Dynamic positron emission tomography (PET) and kinetic modeling are pivotal in advancing tracer development research in small animal studies. Accurate kinetic modeling requires precise input function estimation, traditionally achieved via arterial blood sampling. However, arterial cannulation in small animals like mice, involves intricate, time-consuming, and terminal procedures, precluding longitudinal studies. This work proposes a non-invasive, fully convolutional deep learning-based approach (FC-DLIF) to predict input functions directly from PET imaging, potentially eliminating the need for blood sampling in dynamic small-animal PET. The proposed FC-DLIF model includes a spatial feature extractor acting on the volumetric time frames of the PET sequence, extracting spatial features. These are subsequently further processed in a temporal feature extractor that predicts the arterial input function. The proposed approach is trained and evaluated using images and arterial blood curves from [^18F]FDG data using cross validation. Further, the model applicability is evaluated on imaging data and arterial blood curves collected using two additional radiotracers ([^18F]FDOPA, and [^68Ga]PSMA). The model was further evaluated on data truncated and shifted in time, to simulate shorter, and shifted, PET scans. The proposed FC-DLIF model reliably predicts the arterial input function with respect to mean squared error and correlation. Furthermore, the FC-DLIF model is able to predict the arterial input function even from truncated and shifted samples. The model fails to predict the AIF from samples collected using different radiotracers, as these are not represented in the training data. Our deep learning-based input function offers a non-invasive and reliable alternative to arterial blood sampling, proving robust and flexible to temporal shifts and different scan durations.

Glioblastoma multiforme (GBM) is an aggressive brain tumor with poor prognosis and limited treatment options. Chemokine receptor type 4 (CXCR4) plays a key role in GBM invasion and therapy resistance, making it an attractive target for molecular imaging and theranostics. We developed [68Ga]Ga-TD-01, a next-generation positron emission tomography (PET) radiotracer for CXCR4 imaging, and evaluated its pharmacokinetics, dosimetry, and translational potential. In mice bearing orthotopic GL261 GBM, dynamic PET/MRI, receptor blocking studies, and kinetic modeling demonstrated a specific binding component and high tumor-to-background contrast. [68Ga]Ga-TD-01 showed high tumor uptake (5.4 ± 4.3

Background Glioblastoma multiforme (GBM) is an aggressive brain tumor with poor prognosis and limited treatment options. Chemokine receptor type 4 (CXCR4) plays a key role in GBM invasion and therapy resistance, making it an attractive target for molecular imaging and theranostics. We developed [68Ga]Ga-TD-01, a next-generation positron emission tomography (PET) radiotracer for CXCR4 imaging, and evaluated its pharmacokinetics, dosimetry, and translational potential.Results In mice bearing orthotopic GL261 GBM, dynamic PET/MRI, receptor blocking studies, and kinetic modeling demonstrated a specific binding component and high tumor-to-background contrast. [68Ga]Ga-TD-01 showed high tumor uptake (5.4 +/- 4.3%ID/g at 9 min), low uptake in normal brain (2.2 +/- 1.7%ID/g), and a tumor-to-background ratio of 2.9 +/- 0.2 at 60 min, which was significantly reduced by CXCR4 blockade. PET pharmacokinetic modeling confirmed increased tumor retention (Vt of 0.5 +/- 0.07 ml/cm3 for tumor; Vt of 0.2 +/- 0.05 ml/cm3 for brain). The radiotracer exhibited high in vivo stability, rapid renal clearance, and favorable biodistribution. CXCR4 expression was confirmed in both murine and human GBM tissues by RNAscope. Human dosimetry extrapolation estimated an effective dose of 4.6 mSv for a standard PET scan, comparable to approved tracers.Conclusions When compared with reported data for [68Ga]Ga-Pentixafor, [68Ga]Ga-TD-01 showed tumor uptake within the reported range and low background accumulation; however, no direct head-to-head comparison was performed. These results support [68Ga]Ga-TD-01 as a promising CXCR4-targeted imaging agent and a candidate for further theranostic development.

PURPOSE:Targeted radioligand therapy (TRT) shows promise for treating glioblastoma multiforme (GBM), but its effectiveness is limited by insufficient and heterogeneous tumor uptake. Prostate-specific membrane antigen (PSMA) is being clinically explored as a target in GBM however, low and variable expression restricts effective radioligand delivery. Acoustic Cluster Therapy (ACT) is an ultrasound-mediated platform for targeted therapeutic enhancement that transiently increases vascular permeability to enhance accumulation of co-administered agents. Here, we investigate whether ACT improves the delivery of the PSMA-targeted radioligand [68Ga]Ga-PSMA-617 in a GBM mouse model. RESULTS:Three weeks after orthotopic implantation of GL261 cells in C57BL/6JRj mice (n = 19, female, 8 weeks, 20.1 ± 0.9 g; tumor volume 0.07 ± 0.02 cm3), dynamic [68Ga]Ga-PSMA-617 PET/MRI was performed to evaluate the effect of ACT on radioligand delivery. A cross-over design was used with baseline treatment (saline + ultrasound) on day 21 followed by experimental treatment (PS101 microclusters + ultrasound) 24 h later. Additional controls included perfluorobutane microbubbles with ultrasound, PS101 microclusters without ultrasound, and repeated baseline procedures. A dedicated ultrasound sequence was applied before i.v. injection of 5.9 ± 1.6 MBq [68Ga]Ga-PSMA-617. Pharmacokinetic analyses were performed using 1-tissue and 2-tissue compartment models and Logan analysis. ACT increased the accumulation of [68Ga]Ga-PSMA-617 in the tumor by approximately two-fold and resulted in an increased VT and cumulative activity (103-217%) within the tumor region. Gadolinium-enhanced MRI in six healthy mice (female, 8 weeks, 24.1 ± 0.9 g) demonstrated increased contrast following ACT, indicating a transient ACT-mediated increase in BBB permeability. Exploratory correlation analyses further demonstrate associations between tumor size and baseline VT, indicating that baseline vascular permeability and BBB status influence tracer availability and the response to ACT. CONCLUSIONS:ACT increased tumor accumulation of [68Ga]Ga-PSMA-617 by approximately two-fold. Pharmacokinetic modeling and longitudinal MRI suggest that this effect is primarily driven by ACT-induced increases in vascular permeability across the blood-brain barrier, enhancing radioligand delivery and availability in the brain parenchyma rather than increased receptor-mediated binding. ACT-induced enhancement was most pronounced in tumors with lower baseline tracer accessibility, indicating that the vascular state of the tumor microenvironment strongly influences radioligand delivery. While these findings suggest an increased radioactive burden in the tumor, dedicated therapeutic studies using therapeutic radionuclides will be required to understand the implications ACT may have for dosimetry and therapeutic outcomes in TRT.

Background Dynamic positron emission tomography (PET) combined with tracer kinetic modeling enables noninvasive quantification of biochemical processes. A prerequisite is availability of the arterial input function (AIF), which, in small-animal PET imaging, involves labor-intensive terminal surgery. Deep learning based input function (DLIF) allows estimation of whole-blood tracer concentration from PET data, avoiding arterial cannulation, but require comprehensive validation. The aim of this study was to collect dynamic PET and AIF data under controlled conditions and to evaluate the variability of AIF, image-derived input function (IDIF), and kinetic modeling parameters, which is important for future DLIF model training. Dynamic PET and AIF data were collected prospectively from 112 mice in groups with varying experimental conditions, including radiotracer injection volume, injection time, withdrawal rate, mouse age, strain, radiopharmaceutical, and PET scanner. Brain, myocardium, left ventricle and liver were delineated for kinetic modeling and IDIF generation. Curve features and kinetic modeling parameters were computed, using both AIF and IDIF, and compared across groups using box plots and statistical tests. Intra-subject repeatability was evaluated in six mice using three small-volume radiotracer injections. Results Experimental factors such as mouse strain, injection time, withdrawal rate, PET scanner and radiopharmaceutical significantly affect AIF and IDIF shapes, while injection volume and mouse age, did not introduce bias. AIF measurements within the same subject were highly repeatable. Conclusions This study collected a comprehensive dataset of dynamic PET and AIF measurements under controlled conditions to evaluate the variability of AIF, IDIF, and kinetic modeling parameters. The findings provide valuable insights into input function variability, with potential implications for the future development of DLIF models across diverse experimental conditions.

Understanding the in vivo behavior of nanomedicines is critical for optimizing their therapeutic efficacy and facilitating personalized treatment strategies. In the quest to develop positron emission tomography (PET) methodology for liposome biodistribution studies, we systematically compared three liposome radiolabeling strategies - remote loading of 64Cu into liposomes containing the hydrophilic chelator NOTA, membrane labeling using ATSM, and surface labeling with DSPE-NODAGA (phospholipid DSPE conjugated with chelator NODAGA) - to identify an effective method for liposome radiolabeling with 64Cu. Our results demonstrated that DSPE-NODAGA incorporated in PEGylated liposomes allows for achieving 100 % radiochemical yield of 64Cu at room temperature within just 5 min. Stability studies confirmed liposome integrity and minimal transchelation or dissociation in serum over 24 h, highlighting its suitability for in vivo applications. PET/MR imaging in healthy and tumor-bearing mice revealed prolonged circulation of 64Cu-labeled PEGylated liposomes (PL-NODAGA) and significant tumor accumulation, validating DSPE-NODAGA’s potential for real-time tracking of liposome delivery. These findings establish the incorporation of DSPE-NODAGA as a robust and adaptable platform for PET-based monitoring of lipidic nanomedicine.

Targeted radionuclide therapy (TRT) is an emerging pillar of precision medicine, with prostate-specific membrane antigen (PSMA)-targeted agents like [177Lu]PSMA-617 demonstrating notable clinical success. Achieving optimal therapeutic efficacy while minimizing toxicity requires precise tumor and organ dosimetry based on accurate time-activity curves (TACs) derived from molecular imaging. Glioblastoma (GBM), a highly treatment-resistant brain tumor with limited therapeutic possibilities, has shown PSMA expression, opening new avenues for TRT. Copper-67, a promising theranostic isotope producible in standard hospital-based cyclotrons, offers previously unavailable opportunities for such applications. In this study, we evaluated the feasibility of [67Cu]Cu-rhPSMA-10.1 for TRT analyzed by SPECT and [68Ga]Ga-rhPSMA-10.1 for PET-based imaging and predictive dosimetry in an orthotopic GBM mouse model. METHODS:67Cu was produced via a biomedical cyclotron, purified, and validated using gamma spectrometry and ICP-OES. [67Cu]Cu-rhPSMA-10.1 and [68Ga]Ga-rhPSMA-10.1 were synthesized with high radiochemical purity. GL261-luc2 GBM tumors were implanted in eight C57BL/6JRj mice. One hour PET and up to 72 h SPECT imaging were performed. PET pharmacokinetic modeling analyzed tumor uptake and whole-body biodistribution. Ex vivo gamma counting was applied to validate image-derived organ distribution. Tumor dosimetry was estimated using PET-derived TAC and validated by SPECT, with absorbed dose calculations performed via sphere- and voxel-based models (IDAC-dose 2.1 or Imalytics). Whole-body dosimetry was assessed using OLINDA 2.0. Immunohistochemical (IHC) staining against PSMA and CD31 was performed in human GBM tissue to validate the imaging findings. RESULTS:PET confirmed significantly higher tumor uptake of [68Ga]Ga-rhPSMA-10.1 compared to healthy brain (SUVR 1.9 ± 0.5, 60 min). Pharmacokinetic modeling identified elevated tumor perfusion (K1 = 0.3 ± 0.07 mL/cm3/min) and volume of distribution (Vt = 0.26 ± 0.05 mL/cm3) relative to healthy brain. Predictive tumor dosimetry using [68Ga]Ga-rhPSMA-10.1 PET data extrapolated to [67Cu]Cu-rhPSMA-10.1 estimated a mean tumor dose of up to 31.1 mGy/MBq. Multi-time-point SPECT imaging confirmed tumor uptake of [67Cu]Cu-rhPSMA-10.1, with an absorbed tumor dose of 29.1 mGy/MBq, resembling closely the predicted value. IHC staining confirmed expression of PSMA in human GBM tissue and its localization in vasculature. CONCLUSIONS:This study demonstrates the translational potential of [67Cu]Cu-rhPSMA-10.1 as a targeted radionuclide therapy for PSMA-expressing GBM, supported by predictive PET-based dosimetry using [68Ga]Ga-rhPSMA-10.1. Tumor dose estimates derived from PET closely matched those validated by SPECT imaging, indicating strong concordance between prediction and actual uptake. Additionally, IHC confirmed PSMA expression in human GBM vasculature, reinforcing the clinical relevance. These findings highlight the feasibility of integrating a cyclotron-produced 67Cu-based theranostic strategy for GBM, enabling personalized, image-guided dosimetry and paving the way for future preclinical efficacy studies and potential clinical translation.

Clinical advancements in nuclear medicine theranostics has excited a research interest in exploring novel radionuclides for medical use. The duo of the (3- emitter 67 Cu and the positron emitter 64 Cu, has advantages over the well-established clinical pair 68 Ga and 177 Lu in terms of capability for high-precision therapy. Low availability has hindered the use of 67 Cu whereas 64 Cu has become established at a limited number of sites through production in low-to-medium energy biomedical cyclotrons. Via the reaction 70 Zn(p,alpha) 67 Cu, 67 Cu can also be cyclotron produced, although data on the cross sections of this reaction are sparse. Our aim in this study was three-fold: 1) to establish cross sections for relevant beam energies (14-16 MeV) of the 70 Zn(p,alpha) 67 Cu reaction; 2) determine experimentally the thick target yield for 16.5 MeV proton beam; 3) establish a routine production of 67 Cu for radiochemical and preclinical research. Additionally, our work aims to explore the feasibility of using biomedical cyclotrons for developing of novel therapeutic radionuclides. Thin layers of enriched 70 Zn were electrodeposited onto silver foils to employ the stacked foils technique for assessing the cross section at six energies. The thick target yield was measured experimentally using a pressed [70Zn]ZnO target. Methods were developed for solid phase extraction separation of 67 Cu from the target material, as well as quality control of the product with regards to radionuclidic and radiochemical purity. Radiolabelling of PSMA-617 precursor was performed and the end product injected in a healthy mouse for a kinetic study. As a proof of concept for preclinical applications The animal was then SPECT imaged using the 185 keV gamma emission line. Summarizing, our data confirm that biomedical cyclotrons can contribute in developing novel radionuclides, even of low cross section, for preclinical research.

Cancer immunotherapy has evolved significantly over the last decade, with therapeutics targeting the adaptive immune system showing exciting effects in clinics. Yet, the modulation of the innate immune system, particularly the tumor-associated innate immune cells which are an integral part of immune responses in cancer, remains less understood. The arginase 1 (Arg1) pathway is a pivotal metabolic pathway that tumor-associated innate immune cells exploit to create an immunosuppressive tumor microenvironment, leading to the evasion of immune surveillance. The inhibition of Arg1 presents a therapeutic opportunity to reverse this immunosuppression, and Nω‑hydroxy-l-norarginine (nor-NOHA) has emerged as a potent arginase inhibitor with promising in vivo efficacy. However, the rapid systemic clearance of nor-NOHA poses a significant challenge for its therapeutic application. This study pioneers the encapsulation of nor-NOHA in liposomes, aiming to enhance its bioavailability and prolong its inhibitory activity against Arg1. Historically, the extensive interaction between innate immune cells and nanoparticles has been one of the biggest drawbacks in nanomedicine. Here we seek to utilize this effect and deliver liposomal nor-NOHA to the arginase 1 expressing innate immune cells. We systematically investigated the effect of lipid composition, acyl chain length, manufacturing and loading methodology on the encapsulation efficiency (EE%) and release profile of nor-NOHA. Our results indicate that while the manufacturing method and lipid acyl chain length do not significantly impact EE%, they crucially influence the release kinetics of nor-NOHA, with longer acyl chains demonstrating a more sustained release of nor-NOHA from liposomes enabling continuous inhibition of Arg1. Our findings suggest that liposomal nor-NOHA retains its functional inhibitory activity and could offer improved pharmacokinetic properties, making it a compelling base for iterations for further innovative cancer immunotherapeutic strategies in preclinical and clinical evaluations.

Increasing interest in 67Cu for targeted radionuclide therapy necessitates development of robust and validated analytical methods to ensure compliance with regulatory standards for clinical translation. We validated methods based on inductively coupled plasma optical emission spectroscopy (ICP-OES) and high-purity germanium (HPGe) γ-spectrometry. For ICP-OES, criteria were met for most elements, with Al and Ca, suffering matrix effects. Apparent molar activity calculated by ICP-OES was congruent with DOTA-titration-based effective molar activity when Al and Ca were excluded. HPGe γ-spectrometry was shown to enable accurate discrimination and quantification of co-produced radionuclides (67Ga, 66Ga, 69mZn) from 67Cu at 99.5

Dynamic positron emission tomography and kinetic modeling play a critical role in tracer development research using small animals. Kinetic modeling from dynamic PET imaging requires accurate knowledge of an input function, ideally determined through arterial blood sampling. Arterial cannulation in mice, however, requires complex, time-consuming and terminal surgery, meaning that longitudinal studies are impossible. The aim of the current work was to develop and evaluate a non-invasive, deep learning based prediction model (DLIF), that directly takes the PET data as input to predict a usable input function. We first trained and evaluated the DLIF model on 68 [18F]Fluorodeoxyglucose mouse scans with image-derived targets using cross validation. Subsequently, we evaluated the performance of a trained DLIF model on an external dataset consisting of 8 mouse scans where the input function was measured by continuous arterial blood sampling. The results showed that the predicted DLIF and image-derived targets were similar, and the net influx rate constants following from Patlak modeling using DLIF as input function were strongly correlated to the corresponding values obtained using the image-derived input function. There were somewhat larger discrepancies when evaluating the model on the external dataset, which could be attributed to systematic differences in the experimental setup between the two datasets. In conclusion, our non-invasive DLIF prediction method may be a viable alternative to arterial blood sampling in small animal [18F]FDG imaging. With further validation, DLIF could overcome the need for arterial cannulation and allow fully quantitative and longitudinal experiments in PET imaging studies of mice.

Background:Treatment of castration-resistant metastatic prostate cancer with [¹⁷⁷Lu]PSMA radioligand. Case presentation:A man in his seventies with metastatic prostate cancer received castration therapy for four years, developing castration-resistant disease. PET/CT with [⁶⁸Ga]PSMA-11 showed high uptake in metastatic lymph nodes. The patient received 7.4 GBq [¹⁷⁷Lu]PSMA-I&T (Curium, Finland) as five treatments at five-week intervals. Five weeks after the first treatment, p-PSA dropped from 154 to 53 µg/L. Five weeks after the fifth treatment, p-PSA was 1.8 µg/L. [⁶⁸Ga]PSMA-11 PET/CT showed significant reduction in the size of metastases, with the largest decreasing in diameter from 10 to 4 mm. Seven months after the fifth treatment, p-PSA increased to 14.3 µg/L, and [⁶⁸Ga]PSMA-11 PET/ CT revealed additional skeletal metastases, while the lymph node metastases remained unchanged. Thus, the treatment had a good but temporary effect on the metastases. Interpretation:Treatment with [¹⁷⁷Lu]PSMA radioligand resulted in a temporary regression of the metastases.

The main objective is to discuss why treatment of non-prostate cancers with [177Lu]Lu-PSMA-radioligand achieved only low tumor dose in most published cases, despite high uptake on PSMA PET. We use a patient with renal cell carcinoma as an illustrative example. Furthermore, we discuss how the problem with early washout and low tumor dose might be overcome by using a radionuclide with shorter half-life, matching the target binding residence time. [68Ga]Ga-PSMA-11 PET/CT of a 56-year old man with metastatic renal cell carcinoma showed high lesion uptake. One dose of 6.9 GBq [177Lu]Lu-PSMA-I T was administrated. Post-therapy dosimetry was performed with SPECT/CT and whole-body planar imaging after 5, 24 and 48 h. Doses to target lesions were only 0.2–0.5 Gy. No treatment effect was achieved. Rapid tumor washout of [177Lu]Lu-PSMA-I T and low tumor dose despite high uptake of [68Ga]Ga-PSMA-11 are most likely caused by localization of PSMA-receptors on neovasculature rather than on the tumor cells, and unlike in prostate cancer cells, the PSMA-RL / PSMA-receptor complex is not internalized. To overcome the problem with early washout, the use of a radionuclide with shorter half-life matching the target binding residence time will be needed.

Clinical advancements in nuclear medicine theranostics has excited a research interest in exploring novel radionuclides for medical use. The duo of the β- emitter 67Cu and the positron emitter 64Cu, has advantages over the well-established clinical pair 68Ga and 177Lu in terms of capability for high-precision therapy. Low availability has hindered the use of 67Cu whereas 64Cu has become established at a limited number of sites through production in low-to-medium energy biomedical cyclotrons. Via the reaction 70Zn(p,α)67Cu, 67Cu can also be cyclotron produced, although data on the cross sections of this reaction are sparse. Our aim in this study was three-fold: 1) to establish cross sections for relevant beam energies (14-16 MeV) of the 70Zn(p,α)67Cu reaction; 2) determine experimentally the thick target yield for 16.5 MeV proton beam; 3) establish a routine production of 67Cu for radiochemical and preclinical research. Additionally, our work aims to explore the feasibility of using biomedical cyclotrons for developing of novel therapeutic radionuclides. Thin layers of enriched 70Zn were electrodeposited onto silver foils to employ the stacked foils technique for assessing the cross section at six energies. The thick target yield was measured experimentally using a pressed [70Zn]ZnO target. Methods were developed for solid phase extraction separation of 67Cu from the target material, as well as quality control of the product with regards to radionuclidic and radiochemical purity. Radiolabelling of PSMA-617 precursor was performed and the end product injected in a healthy mouse for a kinetic study. As a proof of concept for preclinical applications The animal was then SPECT imaged using the 185 keV gamma emission line. Summarizing, our data confirm that biomedical cyclotrons can contribute in developing novel radionuclides, even of low cross section, for preclinical research.

Abstract Background This study aimed to develop a novel positron emission tomography (PET) tracer, [68Ga]Ga-TD-01, for CXCR4 imaging. To achieve this goal, the molecular scaffold of TIQ15 was tuned by conjugation with the DOTA chelator to make it suitable for 68Ga radiolabeling. Methods A bifunctional chelator was prepared by conjugating the amine group of TIQ15 with p-NCS-Bz-DOTA, yielding TD-01, with a high yield (68.92%). TD-01 was then radiolabeled with 68Ga using 0.1 M ammonium acetate at 60 °C for 10 min. A 1-h dynamic small animal PET/MRI study of the labeled compound in GL261-luc2 tumor-bearing mice was performed, and brain tumor uptake was assessed. Blocking studies involved pre-administration of TIQ15 (10 mg/kg) 10 min before the PET procedure started. Results [68Ga]Ga-TD-01 exhibited a radiochemical yield (RCY) of 36.33 ± 1.50% (EOS), with a radiochemical purity > 99% and a molar activity of 55.79 ± 1.96 GBq/µmol (EOS). The radiotracer showed in vitro stability in PBS and human plasma for over 4 h. Biodistribution studies in healthy animals revealed favorable kinetics for subsequent PET pharmacokinetic modeling with low uptake in the brain and moderate uptake in lungs, intestines and spleen. Elimination could be assigned to a renal-hepatic pathway as showed by high uptake in kidneys, liver, and urinary bladder. Importantly, [68Ga]Ga-TD-01 uptake in glioblastoma (GBM)-bearing mice significantly decreased upon competition with TIQ15, with a baseline tumor-to-background ratios > 2.5 (20 min p.i.), indicating high specificity. Conclusion The newly developed CXCR4 PET tracer, [68Ga]Ga-TD-01, exhibited a high binding inhibition for CXCR4, excellent in vitro stability, and favorable pharmacokinetics, suggesting that the compound is a promising candidate for full in vivo characterization of CXCR4 expression in GBM, with potential for further development as a tool in cancer diagnosis.

Nanomedicine holds immense potential for therapeutic manipulation of phagocytic immune cells. However, in vitro studies often fail to accurately translate to the complex in vivo environment. To address this gap, we employed an ex vivo human whole-blood assay to evaluate liposome interactions with immune cells. We systematically varied liposome size, PEG-surface densities and sphingomyelin and ganglioside content. We observed differential uptake patterns of the assessed liposomes by neutrophils and monocytes, emphasizing the importance of liposome design. Interestingly, our results aligned closely with published in vivo observations in mice and patients. Moreover, liposome exposure induced changes in cytokine release and cellular responses, highlighting the potential modulation of immune system. Our study highlights the utility of human whole-blood models in assessing nanoparticle-immune cell interactions and provides insights into liposome design for modulating immune responses.