EJNMMI Research最新文献

Background: Patient-derived tumour organoids (PDOs) are highly advanced in vitro models for disease modelling, yet they lack vascularisation. To overcome this shortcoming, organoids can be inoculated onto the chorioallantoic membrane (CAM); the highly vascularised, not innervated extraembryonic membrane of fertilised chicken eggs. Therefore, we aimed to (1) establish a CAM patient-derived xenograft (PDX) model based on PDOs generated from the liver metastasis of a colorectal cancer (CRC) patient and (2) to evaluate the translational pipeline (patient - in vitro PDOs - in vivo CAM-PDX) regarding morphology, histopathology, expression of C-X-C chemokine receptor type 4 (CXCR4), and radiotracer uptake patterns.

Results: The main liver metastasis of the CRC patient exhibited high 2-[¹⁸F]FDG uptake and moderate and focal [⁶⁸Ga]Ga-Pentixafor accumulation in the peripheral part of the metastasis. Inoculation of PDOs derived from this region onto the CAM resulted in large, highly viable, and extensively vascularised xenografts, as demonstrated immunohistochemically and confirmed by high 2-[¹⁸F]FDG uptake. The xenografts showed striking histomorphological similarity to the patient's liver metastasis. The moderate expression of CXCR4 was maintained in ovo and was concordant with the expression levels of the patient's sample and in vitro PDOs. Following in vitro re-culturing of CAM-PDXs, growth, and [⁶⁸Ga]Ga-Pentixafor uptake were unaltered compared to PDOs before transplantation onto the CAM. Although [⁶⁸Ga]Ga-Pentixafor was taken up into CAM-PDXs, the uptake in the baseline and blocking group were comparable and there was only a trend towards blocking.

Conclusions: We successfully established an in vivo CAM-PDX model based on CRC PDOs. The histomorphological features and target protein expression of the original patient's tissue were mirrored in the in vitro PDOs, and particularly in the in vivo CAM-PDXs. The [⁶⁸Ga]Ga-Pentixafor uptake patterns were comparable between in vitro, in ovo and clinical data and 2-[¹⁸F]FDG was avidly taken up in the patient's liver metastasis and CAM-PDXs. We thus propose the CAM-PDX model as an alternative in vivo model with promising translational value for CRC patients.

Background: The orexin receptor (OXR) plays a role in drug addiction and is aberrantly expressed in colorectal tumors. Subtype-selective OXR PET ligands suitable for in vivo use have not yet been reported. This work reports the development of ¹⁸F-labeled OXR PET ligand candidates derived from the OXR antagonist suvorexant and the OX1R-selective antagonist JH112.

Results: Computational analysis predicted that fluorine substitution (1e) and introduction of the fluorobenzothiazole scaffold (1f) would be suitable for maintaining high OX1R affinity. After multi-step synthesis of 1a-1f, in vitro OXR binding studies confirmed the molecular dynamics calculations and revealed single-digit nanomolar OX1R affinities for 1a-f, ranging from 0.69 to 2.5 nM. The benzothiazole 1f showed high OX1R affinity (K_i = 0.69 nM), along with 77-fold subtype selectivity over OX2R. Cu-mediated ¹⁸F-fluorination of boroxine precursors allowed for a shortened reaction time of 5 min to provide the non-selective OXR ligand [¹⁸F]1c and its selective OX1R congener [¹⁸F]1f in activity yields of 14% and 22%, respectively, within a total synthesis time of 52-76 min. [¹⁸F]1c and [¹⁸F]1f were stable in plasma and serum in vitro, with logD_7.4 of 2.28 ([¹⁸F]1c) and 2.37 ([¹⁸F]1f), and high plasma protein binding of 66% and 77%, respectively. Dynamic PET imaging in rats showed similar brain uptake of [¹⁸F]1c (0.17%ID/g) and [¹⁸F]1f (0.15%ID/g). However, preinjection of suvorexant did not significantly block [¹⁸F]1c or [¹⁸F]1f uptake in the rat brain. Pretreatment with cyclosporine A to study the role of P-glycoprotein (P-gp) in limiting brain accumulation moderately increased brain uptake of [¹⁸F]1c and [¹⁸F]1f. Accordingly, in vitro experiments demonstrated that the P-gp inhibitor zosuquidar only moderately inhibited polarized, basal to apical transport of 1c (p < 0.05) and had no effect on the transport of 1f, indicating that P-gp does not play a relevant role in brain accumulation of [¹⁸F]1c and [¹⁸F]1f in vivo.

Conclusions: The in vitro and in vivo results of [¹⁸F]1c and [¹⁸F]1f provide a solid basis for further development of suitable OXR PET ligands for brain imaging.

Background: Neuromelanin is mostly located in dopaminergic neurons in the substantia nigra (SN) pars compacta, and can be detected by magnetic resonance imaging (MRI). It is a promising imaging-base biomarker for neurological diseases. We previously developed a melanin-specific probe N-(2-(diethylamino)-ethyl)-¹⁸F-5-fluoropicolinamide (¹⁸F-P3BZA), which was initially developed for the imaging of melanoma. ¹⁸F-P3BZA exhibited high levels of binding to the melanin in vitro and in vivo with high retention and favorable pharmacokinetics. In this study we further investigated whether ¹⁸F-P3BZA could be used to quantitatively detect neuromelanin in the SN in healthy rhesus macaques.

Results: ¹⁸F-P3BZA exhibited desired hydrophobicity with estimated log Know 5.08 and log D7.4 1.68. ¹⁸F-P3BZA readily crossed the blood-brain barrier with brain transport coefficients (Kin) of 40 ± 8 µL g-1s-1. ¹⁸F-P3BZA accumulated specifically in neuromelanotic PC12 cells, melanin-rich melanoma cells, and melanoma xenografts. Binding of ¹⁸F-P3BZA to B16F10 cells was much higher than to SKOV3 cells at 60 min (6.17 ± 0.53%IA and 0.24 ± 0.05%IA, respectively). In the biodistribution study, ¹⁸F-P3BZA had higher accumulation in B16F10 tumors (6.31 ± 0.99%IA/g) than in SKOV3 tumors (0.25 ± 0.09%IA/g). Meanwhile, ¹⁸F-P3BZA uptake in B16F10 tumors could be blocked by excess cold ¹⁹F-P3BZA (0.81 ± 0.02%IA/g, 88% inhibition, p < 0.05). PET/MRI ¹⁸F-P3BZA provided clear visualization of neuromelanin-rich SN at 30-60 min after injection in healthy macaques. The SN to cerebella ratios were 2.7 and 2.4 times higher at 30 and 60 min after injection. In in vitro autoradiography studies ¹⁸F-P3BZA exhibited high levels of binding to the SN, and almost no binding to surrounding midbrain tissues.

Conclusion: ¹⁸F-P3BZA PET/MRI clearly images neuromelanin in the SN, and may assist in the early diagnosis of neurological diseases associated with abnormal neuromelanin expression.

Background: Parkinson's disease (PD) is a prevalent neurodegenerative disorder worldwide, diagnosed based on classic symptoms like motor dysfunction and cognitive impairments. With the development of various radioactive ligands, positron emission tomography (PET) imaging combined with specific radiolabelling probes has proven to be effective in aiding clinical PD diagnosis. Among these probes, 2β-Carbomethoxy-3β-(4-chlorophenyl)-8-(2-[¹⁸F]-fluoroethyl) nortropane ([¹⁸F]FECNT) has been utilized as a PET tracer to image dopamine transporter (DAT) integrity in striatal presynaptic dopaminergic terminals. However, the presence of brain-penetrant radioactive metabolites produced by [¹⁸F]FECNT may impact the accuracy of PET imaging. In previous research, we developed 2β-Carbomethoxy-3β-(4-chlorophenyl)-8-(2-[¹⁸F]-fluoroethyl-1,1,2,2-d4) nortropane ([¹⁸F]FECNT-d₄), a deuterated derivative with enhanced stability in plasma and the striatum, along with a slower washout rate. In this study, we further investigated the potential of [¹⁸F]FECNT-d₄ to detect dopaminergic neuron degeneration in Parkinson's disease. This involved PET imaging in unilaterally-lesioned PD model rats and in vitro autoradiography conducted on postmortem brain sections.

Results: PET images revealed reduced specific uptake in the ipsilateral striatum of rats stereotactically injected with 6-hydroxydopamine hydrochloride (6-OHDA). Compared to the sham group, the ratio of standardized uptake value (SUV) in the ipsilateral to contralateral striatum decreased by 13%, 23%, and 63% in the mild, moderate, and severe lesioned groups, respectively. Dopaminergic denervation observed in PET imaging was further supported by behavioral assessments, immunostaining, and monoamine concentration tests. Moreover, the microPET results exhibited positive correlations with these measurements, except for the apomorphine-induced rotational behavior test, which showed a negative correlation. Additionally, [¹⁸F]FECNT-d₄ uptake was approximately 40% lower in the postmortem striatal sections of a PD patient compared to a healthy subject. Furthermore, estimated human dosimetry (effective dose equivalent: 5.06 E-03 mSv/MBq), extrapolated from rat biodistribution data, remained below the current Food and Drug Administration limit for radiation exposure.

Conclusion: Our findings demonstrate that [¹⁸F]FECNT-d₄ accurately estimates levels of dopaminergic neuron degeneration in the 6-OHDA-induced PD rat model and effectively distinguishes between PD patients and healthy individuals. This highly sensitive and safe PET probe holds promising potential for clinical application in the diagnosis and monitoring of Parkinson's disease.

Purpose: CXCR3 is expressed on activated T cells and plays a crucial role in T-cell recruitment to the tumor microenvironment (TME) during cell-based and immune checkpoint inhibitor (ICI) immunotherapy. This study utilized a ⁶⁴Cu-labeled NOTA-α-CXCR3 antibody to assess CXCR3 expression in the TME and validate it as a potential T cell activation biomarker in vivo.

Procedures: CXCR3⁺ cells infiltrating MC38 tumors (B57BL/6 mice, untreated and treated with αPD-1/αCTLA-4 ICI) were quantified using fluorescence microscopy and flow cytometry. A commercial anti-mouse CXCR3 antibody (α-CXCR3) was site-specifically conjugated with 2,2,2-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) and radiolabeled with ⁶⁴Cu. Saturation binding of [⁶⁴Cu]Cu-NOTA-α-CXCR3 was investigated using CHO cells stably transfected with murine CXCR3. Biodistribution and PET imaging studies both at baseline and after 1 to 3 cycles of ICI, respectively, were carried out using different molar activities (10 GBq/µmol to 300 GBq/µmol) of [⁶⁴Cu]Cu-NOTA-α-CXCR3.

Results: Flow cytometry analysis at baseline confirmed the presence of CXCR3 + T-cells in MC38 tumors, which was significantly increased at day five after ICI (treated 33.8 ± 17.4 vs. control 8.8 ± 6.2 CD3⁺CXCR3⁺ cells/mg). These results were qualitatively and quantitatively confirmed by immunofluorescence of tumor cryoslices. In vivo PET imaging of MC38 tumor bearing mice before, during and after ICI using [⁶⁴Cu]Cu-NOTA-α-CXCR3 (Kd = 3.3 nM) revealed a strong dependence of CXCR3-specificity of tracer accumulation in secondary lymphoid organs on molar activity. At 300 GBq/µmol (1.5 µg of antibody/mouse), a specific signal was observed in lymph nodes (6.33 ± 1.25 control vs. 3.95 ± 1.23%IA/g blocking) and the spleen (6.04 ± 1.02 control vs. 3.84 ± 0.79%IA/g blocking) at 48 h p.i. Spleen-to-liver ratios indicated a time dependent systemic immune response showing a steady increase from 1.08 ± 0.19 (untreated control) to 1.54 ± 0.14 (three ICI cycles).

Conclusions: This study demonstrates the feasibility of in vivo imaging of CXCR3 upregulation under immunotherapy using antibodies. However, high molar activities and low antibody doses are essential for sensitive detection in lymph nodes and spleen. Detecting therapy-induced changes in CXCR3⁺ T cell numbers in tumors was challenging due to secondary antibody-related effects. Nonetheless, CXCR3 remains a promising target for imaging T cell activation, with anticipated improvements in sensitivity using alternative tracers with high affinities and favorable pharmacokinetics.