Improvement of Crystal Identification Accuracy for Depth-of-Interaction Detector System with Peak-to-Charge Discrimination Method.

In positron emission tomography (PET), parallax errors degrade spatial resolution. The depth of interaction (DOI) information provides the position in the depth of the scintillator interacting with the γ-rays, thus reducing parallax errors. A previous study developed a Peak-to-Charge discrimination (PQD), which can separate spontaneous alpha decay in LaBr3:Ce. Since decay constant of GSO:Ce depends on Ce concentration, the PQD is expected to discriminate GSO:Ce scintillators with different Ce concentration. In this study, the PQD-based DOI detector system was developed, which can be processed online and implemented in PET. A detector was composed of four layers of GSO:Ce crystals and a PS-PMT. The four crystals were obtained from both the top and bottom of ingots with a nominal Ce concentration of 0.5 mol% and 1.5 mol%. The PQD was implemented on the Xilinx Zynq-7000 SoC board with 8ch Flash ADC to gain real-time processing, flexibility, and expandability. The results showed that the mean Figure of Merits in 1D between four scintillators are 1.5, 0.99, 0.91 for layers between 1st-2nd, 2nd-3rd, and 3rd-4th respectively, and the mean Error Rate in 1D between four scintillators are 3.50%, 2.96%, 13.3%, and 1.88% for layers 1, 2, 3, and 4, respectively. In addition, the introduction of the 2D PQDs resulted in the mean Figure of Merits in 2D greater than 0.9 and the mean Error Rate in 2D less than 3% in all layers.

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.

Synthesis, 18F-labelling and radiopharmacological characterisation of the C-terminal 30mer of Clostridium perfringens enterotoxin as a potential claudin-targeting peptide.

The cell surface receptor claudin-4 (Cld-4) is upregulated in various tumours and represents an important emerging target for both diagnosis and treatment of solid tumours of epithelial origin. The C-terminal fragment of the Clostridium perfringens enterotoxin cCPE290-319 appears as a suitable ligand for targeting Cld-4. The synthesis of this 30mer peptide was attempted via several approaches, which has revealed sequential SPPS using three pseudoproline dipeptide building blocks to be the most efficient one. Labelling with fluorine-18 was achieved on solid phase using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) and 4-[18F]fluorobenzoyl chloride as 18F-acylating agents, which was the most advantageous when [18F]SFB was reacted with the resin-bound 30mer containing an N-terminal 6-aminohexanoic spacer. Binding to Cld-4 was demonstrated via surface plasmon resonance using a protein construct containing both extracellular loops of Cld-4. In addition, cell binding experiments were performed for 18F-labelled cCPE290-319 with the Cld-4 expressing tumour cell lines HT-29 and A431 that were complemented by fluorescence microscopy studies using the corresponding fluorescein isothiocyanate-conjugated peptide. The 30mer peptide proved to be sufficiently stable in blood plasma. Studying the in vivo behaviour of 18F-labelled cCPE290-319 in healthy mice and rats by dynamic PET imaging and radiometabolite analyses has revealed that the peptide is subject to substantial liver uptake and rapid metabolic degradation in vivo, which limits its suitability as imaging probe for tumour-associated Cld-4.

Impact of Glutathione Modulation on Stability and Pharmacokinetic Profile of Redox-Sensitive Nanogels.

Nanoparticles degradable upon external stimuli combine pharmacokinetic features of both small molecules as well as large nanoparticles. However, despite promising preclinical results, several redox responsive disulphide-linked nanoparticles failed in clinical translation, mainly due to their unexpected in vivo behavior. Glutathione (GSH) is one of the most evaluated antioxidants responsible for disulfide degradation. Herein, the impact of GSH on the in vivo behavior of redox-sensitive nanogels under physiological and modulated conditions is investigated. Labelling of nanogels with a DNA-intercalating dye and a radioisotope allows visualization of the redox responsiveness at the cellular and the systemic levels, respectively. In vitro, efficient cleavage of disulphide bonds of nanogels is achieved by manipulation of intracellular GSH concentration. While in vivo, the redox-sensitive nanogels undergo, to a certain extent, premature degradation in circulation leading to rapid renal elimination. This instability is modulated by transient inhibition of GSH synthesis with buthioninsulfoximin. Altered GSH concentration significantly changes the in vivo pharmacokinetics. Lower GSH results in higher elimination half-life and altered biodistribution of the nanogels with a different metabolite profile. These data provide strong evidence that decreased nanogel degradation in blood circulation can limit the risk of premature drug release and enhance circulation half-life of the nanogel.

Overexpression of Receptor Tyrosine Kinase EphB4 Triggers Tumor Growth and Hypoxia in A375 Melanoma Xenografts: Insights from Multitracer Small Animal Imaging Experiments.

Experimental evidence has associated receptor tyrosine kinase EphB4 with tumor angiogenesis also in malignant melanoma. Considering the limited in vivo data available, we have conducted a systematic multitracer and multimodal imaging investigation in EphB4-overexpressing and mock-transfected A375 melanoma xenografts. Tumor growth, perfusion, and hypoxia were investigated by positron emission tomography. Vascularization was investigated by fluorescence imaging in vivo and ex vivo. The approach was completed by magnetic resonance imaging, radioluminography ex vivo, and immunohistochemical staining for blood and lymph vessel markers. Results revealed EphB4 to be a positive regulator of A375 melanoma growth, but a negative regulator of tumor vascularization. Resulting in increased hypoxia, this physiological characteristic is considered as highly unfavorable for melanoma prognosis and therapy outcome. Lymphangiogenesis, by contrast, was not influenced by EphB4 overexpression. In order to distinguish between EphB4 forward and EphrinB2, the natural EphB4 ligand, reverse signaling a specific EphB4 kinase inhibitor was applied. Blocking experiments show EphrinB2 reverse signaling rather than EphB4 forward signaling to be responsible for the observed effects. In conclusion, functional expression of EphB4 is considered a promising differentiating characteristic, preferentially determined by non-invasive in vivo imaging, which may improve personalized theranostics of malignant melanoma.

Combined PET Radiotracer Approach Reveals Insights into Stromal Cell-Induced Metabolic Changes in Pancreatic Cancer In Vitro and In Vivo.

Background/Objective Pancreatic stellate cells (PSCs) in pancreatic adenocarcinoma (PDAC) are producing extracellular matrix, which promotes the formation of a dense fibrotic microenvironment. This makes PDAC a highly heterogeneous tumor-stroma-driven entity, associated with reduced perfusion, limited oxygen supply, high interstitial fluid pressure, and limited bioavailability of therapeutic agents. Methods In this study, spheroid and tumor xenograft models of human PSCs and PanC-1 cells were characterized radiopharmacologically using a combined positron emission tomography (PET) radiotracer approach. [18F]FDG, [18F]FMISO, and [18F]FAPI-74 were employed to monitor metabolic activity, hypoxic metabolic state, and functional expression of fibroblast activation protein alpha (FAPα), a marker of activated PSCs. Results In vitro, PanC-1 and multi-cellular tumor spheroids demonstrated comparable glucose uptake and hypoxia, whereas FAPα expression was significantly higher in PSC spheroids. In vivo, glucose uptake as well as the transition to hypoxia were comparable in PanC-1 and multi-cellular xenograft models. In mice injected with PSCs, FAPα expression decreased over a period of four weeks post-injection, which was attributed to the successive death of PSCs. In contrast, FAPα expression increased in both PanC-1 and multi-cellular xenograft models over time due to invasion of mouse fibroblasts. Conclusion The presented models are suitable for subsequently characterizing stromal cell-induced metabolic changes in tumors using noninvasive molecular imaging techniques.

The assessment of left ventricular volume and function in gated small animal 18F-FDG PET/CT imaging: a comparative study of three commercially available software tools.

BACKGROUND: Several software tools have been developed for gated PET imaging that use distinct algorithms to analyze tracer uptake, myocardial perfusion, and left ventricle volumes and function. Studies suggest that different software tools cannot be used interchangeably in humans. In this study, we sought to compare the left ventricular parameters in gated 18F-FDG PET/CT imaging in mice by three commercially available software tools: PMOD, MIM, and QGS. METHODS AND RESULTS: Healthy mice underwent ECG-gated 18F-FDG imaging using a small-animal nanoPET/CT (Mediso) under isoflurane narcosis. Reconstructed gates PET images were subsequently analyzed in three different software tools, and cardiac volume and function (end-diastolic (EDV), end-systolic volumes (ESV), stroke volume (SV), and ejection fraction (EF)) were evaluated. While cardiac volumes correlated well between PMOD, MIM, and QGS, the left ventricular parameters and cardiac function differed in agreement using Bland-Altman analysis. EDV in PMOD vs. QGS: r = 0.85; p < 0.001, MIM vs. QGS: r = 0.92; p < 0.001, and MIM vs. PMOD: r = 0.88; p < 0.001, showed good correlations. Correlation was also found in ESV: PMOD vs. QGS: r = 0.48; p = 0.07, MIM vs QGS: r = 0.79; p < 0.001, and MIM vs. PMOD: r = 0.69; p < 0.01. SV showed good correlations in: PMOD vs. QGS: r = 0.73; p < 0.01, MIM vs. QGS: r = 0.86; p < 0.001, and MIM vs. PMOD: r = 0.92; p < 0.001. However, EF among correlated poorly: PMOD vs. QGS: r = -0.31; p = 0.26, MIM vs. QGS: r = 0.48; p = 0.07, and MIM vs. PMOD: r = 0.23; p = 0.41. Inter-class and intra-class correlation coefficient were > 0.9 underlining repeatability in using PMOD, MIM, and QGS for cardiac volume and function assessment. CONCLUSIONS: All three commercially available software tools are feasible in small animal cardiac volume assessment in gated 18F-FDG PET/CT imaging. However, due to software-related differences in agreement analysis for cardiac volumes and function, PMOD, MIM, and QGS cannot be used interchangeably in murine research.

Longitudinal Studies on Alzheimer Disease Mouse Models with Multiple Tracer PET/CT: Application of Reduction and Refinement Principles in Daily Practice to Safeguard Animal Welfare during Progressive Aging.

Longitudinal studies on mouse models related to Alzheimer disease (AD) pathology play an important role in the investigation of therapeutic targets to help pharmaceutical research in the development of new drugs and in the attempt of an early diagnosis that can contribute to improving people's quality of life. There are several advantages to enriching longitudinal studies in AD models with Positron Emission Tomography (PET); among these advantages, the possibility of following the principle of the 3Rs of animal welfare is fundamental. In this manuscript, good daily experimental practice focusing on animal welfare is described and commented upon, based on the experience attained from studies conducted in our Nuclear Medicine department.

Evaluation of Integrin αvß3 Expression in Murine Xenograft Models: [68Ga]Ga-DOTA-C(RGDfK) PET Study with Immunohistochemical Confirmation.

Tumor blood flow (TBF) is related to drug delivery and hypoxia, both of which can impact the efficacy of anti-cancer therapies. Although integrin αvß3 expression is related to tumor angiogenesis, it remains unclear whether the degree of angiogenesis affects TBF. This study aimed to evaluate the expression of integrin αvß3 in mouse tumor models using [68Ga]Ga-DOTA-c(RGDfK) peptide positron emission tomography (PET) and immunohistochemical staining. PET studies were conducted using mouse C6 glioma models and MIA PaCa-2 (n = 6 each). The [68Ga]Ga-DOTA-c(RGDfK) peptide was injected via the tail vein (2.17 ± 0.28 MBq), and 10 min static PET scans were performed. Immunohistochemical analysis was conducted using an integrin αVß3 antibody. [68Ga]Ga-DOTA-c(RGDfK) peptide PET revealed higher uptake of the radiotracer in C6 gliomas than in MIA PaCa-2 tumors. The mean standardized uptake value was significantly higher in C6 gliomas (0.35 ± 0.058) than in MIA PaCa-2 tumors (0.17 ± 0.045). Histological analysis revealed intense integrin αVß3 expression in the C6 gliomas, whereas the MIA PaCa-2 tumors had low expression levels. This study showed that the expression of integrin αvß3 can be differentiated by the [68Ga]Ga-DOTA-c(RGDfK) peptide, suggesting the potential applicability of this peptide in the evaluation of the relationship between angiogenesis and TBF.

In vivo evaluation of [11C]preladenant positron emission tomography for quantification of adenosine A2A receptors in the rat brain.

[11C]Preladenant was developed as a novel adenosine A2A receptor positron emission tomography radioligand. The present study aims to evaluate the suitability of [11C]preladenant positron emission tomography for the quantification of striatal A2A receptor density and the assessment of striatal A2A receptor occupancy by KW-6002. Sixty- or ninety-minute dynamic positron emission tomography imaging was performed on rats. Tracer kinetics was quantified by the two-tissue compartment model, Logan graphical analysis and several reference tissue-based models. Test-retest reproducibility was assessed by repeated imaging on two consecutive days. Two-tissue compartment model and Logan plot estimated comparable distribution volume ( VT) values of ∼10 in the A2A receptor-rich striatum and substantially lower values in all extra-striatal regions (∼1.5-2.5). The simplified reference tissue model with midbrain or occipital cortex as the reference region proved to be the best non-invasive model for quantification of A2A receptor, showing a striatal binding potential ( BPND) value of ∼5.5, and a test-retest variability of ∼5.5%. The brain metabolite analysis showed that at 60-min post injection, 17% of the radioactivity in the brain was due to radioactive metabolites. The ED50 of KW-6002 in rat striatum for i.p. injection was 0.044-0.062 mg/kg. The study demonstrates that [11C]preladenant is a suitable tracer to quantify striatal A2A receptor density and assess A2A receptor occupancy by A2A receptor-targeting molecules.

Multimodal imaging analysis of an orthotopic head and neck cancer mouse model and application of anti-CD137 tumor immune therapy.

BACKGROUND: Recent technical progress makes sophisticated noninvasive imaging methods available for murine models. For the first time, in this study, we applied fluorodeoxyglucose (FDG)-positron emission tomography (PET)-CT and FDG-PET-MRI to a murine orthotopic model of head and neck cancer immunotherapy. METHODS: Tumor growth of floor of the mouth tumors was evaluated by multimodal small-animal imaging using FDG-PET-CT and FDG-PET-MRI. The immunotherapeutic effects of anti-CD137 antibody therapy were examined on body weight, tumor growth, and tumor-infiltrating immune cells in longitudinal imaging studies and immunohistochemical analyses. RESULTS: Imaging revealed aggressive, fast-growing tumors without evidence of local or distant metastases. CD137 immunotherapy decreased tumor take and growth and stabilized body weight over time. A clear case of tumor regression was demonstrated by longitudinal PET-CT. CONCLUSION: The murine model mimics the characteristics of head and neck cancer in humans and offers excellent opportunities to investigate immunomodulatory anticancer drugs. The CD137 antibody showed antitumor effects in some therapy-responsive mice.

Synthesis and radiopharmacological evaluation of 64Cu-labeled bombesin analogs featuring a bis(2-pyridylmethyl)-1,4,7-triazacyclononane chelator.

The bifunctional chelating agent 2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]acetic acid, DMPTACN-COOH, has been found to bind strongly to copper(II), resulting in a radiocopper(II)-ligand complex that exhibits high in vivo stability. The pendant carboxylic acid group enables this derivative to be conjugated to the N-terminal amino acid residues of peptides. Exploiting this, two stabilized bombesin (BBN) derivatives, ßAla-ßAla-[Cha(13),Nle(14)]BBN(7-14) and ßhomo-Glu-ßAla-ßAla-[Cha(13),Nle(14)]BBN(7-14) have been coupled to DMPTACN-COOH and radiolabeled with the positron emitter copper-64 ((64)Cu-1 and (64)Cu-3). The in vitro binding characteristics of the [(64)Cu]Cu-labeled bombesin conjugates in gastrin-releasing peptide receptor (GRPR) over-expressing prostate cancer (PC-3) cells have been evaluated. Biodistribution studies performed in Wistar rats indicate a specific uptake in the GRPR-rich pancreas and rapid renal elimination for both (64)Cu-1 and (64)Cu-3. Small animal PET imaging studies performed in NMRI nu/nu mice bearing the human prostate tumor PC-3 demonstrated a very high degree of tumor accumulation for (64)Cu-1 and (64)Cu-3. Incorporation of a single additional glutamic acid residue within the spacer between bombesin and the radiolabeled complex ((64)Cu-3) leads to a higher tumor-to-muscle uptake ratio (amounting to >30 at 100 min post injection) compared to (64)Cu-1.