIS protein translation was investigated by PET/MRI imaging. Results revealed rapid increase of ¹²⁴Iodine uptake in the spleen or lung compared to control-RNA-lipoplexes (containing non-coding RNA) with minimal background in other organs except from thyroid, stomach and salivary gland (where NIS is physiologically expressed). The strong organ selectivity and high target-to-background acquisition of NIS-RNA lipoplexes indicate the feasibility of small animal PET/MRI to quantify organ-specific delivery of RNA.

Results from nanoScan PET/MRI

Female BALB/c mice were intravenously injected with RNA-lipoplexes containing 20μg NIS RNA. Six hours later 6.64±0.66MBq ¹²⁴Iodine was injected intravenously. Three hours after ¹²⁴Iodine injection, mice were anesthetized and static imaging was performed over 20min by nanoScan PET/MRI. Additionally, one animal per group was imaged dynamically for one hour.

• PET/MRI of anionic NIS-RNA lipoplexes showed a visually detectable increase of ¹²⁴Iodine uptake in the spleen compared to control-RNA lipoplexes. Due to the high physiological NIS expression in the adjacent gastric wall, this increase was only visually clear with anatomical correlation by MRI. On PET imaging, spleen uptake appeared as an irregularity of the gastric wall which is not detected in control animals
• Lung uptake of NIS-RNA transported by cationic RNA-lipoplexes was depicted more clearly due to larger organ size and no adjacent physiological NIS uptake
• The quantified radioactivity from imaging matched well with the extent of uptake as measured in organs ex vivo, showing enhanced uptake of NIS-RNA and expression of functional NIS-protein in lung or spleen compared to the control RNA
• The uptake in lung was rapid and remained high over the first hour of dynamic acquisition

Validation of Image Qualities of a Novel Four-Mice Bed PET System as an Oncological and Neurological Analysis Tool

Kyung Jun Kang¹, Se Jong Oh¹, Kyung Rok Nam¹, Heesu Ahn^1,2, Ji-Ae Park^1,2, Kyo Chul Lee¹, Jae Yong Choi^1,2
1Division of Applied RI, Korea Institute of Radiological and Medical Sciences, Seoul 01812, Korea
²Radiological and Medico-Oncological Sciences, University of science and technology (UST), Seoul 01812, Korea

https://doi.org/10.3390/jimaging7030043

Summary
Molecular imaging is used for the diagnosis of oncological, neurological, and cardiovascular diseases. Positron emission tomography (PET) is a molecular imaging technique that enables quantitative measurements of biochemical parameters, such as metabolic changes and concentrations of neurotransmitters. The demand for preclinical research is increasing worldwide, as it can predict clinical trials and advance our understanding of the pathophysiological mechanisms underlying a specific disease. For this purpose, researchers use micro-PET (µPET), which is a small-animal dedicated system. Most µPET vendors provide a single bed, thereby allowing imaging of only a single animal at a time. Large-scale research involving many objects thus requires tremendous time and use of radioactivity.
Recently, Mediso developed a multi-bed system dedicated to the nanoScan scanners with the contribution of Tim Witney and his team at UCL and KCL, where the initial validity for research has been investigated. Greenwood et al. (https://doi.org/10.2967/jnumed.119.228692) tested the four-bed mouse system using 2-deoxy-2-(¹⁸F)fluoro-D-glucose (¹⁸F-FDG) in phantom and normal mice and reported a quantitative accuracy similar to that of a single-bed. To date, however, few studies have focused on the validity of oncological and neurological PET imaging of the four-mice bed system.
This study aimed to evaluate the image qualities of oncological and neurological PET imaging using a novel four-mice bed system.

Results from the nanoScan PET/CT
For the small animal imaging, the authors have used a nanoScan PET/CT, to see the differences using the single-bed vs. multi-bed scans regarding the image quality in oncological and neurological studies. The scanner has a peak absolute system sensitivity of >9% in the 400–600 keV energy window, an axial field of view of 28 cm, a transaxial field of view of 35–120 mm, and a transaxial intrinsic resolution of 0.7 mm at 1 cm off-center.
For the phantom studies, following the guidelines of NEMA NU-4 2008, four phantoms were filled with 3.7 MBq of ¹⁸F-FDG in 10 mL of saline and decay-corrected to the start of acquisition. On the first day, four phantoms were imaged in the multi-bed system, and one day later a single phantom was imaged using a single-bed system for comparison. Static PET acquisition was performed using the nanoScan PET/CT over 20 min, followed by CT (480 projections; 50 kVp tube voltage; 630 µA; 300 ms exposure time; 1:4 binning). Whole-body TeraTomo 3D reconstruction with four iterations and six subsets was performed (1–5 coincidence mode) using an isotropic voxel size of 0.4 mm³.
In all the preclinical experiments, four animals were scanned in the multi-bed system, and then, single PET images were acquired using the single-bed system 2 days later. For the oncological study, prior to imaging, all mice fasted overnight. The mice were anesthetized with 2.5% isoflurane in oxygen, and ¹⁸F-FDG (8.8 ± 0.9 MBq/200 µL) was administrated into the tail vein. Based on previous studies, ¹⁸F-FDG images were acquired after 40–60 min. For the brain study, an ¹⁸F-FPEB PET tracer (8.7 ± 0.7 MBq/200 µL) was prepared. ¹⁸F-FPEB PET images were acquired 30–50 min after injection because the radiotracer initially shows specific-to-nonspecific binding values that reach a plateau after 30 min.  Images were reconstructed using TeraTomo 3D algorithm with four iterations. For attenuation correction, CT imaging was performed immediately after PET using 50 kVp of X-ray voltage with 0.16 mAs.
Figure 3. shows the main results from the oncological study, with representative PET/CT images of tumor-bearing mice after ¹⁸F-FDG injection in single- and multi-bed systems (A). The plane was chosen to best reflect the tracer uptake characteristics of each group. Comparison of tumor uptake values in single- and multi-bed systems based on tumor size (B,C), **** p < 0.0001, n.s.= statistically nonsignificant. PET/CT, positron emission tomography/computed tomography; SUV_max, maximum standardized uptake value.

Figure 4. shows the results from the neurological study. Summed ¹⁸F-FPEB brain PET images of the single-bed (n = 12) and each bed of the multi-bed system (A, n = 3 in each bed). Comparison of regional SUV values of single- and multi-bed systems (B,C). STR, striatum; HIP, hippocampus; SUV, standardized uptake value.

• PET images acquired with the multi-bed system showed a lower estimation of radiation uptake compared to that of the single-bed system when images were acquired for small lesions. However, phantom studies showed that there were no differences between the single- and multi-bed systems for volumes > 4 mm and that multi-bed systems can be applied in oncological and neurological studies.
  
• The multi-bed system may improve the efficacy of preclinical research by saving the time taken to acquire PET images.
  

Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates

 https://www.biorxiv.org/content/10.1101/2021.02.10.430696v1

Summary

Despite of the success of messenger RNA vaccines against SARS-CoV-2, a wider portfolio of different vaccine candidates would be needed to stop COVID-19 pandemic. In particular, vaccinating infants and the elderly could benefit from the use of subunit vaccine platforms with a demonstrable history of safety and efficacy in such populations.

Subunit vaccines include only a fragment of a pathogen which can still induce the immune system. Although, the production of these types of vaccines are safer than producing attenuated pathogen, they often require adjuvants to enhance the immune response for long term protection.

In this extensive research collaboration, the authors demonstrate the capacity of a subunit vaccine under clinical development, containing the SARS-CoV-2 Spike protein receptor binding domain displayed on a two component protein nanoparticle (RBD-NP). They assessed the immunogenicity and protective efficacy of RBD-NP vaccination using five different clinically relevant adjuvants in non-human primates.

Among the tested adjuvants, AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion, and CpG 1018 (with Alum), a Toll-like receptor 9 agonist formulated in Alum, were the most promising adjuvants.

The team concluded that the neutralizing antibody response by the RBD-NP/AS03 vaccination was durable. The study results can also help in the development of subunit vaccines to combat the ongoing pandemic.

Results from  MultiScan LFER PET/CT

The researchers in University of Pittsburgh School of Medicine evaluated inflammation in the lung tissues with no adjuvant, AS03 and CpG-Alum on pre and post challenge days using MultiScan LFER PET/CT

• Vaccinated animals showed FDG uptake, to a much lesser extent than the control animals (Fig. 3e and f)

  

  Fig 3 e, Inflammation in the lungs of two animals from each group indicated in the legend, pre-challenge (day 0) and post-challenge (day 4 or 5 after infection), measured using PET-CT scans. f, Representative PET-CT images of lungs from one animal in each group. PET signal is scaled 0 to 15 SUV.

SPECT imaging evaluation of ¹¹¹indium-chelated cetuximab for diagnosing EGFR-positive tumor in an HCT-15-induced colorectal xenograft

Bin-Bin Shih^a, Yi-Fang Chang^b, Chun-Chia Cheng^b, Hao-Jhih Yang^c, Kang-Wei Chang^c, Ai-Sheng Ho^a, Hua-Ching Lin^d, Chun Yeh^a, Chun-Chao Chang^e,f

^a Division of Gastroenterology, Cheng Hsin General Hospital, Taipei, Taiwan, ROC

^b Hematology and Oncology, Mackay Memorial Hospital, Taipei, Taiwan, ROC

^c Institute of Nuclear Energy Research, Atomic Energy Council, Taoyuan, Taiwan, ROC

^d Division of Proctology, Cheng Hsin General Hospital, Taipei, Taiwan, ROC

^e Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan, ROC

^f Division of Gastroenterology and Hepatology, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, ROC

http://dx.doi.org/10.1016/j.jcma.2017.02.010

Summary

Colorectal cancer (CRC) occurs with high incidence worldwide, but is usually diagnosed in late stage with metastasis by the conventional methods. Epidermal growth factor receptor (EGFR) is overexpressed in 97% of CRC cells, serving a promising diagnostic candidate. In the present study, Cetuximab, an anti-EGFR monoclonal antibody was conjugated with an isotope chelator, diethylene triamine penta acetic acid (DTPA), labeled with ¹¹¹indium (¹¹¹In) and injected to tumor bearing mice. Biological distrubution was investigated by SPECT/CT imaging.

Results revealed that ¹¹¹In-Cetuximab accumulated in the both small (50mm³) and large (250mm³) tumors, whereas the ratio of tumor to muscle in the large tumor was 7.5-fold. The biodistribution data indicated that the ¹¹¹In-cetuximab bound to tumor specifically that was higher than that in other organs. Consequently, ¹¹¹In-cetuximab is suggested to be suitable for early diagnosis and prognostic monitor of EGFR-positive CRC in further clinical practice.

Results from nanoSPECT/CT

• The tumor of the ¹¹¹In-Cetuximab group was apparently observed both in 24h and 48h and higher than that in the ¹¹¹In group.
• ¹¹¹In-Cetuximab majorly accumulated in liver and tumor, otherwise, ¹¹¹In accumulated only in the kidney.
• The tumor to muscle ratio of ¹¹¹In-Cetuximab was measured 7.5-fold, which was higher than that of ¹¹¹In group measured as 3.1-fold, indicating that ¹¹¹In-cetuximab specifically bound to EGFR-positive tumors as a reliable diagnosing agent.
• The result also indicated that ¹¹¹In labeled with Cetuximab through chelator DTPA was easily excreted out the mice better than free ¹¹¹In, suggesting that this labeling method may not lead to accumulation of ¹¹¹In metal in mice.

In vivo imaging with ¹⁸F-FDG- and ¹⁸F-Florbetaben-PET/MRI detects pathological changes in the brain of the commonly used 5XFAD mouse model of Alzheimer's Disease

Timon N Franke¹, Caroline Irwin¹, Thomas A Bayer¹, Winfried Brenner², Nicola Beindorff³, Caroline Bouter⁴, Yvonne Bouter^1
1Department of Psychiatry and Psychotherapy, University Medical Center Göttingen (UMG), Georg-August-University, Göttingen, Germany.
²Department of Nuclear Medicine, Charité-Universitätsmedizin Berlin, Berlin, Germany.
³Berlin Experimental Radionuclide Imaging Center (BERIC), Charité-Universitätsmedizin Berlin, Berlin, Germany.
⁴Department of Nuclear Medicine, University Medical Center Göttingen (UMG), Georg-August-University, Göttingen, Germany.

https://doi.org/10.3389/fmed.2020.00529

Summary
Alzheimer's disease (AD) is the most common form of dementia with an estimated number of more than 40 Million cases worldwide. To date, a final diagnosis of the disease can only be made by histopathological detection of amyloid plaques and neurofibrillary tangles post mortem, therefore it is mandator to find proper animal models, on which different in vivo diagnostics can be used.
The imaging biomarkers of AD that are able to detect molecular changes in vivo and transgenic animal models mimicking AD pathologies are essential for the evaluation of new therapeutic strategies. Positron-emission tomography (PET) using either ¹⁸F-Fluorodeoxyglucose (¹⁸F-FDG) or amyloid-tracers is a well-established, non-invasive tool in the clinical diagnostics of AD assessing two major pathological hallmarks. ¹⁸F-FDG-PET is able to detect early changes in cerebral glucose metabolism and amyloid-PET shows cerebral amyloid load. However, the suitability of ¹⁸F-FDG- and amyloid-PET in the widely used 5XFAD mouse model of AD is unclear as only a few studies on the use of PET biomarkers are available showing some conflicting results. The aim of this study was the evaluation of ¹⁸F-FDG-PET and amyloid-PET in 5XFAD mice in comparison to neurological deficits and neuropathological changes.

Results from the nanoScan PET/MRI
For the small animal imaging, the authors have used a nanoScan PET/MRI 1T, which provided a good enough spatial resolution to differentiate the examined brain regions, and creating even 10mm³ large VOIs (which is still above the nanoScan PET intrinsic resolution of 0.7mm).
¹⁸F-FDG-PET/MRI was performed on 7- and 12-month-old male 5XFAD mice as well as age- and sex-matched C57Bl/6J wild type mice (n = 4–6 per group). ¹⁸F-FDG (11.46–20.53 MBq; mean 16.81 MBq) was injected into a tail vein with a maximum volume of 200 μl followed by an uptake period of 45 min. Mice were awake during the uptake process. PET scans were performed for 20 min. MRI-based attenuation correction was conducted with the material map (matrix 144 × 144 × 163 with a voxel size of 0.5 × 0.5 × 0.6 mm³, repetition time: 15 ms, echo time: 2.032 ms and a flip angle of 25°) and the PET images were reconstructed using the following parameters: matrix 136 × 131 × 315, voxel size 0.23 × 0.3 × 0.3 mm3.
For the ¹⁸F-FBB-PET/MRI imaging, it was performed on 7- and 12-month-old male 5XFAD mice as well as age- and sex-matched C57Bl/6J wild type mice after ¹⁸F-FDG-PET imaging (n = 4–6 per group). In isoflurane anesthetized mice ¹⁸F-Florbetaben (7.5–24 MBq; mean 14 MBq) was administered intravenously with a maximum volume of 200 μl. PET acquisition of 30 min duration started after an uptake period of 40 min.
Figure 2. shows the main results from ¹⁸F-FDG-PET images in coronal, transverse, and sagittal view. (A–C) MRI images with predefined brain regions. (D–F) ¹⁸F-FDG images of a representative 7-month-old WT mouse. (G–I) ¹⁸F-FDG-PET images of a 7-month-old 5XFAD mouse. 7-month-old 5XFAD mice showed distinctly lower FDG uptake compared to WT mice. (J–L) ¹⁸F-FDG images of a 12-month-old WT mouse. 12-month-old 5XFAD mice also showed significantly lower ¹⁸F-FDG uptake compared to age-matched WT mice. A, Amygdala; Bs, Brain Stem; C, Cortex; Cb, Cerebellum, H, Hypothalamus; Hc, Hippocampus; Hg, Harderian Glands; M, Midbrain; O, Olfactory Bulb; S, Septum/Basal Forebrain; St, Striatum; T, Thalamus.

Figure 4. shows the results from ¹⁸F-Florbetaben-PET images in coronal view. (A) ¹⁸F-Florbetaben image of a 7-month-old WT mouse. (B) Cerebral ¹⁸F-Florbetaben uptake was higher in 7-month-old 5XFAD mice as well as in 12-month-old 5XFAD mice (C).

• The authors could show that PET biomarkers ¹⁸F-FDG and ¹⁸F-Florbetaben detected cerebral hypometabolism and increased plaque load even before the onset of severe memory deficits.
• Summarizing their results, the ¹⁸F-FDG- and ¹⁸F-Florbetaben-PET, are useful tools for the in vivo detection of cerebral AD pathologies in 5XFAD mice even before the onset of severe memory deficits. The 5XFAD mouse model of AD therefore is a suitable model for preclinical PET studies showing comparable changes to AD patients with the clinically established biomarkers ¹⁸F-FDG and ¹⁸F-Florbetaben. Therefore, PET imaging can be utilized as a readout for therapeutic effects in vivo in future longitudinal therapy studies using the 5XFAD mouse model of AD.

Copper‑67 radioimmunotheranostics for simultaneous immunotherapy and immuno‑SPECT

Guiyang Hao¹, Tara Mastren¹, William Silvers¹, Gedaa Hassan¹, Orhan K. Öz¹ &

Xiankai Sun^1,2

1 Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.

2 Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.

ttps://www.nature.com/articles/s41598-021-82812-1

Introduction

Copper-67 is useful from both therapeutic and diagnostic standpoints due to its medium energy beta particle, gamma emissions, and 2.6-day half-life. Moreover, since copper radioisotopes are chemically identical, the same radiopharmaceutical can be used for ⁶⁴Cu PET imaging to pre-screen of patients and ⁶⁷Cu based radionuclide therapy. For these reasons, the usage of ⁶⁷Cu in radiotherapy has long been arisen. However until now, its widespread use has been limited by unreliable supplies, cost, and difficulty in obtaining therapeutic quantities. The recent breakthrough of copper-67 production provides an opportunity to reassess its use in radiotherapy.

Summary

In this work, Xiankai Sun and his co-workers have evaluated and demonstrated the practical use of ⁶⁷Cu in radioimmunotherapy. To demonstrate the concept, human epidermal growth factor receptor 2 (HER2) antibody, pertuzumab, was labeled with ⁶⁷Cu isotope. The radiolabeling efficiency was two-order of magnitude higher compared to literature reports. Mice bearing HER2+ xenografts showed ⁶⁷Cu-dose dependent tumor-growth inhibition from ⁶⁷Cu-labeled-Pertuzumab co-administered with trastuzumab. Moreover, authors visualized and measured [⁶⁷Cu]Cu-NOTA-Pertuzumab uptake quantitatively by SPECT imaging.

Results from nanoSPECT/CT

Thanks to technological advances of Mediso's SPECT/CT, researchers were able to perform quantitative data analysis.

• All the tumors were clearly visualized with [⁶⁷Cu]Cu-NOTA-Pertuzumab on both day 2 and day 5 post injection (Fig. 4A).
• Quantitative tumor uptake from the SPECT imaging data are presented as absolute radioactivity concentration (μCi/mL) (Fig. 4B)

Figure 4. SPECT/CT imaging results. (A) Representative maximum intensity projection (MIP)  SPECT/CT images of HCC1954 HER2+ tumor-bearing mice injected with [⁶⁷Cu]Cu-NOTA-Pertuzumab (Group 2, 3, 4, and 5) at day 2 and 5 post the start of treatment (yellow arrows indicate the tumors); (B) Actual radioactivity concentration in tumors (MBq/mL) on Day 2 and 5 (without decay correction).

Feasibility of Imaging EpCAM Expression in Ovarian Cancer Using Radiolabeled DARPin Ec1

Anzhelika Vorobyeva^1,2,† , Elena Konovalova^3,†, Tianqi Xu¹, Alexey Schulga^2,3, Mohamed Altai¹, Javad Garousi¹, Sara S. Rinne⁴, Anna Orlova^2,4,5, Vladimir Tolmachev^1,2 and Sergey Deyev^2,3,6,7 

¹ Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden

² Research Centrum for Oncotheranostics, Research School of Chemistry and Applied Biomedical Sciences, Tomsk Polytechnic University, Tomsk, Russia

³ Molecular Immunology Laboratory, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia

⁴ Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden

⁵ Science for Life Laboratory, Uppsala University, Uppsala, Sweden

⁶ Bio-Nanophotonic Lab, Institute of Engineering Physics for Biomedicine (PhysBio), National Research Nuclear University ‘MEPhI’, Moscow, Russia

⁷ Center of Biomedical Engineering, Sechenov University, Moscow, Russia

^† A.V. and E.K. contributed equally

https://doi.org/10.3390/ijms21093310

Summary

Up to 85% of ovarian cancer patients are diagnosed only at advanced stages, when cancer has already spread through the body. The present study aimed to find a more efficient way for diagnosis and treatment using mouse xenograft models.

As epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%–75% of ovarian carcinomas (OC), it might be a promising target. Designed ankyrin repeats protein (DARPin) Ec1 binds to EpCAM with subnanomolar affinity. In the present study, DARPin Ec1 was labeled with ¹²⁵I using N-succinimidyl-para-iodobenzoate (PIB) and injected to mice bearing SKOV-3 or OVCAR-3 xenografts. In vitro experiments showed highly specific binding to ovarian carcinoma cells, moreover, slow internalization, which is essential for in vivo imaging a few hours after injection. In vivo biodistribution analyses of SPECT/CT images suggest that EpCAM on ovarian cancer xenografts is sufficiently accessible to permit DARPin-mediated delivery of cytotoxic payload.

Results from nanoScan SPECT/CT

For establishment of xenografts, 107 of SKOV-3 and OVCAR-3 cells or 5x106 Ramos cells (EpCAM-negative lymphoma xenografts served as specificity control) in 100µl of media were subcutaneously injected in the right hind leg of female Balb/c nu/nu mice. The experiments in mice bearing SKOV-3 and Ramos xenografts were performed 2–3 weeks after implantation. The experiments in mice bearing OVCAR-3 xenografts were performed 7 weeks after implantation.

Mice were injected with ¹²⁵I-PIB-Ec1 (20µg, 1.2MBq for SKOV-3, and 6µg, 2.8MBq for OVCAR-3), SPECT/CT images were acquired 6h pi time later for 20min. with nanoScan SPECT/CT.

• In vitro studies revealed specific binding to SKOV-3 and OVCAR-3 cells; rapid binding and slow dissociation and internalization
• SPECT/CT imaging demonstrated that radiolabeled ¹²⁵I-PIB-Ec1 provided clear visualization of both EpCAM-expressing xenografts. In vivo biodistribution is characterized by high tumor-to-organ ratio, the only organ with noticeable activity were kidneys.

Iodine-124 PET quantification of organ-specific delivery and expression of NIS-encoding RNA

Matthias Miederer¹, Stefanie Pektor¹, Isabelle Miederer¹, Nicole Bausbacher¹, Isabell Sofa Keil², Hossam Hefesha³, Heinrich Haas³, Ugur Sahin^2,3, Mustafa Diken^2,3
1Department of Nuclear Medicine, University Medical Center of Johannes Gutenberg University, Mainz, Germany
²TRON - Translational Oncology at the University Medical Center, Johannes Gutenberg University Mainz gGmbH, Mainz, Germany
³Biopharmaceutical New Technologies (BioNTech) SE, Mainz, Germany

https://doi.org/10.1186/s13550-021-00753-2

Summary
RNA-based vaccination strategies tailoring immune response to specific reactions have become an important pillar for a broad range of applications. Recently, the use of lipid-based nanoparticles opened the possibility to deliver RNA to specific sites within the body, overcoming the limitation of rapid degradation in the bloodstream. 
Nanoparticles show promising potency as delivery vehicles for a variety of molecules, leading to fundamentally new applications and therapeutic strategies. Due to their complex chemical composition and relevant interaction with plasma proteins, the pharmacokinetic properties and delivery properties of nanoparticles are variable and remain challenging to adapt for optimal conditions. Accomplishing precise RNA delivery to target tissue using nanoparticles would serve as a versatile platform that enables easy and transient expression of any protein in principal. RNA is currently already in use to selectively activate the immune system against specific target proteins for cancer therapy. 
In the article, the authors have investigated whether small animal PET/MRI can be employed to image the biodistribution of RNA-encoded protein. For this purpose, a reporter RNA coding for the sodium-iodide-symporter (NIS) was assembled with liposomes at different charge ratios, and functional NIS protein translation was imaged and quantified in vivo and ex vivo by Iodine-124 PET upon intravenous administration in mice.

Results from the nanoScan PET/MRI
For the small animal imaging, the authors have used a nanoScan PET/MRI 1T, which provided a perfect option to follow the uptake of Iodine-124 not just in the thyroids, but also in the NIS-expressing tissues. Moreover, it could be accompanied by the help of MRI, to identify internal organs, like spleen.
Groups of n = 3 animals were intravenously injected with RNA-lipoplexes containing 20 µg NIS RNA. Six hours later 6.64 ± 0.66 MBq Iodine-124 was injected intravenously. Three hours after Iodine-124 injection, mice were anesthetized with 2% isoflurane and static imaging was performed over 20 min. For anatomic imaging MRI measurements (Material Map for coregistration of the PET scan; 3D Gradient Echo External Averaging (GRE-EXT), Multi Field of View (FOV); slice thickness: 0.6 mm; TE: 2 ms; TR: 15 ms; flip angle: 25 deg) were performed afterward. Additionally, one animal per group was imaged dynamically for one hour. PET data were reconstructed with Teratomo 3D (4 iterations, 6 subsets, voxel size 0.4 mm), co-registered to the MR and corrected for decay.
Figure 2. shows the PET/MRI of Iodine-124 distribution in vivo. (A) Coronal slices of PET/MRI fusion and volumes of interests (red) for spleen and lung are shown in representative animals. From left to right: targeting of spleen with non-coding RNA, targeting of spleen with NIS RNA, targeting of lung with non-coding RNA, targeting of lung with NIS-RNA. (B) Calculated organ uptake from the volumes of interests. Data are shown as mean + SD of n = 3 mice. (C) Representative in vivo bioluminescence images of Luc-RNA lipoplexes after targeting the spleen and lung. (D) Maximum intensity projections of PET images after application of lung-targeting NIS-RNA lipoplexes (right) in comparison with non-coding control (left). (E) Time activity curve of Iodine-124 uptake in the lung over 60 min immediately after Iodine-124 injection (6 h after administration of NIS-RNA lipoplexes targeting the lung).

• In this study, two RNA-lipoplex systems for systemic NIS-RNA delivery were compared by small animal PET/MRI of Iodine-124 uptake. One system with net anionic charge is known to mediate translation primarily within the spleen, and the other with net positive charge is known to yield translation primarily within the lungs.
• Tha authors have shown highly specific targeting, delivery and expression of RNA to spleen and lung by anionic and cationic RNA-lipoplex nanoparticles, respectively, through the use of the NIS reporter gene system and Iodine-124 uptake as imaged by PET/MRI. Combining NIS reporter gene imaging with in vivo small animal PET/MRI thus represents a powerful tool to monitor the distribution and extent of expression of RNA targeted specifically to any tissue over time.

PD-1 blockade exacerbates Mycobacterium tuberculosis infection in rhesus macaques

Keith D Kauffman, Shunsuke Sakai, Nickiana E Lora, Sivaranjani Namasivayam, Paul J Baker, Olena Kamenyeva, Taylor W Foreman, Christine E Nelson, Deivide Oliveira-de-Souza, Caian L. Vinhaes, Ziv Yaniv, Cecilia S Lindestam Arleham, Alessandro Sette, Gordon J Freeman, Rashida Moore, the NIAID/DIR Tuberculosis Imaging Program, Alan Sher, Katrin D Mayer-Barber, Bruno B Andrade, Juraj Kabat, Laura E Via, Daniel L Barber

 doi: 10.1126/sciimmunol.abf3861,  BioRxiv 

Summary

PD-1 (programmed death-1) is a coinhibitory receptor primarily expressed on activated CD4 and CD8 T cells that has been shown to limit the function of pathogen-specific T cells during chronic infection. The reactive expression of PD-L1 (PD-1 receptor ligand) on cancer cells turns off the T cells that are trying to attack the tumor. Therefore, blockade of PD-1 receptor or its ligands with monoclonal antibodies (mAbs) has become an attractive target in cancer therapy. Recognition of this pathway has led to suggestions that anti–PD-1 therapy might also boost T cell immunity in chronic infections including tuberculosis.

In this recent Science Immunology publication, Kauffman et al. examined the role of PD-1 during Mycobacterium tuberculosis (Mtb) infection of rhesus macaques. It was shown that the PD-1 blockade increased the number and functionality of Mtb-specific CD8 T cells, but not CD4 cells and was associated with increases in proinflammatory cytokines. However, animals treated with anti–PD-1 monoclonal antibody developed worse disease and higher granuloma bacterial loads compared with isotype control–treated monkeys.

These data indicate that negative regulation of immune responses is a critical aspect of host resistance to Mtb infection. Also, these findings suggest that the anti-PD-1 cancer therapy needs to be used cautiously in patients with a history of Mtb exposure.

Results from MultiScan LFER

Mediso MultiScan™ Large Field of view Extreme Resolution (LFER) PET/CT system was used to follow the course of Mycobacterium tuberculosis (Mtb) infection in rhesus macaques. The animals were imaged before infection and every 2 weeks after infection beginning at 4 weeks for a maximum of eight PET-CT scans. [18F] FDG was injected intravenously (1 mCi/kg), and after 60 minutes incubation time a 20-min PET scan was acquired.

(C) Example PET-CT image from isotype control– (top) or PD-1 (bottom)–treated animals. (D) Fold change over week 4 value of total lung standardized uptake value (SUV) in isotype control (left) and PD-1–treated (right) animals.