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Frontiers in Immunology 2023Non-invasive imaging techniques such as positron emission tomography (PET) are extremely important for cancer detection and characterization especially for difficult to...
High folate receptor expression in gliomas can be detected using folate-based positron emission tomography with high tumor-to-brain uptake ratio divulging potential future targeting possibilities.
INTRODUCTION
Non-invasive imaging techniques such as positron emission tomography (PET) are extremely important for cancer detection and characterization especially for difficult to biopsy or extremely delicate organs such as the brain. The folate analogue 1,4,7-triazacylononane-1,4,7-triacetic acid-conjugated folate radiolabeled with aluminum fluoride-18 ([F]FOL) has been previously shown to accumulate preferentially in tumor cells with an overexpression of folate receptors (FRs) and here was investigated for its ability to detect orthotopic gliomas in a rat model. In addition, we studied the expression of FRs in human glioblastoma samples to investigate if an analogous relationship may exist.
METHODS
Nine BDIX rats were injected with BT4C rat glioma cells into the right hemisphere of the brain. Animals were imaged with gadolinium-enhanced magnetic resonance imaging at on days prior to PET/computed tomography (CT) imaging. Animals were divided into two groups, and were PET/CT imaged with either [F]FOL or 2-deoxy-2-18F-fluoro-D-glucose ([F]FDG) on 19 and 32-days post glioma grafting. Two subjects were also PET/CT imaged with [F]FOL on day 16. Biodistribution was studied and brains were cryosectioned for autoradiography, immunofluorescence, and histological studies. Patient-derived paraffin-embedded glioblastomas were sectioned and stained with similar methods.
RESULTS
PET imaging showed an increase of [18F]FOL tumor-to-brain uptake ratio (TBR) over the study duration from day 16/19 (3.3 ± 0.9) increasing to 5.7 ± 1.0 by day 32. [F]FDG PET-imaged rats had a consistent TBR of 1.6 ± 0.1 throughout the study. Ex vivo autoradiography results revealed an exceptionally high TBR of 116.1 ± 26.9 for [F]FOL while the [18F]FDG values were significantly lower giving 2.9 ± 0.6 (P<0.0001). Immunostaining demonstrated an increased presence of FR-α in the BT4C gliomas versus the contralateral brain tissue, while FR-β was present only on glioma periphery. Human sections assayed showed similar FRs expression characteristics.
CONCLUSION
This study shows upregulation of FR-α inside glioma regions in both human and animal tissue, providing a biochemical basis for the observed increased [F]FOL uptake in animal PET images. These results suggest that FRs targeting imaging and therapeutic compounds may possess clinically relevant translational abilities for the detection and treatment of gliomas.
Topics: Rats; Humans; Animals; Fluorodeoxyglucose F18; Positron Emission Tomography Computed Tomography; Folic Acid; Tissue Distribution; Radiopharmaceuticals; Positron-Emission Tomography; Glioma; Brain; Glioblastoma
PubMed: 37275898
DOI: 10.3389/fimmu.2023.1145473 -
EJNMMI Research Jul 2022Microtubules (MTs) are critical for cell structure, function, and survival. MT instability may contribute to Alzheimer's disease (AD) pathogenesis as evidenced by...
BACKGROUND
Microtubules (MTs) are critical for cell structure, function, and survival. MT instability may contribute to Alzheimer's disease (AD) pathogenesis as evidenced by persistent negative regulation (phosphorylation) of the neuronal microtubule-associated protein tau. Hyperphosphorylated tau, not bound to MTs, forms intraneuronal pathology that correlates with dementia and can be tracked using positron emission tomography (PET) imaging. The contribution of MT instability in AD remains unknown, though it may be more proximal to neuronal dysfunction than tau accumulation. Our lab reported the first brain-penetrant MT-based PET ligand, [C]MPC-6827, and its PET imaging with this ligand in normal rodents and non-human primates demonstrated high brain uptake and excellent pharmacokinetics. Target engagement and mechanism of action using in vitro, in vivo, and ex vivo methods were evaluated here.
METHODS
In vitro cell uptake assay was performed in SH-SY5Y neuronal cells with [C]MPC-6827, with various MT stabilizing and destabilizing agents. To validate the in vitro results, wild type (WT) mice (n = 4) treated with a brain-penetrant MT stabilizing drug (EpoD) underwent microPET/CT brain imaging with [C]MPC-6827. To determine the influence of tau protein on radiotracer binding in the absence of protein accumulation, we utilized tau knockout (KO) mice. In vivo microPET imaging, ex vivo biodistribution, and autoradiography studies were performed in tau KO and WT mice (n = 6/group) with [C]MPC-6827. Additionally, α, β, and acetylated tubulin levels in both brain samples were determined using commercially available cytoskeleton-based MT kit and capillary electrophoresis immunoblotting assays.
RESULTS
Cell uptake demonstrated higher radioactive uptake with MT destabilizing agents and lower uptake with stabilizing agents compared to untreated cells. Similarly, acute treatment with EpoD in WT mice decreased [C]MPC-6827 brain uptake, assessed with microPET/CT imaging. Compared to WT mice, tau KO mice expressed significantly lower β tubulin, which contains the MPC-6827 binding domain, and modestly lower levels of acetylated α tubulin, indicative of unstable MTs. In vivo imaging revealed significantly higher [C]MPC-6827 uptake in tau KOs than WT, particularly in AD-relevant brain regions known to express high levels of tau. Ex vivo post-PET biodistribution and autoradiography confirmed the in vivo results.
CONCLUSIONS
Collectively, our data indicate that [C]MPC-6827 uptake inversely correlates with MT stability and may better reflect the absence of tau than total tubulin levels. Given the radiotracer binding does not require the presence of aggregated tau, we hypothesize that [C]MPC-6827 may be particularly useful in preclinical stages of AD prior to tau deposition. Our study provides immediate clarity on high uptake of the MT-based radiotracer in AD brains, which directly informs clinical utility in MT/tau-based PET imaging studies.
PubMed: 35881263
DOI: 10.1186/s13550-022-00912-z -
Theranostics 2021Myocardial infarction (MI) evokes an organized remodeling process characterized by the activation and transdifferentiation of quiescent cardiac fibroblasts to generate...
Myocardial infarction (MI) evokes an organized remodeling process characterized by the activation and transdifferentiation of quiescent cardiac fibroblasts to generate a stable collagen rich scar. Early fibroblast activation may be amenable to targeted therapy, but is challenging to identify . We aimed to non-invasively image active fibrosis by targeting the fibroblast activation protein (FAP) expressed by activated (myo)fibroblasts, using a novel positron emission tomography (PET) radioligand [Ga]MHLL1 after acute MI. One-step chemical synthesis and manual as well as module-based radiolabeling yielded [Ga]MHLL1. Binding characteristics were evaluated in murine and human FAP-transfected cells, and stability tested in human serum. Biodistribution in healthy animals was interrogated by dynamic PET imaging, and metabolites were measured in blood and urine. The temporal pattern of FAP expression was determined by serial PET imaging at 7 d and 21 d after coronary artery ligation in mice as percent injected dose per gram (%ID/g). PET measurements were validated by autoradiography and immunostaining for FAP and inflammatory macrophages. [Ga]MHLL1 displayed specific uptake in murine and human FAP-positive cells (p = 0.0208). In healthy mice the tracer exhibited favorable imaging characteristics, with low blood pool retention and dominantly renal clearance. At 7 d after coronary artery ligation, [Ga]MHLL1 uptake was elevated in the infarct relative to the non-infarcted remote myocardium (1.3 ± 0.3 1.0 ± 0.2 %ID/g, p < 0.001) which persisted to 21 d after MI (1.3 ± 0.4 1.1 ± 0.4 %ID/g, p = 0.013). Excess unlabeled compound blocked tracer accumulation in both infarct and non-infarct remote myocardium regions (p < 0.001). Autoradiography and histology confirmed the regional uptake of [Ga]MHLL1 in the infarct and especially border zone regions, as identified by Masson trichrome collagen staining. Immunostaining further delineated persistent FAP expression at 7 d and 21 d post-MI in the border zone, consistent with tracer distribution . The simplified synthesis of [Ga]MHLL1 bears promise for non-invasive characterization of fibroblast activation protein early in remodeling after MI.
Topics: Animals; Autoradiography; Cell Line, Tumor; Endopeptidases; Fibroblasts; Fibrosis; Gallium Radioisotopes; Humans; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Molecular Imaging; Myocardial Infarction; Myocardium; Positron-Emission Tomography; Tissue Distribution; Tomography, X-Ray Computed
PubMed: 34335962
DOI: 10.7150/thno.51419 -
In Vivo (Athens, Greece) 2024Since acute myeloid leukemias still represent the most aggressive type of adult acute leukemias, the profound understanding of disease pathology is of paramount...
BACKGROUND/AIM
Since acute myeloid leukemias still represent the most aggressive type of adult acute leukemias, the profound understanding of disease pathology is of paramount importance for diagnostic and therapeutic purposes. Hence, this study aimed to explore the real-time disease fate with the establishment of an experimental myelomonoblastic leukemia (My1/De) rat model using preclinical positron emission tomography (PET) and whole-body autoradiography.
MATERIALS AND METHODS
In vitro [F]F-FDG uptake studies were performed to compare the tracer accumulation in the newly cultured My1/De tumor cell line (blasts) with that in healthy control and My1/De bone marrow suspensions. Post transplantation of My1/De cells under the left renal capsule of Long-Evans rats, primary My1/De tumorigenesis, and metastatic propagation were investigated using [F]F-FDG PET imaging, whole-body autoradiography and phosphorimage analyses. To assess the organ uptake profile of the tumor-carrying animals we accomplished ex vivo biodistribution studies.
RESULTS
The tracer accumulation in the My1/De culture cells exceeded that of both the tumorous and the healthy bone marrow suspensions (p<0.01). Based on in vivo imaging, the subrenally transplanted My1/De cells resulted in the development of leukemia in the abdominal organs, and metastasized to the mesenterial and thoracic parathymic lymph nodes (PTLNs). The lymphatic spread of metastasis was further confirmed by the significantly higher %ID/g values of the metastatic PTLNs (4.25±0.28) compared to the control (0.94±0.34). Cytochemical staining of the peripheral blood, autopsy findings, and wright-Giemsa-stained post-mortem histological sections proved the leukemic involvement of the assessed tissues/organs.
CONCLUSION
The currently established My1/De model appears to be well-suited for further leukemia-related therapeutic and diagnostic investigations.
Topics: Animals; Rats; Disease Models, Animal; Fluorodeoxyglucose F18; Autoradiography; Positron-Emission Tomography; Cell Line, Tumor; Tissue Distribution; Leukemia, Myeloid, Acute; Radiopharmaceuticals; Male; Humans
PubMed: 38688644
DOI: 10.21873/invivo.13540 -
Nuclear Medicine and Biology 2022Antibody-based constructs, engineered to enter the brain using transferrin receptor (TfR) mediated transcytosis, have been successfully used as PET radioligands for...
PURPOSE
Antibody-based constructs, engineered to enter the brain using transferrin receptor (TfR) mediated transcytosis, have been successfully used as PET radioligands for imaging of amyloid-beta (Aβ) in preclinical studies. However, these radioligands have been large and associated with long circulation times, i.e. non-optimal properties for neuroPET radioligands. The aim of this study was to investigate the in vivo brain delivery of the radiolabeled nanobody VHH-E9 that binds to glial fibrillary acidic protein (GFAP) expressed by reactive astrocytes, without and with fusion to a TfR binding moiety, as potential tools to detect neuroinflammation.
METHODS
Three protein constructs were recombinantly expressed: 1) The GFAP specific nanobody VHH-E9, 2) VHH-E9 fused to a single chain variable fragment of the TfR binding antibody 8D3 (scFv8D3) and 3) scFv8D3 alone. Brain delivery of the constructs was investigated at 2 h post injection. Binding to GFAP was studied with autoradiography while in vivo brain retention of [I]VHH-E9 and [I]VHH-E9-scFv8D3 was further investigated at 8 h, 24 h and 48 h in wild-type (WT), and at the same time points in transgenic mice (ArcSwe) that in addition to Aβ pathology also display neuroinflammation.
RESULTS
At 2 h after administration, [I]VHH-E9-scFv8D3 and [I]scFv8D3 displayed 3-fold higher brain concentrations than [I]VHH-E9. In vitro autoradiography showed distinct binding of both [I]VHH-E9-scFv8D3 and [I]VHH-E9 to regions with abundant GFAP in ArcSwe mice. However, in vivo, there was no difference in brain concentrations between WT and ArcSwe at any of the studied time points.
CONCLUSIONS
Fused to scFv8D3, VHH-E9 displayed increased brain delivery. When radiolabeled and applied on brain sections, the bispecific construct was able to discriminate between WT and ArcSwe mice, but in vivo brain uptake and retention over time did not differ between WT and ArcSwe mice.
Topics: Animals; Mice; Glial Fibrillary Acidic Protein; Alzheimer Disease; Neuroinflammatory Diseases; Positron-Emission Tomography; Brain; Amyloid beta-Peptides; Mice, Transgenic
PubMed: 35487832
DOI: 10.1016/j.nucmedbio.2022.04.002 -
Chembiochem : a European Journal of... Dec 2022Tetrazine (Tz)-trans-cyclooctene (TCO) ligation is an ultra-fast and highly selective reaction and it is particularly suited to label biomolecules under physiological...
Tetrazine (Tz)-trans-cyclooctene (TCO) ligation is an ultra-fast and highly selective reaction and it is particularly suited to label biomolecules under physiological conditions. As such, a H-Tz based synthon would have wide applications for in vitro/ex vivo assays. In this study, we developed a H-labeled Tz and characterized its potential for application to pretargeted autoradiography. Several strategies were explored to synthesize such a Tz. However, classical approaches such as reductive halogenation failed. For this reason, we designed a Tz containing an aldehyde and explored the possibility of reducing this group with NaBT . This approach was successful and resulted in [ H]-(4-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)phenyl)methan-t-ol with a radiochemical yield of 22 %, a radiochemical purity of 96 % and a molar activity of 0.437 GBq/μmol (11.8 Ci/mmol). The compound was successfully applied to pretargeted autoradiography. Thus, we report the synthesis of the first H-labeled Tz and its successful application as a labeling building block.
Topics: Cell Line, Tumor; Radiopharmaceuticals; Heterocyclic Compounds; Cyclooctanes
PubMed: 36333105
DOI: 10.1002/cbic.202200539 -
Molecular Imaging and Biology Jun 2023Resection of the tumor-draining lymph -node (TDLN) represents a standard method to identify metastasis for several malignancies. Interestingly, recent preclinical...
PURPOSE
Resection of the tumor-draining lymph -node (TDLN) represents a standard method to identify metastasis for several malignancies. Interestingly, recent preclinical studies indicate that TDLN resection diminishes the efficacy of immune checkpoint inhibitor-based cancer immunotherapies. Thus, accurate preclinical identification of TDLNs is pivotal to uncovering the underlying immunological mechanisms. Therefore, we validated preclinically, and clinically available non-invasive in vivo imaging approaches for precise TDLN identification.
PROCEDURES
For visualization of the lymphatic drainage into the TDLNs by non-invasive in vivo optical imaging, we injected the optical imaging contrast agents Patent Blue V (582.7 g mol) and IRDye® 800CW polyethylene glycol (PEG; 25,000-60,000 g mol), subcutaneously (s.c.) in close proximity to MC38 adenocarcinomas at the right flank of experimental mice. For determination of the lymphatic drainage and the glucose metabolism in TDLNs by non-invasive in vivo PET/magnetic resonance imaging (PET/MRI), we injected the positron emission tomography (PET) tracer (2-deoxy-2[F]fluoro-D-glucose (F-FDG) [181.1 g mol]) in a similar manner. For ex vivo cross-correlation, we isolated TDLNs and contralateral nontumor-draining lymph nodes (NTDLNs) and performed optical imaging, biodistribution, and autoradiography analysis.
RESULTS
The clinically well-established Patent Blue V was superior for intraoperative macroscopic identification of the TDLNs compared with IRDye® 800CW PEG but was not sensitive enough for non-invasive in vivo detection by optical imaging. Ex vivo Patent Blue V biodistribution analysis clearly identified the right accessory axillary and the proper axillary lymph node (LN) as TDLNs, whereas ex vivo IRDye® 800CW PEG completely failed. In contrast, functional non-invasive in vivo F-FDG PET/MRI identified a significantly elevated uptake exclusively within the ipsilateral accessory axillary TDLN of experimental mice and was able to differentiate between the accessory axillary and the proper LN. Ex vivo biodistribution and autoradiography confirmed our in vivo F-FDG PET/MRI results.
CONCLUSIONS
When taken together, our results demonstrate the feasibility of F-FDG-PET/MRI as a valid method for non-invasive in vivo, intraoperative, and ex vivo identification of the lymphatic drainage and glucose metabolism within the TDLNs. In addition, using Patent Blue V provides additive value for the macroscopic localization of the lymphatic drainage both visually and by ex vivo optical imaging analysis. Thus, both methods are valuable, easy to implement, and cost-effective for preclinical identification of the TDLN.
Topics: Animals; Mice; Fluorodeoxyglucose F18; Tissue Distribution; Positron-Emission Tomography; Lymph Nodes; Lymphatic Metastasis; Glucose; Radiopharmaceuticals; Sensitivity and Specificity
PubMed: 36600172
DOI: 10.1007/s11307-022-01797-z -
PloS One 2019In the rat, oxytocin (OT) produces dose-dependent diuretic and natriuretic responses. Post-translational enzymatic conversion of the OT biosynthetic precursor forms both...
In the rat, oxytocin (OT) produces dose-dependent diuretic and natriuretic responses. Post-translational enzymatic conversion of the OT biosynthetic precursor forms both mature and C-terminally extended peptides. The plasma concentrations of these C-terminally extended peptides (OT-G; OT-GK and OT-GKR) are elevated in newborns and pregnant rats. Intravenous injection of OT-GKR to rats inhibits diuresis, whereas injection of amidated OT stimulates diuresis. Since OT and OT-GKR show different effects on the urine flow, we investigated whether OT-GKR modulates renal action by inhibition of the arginine-vasopressin (AVP) receptor V2 (V2R), the receptor involved in renal water reabsorption. Experiments were carried out in the 8-week-old Wistar rats receiving intravenous (iv) injections of vehicle, OT, OT-GKR or OT+OT-GKR combination. OT (10 μmol/kg) increased urine outflow by 40% (P<0.01) and sodium excretion by 47% (P<0.01). Treatment with OT-GKR (10 μmol/kg) decreased diuresis by 50% (P<0.001), decreased sodium excretion by 50% (P<0.05) and lowered potassium by 42% (P<0.05). OT antagonist (OTA) reduced diuresis and natriuresis exerted by OT, whereas the anti-diuretic effect of OT-GKR was unaffected by OTA. The treatment with V2R antagonist (V2A) in the presence and absence of OT induced diuresis, sodium and potassium outflow. V2A in the presence of OT-GKR only partially increased diuresis and natriuresis. Autoradiography and molecular docking analysis showed potent binding of OT-GKR to V2R. Finally, the release of cAMP from CHO cells overexpressing V2 receptor was induced by low concentration of AVP (EC50:4.2e-011), at higher concentrations of OT (EC50:3.2e-010) and by the highest concentrations of OT-GKR (EC50:1.1e-006). OT-GKR potentiated cAMP release when combined with AVP, but blocked cAMP release when combined with OT. These results suggest that OT-GKR by competing for the OT renal receptor (OTR) and binding to V2R in the kidney, induces anti-diuretic, anti-natriuretic, and anti-kaliuretic effects.
Topics: Animals; Autoradiography; Binding, Competitive; CHO Cells; Cell Line; Cricetinae; Cricetulus; Cyclic AMP; Diuresis; Electrolytes; Humans; Kidney; Molecular Docking Simulation; Natriuresis; Oxytocin; Peptides; Rats; Rats, Wistar; Receptors, Vasopressin; Urination; Vasopressins
PubMed: 31269062
DOI: 10.1371/journal.pone.0219205 -
Journal of Nuclear Cardiology :... Dec 2021Imaging Somatostatin Subtype Receptor 2 (SST) expressing macrophages by [DOTA,Tyr]-octreotate (DOTATATE) has proven successful for plaque detection. DOTA-JR11 is a SST...
BACKGROUND
Imaging Somatostatin Subtype Receptor 2 (SST) expressing macrophages by [DOTA,Tyr]-octreotate (DOTATATE) has proven successful for plaque detection. DOTA-JR11 is a SST targeting ligand with a five times higher tumor uptake than DOTATATE, and holds promise to improve plaque imaging. The aim of this study was to evaluate the potential of DOTA-JR11 for plaque detection.
METHODS AND RESULTS
Atherosclerotic ApoE mice (n = 22) fed an atherogenic diet were imaged by SPECT/CT two hours post injection of [In]In-DOTA-JR11 (~ 200 pmol, ~ 50 MBq). In vivo plaque uptake of [In]In-DOTA-JR11 was visible in all mice, with a target-to-background-ratio (TBR) of 2.23 ± 0.35. Post-mortem scans after thymectomy and ex vivo scans of the arteries after excision of the arteries confirmed plaque uptake of the radioligand with TBRs of 2.46 ± 0.52 and 3.43 ± 1.45 respectively. Oil red O lipid-staining and ex vivo autoradiography of excised arteries showed [In]In-DOTA-JR11 uptake at plaque locations. Histological processing showed CD68 (macrophages) and SST expressing cells in plaques. SPECT/CT, in vitro autoradiography and immunohistochemistry performed on slices of a human carotid endarterectomy sample showed [In]In-DOTA-JR11 uptake at plaque locations containing CD68 and SST expressing cells.
CONCLUSIONS
The results of this study indicate DOTA-JR11 as a promising ligand for visualization of atherosclerotic plaque inflammation.
Topics: Animals; Heterocyclic Compounds, 1-Ring; Indium Radioisotopes; Inflammation; Mice; Plaque, Atherosclerotic; Receptors, Somatostatin
PubMed: 32026330
DOI: 10.1007/s12350-020-02046-y -
European Journal of Nuclear Medicine... Apr 2023The recent conditional FDA approval of Aducanumab (Adu) for treating Alzheimer's disease (AD) and the continued discussions around that decision have increased interest...
PURPOSE
The recent conditional FDA approval of Aducanumab (Adu) for treating Alzheimer's disease (AD) and the continued discussions around that decision have increased interest in immunotherapy for AD and other brain diseases. Reliable techniques for brain imaging of antibodies may guide decision-making in the future but needs further development. In this study, we used Zr-immuno-PET to evaluate the targeting and distribution of a bispecific brain-shuttle IgG based on Adu with transferrin receptor protein-1 (TfR1) shuttling mechanism, mAbAdu-scFab8D3, designated Adu-8D3, as a candidate theranostic for AD. We also validated the Zr-immuno-PET platform as an enabling technology for developing new antibody-based theranostics for brain disorders.
METHODS
Adu, Adu-8D3, and the non-binding control construct B12-8D3 were modified with DFO*-NCS and radiolabeled with Zr. APP/PS1 mice were injected with Zr-labeled mAbs and imaged on days 3 and 7 by positron emission tomography (PET). Ex vivo biodistribution was performed on day 7, and ex vivo autoradiography and immunofluorescence staining were done on brain tissue to validate the PET imaging results and target engagement with amyloid-β plaques. Additionally, [Zr]Zr-DFO*-Adu-8D3 was evaluated in 3, 7, and 10-month-old APP/PS1 mice to test its potential in early stage disease.
RESULTS
A 7-fold higher brain uptake was observed for [Zr]Zr-DFO*-Adu-8D3 compared to [Zr]Zr-DFO*-Adu and a 2.7-fold higher uptake compared to [Zr]Zr-DFO*-B12-8D3 on day 7. Autoradiography and immunofluorescence of [Zr]Zr-DFO*-Adu-8D3 showed co-localization with amyloid plaques, which was not the case with the Adu and B12-8D3 conjugates. [Zr]Zr-DFO*-Adu-8D3 was able to detect low plaque load in 3-month-old APP/PS1 mice.
CONCLUSION
Zr-DFO*-immuno-PET revealed high and specific uptake of the bispecific Adu-8D3 in the brain and can be used for the early detection of Aβ plaque pathology. Here, we demonstrate that Zr-DFO*-immuno-PET can be used to visualize and quantify brain uptake of mAbs and contribute to the evaluation of biological therapeutics for brain diseases.
Topics: Mice; Animals; Radioisotopes; Tissue Distribution; Positron-Emission Tomography; Antibodies, Monoclonal; Alzheimer Disease; Amyloid; Zirconium; Cell Line, Tumor
PubMed: 36635462
DOI: 10.1007/s00259-023-06109-3