Staying on track - Keeping things running in a high-end scientific imaging core facility.

Modern life science research is a collaborative effort. Few research groups can single-handedly support the necessary equipment, expertise and personnel needed for the ever-expanding portfolio of technologies that are required across multiple disciplines in today's life science endeavours. Thus, research institutes are increasingly setting up scientific core facilities to provide access and specialised support for cutting-edge technologies. Maintaining the momentum needed to carry out leading research while ensuring high-quality daily operations is an ongoing challenge, regardless of the resources allocated to establish such facilities. Here, we outline and discuss the range of activities required to keep things running once a scientific imaging core facility has been established. These include managing a wide range of equipment and users, handling repairs and service contracts, planning for equipment upgrades, renewals, or decommissioning, and continuously upskilling while balancing innovation and consolidation.

Innovating in a bioimaging core through instrument development.

Developing devices and instrumentation in a bioimaging core facility is an important part of the innovation mandate inherent in the core facility model but is a complex area due to the required skills and investments, and the impossibility of a universally applicable model. Here, we seek to define technological innovation in microscopy and situate it within the wider core facility innovation portfolio, highlighting how strategic development can accelerate access to innovative imaging modalities and increase service range, and thus maintain the cutting edge needed for sustainability. We consider technology development from the perspective of core facility staff and their stakeholders as well as their research environment and aim to present a practical guide to the 'Why, When, and How' of developing and integrating innovative technology in the core facility portfolio. Core facilities need to innovate to stay up to date. However, how to carry out the innovation is not very obvious. One area of innovation in imaging core facilities is the building of optical setups. However, the creation of optical setups requires specific skill sets, time, and investments. Consequently, the topic of whether a core facility should develop optical devices is discussed as controversial. Here, we provide resources that should help get into this topic, and we discuss different options when and how it makes sense to build optical devices in core facilities. We discuss various aspects, including consequences for staff and the relation of the core to the institute, and also broaden the scope toward other areas of innovation.

Synthesis and Preclinical Evaluation of PSMA-Targeted 111In-Radioconjugates Containing a Mitochondria-Tropic Triphenylphosphonium Carrier.

Nuclear DNA is the canonical target for biological damage induced by Auger electrons (AE) in the context of targeted radionuclide therapy (TRT) of cancer, but other subcellular components might also be relevant for this purpose, such as the energized mitochondria of tumor cells. Having this in mind, we have synthesized novel DOTA-based chelators carrying a prostate-specific membrane antigen (PSMA) inhibitor and a triphenyl phosphonium (TPP) group that were used to obtain dual-targeted 111In-radioconjugates ([111In]In-TPP-DOTAGA-PSMA and [111In]In-TPP-DOTAGA-G3-PSMA), aiming to promote a selective uptake of an AE-emitter radiometal (111In) by PSMA+ prostate cancer (PCa) cells and an enhanced accumulation in the mitochondria. These dual-targeted 111In-radiocomplexes are highly stable under physiological conditions and in cell culture media. The complexes showed relatively similar binding affinities toward the PSMA compared to the reference tracer [111In]In-PSMA-617, in line with their high cellular uptake and internalization in PSMA+ PCa cells. The complexes compromised cell survival in a dose-dependent manner and in the case of [111In]In-TPP-DOTAGA-G3-PSMA to a higher extent than observed for the single-targeted congener [111In]In-PSMA-617. µSPECT imaging studies in PSMA+ PCa xenografts showed that the TPP pharmacophore did not interfere with the excellent in vivo tumor uptake of the "golden standard" [111In]In-PSMA-617, although it led to a higher kidney retention. Such kidney retention does not necessarily compromise their usefulness as radiotherapeutics due to the short tissue range of the Auger/conversion electrons emitted by 111In. Overall, our results provide valuable insights into the potential use of mitochondrial targeting by PSMA-based radiocomplexes for efficient use of AE-emitting radionuclides in TRT, giving impetus to extend the studies to other AE-emitting trivalent radiometals (e.g., 161Tb or 165Er) and to further optimize the designed dual-targeting constructs.

In vivo validation of 68Ga-labeled AMD3100 conjugates for PET imaging of CXCR4.

INTRODUCTION: The chemokine receptor CXCR4 has been shown to be over-expressed in multiple types of cancer and is usually associated with aggressive phenotypes and poor prognosis. Successfully targeting and imaging the expression level of this receptor in tumours could inform treatment selection and facilitate patient stratification. METHODS: Known conjugates of AMD3100 that are specific to CXCR4 have been radiolabelled with gallium-68 and evaluated in naïve and tumour-bearing mice. Tumour uptake of the radiotracers was compared to the known CXCR4-specific PET imaging agent, [68Ga]Pentixafor. RESULTS: Ex vivo biodistribution in naïve animals showed CXCR4-mediated uptake in the liver with both radiotracers, confirmed by blocking experiments with the high affinity CXCR4 antagonist Cu2CB-Bicyclam (IC50 = 3 nM). PET/CT imaging studies revealed one tracer to have a higher accumulation in the tumour (SUVMean of 0.89 ± 0.14 vs 0.32 ± 0.11). CXCR4-specificity of the best performing tracer was confirmed by administration of a blocking dose of Cu2CB-Bicyclam, showing a 3- and 6-fold decrease in tumour and liver uptake, respectively. CONCLUSION AND ADVANCES IN KNOWLEDGE: This initial study offers some interesting insights on the impact of some structural features on the pharmacokinetics and metabolic stability of the radiotracer. Additionally, as Pentixafor only binds to human CXCR4, the development of CXCR4-targeted imaging agents that bind to the receptor across different species could significantly help with preclinical evaluation of new CXCR4-specific therapeutics.

Direct comparison of [18F]AlF-NOTA-JR11 and [18F]AlF-NOTA-octreotide for PET imaging of neuroendocrine tumors: Antagonist versus agonist.

BACKGROUND: [18F]AlF-NOTA-octreotide is an 18F-labeled somatostatin analogue which is a good clinical alternative for 68Ga-labeled somatostatin analogues. However, radiolabeled somatostatin receptor (SSTR) antagonists might outperform agonists regarding imaging sensitivity of neuroendocrine tumors (NETs). No direct comparison between the antagonist [18F]AlF-NOTA-JR11 and the agonist [18F]AlF-NOTA-octreotide as SSTR PET probes is available. Herein, we present the radiosynthesis of [18F]AlF-NOTA-JR11 and compare its NETs imaging properties directly with the established agonist radioligand [18F]AlF-NOTA-octreotide preclinically. METHODS: [18F]AlF-NOTA-JR11 was synthesized in an automated synthesis module. The in vitro binding characteristics (IC50) of [natF]AlF-NOTA-JR11 and [natF]AlF-NOTA-octreotide were evaluated and the in vitro stability of [18F]AlF-NOTA-JR11 was determined in human serum. In vitro cell binding and internalization was performed with [18F]AlF-NOTA-JR11 and [18F]AlF-NOTA-octreotide using SSTR2 expressing cells and the pharmacokinetics were evaluated using µPET/CT in mice bearing BON1.SSTR2 tumor xenografts. RESULTS: Excellent binding affinity for SSTR2 was found for [natF]AlF-NOTA-octreotide (IC50 of 25.7 ± 7.9 nM). However, the IC50 value for [natF]AlF-NOTA-JR11 (290.6 ± 71 nM) was 11-fold higher compared to [natF]AlF-NOTA-octreotide, indicating lower affinity for SSTR2. [18F]AlF-NOTA-JR11 was obtained in a good RCY (50 ± 6 %) but with moderate RCP of 94 ± 1 %. [18F]AlF-NOTA-JR11 demonstrated excellent stability in human serum (>95 % after 240 min). 2.7-fold higher cell binding was observed for [18F]AlF-NOTA-JR11 as compared to [18F]AlF-NOTA-octreotide after 60 min. µPET/CT images demonstrated comparable pharmacokinetics and tumor uptake between [18F]AlF-NOTA-JR11 (SUVmax: 3.7 ± 0.8) and [18F]AlF-NOTA-octreotide (SUVmax: 3.6 ± 0.4). CONCLUSIONS: [18F]AlF-NOTA-JR11 was obtained in good RCY, albeit with a moderate RCP. The cell binding study showed significant higher binding of [18F]AlF-NOTA-JR11 compared to [18F]AlF-NOTA-octreotide, despite the higher IC50 value of AlF-NOTA-JR11. However, pharmacokinetics and in vivo tumor uptake was comparable for both radiotracers. Novel Al18F-labeled derivatives of JR11 with higher SSTR2 affinity should be developed for increased tumor uptake and NET imaging sensitivity.

Utilizing PET and MALDI Imaging for Discovery of a Targeted Probe for Brain Endocannabinoid α/ß-Hydrolase Domain 6 (ABHD6).

Multimodal imaging provides rich biological information, which can be exploited to study drug activity, disease associated phenotypes, and pharmacological responses. Here we show discovery and validation of a new probe targeting the endocannabinoid α/ß-hydrolase domain 6 (ABHD6) enzyme by utilizing positron emission tomography (PET) and matrix-assisted laser desorption/ionization (MALDI) imaging. [18F]JZP-MA-11 as the first PET ligand for in vivo imaging of the ABHD6 is reported and specific uptake in ABHD6-rich peripheral tissues and major brain regions was demonstrated using PET. A proof-of-concept study in nonhuman primate confirmed brain uptake. In vivo pharmacological response upon ABHD6 inhibition was observed by MALDI imaging. These synergistic imaging efforts used to identify biological information cannot be obtained by a single imaging modality and hold promise for improving the understanding of ABHD6-mediated endocannabinoid metabolism in peripheral and central nervous system disorders.

3p-C-NETA: A versatile and effective chelator for development of Al18F-labeled and therapeutic radiopharmaceuticals.

Background: Radiolabeled somatostatin analogues (e.g. [68Ga]Ga-DOTATATE and [177Lu]Lu-DOTATATE) have been used to diagnose, monitor, and treat neuroendocrine tumour (NET) patients with great success. [18F]AlF-NOTA-octreotide, a promising 18F-labeled somatostatin analogue and potential alternative for 68Ga-DOTA-peptides, is under clinical evaluation. However, ideally, the same precursor (combination of chelator-linker-vector) can be used for production of both diagnostic and therapeutic radiopharmaceuticals with very similar (e.g. Al18F-method in combination with therapeutic radiometals 213Bi/177Lu) or identical (e.g. complementary Tb-radionuclides) pharmacokinetic properties, allowing for accurate personalised dosimetry estimation and radionuclide therapy of NET patients. In this study we evaluated 3p-C-NETA, as potential theranostic Al18F-chelator and present first results of radiosynthesis and preclinical evaluation of [18F]AlF-3p-C-NETA-TATE. Methods: 3p-C-NETA was synthesized and radiolabeled with diagnostic (68Ga, Al18F) or therapeutic (177Lu, 161Tb, 213Bi, 225Ac and 67Cu) radionuclides at different temperatures (25-95 °C). The in vitro stability of the corresponding radiocomplexes was determined in phosphate-buffered saline (PBS) and human serum. 3p-C-NETA-TATE was synthesized using standard solid/liquid-phase peptide synthesis. [18F]AlF-3p-C-NETA-TATE was synthesized in an automated AllinOne® synthesis module and the in vitro stability of [18F]AlF-3p-C-NETA-TATE was evaluated in formulation buffer, PBS and human serum. [18F]AlF-3p-C-NETA-TATE pharmacokinetics were evaluated using µPET/MRI in healthy rats, with [18F]AlF-NOTA-Octreotide as benchmark. Results: 3p-C-NETA quantitatively sequestered 177Lu, 213Bi and 67Cu at 25 °C while heating was required to bind Al18F, 68Ga, 161Tb and 225Ac efficiently. The [18F]AlF-, [177Lu]Lu- and [161Tb]Tb-3p-C-NETA-complex showed excellent in vitro stability in both PBS and human serum over the study period. In contrast, [67Cu]Cu- and [225Ac]Ac-, [68Ga]Ga-3p-C-NETA were stable in PBS, but not in human serum. [18F]AlF-3p-C-NETA-TATE was obtained in good radiochemical yield and radiochemical purity. [18F]AlF-3p-C-NETA-TATE displayed good in vitro stability for 4 h in all tested conditions. Finally, [18F]AlF-3p-C-NETA-TATE showed excellent pharmacokinetic properties comparable with the results obtained for [18F]AlF-NOTA-Octreotide. Conclusions: 3p-C-NETA is a versatile chelator that can be used for both diagnostic applications (Al18F) and targeted radionuclide therapy (213Bi, 177Lu, 161Tb). It has the potential to be the new theranostic chelator of choice for clinical applications in nuclear medicine.

Challenges and advances in optical 3D mesoscale imaging.

Optical mesoscale imaging is a rapidly developing field that allows the visualisation of larger samples than is possible with standard light microscopy, and fills a gap between cell and organism resolution. It spans from advanced fluorescence imaging of micrometric cell clusters to centimetre-size complete organisms. However, with larger volume specimens, new problems arise. Imaging deeper into tissues at high resolution poses challenges ranging from optical distortions to shadowing from opaque structures. This manuscript discusses the latest developments in mesoscale imaging and highlights limitations, namely labelling, clearing, absorption, scattering, and also sample handling. We then focus on approaches that seek to turn mesoscale imaging into a more quantitative technique, analogous to quantitative tomography in medical imaging, highlighting a future role for digital and physical phantoms as well as artificial intelligence.

This review discusses the state of the art of an emerging field called mesoscale imaging. Mesoscale imaging refers to the trend towards imaging ever-larger samples that exceed the classic microscopy domain and is also referred to as 'mesoscopic imaging'. In optical imaging, this refers to objects between the microscopic and macroscopic scale that are imaged with subcellular resolution; in practice, this implies the imaging of objects from millimetre up to cm size with µm or nm resolution. As such, the mesoscopy field spans the boundary between classic 'biological' imaging and preclinical 'biomedical' imaging, typically utilising lower magnification objective lenses with a bigger field of view. We discuss the types of samples currently imaged with examples, and highlight how this type of imaging fills the gap between microscopic and macroscopic imaging, allowing further insight into the organisation of tissues in an organism. We also discuss the challenges of imaging such large samples, from sample handling to labelling and optical phenomena that stand in the way of quantitative imaging. Finally, we put the current state of the art into context within the neighbouring fields and outline future developments, such as the use of 'phantom' test samples and artificial intelligence for image analysis that will underpin the quality of mesoscale imaging.

Use of Micro-Computed Tomography to Visualize and Quantify COVID-19 Vaccine Efficiency in Free-Breathing Hamsters.

The SARS-CoV-2 pandemic has impacted the health of humanity after the outbreak in Hubei, China in late December 2019. Ever since, it has taken unprecedented proportions and rapidity causing over a million fatal cases. Recently, a robust Syrian golden hamster model recapitulating COVID-19 was developed in search for effective therapeutics and vaccine candidates. However, overt clinical disease symptoms were largely absent despite high levels of virus replication and associated pathology in the respiratory tract. Therefore, we used micro-computed tomography (µCT) to longitudinally visualize lung pathology and to preclinically assess candidate vaccines. µCT proved to be crucial to quantify and noninvasively monitor disease progression, to evaluate candidate vaccine efficacy, and to improve screening efforts by allowing longitudinal data without harming live animals. Here, we give a comprehensive guide on how to use low-dose high-resolution µCT to follow-up SARS-CoV-2-induced disease and test the efficacy of COVID-19 vaccine candidates in hamsters. Our approach can likewise be applied for the preclinical assessment of antiviral and anti-inflammatory drug treatments in vivo.

Preclinical Evaluation of [11C]YC-72-AB85 for In Vivo Visualization of Heat Shock Protein 90 in Brain and Cancer with Positron Emission Tomography.

Aberrant Hsp90 has been implied in cancer and neurodegenerative disorders. The development of a suitable Hsp90 Positron emission tomography (PET) probe can provide in vivo quantification of the expression levels of Hsp90 as a biomarker for diagnosis and follow-up of cancer and central nervous system (CNS) disease progression. In this respect, [11C]YC-72-AB85 was evaluated as an Hsp90 PET probe in B16.F10 melanoma bearing mice and its brain uptake was determined in rats and nonhuman primate. In vitro binding of [11C]YC-72-AB85 to tissue slices of mouse B16.F10 melanoma, PC3 prostate carcinoma, and rodent brain was evaluated using autoradiography. Biodistribution of [11C]YC-72-AB85 was evaluated in healthy and B16.F10 melanoma mice. In vivo brain uptake was assessed by µPET studies in rats and a rhesus monkey. In vitro binding was deemed Hsp90-specific by blocking studies with heterologous Hsp90 inhibitors onalespib and SNX-0723. Saturable Hsp90 binding was observed in brain, tumor, blood, and blood-rich organs in mice. In combined pretreatment and displacement studies, reversible and Hsp90-specific binding of [11C]YC-72-AB85 was observed in rat brain. Dynamic µPET brain scans in baseline and blocking conditions in a rhesus monkey indicated Hsp90-specific binding. [11C]YC-72-AB85 is a promising PET tracer for in vivo visualization of Hsp90 in tumor and brain. Clear differences of Hsp90 binding to blood and blood-rich organs were observed in tumor vs control mice. Further, we clearly demonstrate, for the first time, binding to a saturable Hsp90 pool in brain of rats and a rhesus monkey.

D-Peptide-Based Probe for CXCR4-Targeted Molecular Imaging and Radionuclide Therapy.

Positron emission tomography (PET) imaging of the C-X-C chemokine receptor 4 (CXCR4) with [68Ga]PentixaFor has intrinsic diagnostic value and is used to select patients for personalized CXCR4-targeted radionuclide therapy with its therapeutic radiopharmaceutical companion [177Lu]PentixaTher. However, a CXCR4-targeting radiopharmaceutical labeled with fluorine-18 is still of high value due to its favorable characteristics over gallium-68. Furthermore, clinical results with [177Lu]PentixaTher are promising, but there is still room for improvement regarding pharmacokinetics and dosimetry profile. Therefore, this study aimed to develop innovative CXCR4-targeting radiopharmaceuticals, both for diagnostic and therapeutic purposes, starting from a D-amino acid-based peptide probe (DV1-k-(DV3)) that conserves high CXCR4 binding affinity after radiolabeling. AlF-NOTA-DV1-k-(DV3) showed similar in vitro binding affinity to human CXCR4 (hCXCR4) compared to [natGa]PentixaFor (half-maximal inhibitory concentration (IC50): 5.3 ± 0.9 nM and 8.6 ± 1.1 nM, respectively) and also binds to murine CXCR4 (mCXCR4) (IC50: 33.4 ± 13.5 nM) while [natGa]PentixaFor is selective for hCXCR4 (IC50 > 1000 nM for mCXCR4). Both the diagnostic radiotracers based on the DV1-k-(DV3) vector platform, [18F]AlF-NOTA-DV1-k-(DV3) and [68Ga]Ga-DOTA-DV1-k-(DV3), and their therapeutic companion [177Lu]Lu-DOTA-DV1-k-(DV3) were successfully produced in high yield, demonstrated high in vitro and in vivo stability, and have the same favorable pharmacokinetic profile. Furthermore, in wild-type mice and a hCXCR4-expressing tumor model, [18F]AlF-NOTA-DV1-k-(DV3) shows CXCR4-specific targeting in mCXCR4-expressing organs such as liver (mean standardized uptake value (SUVmean) 8.2 ± 1.0 at 75 min post-injection (p.i.)), spleen (SUVmean 2.5 ± 1.0 at 75 min p.i.), and bone (SUVmean 0.4 ± 0.1 at 75 min p.i., femur harboring bone marrow) that can be blocked with the CXCR4 antagonist AMD3100. However, in a hCXCR4-expressing tumor model, tumor uptake of [18F]AlF-NOTA-DV1-k-(DV3) was significantly lower (SUVmean 0.6 ± 0.2) compared to [68Ga]PentixaFor (SUVmean 2.9). This might be explained by the high affinity of [18F]AlF-NOTA-DV1-k-(DV3) toward both mCXCR4 and hCXCR4. High mCXCR4 expression in mouse liver results in a large fraction of [18F]AlF-NOTA-DV1-k-(DV3) that is sequestered to the liver, resulting despite its similar in vitro affinity for hCXCR4, in lower tumor accumulation compared to [68Ga]PentixaFor. As CXCR4 is not expressed in healthy human liver, the findings in mice are not predictive for the potential clinical performance of this novel class of CXCR4-targeting radiotracers. In conclusion, the DV1-k-(DV3) scaffold is a promising vector platform for translational CXCR4-directed research.

A workflow for streamlined acquisition and correlation of serial regions of interest in array tomography.

BACKGROUND: Array tomography (AT) is a high-resolution imaging method to resolve fine details at the organelle level and has the advantage that it can provide 3D volumes to show the tissue context. AT can be carried out in a correlative way, combing light and electron microscopy (LM, EM) techniques. However, the correlation between modalities can be a challenge and delineating specific regions of interest in consecutive sections can be time-consuming. Integrated light and electron microscopes (iLEMs) offer the possibility to provide well-correlated images and may pose an ideal solution for correlative AT. Here, we report a workflow to automate navigation between regions of interest. RESULTS: We use a targeted approach that allows imaging specific tissue features, like organelles, cell processes, and nuclei at different scales to enable fast, directly correlated in situ AT using an integrated light and electron microscope (iLEM-AT). Our workflow is based on the detection of section boundaries on an initial transmitted light acquisition that serves as a reference space to compensate for changes in shape between sections, and we apply a stepwise refinement of localizations as the magnification increases from LM to EM. With minimal user interaction, this enables autonomous and speedy acquisition of regions containing cells and cellular organelles of interest correlated across different magnifications for LM and EM modalities, providing a more efficient way to obtain 3D images. We provide a proof of concept of our approach and the developed software tools using both Golgi neuronal impregnation staining and fluorescently labeled protein condensates in cells. CONCLUSIONS: Our method facilitates tracing and reconstructing cellular structures over multiple sections, is targeted at high resolution ILEMs, and can be integrated into existing devices, both commercial and custom-built systems.

Human biodistribution and dosimetry of [11C]-UCB-J, a PET radiotracer for imaging synaptic density.

RATIONALE: [11C]-UCB-J is an emerging tool for the noninvasive measurement of synaptic vesicle density in vivo. Here, we report human biodistribution and dosimetry estimates derived from sequential whole-body PET using two versions of the OLINDA dosimetry program. METHODS: Sequential whole-body PET scans were performed in 3 healthy subjects for 2 h after injection of 254 ± 77 MBq [11C]-UCB-J. Volumes of interest were drawn over relevant source organs to generate time-activity curves and calculate time-integrated activity coefficients, with effective dose coefficients calculated using OLINDA 2.1 and compared to values derived from OLINDA 1.1 and those recently reported in the literature. RESULTS: [11C]-UCB-J administration was safe and showed mixed renal and hepatobiliary clearance, with largest organ absorbed dose coefficients for the urinary bladder wall and small intestine (21.7 and 23.5 µGy/MBq, respectively). The average (±SD) effective dose coefficient was 5.4 ± 0.7 and 5.1 ± 0.8 µSv/MBq for OLINDA versions 1.1 and 2.1 respectively. Doses were lower than previously reported in the literature using either software version. CONCLUSIONS: A single IV administration of 370 MBq [11C]-UCB-J corresponds to an effective dose of less than 2.0 mSv, enabling multiple PET examinations to be carried out in the same subject. TRIAL REGISTRATION: EudraCT number: 2016-001190-32. Registered 16 March 2016, no URL available for phase 1 trials.

PI3K inhibition as a novel therapeutic strategy for neoadjuvant chemoradiotherapy resistant oesophageal adenocarcinoma.

OBJECTIVE: Neoadjuvant chemoradiotherapy (neo-CRT) prior to surgery is the standard of care for oesophageal adenocarcinoma (OAC) patients. Unfortunately, most patients fail to respond to treatment. MiR-187 was previously shown to be downregulated in neo-CRT non-responders, whist in vitro miR-187 overexpression enhanced radiosensitivity and upregulated PTEN. This study evaluates the role of miR-187 and downstream PI3K signalling in radiation response in OAC. METHODS: The effect of miR-187 overexpression on downstream PI3K signalling was evaluated in OAC cell lines by qPCR and Western blotting. PTEN expression was analysed in OAC pre-treatment biopsies of neo-CRT responders and non-responders. Pharmacological inhibition of PI3K using GDC-0941 was evaluated in combination with radiotherapy in two-dimensional and three-dimensional OAC models in vitro and as a single agent in vivo. Radiation response in vitro was assessed via clonogenic assay. RESULTS: PTEN expression was significantly decreased in neo-CRT non-responders. MiR-187 overexpression significantly upregulated PTEN expression and inhibited downstream PI3K signalling in vitro. GDC-0941 significantly reduced viability and enhanced radiation response in vitro and led to tumour growth inhibition as a single agent in vivo. CONCLUSION: Targeting of PI3K signalling is a promising therapeutic strategy for OAC patients who have repressed miR-187 expression and do not respond to conventional neo-CRT. ADVANCES IN KNOWLEDGE: This is the first study evaluating the effect of PI3K inhibition on radiosensitivity in OAC, with a particular focus on patients that do not respond to neo-CRT. We have shown for the first time that targeting of PI3K signalling is a promising alternative therapeutic strategy for OAC patients who do not respond to conventional neo-CRT.

Cell proliferation detected using [18F]FLT PET/CT as an early marker of abdominal aortic aneurysm.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a focal aortic dilatation progressing towards rupture. Non-invasive AAA-associated cell proliferation biomarkers are not yet established. We investigated the feasibility of the cell proliferation radiotracer, fluorine-18-fluorothymidine ([18F]FLT) with positron emission tomography/computed tomography (PET/CT) in a progressive pre-clinical AAA model (angiotensin II, AngII infusion). METHODS AND RESULTS: Fourteen-week-old apolipoprotein E-knockout (ApoE-/-) mice received saline or AngII via osmotic mini-pumps for 14 (n = 7 and 5, respectively) or 28 (n = 3 and 4, respectively) days and underwent 90-minute dynamic [18F]FLT PET/CT. Organs were harvested from independent cohorts for gamma counting, ultrasound scanning, and western blotting. [18F]FLT uptake was significantly greater in 14- (n = 5) and 28-day (n = 3) AAA than in saline control aortae (n = 5) (P < 0.001), which reduced between days 14 and 28. Whole-organ gamma counting confirmed greater [18F]FLT uptake in 14-day AAA (n = 9) compared to saline-infused aortae (n = 4) (P < 0.05), correlating positively with aortic volume (r = 0.71, P < 0.01). Fourteen-day AAA tissue showed increased expression of thymidine kinase-1, equilibrative nucleoside transporter (ENT)-1, ENT-2, concentrative nucleoside transporter (CNT)-1, and CNT-3 than 28-day AAA and saline control tissues (n = 3 each) (all P < 0.001). CONCLUSIONS: [18F]FLT uptake is increased during the active growth phase of the AAA model compared to saline control mice and late-stage AAA.

Clinical validation of the novel HDAC6 radiotracer [18F]EKZ-001 in the human brain.

PURPOSE: Histone deacetylase 6 (HDAC6) is a cytoplasmic enzyme that modulates intracellular transport and protein quality control. Inhibition of HDAC6 deacetylase activity has shown beneficial effects in disease models, including Alzheimer's disease and amyotrophic lateral sclerosis. This first-in-human positron emission tomography (PET) study evaluated the brain binding of [18F]EKZ-001 ([18F]Bavarostat), a radiotracer selective for HDAC6, in healthy adult subjects. METHODS: Biodistribution and radiation dosimetry studies were performed in four healthy subjects (2M/2F, 23.5 ± 2.4 years) using sequential whole-body PET/CT. The most appropriate kinetic model to quantify brain uptake was determined in 12 healthy subjects (6M/6F, 57.6 ± 3.7 years) from 120-min dynamic PET/MR scans using a radiometabolite-corrected arterial plasma input function. Four subjects underwent retest scans (2M/2F, 57.3 ± 5.6 years) with a 1-day interscan interval to determine test-retest variability (TRV). Regional volume of distribution (VT) was calculated using one-tissue and two-tissue compartment models (1-2TCM) and Logan graphical analysis (LGA), with time-stability assessed. VT differences between males and females were evaluated using volume of interest and whole-brain voxel-wise approaches. RESULTS: The effective dose was 39.1 ± 7.0 µSv/MBq. Based on the Akaike information criterion, 2TCM was the preferred model compared to 1TCM. Regional LGA VT were in agreement with 2TCM VT, however demonstrated a lower absolute TRV of 7.7 ± 4.9%. Regional VT values were relatively homogeneous with highest values in the hippocampus and entorhinal cortex. Reduction of acquisition time was achieved with a 0 to 60-min scan followed by a 90 to 120-min scan. Males demonstrated significantly higher VT than females in the majority of cortical and subcortical brain regions. No relevant radiotracer related adverse events were reported. CONCLUSION: [18F]EKZ-001 is safe and appropriate for quantifying HDAC6 expression in the human brain with Logan graphical analysis as the preferred quantitative approach. Males showed higher HDAC6 expression across the brain compared to females.

A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response1. Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques (Macaca fascicularis), and-concomitantly-protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model2 and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate.