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99mTc-labeled estradiol as an estrogen receptor probe: Preparation and preclinical evaluation
99m
Tc-labeled estradiol as an estrogen receptor probe: preparation and preclinical evaluation Xiaotian Xia, Hongyan Feng, Chongjiao Li, Chunxia Qin, Yiling Song, Yongxue Zhang, Xiaoli Lan PII: DOI: Reference:
S0969-8051(15)00165-1 doi: 10.1016/j.nucmedbio.2015.09.006 NMB 7770
To appear in:
Nuclear Medicine and Biology
Received date: Revised date: Accepted date:
2 May 2015 22 August 2015 8 September 2015
Please cite this article as: Xia Xiaotian, Feng Hongyan, Li Chongjiao, Qin Chunxia, Song Yiling, Zhang Yongxue, Lan Xiaoli, 99m Tc-labeled estradiol as an estrogen receptor probe: preparation and preclinical evaluation, Nuclear Medicine and Biology (2015), doi: 10.1016/j.nucmedbio.2015.09.006
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ACCEPTED MANUSCRIPT Title Page Title: 99mTc-labeled estradiol as an estrogen receptor probe: preparation and preclinical evaluation
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Running headline: 99mTc-labeled estradiol: an ER probe
Authors: Xiaotian Xia#, Hongyan Feng#, Chongjiao Li, Chunxia Qin, Yiling Song, Yongxue
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Zhang*, Xiaoli Lan*
Affiliations: Department of Nuclear Medicine, Union Hospital, Tongji Medical College,
Imaging, Wuhan 430022, China
#
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Huazhong University of Science and Technology,Hubei Province Key Laboratory of Molecular
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Xiaotian Xia and Hongyan Feng contributed equally to the article.
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*Corresponding author: Prof. Xiaoli Lan PhD, MD, and Prof. Yongxue Zhang MD
430022, China.
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Address: Department of Nuclear Medicine, Wuhan Union Hospital, No. 1277 Jiefang Ave, Wuhan,
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Tel.: +86-27-83692633(O), +86-13886193262 (mobile); Fax: +86-27-85726282. E-mail: [email protected] (X. Lan), and [email protected] (Y. Zhang)
Funding: This work was supported by the National Natural Science Foundation of China (No. 30970853, 81071200), the Natural Science Foundation of Hubei Province of China for Distinguished Young Scholars (No. 2010CDA094).
ACCEPTED MANUSCRIPT Abstract
Introduction: Most breast cancers express estrogen receptors (ERs). Noninvasive imaging of ER
99m
Tc-labeled estradiol, with diethylenetriaminepentaacetic acid (DTPA) as a chelating
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probe,
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expression may be helpful for planning therapy of ER+ tumors. We developed a new ER-binding
ligand, and assessed its targeting ability in vitro and in vivo.
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Methods: 3-aminoethyl estradiol was synthesized in two steps from estrone, followed by
99m
Tc
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labeling. Western blotting and immunofluorescence staining were used to detect ER expression in MCF-7 and MDA-MB-231 breast cancer cells. Saturation binding and specific binding were performed by incubating MCF-7 cells with increasing concentrations of99mTc-DTPA-estradiol.
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Cell uptake, efflux, and blocking assays were also performed. To test
99m
Tc-DTPA-estradiol in
vivo, nude mice bearing either MCF-7- (high ER expression) or MDA-MB-231-derived tumors 99m
Tc-DTPA-estradiol, and underwent single-photon
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(low ER expression) were injected with
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emission-computed tomography (SPECT). Mice injected with excess unlabeled DTPA-estradiol were used as controls. Ex vivo gamma-counting of tissues from normal and tumor-bearing mice
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was used to evaluate 99mTc-DTPA-estradiol biodistribution. Results: The radiochemical purity of 99mTc-DTPA-estradiol was 98.3% ± 1.3% with a specific activity of 33.1 ± 1.5 MBq/µmol (n=3). Western blotting and immunofluorescence staining confirmed extensive expression of ERs by the MCF-7 cells, and less extensive expression by MDA-MB-231 cells. There was high binding affinity of
99m
Tc-DTPA-estradiol to MCF-7 cells
with a > 45% specific rate of total cell uptake. SPECT images and the biodistribution study results showed significantly higher uptake by MCF-7 tumors (6.06 ± 0.38 %ID/g) than by MDA-MB-231 tumors (1.57 ± 0.28 %ID/g). Pre-injection of MCF-7 tumor-bearing nude mice with excess
ACCEPTED MANUSCRIPT unlabeled DTPA-estradiol significantly reduced tumor uptake of
99m
Tc-DTPA-estradiol (2.24 ±
0.28 %ID/g), suggesting that 99mTc-DTPA-estradiol specifically targets ERs in tumors.
stability.
99m
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Tc-DTPA-estradiol can be synthesized with satisfactory labeling efficiency and
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Conclusions: 99m
Tc-DTPA-estradiol specifically targeted ERs in vitro and in vivo with favorable
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pharmacokinetics, allowing ER receptor expression assessment with SPECT imaging.
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Key words: Breast Cancer; Estrogen Receptors; Estradiol; Single-photon Emission-Computed
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Tomography
ACCEPTED MANUSCRIPT INTRODUCTION Breast cancer is one of the most common malignancies of women in China and worldwide. It has
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a high mortality rate [1]. Early diagnosis is imperative for long-term survival. The estrogen receptor (ER) is a very important biomarker in breast cancer, often guiding treatment planning.
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Tumors with higher concentrations of ERs (ER+) tend to respond to anti-estrogen hormone
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therapy, whereas ER-negative (ER-) tumors require a different treatment approach [2].
Quantitation of ERs is prone to sampling error. In the past decade, emphasis has been placed on the development of nuclear imaging approaches.
18
F and other cyclotron-produced radionuclides
F-labeled estrogen derivative, 16α-18F-17β-estradiol (18F-FES), has been evaluated clinically,
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18
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have been used to label ER ligands to develop in vivo imaging agents [3-6]. The most successful
with promising results reported for prediction of treatment response to anti-estrogen drugs such as
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tamoxifen [7]. Subsequent studies have been directed at developing methods for labeling estrogen 99m
Tc for single-photon emission-computed tomography (SPECT) [8, 9].
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imaging agents with
However, these agents have demonstrated suboptimal target tissue selectivity in vivo, possibly as a result of their high lipophilicity or rapid metabolism [2]. The complex chemistry involved in the radiosynthesis of these compounds to obtain high yields and purities has further hampered their development [9-11].
We synthesized and characterized an estradiol analog, labeled with technetium (I)-99m, and chelated it with diethylenetriaminepentaacetic acid (DTPA), a useful chelating agent for lanthanide metal ions that has been used in a multitude of preclinical and clinical
ACCEPTED MANUSCRIPT radiopharmaceuticals. It possesses the requisite chemistry for covalent attachment to targeting vectors such as amino acids[12], peptides [13, 14], proteins [15], and nanoparticles [16, 17], and is
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metabolized by the kidneys, while most estrogen receptor probes undergo hepatic metabolism. We hypothesized that DTPA may be suitable to conjugate estradiol, and DTPA-estradiolcould chelate
99m
Tc-DTPA-estradiol, and assessed its targeting ability with ERs in
vitro and in vivo.
MATERIALS AND METHODS
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radiocompound, named it
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radiometallic isotopes for imaging and therapeutic applications. Therefore, we synthesized a
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Synthesis and radiolabeling of DTPA-estradiol
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Estrone (2.7 g, 10 mmol) was dissolved in acetone (150 mL). NaOH (600 mg, 15 mmol), bromoacetonitrile (Xiyashiji, Chengdu, China) (1.3 mL, 17.3 mmol), and potassium iodide
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(Sigma-Aldrich, St. Louis MO, USA) (120 mg, 0.723 mmol) were added. The reaction mixture
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was heated under reflux for 4 h. It was then evaporated to dryness and ethyl acetate (150 mL) was added. After washing the mixture with double-distilled water in a separatory funnel, the organic layer was dried over magnesium sulfate and filtered. The ethyl acetate was evaporated under reduced pressure, and the solid product washed with ether on filter paper. The 3-cyanomethyl estrone weighed 2.32 g (yield 75%). We confirmed its structure using proton NMR (1H-NMR) spectroscopy (CDCl3) (Bruker Biospin®, Rheinstetten, Germany).
We added lithium aluminum hydride (Xiyashiji, China) (270 mg, 7.1 mmol in THF) to 3-cyanomethyl estrone (440 mg, 1.42 mmol) in tetrahydrofuran (THF, 25 mL), and the reaction
ACCEPTED MANUSCRIPT mixture was stirred at room temperature for 4 h. After the solvent was evaporated, the solid was dissolved in ethyl acetate and washed with water. The ethyl acetate layer was dried over
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magnesium sulfate and filtered. The solvent was again evaporated. The yield of 3-acetonitrile
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estradiol was 68%. Its structure was confirmed using 1H-NMR (CDCl3).
Diethylenetriaminepentaacetic dianhydride (DTPAA) (TCI, Shanghai, China) (357.7 mg, 1 mmol)
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was dissolved in 10 mL of anhydrous dimethylformamide (DMF), and added to 3-acetonitrile estradiol (315 mg, 1 mmol). The mixture was stirred at 60°C for 24 h, then dialyzed with a molecular weight cut-off of 500 Da (MD31®, Spectrum Labs, Los Angeles, CA USA) for 48 h.
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After lyophilization, the product weighed 78.2 mg. The resulting white precipitate was the product
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DTPA-estradiol. Its structure was confirmed using 1H-NMR (CDCl3). The synthetic scheme of
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DTPA-estradiol is shown in Figure 1.
99m
Tc/99Mo generator (Beijing Atom High Tech Co., Ltd., Beijing,
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Na99mTcO4 was eluted from a
China). 99mTc-pertechnetate (37 MBq) was added to a vial containing the DTPA-estradiol (0.5 mg, 720 nmol) and tin (II) chloride (Sigma Aldrich) (SnCl2, 100 μg) in 0.5 mL water. The reaction proceeded for 30 min. Then, the product was purified using Sep-Pak C-18 cartridge (Waters, Milford MA, USA). The radiolabeled mixture was passed through the pre-activated column, washed with saline (5 mL), and eluted with methanol (5 mL). Thin-layer chromatography (TLC) silica-gel paper strips (Gelman Sciences, Rossdorf, Germany) was used to achieve radiochemical purity with saline and a mixture of ammonium acetate: methanol (V:V = 4:1) as eluents.
ACCEPTED MANUSCRIPT Stability of 99mTc-DTPA-estradiol Two hundred microliters of labeled DTPA-estradiol was incubated at 37°C in 500 μL of histidine
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(1 mM), cysteine (1 mM), phosphate-buffered saline (PBS), and human serum for 1, 3, 6 and 24 h. The radiochemical purity was assessed using a radio-TLC scanner. Each time point and condition
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was analyzed in triplicate.
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Cell lines and cell culture
All cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The human breast carcinoma MCF-7 cell line was cultured in
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Minimum Essential Media (MEM®; Gibco, Carlsbad CA, USA) supplemented with 10% fetal
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bovine serum (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Beyotime, Shanghai, China). The human breast cancer MDA-MB-231 cell line was maintained in Leibovitz’s L-15
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medium containing 10% fetal bovine serum and 1% penicillin/streptomycin with high humidity at
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37°C and 5% CO2 for cell culture. Cells were subpassaged in a 1:2 split using 0.25% trypsin/0.02% ethylene diamine tetraacetic acid (EDTA).
Measuring ER expression Immunofluorescence staining was performed. MCF-7 and MDA-MB-231 cells were digested, resuspended, and grown overnight in six-well plates. Cells adhering to the coverslips were fixed in iced acetone for 10 min. For immunofluorescence staining, the fixed cells were rinsed with PBS, blocked with 1% bovine serum albumin (BSA), then incubated with diluted primary antibody (rabbit anti-ER, 1:50) (Epitomics, Abcam, Cambridge, England) overnight at 4°C, followed by
ACCEPTED MANUSCRIPT incubation with diluted secondary antibody (Cy3-labeled goat anti-rabbit IgG, 1:200) (Beyotime, China) for 60 min at 37°C. Finally, the cells were incubated with 4-6-diamidino-2-phenylindole
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(DAPI) for 5 min and a quenching-resisting agent was added before observation by confocal
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microscopy (Nikon, Osaka, Japan).
Western blotting was performed to detect ER protein expression levels in MCF-7 and
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MDA-MB-231 tumor extracts. Briefly, equal amounts of total protein were separated by sodium lauryl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk for 1 h at room
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temperature. After washing, anti-ER monoclonal antibody, diluted 1:1000 (Epitomics, Abcam),
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was incubated with the membranes at 4°C overnight. Actin 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a loading control. The blots were washed with PBS, and then
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horseradish peroxidase (HRP)-conjugated secondary antibody 1:3000 (Goodbio, Wuhan, China)
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was added to the membranes and incubated for 1 h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence (ECL; Beyotime, Wuhan, China).
Cell culture Assay Saturation binding experiments were performed as described previously [18, 19]. Approximately 2 × 105 MCF-7 cells were seeded per well and incubated at 37°C for 24 h, followed by incubation with 99mTc-DTPA-estradiol at various concentrations (1–200 nM) at 37°C for 1 h. After washing, the cells were lysed with 1 N NaOH. Radioactivity was measured with a gamma counter (2470, WIZARD®; PerkinElmer, Waltham, MA, USA) and expressed as the percentage of applied
ACCEPTED MANUSCRIPT activity normalized to 2 × 105 cells. The resulting data were used to calculate the efficiency of surface stripping to normalize results obtained from experiments performed at 37°C. Each assay
99m
Tc-DTPA-estradiol were performed on both cell types. After
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Cell uptake and efflux studies of
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was performed in triplicate.
overnight incubation of 2 × 105 cells in each well of a 24-well plate overnight, the cells were
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incubated with 99mTc-DTPA-estradiol (4 nmol per well, 0.185 MBq) at 37°C for 60, 120, 180, 240, or 360 min. After removing the supernatant, the cells were washed and then lysed with 1 N NaOH at 37°C for 10 min. The radioactivity of the lysates was measured using an automatic
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gamma-counter and the cell uptake rate was expressed as a percentage of the total input
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radioactive dose. For the cell efflux study, MCF-7 and MDA-MB-231 cells were incubated with 99m
Tc-DTPA-estradiol at 37°C for 360 min. Following two washings with PBS, the cells were
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incubated in culture medium for 60, 120, 180, 240, or 360 min, then collected and counted using
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the aforementioned methods.
For the blocking study, MCF-7 cells were incubated with 99mTc-DTPA-estradiol (4 nmol per well, 0.185 MBq) in the presence of no other estradiol, 40 nmol per well unlabeled DTPA-estradiol, or 10-fold estradiol (Xiyashiji, Sichuan, China) (40 nmol per well) at 37°C for 1 h and then treated as before. All tests were performed in triplicate.
Animal models
ACCEPTED MANUSCRIPT BALB/c mice (3–4 weeks) (Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technology), and BALB/c-nu mice (female, 3–4 weeks) (Beijing HFK
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Bioscience Co., Ltd., Beijing, China) were maintained at 22 ± 2°C with an alternating 12-h light/dark cycle. The nude mice were maintained in a barrier system room. All animal experiments
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were performed under a protocol approved by the Institutional Animal Care and Use Committee
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of Tongji Medical College, Huazhong University of Science and Technology.
MCF-7 breast tumor cells (5 × 107 cells) or MDA-MB-231 cells (1 × 107 cells) in 50 μL PBS and 50 μL basement membrane gel (Matrigel®, BD Biosciences, San Jose CA, USA), respectively,
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were injected subcutaneously into the right axilla. Mice underwent in vivo SPECT imaging or ex
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vivo biodistribution studies when the tumor size reached 0.6-1.2 cm in diameter.
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Ex vivo distribution experimentsin healthy and tumor-bearing mice
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For tissue distribution experiments, normal female BALB/c mice (n = 5 per group) and MCF-7and MDA-MB-231-tumor-bearing mice(n = 4 per group) were each injected with a 100-µL bolus containing 3.7–7.4 MBq (100–200 µCi, 0.1–0.2 µmol)
99m
Tc-DTPA-estradiol via the tail vein.
Normal animals were sacrificed under deep ketamine-xylazine anesthesia in groups of five at 0.5, 1, 2, 3, and 4 h post-injection. Tumor-bearing animals were sacrificed at 2 and 4 h post-injection. The tumor and organs or tissues (blood, heart, lung, stomach, liver, spleen, kidney, small intestine, large intestine, ovary, bone and muscle) were harvested, rinsed with PBS, wiped with filter paper, and weighed for the gamma-counter measurements. The radioactivity of each sample was measured, decay corrected to the injection time, normalized for injected dose and organ/tissue
ACCEPTED MANUSCRIPT weight, and expressed as percentage of injected dose per gram tissue (%ID/g). Tumor-targeting specificity was evaluated in the blocking group by the administration of a 10-fold excess of 99m
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Tc-DTPA-estradiol. At 4 h post-injection,
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unlabeled DTPA-estradiol 1 h prior to injection of
the mice were sacrificed and dissected, and the %ID/g was calculated as described above. The
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values were corrected for the injected dose and expressed as mean ± standard deviation (SD).
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SPECT imaging
Nude mice bearing MCF-7 or MDA-MB-231 tumors were injected with
99m
Tc-DTPA-estradiol
(7.4–11.1 MBq, 0.2–0.3 µmol) via the tail vein. For the blocking study, MCF-7 tumor-bearing
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mice were administered a 10-fold excess dose of non-labeled DTPA-estradiol 1 h prior to injection 99m
Tc-DTPA-estradiol. SPECT with a pinhole collimator (Symbia T6®; Siemens, Erlangen,
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of
Germany) was used for obtaining mouse images. Each anesthetized mouse was placed on the
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scanner table in the prone position. SPECT images were acquired at 2 and 4 h post-injection with
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an acquisition time of 10 min.
Statistical analysis
Quantitative data are presented as mean ± SD. For statistical classification, a Student’s t-test (2-tailed, unpaired) was performed using commercial software (Prism 5.0®, GraphPad Software, San Diego CA, USA and SPSS 13.0®, IBM, Armonk NY, USA). Differences were statistically significant when P <0.05. Pearson correlation was carried out between different organs for 99m
Tc-DTPA-estradiol.
ACCEPTED MANUSCRIPT RESULTS Precursor identification
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The structures of 3-acetonitrile estradiol and DTPA-estradiol were confirmed by 1H-NMR spectroscopy. For 3-acetonitrile estradiol, the 1H-NMR (CDCl3) showed peaks at δ 7.21 (dt, J =
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10.8, 5.4 Hz, 1H), 6.71 (td, J = 8.7, 2.6 Hz, 1H), 6.64 (dd, J = 9.1, 2.6 Hz, 1H), 4.20–3.95 (m, 2H), 3.81–3.63 (m, 2H), 3.16–3.02 (m, 1H), 2.90–2.78 (m, 2H), 2.32 (d, J = 13.2 Hz, 1H), 2.24–2.09
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(m, 2H), 2.00–1.62 (m, 7H), 1.54–1.16 (m, 8H), and 0.79 (s, 3H). Data for DTPA-estradiol (400 MHz, CDCl3-added D2O) showed peaks at: δ 7.21 (dd, 1H), 6.81 (t, 2H), 4.21–4.14 (m, 4H), 3.84–3.44 (m, 8H), 3.06 (s, 2H), 2.79 (m, 4H), 2.41–2.18 (m, 8H), 1.99–1.76 (m, 6H), 1.54–1.36
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(m, 6H), and 0.76 (s, 3H).
Radiolabeling identification and stability studies
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The radiolabeling yield of 99mTc-DTPA-estradiol was 64.5% ± 2.8%, and the radiochemical purity
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was 98.3% ± 1.3% (n=3). The specific activity of
MBq/µmol (n=3). The stability of
99m
Tc-DTPA-estradiol was 33.1 ± 1.5
99m
Tc-DTPA-estradiol was assessed by simultaneous
incubation with 1 mM histidine, 1 mM cysteine, PBS (pH = 7.4), and human serum at 37°C at 1, 3, 6, and 24 h (Figure 2). Little decomposition or dissociation of
99m
Tc-DTPA-estradiol into either
[99mTcO4]− or other subsidiary products was detected under these conditions.
Western blotting and immunofluorescence staining Western blotting analysis of ER expression in MCF-7 and MDA-MB-231 cells is shown in Figure 3A, in which the band at 66 kDa belongs to ER expression. Clear and indistinct bands at 66 kDa
ACCEPTED MANUSCRIPT were seen in MCF-7 and MDA-MB-231 cells, respectively, which suggested that ER was overexpressed in MCF-7 cells but not in MDA-MB-231 cells. In addition, in Figure 3B, a strong
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red fluorescence signal distribution was seen from the MCF-7 cells. In contrast, there was almost no red fluorescence signal from the MDA-MB-231 cells, further confirming that MCF-7 cells had
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high ER expression while MDA-MB-231 cells had low ER expression. This result suggested that
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MDA-MB-231 cells could be used as a relative negative control in this study.
In vitro assay
The binding affinity of 99mTc-DTPA-estradiol was also measured by incubating MCF-7 cells with
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increasing concentrations (1–200 nM) at 37°C, showing an equilibrium dissociation constant (KD)
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of 30.0 ± 4.1 nM (n=3) with a calculated maximal number of binding sites (Bmax) of 2.7 × 105 per cell (Figure 4A). The specific binding of
99m
Tc-DTPA-estradiol varied over the concentrations
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tested, ranging from 46.0% ± 4.2% to 90.6% ± 0.2% of total cell-associated uptake.
The blocking study showed that the specific binding of 99mTc-DTPA-estradiol could be blocked in the presence of a 10-fold excess of unlabeled DTPA-estradiol or 10-fold excess of estradiol, in MCF-7 cells (Figure 4B). At 1 h, the tracer uptake rates were only 3.23% ± 0.34% and 3.05% ± 0.23%, which were approximately 51.7% decreased and 54.4% decreased from unblocked conditions (P <0.05), respectively. These data confirm the specificity of binding to ER-positive MCF-7 cells.
99m
Tc-DTPA-estradiol
ACCEPTED MANUSCRIPT The cell uptake study revealed that
99m
Tc-DTPA-estradiol binds strongly to ER-positive MCF-7
cells, and very weakly to MDA-MB-231 cells. For
99m
Tc-DTPA-estradiol, 6.68% ± 0.20% of
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probe uptake in MCF-7 cells was measured during the first hour of incubation, followed by peak uptake at 6 h, with 8.07% ± 0.27% probe uptake identified (Figure 4C). The cell efflux study
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showed that 99mTc-DTPA-estradiol has good cell retention by MCF-7 cells, with only about 1.99% (decreased from 8.07% to 6.08% of total input radioactivity) of
99m
Tc-DTPA-estradiol efflux
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observed (Figure 4D). In contrast, significantly lower levels of total radioactivity were observed in both cellular uptake (4.95% ± 0.48%) and retention (4.21% ± 0.24%) during 6-hour incubation
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with the MDA-MB-231 cells (Figure 4C and4D).
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Biodistribution of 99mTc-DTPA-estradiol in normal and tumor-bearing mice To further evaluate 99mTc-DTPA-estradiol characteristics in vivo, its biodistribution in both tumor
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and non-tumor tissues of normal BALB/c mice and tumor-bearing mice was examined (Figure 5,
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Tables 1 and 2). The ratios of tumor-to-normal tissues or organs were also calculated (Table 2).
In MCF-7 cell models, tumor uptakes were 4.67 ± 0.39 %ID/g and 6.06 ± 0.38 %ID/g at 2 and 4 h post-injection, respectively (Figure 5A and B). In contrast, uptake within MDA-MB-231 tumors peaked 4 h after injection with 1.57 ± 0.28 %ID/g (P <0.05, Figure 5B). The tumor uptake of labeled DTPA-estradiol in the blocked mice was significantly lower than that in the unblocked mice (2.24 ± 0.28 %ID/g vs 6.06 ± 0.38 %ID/g, P <0.05; Figure 5C). To quantify 99m
Tc-DTPA-estradiol uptake in different tissues and therefore assess its specificity,
tumor-to-normal ratios for tissues and organs were calculated (Table 2). At 4 h post-injection,
ACCEPTED MANUSCRIPT 99m
Tc-DTPA-estradiol showed the highest tumor-to-normal ratios, with 2.93 ± 0.30 for blood and
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5.24 ± 1.23 for muscle.
Blood clearance of 99mTc-DTPA-estradiol was 5.14 ± 0.15 %ID/g at 0.5 h and 2.08 ± 0.22 %ID/g
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at 4 h. Large amounts of renal and hepatic accumulation (36.10 ± 3.35 %ID/g and 50.47 ± 4.49 %ID/g at 0.5h, 10.07 ± 0.91 %ID/g and 48.52 ± 3.35 %ID/g at 4 h, respectively) were
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observed, suggesting both renal and hepatic elimination may be involved in the pharmaceutical’s metabolism. Notably, moderate accumulation was also observed in the spleen and ovary.
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SPECT imaging
administration of
99m
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SPECT images of MCF-7 and MDA-MB-231 tumor-bearing mice were acquired at 2 and 4 h after Tc-DTPA-estradiol, and are shown in Figure 6. MCF-7 tumors were clearly
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visible with low background signal as early as 2 h post-injection, compared with little radioactive
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accumulation within MDA-MB-231 tumors at any of the tested time points. Under blocking conditions with excess unlabeled DTPA-estradiol, clearly reduced tumor uptake of 99m
Tc-DTPA-estradiol was observed (Figure 6). These results confirm the specificity of
99m
Tc-DTPA-estradiol for ER-positive tumors.
DISCUSSION Advances in molecular and cellular biology are transforming our understanding of basic physiology and pathology; similar advances in molecular imaging technologies now permit dynamic and quantitative studies in vivo with minimal invasiveness [10]. There is a need for an
ACCEPTED MANUSCRIPT accurate, affordable, and most of all, noninvasive method for assessing extramammary estrogen receptor status, which can avoid unnecessary biopsies and permit serial assessments during 99m
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Tc-based estradiol derivative for ER
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endocrine therapy in breast cancer. We developed a
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imaging, and proved its targeting efficiency with in vitro and in vivo imaging.
To meet the need for quantifiable targeting to estrogen receptor sites, we focused on linear
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polyaminopolycarboxylic acid chelating agents [20]. We intended to enhance the water solubility of the estrogen receptor probe by introducing DTPA groups, expecting to minimize the intrahepatic metabolism. The chelator DTPA was introduced to estradiolvia DTPA anhydride
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under mild conditions of pH and temperatures. Previous studies have suggested that 3-acetonitrile
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estradiol could be produced via an ER-mediated process, and that 99mTc-labeled estradiol could be a useful agent for imaging ER status [21, 22]. Compared with previous estradiol probes,
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DTPA-estradiol is a small molecule with a suitable molecular weight. It does not alter
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biodistribution or tumor uptake patterns [21]. In addition, unlike other studies highlighting the difficulty of preparing 99m
99m
Tc-labeled derivatives with high specific activity or yields [9, 11],
Tc-DTPA-estradiol was prepared easily, with a satisfactory yield and labeling efficiency. The
in vitro experiments demonstrated that
99m
Tc-DTPA-estradiol is stable in histidine, cysteine, PBS
and human serum at 37°C for 24 h, with more than 90% remaining intact after 24 h of incubation.
Although we presented specific activity in this study, we could not present the effective specific activity (ESA), which is one of the most important characteristics of a receptor-binding radiopharmaceutical. ESA has often been emphasized for ER radioligands and has been an
ACCEPTED MANUSCRIPT important parameter for routine quality control to validate the synthesis procedures. There are direct and indirect methods for the measurement of ER binding. They are used routinely to
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determine relative binding affinity (RBA) and ESA of various cold and radiolabeled ER-binding compounds. Some researchers used scintillation proximity technology to develop a rapid
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screening assay for ER ligands with satisfactory results [23]. The assay requires tritiated E2 ([3H]-E2; New England Nuclear, Boston MA, USA). Unfortunately, we were unable to acquire
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this kit, or the instruments needed to measure tritiated E2, so we are unable to present the ESA.
The binding experiments showed that the binding affinity of
99m
Tc-DTPA-estradiol to ER
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receptors was 30.0 ± 4.1 nM, with specific binding resulting in over 45% of total binding.
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Classically, estrogen elicits genomic effects on transcription via α and β ERs, which are mainly located in the nucleus. Performing receptor-binding studies on whole cells with steroid analogs
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poses challenges related to ligand equilibrium, the lipophilic nature of the ligand, estimation of
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free ligand, the processes of receptor activation and degradation, and the presence of multiple estrogen receptor types [24]. As a result, careful consideration is required for data interpretation as one can easily over- or underestimate the KD [10].
99m
Tc-DTPA-estradioldemonstrated a KD
much higher than typically reported values of estradiol itself (0.1–1.0 nM)[25, 26], but was comparable to the affinity of a similar estrogen receptor (11 ± 1.5) nM[10]. Further, 99m
Tc-DTPA-estradiolalso displayed good retention in MCF-7 cells with a maximum of only 1.99%
effluxat 6 h.
SPECT
imaging
and
biodistribution
quantitative
analysis
in
nude
mice
showed
ACCEPTED MANUSCRIPT that99mTc-DTPA-estradiolaccumulated in ER-positive MCF-7 tumors with an uptake range of 4.67 ±0.39 %ID/g – 6.06 ±0.38 %ID/g. The tumor-to-normal ratios peaked at 4 h, with 2.93 ± 0.30 for
contrast,
the
tumor-to-normal
ratios
were
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blood and 5.24 ± 1.23 for muscle, allowing easy visualization of the ER-positive tumors. In significantly
in
masses
in
99m
Tc-DTPA-estradiol exhibited
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MDA-MB-231-tumor-bearing mice or blocked mice, where
lower
minimal tumor uptake at 1.57 ± 0.28%ID/g and 2.24 ± 0.28%ID/g, respectively.Previous research
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has shown that estradiol-based estrogen receptor probes can target the ER with good results. GAP-EDL was found to accumulate into ER+ 13762 (rat mammary carcinoma) tumors with a tumor-to-muscle ratio of 7.92 ± 0.56 at 4 h [21]; and 99mTc-estradiol-pyridin-2-yl has been shown
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to target MCF-7 tumors with a tumor-to-muscle ratio of 5.67 ± 1.06 at 3 h post-injection [10].
We initially intended to use DTPA to improve the metabolic pathways of the probe to overcome
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its high uptake in liver. However, the distribution studies revealed that 99mTc-DTPA-estradiol still
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accumulated mainly in the liver, with significant uptake in the spleen, the kidneys and the ovary. Attempts to develop a neutral
99m
Tc-estradiol derivative suitable for imaging of tumors
encountered problems with excessive lipophilicity [27]. Lipophilic compounds are usually characterized by slow elimination from the blood pool, resulting in high accumulation within extensively-perfused organs such as the liver and spleen [8, 9, 11].Additionally, steroid hormones (including estrogen) have been identified as stimulators of the reticuloendothelial system [28, 29], suggesting that phagocytosis within the liver and spleen may be another possible contributor towards 99m
such
a
phenomenon.
We
encountered
similar
problems.
The
uptake
of
Tc-DTPA-estradiol in the liver was 50.47 ± 4.49 %ID/g at 30 min and 48.52 ± 3.35 %ID/g at 4
ACCEPTED MANUSCRIPT h post injection, whereas the kidney, which we expected tobe the critical metabolic organ of the DTPA-chelated probecontained36.10 ± 3.35 %ID/g at 30 min and 10.07 ± 0.91 %ID/g at 4 h. In 99m
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Tc-DTPA-estradiol for producing
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addition, the ovaries would be expected to accumulate
steroid hormones, especially estradiol [30, 31]. Because this is a preliminary study testing the
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imaging performance of this estrogen receptor targeted probe, ovarian accumulation of this probe
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was not studied, and the estrus cycles of all the female mice were not synchronized.
The major goal for cancer imaging is accurate disease characterization through the application of functional and molecular imaging studies.18F-FES PET cancer imaging has been immensely
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valuable to clinical oncologists for staging and visualizing primary and metastatic carcinomas [32].
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The supplemental molecular characterization and receptor-expression assessment of the tumor has often assisted in determining endocrine therapy options [7, 33]. As an alternative to PET, we
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sought to evaluate the utility of a
99m
Tc-DTPA-chelated estradiol as a SPECT tracer in breast
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cancer, which is much less expensive than 18F-FES.
So far, there are no readily available and easily synthesized SPECT agents for assessing reproductive cancers expressing estrogen-binding activity [10]. Despite the challenges and drawbacks, newer
99m
Tc-based estradiol derivatives have been reported, including the one in this
study. However, to date, all of them have failed to provide an alternative to 18F-FES PET, mainly attributable to low tumor uptake compounded with high background levels. To overcome the problems posed by such steroid analogs, the development of neutral nonsteroidal analogs that would specifically bind to each estrogen receptor subtype with high affinity would be a valuable
ACCEPTED MANUSCRIPT step [34, 35].
We developed a novel estradiol-based probe,
99m
Tc-DTPA-estradiol, that specifically binds to the
99m
Tc-DTPA-estradiol showed favorable characteristics in vitro and in vivo,
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estrogen receptor.
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CONCLUSION
demonstrating potential use in targeted imaging and therapy development in ER-positive tumors.
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Further studies should be performed to optimize the radiocompound for better pharmacokinetics in vivo, especially decreasing the uptake in the liver.
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Acknowledgments
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This work was supported by the National Natural Science Foundation of China (No. 30970853, 81071200), the Natural Science Foundation of Hubei Province of China for Distinguished Young
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Scholars (No. 2010CDA094). The authors are grateful to Prof. Guoping Yan and Dr. Biao Zhao at
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the School of Material Science and Engineering, Wuhan Institute of Technology, for their kind help in preparation of the labeling precursors.
Conflicts of interest The authors declare that they have no conflict of interest.
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ACCEPTED MANUSCRIPT [2] Huang L, Zhu H, Zhang Y, Xu X, Cui W, Yang G, et al. Synthesis and binding affinities of Re(I) and (99m)Tc(I)-containing 16alpha-substituted estradiol complexes: Models for potential breast
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[30] Drummond AE and Fuller PJ. Ovarian actions of estrogen receptor-beta: an update.Semin Reprod Med 2012;30:32-8. [31] Janik ME, Belkot K, and Przybylo M. Is oestrogen an important player in melanoma progression? Contemp Oncol (Pozn) 2014;18:302-6. [32] Gemignani ML, Patil S, Seshan VE, Sampson M, Humm JL, Lewis JS, et al. Feasibility and predictability of perioperative PET and estrogen receptor ligand in patients with invasive breast cancer.J Nucl Med 2013;54:1697-702. [33] Cintolo JA, Tchou J, and Pryma DA. Diagnostic and prognostic application of positron
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ACCEPTED MANUSCRIPT Tables
1
2
4
Blood
5.14±0.15
4.23±0.29
3.50±0.19
2.46±0.34
2.08±0.22
Brain
0.21±0.06
0.22±0.08
0.18±0.09
0.18±0.02
0.15±0.04
Heart
1.78±0.09
0.99±0.21
0.67±0.22
0.68±0.05
0.63±0.16
Lung
5.68±0.32
5.03±0.17
3.83±1.12
3.08±0.22
2.53±0.58
Stomach
2.62±0.29
1.69±0.20
1.71±0.55
1.64±0.13
1.35±0.58
Liver
50.47±4.49
62.58±5.40
54.17±5.91
50.19±1.71
48.52±3.35
Spleen
12.04±2.39
10.45±0.67
8.74±1.46
7.25±0.68
6.50±0.65
Kidney
36.10±3.35
21.13±1.07
13.16±2.40
11.51±0.65
10.07±0.91
Small intestine
2.58±0.24
2.26±0.46
2.06±0.55
1.84±0.20
1.69±0.29
Large intestine
4.60±0.27
4.50±0.59
3.67±0.78
3.46±0.08
3.16±0.57
Ovary
2.20±0.35
2.47±0.10
3.06±0.42
3.16±0.21
3.34±0.08
1.93±0.08
1.86±0.38
1.41±0.30
1.38±0.15
1.07±0.16
1.17±0.24
1.15±0.42
1.12±0.15
1.09±0.13
0.98±0.37
Muscle
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Injected dose (100–200 µCi, 0.1–0.2 µmol per mouse)
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*
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Bone
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3
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0.5
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Time (h) Organs
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Table 1 Biodistribution of 99mTc-DTPA-estradiol in normal BALB/c mice (n = 5, %ID/g ± SD)*
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Table 2 Biodistribution of 99mTc-DTPA-estradiol and blocking group in nude mice bearing MCF-7 and MDA-MB-231 tumor xenografts (n = 4, %ID/g ± SD)a MDA-MB-231
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MCF-7
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Organs 4h
Blockingb (4h)
4h
Tumor
4.67±0.39
6.06±0.38
2.24±0.28
1.57±0.28
Blood
3.84±0.36
2.18±0.31
2.54±0.24
2.07±0.16
Brain
0.19±0.06
0.15±0.08
0.14±0.03
0.16±0.09
Heart
0.68±0.13
0.59±0.14
0.47±0.18
0.56±0.14
Lung
3.57±0.26
2.33±0.29
2.07±0.32
2.42±0.26
Stomach
1.81±0.29
1.02±0.22
0.85±0.22
1.09±0.17
46.33±3.25
38.25±5.25
42.38±2.38
6.97±1.05
6.88±1.47
7.19±0.72
59.91±12.22
Spleen
8.13±0.52
Kidney
12.92±1.10
9.57±0.98
10.27±0.39
10.35±1.71
Small intestine
1.76±0.14
1.40±0.14
1.32±0.17
1.45±0.18
Large intestine
3.26±0.45
3.06±0.18
2.13±0.13
2.98±0.40
3.36±0.23
3.56±0.17
2.43±0.25
3.35±0.40
1.20±0.19
1.19±0.25
1.09±0.07
1.22±0.04
1.21±0.17
1.19±0.19
1.09±0.29
0.98±0.13
Muscle
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Bone
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Ovary
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Liver
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2h
Uptake ratios
Tumor-to-blood
1.22±0.09
2.82±0.39
0.89±0.11
0.76±0.13
Tumor-to-muscle
3.94±0.70
5.24±1.23
2.05±0.12
1.63±0.33
a
Injected dose (100–200 µCi, 0.1–0.2 µmol per mouse) Blocking groupwas performed with 10-fold excess of cold DTPA-estradiol injected 1 h before injection of 99mTc-radiolabeled DTPA-estradiol. b
ACCEPTED MANUSCRIPT Figure Legends
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Figure 1 Synthetic scheme of DTPA-estradiol
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Figure 2 Stability of 99mTc-DTPA-estradiol incubated at 37°C in 1 mM histidine, 1 mM cysteine,
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PBS, and human serum, with radiochemical purity assessed by TLC.
Figure 3 MCF-7 and MDA-MB-231 cells were stained with fluorescent anti-ER antibody (red) and DAPI (blue) and analyzed by microscopy (200×). Western blotting and immunofluorescence
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staining show positive ER expression by MCF-7 and almost no expression by MDA-MB-231
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cells.
99m
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Figure 4 In vitro cell saturation binding, specific binding, uptake, efflux, and blocking study of
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Tc-DTPA-estradiol. (A) Saturation binding (black diamonds) and specific binding (red squares)
curves were generated by measuring the radioactivity in MCF-7 cells incubated with increasing concentrations of
99m
Tc-DTPA-estradiol as indicated. The inset is the Scatchard plot of the
saturation binding experiment. (B) In blocking studies, cell uptake of
99m
Tc-DTPA-estradiol
conjugates at 1 h was blocked in the presence of 10-fold excess of DTPA-estradiol (1, middle) and 10-fold excess of estradiol (2, right), respectively. (C, D) Cell uptake and cell efflux of 99m
Tc-DTPA-estradiol in MCF-7 and MDA-MB-231 cells were measured at the indicated time
points. All data were determined from three independent experiments and expressed as mean ± SD.
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Figure 5 Biodistribution of 99mTc-DTPA-estradiol in mouse xenograft tumors. (A) Biodistribution 99m
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Tc-DTPA-estradiol in nude mice bearing MCF-7 tumors at 2 and 4 h post-injection (n = 4)
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was assessed by uptake assay. (B) Tumor uptake of MCF-7 and MDA-MB-231 tumor-bearing
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mice at 4 h post-injection. (C) Comparison of tumor uptake in MCF-7 tumor-bearing mice at 4 h
Figure 6 SPECT imaging of
99m
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post-injection of 99mTc-DTPA-estradiol with/without excess cold DTPA-estradiol.
Tc-DTPA-estradiol in nude mice bearing MCF-7 and
MDA-MB-231 tumors, and blocked tumors with pre-injection of 10-fold excess unlabeled
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DTPA-estradiol. Arrows indicate tumors.
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Tc-labeled estradiol as an estrogen receptor probe: preparation and preclinical evaluation Xiaotian Xia, Hongyan Feng, Chongjiao Li, Chunxia Qin, Yiling Song, Yongxue Zhang, Xiaoli Lan PII: DOI: Reference:
S0969-8051(15)00165-1 doi: 10.1016/j.nucmedbio.2015.09.006 NMB 7770
To appear in:
Nuclear Medicine and Biology
Received date: Revised date: Accepted date:
2 May 2015 22 August 2015 8 September 2015
Please cite this article as: Xia Xiaotian, Feng Hongyan, Li Chongjiao, Qin Chunxia, Song Yiling, Zhang Yongxue, Lan Xiaoli, 99m Tc-labeled estradiol as an estrogen receptor probe: preparation and preclinical evaluation, Nuclear Medicine and Biology (2015), doi: 10.1016/j.nucmedbio.2015.09.006
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ACCEPTED MANUSCRIPT Title Page Title: 99mTc-labeled estradiol as an estrogen receptor probe: preparation and preclinical evaluation
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Running headline: 99mTc-labeled estradiol: an ER probe
Authors: Xiaotian Xia#, Hongyan Feng#, Chongjiao Li, Chunxia Qin, Yiling Song, Yongxue
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Zhang*, Xiaoli Lan*
Affiliations: Department of Nuclear Medicine, Union Hospital, Tongji Medical College,
Imaging, Wuhan 430022, China
#
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Huazhong University of Science and Technology,Hubei Province Key Laboratory of Molecular
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Xiaotian Xia and Hongyan Feng contributed equally to the article.
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*Corresponding author: Prof. Xiaoli Lan PhD, MD, and Prof. Yongxue Zhang MD
430022, China.
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Address: Department of Nuclear Medicine, Wuhan Union Hospital, No. 1277 Jiefang Ave, Wuhan,
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Tel.: +86-27-83692633(O), +86-13886193262 (mobile); Fax: +86-27-85726282. E-mail: [email protected] (X. Lan), and [email protected] (Y. Zhang)
Funding: This work was supported by the National Natural Science Foundation of China (No. 30970853, 81071200), the Natural Science Foundation of Hubei Province of China for Distinguished Young Scholars (No. 2010CDA094).
ACCEPTED MANUSCRIPT Abstract
Introduction: Most breast cancers express estrogen receptors (ERs). Noninvasive imaging of ER
99m
Tc-labeled estradiol, with diethylenetriaminepentaacetic acid (DTPA) as a chelating
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probe,
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expression may be helpful for planning therapy of ER+ tumors. We developed a new ER-binding
ligand, and assessed its targeting ability in vitro and in vivo.
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Methods: 3-aminoethyl estradiol was synthesized in two steps from estrone, followed by
99m
Tc
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labeling. Western blotting and immunofluorescence staining were used to detect ER expression in MCF-7 and MDA-MB-231 breast cancer cells. Saturation binding and specific binding were performed by incubating MCF-7 cells with increasing concentrations of99mTc-DTPA-estradiol.
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Cell uptake, efflux, and blocking assays were also performed. To test
99m
Tc-DTPA-estradiol in
vivo, nude mice bearing either MCF-7- (high ER expression) or MDA-MB-231-derived tumors 99m
Tc-DTPA-estradiol, and underwent single-photon
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(low ER expression) were injected with
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emission-computed tomography (SPECT). Mice injected with excess unlabeled DTPA-estradiol were used as controls. Ex vivo gamma-counting of tissues from normal and tumor-bearing mice
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was used to evaluate 99mTc-DTPA-estradiol biodistribution. Results: The radiochemical purity of 99mTc-DTPA-estradiol was 98.3% ± 1.3% with a specific activity of 33.1 ± 1.5 MBq/µmol (n=3). Western blotting and immunofluorescence staining confirmed extensive expression of ERs by the MCF-7 cells, and less extensive expression by MDA-MB-231 cells. There was high binding affinity of
99m
Tc-DTPA-estradiol to MCF-7 cells
with a > 45% specific rate of total cell uptake. SPECT images and the biodistribution study results showed significantly higher uptake by MCF-7 tumors (6.06 ± 0.38 %ID/g) than by MDA-MB-231 tumors (1.57 ± 0.28 %ID/g). Pre-injection of MCF-7 tumor-bearing nude mice with excess
ACCEPTED MANUSCRIPT unlabeled DTPA-estradiol significantly reduced tumor uptake of
99m
Tc-DTPA-estradiol (2.24 ±
0.28 %ID/g), suggesting that 99mTc-DTPA-estradiol specifically targets ERs in tumors.
stability.
99m
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Tc-DTPA-estradiol can be synthesized with satisfactory labeling efficiency and
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Conclusions: 99m
Tc-DTPA-estradiol specifically targeted ERs in vitro and in vivo with favorable
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pharmacokinetics, allowing ER receptor expression assessment with SPECT imaging.
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Key words: Breast Cancer; Estrogen Receptors; Estradiol; Single-photon Emission-Computed
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Tomography
ACCEPTED MANUSCRIPT INTRODUCTION Breast cancer is one of the most common malignancies of women in China and worldwide. It has
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a high mortality rate [1]. Early diagnosis is imperative for long-term survival. The estrogen receptor (ER) is a very important biomarker in breast cancer, often guiding treatment planning.
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Tumors with higher concentrations of ERs (ER+) tend to respond to anti-estrogen hormone
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therapy, whereas ER-negative (ER-) tumors require a different treatment approach [2].
Quantitation of ERs is prone to sampling error. In the past decade, emphasis has been placed on the development of nuclear imaging approaches.
18
F and other cyclotron-produced radionuclides
F-labeled estrogen derivative, 16α-18F-17β-estradiol (18F-FES), has been evaluated clinically,
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18
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have been used to label ER ligands to develop in vivo imaging agents [3-6]. The most successful
with promising results reported for prediction of treatment response to anti-estrogen drugs such as
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tamoxifen [7]. Subsequent studies have been directed at developing methods for labeling estrogen 99m
Tc for single-photon emission-computed tomography (SPECT) [8, 9].
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imaging agents with
However, these agents have demonstrated suboptimal target tissue selectivity in vivo, possibly as a result of their high lipophilicity or rapid metabolism [2]. The complex chemistry involved in the radiosynthesis of these compounds to obtain high yields and purities has further hampered their development [9-11].
We synthesized and characterized an estradiol analog, labeled with technetium (I)-99m, and chelated it with diethylenetriaminepentaacetic acid (DTPA), a useful chelating agent for lanthanide metal ions that has been used in a multitude of preclinical and clinical
ACCEPTED MANUSCRIPT radiopharmaceuticals. It possesses the requisite chemistry for covalent attachment to targeting vectors such as amino acids[12], peptides [13, 14], proteins [15], and nanoparticles [16, 17], and is
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metabolized by the kidneys, while most estrogen receptor probes undergo hepatic metabolism. We hypothesized that DTPA may be suitable to conjugate estradiol, and DTPA-estradiolcould chelate
99m
Tc-DTPA-estradiol, and assessed its targeting ability with ERs in
vitro and in vivo.
MATERIALS AND METHODS
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radiocompound, named it
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radiometallic isotopes for imaging and therapeutic applications. Therefore, we synthesized a
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Synthesis and radiolabeling of DTPA-estradiol
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Estrone (2.7 g, 10 mmol) was dissolved in acetone (150 mL). NaOH (600 mg, 15 mmol), bromoacetonitrile (Xiyashiji, Chengdu, China) (1.3 mL, 17.3 mmol), and potassium iodide
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(Sigma-Aldrich, St. Louis MO, USA) (120 mg, 0.723 mmol) were added. The reaction mixture
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was heated under reflux for 4 h. It was then evaporated to dryness and ethyl acetate (150 mL) was added. After washing the mixture with double-distilled water in a separatory funnel, the organic layer was dried over magnesium sulfate and filtered. The ethyl acetate was evaporated under reduced pressure, and the solid product washed with ether on filter paper. The 3-cyanomethyl estrone weighed 2.32 g (yield 75%). We confirmed its structure using proton NMR (1H-NMR) spectroscopy (CDCl3) (Bruker Biospin®, Rheinstetten, Germany).
We added lithium aluminum hydride (Xiyashiji, China) (270 mg, 7.1 mmol in THF) to 3-cyanomethyl estrone (440 mg, 1.42 mmol) in tetrahydrofuran (THF, 25 mL), and the reaction
ACCEPTED MANUSCRIPT mixture was stirred at room temperature for 4 h. After the solvent was evaporated, the solid was dissolved in ethyl acetate and washed with water. The ethyl acetate layer was dried over
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magnesium sulfate and filtered. The solvent was again evaporated. The yield of 3-acetonitrile
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estradiol was 68%. Its structure was confirmed using 1H-NMR (CDCl3).
Diethylenetriaminepentaacetic dianhydride (DTPAA) (TCI, Shanghai, China) (357.7 mg, 1 mmol)
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was dissolved in 10 mL of anhydrous dimethylformamide (DMF), and added to 3-acetonitrile estradiol (315 mg, 1 mmol). The mixture was stirred at 60°C for 24 h, then dialyzed with a molecular weight cut-off of 500 Da (MD31®, Spectrum Labs, Los Angeles, CA USA) for 48 h.
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After lyophilization, the product weighed 78.2 mg. The resulting white precipitate was the product
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DTPA-estradiol. Its structure was confirmed using 1H-NMR (CDCl3). The synthetic scheme of
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DTPA-estradiol is shown in Figure 1.
99m
Tc/99Mo generator (Beijing Atom High Tech Co., Ltd., Beijing,
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Na99mTcO4 was eluted from a
China). 99mTc-pertechnetate (37 MBq) was added to a vial containing the DTPA-estradiol (0.5 mg, 720 nmol) and tin (II) chloride (Sigma Aldrich) (SnCl2, 100 μg) in 0.5 mL water. The reaction proceeded for 30 min. Then, the product was purified using Sep-Pak C-18 cartridge (Waters, Milford MA, USA). The radiolabeled mixture was passed through the pre-activated column, washed with saline (5 mL), and eluted with methanol (5 mL). Thin-layer chromatography (TLC) silica-gel paper strips (Gelman Sciences, Rossdorf, Germany) was used to achieve radiochemical purity with saline and a mixture of ammonium acetate: methanol (V:V = 4:1) as eluents.
ACCEPTED MANUSCRIPT Stability of 99mTc-DTPA-estradiol Two hundred microliters of labeled DTPA-estradiol was incubated at 37°C in 500 μL of histidine
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(1 mM), cysteine (1 mM), phosphate-buffered saline (PBS), and human serum for 1, 3, 6 and 24 h. The radiochemical purity was assessed using a radio-TLC scanner. Each time point and condition
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was analyzed in triplicate.
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Cell lines and cell culture
All cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The human breast carcinoma MCF-7 cell line was cultured in
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Minimum Essential Media (MEM®; Gibco, Carlsbad CA, USA) supplemented with 10% fetal
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bovine serum (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Beyotime, Shanghai, China). The human breast cancer MDA-MB-231 cell line was maintained in Leibovitz’s L-15
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medium containing 10% fetal bovine serum and 1% penicillin/streptomycin with high humidity at
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37°C and 5% CO2 for cell culture. Cells were subpassaged in a 1:2 split using 0.25% trypsin/0.02% ethylene diamine tetraacetic acid (EDTA).
Measuring ER expression Immunofluorescence staining was performed. MCF-7 and MDA-MB-231 cells were digested, resuspended, and grown overnight in six-well plates. Cells adhering to the coverslips were fixed in iced acetone for 10 min. For immunofluorescence staining, the fixed cells were rinsed with PBS, blocked with 1% bovine serum albumin (BSA), then incubated with diluted primary antibody (rabbit anti-ER, 1:50) (Epitomics, Abcam, Cambridge, England) overnight at 4°C, followed by
ACCEPTED MANUSCRIPT incubation with diluted secondary antibody (Cy3-labeled goat anti-rabbit IgG, 1:200) (Beyotime, China) for 60 min at 37°C. Finally, the cells were incubated with 4-6-diamidino-2-phenylindole
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(DAPI) for 5 min and a quenching-resisting agent was added before observation by confocal
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microscopy (Nikon, Osaka, Japan).
Western blotting was performed to detect ER protein expression levels in MCF-7 and
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MDA-MB-231 tumor extracts. Briefly, equal amounts of total protein were separated by sodium lauryl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk for 1 h at room
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temperature. After washing, anti-ER monoclonal antibody, diluted 1:1000 (Epitomics, Abcam),
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was incubated with the membranes at 4°C overnight. Actin 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a loading control. The blots were washed with PBS, and then
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horseradish peroxidase (HRP)-conjugated secondary antibody 1:3000 (Goodbio, Wuhan, China)
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was added to the membranes and incubated for 1 h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence (ECL; Beyotime, Wuhan, China).
Cell culture Assay Saturation binding experiments were performed as described previously [18, 19]. Approximately 2 × 105 MCF-7 cells were seeded per well and incubated at 37°C for 24 h, followed by incubation with 99mTc-DTPA-estradiol at various concentrations (1–200 nM) at 37°C for 1 h. After washing, the cells were lysed with 1 N NaOH. Radioactivity was measured with a gamma counter (2470, WIZARD®; PerkinElmer, Waltham, MA, USA) and expressed as the percentage of applied
ACCEPTED MANUSCRIPT activity normalized to 2 × 105 cells. The resulting data were used to calculate the efficiency of surface stripping to normalize results obtained from experiments performed at 37°C. Each assay
99m
Tc-DTPA-estradiol were performed on both cell types. After
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Cell uptake and efflux studies of
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was performed in triplicate.
overnight incubation of 2 × 105 cells in each well of a 24-well plate overnight, the cells were
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incubated with 99mTc-DTPA-estradiol (4 nmol per well, 0.185 MBq) at 37°C for 60, 120, 180, 240, or 360 min. After removing the supernatant, the cells were washed and then lysed with 1 N NaOH at 37°C for 10 min. The radioactivity of the lysates was measured using an automatic
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gamma-counter and the cell uptake rate was expressed as a percentage of the total input
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radioactive dose. For the cell efflux study, MCF-7 and MDA-MB-231 cells were incubated with 99m
Tc-DTPA-estradiol at 37°C for 360 min. Following two washings with PBS, the cells were
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incubated in culture medium for 60, 120, 180, 240, or 360 min, then collected and counted using
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the aforementioned methods.
For the blocking study, MCF-7 cells were incubated with 99mTc-DTPA-estradiol (4 nmol per well, 0.185 MBq) in the presence of no other estradiol, 40 nmol per well unlabeled DTPA-estradiol, or 10-fold estradiol (Xiyashiji, Sichuan, China) (40 nmol per well) at 37°C for 1 h and then treated as before. All tests were performed in triplicate.
Animal models
ACCEPTED MANUSCRIPT BALB/c mice (3–4 weeks) (Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technology), and BALB/c-nu mice (female, 3–4 weeks) (Beijing HFK
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Bioscience Co., Ltd., Beijing, China) were maintained at 22 ± 2°C with an alternating 12-h light/dark cycle. The nude mice were maintained in a barrier system room. All animal experiments
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were performed under a protocol approved by the Institutional Animal Care and Use Committee
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of Tongji Medical College, Huazhong University of Science and Technology.
MCF-7 breast tumor cells (5 × 107 cells) or MDA-MB-231 cells (1 × 107 cells) in 50 μL PBS and 50 μL basement membrane gel (Matrigel®, BD Biosciences, San Jose CA, USA), respectively,
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were injected subcutaneously into the right axilla. Mice underwent in vivo SPECT imaging or ex
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vivo biodistribution studies when the tumor size reached 0.6-1.2 cm in diameter.
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Ex vivo distribution experimentsin healthy and tumor-bearing mice
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For tissue distribution experiments, normal female BALB/c mice (n = 5 per group) and MCF-7and MDA-MB-231-tumor-bearing mice(n = 4 per group) were each injected with a 100-µL bolus containing 3.7–7.4 MBq (100–200 µCi, 0.1–0.2 µmol)
99m
Tc-DTPA-estradiol via the tail vein.
Normal animals were sacrificed under deep ketamine-xylazine anesthesia in groups of five at 0.5, 1, 2, 3, and 4 h post-injection. Tumor-bearing animals were sacrificed at 2 and 4 h post-injection. The tumor and organs or tissues (blood, heart, lung, stomach, liver, spleen, kidney, small intestine, large intestine, ovary, bone and muscle) were harvested, rinsed with PBS, wiped with filter paper, and weighed for the gamma-counter measurements. The radioactivity of each sample was measured, decay corrected to the injection time, normalized for injected dose and organ/tissue
ACCEPTED MANUSCRIPT weight, and expressed as percentage of injected dose per gram tissue (%ID/g). Tumor-targeting specificity was evaluated in the blocking group by the administration of a 10-fold excess of 99m
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Tc-DTPA-estradiol. At 4 h post-injection,
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unlabeled DTPA-estradiol 1 h prior to injection of
the mice were sacrificed and dissected, and the %ID/g was calculated as described above. The
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values were corrected for the injected dose and expressed as mean ± standard deviation (SD).
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SPECT imaging
Nude mice bearing MCF-7 or MDA-MB-231 tumors were injected with
99m
Tc-DTPA-estradiol
(7.4–11.1 MBq, 0.2–0.3 µmol) via the tail vein. For the blocking study, MCF-7 tumor-bearing
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mice were administered a 10-fold excess dose of non-labeled DTPA-estradiol 1 h prior to injection 99m
Tc-DTPA-estradiol. SPECT with a pinhole collimator (Symbia T6®; Siemens, Erlangen,
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of
Germany) was used for obtaining mouse images. Each anesthetized mouse was placed on the
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scanner table in the prone position. SPECT images were acquired at 2 and 4 h post-injection with
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an acquisition time of 10 min.
Statistical analysis
Quantitative data are presented as mean ± SD. For statistical classification, a Student’s t-test (2-tailed, unpaired) was performed using commercial software (Prism 5.0®, GraphPad Software, San Diego CA, USA and SPSS 13.0®, IBM, Armonk NY, USA). Differences were statistically significant when P <0.05. Pearson correlation was carried out between different organs for 99m
Tc-DTPA-estradiol.
ACCEPTED MANUSCRIPT RESULTS Precursor identification
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The structures of 3-acetonitrile estradiol and DTPA-estradiol were confirmed by 1H-NMR spectroscopy. For 3-acetonitrile estradiol, the 1H-NMR (CDCl3) showed peaks at δ 7.21 (dt, J =
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10.8, 5.4 Hz, 1H), 6.71 (td, J = 8.7, 2.6 Hz, 1H), 6.64 (dd, J = 9.1, 2.6 Hz, 1H), 4.20–3.95 (m, 2H), 3.81–3.63 (m, 2H), 3.16–3.02 (m, 1H), 2.90–2.78 (m, 2H), 2.32 (d, J = 13.2 Hz, 1H), 2.24–2.09
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(m, 2H), 2.00–1.62 (m, 7H), 1.54–1.16 (m, 8H), and 0.79 (s, 3H). Data for DTPA-estradiol (400 MHz, CDCl3-added D2O) showed peaks at: δ 7.21 (dd, 1H), 6.81 (t, 2H), 4.21–4.14 (m, 4H), 3.84–3.44 (m, 8H), 3.06 (s, 2H), 2.79 (m, 4H), 2.41–2.18 (m, 8H), 1.99–1.76 (m, 6H), 1.54–1.36
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(m, 6H), and 0.76 (s, 3H).
Radiolabeling identification and stability studies
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The radiolabeling yield of 99mTc-DTPA-estradiol was 64.5% ± 2.8%, and the radiochemical purity
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was 98.3% ± 1.3% (n=3). The specific activity of
MBq/µmol (n=3). The stability of
99m
Tc-DTPA-estradiol was 33.1 ± 1.5
99m
Tc-DTPA-estradiol was assessed by simultaneous
incubation with 1 mM histidine, 1 mM cysteine, PBS (pH = 7.4), and human serum at 37°C at 1, 3, 6, and 24 h (Figure 2). Little decomposition or dissociation of
99m
Tc-DTPA-estradiol into either
[99mTcO4]− or other subsidiary products was detected under these conditions.
Western blotting and immunofluorescence staining Western blotting analysis of ER expression in MCF-7 and MDA-MB-231 cells is shown in Figure 3A, in which the band at 66 kDa belongs to ER expression. Clear and indistinct bands at 66 kDa
ACCEPTED MANUSCRIPT were seen in MCF-7 and MDA-MB-231 cells, respectively, which suggested that ER was overexpressed in MCF-7 cells but not in MDA-MB-231 cells. In addition, in Figure 3B, a strong
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red fluorescence signal distribution was seen from the MCF-7 cells. In contrast, there was almost no red fluorescence signal from the MDA-MB-231 cells, further confirming that MCF-7 cells had
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high ER expression while MDA-MB-231 cells had low ER expression. This result suggested that
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MDA-MB-231 cells could be used as a relative negative control in this study.
In vitro assay
The binding affinity of 99mTc-DTPA-estradiol was also measured by incubating MCF-7 cells with
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increasing concentrations (1–200 nM) at 37°C, showing an equilibrium dissociation constant (KD)
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of 30.0 ± 4.1 nM (n=3) with a calculated maximal number of binding sites (Bmax) of 2.7 × 105 per cell (Figure 4A). The specific binding of
99m
Tc-DTPA-estradiol varied over the concentrations
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tested, ranging from 46.0% ± 4.2% to 90.6% ± 0.2% of total cell-associated uptake.
The blocking study showed that the specific binding of 99mTc-DTPA-estradiol could be blocked in the presence of a 10-fold excess of unlabeled DTPA-estradiol or 10-fold excess of estradiol, in MCF-7 cells (Figure 4B). At 1 h, the tracer uptake rates were only 3.23% ± 0.34% and 3.05% ± 0.23%, which were approximately 51.7% decreased and 54.4% decreased from unblocked conditions (P <0.05), respectively. These data confirm the specificity of binding to ER-positive MCF-7 cells.
99m
Tc-DTPA-estradiol
ACCEPTED MANUSCRIPT The cell uptake study revealed that
99m
Tc-DTPA-estradiol binds strongly to ER-positive MCF-7
cells, and very weakly to MDA-MB-231 cells. For
99m
Tc-DTPA-estradiol, 6.68% ± 0.20% of
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probe uptake in MCF-7 cells was measured during the first hour of incubation, followed by peak uptake at 6 h, with 8.07% ± 0.27% probe uptake identified (Figure 4C). The cell efflux study
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showed that 99mTc-DTPA-estradiol has good cell retention by MCF-7 cells, with only about 1.99% (decreased from 8.07% to 6.08% of total input radioactivity) of
99m
Tc-DTPA-estradiol efflux
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observed (Figure 4D). In contrast, significantly lower levels of total radioactivity were observed in both cellular uptake (4.95% ± 0.48%) and retention (4.21% ± 0.24%) during 6-hour incubation
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with the MDA-MB-231 cells (Figure 4C and4D).
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Biodistribution of 99mTc-DTPA-estradiol in normal and tumor-bearing mice To further evaluate 99mTc-DTPA-estradiol characteristics in vivo, its biodistribution in both tumor
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and non-tumor tissues of normal BALB/c mice and tumor-bearing mice was examined (Figure 5,
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Tables 1 and 2). The ratios of tumor-to-normal tissues or organs were also calculated (Table 2).
In MCF-7 cell models, tumor uptakes were 4.67 ± 0.39 %ID/g and 6.06 ± 0.38 %ID/g at 2 and 4 h post-injection, respectively (Figure 5A and B). In contrast, uptake within MDA-MB-231 tumors peaked 4 h after injection with 1.57 ± 0.28 %ID/g (P <0.05, Figure 5B). The tumor uptake of labeled DTPA-estradiol in the blocked mice was significantly lower than that in the unblocked mice (2.24 ± 0.28 %ID/g vs 6.06 ± 0.38 %ID/g, P <0.05; Figure 5C). To quantify 99m
Tc-DTPA-estradiol uptake in different tissues and therefore assess its specificity,
tumor-to-normal ratios for tissues and organs were calculated (Table 2). At 4 h post-injection,
ACCEPTED MANUSCRIPT 99m
Tc-DTPA-estradiol showed the highest tumor-to-normal ratios, with 2.93 ± 0.30 for blood and
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5.24 ± 1.23 for muscle.
Blood clearance of 99mTc-DTPA-estradiol was 5.14 ± 0.15 %ID/g at 0.5 h and 2.08 ± 0.22 %ID/g
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at 4 h. Large amounts of renal and hepatic accumulation (36.10 ± 3.35 %ID/g and 50.47 ± 4.49 %ID/g at 0.5h, 10.07 ± 0.91 %ID/g and 48.52 ± 3.35 %ID/g at 4 h, respectively) were
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observed, suggesting both renal and hepatic elimination may be involved in the pharmaceutical’s metabolism. Notably, moderate accumulation was also observed in the spleen and ovary.
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SPECT imaging
administration of
99m
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SPECT images of MCF-7 and MDA-MB-231 tumor-bearing mice were acquired at 2 and 4 h after Tc-DTPA-estradiol, and are shown in Figure 6. MCF-7 tumors were clearly
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visible with low background signal as early as 2 h post-injection, compared with little radioactive
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accumulation within MDA-MB-231 tumors at any of the tested time points. Under blocking conditions with excess unlabeled DTPA-estradiol, clearly reduced tumor uptake of 99m
Tc-DTPA-estradiol was observed (Figure 6). These results confirm the specificity of
99m
Tc-DTPA-estradiol for ER-positive tumors.
DISCUSSION Advances in molecular and cellular biology are transforming our understanding of basic physiology and pathology; similar advances in molecular imaging technologies now permit dynamic and quantitative studies in vivo with minimal invasiveness [10]. There is a need for an
ACCEPTED MANUSCRIPT accurate, affordable, and most of all, noninvasive method for assessing extramammary estrogen receptor status, which can avoid unnecessary biopsies and permit serial assessments during 99m
T
Tc-based estradiol derivative for ER
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endocrine therapy in breast cancer. We developed a
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imaging, and proved its targeting efficiency with in vitro and in vivo imaging.
To meet the need for quantifiable targeting to estrogen receptor sites, we focused on linear
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polyaminopolycarboxylic acid chelating agents [20]. We intended to enhance the water solubility of the estrogen receptor probe by introducing DTPA groups, expecting to minimize the intrahepatic metabolism. The chelator DTPA was introduced to estradiolvia DTPA anhydride
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under mild conditions of pH and temperatures. Previous studies have suggested that 3-acetonitrile
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estradiol could be produced via an ER-mediated process, and that 99mTc-labeled estradiol could be a useful agent for imaging ER status [21, 22]. Compared with previous estradiol probes,
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DTPA-estradiol is a small molecule with a suitable molecular weight. It does not alter
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biodistribution or tumor uptake patterns [21]. In addition, unlike other studies highlighting the difficulty of preparing 99m
99m
Tc-labeled derivatives with high specific activity or yields [9, 11],
Tc-DTPA-estradiol was prepared easily, with a satisfactory yield and labeling efficiency. The
in vitro experiments demonstrated that
99m
Tc-DTPA-estradiol is stable in histidine, cysteine, PBS
and human serum at 37°C for 24 h, with more than 90% remaining intact after 24 h of incubation.
Although we presented specific activity in this study, we could not present the effective specific activity (ESA), which is one of the most important characteristics of a receptor-binding radiopharmaceutical. ESA has often been emphasized for ER radioligands and has been an
ACCEPTED MANUSCRIPT important parameter for routine quality control to validate the synthesis procedures. There are direct and indirect methods for the measurement of ER binding. They are used routinely to
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determine relative binding affinity (RBA) and ESA of various cold and radiolabeled ER-binding compounds. Some researchers used scintillation proximity technology to develop a rapid
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screening assay for ER ligands with satisfactory results [23]. The assay requires tritiated E2 ([3H]-E2; New England Nuclear, Boston MA, USA). Unfortunately, we were unable to acquire
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this kit, or the instruments needed to measure tritiated E2, so we are unable to present the ESA.
The binding experiments showed that the binding affinity of
99m
Tc-DTPA-estradiol to ER
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receptors was 30.0 ± 4.1 nM, with specific binding resulting in over 45% of total binding.
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Classically, estrogen elicits genomic effects on transcription via α and β ERs, which are mainly located in the nucleus. Performing receptor-binding studies on whole cells with steroid analogs
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poses challenges related to ligand equilibrium, the lipophilic nature of the ligand, estimation of
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free ligand, the processes of receptor activation and degradation, and the presence of multiple estrogen receptor types [24]. As a result, careful consideration is required for data interpretation as one can easily over- or underestimate the KD [10].
99m
Tc-DTPA-estradioldemonstrated a KD
much higher than typically reported values of estradiol itself (0.1–1.0 nM)[25, 26], but was comparable to the affinity of a similar estrogen receptor (11 ± 1.5) nM[10]. Further, 99m
Tc-DTPA-estradiolalso displayed good retention in MCF-7 cells with a maximum of only 1.99%
effluxat 6 h.
SPECT
imaging
and
biodistribution
quantitative
analysis
in
nude
mice
showed
ACCEPTED MANUSCRIPT that99mTc-DTPA-estradiolaccumulated in ER-positive MCF-7 tumors with an uptake range of 4.67 ±0.39 %ID/g – 6.06 ±0.38 %ID/g. The tumor-to-normal ratios peaked at 4 h, with 2.93 ± 0.30 for
contrast,
the
tumor-to-normal
ratios
were
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blood and 5.24 ± 1.23 for muscle, allowing easy visualization of the ER-positive tumors. In significantly
in
masses
in
99m
Tc-DTPA-estradiol exhibited
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MDA-MB-231-tumor-bearing mice or blocked mice, where
lower
minimal tumor uptake at 1.57 ± 0.28%ID/g and 2.24 ± 0.28%ID/g, respectively.Previous research
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has shown that estradiol-based estrogen receptor probes can target the ER with good results. GAP-EDL was found to accumulate into ER+ 13762 (rat mammary carcinoma) tumors with a tumor-to-muscle ratio of 7.92 ± 0.56 at 4 h [21]; and 99mTc-estradiol-pyridin-2-yl has been shown
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to target MCF-7 tumors with a tumor-to-muscle ratio of 5.67 ± 1.06 at 3 h post-injection [10].
We initially intended to use DTPA to improve the metabolic pathways of the probe to overcome
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its high uptake in liver. However, the distribution studies revealed that 99mTc-DTPA-estradiol still
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accumulated mainly in the liver, with significant uptake in the spleen, the kidneys and the ovary. Attempts to develop a neutral
99m
Tc-estradiol derivative suitable for imaging of tumors
encountered problems with excessive lipophilicity [27]. Lipophilic compounds are usually characterized by slow elimination from the blood pool, resulting in high accumulation within extensively-perfused organs such as the liver and spleen [8, 9, 11].Additionally, steroid hormones (including estrogen) have been identified as stimulators of the reticuloendothelial system [28, 29], suggesting that phagocytosis within the liver and spleen may be another possible contributor towards 99m
such
a
phenomenon.
We
encountered
similar
problems.
The
uptake
of
Tc-DTPA-estradiol in the liver was 50.47 ± 4.49 %ID/g at 30 min and 48.52 ± 3.35 %ID/g at 4
ACCEPTED MANUSCRIPT h post injection, whereas the kidney, which we expected tobe the critical metabolic organ of the DTPA-chelated probecontained36.10 ± 3.35 %ID/g at 30 min and 10.07 ± 0.91 %ID/g at 4 h. In 99m
T
Tc-DTPA-estradiol for producing
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addition, the ovaries would be expected to accumulate
steroid hormones, especially estradiol [30, 31]. Because this is a preliminary study testing the
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imaging performance of this estrogen receptor targeted probe, ovarian accumulation of this probe
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was not studied, and the estrus cycles of all the female mice were not synchronized.
The major goal for cancer imaging is accurate disease characterization through the application of functional and molecular imaging studies.18F-FES PET cancer imaging has been immensely
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valuable to clinical oncologists for staging and visualizing primary and metastatic carcinomas [32].
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The supplemental molecular characterization and receptor-expression assessment of the tumor has often assisted in determining endocrine therapy options [7, 33]. As an alternative to PET, we
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sought to evaluate the utility of a
99m
Tc-DTPA-chelated estradiol as a SPECT tracer in breast
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cancer, which is much less expensive than 18F-FES.
So far, there are no readily available and easily synthesized SPECT agents for assessing reproductive cancers expressing estrogen-binding activity [10]. Despite the challenges and drawbacks, newer
99m
Tc-based estradiol derivatives have been reported, including the one in this
study. However, to date, all of them have failed to provide an alternative to 18F-FES PET, mainly attributable to low tumor uptake compounded with high background levels. To overcome the problems posed by such steroid analogs, the development of neutral nonsteroidal analogs that would specifically bind to each estrogen receptor subtype with high affinity would be a valuable
ACCEPTED MANUSCRIPT step [34, 35].
We developed a novel estradiol-based probe,
99m
Tc-DTPA-estradiol, that specifically binds to the
99m
Tc-DTPA-estradiol showed favorable characteristics in vitro and in vivo,
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estrogen receptor.
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CONCLUSION
demonstrating potential use in targeted imaging and therapy development in ER-positive tumors.
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Further studies should be performed to optimize the radiocompound for better pharmacokinetics in vivo, especially decreasing the uptake in the liver.
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Acknowledgments
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This work was supported by the National Natural Science Foundation of China (No. 30970853, 81071200), the Natural Science Foundation of Hubei Province of China for Distinguished Young
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Scholars (No. 2010CDA094). The authors are grateful to Prof. Guoping Yan and Dr. Biao Zhao at
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the School of Material Science and Engineering, Wuhan Institute of Technology, for their kind help in preparation of the labeling precursors.
Conflicts of interest The authors declare that they have no conflict of interest.
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ACCEPTED MANUSCRIPT [2] Huang L, Zhu H, Zhang Y, Xu X, Cui W, Yang G, et al. Synthesis and binding affinities of Re(I) and (99m)Tc(I)-containing 16alpha-substituted estradiol complexes: Models for potential breast
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ACCEPTED MANUSCRIPT Tables
1
2
4
Blood
5.14±0.15
4.23±0.29
3.50±0.19
2.46±0.34
2.08±0.22
Brain
0.21±0.06
0.22±0.08
0.18±0.09
0.18±0.02
0.15±0.04
Heart
1.78±0.09
0.99±0.21
0.67±0.22
0.68±0.05
0.63±0.16
Lung
5.68±0.32
5.03±0.17
3.83±1.12
3.08±0.22
2.53±0.58
Stomach
2.62±0.29
1.69±0.20
1.71±0.55
1.64±0.13
1.35±0.58
Liver
50.47±4.49
62.58±5.40
54.17±5.91
50.19±1.71
48.52±3.35
Spleen
12.04±2.39
10.45±0.67
8.74±1.46
7.25±0.68
6.50±0.65
Kidney
36.10±3.35
21.13±1.07
13.16±2.40
11.51±0.65
10.07±0.91
Small intestine
2.58±0.24
2.26±0.46
2.06±0.55
1.84±0.20
1.69±0.29
Large intestine
4.60±0.27
4.50±0.59
3.67±0.78
3.46±0.08
3.16±0.57
Ovary
2.20±0.35
2.47±0.10
3.06±0.42
3.16±0.21
3.34±0.08
1.93±0.08
1.86±0.38
1.41±0.30
1.38±0.15
1.07±0.16
1.17±0.24
1.15±0.42
1.12±0.15
1.09±0.13
0.98±0.37
Muscle
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Injected dose (100–200 µCi, 0.1–0.2 µmol per mouse)
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Bone
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Table 1 Biodistribution of 99mTc-DTPA-estradiol in normal BALB/c mice (n = 5, %ID/g ± SD)*
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Table 2 Biodistribution of 99mTc-DTPA-estradiol and blocking group in nude mice bearing MCF-7 and MDA-MB-231 tumor xenografts (n = 4, %ID/g ± SD)a MDA-MB-231
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MCF-7
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Blockingb (4h)
4h
Tumor
4.67±0.39
6.06±0.38
2.24±0.28
1.57±0.28
Blood
3.84±0.36
2.18±0.31
2.54±0.24
2.07±0.16
Brain
0.19±0.06
0.15±0.08
0.14±0.03
0.16±0.09
Heart
0.68±0.13
0.59±0.14
0.47±0.18
0.56±0.14
Lung
3.57±0.26
2.33±0.29
2.07±0.32
2.42±0.26
Stomach
1.81±0.29
1.02±0.22
0.85±0.22
1.09±0.17
46.33±3.25
38.25±5.25
42.38±2.38
6.97±1.05
6.88±1.47
7.19±0.72
59.91±12.22
Spleen
8.13±0.52
Kidney
12.92±1.10
9.57±0.98
10.27±0.39
10.35±1.71
Small intestine
1.76±0.14
1.40±0.14
1.32±0.17
1.45±0.18
Large intestine
3.26±0.45
3.06±0.18
2.13±0.13
2.98±0.40
3.36±0.23
3.56±0.17
2.43±0.25
3.35±0.40
1.20±0.19
1.19±0.25
1.09±0.07
1.22±0.04
1.21±0.17
1.19±0.19
1.09±0.29
0.98±0.13
Muscle
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Bone
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Liver
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2h
Uptake ratios
Tumor-to-blood
1.22±0.09
2.82±0.39
0.89±0.11
0.76±0.13
Tumor-to-muscle
3.94±0.70
5.24±1.23
2.05±0.12
1.63±0.33
a
Injected dose (100–200 µCi, 0.1–0.2 µmol per mouse) Blocking groupwas performed with 10-fold excess of cold DTPA-estradiol injected 1 h before injection of 99mTc-radiolabeled DTPA-estradiol. b
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Figure 1 Synthetic scheme of DTPA-estradiol
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Figure 2 Stability of 99mTc-DTPA-estradiol incubated at 37°C in 1 mM histidine, 1 mM cysteine,
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PBS, and human serum, with radiochemical purity assessed by TLC.
Figure 3 MCF-7 and MDA-MB-231 cells were stained with fluorescent anti-ER antibody (red) and DAPI (blue) and analyzed by microscopy (200×). Western blotting and immunofluorescence
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staining show positive ER expression by MCF-7 and almost no expression by MDA-MB-231
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cells.
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Figure 4 In vitro cell saturation binding, specific binding, uptake, efflux, and blocking study of
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Tc-DTPA-estradiol. (A) Saturation binding (black diamonds) and specific binding (red squares)
curves were generated by measuring the radioactivity in MCF-7 cells incubated with increasing concentrations of
99m
Tc-DTPA-estradiol as indicated. The inset is the Scatchard plot of the
saturation binding experiment. (B) In blocking studies, cell uptake of
99m
Tc-DTPA-estradiol
conjugates at 1 h was blocked in the presence of 10-fold excess of DTPA-estradiol (1, middle) and 10-fold excess of estradiol (2, right), respectively. (C, D) Cell uptake and cell efflux of 99m
Tc-DTPA-estradiol in MCF-7 and MDA-MB-231 cells were measured at the indicated time
points. All data were determined from three independent experiments and expressed as mean ± SD.
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Figure 5 Biodistribution of 99mTc-DTPA-estradiol in mouse xenograft tumors. (A) Biodistribution 99m
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Tc-DTPA-estradiol in nude mice bearing MCF-7 tumors at 2 and 4 h post-injection (n = 4)
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was assessed by uptake assay. (B) Tumor uptake of MCF-7 and MDA-MB-231 tumor-bearing
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mice at 4 h post-injection. (C) Comparison of tumor uptake in MCF-7 tumor-bearing mice at 4 h
Figure 6 SPECT imaging of
99m
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post-injection of 99mTc-DTPA-estradiol with/without excess cold DTPA-estradiol.
Tc-DTPA-estradiol in nude mice bearing MCF-7 and
MDA-MB-231 tumors, and blocked tumors with pre-injection of 10-fold excess unlabeled
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DTPA-estradiol. Arrows indicate tumors.
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Figure 5
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Figure 6