Summary

As estimated by the World Health Organization (WHO) in 2019, cancer remains the primary or secondary cause of mortality before the age of 70 years in over 60% of countries globally, and its incidence is expected to increase over the next 50 years. For an individual diagnosed with cancer, delayed diagnosis and incomplete treatment may result in poor prognosis and increased mortality. Consequently, an early detection of primary lesions and metastases, in addition to effective surgical intervention, can significantly improve patient outcomes.
In recent decades, molecular imaging, which includes a range of techniques such as magnetic resonance imaging (MRI), optical and near-infrared fluorescence imaging (FI), ultrasound imaging (USI), and nuclear medicine imaging techniques, including positron emission tomography (PET) and single-photon emission computed tomography (SPECT), has attained a crucial role in the field of cancer diagnostics and therapeutics.
In order to fully exploit their distinctive and complementary strengths, PET and FI can be employed in a synergistic approach to visualize tumour lesions pre- and intra-operatively, respectively. PET offers detection sensitivity at the subnanomolar level without penetration-related limitations in living tissues. A single radiotracer injection provides non-invasive, functional, and quantitative whole-body data.
Fluorescence imaging in the near-infrared region of 650–900 nm is well suited to surgical guidance, as it enables a real-time visualization of tumour margins with a high spatial resolution (down to tens of nanometers) and a sensitivity of detection in the nanomolar range. This enables the complete and minimally invasive resection of cancerous tissue. Nevertheless, the low penetration of the fluorescent signal precludes quantitative information and whole-body observation. So far, cyanine dyes have been successfully employed for fluorescence-guided surgery. Recently, OTL38, a folate receptor-targeted FI agent featuring an indocyanine green-like near-infrared dye, has gained FDA approval. This achievement substantiates the clinical utility of this approach.
Given the comparable sensitivity and complementary information provided, nuclear and fluorescence imaging capabilities can be combined in a single bimodal PET/FI agent. This approach ensures the optimal correlation of the radioactive and fluorescent signals, thereby also facilitating cross-validation of the two techniques. A bimodal PET/FI agent can therefore be used to enable preoperative nuclear imaging for surgical planning and intraoperative real-time guidance aiming to complete tumour resection, ideally via a single probe injection.
Over the last twenty years, CCK2R, expressed at high incidence and density in medullary thyroid carcinoma (MTC) and small-cell lung cancer (SCLC) among other malignancies, emerged as an attractive target for the development of diagnostic and therapeutic targeted radiopharmaceuticals based on gastrin and cholecystokinin analogues. In the specific context of MTC, local lymph node metastases are prevalent and frequently undetected during preoperative neck ultrasonography. Consequently, despite total thyroidectomy and central lymph node dissection, approximately 50% of patients exhibit persistent disease or develop recurrent lesions.
To enable complete resection, fluorescent imaging agents based on peptide agonists and small-molecule antagonists for the intraoperative surgical guidance of MTC have been investigated. The authors' group reported the first dimeric bimodal PET/FI agent targeting the CCK2R, [⁶⁸Ga]Ga-Sulfo-Cy7-FSC-MG. In this proof-of-principle study, the Fusarinine C (FSC) chelator was used as a central scaffold and was conjugated to a near-infrared fluorophore (Sulfo-Cyanine7) and to two units of a minigastrin analogue (MG11). In vivo, this dimeric bimodal probe demonstrated specific but low tumour uptake accompanied by slow blood clearance and retention in non-targeted tissues, resulting in suboptimal imaging contrast at early timepoints.
In this study, the authors aimed to improve this initial approach and investigated the properties of a pair of novel candidates based on a more hydrophilic fluorophore (SulfoCy5.5) and using two units of a modified CCK2R-binding motif with reduced susceptibility to enzymatic degradation. These probes, designated CyTMG and CyFMG, differ exclusively for the multifunctional chelator used as the core scaffold, respectively, TRAP and FSC. In vitro characterization included internalization experiments with A431 cells stably transfected with human CCK2R (A431-CCK2R) and mock-transfected cells (A431-mock), as well as competitive binding assays. Subsequently, the authors validated the in vivo targeting ability using tumour xenografted mice. In addition, by performing imaging studies, they assessed the potential for preoperative lesion detection and intraoperative tumour margin delineation.

Results from nanoScan® PET/CT

For the comparative imaging study (PET/CT and in vivo fluorescence imaging), 2 xenograft-bearing mice were injected with 0.3 nmol (6 MBq) of radiotracer. Static PET/CT images of the anesthetized animals in prone position were acquired with the Mediso nanoScan PET/CT small-animal imaging system at 1, 2, and 3 h p.i. Image reconstruction was performed via Mediso Tera-Tomo 3D PET iterative reconstruction. The images were visualized, processed, and quantified in Mediso InterView FUSION. Quantitative analyses were performed on images of 3 mouse xenografts per compound. The images were normalized to injected activity and animal weight. The results were expressed as percentages of injected dose per gram tissue (% ID/g).

The PET/CT images obtained with the two radiotracers confirmed the results from the ex vivo biodistribution profile, demonstrating an elevated accumulation of radioactivity in the kidneys, together with moderate uptake in the liver and in the heart (Figure 8a). Receptor-specific uptake could be clearly visualized in A431-CCK2R xenografts, while no uptake was visible in A431-mock tumours. Quantitative analysis of the images confirmed a good tumour retention of [⁶⁸Ga]Ga-CyTMG and [⁶⁸Ga]Ga-CyFMG, with uptake values slightly increasing over time (Figure 8b) . Due to the decreasing signal in non-targeted tissue, the best contrast for PET/CT images was achieved at 3 h after injection. The A431-CCK2R xenografts were clearly visualized also by fluorescence imaging (Figure 8a). For [⁶⁸Ga]Ga-CyTMG, the cancerous tissue could be well delineated starting from 1 h after injection, while in the case of [⁶⁸Ga]Ga-CyFMG, this was possible starting from 3 h after injection. The best target-to-background contrast was reached from 3 h to 24 h and from 5 h to 72 h after administration, respectively, for [⁶⁸Ga]Ga-CyTMG and [⁶⁸Ga]Ga-CyFMG. A noteworthy observation derived from these results is that in the case of [⁶⁸Ga]Ga-CyTMG, the signal in the kidneys persisted for up to 72 h post-injection. In contrast, in the case of [⁶⁸Ga]Ga-CyFMG, this uptake visibly declined from 24 h to disappear completely at 72 h after injection.

• In this study, the authors investigated the possibility of improving the slow blood clearance and non-specific accumulation showed by [⁶⁸Ga]Ga-Sulfo-Cy7-FSC-MG, the first example of a CCK2R-targeting dual-modality imaging agent. For this, they considered a stabilized and higher-affinity targeting unit, a more hydrophilic fluorophore, and considered TRAP as an alternative core scaffold and provided for the first time a head-to-head comparison with Fusarinine C (FSC).
• The developed [⁶⁸Ga]Ga-CyTMG and [⁶⁸Ga]Ga-CyFMG showed promising in vitro and in vivo targeting properties along with improvements in the off target uptake, especially in the liver. It was also demonstrated for both compounds that other than PET, fluorescence imaging is also possible with one single injection even at later timepoints, validating the feasibility of the dual modality imaging approach in a murine model. However, further probe optimization, especially in terms of the fluorophore, may improve the imaging performance at early timepoints increasing the potential for clinical translation.
• Lastly, the major difference observed between [⁶⁸Ga]Ga-CyTMG and [⁶⁸Ga]Ga-CyFMG was the different renal accumulation and retention. This shows that the choice of the chelator for such an approach can have a substantial effect on the pharmacokinetic profile of the final compound and, in particular, influence the biodistribution at late timepoints.

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