Glioblastoma PET/MRI: kinetic investigation of [18F]rhPSMA-7.3, [18F]FET and [18F]fluciclovine in an orthotopic mouse model of cancer.

PURPOSE: Glioblastoma multiforme (GBM) is the most common glioma and standard therapies can only slightly prolong the survival. Neo-vascularization is a potential target to image tumor microenvironment, as it defines its brain invasion. We investigate [18F]rhPSMA-7.3 with PET/MRI for quantitative imaging of neo-vascularization in GBM bearing mice and human tumor tissue and compare it to [18F]FET and [18F]fluciclovine using PET pharmacokinetic modeling (PKM). METHODS: [18F]rhPSMA-7.3, [18F]FET, and [18F]fluciclovine were i.v. injected with 10.5 ± 3.1 MBq, 8.0 ± 2.2 MBq, 11.5 ± 1.9 MBq (n = 28, GL261-luc2) and up to 90 min PET/MR imaged 21/28 days after surgery. Regions of interest were delineated on T2-weighted MRI for (i) tumor, (ii) brain, and (iii) the inferior vena cava. Time-activity curves were expressed as SUV mean, SUVR and PKM performed using 1-/2-tissue-compartment models (1TCM, 2TCM), Patlak and Logan analysis (LA). Immunofluorescent staining (IFS), western blotting, and autoradiography of tumor tissue were performed for result validation. RESULTS: [18F]rhPSMA-7.3 showed a tumor uptake with a tumor-to-background-ratio (TBR) = 2.1-2.5, in 15-60 min. PKM (2TCM) confirmed higher K1 (0.34/0.08, p = 0.0012) and volume of distribution VT (0.24/0.1, p = 0.0017) in the tumor region compared to the brain. Linearity in LA and similar k3 = 0.6 and k4 = 0.47 (2TCM, tumor, p = ns) indicated reversible binding. K1, an indicator for vascularization, increased (0.1/0.34, 21 to 28 days, p < 0.005). IFS confirmed co-expression of PSMA and tumor vascularization. [18F]fluciclovine showed higher TBR (2.5/1.8, p < 0.001, 60 min) and VS (1.3/0.7, p < 0.05, tumor) compared to [18F]FET and LA indicated reversible binding. VT increased (p < 0.001, tumor, 21 to 28 days) for [18F]FET (0.5-1.4) and [18F]fluciclovine (0.84-1.5). CONCLUSION: [18F]rhPSMA-7.3 showed to be a potential candidate to investigate the tumor microenvironment of GBM. Following PKM, this uptake was associated with tumor vascularization. In contrast to what is known from PSMA-PET in prostate cancer, reversible binding was found for [18F]rhPSMA-7.3 in GBM, contradicting cellular trapping. Finally, [18F]fluciclovine was superior to [18F]FET rendering it more suitable for PET imaging of GBM.

Synthesis and Biological Evaluation of a Novel 18F-Labeled Radiotracer for PET Imaging of the Adenosine A2A Receptor.

The adenosine A2A receptor (A2AR) has emerged as a potential non-dopaminergic target for the treatment of Parkinson's disease and, thus, the non-invasive imaging with positron emission tomography (PET) is of utmost importance to monitor the receptor expression and occupancy during an A2AR-tailored therapy. Aiming at the development of a PET radiotracer, we herein report the design of a series of novel fluorinated analogs (TOZ1-TOZ7) based on the structure of the A2AR antagonist tozadenant, and the preclinical evaluation of [18F]TOZ1. Autoradiography proved A2AR-specific in vitro binding of [18F]TOZ1 to striatum of mouse and pig brain. Investigations of the metabolic stability in mice revealed parent fractions of more than 76% and 92% of total activity in plasma and brain samples, respectively. Dynamic PET/magnetic resonance imaging (MRI) studies in mice revealed a brain uptake but no A2AR-specific in vivo binding.

Development of 18F-Labeled Radiotracers for PET Imaging of the Adenosine A2A Receptor: Synthesis, Radiolabeling and Preliminary Biological Evaluation.

The adenosine A2A receptor (A2AR) represents a potential therapeutic target for neurodegenerative diseases. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor changes of receptor density and/or occupancy during the A2AR-tailored therapy, we designed a library of fluorinated analogs based on a recently published lead compound (PPY). Among those, the highly affine 4-fluorobenzyl derivate (PPY1; Ki(hA2AR) = 5.3 nM) and the 2-fluorobenzyl derivate (PPY2; Ki(hA2AR) = 2.1 nM) were chosen for 18F-labeling via an alcohol-enhanced copper-mediated procedure starting from the corresponding boronic acid pinacol ester precursors. Investigations of the metabolic stability of [18F]PPY1 and [18F]PPY2 in CD-1 mice by radio-HPLC analysis revealed parent fractions of more than 76% of total activity in the brain. Specific binding of [18F]PPY2 on mice brain slices was demonstrated by in vitro autoradiography. In vivo PET/magnetic resonance imaging (MRI) studies in CD-1 mice revealed a reasonable high initial brain uptake for both radiotracers, followed by a fast clearance.

Preclinical Evaluation of [18F]FACH in Healthy Mice and Piglets: An 18F-Labeled Ligand for Imaging of Monocarboxylate Transporters with PET.

The expression of monocarboxylate transporters (MCTs) is linked to pathophysiological changes in diseases, including cancer, such that MCTs could potentially serve as diagnostic markers or therapeutic targets. We recently developed [18F]FACH as a radiotracer for non-invasive molecular imaging of MCTs by positron emission tomography (PET). The aim of this study was to evaluate further the specificity, metabolic stability, and pharmacokinetics of [18F]FACH in healthy mice and piglets. We measured the [18F]FACH plasma protein binding fractions in mice and piglets and the specific binding in cryosections of murine kidney and lung. The biodistribution of [18F]FACH was evaluated by tissue sampling ex vivo and by dynamic PET/MRI in vivo, with and without pre-treatment by the MCT inhibitor α-CCA-Na or the reference compound, FACH-Na. Additionally, we performed compartmental modelling of the PET signal in kidney cortex and liver. Saturation binding studies in kidney cortex cryosections indicated a KD of 118 ± 12 nM and Bmax of 6.0 pmol/mg wet weight. The specificity of [18F]FACH uptake in the kidney cortex was confirmed in vivo by reductions in AUC0-60min after pre-treatment with α-CCA-Na in mice (-47%) and in piglets (-66%). [18F]FACH was metabolically stable in mouse, but polar radio-metabolites were present in plasma and tissues of piglets. The [18F]FACH binding potential (BPND) in the kidney cortex was approximately 1.3 in mice. The MCT1 specificity of [18F]FACH uptake was confirmed by displacement studies in 4T1 cells. [18F]FACH has suitable properties for the detection of the MCTs in kidney, and thus has potential as a molecular imaging tool for MCT-related pathologies, which should next be assessed in relevant disease models.

Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors.

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of ß-adrenergic receptors. Because BAT therapies based on cold exposure or ß-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells-so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Preclinical Incorporation Dosimetry of [18F]FACH-A Novel 18F-Labeled MCT1/MCT4 Lactate Transporter Inhibitor for Imaging Cancer Metabolism with PET.

Overexpression of monocarboxylate transporters (MCTs) has been shown for a variety of human cancers (e.g., colon, brain, breast, and kidney) and inhibition resulted in intracellular lactate accumulation, acidosis, and cell death. Thus, MCTs are promising targets to investigate tumor cancer metabolism with positron emission tomography (PET). Here, the organ doses (ODs) and the effective dose (ED) of the first 18F-labeled MCT1/MCT4 inhibitor were estimated in juvenile pigs. Whole-body dosimetry was performed in three piglets (age: ~6 weeks, weight: ~13-15 kg). The animals were anesthetized and subjected to sequential hybrid Positron Emission Tomography and Computed Tomography (PET/CT) up to 5 h after an intravenous (iv) injection of 156 ± 54 MBq [18F]FACH. All relevant organs were defined by volumes of interest. Exponential curves were fitted to the time-activity data. Time and mass scales were adapted to the human order of magnitude and the ODs calculated using the ICRP 89 adult male phantom with OLINDA 2.1. The ED was calculated using tissue weighting factors as published in Publication 103 of the International Commission of Radiation Protection (ICRP103). The highest organ dose was received by the urinary bladder (62.6 ± 28.9 µSv/MBq), followed by the gall bladder (50.4 ± 37.5 µSv/MBq) and the pancreas (30.5 ± 27.3 µSv/MBq). The highest contribution to the ED was by the urinary bladder (2.5 ± 1.1 µSv/MBq), followed by the red marrow (1.7 ± 0.3 µSv/MBq) and the stomach (1.3 ± 0.4 µSv/MBq). According to this preclinical analysis, the ED to humans is 12.4 µSv/MBq when applying the ICRP103 tissue weighting factors. Taking into account that preclinical dosimetry underestimates the dose to humans by up to 40%, the conversion factor applied for estimation of the ED to humans would rise to 20.6 µSv/MBq. In this case, the ED to humans upon an iv application of ~300 MBq [18F]FACH would be about 6.2 mSv. This risk assessment encourages the translation of [18F]FACH into clinical study phases and the further investigation of its potential as a clinical tool for cancer imaging with PET.

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

Glioblastoma multiforme (GBM) is the most devastating primary brain tumour characterised by infiltrative growth and resistance to therapies. According to recent research, 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 characterisation of GBM. To test this hypothesis, we used the clinically applied sig1R-ligand (S)-(-)-[18F]fluspidine in imaging studies in an orthotopic mouse model of GBM (U87-MG) as well as in human GBM tissue. A tumour-specific overexpression of sig1R in the U87-MG model was revealed in vitro by autoradiography. The binding parameters demonstrated target-selective binding according to identical KD values in the tumour area and the contralateral side, but a higher density of sig1R in the tumour. Different kinetic profiles were observed in both areas, with a slower washout in the tumour tissue compared to the contralateral side. The translational relevance of sig1R imaging in oncology is reflected by the autoradiographic detection of tumour-specific expression of sig1R in samples obtained from patients with glioblastoma. Thus, the herein presented data support further research on sig1R in neuro-oncology.

PET Imaging of the Adenosine A2A Receptor in the Rotenone-Based Mouse Model of Parkinson's Disease with [18F]FESCH Synthesized by a Simplified Two-Step One-Pot Radiolabeling Strategy.

The adenosine A2A receptor (A2AR) is regarded as a particularly appropriate target for non-dopaminergic treatment of Parkinson's disease (PD). An increased A2AR availability has been found in the human striatum at early stages of PD and in patients with PD and dyskinesias. The aim of this small animal positron emission tomography/magnetic resonance (PET/MR) imaging study was to investigate whether rotenone-treated mice reflect the aspect of striatal A2AR upregulation in PD. For that purpose, we selected the known A2AR-specific radiotracer [18F]FESCH and developed a simplified two-step one-pot radiosynthesis. PET images showed a high uptake of [18F]FESCH in the mouse striatum. Concomitantly, metabolism studies with [18F]FESCH revealed the presence of a brain-penetrant radiometabolite. In rotenone-treated mice, a slightly higher striatal A2AR binding of [18F]FESCH was found. Nonetheless, the correlation between the increased A2AR levels within the proposed PD animal model remains to be further investigated.

Radiosynthesis and Biological Investigation of a Novel Fluorine-18 Labeled Benzoimidazotriazine- Based Radioligand for the Imaging of Phosphodiesterase 2A with Positron Emission Tomography.

A specific radioligand for the imaging of cyclic nucleotide phosphodiesterase 2A (PDE2A) via positron emission tomography (PET) would be helpful for research on the physiology and disease-related changes in the expression of this enzyme in the brain. In this report, the radiosynthesis of a novel PDE2A radioligand and the subsequent biological evaluation were described. Our prospective compound 1-(2-chloro-5-methoxy phenyl)-8-(2-fluoropyridin-4-yl)-3- methylbenzo[e]imidazo[5,1-c][1,2,4]triazine, benzoimidazotriazine (BIT1) (IC50 PDE2A = 3.33 nM; 16-fold selectivity over PDE10A) was fluorine-18 labeled via aromatic nucleophilic substitution of the corresponding nitro precursor using the K[18F]F-K2.2.2-carbonate complex system. The new radioligand [18F]BIT1 was obtained with a high radiochemical yield (54 ± 2%, n = 3), a high radiochemical purity (≥99%), and high molar activities (155-175 GBq/µmol, n = 3). In vitro autoradiography on pig brain cryosections exhibited a heterogeneous spatial distribution of [18F]BIT1 corresponding to the known pattern of expression of PDE2A. The investigation of in vivo metabolism of [18F]BIT1 in a mouse revealed sufficient metabolic stability. PET studies in mouse exhibited a moderate brain uptake of [18F]BIT1 with a maximum standardized uptake value of ~0.7 at 5 minutes p.i. However, in vivo blocking studies revealed a non-target specific binding of [18F]BIT1. Therefore, further structural modifications are needed to improve target selectivity.

Radiosynthesis and in vivo evaluation of a fluorine-18 labeled pyrazine based radioligand for PET imaging of the adenosine A2B receptor.

On the basis of a pyrazine core structure, three new adenosine A2B receptor ligands (7a-c) were synthesized containing a 2-fluoropyridine moiety suitable for 18F-labeling. Compound 7a was docked into a homology model of the A2B receptor based on X-ray structures of the related A2A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A1 receptor, 7a was radiolabeled as the most suitable candidate (Ki(A2B) = 4.24 nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [18F]7a with a standardized uptake value (SUV) of ≈1 at 5 min post injection followed by a fast wash out. Metabolism studies of [18F]7a in mice revealed the formation of a blood-brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo.

Bridging from Brain to Tumor Imaging: (S)-(-)- and (R)-(+)-[18F]Fluspidine for Investigation of Sigma-1 Receptors in Tumor-Bearing Mice.

Sigma-1 receptors (Sig1R) are highly expressed in various human cancer cells and hence imaging of this target with positron emission tomography (PET) can contribute to a better understanding of tumor pathophysiology and support the development of antineoplastic drugs. Two Sig1R-specific radiolabeled enantiomers (S)-(-)- and (R)-(+)-[18F]fluspidine were investigated in several tumor cell lines including melanoma, squamous cell/epidermoid carcinoma, prostate carcinoma, and glioblastoma. Dynamic PET scans were performed in mice to investigate the suitability of both radiotracers for tumor imaging. The Sig1R expression in the respective tumors was confirmed by Western blot. Rather low radiotracer uptake was found in heterotopically (subcutaneously) implanted tumors. Therefore, a brain tumor model (U87-MG) with orthotopic implantation was chosen to investigate the suitability of the two Sig1R radiotracers for brain tumor imaging. High tumor uptake as well as a favorable tumor-to-background ratio was found. These results suggest that Sig1R PET imaging of brain tumors with [18F]fluspidine could be possible. Further studies with this tumor model will be performed to confirm specific binding and the integrity of the blood-brain barrier (BBB).

Investigation of an 18F-labelled Imidazopyridotriazine for Molecular Imaging of Cyclic Nucleotide Phosphodiesterase 2A.

Specific radioligands for in vivo visualization and quantification of cyclic nucleotide phosphodiesterase 2A (PDE2A) by positron emission tomography (PET) are increasingly gaining interest in brain research. Herein we describe the synthesis, the 18F-labelling as well as the biological evaluation of our latest PDE2A (radio-)ligand 9-(5-Butoxy-2-fluorophenyl)-2-(2-([18F])fluoroethoxy)-7-methylimidazo[5,1-c]pyrido[2,3-e][1,2,4]triazine (([18F])TA5). It is the most potent PDE2A ligand out of our series of imidazopyridotriazine-based derivatives so far (IC50 hPDE2A = 3.0 nM; IC50 hPDE10A > 1000 nM). Radiolabelling was performed in a one-step procedure starting from the corresponding tosylate precursor. In vitro autoradiography on rat and pig brain slices displayed a homogenous and non-specific binding of the radioligand. Investigation of stability in vivo by reversed-phase HPLC (RP-HPLC) and micellar liquid chromatography (MLC) analyses of plasma and brain samples obtained from mice revealed a high fraction of one main radiometabolite. Hence, we concluded that [18F]TA5 is not appropriate for molecular imaging of PDE2A neither in vitro nor in vivo. Our ongoing work is focusing on further structurally modified compounds with enhanced metabolic stability.

Radiosynthesis and biological evaluation of the new PDE10A radioligand [18 F]AQ28A.

Cyclic nucleotide phosphodiesterase 10A (PDE10A) regulates the level of the second messengers cAMP and cGMP in particular in brain regions assumed to be associated with neurodegenerative and psychiatric diseases. A better understanding of the pathophysiological role of the expression of PDE10A could be obtained by quantitative imaging of the enzyme by positron emission tomography (PET). Thus, in this study we developed, radiolabeled, and evaluated a new PDE10A radioligand, 8-bromo-1-(6-[18 F]fluoropyridin-3-yl)-3,4-dimethylimidazo[1,5-a]quinoxaline ([18 F]AQ28A). [18 F]AQ28A was radiolabeled by both nucleophilic bromo-to-fluoro or nitro-to-fluoro exchange using K[18 F]F-K2.2.2 -carbonate complex with different yields. Using the superior nitro precursor, we developed an automated synthesis on a Tracerlab FX F-N module and obtained [18 F]AQ28A with high radiochemical yields (33 ± 6%) and specific activities (96-145 GBq·µmol-1 ) for further evaluation. Initially, we investigated the binding of [18 F]AQ28A to the brain of different species by autoradiography and observed the highest density of binding sites in striatum, the brain region with the highest PDE10A expression. Subsequent dynamic PET studies in mice revealed a region-specific accumulation of [18 F]AQ28A in this region, which could be blocked by preinjection of the selective PDE10A ligand MP-10. In conclusion, the data suggest [18 F]AQ28A is a suitable candidate for imaging of PDE10A in rodent brain by PET.

Evaluation of the Enantiomer Specific Biokinetics and Radiation Doses of [(18)F]Fluspidine-A New Tracer in Clinical Translation for Imaging of σ1 Receptors.

The enantiomers of [(18)F]fluspidine, recently developed for imaging of σ1 receptors, possess distinct pharmacokinetics facilitating their use in different clinical settings. To support their translational potential, we estimated the human radiation dose of (S)-(-)-[(18)F]fluspidine and (R)-(+)-[(18)F]fluspidine from ex vivo biodistribution and PET/MRI data in mice after extrapolation to the human scale. In addition, we validated the preclinical results by performing a first-in-human PET/CT study using (S)-(-)-[(18)F]fluspidine. Based on the respective time-activity curves, we calculated using OLINDA the particular organ doses (ODs) and effective doses (EDs). The ED values of (S)-(-)-[(18)F]fluspidine and (R)-(+)-[(18)F]fluspidine differed significantly with image-derived values obtained in mice with 12.9 µSv/MBq and 14.0 µSv/MBq (p < 0.025), respectively. A comparable ratio was estimated from the biodistribution data. In the human study, the ED of (S)-(-)-[(18)F]fluspidine was calculated as 21.0 µSv/MBq. Altogether, the ED values for both [(18)F]fluspidine enantiomers determined from the preclinical studies are comparable with other (18)F-labeled PET imaging agents. In addition, the first-in-human study confirmed that the radiation risk of (S)-(-)-[(18)F]fluspidine imaging is within acceptable limits. However, as already shown for other PET tracers, the actual ED of (S)-(-)-[(18)F]fluspidine in humans was underestimated by preclinical imaging which needs to be considered in other first-in-human studies.

Synthesis, 18F-Radiolabelling and Biological Characterization of Novel Fluoroalkylated Triazine Derivatives for in Vivo Imaging of Phosphodiesterase 2A in Brain via Positron Emission Tomography.

Phosphodiesterase 2A (PDE2A) is highly and specifically expressed in particular brain regions that are affected by neurological disorders and in certain tumors. Development of a specific PDE2A radioligand would enable molecular imaging of the PDE2A protein via positron emission tomography (PET). Herein we report on the syntheses of three novel fluoroalkylated triazine derivatives (TA2-4) and on the evaluation of their effect on the enzymatic activity of human PDE2A. The most potent PDE2A inhibitors were 18F-radiolabelled ([18F]TA3 and [18F]TA4) and investigated regarding their potential as PET radioligands for imaging of PDE2A in mouse brain. In vitro autoradiography on rat brain displayed region-specific distribution of [18F]TA3 and [18F]TA4, which is consistent with the expression pattern of PDE2A protein. Metabolism studies of both [18F]TA3 and [18F]TA4 in mice showed a significant accumulation of two major radiometabolites of each radioligand in brain as investigated by micellar radio-chromatography. Small-animal PET/MR studies in mice using [18F]TA3 revealed a constantly increasing uptake of activity in the non-target region cerebellum, which may be caused by the accumulation of brain penetrating radiometabolites. Hence, [18F]TA3 and [18F]TA4 are exclusively suitable for in vitro investigation of PDE2A. Nevertheless, further structural modification of these promising radioligands might result in metabolically stable derivatives.

A Promising PET Tracer for Imaging of α7 Nicotinic Acetylcholine Receptors in the Brain: Design, Synthesis, and in Vivo Evaluation of a Dibenzothiophene-Based Radioligand.

Changes in the expression of α7 nicotinic acetylcholine receptors (α7 nAChRs) in the human brain are widely assumed to be associated with neurological and neurooncological processes. Investigation of these receptors in vivo depends on the availability of imaging agents such as radioactively labelled ligands applicable in positron emission tomography (PET). We report on a series of new ligands for α7 nAChRs designed by the combination of dibenzothiophene dioxide as a novel hydrogen bond acceptor functionality with diazabicyclononane as an established cationic center. To assess the structure-activity relationship (SAR) of this new basic structure, we further modified the cationic center systematically by introduction of three different piperazine-based scaffolds. Based on in vitro binding affinity and selectivity, assessed by radioligand displacement studies at different rat and human nAChR subtypes and at the structurally related human 5-HT3 receptor, we selected the compound 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-2-fluorodibenzo-[b,d]thiophene 5,5-dioxide (10a) for radiolabeling and further evaluation in vivo. Radiosynthesis of [18F]10a was optimized and transferred to an automated module. Dynamic PET imaging studies with [18F]10a in piglets and a monkey demonstrated high uptake of radioactivity in the brain, followed by washout and target-region specific accumulation under baseline conditions. Kinetic analysis of [18F]10a in pig was performed using a two-tissue compartment model with arterial-derived input function. Our initial evaluation revealed that the dibenzothiophene-based PET radioligand [18F]10a ([18F]DBT-10) has high potential to provide clinically relevant information about the expression and availability of α7 nAChR in the brain.

4-nitroquinoline 1-oxide-induced oral epithelial lesions exhibit time- and stage-dependent changes in the tumor immune microenvironment.

Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity and is associated with high morbidity due to local invasion and lymph node metastasis. Tumor infiltrating lymphocytes (TILs) are associated with good prognosis in oral cancer patients and dictate response to treatment. Ectopic sites for immune activation in tumors, known as tertiary lymphoid structures (TLS), and tumor-associated high-endothelial venules (TA-HEVs), which are specialized lymphocyte recruiting vessels, are associated with a favorable prognosis in OSCC. Why only some tumors support the development of TLS and HEVs is poorly understood. In the current study we explored the infiltration of lymphocyte subsets and the development of TLS and HEVs in oral epithelial lesions using the 4-nitroquinoline 1-oxide (4NQO)-induced mouse model of oral carcinogenesis. We found that the immune response to 4NQO-induced oral epithelial lesions was dominated by T cell subsets. The number of T cells (CD4+, FoxP3+, and CD8+), B cells (B220+) and PNAd+ HEVs increased from the earliest to the latest endpoints. All the immune markers increased with the severity of the dysplasia, while the number of HEVs and B cells further increased in SCCs. HEVs were present already in early-stage lesions, while TLS did not develop at any timepoint. This suggests that the 4NQO model is applicable to study the dynamics of the tumor immune microenvironment at early phases of oral cancer development, including the regulation of TA-HEVs in OTSCC.

Weakly supervised semantic segmentation for MRI: exploring the advantages and disadvantages of class activation maps for biological image segmentation with soft boundaries.

Fully supervised semantic segmentation models require pixel-level annotations that are costly to obtain. As a remedy, weakly supervised semantic segmentation has been proposed, where image-level labels and class activation maps (CAM) can detect discriminative regions for specific class objects. In this paper, we evaluated several CAM methods applied to different convolutional neural networks (CNN) to highlight tissue damage of cod fillets with soft boundaries in MRI. Our results show that different CAM methods produce very different CAM regions, even when applying them to the same CNN model. CAM methods that claim to highlight more of the class object do not necessarily highlight more damaged regions or originate from the same high discriminatory regions, nor do these damaged regions show high agreement across the different CAM methods. Additionally, CAM methods produce damaged regions that do not align with external reference metrics, and even show correlations contrary to what can be expected.

Magnetic resonance imaging for non-invasive measurement of plastic ingestion in marine wildlife.

Monitoring plastic ingestion by marine wildlife is important for both characterizing the extent of plastic pollution in the environment and understanding its effect on species and ecosystems. Current methods to detect plastic in the digestive system of animals are slow and invasive, such that the number of animals that can be screened is limited. In this article, magnetic resonance imaging (MRI) is investigated as a possible technology to perform rapid, non-invasive detection of plastic ingestion. Standard MRI methods were able to directly measure one type of plastic in a fulmar stomach and another type was able to be indirectly detected. In addition to MRI, other standard nuclear magnetic resonance (NMR) measurements were made. Different types of plastic were tested, and distinctive NMR signal characteristics were found in common for each type, allowing them to be distinguished from one another. The NMR results indicate specialized MRI sequences could be used to directly image several types of plastic. Although current commercial MRI technology is not suitable for field use, existing single-sided MRI research systems could be adapted for use outside the laboratory and become an important tool for future monitoring of wild animals.

Diffusion tensor imaging for spatially-resolved characterization of muscle fiber structure in seafood.

The fiber structure of tissue in meat and seafood has a significant impact on their perceived quality. However, quantifiable description of muscle structure is challenging. We investigate diffusion tensor imaging (DTI) magnetic resonance imaging (MRI) as a method to quantitatively describe tissue structure. DTI measures the anisotropy of water molecule diffusion within muscle fibers. A pilot study evaluated three different cod loin samples: one of high-quality, one of medium-quality, and one of poor-quality. DTI parameters such as fractional anisotropy, axial diffusion and radial diffusion showed clear differences between the sample qualities. Changes in the DTI metrics consistent with freezing and thawing damage to the tissue were observed. The DTI maps were compared to T2-weighted images and DTI detected significant details that were not visible in T2-weighted images. Overall, these results indicate that DTI is a promising method for spatially-resolved characterization of tissue structure in seafood and meat.