Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous malignancy with limited predictive markers to guide personalized treatment, particularly in human papillomavirus (HPV)-negative cases, which exhibit poor outcomes. Identifying reliable biomarkers for prognosis and therapeutic response remains a critical challenge. In a retrospective cohort of 51 patients with primary HPV-negative HNSCC, we investigated the prognostic significance of the Hedgehog (HH) signaling pathway and its association with imaging biomarkers. Genomic and transcriptomic analysis revealed that HH pathway activation correlated with distinct [18F]FDG PET/CT radiomic features, notably the PET-derived “histogram:ih.max”, a surrogate for peak [18F]FDG uptake that was associated with inferior survival outcomes. Functionally, pharmacologic inhibition of HH signaling demonstrated anticancer efficacy across multiple models, including HNSCC cell lines, patient-derived tumoroids, and in vivo xenograft models. Importantly, HH pathway inhibition induced reproducible changes in imaging characteristics in xenografts, including a measurable reduction in [1⁸F]FDG uptake, closely mirroring patterns observed in patient tumors. Together these findings demonstrate that integration of multi-level molecular profiling with functional imaging captures HH-driven tumor biology in HPV-negative HNSCC. Our study underscores the value of [18F]FDG PET/CT multiomics in linking tumor biology with imaging features, providing a framework for biologically-informed patient stratification and hypothesis-driven evaluation of treatment response. These results support further translational validation of HH pathway inhibition in HPV-negative HNSCC within appropriately designed preclinical and clinical studies.

PurposeDopamine D2/3 receptors are expressed throughout the human brain, but synchronous receptor quantification in subcortical and cortical brain areas remains difficult to achieve with a single radioligand. Here, we assessed the suitability of a bolus plus constant infusion (B/I) paradigm for brain-wide dopamine D2/3 receptor imaging with the agonist radioligand [11C]-(+)-PHNO.MethodsFive healthy male volunteers underwent two B/I [11C]-(+)-PHNO positron emission tomography (PET) scans combined with a mid-scan intravenous amphetamine challenge. Non-displaceable binding potentials (BPND) were calculated using the equilibrium ratio method and an algorithmically constrained cerebellum as the reference region.ResultsReproducible baseline BPND values were obtained for the majority of regions examined, with intraclass correlation coefficients ranging from 0.5 to 0.93, and test-retest variability remaining below 10%. Moreover, amphetamine-induced decreases in receptor binding were observed in both scans in cortical and a number of subcortical regions. In the globus pallidus and ventral striatum, however, equilibrium had not been reached prior to amphetamine injection, suggesting that further optimization of the protocol might be needed to quantify amphetamine effects in D3 receptor-rich regions.ConclusionsThese results indicate that, within certain limitations, synchronous cortical and subcortical measurements of D2/3 receptors at baseline and under challenge conditions can be achieved using the B/I approach and [11C]-(+)-PHNO PET.

Pt (IV) complexes have emerged as promising redox-activated prodrugs with improved pharmacological properties compared to Pt(II) and the potential for functionalising additional binding sites. In this study, four Pt(IV)-deferoxamine (DFO) complexes (A-D) were evaluated as gallium-68 radiolabelled imaging agents in healthy mice and in a murine osteosarcoma model using positron emission tomography (PET). All complexes presented quantitative 68Ga-labelling yields with high radiochemical purity (>95%) and excellent stability in aqueous buffer and human serum after 1h incubation. In vivo evaluation in naïve mice demonstrated that pharmacokinetics of the complexes were largely governed by their hydrophilicity and lipophilicity, displaying predominant renal or hepatobiliary clearance, respectively. In the orthotopic osteosarcoma model, the complexes showed tumour-to-healthy tissue ratios ranging from 1.1 ± 0.7 to 4.5 ± 3.2 at 55 min post-injection. Ex vivo biodistribution confirmed relatively low tumour uptake, ranging from 0.3 ± 0.1 to 1.1 ± 0.9%IA/g at 60 min post-injection. Radio-metabolite studies revealed in vivo formation of chelator [68Ga]Ga-1 (DFO-succinic acid), consistent with ester bond hydrolysis, with variable systemic stability across all complexes. Cellular radioFlow analysis of [68Ga]Ga-D accumulation in tumours confirmed predominant retention in tumour and stromal cells. Among the investigated complexes, [68Ga]Ga-D provided the highest tumour-to-background contrast with the most desirable pharmacokinetic profile and in vivo stability, making it the most promising candidate for future studies. Overall, these results demonstrate that Pt(IV)-DFO complexes retain their in vivo tumour-targeting capabilities, supporting their potential use as PET-based imaging agents for osteosarcoma.

[¹¹C]Acetate-positron emission tomography (PET), widely applied in cardiology and oncology, offers unique potential as a noninvasive imaging method for assessing oxidative metabolism in metabolic disorders. In this study, we investigated its ability to detect early metabolic changes in the diet-induced model of metabolic dysfunction-associated fatty liver disease (MAFLD). Sprague Dawley rats were maintained on either a standard diet or a high-fat diet for 10 weeks before undergoing dynamic [11C]acetate-PET/computed tomography measurement. Four weeks later, radiometabolite analysis was performed in half of the animals, while the remainder underwent nonradioactive blood gas measurements. Uptake of [11C]acetate at 60 min after administration was quantified in the kidneys, myocardium, and liver (SUVmean and SUVmax), followed by analysis of time activity curves (employing AUC), alongside monoexponential clearance rates (kmono) and kinetic modeling using 1- and 2-tissue compartment models (Vt). MAFLD animals exhibited altered [¹¹C]acetate metabolism, with [11C]CO2 excretion patterns validated by non-radiative blood gas analyses. While healthy rats showed a radiometabolite peak at ∼30 min postinjection, MAFLD rats displayed an earlier maximum at 5 min and a secondary peak at 40 min, indicating a shift in longitudinal oxidative metabolism. Despite contradictions between SUVs and kmono, compartmental modeling demonstrated a clear separation of healthy from MAFLD groups, solely in hepatic volume of distribution (Vt). These results establish repurposing of [11C]acetate-PET, particularly when combined with metabolite correction, as a sensitive approach for phenotyping and identifying metabolic alterations in MAFLD and also hold translational promise for understanding and monitoring of other obesity-related liver dysfunctions.

Abstract Tissue regeneration requires careful allocation of metabolic resources, yet how organisms adjust this allocation in response to varying amounts of tissue loss remains poorly understood. Here, we show that the regenerative metabolic response is not fixed: the size of an injury regulates how glucose is allocated at both local and organism-wide levels. We first demonstrate that tail regeneration requires glucose metabolism in the axolotl ( Ambystoma mexicanum ), a salamander capable of regenerating centimetre-scale tissues. We then mapped glucose uptake in axolotls regenerating from small or large tail injuries using positron emission tomography/magnetic resonance imaging (PET/MRI) and the radiolabelled glucose analogue [ 18 F]FDG. Glucose uptake was elevated in regenerating tails compared to uninjured tails. During early regeneration, larger injuries induced higher glucose uptake than smaller injuries, correlating with faster regenerative outgrowth. Larger injuries also increased glucose uptake in distant organs, indicating a systemic metabolic response. Together, our findings suggest that metabolic responses tuned to injury size underlie faithful tissue regeneration and establish PET/MRI as a powerful approach for studying whole-body metabolic dynamics in large regenerating vertebrates.

Abstract Despite the widespread environmental contamination by micro- and nanoplastics (MNPs), the cellular toxicity of polyethylene terephthalate (PET) particles is poorly understood. Here, we assessed pristine PET-Micro- and nanoplastic particles (MNPs) (diameter: 1.24 ± 0.52 µm) and fluorescently labeled PET-MNPs (Fluo-PET, diameter: 1.25 ± 0.97 µm) in both 2D (monolayer cultures) and 3D models (multicellular spheroids) across different mammalian cell types. PET exposure caused dose- and cell type–dependent reductions in cell viability at concentrations of 2.5–5 µg/ml, with uptake rates of up to 16%. In 3D models, optical photothermal infrared spectroscopy (O-PTIR) enabled the detection and biodistribution analysis of non-labeled particles, highlighting its value as a polymer-specific, label-free technique. Short-term exposure (10 min − 120 min) induced rapid, concentration-dependent ROS generation across all cell types, while reactive nitrogen species (RNS) levels remained unchanged. Due to numerous potential interferences, particular attention was given to identifying artifacts caused by MNP interactions with conventional assays, including plastic-plate interferences due to MNP adsorption during spheroid formation, distortions in fluorescence-based assays due to light scattering, dye adsorption, or surfactant effects, all of which can yield false-positive or false-negative results if appropriate controls are not implemented. Our findings emphasize the importance of accounting for such artifacts and interferences, and the need to report and discuss these effects explicitly. In summary, PET particles induce significant cytotoxicity and oxidative stress, which is confirmed after correction for experimental artifacts. Graphical Abstract Created in BioRender.com and adapted in MS Power Point

Abstract The unique capability of deuterium metabolic imaging (DMI) to detect downstream metabolic products and trace substrates’ transport within tissues using conventional magnetic resonance (MR) scanners can, in theory, be employed with routine positron emission tomography (PET)/MR equipment. Our technical proof-of-concept study proposes a protocol for the simultaneous acquisition of DMI and [¹⁸F]FDG-PET data to enable dual assessment of hepatic glucose metabolism. A protocol that integrates high-dose glucose administration, required for DMI, with [¹⁸F]FDG-PET imaging was applied in a spectroscopy-validated rodent model of metabolic dysfunction-associated fatty liver disease (MAFLD). We acquired and quantified high-quality DMI and PET data of the liver that could provide a distinction between healthy and MAFLD cohorts in the future. Relevance statement This proof-of-concept study demonstrates the simultaneous DMI-[18F]FDG-PET acquisition for assessing hepatic glucose metabolism. With its proven viability in healthy and MAFLD livers, hybrid DMI-PET imaging shows promise as a prospective non-invasive tool to improve metabolic disease characterization and support future research applications. Key Points Simultaneous DMI and [¹⁸F]FDG-PET acquisition is technically feasible on a standard PET/MR system. Hybrid DMI-PET imaging enables dual assessment of hepatic glucose transport and metabolism, offering the advantage of reduced assessment duration. High-dose glucose administration for DMI is compatible with PET imaging, thereby reducing the subject’s burden and enhancing liver metabolic evaluation. Graphical Abstract

The fertilised chicken egg (in ovo) is a promising, ethically acceptable intermediate model for preselection of radiotracers prior to testing in rodents. The behaviour of monoclonal antibodies in ovo for application in positron emission tomography is yet to be explored. Here, we investigated the potential of this model for development of radiolabelled antibodies using simultaneous PET and magnetic resonance imaging. The biodistribution of the model antibody [89Zr]Zr-nSuc-Df-Denosumab was assessed at different time points and specific activities. The study was complemented with the more established [89Zr]Zr-nSuc-Df-Atezolizumab.Both 89Zr-labelled antibodies were synthesised with radiochemical yields >70%, radiochemical purities >98% and immune reactive fractions greater than 99%. [89Zr]Zr-nSuc-Df-Denosumab showed initial uptake in the chick embryonic heart, spleen and liver, while brain accumulation was negligible at all investigated time points. Using radiotracer in lower specific activities appeared to slow down biodistribution and clearance. By 23 h p.i., most activity was localised in the gallbladder, suggesting hepatobiliary excretion. A comparable biodistribution profile was observed for [89Zr]Zr-nSuc-Df-Atezolizumab at 19 h p.i. The rapid clearance of both IgG antibodies is likely caused by the absence of neonatal Fc receptors (FcRn) in birds, which in mammals mediate IgG recycling and thereby prolong antibody half-life.In conclusion, while the overall biodistribution patterns were broadly comparable to those reported in mammals, this first exploratory investigation of 89Zr-labelled antibodies in ovo revealed markedly altered pharmacokinetics in chick embryos, most likely due to the absence of FcRn. These findings indicate predominantly non-specific distribution and clearance of these radiolabelled antibodies in fertilised chicken eggs. This study underscores the importance of considering species-specific differences when interpreting in ovo data and the necessity of further research into the suitability of the in ovo model for evaluating different classes of radiotracers.

Thyroid hormone (TH) signaling plays a major role in the development, energy homeostasis, and metabolism of most tissues. Recent observations have identified THs as drivers of prostate cancer (PCa) tumor development and progression. We reported that the T3-scavenger protein µ-crystallin (CRYM) regulates the development and progression of PCa and that this involved crosstalk with the androgen receptor (AR) signaling. However, the mechanisms remain incompletely understood. Here, we explored the role of thyroid hormone receptor β (TRβ), which is the main effector of TH signaling, in the context of PCa. The use of the TRβ-selective antagonist NH-3 inhibited PCa cell proliferation in vitro and reduced tumor size in PCa xenograft models. Notably, NH-3 was highly effective in the engrafted 22Rv1 cell line, a model for castration-resistant PCa (CRPC). Mechanistic studies revealed that NH-3 downregulates AR and the AR target genes Nkx3.1 and KLK3 ( PSA ). NH-3 was a more effective anticancer agent than enzalutamide and showed synergistic properties in combined use. Evidence from human datasets corroborates our findings whereby elevated TRβ expression and mutations in TH signaling pathways are associated with the onset of PCa. Collectively, these results establish TRβ as a mediator of tumorigenesis in PCa and identify NH-3 as a promising therapeutic agent for targeting AR signaling, particularly in CRPC.

Tumor metabolism is a hallmark of cancer, yet cellular heterogeneity within the tumor microenvironment presents a significant challenge, as bulk analysis masks the diverse metabolic profiles of individual cell populations. This complexity complicates our understanding of [18F]FDG uptake by distinct cell types in the tumor microenvironment. This study aims to investigate [18F]FDG uptake at the single-cell level in the lung of Kirsten rat sarcoma virus-driven cancer mouse models using the novel technique radio-flow cytometry (radioFlow). Methods: Two Kirsten rat sarcoma virus-driven lung cancer mouse models were injected with [18F]FDG for small-animal PET/CT and subsequent fluorescence-activated cell sorting of the lung. For radioFlow, the sorted cell fractions were then measured in a γ-counter and their radioactivity was normalized to the number of cells. Results: RadioFlow analysis of the lung tissue of both models showed a robust cell type-specific uptake pattern across experiments. Our key findings indicate that the [18F]FDG PET signal predominantly derives from immune cells (CD45+, F4/80-, 78.3% ± 6.6%; macrophage, 13.9% ± 4.3%), whereas tumor cells contributed only with 2.8% ± 1.0%, similar to the uptake of structural cells (CD45-; tumor cells, 5.0% ± 2.3%). Normalization showed that macrophages exhibited the highest glucose metabolism in both tumor models (57% ± 8%), followed by the remaining immune cells (27% ± 3%). Conclusion: These findings highlight the critical influence of immune cell metabolism on [18F]FDG imaging, emphasizing the need to account for immune contributions when interpreting [18F]FDG imaging in cancer.

[11C]Acetate and [11C]acetoacetate are PET radiotracers widely used to assess oxidative metabolism and ketone body utilization, respectively. This study aimed to establish robust, high-yield syntheses of both tracers using a GE TRACERlab FX2 C module, with an emphasis on improving radiochemical purity (RCP), radiochemical yield (RCY), optimizing operational parameters, and developing accurate quality control methods. [11C]Acetate was synthesized via Grignard carboxylation using [11C]CO2 and purified with a cartridge-based system. [11C]Acetoacetate was produced via in-loop [11C]CO2 carboxylation of a lithium enolate precursor, followed by semi-preparative reversed-phase HPLC purification. Quality control was performed by reported ion-exchange chromatography (IEX-HPLC) and novel reversed-phase HPLC (RP-HPLC). A systematic literature review was conducted to evaluate prior quality control methods for [11C]acetoacetate. Omission of helium flow during [11C]CO2 trap bake-out significantly improved activity recovery from the [11C]CO2 trap (from 63 to 89

Prostate cancer (PCa) and Type 2 diabetes (T2D) often co-occur, yet their relationship remains elusive. While some studies suggest that T2D lowers PCa risk, others report conflicting data. This study investigates the effects of peroxisome proliferator-activated receptor (PPAR) agonists Bezafibrate, Tesaglitazar, and Pioglitazone on PCa tumorigenesis. Analysis of patient datasets revealed that high PPARG expression correlates with advanced PCa and poor survival. The PPARγ agonists Pioglitazone and Tesaglitazar notably reduced cell proliferation and PPARγ protein levels in primary and metastatic PCa-derived cells. Proteomic analysis identified intrinsic differences in mTORC1 and mitochondrial fatty acid oxidation (FAO) pathways between primary and metastatic PCa cells, which were further disrupted by Tesaglitazar and Pioglitazone. Moreover, metabolomics, Seahorse Assay-based metabolic profiling, and radiotracer uptake assays revealed that Pioglitazone shifted primary PCa cells' metabolism towards glycolysis and increased FAO in metastatic cells, reducing mitochondrial ATP production. Furthermore, Pioglitazone suppressed cell migration in primary and metastatic PCa cells and induced the epithelial marker E-Cadherin in primary PCa cells. In vivo , Pioglitazone reduced tumor growth in a metastatic PC3 xenograft model, increased phosho AMPKα and decreased phospho mTOR levels. In addition, diabetic PCa patients treated with PPAR agonists post-radical prostatectomy implied no biochemical recurrence over five to ten years compared to non-diabetic PCa patients. Our findings suggest that Pioglitazone reduces PCa cell proliferation and induces metabolic and epithelial changes, highlighting the potential of repurposing metabolic drugs for PCa therapy. Graphical Abstract

Background/Objectives: Radiolabeled fibroblast activation protein inhibitors (FAPIs) are emerging as promising imaging agents assessing fibrotic diseases. This study evaluates [68Ga]Ga-DATA5m.SA.FAPi for imaging pulmonary fibrosis in two mouse models, bleomycin-induced (BLM) and a transgenic (fra-2tg) model, both displaying characteristics of human pulmonary fibrotic diseases. Methods: In the BLM model, C57BL/6 mice were treated with bleomycin or isotonic sodium chloride (controls) for 4, 5, and 6 weeks, followed by [68Ga]Ga-DATA5m.SA.FAPi PET/CT scans. Fra-2tg mice and wildtype (WT) littermates underwent at 7, 11, and 18/19 weeks of age a PET/CT scan. The selected timepoints correspond to early, middle, and late disease stages for each model. Imaging was complemented by ex vivo quantification, histological, and immunohistochemical (IHC) analyses. Results: In BLM mice, pulmonary [68Ga]Ga-DATA5m.SA.FAPi uptake showed a trend toward increase as early as 5 weeks of treatment compared with the controls, which was confirmed by ex vivo analysis (BLM: 3.31 ± 0.29%ID/g, n = 5; control: 1.61 ± 0.29%ID/g, n = 4; p = 0.0035). In fra-2tg mice, no significant differences could be detected. IHC revealed elevated pulmonary FAP expression specifically at early (BLM) and mild (fra-2tg) disease stages, whereas for BLM, tracer uptake was more pronounced at later stages. Conclusions: Our findings complement and extend observations from previous studies and support the potential of FAPI tracers as molecular imaging agents for pulmonary fibrosis.

Alterations in tricarboxylic acid (TCA) cycle metabolism are associated with hepatic metabolic disorders. Elevated hepatic acetate concentrations, often attributed to high caloric intake, are recognized as a pivotal factor in the etiology of obesity and metabolic syndrome. Therefore, the assessment of acetate breakdown and TCA cycle activity plays a central role in understanding the impact of diet-induced alterations on liver metabolism. Magnetic resonance-based deuterium metabolic imaging (DMI) could help to unravel the underlying mechanisms involved in disease development and progression, however, the application of conventional deuterated glucose does not lead to substantial enrichment in hepatic glutamine and glutamate. This study aimed to demonstrate the feasibility of DMI for tracking deuterated acetate breakdown via the TCA cycle in lean and diet-induced fatty liver (FL) rats using 3D DMI after an intraperitoneal infusion of sodium acetate-d3 at 9.4T. Localized and nonlocalized liver spectra acquired at 10 time points post-injection over a 130-min study revealed similar intrahepatic acetate uptake in both animal groups (AUCFL = 717.9 ± 131.1 mM▯min-1, AUClean = 605.1 ± 119.9 mM▯min-1, p = 0.62). Metabolic breakdown could be observed in both groups with an emerging glutamine/glutamate (Glx) peak as a downstream metabolic product (AUCFL = 113.6 ± 23.8 mM▯min-1, AUClean = 136.7 ± 41.7 mM▯min-1, p = 0.68). This study showed the viability of DMI for tracking substrate flux through the TCA cycle, underscoring its methodological potential for imaging metabolic processes in the body.

The complex radiosynthesis of alpha-[11C]methyl-L-tryptophan ([11C]AMT) involves harsh chemicals and conditions, posing challenges for its implementation on commercially available synthesis modules. This study describes the adaptation of the GE TRACERlab FX2 C module for [11C]AMT production using both a half-manual approach and a semi-automated method incorporating a 16-way valve system. [11C]AMT was synthesized with decay-corrected radiochemical yields of 13 ± 7.5 % (half-manual) and 10.4 ± 4.1 % (semi-automated), with radiochemical purities exceeding 95 %. The half-manual approach demonstrated higher reliability in synthesis success but required increased operator intervention, while the semi-automated method minimized radiation exposure to the operator. Key factors influencing synthesis success included the preparation and precise addition of lithium diisopropylamide and the use of a soda lime column to mitigate iodine contamination during [11C]CH3I transfer. This work presents a practical and scalable solution for producing [11C]AMT on a commercially available module, enabling its broader application in clinical research, particularly in brain imaging and pediatric oncology.

Poly (ADP-ribose) polymerase (PARP) enzymes are crucial for the repair of DNA single-strand breaks and have become key therapeutic targets in homologous recombination-deficient cancers, including prostate cancer. To enable non-invasive monitoring of PARP-1 expression, several PARP-1-targeting positron emission tomography (PET) tracers have been developed. Here, we aimed to preclinically investigate [carbonyl-11C]DPQ as an alternative PARP-1 PET tracer as it features a strongly distinct chemotype compared to the frontrunners [18F]FluorThanatrace and [18F]PARPi. [carbonyl-11C]DPQ was synthesised in a GE TracerLab FXC2 module, yielding sufficient activity (940 ± 410 MBq), molar activity (53 ± 16 GBq/µmol) and radiochemical purity (> 97

Background/Objectives: Fibroblast activation protein (FAP) has gained tremendous traction as a target for tumor imaging and cancer treatment, while also playing a key role in fibrosis. Our study aimed to evaluate [68Ga]Ga-DATA5m.SA.FAPi for PET imaging of replacement fibrosis following myocardial infarction (MI) or interstitial fibrosis associated with hypertrophy. Methods: MI or transverse aortic constriction (TAC)-induced hypertrophy was induced in C57BL/6 mice, with sham-operated animals serving as controls. At multiple time points during disease progression (1, 2, and 6 weeks post-surgery), [68Ga]Ga-DATA5m.SA.FAPi PET/CT scans were performed, followed by ex vivo investigations. Additionally, in vitro cell uptake experiments simulating hypertrophy were conducted. Results: Cardiac uptake of [68Ga]Ga-DATA5m.SA.FAPi significantly increased two weeks after MI induction (MI: 2.1 ± 0.2%ID/g, n = 7 vs. SHAM: 1.1 ± 0.2%ID/g, n = 5; p = 0.002), confirmed by ex vivo autoradiography. No significant difference was observed at six weeks post-MI (MI: 1.1 ± 0.1%ID/g, n = 4 vs. SHAM: 0.8 ± 0.0%ID/g, n = 3), indicating infarct healing completion. In contrast, TAC mice showed increased uptake after six weeks (TAC: 1.8 ± 0.2%ID/g, n = 6; p = 0.007), related to interstitial fibrosis progression. Consistently, high-stretched cardiac fibroblasts demonstrated a higher uptake compared to low-stretched conditioned ones, suggesting the stretch mediates regulation of FAP. Conclusions: This study demonstrated the efficacy of [68Ga]Ga-DATA5m.SA.FAPi for longitudinal imaging of cardiac fibrosis in response to different cardiac injuries. In vivo FAP imaging during cardiac remodeling may serve as a valuable tool for diagnosing and predicting disease progression, ultimately aiding in the clinical management of patients.