Positron emission tomography-magnetic resonance imaging as a research tool in musculoskeletal conditions.

Compared to positron emission tomography/computed tomography (PET/CT), the uptake of PET- magnetic resonance imaging (MRI) has been slow, even more so in clinical practice compared to the (pre-)clinical research setting. However, for applications in musculoskeletal (MSK) research, the combination of PET and MRI into a single modality offers attractive advantages over other imaging modalities. Most importantly, MRI has exquisite soft-tissue detail without the use of contrast agents or ionizing radiation, superior bone marrow visualization, and an extensive spectrum of distinct multiparametric assessment methods. In the preclinical setting, the introduction of PET inserts for small-animal MRI machines has proven to be a successful concept in bringing this technology to the lab. Initial hurdles in quantification have been mainly overcome in this setting. In parallel, a promising range of radiochemistry techniques has been developed to create multimodality probes that offer the possibility of simultaneously querying different metabolic pathways. Not only will these applications help in elucidating disease mechanisms, but they can also facilitate drug development. The clinical applications of PET/MRI in MSK are still limited, but encouraging initial results with novel radiotracers suggest a high potential for use in various MSK conditions, including osteoarthritis, rheumatoid arthritis, ankylosing spondylitis and inflammation and infection. Further innovations will be required to bring down the cost of PET/MRI to justify a broader clinical implementation, and remaining issues with quality control and standardization also need to be addressed. Nevertheless, PET/MRI is a powerful platform for MSK research with distinct qualities that are not offered by other techniques.

Molecular Imaging of Apoptosis: The Case of Caspase-3 Radiotracers.

The molecular imaging of apoptosis remains an important method for the diagnosis and monitoring of the progression of certain diseases and the evaluation of the efficacy of anticancer apoptosis-inducing therapies. Among the multiple biomarkers involved in apoptosis, activated caspase-3 is an attractive target, as it is the most abundant of the executioner caspases. Nuclear imaging is a good candidate, as it combines a high depth of tissue penetration and high sensitivity, features necessary to detect small changes in levels of apoptosis. However, designing a caspase-3 radiotracer comes with challenges, such as selectivity, cell permeability and transient caspase-3 activation. In this review, we discuss the different caspase-3 radiotracers for the imaging of apoptosis together with the challenges of the translation of various apoptosis-imaging strategies in clinical trials.

Bone metastases in the era of targeted treatments: insights from molecular biology.

Bone metastases remain a common feature of advanced cancers and are associated with significant morbidity and mortality. Recent research has identified promising novel treatment targets to improve current treatment strategies for bone metastatic disease. This review summarizes the well-known and recently discovered molecular biology pathways in bone that govern normal physiological remodeling or drive the pathophysiological changes observed when bone metastases are present. In the rapidly changing world of targeted cancer treatments, it is important to recognize the specific treatment effects induced in bone by these agents and the potential impact on common imaging strategies. The osteoclastic targets (bisphosphonates, LGR4, RANKL, mTOR, MET-VEGFR, cathepsin K, Src, Dock 5) and the osteoblastic targets (Wnt and endothelin) are discussed, and the emerging field of osteo-immunity is introduced as potential future therapeutic target. Finally, a summary is provided of available trial data for agents that target these pathways and that have been assessed in patients. The ultimate goal of research into novel pathways and targets involved in the tumor-bone microenvironment is to tackle one of the great remaining unmet needs in oncology, that is finding a cure for bone metastatic disease.

Caspase-3 probes for PET imaging of apoptotic tumor response to anticancer therapy.

Apoptosis is a highly regulated process involved in the normal organism development and homeostasis. In the context of anticancer therapy, apoptosis is also studied intensively in an attempt to induce cell death in cancer cells. Caspase activation is a known key event in the apoptotic process. In particular, active caspase-3 and -7 are the common effectors in several apoptotic pathways, therefore effector caspase activation may be a promising biomarker for response evaluation to anticancer therapy. Quantitative imaging of apoptosis in vivo could provide early assessment of therapeutic effectiveness and could also be used in drug development to evaluate the efficacy as well as potential toxicity of novel treatments. Positron Emission Tomography (PET) is a highly sensitive molecular imaging modality that allows non-invasive in vivo imaging of biological processes such as apoptosis by using radiolabeled probes. Here we describe the development and evaluation of fluorine-18-labeled caspase-3 activity-based probes (ABPs) for PET imaging of apoptosis. ABPs were selected by screening of a small library of fluorine-19-labeled DEVD peptides containing different electrophilic warhead groups. An acyloxymethyl ketone was identified with low nanomolar affinity for caspase-3 and was radiolabeled with fluorine-18. The resulting radiotracer, [18F]MICA-302, showed good labeling of active caspase-3 in vitro and favorable pharmacokinetic properties. A µPET imaging experiment in colorectal tumor xenografts demonstrated an increased tumor accumulation of [18F]MICA-302 in drug-treated versus control animals. Therefore, our data suggest this radiotracer may be useful for clinical PET imaging of response to anticancer therapy.

Phosphatidylethanolamine targeting for cell death imaging in early treatment response evaluation and disease diagnosis.

Phosphatidylethanolamine (PE) is one of the most abundant phospholipids in mammalian plasma membranes. In healthy cells, PE resides predominantly in the inner leaflet of the cell membrane. In dead or dying cells on the other hand, PE is externalized to the outer leaflet of the plasma membrane. The exposure of PE on the cell surface has therefore become an attractive target for the molecular imaging of cell death using single-photon emission computed tomography (SPECT) and positron emission tomography (PET). This has motivated the development of PE-specific probes to measure cell death in vitro and non-invasively in vivo. In this review, we highlight the biological roles of PE on cell membranes, and PE exposure as a biomarker of cell death in disease processes, along with the use of PE-binding molecular probes to target PE for the characterization of cell death on a cellular and tissue level. We specifically emphasize the preclinical applications of radiolabeled duramycin for the non-invasive imaging of cell death in animal models of disease and in tumors after therapy. In addition, we discuss the clinical relevance, limitations and future perspectives of this imaging approach of cell death.

Adenosine A2AR blockade prevents neuroinflammation-induced death of retinal ganglion cells caused by elevated pressure.

BACKGROUND: Elevated intraocular pressure (IOP) is a major risk factor for glaucoma, a degenerative disease characterized by the loss of retinal ganglion cells (RGCs). There is clinical and experimental evidence that neuroinflammation is involved in the pathogenesis of glaucoma. Since the blockade of adenosine A2A receptor (A2AR) confers robust neuroprotection and controls microglia reactivity in the brain, we now investigated the ability of A2AR blockade to control the reactivity of microglia and neuroinflammation as well as RGC loss in retinal organotypic cultures exposed to elevated hydrostatic pressure (EHP) or lipopolysaccharide (LPS). METHODS: Retinal organotypic cultures were either incubated with LPS (3 µg/mL), to elicit a pro-inflammatory response, or exposed to EHP (+70 mmHg), to mimic increased IOP, for 4 or 24 h, in the presence or absence of the A2AR antagonist SCH 58261 (50 nM). A2AR expression, microglial reactivity and neuroinflammatory response were evaluated by immunohistochemistry, quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). RGC loss was assessed by immunohistochemistry. In order to investigate the contribution of pro-inflammatory mediators to RGC loss, the organotypic retinal cultures were incubated with rabbit anti-tumour necrosis factor (TNF) (2 µg/mL) and goat anti-interleukin-1ß (IL-1ß) (1 µg/mL) antibodies. RESULTS: We report that the A2AR antagonist (SCH 58261) prevented microglia reactivity, increase in pro-inflammatory mediators as well as RGC loss upon exposure to either LPS or EHP. Additionally, neutralization of TNF and IL-1ß prevented RGC loss induced by LPS or EHP. CONCLUSIONS: This work demonstrates that A2AR blockade confers neuroprotection to RGCs by controlling microglia-mediated retinal neuroinflammation and prompts the hypothesis that A2AR antagonists may be a novel therapeutic option to manage glaucomatous disorders.

Adenosine A3 receptor activation is neuroprotective against retinal neurodegeneration.

Death of retinal neural cells, namely retinal ganglion cells (RGCs), is a characteristic of several retinal neurodegenerative diseases. Although the role of adenosine A3 receptor (A3R) in neuroprotection is controversial, A3R activation has been reported to afford protection against several brain insults, with few studies in the retina. In vitro models (retinal neural and organotypic cultures) and animal models [ischemia-reperfusion (I-R) and partial optic nerve transection (pONT)] were used to study the neuroprotective properties of A3R activation against retinal neurodegeneration. The A3R selective agonist (2-Cl-IB-MECA, 1 µM) prevented apoptosis (TUNEL(+)-cells) induced by kainate and cyclothiazide (KA + CTZ) in retinal neural cultures (86.5 ± 7.4 and 37.2 ± 6.1 TUNEL(+)-cells/field, in KA + CTZ and KA + CTZ + 2-Cl-IB-MECA, respectively). In retinal organotypic cultures, 2-Cl-IB-MECA attenuated NMDA-induced cell death, assessed by TUNEL (17.3 ± 2.3 and 8.3 ± 1.2 TUNEL(+)-cells/mm(2) in NMDA and NMDA+2-Cl-IB-MECA, respectively) and PI incorporation (ratio DIV4/DIV2 3.3 ± 0.3 and 1.3 ± 0.1 in NMDA and NMDA+2-Cl-IB-MECA, respectively) assays. Intravitreal 2-Cl-IB-MECA administration afforded protection against I-R injury decreasing the number of TUNEL(+) cells by 72%, and increased RGC survival by 57%. Also, intravitreal administration of 2-Cl-IB-MECA inhibited apoptosis (from 449.4 ± 37.8 to 207.6 ± 48.9 annexin-V(+)-cells) and RGC loss (from 1.2 ± 0.6 to 8.1 ± 1.7 cells/mm) induced by pONT. This study demonstrates that 2-Cl-IB-MECA is neuroprotective to the retina, both in vitro and in vivo. Activation of A3R may have great potential in the management of retinal neurodegenerative diseases characterized by RGC death, as glaucoma and diabetic retinopathy, and ischemic diseases.

Diabetes induces changes in KIF1A, KIF5B and dynein distribution in the rat retina: implications for axonal transport.

Diabetic retinopathy is a leading cause of vision loss and blindness. Disruption of axonal transport is associated with many neurodegenerative diseases and might also play a role in diabetes-associated disorders affecting nervous system. We investigated the impact of type 1 diabetes (2 and 8 weeks duration) on KIF1A, KIF5B and dynein motor proteins in the retina. Additionally, since hyperglycemia is considered the main trigger of diabetic complications, we investigated whether prolonged exposure to elevated glucose could affect the content and distribution of motor proteins in retinal cultures. The immunoreactivity of motor proteins was evaluated by immunohistochemistry in retinal sections and by immunoblotting in total retinal extracts from streptozotocin-induced diabetic and age-matched control animals. Primary retinal cultures were exposed to high glucose (30 mM) or mannitol (osmotic control; 24.5 mM plus 5.5 mM glucose), for seven days. Diabetes decreased the content of KIF1A at 8 weeks of diabetes as well as KIF1A immunoreactivity in the majority of retinal layers, except for the photoreceptor and outer nuclear layer. Changes in KIF5B immunoreactivity were also detected by immunohistochemistry in the retina at 8 weeks of diabetes, being increased at the photoreceptor and outer nuclear layer, and decreased in the ganglion cell layer. Regarding dynein immunoreactivity there was an increase in the ganglion cell layer after 8 weeks of diabetes. No changes were detected in retinal cultures. These alterations suggest that axonal transport may be impaired under diabetes, which might contribute to early signs of neural dysfunction in the retina of diabetic patients and animal models.

Trans-cyclooctene-a Swiss army knife for bioorthogonal chemistry: exploring the synthesis, reactivity, and applications in biomedical breakthroughs.

BACKGROUND: Trans-cyclooctenes (TCOs) are highly strained alkenes with remarkable reactivity towards tetrazines (Tzs) in inverse electron-demand Diels-Alder reactions. Since their discovery as bioorthogonal reaction partners, novel TCO derivatives have been developed to improve their reactivity, stability, and hydrophilicity, thus expanding their utility in diverse applications. MAIN BODY: TCOs have garnered significant interest for their applications in biomedical settings. In chemical biology, TCOs serve as tools for bioconjugation, enabling the precise labeling and manipulation of biomolecules. Moreover, their role in nuclear medicine is substantial, with TCOs employed in the radiolabeling of peptides and other biomolecules. This has led to their utilization in pretargeted nuclear imaging and therapy, where they function as both bioorthogonal tags and radiotracers, facilitating targeted disease diagnosis and treatment. Beyond these applications, TCOs have been used in targeted cancer therapy through a "click-to-release" approach, in which they act as key components to selectively deliver therapeutic agents to cancer cells, thereby enhancing treatment efficacy while minimizing off-target effects. However, the search for a suitable TCO scaffold with an appropriate balance between stability and reactivity remains a challenge. CONCLUSIONS: This review paper provides a comprehensive overview of the current state of knowledge regarding the synthesis of TCOs, and its challenges, and their development throughout the years. We describe their wide ranging applications as radiolabeled prosthetic groups for radiolabeling, as bioorthogonal tags for pretargeted imaging and therapy, and targeted drug delivery, with the aim of showcasing the versatility and potential of TCOs as valuable tools in advancing biomedical research and applications.

Optimizing SUV Analysis: A Multicenter Study on Preclinical FDG-PET/CT Highlights the Impact of Standardization.

PURPOSE: Preclinical imaging, with translational potential, lacks a standardized method for defining volumes of interest (VOIs), impacting data reproducibility. The aim of this study was to determine the interobserver variability of VOI sizes and standard uptake values (SUVmean and SUVmax) of different organs using the same [18F]FDG-PET and PET/CT datasets analyzed by multiple observers. In addition, the effect of a standardized analysis approach was evaluated. PROCEDURES: In total, 12 observers (4 beginners and 8 experts) analyzed identical preclinical [18F]FDG-PET-only and PET/CT datasets according to their local default image analysis protocols for multiple organs. Furthermore, a standardized protocol was defined, including detailed information on the respective VOI size and position for multiple organs, and all observers reanalyzed the PET/CT datasets following this protocol. RESULTS: Without standardization, significant differences in the SUVmean and SUVmax were found among the observers. Coregistering CT images with PET images improved the comparability to a limited extent. The introduction of a standardized protocol that details the VOI size and position for multiple organs reduced interobserver variability and enhanced comparability. CONCLUSIONS: The protocol offered clear guidelines and was particularly beneficial for beginners, resulting in improved comparability of SUVmean and SUVmax values for various organs. The study suggested that incorporating an additional VOI template could further enhance the comparability of the findings in preclinical imaging analyses.

Druglike, 18F-labeled PET Tracers Targeting Fibroblast Activation Protein.

Fibroblast activation protein (FAP) is a very reliable biomarker for tissue remodeling. FAP has so far mainly been studied in oncology, but there is growing interest in the enzyme in other diseases like fibrosis. Recently, FAP-targeting diagnostics and therapeutics have emerged, of which the so-called FAPIs are among the most promising representatives. FAPIs typically have a relatively high molecular weight and contain very polar, multicharged chelator moieties. While this is not limiting the application of FAPIs in oncology, more druglike FAPIs could be required to optimally study diseases characterized by denser, less permeable tissue. In response, we designed the first druglike 18F-labeled FAPIs. We report target potencies, biodistribution, and pharmacokinetics and demonstrate FAP-dependent uptake in murine tumor xenografts. Finally, this paper puts forward compound 10 as a highly promising, druglike FAPI for 18F-PET imaging. This molecule is fit for additional studies in fibrosis and its preclinical profile warrants clinical investigation.

Development of caspase-3-selective activity-based probes for PET imaging of apoptosis.

BACKGROUND: The cysteine-aspartic acid protease caspase-3 is recognized as the main executioner of apoptosis in cells responding to specific extrinsic and intrinsic stimuli. Caspase-3 represents an interesting biomarker to evaluate treatment response, as many cancer therapies exert their effect by inducing tumour cell death. Previously developed caspase-3 PET tracers were unable to reach routine clinical use due to low tumour uptake or lack of target selectivity, which are two important requirements for effective treatment response evaluation in cancer patients. Therefore, the goal of this study was to develop and preclinically evaluate novel caspase-3-selective activity-based probes (ABPs) for apoptosis imaging. RESULTS: A library of caspase-3-selective ABPs was developed for tumour apoptosis detection. In a first attempt, the inhibitor Ac-DW3-KE (Ac-3Pal-Asp-ßhLeu-Phe-Asp-KE) was 18F-labelled on the N-terminus to generate a radiotracer that was incapable of adequately detecting an increase in apoptosis in vivo. The inability to effectively detect active caspase-3 in vivo was likely attributable to slow binding, as demonstrated with in vitro inhibition kinetics. Hence, a second generation of caspase-3 selective ABPs was developed based on the Ac-ATS010-KE (Ac-3Pal-Asp-Phe(F5)-Phe-Asp-KE) with greatly improved binding kinetics over Ac-DW3-KE. Our probes based on Ac-ATS010-KE were made by modifying the N-terminus with 6 different linkers. All the linker modifications had limited effect on the binding kinetics, target selectivity, and pharmacokinetic profile in healthy mice. In an in vitro apoptosis model, the least hydrophilic tracer [18F]MICA-316 showed an increased uptake in apoptotic cells in comparison to the control group. Finally, [18F]MICA-316 was tested in an in vivo colorectal cancer model, where it showed a limited tumour uptake and was unable to discriminate treated tumours from the untreated group, despite demonstrating that the radiotracer was able to bind caspase-3 in complex mixtures in vitro. In contrast, the phosphatidylethanolamine (PE)-binding radiotracer [99mTc]Tc-duramycin was able to recognize the increased cell death in the disease model, making it the best performing treatment response assessment tracer developed thus far. CONCLUSIONS: In conclusion, a novel library of caspase-3-binding PET tracers retaining similar binding kinetics as the original inhibitor was developed. The most promising tracer, [18F]MICA-316, showed an increase uptake in an in vitro apoptosis model and was able to selectively bind caspase-3 in apoptotic tumour cells. In order to distinguish therapy-responsive from non-responsive tumours, the next generation of caspase-3-selective ABPs will be developed with higher tumour accumulation and in vivo stability.

Characterization of Structurally Diverse 18F-Labeled d-TCO Derivatives as a PET Probe for Bioorthogonal Pretargeted Imaging.

Background: The pretargeted imaging strategy using inverse electron demand Diels-Alder (IEDDA) cycloaddition between a trans-cyclooctene (TCO) and tetrazine (Tz) has emerged and rapidly grown as a promising concept to improve radionuclide imaging and therapy in oncology. This strategy has mostly relied on the use of radiolabeled Tz together with TCO-modified targeting vectors leading to a rapid growth of the number of available radiolabeled tetrazines, while only a few radiolabeled TCOs are currently reported. Here, we aim to develop novel and structurally diverse 18F-labeled cis-dioxolane-fused TCO (d-TCO) derivatives to further expand the bioorthogonal toolbox for in vivo ligation and evaluate their potential for positron emission tomography (PET) pretargeted imaging. Results: A small series of d-TCO derivatives were synthesized and tested for their reactivity against tetrazines, with all compounds showing fast reaction kinetics with tetrazines. A fluorescence-based pretargeted blocking study was developed to investigate the in vivo ligation of these compounds without labor-intensive prior radiochemical development. Two compounds showed excellent in vivo ligation results with blocking efficiencies of 95 and 97%. Two novel 18F-labeled d-TCO radiotracers were developed, from which [18F]MICA-214 showed good in vitro stability, favorable pharmacokinetics, and moderate in vivo stability. Micro-PET pretargeted imaging with [18F]MICA-214 in mice bearing LS174T tumors treated with tetrazine-modified CC49 monoclonal antibody (mAb) (CC49-Tz) showed significantly higher uptake in tumor tissue in the pretargeted group (CC49-Tz 2.16 ± 0.08% ID/mL) when compared to the control group with nonmodified mAb (CC49 1.34 ± 0.07% ID/mL). Conclusions: A diverse series of fast-reacting fluorinated d-TCOs were synthesized. A pretargeted blocking approach in tumor-bearing mice allowed the choice of a lead compound with fast reaction kinetics with Tz. A novel 18F-labeled d-TCO tracer was developed and used in a pretargeted PET imaging approach, allowing specific tumor visualization in a mouse model of colorectal cancer. Although further optimization of the radiotracer is needed to enhance the tumor-to-background ratios for pretargeted imaging, we anticipate that the 18F-labeled d-TCO will find use in studies where increased hydrophilicity and fast bioconjugation are required.

Site-specific 68Ga-labeled nanobody for PET imaging of CD70 expression in preclinical tumor models.

BACKGROUND: CD70-CD27 is a costimulatory ligand-receptor pair in the tumor necrosis factor receptor family. With only limited expression in normal tissues, CD70 is constitutively expressed in a variety of solid tumors and hematologic malignancies, facilitating immunosuppression through CD27 signaling in the tumor microenvironment by enhanced survival of regulatory T cells, induction of T cell apoptosis, and T cell exhaustion. Consequently, CD70 is an increasingly recognized target for developing antibody-based therapies, but its expression patterns vary among different tumor types in spatial distribution, magnitude of expression and percentage of positive cells. In that regard, individual confirmation of CD70 expression at screening and during treatment could enhance the successful implementation of anti-CD70 therapies. Here, we developed a gallium-68 (68Ga) radiolabeled single-domain antibody-fragment targeting CD70 for in vivo positron emission tomography (PET) imaging. RESULTS: An anti-CD70 VHH construct containing a C-direct-tag with a free thiol was developed to enable site-specific conjugation to a NOTA bifunctional chelator for 68Ga radiolabeling. [68Ga]Ga-NOTA-anti-CD70 VHH was obtained in good radiochemical yield of 30.4 ± 1.7% and high radiochemical purity (> 94%). The radiolabeled VHH showed excellent in vitro and in vivo stability. Specific binding of [68Ga]Ga-NOTA-anti-CD70 VHH was observed on CD70high 786-O cells, showing significantly higher cell-associated activity when compared to the blocking condition (p < 0.0001) and CD70low NCl-H1975 cells (p < 0.0001). PET imaging showed specific radiotracer accumulation in CD70 expressing human tumor xenografts, which was efficiently blocked by prior injection of unlabeled anti-CD70 VHH (p = 0.0029). In addition, radiotracer uptake in CD70high tumors was significantly higher when compared with CD70low tumors (p < 0.0001). The distribution of the radioactivity in the tumors using autoradiography was spatially matched with immunohistochemistry analysis of CD70 expression. CONCLUSION: [68Ga]Ga-NOTA-anti-CD70 VHH showed excellent in vivo targeting of CD70 in human cancer xenografts. PET imaging using this radioimmunoconjugate holds promise as a non-invasive method to identify and longitudinally follow-up patients who will benefit most from anti-CD70 therapies.

First-in-human study of a novel cell death tracer [99mTc]Tc-Duramycin: safety, biodistribution and radiation dosimetry in healthy volunteers.

BACKGROUND: Imaging of cell death can provide an early indication of treatment response in cancer. [99mTc]Tc-Duramycin is a small-peptide SPECT tracer that recognizes both apoptotic and necrotic cells by binding to phosphatidylethanolamine present in the cell membrane. Preclinically, this tracer has shown to have favorable pharmacokinetics and selective tumor accumulation early after the onset of anticancer therapy. In this first-in-human study, we report the safety, biodistribution and internal radiation dosimetry of [99mTc]Tc-Duramycin in healthy human volunteers. RESULTS: Six healthy volunteers (3 males, 3 females) were injected intravenously with [99mTc]Tc-Duramycin (dose: 6 MBq/kg; 473 ± 36 MBq). [99mTc]Tc-Duramycin was well tolerated in all subjects, with no serious adverse events reported. Following injection, a 30-min dynamic planar imaging of the abdomen was performed, and whole-body (WB) planar scans were acquired at 1, 2, 3, 6 and 23 h post-injection (PI), with SPECT acquisitions after each WB scan and one low-dose CT after the first SPECT. In vivo 99mTc activities were determined from semi-quantitative analysis of the images, and time-activity curves were generated. Residence times were calculated from the dynamic and WB planar scans. The mean effective dose was 7.61 ± 0.75 µSv/MBq, with the kidneys receiving the highest absorbed dose (planar analysis: 43.82 ± 4.07 µGy/MBq, SPECT analysis: 19.72 ± 3.42 µGy/MBq), followed by liver and spleen. The median effective dose was 3.61 mSv (range, 2.85-4.14). The tracer cleared slowly from the blood (effective half-life of 2.0 ± 0.4 h) due to high plasma protein binding with < 5% free tracer 3 h PI. Excretion was almost exclusively renal. CONCLUSION: [99mTc]Tc-Duramycin demonstrated acceptable dosimetry (< 5 mSv) and a favorable safety profile. Due to slow blood clearance, optimal target-to-background ratios are expected 5 h PI. These data support the further assessment of [99mTc]Tc-Duramycin for clinical treatment response evaluation. TRIAL REGISTRATION: NCT05177640, Registered April 30, 2021, https://clinicaltrials.gov/study/NCT05177640 .

Next generation fibroblast activation protein (FAP) targeting PET tracers - The tetrazine ligation allows an easy and convenient way to 18F-labeled (4-quinolinoyl)glycyl-2-cyanopyrrolidines.

Small-molecular fibroblast activation protein inhibitor (FAPI)-based tracer have been shown to be promising Positron Emission Tomography (PET) 68Ga-labeled radiopharmaceuticals to image a variety of tumors including pancreatic, breast, and colorectal cancers, among others. In this study, we developed a novel 18F-labeled FAPI derivative. [18F]6 was labeled using a synthon approach based on the tetrazine ligation. It showed subnanomolar affinity for the FAP protein and a good selectivity profile against known off-target proteases. Small animal PET studies revealed high tumor uptake and good target-to-background ratios. [18F]6 was excreted via the liver. Overall, [18F]6 showed promising characteristics to be used as a PET tracer and could serve as a lead for further development of halogen-based theranostic FAPI radiopharmaceuticals.

Radionuclide Imaging of Cytotoxic Immune Cell Responses to Anti-Cancer Immunotherapy.

Cancer immunotherapy is an evolving and promising cancer treatment that takes advantage of the body's immune system to yield effective tumor elimination. Importantly, immunotherapy has changed the treatment landscape for many cancers, resulting in remarkable tumor responses and improvements in patient survival. However, despite impressive tumor effects and extended patient survival, only a small proportion of patients respond, and others can develop immune-related adverse events associated with these therapies, which are associated with considerable costs. Therefore, strategies to increase the proportion of patients gaining a benefit from these treatments and/or increasing the durability of immune-mediated tumor response are still urgently needed. Currently, measurement of blood or tissue biomarkers has demonstrated sampling limitations, due to intrinsic tumor heterogeneity and the latter being invasive. In addition, the unique response patterns of these therapies are not adequately captured by conventional imaging modalities. Consequently, non-invasive, sensitive, and quantitative molecular imaging techniques, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) using specific radiotracers, have been increasingly used for longitudinal whole-body monitoring of immune responses. Immunotherapies rely on the effector function of CD8+ T cells and natural killer cells (NK) at tumor lesions; therefore, the monitoring of these cytotoxic immune cells is of value for therapy response assessment. Different immune cell targets have been investigated as surrogate markers of response to immunotherapy, which motivated the development of multiple imaging agents. In this review, the targets and radiotracers being investigated for monitoring the functional status of immune effector cells are summarized, and their use for imaging of immune-related responses are reviewed along their limitations and pitfalls, of which multiple have already been translated to the clinic. Finally, emerging effector immune cell imaging strategies and future directions are provided.

Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments.

PURPOSE: RANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [89Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [64Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL. EXPERIMENTAL DESIGN: Fab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with 64Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated tumor targeting and biodistribution in transduced RANKL-ME-180 xenografts. RESULTS: The radiolabeling yield of [64Cu]Cu-NOTA-denos-Fab was 58 ± 9.2%, with a specific activity of 0.79 ± 0.11 MBq/µg (n = 3). A radiotracer binding assay proved specific targeting of RANKL in vitro. PET imaging showed fast blood clearance and high tumor accumulation as early as 1 h p.i. (2.14 ± 0.21% ID/mL), which peaked at 5 h p.i. (2.72 ± 0.61% ID/mL). In contrast, [64Cu]Cu-NOTA-denosumab reached its highest tumor uptake at 24 h p.i. (6.88 ± 1.12% ID/mL). [64Cu]Cu-NOTA-denos-Fab specifically targeted human RANKL in transduced ME-180 xenografts compared with the blocking group and negative ME-180 xenograft model. Histological analysis confirmed RANKL expression in RANKL-ME-180 xenografts. CONCLUSIONS: Here, we report on a novel RANKL PET imaging agent, [64Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications.

Immuno-PET Molecular Imaging of RANKL in Cancer.

PURPOSE: The involvement of RANK/RANKL signaling in the tumor microenvironment (TME) in driving response or resistance to immunotherapy has only very recently been recognized. Current quantification methods of RANKL expression suffer from issues such as sensitivity, variability, and uncertainty on the spatial heterogeneity within the TME, resulting in conflicting reports on its reliability and limited use in clinical practice. Non-invasive molecular imaging using immuno-PET is a promising approach combining superior targeting specificity of monoclonal antibodies (mAb) and spatial, temporal and functional information of PET. Here, we evaluated radiolabeled anti-RANKL mAbs as a non-invasive biomarker of RANKL expression in the TME. EXPERIMENTAL DESIGN: Anti-human RANKL mAbs (AMG161 and AMG162) were radiolabeled with 89Zr using the bifunctional chelator DFO in high yield, purity and with intact binding affinity. After assessing the biodistribution in healthy CD-1 nude mice, [89Zr]Zr-DFO-AMG162 was selected for further evaluation in ME-180 (RANKL-transduced), UM-SCC-22B (RANKL-positive) and HCT-116 (RANKL-negative) human cancer xenografts to assess the feasibility of in vivo immuno-PET imaging of RANKL. RESULTS: [89Zr]Zr-DFO-AMG162 was selected as the most promising tracer for further validation based on biodistribution experiments. We demonstrated specific accumulation of [89Zr]Zr-DFO-AMG162 in RANKL transduced ME-180 xenografts. In UM-SCC-22B xenograft models expressing physiological RANKL levels, [89Zr]Zr-DFO-AMG162 imaging detected significantly higher signal compared to control [89Zr]Zr-DFO-IgG2 and to RANKL negative HCT-116 xenografts. There was good visual agreement with tumor autoradiography and immunohistochemistry on adjacent slides, confirming these findings. CONCLUSIONS: [89Zr]Zr-DFO-AMG162 can detect heterogeneous RANKL expression in the TME of human cancer xenografts, supporting further translation of RANKL immuno-PET to evaluate tumor RANKL distribution in patients.

SPECT/CT: Standing on the Shoulders of Giants, It Is Time to Reach for the Sky!

Twenty years ago, SPECT/CT became commercially available, combining the strengths of both techniques: the diagnostic sensitivity of SPECT and the anatomic detail of CT. Other benefits initially included attenuation correction of SPECT reconstructions, ultimately evolving to correction techniques that would enable absolute tracer uptake quantification. Recent developments in SPECT hardware include solid-state digital systems with higher sensitivity and resolution, using novel collimator designs based on tungsten. Similar advances in CT technology have been introduced in hybrid SPECT/CT systems, replacing low-end x-ray tubes with high-end multislice CT scanners equipped with iterative reconstruction, metal artifact reduction algorithms, and dual-energy capabilities. More recently, the design of whole-body SPECT/CT systems has taken another major leap with the introduction of a ring-shaped gantry equipped with multiple movable detectors surrounding the patient. These exciting developments have fueled efforts to develop novel SPECT radiopharmaceuticals, creating new chelators and prosthetic groups for radiolabeling. Innovative SPECT radionuclide pairs have now become available for radiolabeling with the potential for use as theranostic agents. The growth of precision medicine and the associated need for accurate radionuclide treatment dosimetry will likely drive the use of SPECT/CT in the near future. In addition, expanding clinical applications of SPECT/CT in other areas such as orthopedics offer exciting opportunities. Although it is true that the SPECT/CT ecosystem has seen several challenges during its development over the past 2 decades, it is now a feature-rich and mature tool ready for clinical prime time.