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1.  Effects of Chelator Modifications on 68Ga-Labeled [Tyr3]Octreotide Conjugates 
Purpose
Somatostatin receptors (SSTR) have been reported as promising targets for imaging agents for cancer. Recently, 68Ga-DOTATOC-based PET imaging has been used successfully for diagnosis and management of SSTR-expressing tumors. The purpose of this study was to evaluate the influence of chelator modifications and charge on 68Ga-labeled peptide conjugates.
Procedures
We have synthesized a series of [Tyr3]octreotide conjugates that consisted of different NOTA-based chelators with two to five carboxylate moieties, and compared our results with 68Ga-DOTATOC in both in vitro and in vivo studies.
Results
With the exception of 68Ga-1 (three carboxylates), the increased number of carboxylates on the NOTA-based chelators resulted in a reduced binding affinity and internalization. Additionally, the tumor uptake for 68Ga-2 (four carboxylates) and 68Ga-3 (five carboxylates) was reduced compared to that of 68Ga-DOTATOC (three carboxylates) and 68Ga-NO2ATOC (two carboxylates) and 68Ga-1 (three carboxylates) at 2 h p.i. suggesting the presence of an optimal charge for this compound.
Conclusions
Chelator modifications can lead to the altered pharmacokinetics. These results may impact further design considerations for peptide-based imaging agents.
doi:10.1007/s11307-013-0627-x
PMCID: PMC4329983  PMID: 23529373
68Ga; Somatostatin receptor; [Tyr3]octreotide; Positron emission tomography; Peptide
2.  IND-Directed Safety and Biodistribution Study of Intravenously Injected Cetuximab-IRDye800 in Cynomolgus Macaques 
Purpose
The use of receptor-targeted antibodies conjugated to fluorophores is actively being explored for real-time imaging of disease states, however, the toxicity of the bioconjugate has not been assessed in non-human primates.
Procedures
To this end, the in vivo toxicity and pharmacokinetics of IRDye800 conjugated to cetuximab (cetuximab-IRDye800; 21 mg/kg; equivalent to 250 mg/m2 human dose) was assessed in male cynomolgus monkeysover15 days following intravenous injection and compared with an unlabeled cetuximab-dosed control group.
Results
Cetuximab-IRDye800 was well tolerated. There were no infusion reactions, adverse clinical signs, mortality, weight loss, or clinical histopathology findings. The plasma half-life for the cetuximab-IRDye800 and cetuximab groups were equivalent (2.5 days). The total recovered cetuximab-IRDye800 in all tissues at study termination was estimated to be 12% of the total dose. Both cetuximab-IRDye800 and cetuximab groups showed increased QTc after dosing. The QTc for the cetuximab-dosed group returned to baseline by day 15, while the QTc of the cetuximab-IRDye800 remained elevated compared to baseline.
Conclusion
IRDye800 in low molar ratios does not significantly impact cetuximab half-life or result in organ toxicity. These studies support careful cardiac monitoring (ECG) for human studies using fluorescent dyes.
doi:10.1007/s11307-014-0773-9
PMCID: PMC4318248  PMID: 25080323
cetuximab; IRDye800; toxicity; macaques; pharmacokinetics
3.  [No title available] 
PMCID: PMC3947091  PMID: 23918654
4.  Utility of 18FLT-PET to Assess Treatment Response in Trastuzumab-resistant and Sensitive HER2-overexpressingHuman Breast Cancer Xenografts 
Purpose
Evaluate 3’-deoxy-3’-[18F]-fluorothymidine (18FLT) PET as an early marker of trastuzumab response in HER2-overexpressing xenografts.
Procedures
Tumor-to-muscle ratios were compared between both trastuzumab-sensitive and resistant cohorts prior to and after one and two treatments.
Results
A significant difference (P=0.03) was observed between treated and control trastuzumab-sensitive xenografts after one treatment, which preceded between-group differences in tumor volume. Reduced Ki67 (P=0.02) and thymidine kinase 1 (TK1) (P=0.35) immunoreactivity was observed in the treated xenografts. No significant differences in volume, tumor-to-muscle ratio, or immunoreactivity were observed between treated and control trastuzumab-resistant cohorts. A significant difference (P=0.02) in tumor-to-muscle ratio was observed between trastuzumab-sensitive and resistant cohorts after two treatments; however, tumor volumes were also different (P=0.04). Ki67 (P=0.04) and TK1 (P=0.24) immunoreactivity was ~50% less in trastuzumab-sensitive xenografts..
Conclusions
18FLT-PET provided early response assessment in trastuzumab-sensitive xenografts, but only differentiated between trastuzumab-resistant and sensitive xenografts concurrent with differences in tumor size.
doi:10.1007/s11307-014-0770-z
PMCID: PMC4311727  PMID: 25034624
18FLT-PET; assessing treatment response; HER2+ xenografts; breast cancer; trastuzumab
5.  Detection of the prodrug-activating enzyme carboxypeptidase G2 activity with chemical exchange saturation transfer magnetic resonance 
Purpose
To evaluate if the differential exchange rates with bulk water between amine and amide protons can be exploited using chemical exchange saturation transfer magnetic resonance (CEST-MR) to monitor the release of glutamate induced by carboxypeptidase G2 (CPG2), an enzyme utilized in cancer gene therapy.
Procedures
The CEST properties of glutamate (amine) and the CPG2 substrate (amide), 3,5-difluorobenzoyl-L-glutamate (3,5-DFBGlu), were evaluated at 11.7T, 37°C and varying pH. The ability of CEST-MR to monitor CPG2-mediated release of glutamate was assessed in extracts of CPG2-expressing cancer cells and purified solution of CPG2.
Results
The addition of CPG2 to a solution containing 3,5-DFBGlu led to a marked and progressively increasing CEST effect (+3ppm), concomitant with the time-dependent release of glutamate induced by CPG2.
Conclusion
CEST-MR affords the detection of CPG2 activity in vitro and supports the translation of CEST-MRI to assess CPG2-based gene therapy in vivo.
doi:10.1007/s11307-013-0680-5
PMCID: PMC4298162  PMID: 23955100
CEST MRI; Carboxypeptidase G2; Gene therapy; GDEPT
6.  Single-Input-Dual-Output Modeling of Image-Based Input Function Estimation 
Purpose
Quantification of small animal PET images necessitates knowledge of the plasma input function (PIF). We propose and validate a simplified hybrid single-input-dual-output (HSIDO) algorithm to estimate the PIF.
Procedures
The HSIDO algorithm integrates the peak of the input function from two ROI TACs with a tail segment expressed by a sum of two exponentials. Partial volume parameters are optimized simultaneously. The algorithm is validated using both simulated and real small animal PET images. In addition, the algorithm is compared to existing techniques in terms of area under curve (AUC) error, bias and precision of compartmental model micro-parameters.
Results
In general, the HSIDO method generated PIF with significantly (P<0.05) less AUC error, lower bias, and improved precision of kinetic estimates in comparison to the reference method.
Conclusions
HISDO is an improved modeling based PIF estimation method. This method can be applied for quantitative analysis of small animal dynamic PET studies.
doi:10.1007/s11307-009-0273-5
PMCID: PMC4286178  PMID: 19949986
input function; PET; small animal imaging; compartment model
7.  Integrin αvβ3 as a PET Imaging Biomarker for Osteoclast Number in Mouse Models of Negative and Positive Osteoclast Regulation 
Purpose
The goal of this study was to determine the specificity of 64Cu-CB-TE2A-c(RGDyK) (64Cu-RGD) for osteoclast-related diseases, such as Paget's disease or rheumatoid arthritis.
Procedures
C57BL/6 mice were treated systemically with osteoprotegerin (OPG) for 15 days or RANKL for 11 days to suppress and stimulate osteoclastogenesis, respectively. The mice were then imaged by positron emission tomography/computed tomography using 64Cu-RGD, followed by determination of serum TRAP5b and bone histology. Standard uptake values were determined to quantify 64Cu-RGD in bones and other tissues.
Results
Mice treated with OPG showed decreased bone uptake of 64Cu-RGD at 1, 2, and 24 h post-injection of the tracer (p<0.01 for all time points) compared to vehicle controls, which correlated with a post-treatment decrease in serum TRAP5b. In contrast, mice treated with RANKL showed significantly increased bone uptake at 2 h post-injection of 64Cu-RGD (p<0.05) compared to the vehicle control group, corresponding to increased serum TRAP5b and OC numbers as determined by bone histology.
Conclusions
These data demonstrate that 64Cu-RGD localizes to areas in bone with increased osteoclast numbers and support the use of 64Cu-RGD as an imaging biomarker for osteoclast number that could be used to monitor osteoclast-related pathologies and their treatments.
doi:10.1007/s11307-011-0512-4
PMCID: PMC4277818  PMID: 21853370
PET imaging; Copper-64; Osteoclast regulation; Integrin
8.  Characterization and Evaluation of 64Cu-Labeled A20FMDV2 Conjugates for Imaging the Integrin αvβ6 
Purpose
The integrin αvβ6 is overexpressed in a variety of aggressive cancers and serves as a prognosis marker. This study describes the conjugation, radiolabeling, and in vitro and in vivo evaluation of four chelators to determine the best candidate for 64Cu radiolabeling of A20FMDV2, an αvβ6 targeting peptide.
Procedures
Four chelators were conjugated onto PEG28-A20FMDV2 (1): 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 4,4′-((3,6,10,13,16,19-hexazazbicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl)) bis(methylene)dibenzoic acid (BaBaSar). All peptides were radiolabeled with 64Cu in ammonium acetate buffer at pH 6 and formulated to pH 7.2 in PBS for use. The radiotracers were evaluated using in vitro cell binding and internalization assays and serum stability assays. In vivo studies conducted include blocking, biodistribution, and small animal PET imaging. Autoradiography and histology were also conducted.
Results
All radiotracers were radiolabeled in good radiochemical purity (>95 %) under mild conditions (37–50 °C for 15 min) with high specific activity (0.58–0.60 Ci/µmol). All radiotracers demonstrated αvβ6-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers 64Cu-CB-TE1A1P-1 and 64Cu-BaBaSar-1 showed improved specificity for the αvβ6 positive tumor in vivo over 64Cu-DOTA-1 and 64Cu-NOTA-1 (+/− tumor uptake ratios—3.82 +/−0.44, 3.82±0.41, 2.58±0.58, and 1.29±0.14, respectively). Of the four radiotracers, 64Cu-NOTA-1 exhibited the highest liver uptake (10.83±0.1 % ID/g at 4 h).
Conclusions
We have successfully conjugated, radiolabeled, and assessed the four chelates CB-TE1A1P, DOTA, NOTA, and BaBaSar both in vitro and in vivo. However, the data suggests no clear “best candidate” for the 64Cu-radiolabeling of A20FMDV2, but instead a trade-off between the different properties (e.g., stability, selectivity, pharmacokinetics, etc.) with no obvious effects of the individual chelators.
doi:10.1007/s11307-013-0717-9
PMCID: PMC4277820  PMID: 24448825
Integrin αvβ6; Copper-64; PET imaging
9.  Breast Cancer Redox Heterogeneity Detectable with Chemical Exchange Satruation Transfer (CEST) MRI 
Purpose
Tissue redox state is an important mediator of various biological processes in health and diseases such as cancer. Previously, we discovered that the mitochondrial redox state of ex vivo tissues detected by redox scanning (an optical imaging method) revealed interesting tumor redox state heterogeneity that could differentiate tumor aggressiveness. Because the noninvasive chemical exchange saturation transfer (CEST) MRI can probe the proton transfer and generate contrasts from endogenous metabolites, we aim to investigate if the in vivo CEST contrast is sensitive to proton transfer of the redox reactions so as to reveal the tissue redox states in breast cancer animal models.
Procedures
CEST MRI has been employed to characterize tumor metabolic heterogeneity and correlated with the redox states measured by the redox scanning in two human breast cancer mouse xenograft models, MDA-MB-231 and MCF-7. The possible biological mechanism on the correlation between the two imaging modalities was further investigated by phantom studies where the reductants and the oxidants of the representative redox reactions were measured.
Results
The CEST contrast is found linearly correlated with NADH concentration and the NADH redox ratio with high statistical significance, where NADH is the reduced form of nicotinamide adenine dinucleotide. The phantom studies showed that the reductants of the redox reactions have more CEST contrast than the corresponding oxidants, indicating that higher CEST effect corresponds to the more reduced redox state.
Conclusions
This preliminary study suggests that CEST MRI, once calibrated, might provide a novel noninvasive imaging surrogate for the tissue redox state and a possible diagnostic biomarker for breast cancer in the clinic.
doi:10.1007/s11307-014-0739-y
PMCID: PMC4251869  PMID: 24811957
Cancer metabolism; Redox state; Intratumor heterogeneity; CEST; NADH
10.  Microscopic Detection of Quenched Activity Based Optical Imaging Probes Using an Antibody Detection System: Localizing Protease Activity 
Purpose
The cysteine cathepsins are a family of proteases that play important physiological roles in both normal cellular physiology and many human diseases. In cancer, the activity of many of the cysteine cathepsins is up-regulated and can be exploited for tumor imaging both in vivo and ex vivo. To characterize the behavior of a topically applied quenched fluorescent activity-based probe, GB119, ex vivo, we developed a basic immunohistochemistry technique to identify unquenched GB119 within tissue.
Procedures
Immunoblot assays were used to validate the utility of an anit-Cy5 antibody for the detection of unquenched GB119 generated by cathepsin-L. Following validation the anti-Cy5 antibody an immunohistochemical procedure was developed to detect the presence of unquenched GB119 in frozen sections of brain tumors derived from an orthotopic mouse model.
Results
These studies demonstrate that the anti-Cy5 antibody preferentially recognizes unquenched GB119 and that this differential can be used to identify the regions within the brain and the tumor that contained unquenched GB119. Using H&E staining and antibodies against other biochemical markers it was further determined that unquenched GB119 was localized to the peri-tumor space and co-localized with cathepsin-L expression.
Conclusion
Our data indicate that this methodology allows high-resolution detection of unquenched GB119 that can be correlated with other immunohistological stains.
doi:10.1007/s11307-014-0736-1
PMCID: PMC4240007  PMID: 24705781
Molecular Optical Imaging; Quenched Activity Based Probe; Topical Application; Cathepsin-L; Brain cancer
11.  Radiolabeling and In Vivo Imaging of Transplanted Renal Lineages Differentiated from Human Embryonic Stem Cells in Fetal Rhesus Monkeys 
Purpose
The goals of this study were to optimize radiolabeling of renal lineages differentiated from human embryonic stem (hES) cells and use noninvasive imaging (positron emission tomography (PET) and bioluminescence imaging (BLI)) to detect the cells in fetal monkeys post-transplant.
Procedures
hES cells expressing firefly luciferase (5×106) were radiolabeled with the optimized concentration of 10 μCi/ml 64Cu-PTSM then transplanted under ultrasound guidance into early second trimester fetal monkey kidneys. Fetuses were imaged in utero with PET and tissues collected for analysis 3 days post-transplant. Fetal kidneys were imaged ex vivo (PET and BLI) post-tissue harvest, and serial kidney sections were assessed by PCR for human-specific DNA sequences, fluorescent in situ hybridization (FISH) for human-specific centromere probes, and immunohistochemistry (IHC) to assess engrafted cells.
Results
Transplanted cells were readily imaged in vivo and identified at the site of injection; tissue analyses confirmed the imaging findings. Using a semi-quantitative method, one in approximately 650 cells in the kidney was shown to be of human origin by PCR and FISH.
Conclusions
These studies suggest that hES cells differentiated toward renal lineages can be effectively radiolabeled, transplanted into fetal monkey kidneys under ultrasound guidance, monitored with PET post-transplant, and identified by PET, BLI, PCR, FISH, and IHC post-tissue harvest.
doi:10.1007/s11307-011-0487-1
PMCID: PMC4224287  PMID: 21479709
PET; Bioluminescence imaging; Human embryonic stem cells; Fetal transplant; Rhesus monkey
12.  Pharmacokinetic Issues of Imaging with Nanoparticles: Focusing on Carbon Nanotubes and Quantum Dots 
With many desirable properties, nanoparticles hold tremendous potential for non-invasive molecular imaging and improving the efficacy of small molecule drugs. The pharmacokinetics (PK) and tissue distribution of nanoparticles largely define their in vivo performance and potential toxicity, which are fundamental issues that need to be elucidated. In this review article, we summarized how molecular imaging techniques (e.g. positron emission tomography, fluorescence imaging, etc.) can facilitate the investigation of PK profiles of nanoparticles, using carbon nanotubes (CNTs) and quantum dots (QDs) as representative examples. Different imaging techniques can provide useful insights in monitoring the in vivo behavior and tissue distribution of these nanoparticles, and a number of strategies were employed to improve the PK profiles of CNTs and QDs. Based on the available literature reports, it can be concluded that chemical and physical properties of the nanoparticles (e.g. surface functionalization, hydrodynamic size, shape, surface charge, etc.), along with the administration routes/doses, can play important roles in determining the PK and biodistribution pattern of nanoparticles. Robust chemistry for surface modification of nanoparticles is the key to success in future biomedical and clinical applications.
doi:10.1007/s11307-013-0648-5
PMCID: PMC3773279  PMID: 23715931
Pharmacokinetics (PK); molecular imaging; nanoparticles; carbon nanotube (CNT); quantum dot (QD); cancer; angiogenesis; positron emission tomography (PET); fluorescence
13.  Tumor Uptake of Hollow Gold Nanospheres after Intravenous and Intra-arterial Injection: PET/CT Study in a Rabbit VX2 Liver Cancer Model 
Purpose
This study was designed to investigate the intratumoral uptake of hollow gold nanospheres (HAuNS) after hepatic intra-arterial (IA) and intravenous (IV) injection in a liver tumor model.
Materials and Methods
Fifteen VX2 tumor-bearing rabbits were randomized into five groups (N=3 in each group) that received either IV 64Cu-labeled PEG-HAuNS (IV-PEG-HAuNS), IA 64Cu-labeled PEG-HAuNS (IA-PEG-HAuNS), IV cyclic peptide (RGD)-conjugated 64Cu-labeled PEG-HAuNS (IV-RGD-PEG-HAuNS), IA RGD-conjugated 64Cu-labeled PEG-HAuNS (IA-RGD-PEG-HAuNS), or IA 64Cu-labeled PEG-HAuNS with lipiodol (IA-PEG-HAuNS-lipiodol). The animals underwent PET/CT 1 hour after injection, and uptake expressed as percentage of injected dose per gram of tissue (%ID/g) was measured in tumor and major organs. The animals were euthanized 24 hours after injection, and tissues were evaluated for radioactivity.
Results
At 1 hour after injection, animals in the IA-PEG-HAuNS-lipiodol group showed significantly higher tumor uptake (P < 0.001) and higher ratios of tumor-to-normal liver uptake (P < 0.001) than those in all other groups. The biodistribution of radioactivity 24 hours after injection showed that IA delivery of PEG-HAuNS with lipiodol resulted in the highest tumor uptake (0.33 %ID/g; P < 0.001) and tumor-to-normal liver ratio (P < 0.001) among all delivery methods. At 24 hours, the IA-RGD-PEG-HAuNS group showed higher tumor uptake than the IA-PEG-HAuNS group (0.20 %ID/g vs. 0.099 %ID/g; P < 0.001).
Conclusion
Adding iodized oil to IA-PEG-HAuNS maximizes nanoparticle delivery to hepatic tumors and therefore may be useful in targeted chemotherapy and photoablative therapy. PET/CT can be used to noninvasively monitor the biodistribution of radiolabeled HAuNS after IV or IA injection.
doi:10.1007/s11307-013-0635-x
PMCID: PMC4164961  PMID: 23608932
Hollow gold nanospheres; liver tumor; intraarterial injection; PET/CT; copper-64; lipiodol
14.  Direct, quantitative, and noninvasive imaging of the transport of active agents through intact brain with Positron Emission Tomography 
Purpose
Our goal was to use Positron Emission Tomography (PET) to analyze the movement of radiolabeled agents in tissue to enable direct measurement of drug delivery to the brain.
Procedures
A variety of 11C- and 18F-labeled compounds were delivered directly to an agarose phantom or rat striatum. Concentration profiles were extracted for analysis and fitted to diffusion models.
Results
Diffusion coefficients ranged from 0.075±0.0026 mm2/min ([18F]Fluoride, 18 Da) to 0.0016±0.0018 mm2/min ([18F]NPB4-avidin, 68 kDa) and matched well with predictions based on molecular weight (R2=0.965). The tortuosity of the brain extracellular space was estimated to be 1.56, with the tissue clearance halftime of each tracer in the brain varying from 19 to 41 minutes.
Conclusions
PET is an effective modality to directly quantify the movement of locally delivered drugs or drug-carriers. This continuous, non-invasive assessment of delivery will aid the design of better drug delivery methods.
doi:10.1007/s11307-013-0636-9
PMCID: PMC3942322  PMID: 23624949
PET; diffusion; drug delivery to the brain; mathematical modeling; radiolabeling; polymer
15.  A Novel In Vitro Assay to Assess Phosphorylation of 3′-[18F]fluoro-3′-Deoxythymidine 
Purpose
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.
Procedures
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.
Results
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.
Conclusions
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.
doi:10.1007/s11307-010-0351-8
PMCID: PMC4180216  PMID: 20532643
3′-[18F]fluoro-3′-deoxythymidine ([18F]FLT); Thymidine kinase 1; Positron emission tomography (PET); PET tracers; Molecular imaging
16.  A Phase 2 Study of 16α-[18F]-fluoro-17β-estradiol Positron Emission Tomography (FES-PET) as a Marker of Hormone Sensitivity in Metastatic Breast Cancer (MBC) 
Purpose
16α-[18F]-fluoro-17β-estradiol positron emission tomography (FES-PET) quantifies estrogen receptor (ER) expression in tumors and may provide diagnostic benefit.
Procedures
Women with newly diagnosed metastatic breast cancer (MBC) from an ER-positive primary tumor were imaged before starting endocrine therapy. FES uptake was evaluated qualitatively and quantitatively, and associated with response and with ER expression.
Results
Nineteen patients underwent FES imaging. Fifteen had a biopsy of a metastasis and 15 were evaluable for response. Five patients had quantitatively low FES uptake, six had at least one site of qualitatively FES-negative disease. All patients with an ER-negative biopsy had both low uptake and at least one site of FES-negative disease. Of response-evaluable patients, 2/2 with low FES standard uptake value tumors had progressive disease within 6 months, as did 2/3 with qualitatively FES-negative tumors.
Conclusions
Low/absent FES uptake correlates with lack of ER expression. FES-positron emission tomography can help identify patients with endocrine resistant disease and safely measures ER in MBC.
doi:10.1007/s11307-013-0699-7
PMCID: PMC4169237  PMID: 24170452
FES-PET; FDG-PET; Metastatic breast cancer; ER expression; Endocrine therapy; Response
17.  In Vivo Bioluminescence Tumor Imaging of RGD Peptide-modified Adenoviral Vector Encoding Firefly Luciferase Reporter Gene 
Purpose
The goal of this study is to demonstrate the feasibility of chemically modified human adenovirus (Ad) vectors for tumor retargeting.
Procedures
E1- and E3-deleted Ad vectors carrying firefly luciferase reporter gene under cytomegalovirus promoter (AdLuc) was surface-modified with cyclic arginine–glycine–aspartic acid (RGD) peptides through a bifunctional poly(ethyleneglycol) linker (RGD-PEG-AdLuc) for integrin αvβ3 specific delivery. The Coxsackie and adenovirus viral receptor (CAR) and integrin αvβ3 expression in various tumor cell lines was determined by reverse transcriptase PCR and fluorescence-activated cell sorting. Bioluminescence imaging was performed in vitro and in vivo to evaluate RGD-modified AdLuc infectivity.
Results
RGD-PEG-AdLuc abrogated the native CAR tropism and exhibited significantly enhanced transduction efficiency of integrin-positive tumors than AdLuc through intravenous administration.
Conclusion
This approach provides a robust platform for site-specific gene delivery and noninvasive monitoring of the transgene delivery efficacy and homing.
doi:10.1007/s11307-007-0079-2
PMCID: PMC4165526  PMID: 17297551
Adenovirus; Firefly luciferase; Reporter gene; Integrin αvβ3; RGD; Bioluminescence imaging
18.  Noninvasive Evaluation of Immunosuppressive Drug Efficacy on Acute Donor Cell Survival 
Purpose
The therapeutic benefits of cell transplantation may depend on the survival of sufficient numbers of grafted cells. We evaluate four potent immunosuppressive medications aimed at preventing acute donor cell death.
Procedures and Results
Embryonic rat H9c2 myoblasts were stably transduced to express firefly luciferase reporter gene (H9c2-Fluc). H9c2-Fluc cells (3 × 106) were injected into thigh muscles of Sprague–Dawley rats (N = 30) treated with cyclosporine, dexamethasone, mycophenolate mofetil, tacrolimus, or saline from day −3 to day +14. Longitudinal optical bioluminescence imaging was performed over two weeks. Fluc activity was 40.0 ± 12.1% (dexamethasone), 30.5 ± 12.5% (tacrolimus), and 21.5 ± 3.5% (mycophenolate) vs. 12.0 ± 5.0% (control) and 8.3 ± 5.0% (cyclosporine) at day 4 (P < 0.05). However, by day 14, cell signals had decreased drastically to <10% for all groups despite drug therapy.
Conclusion
This study demonstrates the ability of optical molecular imaging for tracking cell survival noninvasively and raises important questions with regard to the overall efficacy of immunosuppressives for prolonging transplanted cell survival.
doi:10.1007/s11307-006-0038-3
PMCID: PMC4161130  PMID: 16555032
Molecular imaging; Cell transplant; Cyclosporine; Tacrolimus; Mycophenolate
19.  Comparison of Optical Bioluminescence Reporter Gene and Superparamagnetic Iron Oxide MR Contrast Agent as Cell Markers for Noninvasive Imaging of Cardiac Cell Transplantation 
Purpose
In this study, we compared firefly luciferase (Fluc) reporter gene and super-paramagnetic iron oxide (Feridex) as cell markers for longitudinal monitoring of cardiomyoblast graft survival using optical bioluminescence imaging (BLI) and magnetic resonance imaging (MRI), respectively.
Procedures
Rats (n=31) underwent an intramyocardial injection of cardiomyoblasts (2×106) labeled with Fluc, Feridex, or no marker (control) or an injection of Feridex alone (75 μg). Afterward, rats were serially imaged with BLI or MRI and killed at different time points for histological analysis.
Results
BLI revealed a drastically different cell survival kinetics (half-life = 2.65 days over 6 days) than that revealed by MRI (half-life = 16.8 days over 80 days). Injection of Feridex alone led to prolonged tissue retention of Feridex (≥16 days) and persistent MR signal (≥42 days).
Conclusions
Fluc BLI reporter gene imaging is a more accurate gauge of transplanted cell survival as compared to MRI of Feridex-labeled cells.
doi:10.1007/s11307-008-0182-z
PMCID: PMC4155941  PMID: 19034584
Optical bioluminescence imaging; Magnetic resonance imaging; Reporter gene; Contrast agent; Cell marker; Cell transplantation
20.  Direct Site-Specific Radiolabeling of an Affibody Protein with 4-[18F]Fluorobenzaldehyde via Oxime Chemistry 
Purpose
In this study, we introduce a methodology for preparing 18F-labeled Affibody protein, specifically 18F-Anti-HER2 dimeric Affibody (14 kDa), for in vivo imaging of HER2neu with positron emission tomography (PET).
Procedures
We have used 4-[18F]fluorobenzaldehyde as a synthon to prepare 18F-Anti-HER2 Affibody. Aminooxy-functionalized Affibody (Anti-HER2-ONH2) was incubated with 4-[18F] fluorobenzaldehyde in ammonium acetate buffer at pH 4 in the presence of methanol at 70°C for 15 min. The resulting 18F-labeled Affibody molecule was evaluated as a PET probe in xenograft models expressing HER2.
Results
We have successfully prepared 18F-Anti-HER2 dimeric Affibody (14 kDa), N-(4-[18F] fluorobenzylidine)oxime-Anti-HER2 Affibody, [18F]FBO-Anti-HER2, in 26–30% radiochemical yields (decay corrected). High-contrast small-animal PET images with relatively moderate tumor uptake (1.79±0.40% ID/g) were observed for the 18F-Anti-HER2 Affibody.
Conclusion
Site-specific 18F-labeled Affibody against HER2 has been synthesized via chemo-selective oxime formation between an aminooxy-functionalized Affibody and 18F-fluorobenzaldehyde. The results have implications for radiolabeling of other affibodies and macromolecules and should also be important for advancing Affibody imaging with PET.
doi:10.1007/s11307-008-0142-7
PMCID: PMC4155982  PMID: 18481153
Affibody; HER2; Positron emission tomography (PET); Imaging; 18F
21.  64Cu-Labeled Affibody Molecules for Imaging of HER2 Expressing Tumors 
Introduction
The development of molecular probes based on novel engineered protein constructs is under active investigation due to the great potential of this generalizable strategy for imaging a variety of tumor targets.
Discussion
In this report, human epidermal growth factor receptor type 2 (HER2)-binding Affibody molecules were radiolabeled with 64Cu and their imaging ability was further evaluated in tumor mice models to understand the promise and limitations of such probes. The anti-HER2 Affibody molecules in monomeric (ZHER2:477) and dimeric [(ZHER2:477)2] forms were site specifically modified with the maleimide-functionalized chelator, 1,4,7,10-tetraazacyclodode-cane-1,4,7-tris(acetic acid)-10-acetate mono (N-ethylmaleimide amide) (Mal-DOTA). The resulting DOTA–Affibody conjugates were radiolabeled with 64Cu and evaluated in nude mice bearing subcutaneous SKOV3 tumors. Biodistribution experiments showed that tumor uptake values of 64Cu-DOTA-ZHER2:477 and 64Cu-DOTA-(ZHER2:477)2 were 6.12±1.44% and 1.46±0.50% ID/g, respectively, in nude mice (n=3 each) at 4 h postinjection. Moreover, 64Cu-labeled monomer exhibited significantly higher tumor/blood ratio than that of radiolabeled dimeric counterpart at all time points examined in this study. MicroPET imaging of 64Cu-DOTA-ZHER2:477 in SKOV3 tumor mice clearly showed good and specific tumor localization. This study demonstrates that 64Cu-labeled ZHER2:477 is a promising targeted molecular probe for imaging HER2 receptor expression in living mice. Further work is needed to improve the excretion properties, hence dosimetry and imaging efficacy, of the radiometal-based probe.
doi:10.1007/s11307-009-0256-6
PMCID: PMC4155984  PMID: 19779897
Affibody; HER2; PET; Imaging; 64Cu
22.  Molecular Imaging Using Light-Absorbing Imaging Agents and a Clinical Optical Breast Imaging System—a Phantom Study 
Purpose
The aim of the study was to determine the feasibility of using a clinical optical breast scanner with molecular imaging strategies based on modulating light transmission.
Procedures
Different concentrations of single-walled carbon nanotubes (SWNT; 0.8–20.0 nM) and black hole quencher-3 (BHQ-3; 2.0–32.0 μM) were studied in specifically designed phantoms (200–1,570 mm3) with a clinical optical breast scanner using four wavelengths. Each phantom was placed in the scanner tank filled with optical matching medium. Background scans were compared to absorption scans, and reproducibility was assessed.
Results
All SWNT phantoms were detected at four wavelengths, with best results at 684 nm. Higher concentrations (≥8.0 μM) were needed for BHQ-3 detection, with the largest contrast at 684 nm. The optical absorption signal was dependent on phantom size and concentration. Reproducibility was excellent (intraclass correlation 0.93–0.98).
Conclusion
Nanomolar concentrations of SWNT and micromolar concentrations of BHQ-3 in phantoms were reproducibly detected, showing the potential of light absorbers, with appropriate targeting ligands, as molecular imaging agents for clinical optical breast imaging.
doi:10.1007/s11307-010-0356-3
PMCID: PMC4156007  PMID: 20532642
Optical imaging; Molecular imaging; Breast; Transmission
23.  Molecular Imaging of Phosphorylation Events for Drug Development 
Purpose
Protein phosphorylation mediated by protein kinases controls numerous cellular processes. A genetically encoded, generalizable split firefly luciferase (FL)-assisted complementation system was developed for noninvasive monitoring phosphorylation events and efficacies of kinase inhibitors in cell culture and in small living subjects by optical bioluminescence imaging.
Procedures
An Akt sensor (AST) was constructed to monitor Akt phosphorylation and the effect of different PI-3K and Akt inhibitors. Specificity of AST was determined using a non-phosphorylable mutant sensor containing an alanine substitution (ASA).
Results
The PI-3K inhibitor LY294002 and Akt kinase inhibitor perifosine led to temporal- and dose-dependent increases in complemented FL activities in 293T human kidney cancer cells stably expressing AST (293T/AST) but not in 293T/ASA cells. Inhibition of endogenous Akt phosphorylation and kinase activities by perifosine also correlated with increase in complemented FL activities in 293T/AST cells but not in 293T/ASA cells. Treatment of nude mice bearing 293T/AST xenografts with perifosine led to a 2-fold increase in complemented FL activities compared to that of 293T/ASA xenografts. Our system was used to screen a small chemical library for novel modulators of Akt kinase activity.
Conclusion
This generalizable approach for noninvasive monitoring of phosphorylation events will accelerate the discovery and validation of novel kinase inhibitors and modulators of phosphorylation events.
doi:10.1007/s11307-008-0187-7
PMCID: PMC4154800  PMID: 19048345
Phosphorylation; Kinases; Noninvasive; Repetitive imaging in living subjects; Optical bioluminescence imaging in living subjects; Drug development; Akt
24.  A Titratable Two-Step Transcriptional Amplification Strategy for Targeted Gene Therapy Based on Ligand-Induced Intramolecular Folding of a Mutant Human Estrogen Receptor 
Purpose
The efficacy and safety of cardiac gene therapy depend critically on the level and the distribution of therapeutic gene expression following vector administration. We aimed to develop a titratable two-step transcriptional amplification (tTSTA) vector strategy, which allows modulation of transcriptionally targeted gene expression in the myocardium.
Procedures
We constructed a tTSTA plasmid vector (pcTnT-tTSTA-fluc), which uses the cardiac troponin T (cTnT) promoter to drive the expression of the recombinant transcriptional activator GAL4-mER(LBD)-VP2, whose ability to transactivate the downstream firefly luciferase reporter gene (fluc) depends on the binding of its mutant estrogen receptor (ERG521T) ligand binding domain (LBD) to an ER ligand such as raloxifene. Mice underwent either intramyocardial or hydrodynamic tail vein (HTV) injection of pcTnT-tTSTA-fluc, followed by differential modulation of fluc expression with varying doses of intraperitoneal raloxifene prior to bioluminescence imaging to assess the kinetics of myocardial or hepatic fluc expression.
Results
Intramyocardial injection of pcTnT-tTSTA-fluc followed by titration with intraperitoneal raloxifene led to up to tenfold induction of myocardial fluc expression. HTV injection of pcTnT-tTSTA-fluc led to negligible long-term hepatic fluc expression, regardless of the raloxifene dose given.
Conclusions
The tTSTA vector strategy can effectively modulate transgene expression in a tissue-specific manner. Further refinement of this strategy should help maximize the benefit-to-risk ratio of cardiac gene therapy.
doi:10.1007/s11307-013-0673-4
PMCID: PMC4154804  PMID: 23955099
Gene therapy; Drug-regulated gene expression; Transcriptional amplification; Transcriptional targeting; Intramolecular folding; Bioluminescence imaging
25.  The Synthesis of 18F-FDS and Its Potential Application in Molecular Imaging 
Purpose
2-Deoxy-2-[18F]fluoro-d-glucose (FDG) is the most commonly used positron emission tomography (PET) tracer for oncological and neurological imaging, but it has limitations on detecting tumor or inflammation in brain gray matter. In this study, we describe the development of 2-deoxy-2-[18F]fluorosorbitol (18F-FDS) and its possible application in lesion detection around brain area.
Procedures
18F-FDS was obtained by reduction of FDG using NaBH4 (81±4% yield in 30 min). Cell uptake/efflux experiments in cell culture and small animal PET imaging on tumor and inflammation models were performed.
Results
Despite the low accumulation in cell culture, 18F-FDS had good tumor uptake and contrast in the subcutaneous U87MG tumor model (4.54%ID/g at 30 min post-injection). Minimal uptake in the normal mouse brain facilitated good tumor contrast in both U87MG and GL-26 orthotopic tumor models. 18F-FDS also had increased uptake in the inflamed foci of the TPA-induced acute inflammation model.
Conclusions
Because of the ease of synthesis and favorable in vivo kinetics, 18F-FDS may have potential applications in certain cases where FDG is inadequate (e.g., brain tumor).
doi:10.1007/s11307-007-0125-0
PMCID: PMC4143166  PMID: 18097725
Positron emission tomography (PET); 2-Deoxy-2-[18F]fluoro-sorbitol (18F-FDS); 2-Deoxy-2-[18F]fluoro-d-glucose (FDG); Brain tumor; Inflammation

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