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1.  18F-Alfatide II and 18F-FDG Dual Tracer Dynamic PET for Parametric, Early Prediction of Tumor Response to Therapy 
A single dynamic PET acquisition using multiple tracers administered closely in time could provide valuable complementary information about a tumor’s status under quasi-constant conditions. This study aims to investigate the utility of dual-tracer dynamic PET imaging with 18F-Alfatide II (18F-AlF-NOTA-E[PEG4-c(RGDfk)]2) and 18F-FDG for parametric monitoring of tumor responses to therapy.
We administered doxorubicin to one group of athymic nude mice with U87MG tumors and Abraxane to another group of mice with MDA-MB-435 tumors. To monitor therapeutic responses, we performed dual-tracer dynamic imaging, in sessions that lasted 90 min, starting by injecting the mice via tail vein catheters with 18F-Alfatide II, followed 40 minutes later by 18F-FDG. To achieve signal separation of the two tracers, we fit a three-compartment reversible model to the time activity curve (TAC) of 18F-Alfatide II for the 40 min prior to 18F-FDG injection, and then extrapolated to 90 min. The 18F-FDG tumor TAC was isolated from the 90 min dual tracer tumor TAC by subtracting the fitted 18F-Alfatide II tumor TAC. With separated tumor TACs, the 18F-Alfatide II binding potential (Bp=k3/k4) and volume of distribution (VD), and 18F-FDG influx rate ((K1×k3)/(k2 + k3)) based on the Patlak method were calculated to validate the signal recovery in a comparison with 60-min single tracer imaging and to monitor therapeutic response.
The transport and binding rate parameters K1-k3 of 18F-Alfatide II, calculated from the first 40 min of dual tracer dynamic scan, as well as Bp and VD, correlated well with the parameters from the 60 min single tracer scan (R2 > 0.95). Compared with the results of single tracer PET imaging, FDG tumor uptake and influx were recovered well from dual tracer imaging. Upon doxorubicin treatment, while no significant changes in static tracer uptake values of 18F-Alfatide II or 18F-FDG were observed, both 18F-Alfatide II Bp and 18F-FDG influx from kinetic analysis in tumors showed significant decreases. For Abraxane therapy of MDA-MB-435 tumors, significant decrease was only observed with 18F-Alfatide II Bp value from kinetic analysis but not 18F-FDG influx.
The parameters fitted with compartmental modeling from the dual tracer dynamic imaging are consistent with those from single tracer imaging, substantiating the feasibility of this methodology. Even though no significant differences in tumor size were found until 5 days after doxorubicin treatment started, at day 3 there were already substantial differences in 18F-Alfatide II Bp and 18F-FDG influx rate. Dual tracer imaging can measure 18F-Alfatide II Bp value and 18F-FDG influx simultaneously to evaluate tumor angiogenesis and metabolism. Such changes are known to precede anatomical changes, and thus parametric imaging may offer the promise of early prediction of therapy response.
PMCID: PMC4209961  PMID: 24232871
dual-tracer dynamic PET; parametric imaging; 18F-Alfatide II; 18F-FDG; therapy response
2.  A Novel In Vitro Assay to Assess Phosphorylation of 3′-[18F]fluoro-3′-Deoxythymidine 
3′-[18F]fluoro-3’-deoxythymidine ([18F]FLT) is phosphorylated by thymidine kinase 1 (TK-1), a cell cycle regulated enzyme. Appropriate use of [18F]FLT tracer requires validation of the TK-1 activity. Here, we report development of a novel phosphoryl-transfer assay to assess phosphorylation of [18F]FLT both in tumor cell lysates and tumor cells.
The intrinsic F-18 radioactivity was used to quantify both substrate and phosphorylated products using a rapid thin layer chromatography method. Phosphorylation kinetics of [18F]FLT in SW480 and DiFi tumor cell lysates and cellular uptake were measured.
The apparent Michaelis–Menten kinetic parameters for [18F]FLT are Km = 4.8 ± 0.3 μM and Vmax=7.4 pmol min−1per 1×106 cells with ~2-fold higher TK-1 activity in DiFi versus SW480 lysates.
The apparent Km of [18F]FLT was comparable to the value reported with purified recombinant TK-1. The uptake of [18F]FLT by SW480 cells is inhibited by nitrobenzylthioinosine or dipyridamole indicating that uptake is mediated predominantly by the equilibrative nucleoside transporters in these tumor cells.
PMCID: PMC4180216  PMID: 20532643
3′-[18F]fluoro-3′-deoxythymidine ([18F]FLT); Thymidine kinase 1; Positron emission tomography (PET); PET tracers; Molecular imaging
3.  Differentially expressed genes and signalling pathways are involved in mouse osteoblast-like MC3T3-E1 cells exposed to 17-β estradiol 
Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation. In this study, complementary DNA (cDNA) microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells' response to 17-β estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha (α-MEM) cell culture supplemented with 17-β estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with 10−8 mol⋅L−1 17-β estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase (ALP) activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction (RT-PCR) to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes, of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology (GO) analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta (TGF-β)-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to α-MEM supplemented with 17-β estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.
PMCID: PMC4170150  PMID: 24556956
17-β estradiol; MC3T3-E1 cell; microarray; signal transduction
4.  The Effect of Injection Routes on the Biodistribution, Clearance and Tumor Uptake of Carbon Dots 
ACS nano  2013;7(7):5684-5693.
The emergence of photoluminescent carbon-based nanomaterials has shown exciting potential in the development of benign nanoprobes. However, the in vivo kinetic behaviors of these particles that are necessary for clinical translation are poorly understood to date. In this study, fluorescent carbon dots (C-dots) were synthesized and the effect of three injection routes on their fate in vivo was explored by using both near-infrared fluorescence (NIRF) and positron emission tomography (PET) imaging techniques. We found that C-dots are efficiently and rapidly excreted from the body after all three injection routes. The clearance rate of C-dots is ranked as: intravenous > intramuscular > subcutaneous. The particles had relatively low retention in the reticuloendothelial system (RES) and showed high tumor-to-background contrast. Furthermore, different injection routes also resulted in different blood clearance patterns and tumor uptakes of C-dots. These results satisfy the need for clinical translation and should promote efforts to further investigate the possibility of using carbon-based nanoprobes in a clinical setting. More broadly, we provide a testing blueprint for in vivo behavior of nanoplatforms under various injection routes, an important step forward towards safety and efficacy analysis of nanoparticles.
PMCID: PMC3725601  PMID: 23731122
Biodistribution; carbon dots; clearance; injection routes; translation; tumor uptake
5.  Anthocyanin biosynthetic genes in Brassica rapa 
BMC Genomics  2014;15(1):426.
Anthocyanins are a group of flavonoid compounds. As a group of important secondary metabolites, they perform several key biological functions in plants. Anthocyanins also play beneficial health roles as potentially protective factors against cancer and heart disease. To elucidate the anthocyanin biosynthetic pathway in Brassica rapa, we conducted comparative genomic analyses between Arabidopsis thaliana and B. rapa on a genome-wide level.
In total, we identified 73 genes in B. rapa as orthologs of 41 anthocyanin biosynthetic genes in A. thaliana. In B. rapa, the anthocyanin biosynthetic genes (ABGs) have expanded and most genes exist in more than one copy. The anthocyanin biosynthetic structural genes have expanded through whole genome and tandem duplication in B. rapa. More structural genes located upstream of the anthocyanin biosynthetic pathway have been retained than downstream. More negative regulatory genes are retained in the anthocyanin biosynthesis regulatory system of B. rapa.
These results will promote an understanding of the genetic mechanism of anthocyanin biosynthesis, as well as help the improvement of the nutritional quality of B. rapa through the breeding of high anthocyanin content varieties.
Electronic supplementary material
The online version of this article (doi: 10.1186/1471-2164-15-426) contains supplementary material, which is available to authorized users.
PMCID: PMC4072887  PMID: 24893600
Comparative genomics; Anthocyanin biosynthetic genes; Whole genome duplication; Brassica rapa; Cruciferae
6.  RING3 Kinase Transactivates Promoters of Cell Cycle Regulatory Genes through E2F1 
RING3 is a novel, nuclear-localized, serine-threonine kinase that has elevated activity in human leukemias. RING3 transforms NIH/3T3 cells and is activated by mitogenic signals, all of which suggest that it may play a role in cell cycle-responsive transcription. We tested this hypothesis with transient transfection of RING3 into fibroblasts and assayed transactivation of the promoters of cyclin D1, cyclin A, cyclin E, and dihydrofolate reductase (dhfr) genes. RING3 transactivates these promoters in a manner dependent on ras signaling. A kinase-deficient point mutant of RING3 does not transactivate. Mutational analysis of the dhfr promoter reveals that transactivation also depends on the presence of a functional E2F binding site. Furthermore, ectopic expression of Rb protein, a negative regulator of E2F activity, suppresses the RING3-dependent transactivation of this promoter. Consistent with a potential role of E2F in RING3-dependent transcription, anti-RING3 immunoaffinity chromatography or recombinant RING3 protein affinity chromatography of nuclear extracts copurified a protein complex that contains E2F-1 and E2F-2. These data suggest that RING3 is a potentially important regulator of E2F-dependent cell cycle genes.
PMCID: PMC3968681  PMID: 10965846
7.  Activation-induced nuclear translocation of RING3 
Journal of cell science  2000;113(0 17):3085-3091.
RING3 is a novel protein kinase linked to human leukaemia. Its Drosophila homologue female sterile homeotic is a developmental regulator that interacts genetically with trithorax, a human homologue of which is also associated with leukaemia. The RING3 structure contains two mutually related bromodomains that probably assist in the remodelling of chromatin and thereby affect transcription. Consistent with this hypothesis, a RING3-like protein has been identified in the mouse Mediator complex, where it is associated with transcription factors. We show that, whilst RING3 is constitutively localised to the nucleus of exponentially growing HeLa cells, it is delocalised throughout serumstarved fibroblasts. We use immunostaining and confocal microscopy to demonstrate that RING3 translocates to the fibroblast nucleus upon serum stimulation. After translocation, RING3 participates in nuclear protein complexes that include E2F proteins; it transactivates the promoters of several important mammalian cell cycle genes that are dependent on E2F, including dihydrofolate reductase, cyclin D1, cyclin A and cyclin E. We use site-directed mutagenesis of a putative nuclear localisation motif to show that the activation-induced nuclear localisation and consequent transcriptional activity of RING3 depends on a monopartite, classical nuclear localisation sequence. These observations refine and extend the mechanism by which RING3 contributes to E2Fregulated cell cycle progression. Deregulation of this mechanism may be leukaemogenic.
PMCID: PMC3936601  PMID: 10934046
RING3; Nuclear translocation; Immunolocalisation; Confocal microscopy; Leukaemia
8.  Longitudinal PET Imaging of Doxorubicin Induced Cell Death with 18F-Annexin V 
This study aims to apply longitudinal positron emission tomography (PET) imaging with 18F-Annexin V to visualize and evaluate cell death induced by doxorubicin in a human head and neck squamous cell cancer UM-SCC-22B tumor xenograft model.
In vitro toxicity of doxorubicin to UM-SCC-22B cells was determined by a colorimetric assay. Recombinant human Annexin V protein was expressed and purified. The protein was labeled with fluorescein isothiocyanate (FITC) for fluorescence staining and 18F for PET imaging. Established UM-SCC-22B tumors in nude mice were treated with two doses of doxorubicin (10 mg/kg each dose) with 1 day interval. Longitudinal 18F-Annexin V PET was performed at 6 h, 24 h, 3 days, and 7 days after the treatment started. Following PET imaging, direct tissue biodistribution study was performed to confirm the accuracy of PET quantification.
Two doses of doxorubicin effectively inhibited the growth of UM-SCC-22B tumors by inducing cell death including apoptosis. The cell death was clearly visualized by 18F-Annexin V PET. The peak tumor uptake, which was observed at day 3 after treatment started, was significantly higher than that in the untreated tumors (1.56 ± 0.23 vs. 0.89 ± 0.31 %ID/g, p < 0.05). Moreover, the tumor uptake could be blocked by co-injection of excess amount of unlabeled Annexin V protein. At day 7 after treatment, the tumor uptake of 18F-Annexin had returned to baseline level.
18F-Annexin V PET imaging is sensitive enough to allow visualization of doxorubicin induced cell death in UM-SCC-22B xenograft model. The longitudinal imaging with 18F-Annexin will be helpful to monitor early response to chemotherapeutic anti-cancer drugs.
PMCID: PMC3387344  PMID: 22392643
18F-Annexin V; doxorubicin; apoptosis; PET; chemotherapy
9.  Quantitative Analysis and Parametric Imaging of 18F-Labeled Monomeric and Dimeric RGD Peptides Using Compartment Model 
Non-invasive PET imaging with radiolabeled RGD peptides for αvβ3 integrin targeting has become an important tool for tumor diagnosis and treatment monitoring in both pre-clinical and clinical studies. To better understand the molecular process and tracer pharmacokinetics, we introduced kinetic modeling in the investigation of 18F-labeled RGD peptide monomer 18F-FP-c(RGDyK) (denoted as 18F-FPRGD) and dimer 18F-FP-PEG3-E[c(RGDyK)]2 (denoted as 18F-FPPRGD2).
MDA-MB-435 tumor-bearing mice underwent 60 min dynamic PET scans following the injection of either 18F-FPRGD or 18F-FPPRGD2. Blocking studies with pre-injection of a blocking mass dose were performed for both monomeric and dimeric RGD groups. 18F-FPRAD (RAD) was used as a negative control. Kinetic parameters (K1, k2, k3, k4) of a three-compartment model were fitted to the dynamic data to allow quantitative comparisons between the monomeric and dimeric RGD peptides.
Dimeric RGD peptide tracer showed significantly higher binding potential (BpND = k3/k4, 5.87 ± 0.31) than that of the monomeric analog (2.75 ± 0.48, p = 0.0022, n = 4/group). The BpND values showed a significantly greater ratio (dimer/monomer ~2.1) than the difference in %ID/g uptake measured from static images (dimer/monomer ~1.5, p = 0.0045). Significant decrease in BpND was found in the blocked groups compared with the unblocked ones (dimer p = 0.00024, monomer p = 0.005, n = 4/group). Similarly, the RAD control group showed the lowest BpND value among all the test groups, as the RAD peptide does not bind to integrin αvβ3. Volume of distribution (VT = K1/k2(1+k3/k4)) could be separated into non-specific (VND = K1/k2) and specific (VS = K1k3/(k2k4)) components. Specific distribution volume (VS) was the dominant component of VT in the unblocked groups and decreased in the blocked groups. Unblocked RGD dimer also showed higher VS than that of the monomer (dimer VS = 2.38 ± 0.15, monomer VS = 0.90 ± 0.17, p = 0.0013, n = 4/group), well correlated with BpND calculations. Little difference in VND was found among all groups. Moreover, parametric maps allowed quantitative analysis at voxel level and provided higher tumor-to-background contrast for BpND maps than the static images. Tumor heterogeneity in kinetic parameters was found in parametric images, which couldn’t be clearly identified in static intensity images.
The pharmacokinetics of both monomeric and dimeric RGD peptide tracers was compared, and the RGD dimers showed significantly higher binding affinity than the monomeric analogs. Kinetic parameters were demonstrated to be valuable for separating specific and non-specific binding and may allow more sensitive and detailed quantification than simple standard uptake value (SUV) analysis.
PMCID: PMC3401513  PMID: 22437879
Positron Emission Tomography; kinetic modeling; quantitative analysis; RGD peptide; Integrin
10.  N-Succinimidyl 4-[18F]-fluoromethylbenzoate-labeled dimeric RGD peptide for imaging tumor integrin expression 
Amino acids  2011;43(3):1349-1357.
RGD peptides, radiolabeled with 18F, have been used in the clinic for PET imaging of tumor angiogenesis in cancer patients. RGD peptides are typically labeled using a prosthetic group such as N-succinimidyl 4-[18F]-fluorobenzoate ([18F]SFB) or 4-nitrophenyl 2-[18F]-fluoropropionate ([18F]NPFP). However, the complex radiosynthetic procedures have impeded their broad application in clinical studies. We previously radiolabeled proteins and peptides with the prosthetic group, N-succinimidyl 4-[18F]-fluoromethylbenzoate ([18F]SFMB), which was prepared in a simple one-step procedure. In this study, we labeled a PEGylated cyclic RGD peptide dimer, PEG3-E[c(RGDy K)]2 (PRGD2), using [18F]SFMB and evaluated for imaging tumor αvβ3 integrin expression with positron emission tomography (PET). [18F]SFMB was prepared in one step using [18F]fluoride displacement of a nitrobenzenesulfonate leaving group under mild reaction conditions followed by HPLC purification. The 18F-labeled peptide, [18F]FMBPR GD2 was prepared by coupling PRGD2 with [18F]SFMB in pH 8.6 borate buffer and purified with HPLC. The direct labeling on BMBPRGD2 was also attempted. A Siemens Inveon PET was used to image the uptake of the [18F]FMBPRGD2 into a U87MG xenograft mouse model. [18F]FMBPRGD2, was prepared with a 15% overall radiochemical yield (uncorrected) in a total synthesis time of 90 min, which was considerably shorter than the preparation of [18F]SFB- and [18F]NPFP-labeled RGD peptides. The direct labeling, however, was not successful. High quality microPET images using [18F]FMBPRGD2 clearly visualized tumors by 15 min with good target to background ratio. Early tracer accumulation in the bladder suggests fast renal clearance. No obvious bone uptake can be detected even at 4-h time point indicating that fluorine attachment is stable in mice. In conclusion, N-succinimidyl 4-[18F]-fluoromethylbenzoate ([18F]SFMB) prosthetic group can be a good alternative for labeling RGD peptides to image αvβ3 integrin expression and for labeling other peptides.
PMCID: PMC3577934  PMID: 22209865
Integrin αvβ3; RGD peptide dimer; Positron emission tomography; N-Succinimidyl 4-[18F]-fluoromethylbenzoate ([18F]SFMB)
11.  High Platelet Reactivity on Clopidogrel Therapy Correlates With Increased Coronary Atherosclerosis and Calcification: A Volumetric Intravascular Ultrasound Study 
JACC. Cardiovascular imaging  2012;5(5):540-549.
To evaluate the relationship between platelet reactivity and atherosclerotic burden in patients undergoing percutaneous coronary intervention (PCI) with pre-intervention volumetric intravascular ultrasound (IVUS) imaging.
Atherosclerosis progresses by the pathologic sequence of sub-clinical plaque rupture, thrombosis and healing. In this setting, increased platelet reactivity may lead to more extensive arterial thrombosis at the time of plaque rupture, leading to a more rapid progression of the disease. Alternatively, abnormal vessel wall biology with advanced atherosclerosis is known to enhance platelet reactivity. Therefore, it is possible that by either mechanism, increased platelet reactivity may be associated with greater atherosclerotic burden.
We analyzed patients who underwent PCI with pre-intervention IVUS imaging and platelet reactivity functional assay (P2Y12 reaction-units [PRU]) performed >16 hours post-PCI after stabilization of clopidogrel therapy (administered pre-PCI). A PRU value of >230 defined high on-treatment platelet reactivity (HPR).
Among 335 patients (mean age 65.0; 71% male), there were 109 patients with HPR (32.5%) and 226 without HPR (67.5%), with HPR being associated with diabetes and chronic renal insufficiency. By IVUS analysis, HPR patients had significantly greater target lesion calcium length, calcium arc, and calcium index. Furthermore, HPR patients tended to have longer lesions and greater volumetric dimensions, indicating higher plaque volume, larger total vessel volume and also greater lumen volume, despite similar plaque burden. By multivariable analysis controlling for baseline clinical variables, HPR was the single consistent predictor of all IVUS parameters examined, including plaque volume, calcium length and calcium arc.
Increased platelet reactivity on clopidogrel treatment, as defined by a PRU value of >230, is associated with greater coronary artery atherosclerotic disease burden and plaque calcification.
PMCID: PMC3753810  PMID: 22595163
atherosclerosis; clopidogrel; plaque progression; platelets; platelet reactivity
12.  Molecular Targeting of CEACAM6 Using Antibody Probes of Different Sizes 
Journal of Controlled Release  2012;161(1):18-24.
Carcinocinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is overexpressed in a number of human malignancies, especially in pancreatic cancer. It has been demonstrated that CEACAM6 is a potential target for monoclonal antibody (mAb) therapy with a safe therapeutic index. Here, we labeled three anti-CEACAM6 antibodies of different sizes, including a single-domain antibody 2A3 (16 kDa), a heavy chain antibody 2A3-mFc (80 kDa) and a full length antibody 9A6 (150 kDa), with 64Cu to image CEACAM6 expression in a xenografted pancreatic tumor model. For positron emission tomography (PET) imaging, the tumor mice were intravenously injected with 64Cu-DOTA-antibodies and static scans were obtained at 5 min, 0.5, 1, 2, 4, 8 and 24 h post-injection (p.i.). All three antibodies showed strong CEACAM6 binding. Ex vivo immunostaining on tumor sections at 24 h after Ab injection demonstrated specific tumor targeting of both 2A3-mFc and 9A6. 64Cu-DOTA-2A3 showed fast BxPC3 tumor uptake and rapid whole-body clearance. At 24 h p.i., the tumor uptakes were 98.2 ± 6.12 %ID/g for 64Cu-DOTA-2A3-mFc and 57.8 ± 3.73 %ID/g for 64Cu-DOTA-9A6, respectively. Compared with the full length antibody 9A6, the heavy chain antibody 2A3-mFc showed higher tumor uptake, lower liver uptake and shorter circulation half-life. All the data supported that the heavy chain antibody 2A3-mFc is superior to the single domain antibody and the full-length antibody with regard to tumor detection and pharmacokinetics, which has great potential to be developed for CEACAM6-targeted pancreatic cancer imaging and therapy.
PMCID: PMC3378815  PMID: 22568933
CEACAM6; pancreatic cancer; heavy chain antibody; 64Cu; positron emission tomography (PET)
13.  Noninvasive monitoring of orthotopic glioblastoma therapy response using RGD-conjugated iron oxide nanoparticles 
Biomaterials  2012;33(21):5414-5422.
Noninvasive imaging techniques have been considered important strategies in the clinic to monitor tumor early response to therapy. In the present study, we applied RGD peptides conjugated to iron oxide nanoparticles (IONP-RGD) as contrast agents in magnetic resonance imaging (MRI) to noninvasively monitor the response of a vascular disrupting agent VEGF121/rGel in an orthotopic glioblastoma model. RGD peptides were firstly coupled to IONPs coated with a crosslinked PEGylated amphiphilic triblock copolymer. In vitro binding assays confirmed that cellular uptake of particles was mainly dependent on the interaction between RGD and integrin αvβ3 of human umbilical vein endothelial cells (HUVEC). The tumor targeting of IONP-RGD was observed in an orthotopic U87 glioblastoma model. Finally, noninvasive monitoring of the tumor response to VEGF121/rGel therapy at early stages of treatment was successfully accomplished using IONP-RGD as a contrast agent for MRI, a superior method over common anatomical approaches which are based on tumor size measurements. This preclinical study can accelerate anticancer drug development and promote clinical translation of nanoprobes.
PMCID: PMC3577933  PMID: 22560667
Magnetic resonance imaging (MRI); Iron oxide nanoparticles (IONPs); RGD peptides; Tumor targeting; Therapy response
14.  First Experience of 18F-Alfatide in Lung Cancer Patients Using a New Lyophilized Kit for Rapid Radiofluorination 
18F-FPPRGD2, which was approved for clinical study recently, has favorable properties for integrin targeting and showed potential for antiangiogenic therapy and early response monitoring. However, the time-consuming multiple-step synthesis may limit its widespread applications in the clinic. In this study, we developed a simple lyophilized kit for labeling PRGD2 peptide (18F-AlF-NOTA-PRGD2, denoted as 18F-alfatide) using a fluo-ride–aluminum complex that significantly simplified the labeling procedure.
Nine patients with a primary diagnosis of lung cancer were examined by both static and dynamic PET imaging with 18F-alfatide, and 1 tuberculosis patient was investigated using both 18F-alfatide and 18F-FDG imaging. Standardized uptake values were measured in tumors and other main organs at 30 min and 1 h after injection. Kinetic parameters were calculated by Logan graphical analysis. Immunohisto-chemistry and staining intensity quantification were performed to confirm the expression of integrin αvβ3.
Under the optimal conditions, the whole radiosynthesis including purifica-tion was accomplished within 20 min with a decay-corrected yield of 42.1% ± 2.0% and radiochemical purity of more than 95%. 18F-alfatide PET imaging identified all tumors, with mean standardized uptake values of 2.90 ± 0.10. Tumor-to-muscle and tumor-to-blood ratios were 5.87 ± 2.02 and 2.71 ± 0.92, respectively.
18F-alfatide can be produced with excellent radiochemical yield and purity via a simple, 1-step, lyophilized kit. PET scanning with 18F-alfatide allows specific imaging of αvβ3 expression with good contrast in lung cancer patients. This technique might be used for the assessment of angiogene-sis and for planning and response evaluation of cancer therapies that would affect angiogenesis status and integrin expression levels.
PMCID: PMC3683452  PMID: 23554506
RGD peptide; alfatide; aluminum fluoride; PET; lung cancer
15.  18F-radiolabeled analogs of exendin-4 for PET imaging of GLP-1 in insulinoma 
Glucagon-like peptide type 1 (GLP-1) is an incretin peptide that augments glucose-stimulated insulin release following oral consumption of nutrients. Its message is transmitted via a G protein-coupled receptor called GLP-1R, which is colocalized with pancreatic β-cells. The GLP-1 system is responsible for enhancing insulin release, inhibiting glucagon production, inhibiting hepatic gluconeogenesis, inhibiting gastric mobility, and suppression of appetite. The abundance of GLP-1R in pancreatic β-cells in insulinoma, a cancer of the pancreas, and the activity of GLP-1 in the cardiovascular system have made GLP-1R a target for molecular imaging.
We prepared 18F radioligands for GLP-1R by the reaction of [18F]FBEM, a maleimide prosthetic group, with [Cys0] and [Cys40] analogs of exendin-4. The binding affinity, cellular uptake and internalization, in vitro stability, and uptake and specificity of uptake of the resulting compounds were determined in an INS-1 xenograft model in nude mice.
The [18F]FBEM-[Cysx]-exendin-4 analogs were obtained in good yield (34.3±3.4%, n=11), based on the starting compound [18F]FBEM), and had a specific activity of 45.51±16.28 GBq/μmol (1.23±0.44 Ci/μmol, n=7) at the end of synthesis. The C-terminal isomer, [18F]FBEM-[Cys40]-exendin-4, had higher affinity for INS-1 tumor cells (IC50 1.11±0.057 nM) and higher tumor uptake (25.25±3.39 %ID/g at 1 h) than the N-terminal isomer, [18F]FBEM-[Cys0]-exendin-4 (IC50 2.99±0.06 nM, uptake 7.20±1.26 %ID/g at 1 h). Uptake of both isomers into INS-1 tumor, pancreas, stomach, and lung could be blocked by preinjection of nonradiolabeled [Cysx]-exendin-4 (p<0.05).
[18F]FBEM-[Cys40]-exendin-4 and [18F]FBEM-[Cys0]-exendin-4 have high affinity for GLP-1R and display similar in vitro cell internalization. The higher uptake into INS-1 xenograft tumors exhibited by [18F]FBEM-[Cys40]-exendin-4 suggests that this compound would be the better tracer for imaging GLP-1R.
PMCID: PMC3617488  PMID: 22170321
Exendin-4; GLP-1R; Insulinoma; 18F; PET
16.  Polyaspartic acid coated manganese oxide nanoparticles for efficient liver MRI† 
Nanoscale  2011;3(12):4943-4945.
We report in this communication a simple, facile surface modification strategy to transfer hydrophobic manganese oxide nanoparticles (MONPs) into water by using polyaspartic acid (PASP). We systematically investigated the effect of the size of PASP-MONPs on MRI of normal liver and found that the particles with a core size of 10 nm exhibited greater enhancement than those with larger core sizes.
PMCID: PMC3617494  PMID: 22064945
17.  PET Imaging of Angiogenesis after Myocardial Infarction/Reperfusion using a One-Step Labeled Integrin Targeted Tracer 18F-AlF-NOTA-PRGD2 
The αvβ3 integrin represents a potential target for noninvasive imaging of angiogenesis. The purpose of this study was to evaluate a novel one-step labeled integrin αvβ3 targeting PET probe, 18F-AlF-NOTA-PRGD2, for angiogenesis imaging in a myocardial infract/reperfusion (MI/R) animal model.
Male SD rats underwent 45 min transient left coronary artery occlusion followed by reperfusion. The myocardial infarction was confirmed by ECG, 18F-FDG imaging and cardiac ultrasound. In vivo PET imaging were used to determine myocardial uptake of 18F-AlF-NOTA-PRGD2 at different time points following reperfusion. The control peptide RAD was labeled with a similar procedure and used to confirm the specificity. Ex vivo autoradiographic analysis and CD31/CD61 double immunofluoresence staining were performed to validate the PET results.
Myocardial origin of the 18F-AlF-NOTA-PRGD2 accumulation was confirmed by 18F-FDG and autoradiography. PET imaging demonstrated increased focal accumulation of 18F-AlF-NOTA-PRGD2 in the infarcted area started at day 3 (0.28 ± 0.03 %ID/g, p < 0.05), peaked between 1 and 3 weeks (0.59 ± 0.16 and 0.55 ± 0.13 %ID/g, respectively). The focal accumulation decreased but still kept at a higher level than the sham group after 4 months of reperfusion (0.31 ± 0.01 %ID/g, p < 0.05). Pretreatment with unlabeled RGD peptide significantly decreased tracer uptake, indicating integrin specificity of this tracer. At 1 week after MI/R, uptake of the control tracer 18F-AlF-NOTA-RAD that does not bind to integrin, in the infarcted area, was only 0.21 ± 0.01 %ID/g. Autoradiographic imaging showed the same trend of uptake in myocardial infarction area. The time course of focal tracer uptake was consistent with the pattern of vascular density and integrin β3 expression as measured by CD31 and CD61 immunostaining analysis.
PET imaging using one-step labeled 18F-AlF-NOTA-PRGD2 allows noninvasive visualization of ischemia-reperfusion induced myocardial angiogenesis longitudinally. The favorable in vivo kinetics and easy production method of this integrin targeted PET tracer facilitates its future clinical translation for lesion evaluation and therapy response monitoring in patients with occlusive cardiovascular diseases.
PMCID: PMC3319105  PMID: 22274731
PET imaging; angiogenesis; myocardial infarction; integrin; RGD peptide
18.  PET imaging of EGF receptors using [18F]FBEM-EGF in a Head and Neck Squamous Cell Carcinoma model 
To prepare and evaluate a new radiotracer for molecular imaging of cell surface receptors for epidermal growth factor (EGF).
Cys tagged EGF (cEGF) was labeled with 18F by coupling the free thiol group of the Cys tag with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM) to form [18F]FBEM-cEGF. Cell uptake, internalization and efflux of [18F]FBEM-cEGF were tested in human head and neck squamous carcinoma UM-SCC1 cells. In vivo tumor targeting and pharmacokinetics of the radiotracers were evaluated in UM-SCC1 tumor-bearing athymic nude mice by static and dynamic microPET imaging. Ex vivo biodistribution assays were performed to confirm the noninvasive imaging results.
The radiolabeling yield for [18F]FBEM-cEGF was over 60%, based on starting [18F]FBEM. [18F]FBEM-cEGF exhibited rapid blood clearance through both hepatobiliary and renal excretion. UM-SCC1 tumors were clearly visualized and showed modest tracer uptake of 2.60 ± 0.59 %ID/g at 30 min post injection. Significantly higher tumor uptake of [18F]FBEM-cEGF (5.99 ± 1.61 %ID/g at 30 min p.i., p < 0.01) and tumor/non-tumor ratio were achieved by co-injection of 50 μg of unlabeled EGF. Decreased liver uptake of [18F]FBEM-cEGF was observed when unlabeled EGF was co-administered.
With optimized liver blocking, [18F]FBEM-cEGF has the potential to be used in a non-invasive and quantitative manner for detection of malignant lesions and evaluation of EGFR activity.
PMCID: PMC3262064  PMID: 22109665
19.  All-Trans-Retinoic Acid Modulates ICAM-1 N-Glycan Composition by Influencing GnT-III Levels and Inhibits Cell Adhesion and Trans-Endothelial Migration 
PLoS ONE  2012;7(12):e52975.
Changes in the expression of glycosyltransferases directly influence the oligosaccharide structures and conformations of cell surface glycoproteins and consequently cellular phenotype transitions and biological behaviors. In the present study, we show that all-trans-retinoic acid (ATRA) modulates the N-glycan composition of intercellular adhesion molecule-1 (ICAM-1) by manipulating the expression of two N-acetylglucosaminyltransferases, GnT-III and GnT-V, via the ERK signaling pathway. Exposure of various cells to ATRA caused a remarkable gel mobility down-shift of ICAM-1. Treatment with PNGase F confirmed that the reduction of the ICAM-1 molecular mass is attributed to the decreased complexity of N-glycans. We noticed that the expression of the mRNA encoding GnT-III, which stops branching, was significantly enhanced following ATRA exposure. In contrast, the level of the mRNA encoding GnT-V, which promotes branching, was reduced following ATRA exposure. Silencing of GnT-III prevented the molecular mass shift of ICAM-1. Moreover, ATRA induction greatly inhibited the adhesion of SW480 and U937 cells to the HUVEC monolayer, whereas knock-down of GnT-III expression effectively restored cell adhesion function. Treatment with ATRA also dramatically reduced the trans-endothelial migration of U937 cells. These data indicate that the alteration of ICAM-1 N-glycan composition by ATRA-induced GnT-III activities hindered cell adhesion and cell migration functions simultaneously, pinpointing a unique regulatory role of specific glycosyltransferases in the biological behaviors of tumor cells and a novel function of ATRA in the modulation of ICAM-1 N-glycan composition.
PMCID: PMC3530489  PMID: 23300837
20.  Comparison Study of [18F]FAl-NOTA-PRGD2, [18F]FPPRGD2 and [68Ga]Ga-NOTA-PRGD2 for PET Imaging of U87MG Tumors in Mice 
Bioconjugate chemistry  2011;22(12):2415-2422.
[18F]FPPRGD2, an F-18 labeled dimeric cyclic RGDyK peptide, has favorable properties for PET imaging of angiogenesis by targeting the αvβ3 integrin receptor. This radiotracer has been approved by the FDA for use in clinical trials. However, the time-consuming multiple-step synthetic procedure required for its preparation may hinder the widespread usage of this tracer. The recent development of a method using an F-18 fluoride-aluminum complex to radiolabel peptides provides a strategy for simplifying the labeling procedure. On the other hand, the easy-to-prepare [68Ga]-labeled NOTA-RGD derivatives have also been reported to have promising properties for imaging αvβ3 integrin receptors. The purpose of this study was to prepare [18F]FPPRDG2, [18F]FAl-NOTA-PRGD2, and [68Ga]Ga-NOTA-PRGD2 and to compare their pharmacokinetics and tumor imaging properties using small animal PET. All three compounds showed rapid and high tracer uptake in U87MG tumors with high target-to-background ratios. The uptake in the liver, kidneys and muscle were similar for all three tracers and they all showed predominant renal clearance. In conclusion, [18F]FAl-NOTA-PRGD2 and [68Ga]Ga-NOTA-PRGD2 have imaging properties and pharmacokinetics comparable to those of [18F]FPPRGD2. Considering their ease of preparation and good imaging qualities, [18F]FAl-NOTA-PRGD2 and [68Ga]NOTA-PRGD2 are promising alternatives to [18F]FPPRGD2 for PET imaging of tumor αvβ3 integrin expression.
PMCID: PMC3244506  PMID: 22026940
positron emission tomography (PET); integrin αvβ3; Arg-Gly-Asp (RGD) peptide; fluorine-aluminum complex; Ga-68; F-18
21.  Maxillary nerve compression in cynomolgus monkey Macaca fascicularis: altered somatic sensation and peripheral nerve firing 
BMC Neuroscience  2012;13:150.
Trigeminal nerve is a major source of the sensory input of the face, and trigeminal neuropathology models have been reported in rodents with injury to branches of the maxillary or mandibular division of the trigeminal nerve. Non-human primates are neuroanatomically more closely related to human than rodents; however, nerve injury studies in non-human primates are limited.
We describe here a nerve injury model of maxillary nerve compression (MNC) in the cynomolgus macaque monkey, Macaca fascicularis, and the initial characterization of the consequences of damage to this trigeminal nerve branch. The nerve injury from the compression appeared to be mild, as we did not observe overt changes in home-cage behavior in the monkeys. When mechanical stimulation was applied to the facial area, monkeys with MNC displayed increased mechanical sensitivity, as the avoidance response scores were lower than those from the control animals. Such a change in mechanical sensitivity appeared to be somewhat bilateral, as the contralateral side also showed increased mechanical sensitivity, although the change on the ipsilateral side was more robust. Multiple-unit recording of the maxillary nerve showed a general pattern of increasing responsiveness to escalating force in mechanical stimulation on the contralateral side. Ipsilateral side of the maxillary nerve showed a lack of responsiveness to escalating force in mechanical stimulation, possibly reflecting a maximum stimulation threshold effect from sensitized nerve due to MNC injury.
These results suggest that MNC may produce increased sensitivity of the ipsilateral maxillary nerve, and that this model may serve as a non-human primate model to evaluate the effect of injury to trigeminal nerve branches.
PMCID: PMC3554490  PMID: 23234480
Maxillary nerve; Trigeminal nerve; Compression; Multiple-unit recording; Non-human primates
22.  Effects of cumulus cells removal after 6 h co-incubation of gametes on the outcomes of human IVF 
To investigate the effects of cumulus cells removal after 6 h co-incubation of gametes on the fertilization, polyspermy, multinucleation and clinical pregnancy rates in human IVF.
A total of 1,200 IVF-ET cycles undergoing 6 h co-incubation of gametes in 2009 were included in this study. Inclusion criteria were: female age <38 years, first IVF treatment, with bi-ovary and normal ovarian response, e.g., 4 ~ 20 oocytes could be obtained. A 6 h period of co-incubation was applied in all IVF cycles. According to the history of infertility, cumulus cells were mechanically removed either 6 h post-insemination or 20 h post-insemination. For couples with primary infertility, or unexplained infertility, or mild oligospermia or asthenospermia, the cumulus cells were removed at 6 h of insemination for the polar body observation (6 h group, n = 565). Of these, 80 cycles received early rescue ICSI due to fertilization failure or low fertilization rate at 6 h of insemination. For couples with secondary infertility and normal semen analysis, the cumulus cells were removed at 20 h of insemination as routine (20 h group, n = 635). Of these, three cycles received late rescue ICSI due to fertilization failure at 20 h of insemination. Normal fertilization, polyspermy (≥3PN), multinucleation and clinical pregnancy rates were compared between the two groups (rescue ICSI cycles were not included in the comparison in both groups).
Significant difference (P < 0.05) was observed between the groups regarding polyspermy rates (7.48% in 6 h group and 9.22% in 20 h group). No difference was observed between the groups regarding normal fertilization rates (2PN rate) (64.89% in 6 h group and 65.74% in 20 h group). No difference was observed between the groups regarding multinucleation and clinical pregnancy rates (11.01% and 65.15% in 6 h group, 10.75% and 66.93% in 20 h group, respectively). The clinical pregnancy rate was 51.43% in cycles receiving early rescue ICSI, while no clinical pregnancy was obtained in cycles receiving late rescue ICSI.
The present results indicate that cumulus cells removal at 6 h of insemination is a relatively safe operation, which yielded comparable normal fertilization rate, multinucleation and clinical pregnancy rates compared with 20 h group. This protocol may be beneficial for early obsevation of fertilization failure and make early rescue ICSI possible.
PMCID: PMC3241845  PMID: 21898104
In-vitro fertilization; Short co-incubation; Cumulus cells removal; Polyspermy; Clinical pregnancy; Rescue ICSI
23.  Imaging tumor endothelial marker 8 using an 18F-labeled peptide 
Tumor endothelial marker 8 (TEM8) has been reported to be upregulated in both tumor cells and tumor-associated endothelial cells in several cancer types. TEM8 antagonists and TEM8-targeted delivery of toxins have been developed as effective cancer therapeutics. The ability to image TEM8 expression would be of use in evaluating TEM8-targeted cancer therapy.
A 13-meric peptide, KYNDRLPLYISNP (QQM), identified from the small loop in domain IV of protective antigen of anthrax toxin was evaluated for TEM8 binding and labeled with 18F for small-animal PET imaging in both UM-SCC1 head-and-neck cancer and MDA-MB-435 melanoma models.
A modified ELISA showed that QQM peptide bound specifically to the extracellular vWA domain of TEM8 with an IC50 value of 304 nM. Coupling 4-nitrophenyl 2-18F-fluoropropionate with QQM gave almost quantitative yield and a high specific activity (79.2±7.4 TBq/mmol, n=5) of 18F-FP-QQM at the end of synthesis. 18F-FP-QQM showed predominantly renal clearance and had significantly higher accumulation in TEM8 high-expressing UM-SCC1 tumors (2.96±0.84 %ID/g at 1 h after injection) than TEM8 low-expressing MDA-MB-435 tumors (1.38±0.56 %ID/g at 1 h after injection).
QQM peptide bound specifically to the extracellular domain of TEM8. 18F-FP-QQM peptide tracer would be a promising lead compound for measuring TEM8 expression. Further efforts to improve the affinity and specificity of the tracer and to increase its metabolic stability are warranted.
PMCID: PMC3200564  PMID: 21814853
Tumor endothelial marker 8 (TEM8); Small-animal PET; 18F; Peptide
24.  Molecular imaging probe development: a chemistry perspective 
Molecular imaging is an attractive modality that has been widely employed in many aspects of biomedical research; especially those aimed at the early detection of diseases such as cancer, inflammation and neurodegenerative disorders. The field emerged in response to a new research paradigm in healthcare that seeks to integrate detection capabilities for the prediction and prevention of diseases. This approach made a distinct impact in biomedical research as it enabled researchers to leverage the capabilities of molecular imaging probes to visualize a targeted molecular event non-invasively, repeatedly and continuously in a living system. In addition, since such probes are inherently compact, robust, and amenable to high-throughput production, these probes could potentially facilitate screening of preclinical drug discovery, therapeutic assessment and validation of disease biomarkers. They could also be useful in drug discovery and safety evaluations. In this review, major trends in the chemical synthesis and development of positron emission tomography (PET), optical and magnetic resonance imaging (MRI) probes are discussed.
PMCID: PMC3430472  PMID: 22943038
Positron emission tomography; radiochemistry; MRI; optical probes; molecular imaging
25.  c-Myb Regulates Cell Cycle-Dependent Expression of Erbin: An Implication for a Novel Function of Erbin 
PLoS ONE  2012;7(8):e42903.
In the present study, we demonstrated the cell cycle periodicity of Erbin expression with the maximal expression of Erbin in G2/M phase. A significant increase in Erbin promoter activity was observed in G2/M phase-synchronized cells. Sequence analysis revealed a c-Myb site in the core promoter region of Erbin. Mutagenesis of c-Myb consensus sequences abrogated the increased Erbin promoter activity in G2/M phase. ChIP and oligonucleotide pull-down assays validated that the recruitment of c-Myb to the consensus sequences was specific. The interaction of c-Myb with c-Myb site in the Erbin promoter was significantly enhanced in G2/M phase. Ectopic overexpression of c-Myb led to the up-regulation of Erbin promoter activity and c-Myb silencing by small interfering RNA significantly decreased Erbin protein level. Transfection of c-Myb rescued Erbin expression that was impaired by c-Myb knockdown. It proves that c-Myb and the c-Myb response element mediate the cell cycle-dependent expression of Erbin. Inactivation of Erbin causes an acceleration of the G1/S transition, the formation of multipolar spindles and abnormal chromosome congression. These results unravel a critical role of c-Myb in promoting Erbin transcription in G2/M phase and also predict an unappreciated function of Erbin in cell cycle progression.
PMCID: PMC3413663  PMID: 22880131

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