[177Lu]Lu-PSMA-617 theranostic probe for hepatocellular carcinoma imaging and therapy.

PURPOSE: This study aimed to explore the feasibility of using [177Lu]Lu-prostate-specific membrane antigen (PSMA)-617 and [177Lu]Lu-Evans blue (EB)-PSMA-617 for in vivo radioligand therapy by single-dose administration in a PSMA-positive hepatocellular carcinoma (HCC) xenograft mouse model. METHODS: [177Lu]Lu-PSMA-617 and [177Lu]Lu-EB-PSMA-617 were prepared, and labelling efficiency and radiochemical purity were determined. A HepG2 human HCC subcutaneous xenograft mouse model was established. After intravenous injection of [177Lu]Lu-PSMA-617 or [177Lu]Lu-EB-PSMA-617 (37 MBq) into the mouse model, single-photon emission computed tomography/computed tomography (SPECT/CT) was performed. Biodistribution studies were conducted to verify targeting specificity and pharmacokinetics. In the radioligand therapy study, mice were randomized into 4 groups: 37 MBq [177Lu]Lu-PSMA-617, 18.5 MBq [177Lu]Lu-PSMA-617, 7.4 MBq [177Lu]Lu-EB-PSMA-617, and saline (control). A single-dose administration was applied at the beginning of therapy studies. Tumor volume, body weight, and survival were monitored every 2 days. After the end of therapy, mice were euthanized. Tumors were then weighed, and systemic toxicity was evaluated via blood testing and histological examination of healthy organs. RESULTS: [177Lu]Lu-PSMA-617 and [177Lu]Lu-EB-PSMA-617 were successfully prepared with high purity and stability. SPECT/CT and biodistribution showed that tumor uptake was higher and persisted longer for [177Lu]Lu-EB-PSMA-617 compared with [177Lu]Lu-PSMA-617. [177Lu]Lu-PSMA-617 was rapidly cleared from the blood, while [177Lu]Lu-EB-PSMA-617 persisted for significantly longer. In radioligand therapy studies, tumor growth was significantly suppressed in the 37 MBq [177Lu]Lu-PSMA-617, 18.5 MBq [177Lu]Lu-PSMA-617, and 7.4 MBq [177Lu]Lu-EB-PSMA-617 groups compared to the saline group. Median survival was 40, 44, 43, and 30 days, respectively. No healthy organ toxicity was observed in safety and tolerability evaluation. CONCLUSIONS: Radioligand therapy using [177Lu]Lu-PSMA-617 and [177Lu]Lu-EB-PSMA-617 significantly suppressed tumor growth and prolonged survival time in PSMA-positive HCC xenograft mice without obvious toxicity. These radioligands appear promising for clinical use in humans, and future studies are warranted.

Q.Clear reconstruction for reducing the scanning time for 68 Ga-DOTA-FAPI-04 PET/MR imaging.

PURPOSE: This study aims to determine whether Q.Clear positron emission tomography (PET) reconstruction may reduce tracer injection dose or shorten scanning time in 68Gallium-labelled fibroblast activation protein inhibitor (68 Ga-FAPI) PET/magnetic resonance (MR) imaging. METHODS: We retrospectively collected cases of 68 Ga-FAPI whole-body imaging performed on integrated PET/MR. PET images were reconstructed using three different methods: ordered subset expectation maximization (OSEM) reconstruction with full scanning time, OSEM reconstruction with half scanning time, and Q.Clear reconstruction with half scanning time. We then measured standardized uptake values (SUVs) within and around lesions, alongside their volumes. We also evaluated image quality using lesion-to-background (L/B) ratio and signal-to-noise ratio (SNR). We then compared these metrics across the three reconstruction techniques using statistical methods. RESULTS: Q.Clear reconstruction significantly increased SUVmax and SUVmean within lesions (more than 30%) and reduced their volumes in comparison with OSEM reconstruction. Background SUVmax also increased significantly, while background SUVmean showed no difference. Average L/B values for Q.Clear reconstruction were only marginally higher than those from OSME reconstruction with half-time. SNR decreased significantly in Q.Clear reconstruction compared with OSEM reconstruction with full time (but not half time). Differences between Q.Clear and OSEM reconstructions in SUVmax and SUVmean values within lesions were significantly correlated with SUVs within lesions. CONCLUSIONS: Q.Clear reconstruction was useful for reducing PET injection dose or scanning time while maintaining the image quality. Q.Clear may affect PET quantification, and it is necessary to establish diagnostic recommendations based on Q.Clear results for Q.Clear application.

PET imaging of hepatocellular carcinoma by targeting tumor-associated endothelium using [68Ga]Ga-PSMA-617.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a malignant tumor associated with high morbidity and mortality rates. In many non-prostate solid tumors such as HCC, prostate-specific membrane antigens (PSMA) are overexpressed in tumor-associated endothelial cells. Therefore, the aim of this study was to evaluate the performance of [68Ga]Ga-PSMA-617 PET imaging on HCC with different animal models, including cell line-derived xenografts (CDX) and patient-derived xenografts (PDX), and to explore its mechanisms of function. METHODS: [68Ga]Ga-PSMA-617 was prepared. The expression level of PSMA in two human hepatocellular cancer cells (HepG2 and HuH-7) was evaluated, and the cellular uptakes of [68Ga]Ga-PSMA-617 were assayed. HepG2 and HuH-7 subcutaneous xenograft models, HepG2 orthotopic xenograft models, and four different groups of PDX models were prepared. Preclinical pharmacokinetics and performance of [68Ga]Ga-PSMA-617 were evaluated in different types of HCC xenografts models using small animal PET and biodistribution studies. RESULTS: Low PSMA expression level of HepG2 and HuH-7 cells was observed, and the cellular uptake and blocking study confirmed the non-specificity of the PSMA-targeted probe binding to HepG2 and HuH-7 cells. In the subcutaneous xenograft models, the tumor uptakes at 0.5 h were 0.76 ± 0.12%ID/g (HepG2 tumors) and 0.78 ± 0.08%ID/g (HuH-7 tumors), respectively, which were significantly higher than those of the blocking groups (0.23 ± 0.04%ID/g and 0.20 ± 0.04%ID/g, respectively). In the orthotopic xenograft models, PET images clearly displayed the tumor locations based on the preferential accumulation of [68Ga]Ga-PSMA-617 in tumor tissue versus normal liver tissue, suggesting the possibility of using [68Ga]Ga-PSMA-617 PET imaging to detect primary HCC lesions in deep tissue. In the four different groups of HCC PDX models, PET imaging with [68Ga]Ga-PSMA-617 provided clear tumor uptakes with prominent tumor-to-background contrast, further demonstrating its potential for the clinical imaging of PSMA-positive HCC lesions. The staining of tumor tissue sections with CD31- and PSMA-specific antibodies visualized the tumor-associated blood vessels and PSMA expression on endothelial cells in subcutaneous, orthotopic tissues, and PDX tissues, confirming the imaging with [68Ga]Ga-PSMA-617 might be mediated by targeting tumor associated endothelium. CONCLUSION: In this study, in vivo PET on different types of HCC xenograft models illustrated high uptake within tumors, which confirmed that [68Ga]Ga-PSMA-617 PET may be a promising imaging modality for HCC by targeting tumor associated endothelium.

Increased uptake of 68Ga-DOTA-FAPI-04 in bones and joints: metastases and beyond.

PURPOSE: To describe the uptake of 68Gallium-labelled fibroblast activation protein inhibitor (68Ga-FAPI) in the bones and joints for better understanding of the role of 68Ga-FAPI PET in benign and malignant bone lesions and joint diseases. METHODS: All 129 68Ga-FAPI PET/MR or PET/CT scans from June 1, 2020, to February 20, 2021, performed at our PET center were retrospectively reviewed. Foci of elevated 68Ga-FAPI uptake in the bones and joints were identified. All lesions were divided into malignant and benign diseases. Benign lesions included osteofibrous dysplasia, periodontitis, degenerative bone diseases, arthritis, and other inflammatory or trauma-related abnormalities. The number, locations, and SUVmax of all lesions were recorded and analyzed. The detectability of 68Ga-FAPI PET and 18F-FDG PET in patients who had two scans was also compared. RESULTS: Elevated uptake of 68Ga-FAPI in/around the bones and joints was found in 82 cases (63.57%). A total of 295 lesions were identified, including 94 (31.9%) malignant lesions (all were metastases) and 201 (68.1%) benign lesions. The benign lesions consisted of 13 osteofibrous dysplasia, 48 degenerative bone disease, 33 periodontitis, 56 arthritis, and 51 other inflammatory or trauma-related abnormalities. The spine, shoulder joint, alveolar ridge, and pelvis were the most commonly involved locations. Bone metastases were mainly distributed in the spine, pelvis, and ribs. Among benign diseases, periodontitis and arthritis are site-specific. The mean SUVmax of bone metastases was significantly higher than that of benign diseases (7.14 ± 4.33 vs. 3.57 ± 1.60, p < 0.001), but overlap existed. The differences in SUVmax among subgroups of benign diseases were statistically significant (p < 0.001), with much higher uptake in periodontitis (4.45 ± 1.17). 68Ga-FAPI PET identified much more lesions than 18F-FDG PET (104 vs. 48) with higher uptake value. CONCLUSION: 68Ga-FAPI accumulated in both bone metastases and some benign diseases of the bones and joints. Although the uptake of 68Ga-FAPI was often higher in bone metastases, this finding cannot be used to distinguish between benign and malignant lesions. 68Ga-FAPI PET also has the potential to locate and evaluate the extent of both malignant tumor and benign diseases in bones and joints. TRIAL REGISTRATION: NCT04554719, NCT04605939. Registered September 8, 2020 and October 25, 2020-retrospectively registered, http://clinicaltrails.gov/show/NCT04554719 ; http://clinicaltrails.gov/show/NCT04605939.

Gallium-68-labeled fibroblast activation protein inhibitor PET in gastrointestinal cancer: insights into diagnosis and management.

PURPOSE: Gallium-68-labeled fibroblast activation protein inhibitor (68Ga-FAPI) is an emerging promising tumor tracer. This study aims to evaluate the diagnostic efficiency of 68Ga-FAPI PET in gastrointestinal cancer, and to determine its potential impact on clinical management. METHODS: Patients with malignancies were prospectively enrolled in a clinical trial to evaluate the diagnostic value of 68Ga-FAPI PET. One hundred twenty patients with gastrointestinal malignancies (121 68Ga-FAPI PET scans) between June 2020 and May 2021 were retrospectively analyzed. Initial staging of untreated patients and restaging of treated patients were evaluated. The treatment scheme promoted by imaging was determined according to NCCN guidelines. Final diagnosis and treatment reference standards were determined by a dedicated multidisciplinary team. The diagnostic performance and treatment guidance of 68Ga-FAPI PET were compared with those of conventional imaging (CI) and 18F-FDG PET. RESULTS: The diagnostic accuracy of 68Ga-FAPI PET was much higher than that of CI and 18F-FDG PET (95.0% vs. 65.1% and 69.0%, respectively, both p < 0.001). 68Ga-FAPI PET revised diagnosis in 30.3% and 26.2% of patients compared with CI and 18F-FDG PET. The accordance rate of 68Ga-FAPI PET-guided treatment in comparison with the reference standard was significantly higher than that of CI and 18F-FDG PET (96.7% vs. 75.2% and 76.2%, respectively, both p < 0.001). 68Ga-FAPI PET changed treatment in 22.9% and 23.8% of patients compared with CI and 18F-FDG PET. CONCLUSIONS: 68Ga-FAPI PET showed remarkable diagnostic performance in gastrointestinal cancer, resulting in more accurate staging and guidance for timely treatment revision, thereby having a critical impact on clinical management. TRIAL REGISTRATION: NCT04554719. Registered September 8, 2020-retrospectively registered, http://clinicaltrails.gov/show/NCT04554719.

Visualizing γδ T cells by very late antigen-4-targeted positron emission tomography.

PURPOSE: γδ T cell-based immunotherapy has been rolled out as a promising treatment strategy for malignant tumors due to their potent anti-tumor cytotoxicity, ease of expansion, and unrestricted MHC feature. However, the dynamics and outcomes of γδ T cells in tumor sites are poorly understood. Reported strategies rely on ex vivo biolabeling, significantly limiting the application of γδ T cell molecular imaging. Herein, we investigated whether VLA-4 (very late antigen-4), a crucial component in the effective trafficking of lymphocytes, could serve as a biomarker to non-invasively visualize γδ T cells. METHODS: VLA-4-targeted tracer, 68 Ga-LLP2A, was evaluated in MDA-MB-231- and A549-bearing mice with adoptive transfer of γδ T cells by longitudinal PET/CT imaging. Imaging data were verified by ex vivo biodistribution studies, and the co-localization of CD3 and VLA-4 was validated by immunohistochemistry studies. RESULTS: 68 Ga-LLP2A showed high specificity to VLA-4-expressing γδ T cells in both in vitro and tumor-bearing mice with adoptive transfer of γδ T cells. Longitudinal PET imaging of 68 Ga-LLP2A in tumor-bearing mice with adoptive transfer of γδ T cells showed an increasing tumor tracer uptake, revealing the tumor-specific homing of γδ T cells. The presence of VLA-4-expressing γδ T cells in tumors was confirmed via histological analysis. CONCLUSION: To the best of our knowledge, we reported the first molecular probe, 68 Ga-LLP2A, for in vivo imaging of γδ T cells in live tumors, which advances PET imaging of γδ T cells and supports the translation of imaging agents for immunotherapeutic monitoring.

Nectin-4-targeted immunoSPECT/CT imaging and photothermal therapy of triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is more prone to distant metastasis and visceral recurrence in comparison to other breast cancer subtypes, and is related to dismal prognosis. Nevertheless, TNBC has an undesirable response to targeted therapies. Therefore, to tackle the huge challenges in the diagnosis and treatment of TNBC, Nectin-4 was selected as a theranostic target because it was recently found to be highly expressed in TNBC. We developed anti-Nectin-4 monoclonal antibody (mAbNectin-4)-based theranostic pair, 99mTc-HYNIC-mAbNectin-4 and mAbNectin-4-ICG. 99mTc-HYNIC-mAbNectin-4 was applied to conduct immuno-single photon emission computed tomography (SPECT) for TNBC diagnosis and classification, and mAbNectin-4-ICG to mediate photothermal therapy (PTT) for relieving TNBC tumor growth. METHODS: Nectin-4 expression levels of breast cancer cells (MDA-MB-468: TNBC cells; and MCF-7, non-TNBC cells) were proved by western blot, flow cytometry, and immunofluorescence imagning. Cell uptake assays, SPECT imaging, and biodistribution were performed to evaluate Nectin-4 targeting of 99mTc-HYNIC-mAbNectin-4. A photothermal agent (PTA) mAbNectin-4-ICG was generated and characterized. In vitro photothermal therapy (PTT) mediated by mAbNectin-4-ICG was conducted under an 808 nm laser. Fluorescence (FL) imaging was performed for mAbNectin-4-ICG mapping in vivo. In vivo PTT treatment effects on TNBC tumors and corresponding systematic toxicity were evaluated. RESULTS: Nectin-4 is overexpressed in MDA-MB-468 TNBC cells, which could specifically uptake 99mTc-HYNIC-mAbNectin-4 with high targeting in vitro. The corresponding immunoSPECT imaging demonstrated exceptional performance in TNBC diagnosis and molecular classification. mAbNectin-4-ICG exhibited favourable biocompatibility, photothermal effects, and Nectin-4 targeting. FL imaging mapped biodistribution of mAbNectin-4-ICG with excellent tumor-targeting and retention in vivo. Moreover, mAbNectin-4-ICG-mediated PTT provided advanced TNBC tumor destruction efficiency with low systematic toxicity. CONCLUSION: mAbNectin-4-based radioimmunoimaging provides visualization tools for the stratification and diagnosis for TNBC, and the corresponding mAbNectin-4-mediated PTT shows a powerful anti-tumor effect. Our findings demonstrate that this Nectin-4 targeting strategy offers a simple theranostic platform for TNBC.

Framework Nucleic Acids in Nuclear Medicine Imaging: Shedding Light on Nano-Bio Interactions.

Framework nucleic acids (FNAs) represent nanoscale oligonucleotide assemblies with unique physical, chemical, and biological properties that are different from their building blocks. Following simple Watson-Crick base-pairing rules, arbitrary DNA frameworks with diverse shapes, sizes, and dimensions can be prepared with high reproducibility and stability. The programmable assembly of nucleic acids into FNAs presents a highly controllable model for studies on nano-bio interactions and allows scrutiny of "nanostructure-activity relationships." Herein, we present an overview of recent progress with FNAs in the hope of deepening our understanding of nano-bio interfacing. We summarize the biological profiles and immune responses of various FNAs as functions of their shape, sizes, and surface charges. We then highlight recent efforts to apply FNAs for biomedical applications and discuss the challenges of FNAs for potential clinical translation. We believe that this Minireview can bring up-to-date information on FNAs and shed light on how their design may be harnessed for selective biomedical applications.

A head-to-head comparison of 68Ga-DOTA-FAPI-04 and 18F-FDG PET/MR in patients with nasopharyngeal carcinoma: a prospective study.

PURPOSE: To conduct a head-to-head comparison of the diagnostic ability of 68Ga-DOTA-FAPI-04 (68Ga-FAPI) and 18F-FDG PET/MR in nasopharyngeal carcinoma (NPC) patients. METHODS: Patients diagnosed with NPC were prospectively enrolled. All patients underwent head-and-neck 68Ga-FAPI PET/MR and 18F-FDG PET/MR within 1 week. Primary tumor, lymph node numbers, and tracer uptake were compared by SUVmax and visual evaluation. The primary tumor volumes derived from 68Ga-FAPI, 18F-FDG PET, and MRI were also compared. RESULTS: Fifteen patients were enrolled from June to August 2020. Both 68Ga-FAPI and 18F-FDG PET had 100% detection rate of the primary tumor. The 68Ga-FAPI SUVmax of primary tumors (13.87 ± 5.13) was lower than that of 18F-FDG (17.73 ± 6.84), but the difference was not significant (p = 0.078). Compared with 18F-FDG, 68Ga-FAPI PET improved the delineation of skull-base invasion in eight out of eight patients and intracranial invasion in four out of four patients. When 25%SUVmax of 68Ga-FAPI or 20%SUVmax of 18F-FDG was utilized as a threshold for determining tumor volume, it was highly consistent with MRI. 18F-FDG PET detected much more positive lymph nodes than 68Ga-FAPI (100 vs 48). The SUVmax of 48 paired lymph nodes was significantly lower on 68Ga-FAPI than 18F-FDG (8.67 ± 3.88 vs 11.79 ± 6.17, p < 0.001). Additionally, 68Ga-FAPI further detected four highly suspected small, distant metastases in three patients. Compared with 18F-FDG, 68Ga-FAPI changed overall staging in six of fifteen patients, with three patients being up-staged, and three down-staged. CONCLUSION: 68Ga-FAPI outperforms 18F-FDG in delineating the primary tumor and detecting suspected distant metastases, particularly in the evaluation of skull-base and intracranial invasion, suggesting 68Ga-FAPI hybrid PET/MR has the potential to serve as a single-step staging modality for patients with NPC. However, its value regarding lymph node and distant metastases evaluation needs further study. TRIAL REGISTRATION: NCT04554719. Registered September 8, 2020 - retrospectively registered, http://clinicaltrails.gov/show/NCT04554719.

[18F]F-ET-OTSSP167 Targets Maternal Embryo Leucine Zipper Kinase for PET Imaging of Triple-Negative Breast Cancer.

Maternal embryo leucine zipper kinase (MELK) is a serine/threonine kinase and is highly expressed in triple-negative breast cancer (TNBC). This study aimed to develop a 18F-radiolabeled tracer based on the structure of a small-molecule MELK inhibitor OTSSP167 and evaluate its application for PET imaging of MELK expression in TNBC. OTSSP167 was modified with ethylene glycol to adjust its pharmacokinetics and was then radiolabeled with 18F to obtain [18F]F-ET-OTSSP167 at a labeling yield of 7.14 ± 2.19% and a molar activity of 16.23 ± 1.13 MBq/nmol. In vitro binding assays showed differentiated binding affinities of [18F]F-ET-OTSSP167 in different breast cancer cell lines, with high uptake in MDA-MB-231 (mild MELK expression) and low uptake in MCF-7 (negative MELK expression). PET imaging revealed that MDA-MB-231 tumors could be clearly delineated in vivo, while low tracer uptake was observed in MCF-7 tumors. These findings were confirmed by ex vivo biodistribution studies and were consistent with the immunohistochemistry and tissue staining results. Tracer accumulation in MDA-MB-231 tumors was significantly inhibited by excess amounts of OTSSP167, indicating high specificity of the tracer. In summary, [18F]F-ET-OTSSP167, an easily-prepared probe, can be used to visualize MELK positive tumors, demonstrating its promising clinical potential in selecting patients for MELK inhibitor therapy.

Biomimetic oxygen delivery nanoparticles for enhancing photodynamic therapy in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a kind of aggressive breast cancer with a high rate of metastasis, poor overall survival time, and a low response to targeted therapies. To improve the therapeutic efficacy and overcome the drug resistance of TNBC treatments, here we developed the cancer cell membrane-coated oxygen delivery nanoprobe, CCm-HSA-ICG-PFTBA, which can improve the hypoxia at tumor sites and enhance the therapeutic efficacy of the photodynamic therapy (PDT), resulting in relieving the tumor growth in TNBC xenografts. RESULTS: The size of the CCm-HSA-ICG-PFTBA was 131.3 ± 1.08 nm. The in vitro 1O2 and ROS concentrations of the CCm-HSA-ICG-PFTBA group were both significantly higher than those of the other groups (P < 0.001). In vivo fluorescence imaging revealed that the best time window was at 24 h post-injection of the CCm-HSA-ICG-PFTBA. Both in vivo 18F-FMISO PET imaging and ex vivo immunofluorescence staining results exhibited that the tumor hypoxia was significantly improved at 24 h post-injection of the CCm-HSA-ICG-PFTBA. For in vivo PDT treatment, the tumor volume and weight of the CCm-HSA-ICG-PFTBA with NIR group were both the smallest among all the groups and significantly decreased compared to the untreated group (P < 0.01). No obvious biotoxicity was observed by the injection of CCm-HSA-ICG-PFTBA till 14 days. CONCLUSIONS: By using the high oxygen solubility of perfluorocarbon (PFC) and the homologous targeting ability of cancer cell membranes, CCm-HSA-ICG-PFTBA can target tumor tissues, mitigate the hypoxia of the tumor microenvironment, and enhance the PDT efficacy in TNBC xenografts. Furthermore, the HSA, ICG, and PFC are all FDA-approved materials, which render the nanoparticles highly biocompatible and enhance the potential for clinical translation in the treatment of TNBC patients.

Application and Evaluation of [99mTc]-Labeled Peptide Nucleic Acid Targeting MicroRNA-155 in Breast Cancer Imaging.

It has been reported that dysregulation of microRNA-155 expression and function is associated with tumorigenesis, growth, tumor subtypes, invasion, and poor survival rates. Peptide nucleic acid (PNA), an artificially synthesized nucleic acid mimic, has been applied for molecular diagnosis. In this study, a PNA sequence that undergoes complementary binding to miR-155 was labeled with 99mTc to evaluate whether the tracer could visualize the expression of miR-155 in breast cancer. Both antisense PNA (anti-PNA, fully complementary bound to human mature miR-155, referred to as "anti-PNA-155") and mismatched PNA (referred to as "mis-PNA") single strands containing 23-mer were synthesized. The relative expression of miR-155 in MCF-7 cells and tumors was higher than that in MDA-MB-231 cells and tumors. Single-photon emission computed tomography (SPECT) scan showed that radioactivity mainly accumulated in kidney. MCF-7 tumors, but not MDA-MB-231 tumors, were clearly visualized after [99mTc]anti-PNA-155 injection. MCF-7 tumors were less visible when coinjected with 100-fold excess of anti-PNA-155 or injected with [99mTc]mis-PNA, which suggested specific binding. Biodistribution study results were consistent with SPECT imaging. We successfully demonstrated that [99mTc]anti-PNA-155 could visualize miR-155 expression in vivo, suggesting it may be a promising probe applied in breast cancer.

Comparison of Al18F- and 68Ga-labeled NOTA-PEG4-LLP2A for PET imaging of very late antigen-4 in melanoma.

Malignant melanoma is an aggressive cancer with poor prognosis. Very late antigen-4 (VLA-4) is overexpressed in melanoma and many other tumors, making it an attractive target for developing molecular diagnostic and therapeutic agents. We compared Al18F- and 68Ga-labeled LLP2A peptides for PET imaging of VLA-4 expression in melanoma. The peptidomimetic ligand LLP2A was modified with chelator 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA), and the resulting NOTA-PEG4-LLP2A peptide was then radiolabeled with Al18F or 68Ga. The two labeled peptides were assayed for in vitro and in vivo VLA-4 targeting efficiency. Good Al18F and 68Ga radiolabeling yields were achieved, and the resulting PET tracers showed good serum stability. In the in vivo evaluation of the B16F10 xenograft mouse model, both tracers exhibited high accumulation with good contrast in static PET images. Compared with 68Ga-NOTA-PEG4-LLP2A, Al18F-NOTA-PEG4-LLP2A resulted in relatively higher background, including higher liver uptake (1 h: 20.1 ± 2.6 vs. 15.3 ± 1.7%ID/g, P < 0.05; 2 h: 11.0 ± 1.2 vs. 8.0 ± 0.8%ID/g, P < 0.05) and lower tumor-to-blood ratios (2.5 ± 0.4 vs. 3.3 ± 0.5 at 1 h, P < 0.05; 5.1 ± 0.9 vs. 7.3 ± 0.6 at 2 h, P < 0.01) at some time points. The results obtained from the mice blocked with unlabeled peptides and VLA-4-negative A375 xenografts groups confirmed the high specificity of the developed tracers. Despite the relatively high liver uptake, both Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-PEG4-LLP2A exhibited high VLA-4 targeting efficacy with comparable in vivo performance, rendering them promising candidates for imaging tumors that overexpress VLA-4.

Synthesis and Preclinical Evaluation of a 68Ga-Radiolabeled Peptide Targeting Very Late Antigen-3 for PET Imaging of Pancreatic Cancer.

Pancreatic cancer is highly malignant and has a five-year survival rate of 5% due to an early lymph node, nerve, and vascular metastasis. Integrin α3ß1 (also called very late antigen-3, VLA-3) is overexpressed in many tumors and plays a vital role in tumor formation, recurrence, and metastasis. In this study, we developed a 68Ga-radiolabeled peptide tracer targeting the α3 unit of VLA-3 and evaluated its potential application in positron emission computed tomography (PET) imaging of pancreatic cancer. NOTA-CK11 was prepared by solid-phase synthesis and successfully radiolabeled with 68Ga with greater than 99% radiochemical purity and a specific activity of 37 ± 5 MBq/nmol (n = 5). The expression level of integrin α3 in three human pancreatic cancer cells was evaluated with the order of SW1990, BXPC-3, and PANC-1 from high to low, while the expression level of integrin ß1 was relatively close. When SW1990 cells with the highest expression level of VLA-3 were stained with FITC-CK11, strong fluorescence was observed by flow cytometry and under a laser confocal microscope. However, no significant fluorescence was observed in the blocking group when treated with excessive CK11. 68Ga-NOTA-CK11 showed significant radioactivity accumulation in SW1990 cells and was blocked by CK11 successfully. Subsequent small-animal PET imaging and biodistribution studies in mice bearing SW1990 xenografts confirmed its high tumor uptake with a good tumor-to-blood ratio and tumor-to-muscle ratio (2.45 ± 0.31 and 3.65 ± 0.33, respectively) at 1 h post injection of the probe. In summary, we successfully developed a peptide-based imaging agent, 68Ga-NOTA-CK11, that showed a strong binding affinity with VLA-3 and good target specificity for SW1990 cells and xenografted pancreatic tumor, rending it a promising radiotracer for PET imaging of VLA-3 expression in pancreatic cancer.

Evaluation of an Integrin αvß3 and Aminopeptidase N Dual-Receptor Targeting Tracer for Breast Cancer Imaging.

Integrin αvß3 and aminopeptidase N (APN, also known as CD13) are two important targets involved in the regulation of angiogenesis, tumor proliferation, invasion, and metastasis. In this study, we developed a heterodimeric tracer consisting of arginine-glycine-aspartic (RGD) and asparagine-glycine-arginine (NGR) peptides targeting αvß3 and CD13, respectively, for PET imaging of breast cancer. The NGR peptide was first modified with N3-NOtB2 and then conjugated to BCN-PEG4-c(RGDyK) via copper-free click chemistry. The resulting precursor was purified and radiolabeled with gallium-68. Small-animal PET/CT imaging and post-imaging biodistribution studies were performed in mice bearing human breast cancer MCF-7, MDA-MB-231, MDA-MB-468, and MX-1 xenografts and pulmonary metastases models. The expression levels of αvß3 and CD13 in tumors were checked via immunochemical staining. The heterodimeric tracer was successfully synthesized and radiolabeled with gallium-68 at a molar activity of 45-100 MBq/nmol at the end of synthesis. It demonstrated high in vitro and in vivo stability. In static PET/CT imaging studies, the MCF-7 tumor could be clearly visualized and exhibited higher uptake at 30 min post injection of 68Ga-NGR-RGD than that of either 68Ga-RGD or 68Ga-NGR alone. High specificity was shown in blocking studies using Arg-Gly-Asp (RGD) and Asp-Gly-Arg (NGR) peptides. The MCF-7 tumor exhibited the highest uptake of 68Ga-NGR-RGD followed by MDA-MB-231, MDA-MB-468, and MX-1 tumors. This was consistent with their expression levels of CD13 and αvß3 as confirmed by western blot and immunohistochemical staining. Metastatic lesions in the lungs were clearly detectable on 68Ga-NGR-RGD PET/CT imaging in mouse models of pulmonary metastases. 68Ga-NGR-RGD, a CD13 and αvß3 dual-receptor targeting tracer, showed higher binding avidities, targeting efficiency, and longer tumor retention time compared with monomeric 68Ga-NGR and 68Ga-RGD. Its promising in vivo performance makes it an ideal candidate for future clinical translation.

Preclinical Evaluation of a Fluorine-18 (18F)-Labeled Phosphatidylinositol 3-Kinase Inhibitor for Breast Cancer Imaging.

Breast cancer is one of the commonest malignancies in women, especially in middle-aged and elderly women. Abnormal activation of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKt/mTOR) pathway has been found to be involved in breast cancer proliferation. Pictilisib (GDC-0941) is a potent inhibitor of PI3K with high affinity and is undergoing phase 2 clinical trials. In this study, we aimed to develop a noninvasive PI3K radiotracer to help determine the mechanism of the PI3K/AKt/mTOR pathway to aid in diagnosis. We designed a new 18F-radiolabeled radiotracer based on the structure of pictilisib, to evaluate noninvasively abnormal activation of the PI3K/AKT/mTOR pathway. To increase the water solubility, and to decrease hepatobiliary and gastrointestinal uptake of the tracer, pictilisib was modified with triethylene glycol di(p-toluenesulfonate) (TsO-PEG3-OTs) to obtain TsO-PEG3-GDC-0941 as the precursor for 18F labeling. A nonradiolabeled reference compound [19F]-PEG3-GDC-0941 was also prepared. Breast cancer cell lines, MCF-7 and MDA-MB-231, were used as high- and low-expression PI3K models, respectively. PET imaging and ex vivo biodistribution assays of [18F]-PEG3-GDC-0941 in MCF-7 and MDA-MB-231 xenografts were also performed, and the results were compared. The precursor compound and reference standard compound were successfully synthesized and identified using NMR and mass spectroscopy. The 18F radiolabeling was achieved with a high yield (61 ± 1%) at a high molar activity (2100 ± 100 MBq/mg). MicroPET images and biodistribution studies showed a higher uptake of the radiotracer in MCF-7 tumors than in MDA-MB-231 tumors (7.56 ± 1.01%ID/g vs 4.07 ± 0.68%ID/g, 1 h postinjection). Additionally, the MCF-7 tumor uptake was significantly decreased when a blocking dose of GDC-0941 was coinjected, indicating high specificity. The liver was found to be the major excretory organ with 5.82 ± 0.88%ID/g at 30 min postinjection for MCF-7 mice. This radiotracer holds great potential for patient screening, diagnosis, and therapy prediction of PI3K-related diseases.

Synthesis and Preclinical Evaluation of 18F-PEG3-FPN for the Detection of Metastatic Pigmented Melanoma.

Although 18F-5-fluoro-N-(2-[diethylamino]ethyl)picolinamide (18F-5-FPN) is considered a promising radiopharmaceutical for PET imaging of melanoma, it accumulates at high concentrations in the liver. The aim in this research was to optimize the structure of 18F-5-FPN with triethylene glycol to reduce liver uptake as well as improve pharmacokinetics, and to evaluate its performance in detection of melanoma liver and lung metastases. 18F-PEG3-FPN was successfully prepared with a high radiolabeling yield (44.68% ± 5.99%) and radiochemical purity (>99%). The uptake of 18F-PEG3-FPN by pigmented B16F10 melanoma cells was significantly higher than that by amelanotic melanoma A375 cells. The binding to B16F10 cells could be blocked by excess 19F-PEG3-FPN. On small animal PET images, B16F10 tumors, but not A375 tumors, were clearly delineated after 18F-PEG3-FPN injection. More importantly, 18F-PEG3-FPN uptake by liver (2.27 ± 0.45 and 1.74 ± 0.35% ID/g, at 1 and 2 h) was significantly lower than that of 18F-5-FPN, and the lesions in lung and liver could be clearly detected by 18F-PEG3-FPN PET imaging in mouse models of pulmonary or hepatic metastases. Overall, we successfully synthesized 18F-PEG3-FPN, which has higher labeling efficacy and better in vivo pharmacokinetics along with lower liver uptake compared to 18F-5-FPN. This suggests 18F-PEG3-FPN as a candidate for pigmented melanoma liver and lung metastasis detection.

Imaging malignant melanoma with (18)F-5-FPN.

PURPOSE: Radiolabelled benzamides are attractive candidates for targeting melanoma because they bind to melanin and exhibit high tumour uptake and retention. (18)F-5-Fluoro-N-(2-[diethylamino]ethyl)picolinamide ((18)F-5-FPN), a benzamide analogue, was prepared and its pharmacokinetics and binding affinity evaluated both in vitro and in vivo to assess its clinical potential in the diagnosis and staging of melanoma. METHODS: (18)F-5-FPN was prepared and purified. Its binding specificity was measured in vitro in two different melanoma cell lines, one pigmented (B16F10 cells) and one nonpigmented (A375m cells), and in vivo in mice xenografted with the same cell lines. Dynamic and static PET images using (18)F-5-FPN were obtained in the tumour-bearing mice, and the static images were also compared with those acquired with (18)F-FDG. PET imaging with (18)F-5-FPN was also performed in B16F10 tumour-bearing mice with lung metastases. RESULTS: (18)F-5-FPN was successfully prepared with radiochemical yields of 5 - 10 %. Binding of (18)F-5-FPN to B16F10 cells was much higher than to A375m cells. On dynamic PET imaging B16F10 tumours were visible about 1 min after injection of the tracer, and the uptake gradually increased over time. (18)F-5-FPN was rapidly excreted via the kidneys. B16F10 tumours were clearly visible on static images acquired 1 and 2 h after injection, with high uptake values of 24.34 ± 6.32 %ID/g and 16.63 ± 5.41 %ID/g, respectively, in the biodistribution study (five mice). However, there was no visible uptake by A375m tumours. (18)F-5-FPN and (18)F-FDG PET imaging were compared in B16F10 tumour xenografts, and the tumour-to-background ratio of (18)F-5-FPN was ten times higher than that of (18)F-FDG (35.22 ± 7.02 vs. 3.29 ± 0.53, five mice). (18)F-5-FPN PET imaging also detected simulated lung metastases measuring 1 - 2 mm. CONCLUSION: (18)F-5-FPN specifically targeted melanin in vitro and in vivo with high retention and affinity and favourable pharmacokinetics. (18)F-5-FPN may be an ideal molecular probe for melanoma diagnosis and staging.