Development of a Novel 18F-labeled Probe for PET imaging of Estrogen Receptor β

doi:10.21203/rs.3.rs-1334238/v1

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Development of a Novel 18F-labeled Probe for PET imaging of Estrogen Receptor β

https://doi.org/10.21203/rs.3.rs-1334238/v1

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Purpose: Estrogen receptors beta (ERβ) is an important ER subtype and plays crucial roles in many physiological and pathological disorders. Herein, we aim to develop a probe, ¹⁸F-PVBO, for in vivo ERβ targeted PET imaging with promising results.

Methods:¹⁸F-PVBO was synthesized using a two-step radiolabeling method. The relative binding affinities of the reference compound PVBO towards ERα and ERβ were determined by a competitive radiometric binding assay using ³H-estradiol. Cytotoxicity and cell uptake were evaluated in ERβ-positive DU145 cells. PET imaging, including blocking study, was performed in DU145 tumor-bearing nude mice (n = 3 per group), and the biodistribution study of ¹⁸F-PVBO was also performed.

Results: The non-radioactive PVBO showed 12.46-fold stronger binding affinity to ERβ than to ERα in vitro. ¹⁸F-PVBO was synthesized with a 15-28% radiochemical yield (n = 5) within 40 min, and the radiochemical purity was >98%. The uptake of ¹⁸F-PVBO in DU145 cells was significantly blocked by ERB-041 (p < 0.05). The uptake of ¹⁸F-PVBO in DU145 xenografts increased during the 120 min dynamic scanning, with a maximum uptake of 2.80 ± 0.30% ID/g at 120 min. Based on time active curves (TACs), injection of ¹⁸F-PVBO with unlabeled PVBO or ERB-041 resulted in a significant signals reduction with the T/M ratio <1 at 30, 60, 75, and 120 min post-injection (p <0.05). Comparison of the %ID/g showed ¹⁸F-PVBO had a higher T/M ratio compared to ¹⁸F-FES in DU145 tumor-bearing mice at 60 min (1.65 vs. 1.28), 75 min (1.76 vs. 1.35), and 120 min (1.80 vs. 1.37) (p < 0.05).

Conclusion: ¹⁸F-PVBO shows 12.46-fold stronger binding to ERβ over ERα, with high radiochemical stability. It demonstrates the feasibility of noninvasively imaging ERβ positive tumors by small-animal PET and provides a new strategy for visualizing of ERβ in vivo.

Estrogen receptor β

18F

Positron emission tomography

Nuclear receptor

For decades, molecular imaging has brought significant impact to clinical diagnosis and treatment strategies. Functional receptors are the primary theranostic targets in nuclear medicine and have been actively investigated for targeted imaging and therapy in preclinical research and clinical practice. It is well established that estrogen receptor (ER), function as an essential nuclear receptor, is a valuable biomarker for noninvasive imaging of hormonal status[1]. ERα is the most widely studied ER at present[2]. Evidence proves the significance of ERα in determining the diagnosis and treatment of patients with estrogen-dependent cancers[3, 4]. Notably, ERα was regarded as the only ER form until ERβ was firstly cloned in 1996[2]. ERβ has a DNA-binding domain and ligand-binding domain that is 96% and 60% homologous with those of ERα, respectively[2], indicating that it may have similar but not identical functions. In addition to presenting in mammary epithelial and ductal cells, ERβ is detected in various tissues, such as subcutaneous adipose tissue[5], brain, and prostate[6]. Furthermore, changes in ERβ expression levels and downstream pathways are involved in regulating many physiological and pathological processes[7–9], such as the occurrence and development of the Parkinson’s disease[10], endometriosis[11], coronary heart disease[12], and diabetes[13]. All these findings highlight ERβ to be a promising biomarker for the early diagnosis of a variety of diseases.

With more evidence showing different functions of ERα and ERβ, independent imaging and quantification of them using molecular imaging technique are desired for defining their roles in living subjects. The food drug administration (FDA) - approved positron emission tomography (PET) probe, 16α-¹⁸F-17β-fluoroestradiol (¹⁸F-FES), for ER targeting shows 6.3 times binding affinity for ERα than that of ERβ[14]. It has been recognized as an ERα specific PET probe[15] and widely used to screen and evaluate breast cancer patients receiving endocrine therapy[16]. Given its much higher binding to ERα and relatively narrow indications, ¹⁸F-FES has limitations in the molecular imaging of ERβ and ERβ related diseases.

At present, applications for ERβ PET imaging have been rarely studied. Some attempts have been spent to develop ERβ PET probes, while all the results are disappointing. For example, Yoo J. et al. developed ¹⁸F-FEDNP, which has an 8.3-fold binding affinity preference for ERβ over ERα. Biodistribution studies were performed in the ER knockout mice, but the probe’s specific uptake levels were modest[17]. Lee J. K. et al. reported ¹⁸F-8BFEE₂ with high ERβ selectivity, but the attempts to improve in vivo targeting ability and biodistribution profile failed[18]. Antunes I. F. et al. reported ¹⁸F-FHNP had 3.5 times higher binding affinity for ERβ over ERα. The PET imaging was performed in ERα positive SKOV3 xenograft model, and ¹⁸F-FHNP showed 2-fold lower tumor uptake than ¹⁸F-FES[19]. The previous studies highlight the strong need to develop new generation PET probes for evaluating ERβ levels in vivo.

Previously, a series of diphenolic azoles were studied as highly selective ERβ agonists. The 7-position-substituted benzoxazoles were reported to have high ERβ binding selectivity. Among them, 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3-benzoxazol-5-ol (ERB-041) was reported to have the best selectivity (255.5-fold higher selectivity for ERβ over ERα)[20]. Herein, inspired by the above study, we developed a ¹⁸F-labeled small molecule PET probe based on ERB-041, ¹⁸F-2-(3-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-4-hydroxyphenyl)-4-vinylbenzo[d]oxazol-6-ol (¹⁸F-PVBO, Scheme 1), for ERβ imaging with a 12.46-fold higher selectivity over ERα. After getting satisfactory in vitro relative binding affinities of PVBO, ¹⁸F-PVBO was synthesized in high radiochemical yield and purity. The in vitro cell uptake in DU145 cells and in vivo DU145 xenografts PET/CT imaging and blocking experiment confirmed specific ERβ targeting ability of ¹⁸F-PVBO. In summary, by biological evaluation of ERβ-targeted PET probe, it has been found that ¹⁸F-PVBO performs as a specific targeting PET probe for ERβ in vivo imaging. Our finding provides a promising method for noninvasive imaging of ERβ.

Chemistry-General Methods

All of the reagents and materials were purchased from Sigma Chemical Co. (St. Louis, MO, USA), Bide Pharmatech Co. (Shanghai, China), and Energy Chemical Co. (Shanghai, China). Reactions took place opening to the atmosphere unless otherwise specified. The reactions were monitored by thin-layer chromatography (TLC) using 200 μM silica gel (China National Pharmaceutical Group Co., China) or Dionex UltiMate 3000 HPLC. ¹H and ¹³C NMR spectra were recorded on a Bruker Avance III 400 spectrometer. High-resolution mass spectra (HRMS) were performed on an Agilent G6520 Q-TOF mass spectrometer. ¹⁸F-Fluoride was produced using a Sumitomo 10MeV cyclotron. ¹⁸F-Fluoride was produced from ¹⁸O-water and the ¹⁸O(p,n)¹⁸F reaction. The synthesis and characterization of PVBO and the precursor are presented in supplemental materials.

In vitro relative binding affinity of the reference compound PVBO towards ERα and ERβ

Relative binding affinities were determined by a competitive radiometric binding assay using ³H-estradiol ([2,4,6,7-³H]-17β-estradiol, 70-115 Ci/mmol, Perkin-Elmer, Waltham, MA). Data are presented as the relative binding affinity (RBA) with the RBA of estradiol towards ERα and ERβ set to 100%. The experiment and data analysis were performed according to a previously described binding assay protocol[21].

Computational Details

Molecular docking for PVBO was performed against ERα using the highest resolution X-ray crystal structure (PDB entry 6VIG, 1.45 Å) by Schrodinger suit[22]. As for ERβ, molecular docking was performed with the crystal structure of ERβ complexed with ERB-041 (2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3-benzoxazol-5-ol) (PDB entry 1X7B), because PVBO was derived from ERB-041. All the water molecules as well as the ligand were removed by PyMOL. Before docking, the Schrödinger protein preparation wizard module was used to prepare the 3D structures of protein, optimize 3D structures with pH = 7.0, and restrain minimization converge heavy atoms to RMSD of 0.30 Å, using force field OPLS_2005. The docking grid of 20 Å was generated over the co-crystallized ligand with the Receptor grid generation module. PVBO and ERB-041 were pre-processed by the Ligprep, with Epik to generate the proper protonation states at pH 7.0, with at most 32 stereoisomers generated. The compounds were then docked to the protein using the “extra precision” glide docking.

Radiosynthesis

Starting from unlabeled precursor 12, ¹⁸F-PVBO was obtained using a two-step radiosynthetic method. The ¹⁸F-fluoride solution was passed through a QMA SepPak Light anion exchange cartridge (Waters) and eluted from the cartridge into a vial with MeCN (aq) (1.1 ml) containing Krypotofix 222 (13 mg) and K₂CO₃ (3 mg). The solvents were evaporated at 120 °C and then dried 3 more times by adding 1 ml of MeCN. Then, precursor 12 was dissolved in 1 ml of MeCN and added to the vial. The reaction system was sealed and heated at 120 °C for 20 min. After the solvents were evaporated, 1 ml of DCM and 0.25 ml of TFA were added, and the mixture was heated at 60 °C for 5 min to remove the methoxymethyl ether group. The product was purified by HPLC (Column: XBridge BEH C18 OBD Prep Column, 5 μm, 10 mm × 250 mm; eluent: acetonitrile in 0.3% phosphoric acid solution; flow: 3 ml/min.¹⁸F-FES was produced as previously described[23].

Distribution coefficients (Log D_7.4)

HPLC-purified ¹⁸F-PVBO (100 μCi/tube) was added to a mixture of n-octanol/PBS pH 7.4 (0.5 mL/0.5 mL). After vortexing at room temperature for 10 min, the tubes were shaken in a water bath at 37°C for 30 min. After standing for stratification, 0.2 mL of solution was drawn from both phases. The radioactivity was counted using an automated gamma counter (Wizard 2, model 2480, PerkinElmer). The experiments were performed in triplicate.

In vitro stability

Thestabilityof ¹⁸F-PVBO was measured in PBS or fetal bovine serum (FBS). Briefly, ¹⁸F-PVBO (100 μL, 37 MBq/mL) was added to 500 μL of PBS or FBS and incubated at 37 °C for 1 h or 2 h. Stability was evaluated by HPLC.

Cell culture and animal models

The human prostate cancer cell line DU-145 and the mouse embryonic fibroblast cell line NIH/3T3 were purchased from the American Type Culture Collection (Manassas, USA). Cells were cultured in the Minimum Essential Medium (MEM, Gibco, Carlsbad, USA) or Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, USA) supplemented with 10% FBS (Gibco, Carlsbad, USA) in a 37°C incubator with 5% CO₂.

Athymic female nude mice (6-8 weeks, n = 10, Slaccas, Shanghai, China) and female BALB/c mice (10-12 weeks, n = 15, Slaccas, Shanghai, China) were used in this study. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. After one week of acclimatization, DU-145 cells (1 ×10⁶ cells/mouse) were injected into the right thigh subcutaneously.

Cytotoxicity assay

The DU-145 cell line and NIH/3T3 cell line were cultured in MEM or DMEM supplemented with 10% FBS in a 37°C incubator with 5% CO₂. MTT was used to assess the cytotoxicity of different concentrations (0, 10, 25, 50, 75, and 100 μM) of the unlabeled PVBO on these two cell lines. Briefly, 100 μL of cells were seeded in a 96-well plate at a cell density of 5000 cells/well and incubated for 24 h. The cells were treated with PVBO at different concentrations (0, 10, 25, 50, 75, and 100 μM) and incubated for 24 h. Ten microliters of MTT solution were added to each well and incubated for another 4 h. Then, the MTT solution was discarded, 150 μL of DMSO was added to each well, and the absorbance at 490 nm was measured with a microplate reader (Synergy H1 Hybrid, Biotek, Winooski, USA).

Cell uptake

Cell uptake was performed using DU145 cells in the presence and absence of the ERβ selective drug ERB-041[20]. DU145 cells were seeded in 12-well plates at a cell density of 1×10⁵ cells/well and incubated for 24 h. Fresh serum-free medium (1 ml) containing ¹⁸F-PVBO (37 MBq) was added to each well. To inhibit the uptake of ¹⁸F-PVBO, DU145 cells were pretreated with the ERβ-selective ERB-041 for 1 h and then incubated with ¹⁸F-PVBO (37 MBq) at 37°C for 15, 30, 60, and 120 min. Each well was washed with PBS three times before cell lysis with NaOH. The results were measured using an automatic gamma counter (Wizard 2, model 2480, PerkinElmer). Cell uptake is presented as the percentage of the total added radioactivity dose.

Immunohistochemical (IHC) staining

DU145 tumors were fixed with formalin. IHC staining was performed on 4 μm sections taken from paraffin-embedded tumor tissues. After rehydration, the sections were incubated with a 1:200 dilution of the primary antibody, rabbit anti-estrogen receptor alpha antibody (ab3575, Abcam, Cambridge, UK) and rabbit anti-estrogen receptor β antibody (ab3576, Abcam, Cambridge, UK) at 4°C overnight. Then, the sections were incubated with a biotinylated secondary antibody (ab205718, Abcam, Cambridge, UK) and treated with Avidin-biotin-peroxidase. The 3-amino-9-ethylcarbazole substrate chromogen was used, followed by tissue counterstaining with hematoxylin. Sections were examined and photographed using a confocal microscope (Olympus, Tokyo, Japan)

Western blot

The membranes were incubated with 1:1000 dilutions of primary rabbit anti-estrogen receptor alpha (ab3575, Abcam, Cambridge, UK), rabbit anti-estrogen receptor β (ab3576, Abcam, Cambridge, UK) and anti-GAPDH (1:50000, 60004-1-lg, Proteintech, Rosemont, USA) antibodies at 4 °C overnight. The next day, after washing with PBS three times for 10 min each, the membranes were incubated with the secondary antibodies for 1 h at room temperature. Finally, the target proteins were visualized using a LAS4000 enhanced chemiluminescence system (GE Healthcare, Wisconsin, USA).

Micro-PET/CT

Micro-PET/CT (Inveon, Siemens Company, Germany) studies were carried out in DU145-bearing athymic female nude mice using ¹⁸F-PVBO or ¹⁸F-FES. The in vivo targeting specificity of ¹⁸F-PVBO was evaluated in a blocking study with the administration of unlabeled PVBO (5 μmol/kg) or ERB-041 (5 μmol/kg) 30 min before ¹⁸F-PVBO injection. The dose of ¹⁸F-PVBO was 7.02 ± 0.93 MBq per mouse. The dose of ¹⁸F-FES was 6.51 ± 0.64 MBq per mouse. A 120 min dynamic PET scan was immediately performed for 2 h after injecting the radiotracerthrough the tail vein. A 10 min CT scan was acquired for anatomic localization. The relative position of the organ was defined based on the anatomy of CT. The uptake values were expressed as the percentage of the injected dose per gram of tissue (%ID/g).

Biodistribution

BALB/c mice were anesthetized via inhalation of 2% isoflurane and injected with 3-5 MBq radiotracer through the tail vein for the biodistribution study. The mice were sacrificed at 30 min, 60 min and 120 min after injection. Blood was withdrawn by cardiac puncture, and the organs/tissues were collected, weighed and evaluated using an automatic gamma counter (Wizard 2, model 2480, PerkinElmer). Uptake values were expressed as percentage of injected dose per gram (%ID/g).

Histological analysis of major organs

The mice were sacrificed at 7 days after ¹⁸F-PVBO PET imaging. Tissue samples from the heart, liver, spleen, lung, kidney, and brain were collected for pathological examination. H&E staining of the major organs of nude mice was performed based on a standard procedure[24].

Statistical analysis

GraphPad Prism 5 (GraphPad Software, CA, USA) and SPSS 24.0 software (SPSS, Inc., Chicago, IL) were used for statistical analyses. The data are expressed as the mean ± standard deviation (SD). Differences between groups were analyzed using a two-sided unpaired Student’s t-test. Differences were considered statistically significant at p < 0.05 (*p < 0.05; ** p < 0.01; *** p < 0.001).

Probe synthesis

The tosylate-precursor compound 12 for radiofluorination and the non-radioactive reference compound PVBO were obtained by multiple organic synthesis steps including MOMBr and TBSCl hydroxyl group protection, nitration reaction, Wittig reaction, reduction reaction, aldoamine condensation, and fluorination reaction (Fig. 1). The products were characterized and confirmed by mass spectroscopy (MS) and nuclear magnetic resonance (NMR), including ¹H-NMR and ¹³C-NMR as listed in the experimental section. The purification of the compounds was performed by Semi-Preparative HPLC. The purity of the product was above 98%.

Radiochemistry and stability of ¹⁸F-PVBO

Starting from unlabeled precursor 12, ¹⁸F-PVBO was obtained using a two-step radiosynthetic method (Fig. 2a). The product was purified by HPLC (retention time of unlabeled PVBO = 13.91 min; retention time of ¹⁸F-PVBO = 14.14 min). ¹⁸F-PVBO was synthesized with a 15-28% radiochemical yield within 40 min (n = 5), and the radiochemical purity was higher than 98% (Fig. 2b). The specific activity was 7.5 GBq/μmol. The partition coefficient (LogD_7.4) was 0.96 ± 0.03.

HPLC analysis showed no other peaks after incubating ¹⁸F-PVBO in PBS or FBS for up to 2 h (Fig. 2c), indicating that the probe maintained high stability under physiological conditions.

In vitro binding affinity and cytotoxicity

The relative binding affinity (RBA) of the reference compound PVBO for ERα and ERβ was 0.52% and 6.48% relative to estradiol, respectively. PVBO showed a 12.46-fold higher selectivity for ERβ than for ERα. Cell viability was found to be over 90% after treatment with PVBO at concentrations as high as 100 μΜ, suggesting PVBO displayed low cytotoxicity towards DU145 cells and NIH/3T3 cells (Supporting fig. S1).

Molecular docking study

The docking score of PVBO against ERα (Fig. 3a) was -8.11 Kcal/mol, weaker than that of ERβ (Fig. 3b), -8.25 Kcal/mol, which was in consistent with the relative binding affinity data. Docking of ERB-041 against its own crystal structure receptor (self-docking) yielded the docking score of -8.30 Kcal/mol with the binding mode comparatively consistent with the crystal conformation (RMSD = 0.33) (Fig. 3c).

ERβ expression in DU145 cells and cell uptake of ¹⁸F-PVBO

The results of IHC showed that in DU145 xenografts, the expression of ERα was barely detectable, while the expression of ERβ was much higher (Supporting fig. S2). Western blotting results showed that compared with NIH/3T3 cells, DU145 cells had a significantly higher ERβ expression (Fig. 4a). The uptake of ¹⁸F-PVBO in DU145 cells increased with extending incubation times and reached 4.17±0.29% after 120 min. Importantly, in the presence of ERB-041, there was significant inhibition uptake of ¹⁸F-PVBO at 15, 30, 60, and 120 min (Fig. 4b, p < 0.05).

Micro-PET/CT in vivo imaging

Representative PET/CT images of ¹⁸F-PVBO and ¹⁸F-FES in DU145 tumor-bearing mice are shown in Fig. 5a and 5b. Based on the time activity curves (TACs) of ¹⁸F-PVBO, the tumor uptake showed a gradual upward trend over 120 min dynamic scan (Fig. 5d, n = 3). The maximum uptake was 2.80 ± 0.30% ID/g at 120 min. All images also showed high liver uptake along with intestinal excretion. As a comparison, ¹⁸F-FES showed maximum accumulation in the tumor at 10 min post-injection (PI), with a gradual decrease during 120 min of scanning (1.80 ± 0.13% ID/g at 120 min) (Fig. 5e, n = 3). As shown in Fig. 5c, in the pre-injected PVBO and ERB-041 groups, only weak tumor signals were recorded in PET images at 120 min. The TACs of the blocking experiment are shown in Fig. 5f and 5g (n = 3 per group). Based on the T/M ratio, co-injection of ¹⁸F-PVBO with unlabeled PVBO or ERB-041 resulted in a significant reduction of the T/M ratio at each time point (p < 0.05). Importantly, T/M ratios of ¹⁸F-PVBO were higher than ¹⁸F-FES in DU145 tumor-bearing mice at 60 min (1.65 vs. 1.28), 75 min (1.76 vs. 1.35), and 120 min (1.80 vs. 1.37) PI (Fig. 5h, p < 0.05).

Biodistribution of ¹⁸F-PVBO

To further evaluate the in vivo characteristics of ¹⁸F-PVBO, a biodistribution experiment was performed in BALB/c mice. ¹⁸F-PVBO showed high accumulation in the liver and heart, with a slow clearance in 120 min. The liver uptake was 21.21 ± 9.79% ID/g at 30 min PI and decreased to 14.02 ± 8.65% ID/g at 120 min PI. The uptake of the small intestine was 2.00 ± 0.89% ID/g at 30 min and increased to 12.02 ± 11.86% ID/g at 60 min PI, indicating clearance of ¹⁸F-PVBO through the liver-hepatobiliary system. The radioactive signals of the kidney and bladder were at a low level. Accumulation of ¹⁸F-PVBO in the brain was observed. Uptake in bone was found and reached 6.01 ± 4.58% ID/g at 120 min PI, indicating moderate defluorination of ¹⁸F-PVBO in mice (Fig. 6, Supporting table S1).

H&E staining

The mice were sacrificed 7 days after ¹⁸F-PVBO PET imaging. Tissue samples from the heart, liver, spleen, lung, kidney, and brain were collected for pathological examination. Based on H&E staining, no acute pathological changes were observed in these tissues (Supporting fig. S3).

The discovery of PET was a milestone in the development of modern imaging technology. At present, it is a highly powerful imaging method to detect cellular and molecular events in vivo, which reveals metabolic function through uptake of the radionuclide-labeled probe[25]. Although research on hormone ligands in nuclear medicine is in the ascendant, ERβ-targeted radioactive probes are not intensively investigated. Meanwhile, because of limited pathological information of ERβ, the choice of animal models for PET imaging of ERβ is difficult. Researchers even performed in vivo ERβ PET imaging using ERα positive SKOV3 xenograft model, but the result was not satisfactory because of the high expression of ERα in SKOV3 cells[19]. Similarly, the previously reported PET probe ¹⁸F-FEDNP tested in ER knockout mice, was regarded as not suitable for ERβ PET imaging[17]. Another study introduced ¹⁸F-8BFEE₂ to achieve good ERβ selectivity, it did not conduct further in vivo imaging[18]. To the best of our knowledge, no in vivo imaging study using ERβ positive model has been carried out, although some ligands have been reported to possess good ERβ selectivity[18]. DU145 cells were reported to express only ERβ[26, 27], and in our study, ERβ expression was verified by western blot and immunohistochemistry, implying DU145 xenografts can be used a model for ERβ PET imaging.

The goal of our work is to develop an ¹⁸F-labeled probe to enable noninvasive imaging of ERβ expression in living tissues. Based on previous research, the 7-position-substituted benzoxazoles attracted our attention[20]. Although Zhou et al. successfully converted the 7-position-substituted benzoxazoles compound 92 into ERβ selective probe through radiobromination, there was no further cell tests and in vivo PET imaging to investigate the performance of the probe[28]. In our study, before the radiosynthesis, a competitive radiometric binding assay was performed using ³H-estradiol, revealing a 12.46-fold higher selectivity of PVBO for ERβ over ERα. This value is lower than the reported value for ERB-041 (255.5-fold higher selectivity for ERβ)[29]. To gain insight into the binding nature of PVBO to ERβ, molecular docking simulations were performed. The docking score of PVBO against ERβ is worse than that of ERB-041, -8.25 Kcal/mol vs. -8.30 Kcal/mol, suggesting that the chemical modification of 7-position-substituted of benzoxazoles decreases the ERβ selectivity. Although PVBO adopts a similar binding mode to ERB-041 against ERβ regarding the ERB-041 part, the extended side chain of PVBO abolishes the hydrogen bond between Arg-346 in the ERβ and phenolic hydroxyl in the PVBO. Meanwhile, no significant cytotoxicity was found at a concentration as high as 100 µΜ, inspiring us to carry further radiolabeling and cell uptake studies.

¹⁸F-PVBO was successfully prepared by two-step radiosynthetic method based on the precursor 12. ¹⁸F-PVBO meets the requirements of PET/CT imaging as its high radiochemical purity, specific activity, high stability and low cytotoxicity. The uptake of ¹⁸F-PVBO in DU145 cells increased with extending incubation times due to their ERβ expression and could be significantly blocked by ERB-041, demonstrating the ERβ binding specificity of ¹⁸F-PVBO.

Inspired by the specific ERβ binding ability of ¹⁸F-PVBO, we further explored its PET imaging performance in DU145 tumor-bearing nude mice models. In vivo PET imaging successfully visualized ERβ positive DU145 xenograft tumors in mice models. The uptake of ¹⁸F-PVBO gradually accumulated in the tumor during dynamic scanning. Co-administration of PVBO or ERB-041 reduced tumor uptake at 2h PI by approximated 50% and 75%, respectively, demonstrating the ERβ targeting specificity of ¹⁸F-PVBO. ¹⁸F-FES possessed 2.5-fold higher selectivity for ERα and was considered to provide accurate information about ER expression[30]. In our study, at 2h PI, a 1.3-fold higher T/M ratio was observed of ¹⁸F-PVBO than ¹⁸F-FES, indicating that ¹⁸F-PVBO provides better tumor visualization ability for monitoring ERβ-positive tumors. Collectively, the in vitro and in vivo results showed that ¹⁸F-PVBO can be used to image ERβ positive tumors.

To further clarify the distribution of ¹⁸F-PVBO, a biodistribution study was performed using BALC/c mice. After injection of ¹⁸F-PVBO, the radioactive signal in the blood reached a high level at 30 min and then decreased rapidly at 60 and 120 min. The uptake in the bone was detected 30 min PI and reached a moderate level at 60 min PI, suggesting that ¹⁸F-PVBO was defluorinated in vivo[31], which may be related to the liver metabolism[30, 32]. Interestingly, it has been found that the bone uptake of ¹⁸F-labeled probes in mice is much higher than in primates[33]. Whether ¹⁸F-PVBO will be seriously defluorinated in primates needs further investigation.

Our study meets the urgent needs of noninvasive ERβ detection in living systems. Significant uptake and slow clearance were observed in the heart, which confirmed its future application in ERβ-related heart disease. The imaging data also showed that ¹⁸F-PVBO passed the blood-brain barrier, implying ¹⁸F-PVBO may provide an advantage for the diagnosis of ERβ-related intracranial diseases. Because of the lack of in vivo ERβ quantification method, research on the gradual change of ERβ in different disease models is complicated. ¹⁸F-PVBO has the potential to change such situation.

A novel ¹⁸F-labeled small molecule, ¹⁸F-PVBO, has been developed for PET imaging of ERβ expression. The probe shows 12.46-fold stronger binding to ERβ over ERα, with high radiochemical stability and low cytotoxicity. ¹⁸F-PVBO demonstrates the feasibility of noninvasively imaging ERβ positive tumors by small-animal PET. It may provide a new strategy for visualizing ERβ in vivo. Molecular imaging with PET/CT using ERβ-specific PET probes is expected to help understand the roles of ERβ in various diseases and facilitate the detection of ERβ in clinical setting.

Funding

This work was partially supported by the National Key Research and Development Program of China 2021YFC2100300 (JM), the National Natural Science Foundation of China under Grant No. 82071976 (HC) and 81901799 (CQ), Shanghai Pujiang Program (19PJ1411100) (HC), and Shanghai Municipal Science and Technology Major Project (HC, ZC), National Key Research and Development Plan Digital Diagnosis and Treatment Equipment Research and Development Project (2017YFC0113300) (XL) and Pudong Hospital, Fudan University, college project (YJYJRC202108/ YJYJRC202101/Zdzk2020-14) (XL). The authors wish to thank Haibing Zhou (School of Pharmaceutical sciences, Wuhan university) to provide ERβ protein. We thank Sheng Liang (Department of nuclear medicine, Xinhua hospital, Shanghai jiao tong university) and Shaoli Song (Department of nuclear medicine, Shanghai cancer center, Fudan University) for technical assistance in radiosynthesis of PET probes.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.

Author contribution

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Yujing Zhou, Peng Lei, Jiaxin Han, Zhiming Wang, Aiyan Ji, Yuyang Wu, Lingling Zheng and Xiaoqing Zhang. The first draft of the manuscript was written by Yujing Zhou, Peng Lei, Jiaxin Han and Zhiming Wang. The review and editing of the manuscript was completed by Jian Min, Weiliang Zhu, Zhijian Xu, Hao Chen and Zhen Cheng. Fundings were obtained by Chunrong Qu, Jian Min, Xingdang Liu, Hao Chen and Zhen Cheng. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics approval

Approval was obtained from the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Consent for publication

Not applicable.

Consent for interest

The authors have no relevant financial or non-financial interests to disclose.

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Scheme 1 is only available as a download in the Supplemental Files section.

Scheme1.jpg
Scheme 1 Design strategy for the ERβ-targeted PET imaging probe ¹⁸F-PVBO
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Development of a Novel 18F-labeled Probe for PET imaging of Estrogen Receptor β

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Abstract

Figures

Introduction

Material And Methods

Chemistry-General Methods

In vitro relative binding affinity of the reference compound PVBO towards ERα and ERβ

Computational Details

Radiosynthesis

Distribution coefficients (Log D7.4)

In vitro stability

Cell culture and animal models

Cytotoxicity assay

Cell uptake

Immunohistochemical (IHC) staining

Western blot

Micro-PET/CT

Biodistribution

Histological analysis of major organs

Statistical analysis

Results

Probe synthesis

Radiochemistry and stability of 18F-PVBO

In vitro binding affinity and cytotoxicity

Molecular docking study

ERβ expression in DU145 cells and cell uptake of 18F-PVBO

Micro-PET/CT in vivo imaging

Biodistribution of 18F-PVBO

H&E staining

Discussion

Conclusion

Declarations

Funding

Data availability

Code availability

Author contribution

Ethics approval

Consent for publication

Consent for interest

References

Scheme

Supplementary Files

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Distribution coefficients (Log D_7.4)

Radiochemistry and stability of ¹⁸F-PVBO

ERβ expression in DU145 cells and cell uptake of ¹⁸F-PVBO

Biodistribution of ¹⁸F-PVBO