Xiaotian Xia1, Hongyan Feng1, Chongjiao Li1, Chunxia Qin1, Yiling Song1, Yongxue Zhang2, Xiaoli Lan3. 1. Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022, China. 2. Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022, China. Electronic address: zhyx1229@163.com. 3. Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022, China. Electronic address: LXL730724@hotmail.com.
Abstract
INTRODUCTION: Most breast cancers express estrogen receptors (ERs). Noninvasive imaging of ER expression may be helpful for planning therapy of ER+ tumors. We developed a new ER- binding probe, (99m)Tc-labeled estradiol, with diethylenetriaminepentaacetic acid (DTPA) as a chelating ligand, and assessed its targeting ability in vitro and in vivo. METHODS: 3-Aminoethyl estradiol was synthesized in two steps from estrone, followed by (99m)Tc 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 of (99m)Tc-DTPA-estradiol. 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 (low ER expression) were injected with (99m)Tc-DTPA-estradiol, and underwent single-photon 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 was used to evaluate (99m)Tc-DTPA-estradiol biodistribution. RESULTS: The radiochemical purity of (99m)Tc-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 unlabeled DTPA-estradiol significantly reduced tumor uptake of (99m)Tc-DTPA-estradiol (2.24±0.28 %ID/g), suggesting that (99m)Tc-DTPA-estradiol specifically targets ERs in tumors. CONCLUSIONS: (99m)Tc-DTPA-estradiol can be synthesized with satisfactory labeling efficiency and stability. (99m)Tc-DTPA-estradiol specifically targeted ERs in vitro and in vivo with favorable pharmacokinetics, allowing ER receptor expression assessment with SPECT imaging.
INTRODUCTION: Most breast cancers express estrogen receptors (ERs). Noninvasive imaging of ER expression may be helpful for planning therapy of ER+ tumors. We developed a new ER- binding probe, (99m)Tc-labeled estradiol, with diethylenetriaminepentaacetic acid (DTPA) as a chelating ligand, and assessed its targeting ability in vitro and in vivo. METHODS:3-Aminoethyl estradiol was synthesized in two steps from estrone, followed by (99m)Tc 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 of (99m)Tc-DTPA-estradiol. 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 (low ER expression) were injected with (99m)Tc-DTPA-estradiol, and underwent single-photon 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 was used to evaluate (99m)Tc-DTPA-estradiol biodistribution. RESULTS: The radiochemical purity of (99m)Tc-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 unlabeled DTPA-estradiol significantly reduced tumor uptake of (99m)Tc-DTPA-estradiol (2.24±0.28 %ID/g), suggesting that (99m)Tc-DTPA-estradiol specifically targets ERs in tumors. CONCLUSIONS: (99m)Tc-DTPA-estradiol can be synthesized with satisfactory labeling efficiency and stability. (99m)Tc-DTPA-estradiol specifically targeted ERs in vitro and in vivo with favorable pharmacokinetics, allowing ER receptor expression assessment with SPECT imaging.