A Novel Estrogen Receptor α-Targeted Near-Infrared Fluorescent Probe for in Vivo Detection of Breast Tumor

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00684

The ability to detect breast cancer early in its progression is essential to improve patient survival and quality of life. The noninvasive and dynamic imaging and functional assessments of estrogen receptor-alpha (ERα), which is commonly expressed at high levels in breast cancer, are important for effective diagnosis and treatment. Hence, the development of a specific ERα-targeted probe is a major research goal. To that end, in the present study, we created a novel near-infrared (NIR) fluorescent probe, IRDye800CW-E₂, for targeted ERα imaging in breast-tumor-bearing mice. IRDye800CW-E₂ consisted of a cyanine dye IRDye800CW as the NIR fluorophore and the E₂ analogue ethinyl estradiol amine as an ERα targeting ligand. The ethinyl estradiol amine was initially labeled with fluorescein isothiocyanate (FITC) to evaluate the binding specificity to human breast-tumor cells in vitro. Flow chamber and in vitro confocal laser endomicroscopy imaging experiments demonstrated that FITC-E₂ was specifically taken up by MCF-7 cells. Furthermore, NIR fluorescence imaging revealed the ability of IRDye800CW-E₂ to rapidly target tumors and to achieve good contrast between tumors and background signal 4-48 h postinjection. The fluorescent signal of IRDye800CW-E₂ in tumors was successfully blocked by the coinjection of the endogenous ERα-ligand 17β-estradiol (E₂) and the probe. Ex vivo fluorescent imaging further confirmed high uptake of the probe by tumors. These results indicated that IRDye800CW-E₂ has great potential as an ERα-targeted imaging probe for early breast-tumor detection and has potential for clinical translation.

Full text of DOI:10.1021/acs.molpharmaceut.8b00684

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Article
A novel estrogen receptor #-targeted near-infrared
fluorescent probe for in vivo detection of breast tumor
Chu Tang, Yang Du, Qian Liang, Zhen Cheng, and Jie Tian
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00684 • Publication Date (Web): 04 Sep 2018
Downloaded from http://pubs.acs.org on September 6, 2018
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A novel estrogen receptor αꢀtargeted nearꢀinfrared fluorescent probe
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for in vivo detection of breast tumor
Chu Tang1,2‡, Yang Du2,3,4‡, Qian Liang 2, Zhen Cheng5*, Jie Tian1,2,3,4
*
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1Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education, School
of Life Science and Technology, Xidian University, Xi’an, Shaanxi 710126, China;
2CAS Key Laboratory of Molecular Imaging, The State Key Laboratory of Management and
Control for Complex Systems, Institute of Automation, Chinese Academy of Sciences, Beijing
100190, China;
3 Beijing Key Laboratory of Molecular Imaging, Beijing, 100190, China
4 University of Chinese Academy of Sciences, Beijing, 100080, China
5Molecular Imaging Program at Stanford (MIPS), Department of Radiology, and BioꢀX Program,
Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California,
94305ꢀ5344;
‡ These two authors contributed equally to this work
*Corresponding author: Jie Tian, Eꢀmails: jie.tian@ia.ac.cn, tian@ieee.org. Phone.: 8610
62527995, Fax: 86 10 62527995, Website: http://www.3dmed.net; Zhen Cheng, Eꢀmail:
zcheng@stanford.edu. Phone: 650ꢀ723ꢀ7866, Fax: 650ꢀ736ꢀ7925.
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ABSTRACT:
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The ability to detect breast cancer early in its progression is essential to improve
patient survival and quality of life. The noninvasive and dynamic in vivo imaging and
functional assessments of estrogen receptorꢀalpha (ERα), which is commonly
expressed at high levels in breast cancer, are important for effective diagnosis and
treatment. Hence, the development of a specific ERαꢀtargeted probe is a major
research goal. To that end, in the present study we created a novel nearꢀinfrared (NIR)
fluorescent probe, IRDye800CWꢀE2, for the targeted in vivo ERα imaging in breast
tumorꢀbearing mice. IRDye800CWꢀE2 consisted a cyanine dye IRDye800CW as the
NIR fluorophore and the E2 analogue ethinyl estradiol amine as an ERα targeting
ligand. The ethinyl estradiol amine was initially labeled with fluorescein
isothiocyanate (FITC) to evaluate the binding specificity to human breast tumor cells
in vitro. Flow chamber and in vitro confocal laser endomicroscopy imaging
experiments demonstrated FITCꢀE2 was specifically taken up by MCFꢀ7 cells.
Furthermore, in vivo NIR fluorescence imaging revealed the ability of
IRDye800CWꢀE2 to rapidly target tumors and to achieve good contrast between
tumors and background signal 4 ꢀ 48 h postꢀinjection. The fluorescent signal of
IRDye800CWꢀE2 in tumors was successfully blocked by the coinjection of the
endogenous ERα ligand 17βꢀestradiol (E2) and the probe. Ex vivo fluorescent imaging
further confirmed high uptake of the probe by tumors. These results indicated that
IRDye800CWꢀE2 has great potential as an ERαꢀtargeted imaging probe for the early
breast tumor detection and has potential for clinical translation.
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KEY WORDS: breast cancer, NIR fluorescent probe, ERαꢀtargeted imaging, in vivo
detection
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INTRODUCTION
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Estrogen receptorꢀalpha (ERα) is a nuclear hormone receptor responsible for
regulating physiological effects of signaling associated with endogenous estrogens,
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such as 17β
ꢀestradiol (E2), in diverse target tissues.1ꢀ3 Although the binding of ERα to
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its ligand E2 plays a critical role in female reproductive organs, aberrant ERα
signaling can mediate a hormone imbalance and is associated with several cancers,4,5
especially breast cancer.6,7 Nearly 75% of patients with breast cancer demonstrate
abnormally high expression of ERα.8 Therefore, ERα has emerged as a key target for
breast tumor therapy, and several ERα ligands have been developed as antagonists
against ERαꢀpositive [ERα (+)] breast cancer.9ꢀ11 Despite improvements in overall
survival for patients who receive endocrinotherapy, recurrence and metastasis are
serious problems.12,13 ERαꢀtargeted imaging can be used to identify suitable treatment
regimens, and hence can improve patient outcomes.
Currently, the primary molecular imaging techniques for ERα are single photon
emission computed tomography (SPECT), positron emission tomography (PET), and
magnetic resonance imaging (MRI) with the application of radiolabeling or
metalꢀchelating selective estrogen receptor modulators.14ꢀ17 These methods offer
excellent potential for quantitative imaging to determine the levels of ERα
expression18,19 but are not routinely used for breast cancer detection and diagnosis
owing to the poor specificity and high costs. Optical imaging is a promising molecular
imaging technology for cancer owing to its high sensitivity, high specificity, safety,
low cost,20ꢀ22 and ease compared to nuclear imaging and magnetic resonance imaging
modalities.23 Recently, a few ERαꢀtargeted fluorescent probes have been developed,
but they are mainly used to determine the binding affinity of ligands or the
localization of ER proteins in cells;24ꢀ26 the in vivo targeted imaging of ERα for breast
tumor detection has not been performed.
In the present study, we developed an ERαꢀtargeted molecular probe for ERα (+)
breast tumorꢀtargeted imaging in vivo. Based on an analysis of the crystal structure of
the ERαꢀligand binding domain (ERαꢀLBD) complexed with E2, the Aꢀring phenolic
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hydroxyl and Dꢀring hydroxyl of E2 form hydrogen bonds with helix 3/E353 and helix
11/H524, which are the key elements for maintaining highꢀaffinity ligand binding.1
Hence, the fluorophores cannot be directly conjugated to the hydroxyl groups of E2
but are coupled by the linking group. Herein, an E2 derivative (namely, ethinyl
estradiol amine, 4) with an amino linking group was prepared by click chemistry. To
quantitatively analyze the cellular uptake of 4 by ERαꢀoverexpressing breast tumors at
the cellular level, due to current detection equipment limitations, 4 was labeled with
fluorescein isothiocyanate (FITC) to be used in flow cytometry and the confocal
experiments. Fluorophores with emission in the nearꢀinfrared (NIR) region possess
less absorption and scatter from tissues much more efficiently than fluorophores
based on visible light;27,28 thus, for in vivo imaging using MCFꢀ7 [ERα (+)] tumor
xenografts, 4 was labeled with the NIR dye IRDye800CW to yield IRDye800CWꢀE2.
Our results demonstrated that IRDye800CWꢀE2 is a sensitive and efficient NIR
fluorescent probe for ERα expression visualization in ERα (+) breast tumors in vivo
with high clinical translation potential.
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MATERIALS AND METHODS
Materials. All reagents were obtained from Aldrich (St. Louis, MO, USA), Acros
(Geel, Belgium), Aladdin Reagent (Shanghai, China), and AlfaꢀAesar (Haverhill, MA,
USA). No additional purification was performed. Analytical thinꢀlayer
chromatography (TLC) was used for reaction monitoring, with visualization
performed via UV light (254 nm).
A Bruker Biospin AV400 (400 MHz, 1H NMR; 100 MHz, 13C NMR) instrument
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was utilized to measure the 1H NMR and C NMR spectra. Chemical shifts (in ppM)
were in reference either to tetramethylsilane or the solvent. Highꢀperformance liquid
chromatography (HPLC) allowed for confirmation of >95% purity of all compounds
used in a biological context.
Cell lines. MCFꢀ7 cells (ATCC, USA) and normal breast epithelial MCFꢀ10A cells
(Chinese Academy of Sciences) were grown in a 37 °C 5% CO2 incubator in
Dulbecco’s Minimum Essential Medium (DMEM, Hyclone, Thermo Scientific)
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supplemented with 10 % fetal bovine serum (FBS, Hyclone, Thermo Scientific) as
well as penicillin and streptomycin.
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In vivo tumor model. The Institutional Animal Care and Use Committee of Peking
University approved this study (Permit No: 2011ꢀ0039). For in vivo tumor assays,
female BALB/c mice (6ꢀweekꢀold, 15ꢀ18 g; Beijing Vital River Laboratory Animal
Technology Co., Ltd) received axillary subcutaneous transplants of MCFꢀ7 cells.
Synthesis of ethinyl estradiol amine (4). NaN3 (90 mg, 1.38 mmol) was mixed
into a solution of 3ꢀchloropropylamine hydrochloride 2 (60 mg, 0.46 mmol) in H2O2
(2 mL). This solution was brought to 80 °C for 12 h, cooled, basified by KOH (1
mol/L) to pH 9, and diethyl ether was employed for extraction (3 × 10 mL). Na2SO4
was utilized for organic layer drying, and this layer was concentrated with a flow of
nitrogen to obtain azide 3 as a white oil. The unpurified residue was used in the
following reaction.
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A mixture of azide 3 (10 mg, 0.10 mmol) and ethinyl estradiol 1 (30 mg, 0.10
mmol) in tBuOH (500 ꢁL) was supplemented with H2O (500 ꢁL), ascorbic acid (2 mg,
11 ꢁmol), and CuSO4⋅5H2O (3 mg, 12 ꢁmol), and was stirred at 25 ºC for 12 h. This
crude mixture was diluted using ethyl acetate (40 mL) and 4:1 saturated
NH4Cl/NH4OH (40 mL). The organic layer was separated and washed with 4:1
saturated NH4Cl/NH4OH (3 × 40 mL), dried, and concentrated under a vacuum to
yield 24 mg (61.7%) of ethinyl estradiol amine 4 as a white solid. 1H NMR (400 MHz,
CD3OD): δ 7.84 (s, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.50 (d, J = 8.4 Hz, 1H), 6.47 (s,
1H), 4.44ꢀ4.49 (m, 2H), 2.71ꢀ2.78 (m, 3H), 2.43ꢀ2.501 (m, 1H), 2.072.12 (m, 4H),
1.80ꢀ2.004 (m, 43H), 1.51ꢀ1.64 (m, 3H), 1.35ꢀ1.43 (m, 2H), 1.24ꢀ1.32 (m, 1H), 1.04
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(s, 3H), 0.65ꢀ0.70 (m, 1H); C NMR (100 MHz, CD3OD): δ 154.53, 154.12, 137.42,
131.03, 125.75, 122.76, 114.70, 112.32, 81.82, 48.20, 46.96, 43.50, 39.64, 37.08,
32.97, 29.33, 27.33, 26.13, 23.25, 13.56. MS (ESI): m/z 317.26 ([M+H]+).
Synthesis of FITCꢀE2 conjugate (5). A FITC solution (0.8 mg, 2.1 ꢁmol) in dry
DMSO (2 mL), 4 (0.9 mg, 3.1 ꢁmol) and Et3N (50 ꢁL) were added and stirred in the
dark at 37 ºC for 12 h. Subsequently, a Cꢀ18 HPLC column (250 mm × 10 mm) was
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used to purify this crude product using gradient elution with CH3CN and H2O as
solvents A and B, respectively. The purified FITCꢀE2 was obtained as a yellow solid
with 81.9% yield and over 98% purity by analytical HPLC. MS (ESI): m/z 699.78
([M]+).
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Synthesis of IRDye800CWꢀE2 conjugate (6).
To
a
solution of
IRDye800CWꢀNHS (0.5 mg, 0.43 ꢁmol) in dry DMSO (2 mL), 4 (0.2 mg, 0.64 ꢁmol)
and Et3N (30 ꢁL) were added, stirring protected from light at 37 ºC for 24 h, followed
by trifluoroacetic acid (50 ꢁL) quenching. Subsequently, a Cꢀ18 HPLC column (250
mm × 10 mm) was used for crude product purification using gradient elution with
CH3CN (0.1% TFA) and H2O as solvents A and B, respectively. The purified
IRDye800CWꢀE2 was obtained as a yellow solid with 81.9% yield and over 98%
purity by analytical HPLC. MS (ESI): m/z 1295.418 ([MꢀH]ꢀ).
In Vitro Cellular Uptake Assays. In order to quantitatively assess breast cancer
cell FITCꢀE2 uptake, MCFꢀ7 and MCFꢀ10A cells received several concentrations of
FITCꢀE2 or FITC for 3 h, followed by washing with iceꢀcold PBS. Finally, the cells
were dispersed in iceꢀcold PBS and used for flow cytometry, cellular uptake, and
fluorescence measurements.
In vitro Confocal Microscopy Images. 4 was labeled with FITC to yield probe
FITCꢀE2, which was used for the flow chamber and confocal microscopic experiments.
MCFꢀ7 cells grown in sixꢀwell plates were treated with 0.5 ꢁM FITCꢀE2 or 50ꢀfold
excess E2 was added as blocking agent for 3 h. The cells were then washed using
iceꢀcold PBS prior to rhodamine phalloidin and DAPI staining. The fluorescence
signals were then recorded via confocal laser scanning microscopy (Leica
Microsystems).
In vivo Confocal Laser Endomicroscopy (CLE) Imaging. Cellvizio was used for
CLE imaging with dual band excitation wavelengths of 488 and 660 nm. To conduct
in vivo CLE imaging, FITCꢀE2 was injected into mice bearing MCFꢀ7 tumors.
Fluorescence images were acquired before injection and at 2, 4, 6, 12, 24, and 48 h
later with the UltraMiniO microprobe. Moreover, 12 h after the probe injection, Evans
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blue was injected via the tail vein to the blood vessel for 15 min, and then the
confocal probe was directly inserted into the tumor by a superficial skin incision to
scan the signal with a Sꢀ1500 microprobe.
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Estrogen Receptor Binding Affinity. A fluorometric binding assay was employed
to assess binding affinities, as described in past studies29,30. Relative binding affinity
(RBA) values were used to report these results, based on an E2 RBA of 100%. Each
measurement was performed in triplicate.
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Molecular modeling. The Protein Data Bank was queried to obtain the ERα crystal
structure (PDB: 2P15)31, and water molecules were removed. The crystallographic
coordinates of 6 was prepared using Biochemoffice. AutoDockTools (ADT) were
used for ligand and protein performance, with AutoDock (version 4.2) used for all
molecular docking studies.32,33 PyMOL (Erwin Schrödinger) was used to prepare
figures.
In vivo Fluorescent Imaging. Fluorescent imaging was conducted to assess the
tumorꢀtargeting effects and the in vivo IRDye800CWꢀE2 probe biodistribution. For
fluorescence imaging studies, 10 nmol IRDye800CWꢀE2 was intravenously injected
into the tumorꢀbearing mice (n = 3); images were acquired prior to injection and at 2,
4, 6, 12, 24, and 48 h after injection via IVIS spectrum imaging system. For blocking
studies, tumorꢀbearing mice (n = 3) were coinjected with 10 nmol IRDye800CWꢀE2
and 100 nmol endogenous E2. Regions of interest (ROI) of the tumors and adjacent
normal tissues were measured, and then the ratios of tumor to normal tissue
fluorescence signals were determined. After 48 of imaging, dissection of the tumor
and major organs was performed to facilitate ex vivo imaging acquisition. ROI
analyses of the average fluorescence signals were also performed with the IVIS
spectrum imaging system.
Statistics. Data are given as means ± SD of triplicate experiments. Oneꢀway
ANOVAs followed by Tukey test were used to compare differences between groups,
while twoꢀtailed Student’s t tests were used to compare two groups. *P < 0.05 was the
threshold of significance.
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RESULTS AND DISCUSSION
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Chemical Synthesis. FITCꢀE2 5 and IRDye800CWꢀE2 6 were synthesized by
conjugating FITC or the NHSꢀactivated ester of IRDye800CW to the amino of ethinyl
estradiol amine 4 in the presence of triethylamine, as shown in Scheme 1. Key
intermediate 4 was prepared by utilizing the azideꢀalkyne click chemistry reaction of
ethinyl estradiol 1 and azide 3 in the presence of the Cu(I) catalyst, and organic azide
3 was synthesized by reacting 3ꢀchloropropylamine 2 with NaN3 at 80 °C in H2O for
12 h.
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Scheme 1. Synthesis of FITCꢀE2 and IRDye800CWꢀE2. Reagents and conditions: (a) NaN3, H2O,
80 ºC, 12 h; (b) ascorbic acid, CuSO4·5H2O, tBuOH/H2O, 25 ºC, 12 h; (c) FITC, TEA, DMSO, 37
ºC, 12 h; (d) IRDye 800CWꢀNHS, TEA, DMSO, 37 ºC, 12 h.
Cellular uptake study. Flow cytometry was used for the quantitative analysis of
cellular uptake and fluorescence enhancement by 4. Flow chamber experiments
showed that the percentage of cellular uptake and the mean fluorescence intensity of
FITCꢀE2 by MCFꢀ7 cells were significantly higher than free FITC (Fig. 1A, 1B), but
there were no statistically significant difference between FITCꢀE2 and FITC in
MCFꢀ10A (Fig. 1C, 1D). These results indicated that 4 possessed active breast
tumorꢀtargeting effects, and FITCꢀE2 can specifically bind to breast cancer cells.
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Figure 1. Examination of the cellular uptake of FITCꢀE2 by flow cytometry. (A) Quantitative
analysis of FITCꢀE2 and FITC uptake in MCFꢀ7 cells by flow cytometry; (B) Fluorescence
intensity of FITCꢀE2 and FITC in MCFꢀ7 cells; (C) Quantitative FITCꢀE2 uptake analysis in
MCFꢀ10A cells; (D) Fluorescence intensity of FITCꢀE2 in MCFꢀ10A cells. *P < 0.05, **P < 0.01.
In vitro confocal imaging. To further study the specific intracellular uptake of
FITCꢀE2, confocal laser microscopy was applied to visualize and track FITCꢀE2
within MCFꢀ7 tumor cells. After exposure of tumor cells to FITCꢀE2 for 4 h, the actin
cytoskeleton and nuclei of MCFꢀ7 cells were stained with rhodamine phalloidin and
DAPI. FITCꢀE2 was taken up by MCFꢀ7 cells (Fig. 2), as seen in the flow cytometry
results. The cellular uptake of FITCꢀE2 can be effectively blocked by coꢀincubation
with E2 (Fig. 2B), which further confirmed the targeting specificity of FITCꢀE2 for
MCFꢀ7 cells.
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Figure 2. Targeting specificity of FITCꢀE2 in vitro. Rhodamine phalloidin (red) and DAPI (blue)
were used to stain the actin cytoskeleton and nuclei, respectively. (A) MCFꢀ7 cells incubated in
FITCꢀE2; (B) MCFꢀ7 cells coꢀincubated in FITCꢀE2 with 50ꢀfold excess E2. Scale bar represents
10 µm.
In vivo confocal laser endomicroscopy (CLE) imaging. To further verify the
specific uptake of FITCꢀE2 by breast tumor tissues, probeꢀbased confocal laser
endomicroscopy (pCLE), a recently developed approach offering realꢀtime, in vivo
and very high magnification images, was used to evaluate MCFꢀ7 tumor xenografts
(Fig. 3 and 4). After the intravenous injection of FITCꢀE2, fluorescence signals can be
detected at the tumor region as early as 2 h, and the fluorescence signal can be
detected for 48 h postꢀinjection (Fig. 3A). Most FITCꢀE2 was rapidly cleared from
adjacent healthy tissues within 4 h (Fig. S1, Fig. 3B). At 12 h, there was a sufficiently
high tumor/normal tissue fluorescence signal ratio to clearly discern the tumor region,
and the probe FITCꢀE2 could be observed mainly in the tumor cytoplasm (Fig. 4A). In
contrast, there was no obvious signal in adjacent healthy tissues (Fig. 4B). These
results indicated that the FITCꢀE2 can efficiently traffic via blood vessels to target
tumor tissues and possessed in vivo targeting capability.
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Figure 3. Probeꢀbased confocal laser endomicroscopy fluorescent images for the UltraMiniO
microprobe; green fluorescence is the FITCꢀE2 signal. (A) Tumor tissue; (B) adjacent normal
tissues. Scale bar represents 20 µm.
Figure 4. The probeꢀbased confocal laser endomicroscopy fluorescent images of tumor with
Sꢀ1500 microprobe at 12 h postꢀinjection of probe FITCꢀE2. The green signal is the fluorescence
of FITCꢀE2, and blood vessel was stained with Evans blue shown in red. (A) Tumor tissue; (B)
adjacent normal tissues. Scale bar = 50 µm.
The Binding Affinity of the NIR Probe IRDye800CWꢀE2. These results
suggested the possible application of 4 for in vivo fluorescence imaging. To fully
examine the in vivo tumor targeting effects and biodistribution of 4, conjugation to the
IRDye800CW was conducted yielding IRDye800CWꢀE2 as a NIR probe.
Currently, a major challenge in the development of steroid (steroidal)ꢀbased
ERꢀtargeted fluorescent probes or drug delivery systems is the maintenance of a high
affinity for ER and subtype selectivity. Most steroidalꢀconjugates show extremely low
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ER binding affinity and weak subtype selectivity because conjugating a functional
group to steroids often decreases the ER binding affinity,34ꢀ36 and can even lead to the
loss of affinity for small linkers to steroid molecules.37 Katzenellenbogen et al.
proposed that the relative binding affinity (RBA) for such ERꢀtargeted conjugates
should be at least 1% of the value for E2 (RBA = 100%).38 Therefore, we first used a
competitive fluorometric receptor binding assay to test the ERα and ERβ binding
affinity of IRDye800CWꢀE2 probe (Table 1). The RBA is used to present these
affinities, where E2 was set as 100%. Our results showed that 4 had good ERα binding
affinity, with ERα and ERβ RBA values of 4.44 and 2.53, respectively, and an overall
ERβ/ERα selectivity of 1.7 (Table 1). More interestingly, conjugating the
IRDye800CW moiety to 4 not only obviously increased the ERα binding affinity of
the probe IRDye800CWꢀE2, but also exhibited higher subtype selectivity for ERα
(RBA values were 8.06 for ERα and 0.53 for ERβ, α/β was as high as 15.2, Table 1).
Table 1. ERα and ERβ relative binding affinitiesa
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RBAa (%)
ERα ERβ
Kib (nM)
Compound
α/β
1.7
ERα
ERβ
α/β
1.9
4.44 ± 0.18 2.53 ± 0.32
8.06 ± 0.32 0.53 ± 0.05
69.82
38.46
134.39
641.51
4
15.2
16.6
IRDye800CWꢀE2
aRelative binding affinity (RBA) values were calculated based on the results of a competitive
compound
fluorometric receptor binding assay and are expressed as IC50estradiol/IC50
× 100 ± the
standard deviation (RBA, estradiol = 100%). bKi values of each compound for each receptor were
obtained for the RBA values by formula Ki = (100/RBA) × Kd. The estradiol Kd values are 3.1 nM
on ERα and 3.4 nM on ERβ.29
Molecular Docking analysis of probe binding. To understand how the probe fit
into the ERα ligand pocket, we performed a molecular docking study with
IRDye800CWꢀE2 in ERα (PDB: 2P15) to examine the binding mode.31 Consistent
with E2 (Fig. 5A), IRDye800CWꢀE2 also can bind in an optimal conformation in the
ERα active pocket (Fig. 5B), and phenolic hydroxyl of the steroidal ring forms critical
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hydrogen bond contacts with helix 3/E353 and the fluorophore unit also exhibits a key
interaction with helix 11/H524 (Fig. 5C); in addition, the IRDye800CW group forms
additional hydrogen bonds with A340 residue (Fig. 5C). This may explain why
conjugating the fluorophore IRDye800CW to E2 does not affect the ERα binding
affinity.
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Figure 5. Molecular modeling of IRDye800CWꢀE2 bound to ERα, and E2 comparisons. (A) ERα
LBD in complex with E2 crystal structure (1ERE).1 Hydrogen bonds are present between E2 and
E353, R394, H524. (B) Computer modeling of the complex of IRDye800CWꢀE2 bound to ERα
with the conserved Hꢀbond to A340, T347, E353 and H524. (C) IRDye800CWꢀE2 binding deep
into the ERα active pocket.
In vivo NIR fluorescent imaging of IRDye800CWꢀE2. After the intravenous
injection of 10 nmol IRDye800CWꢀE2 in nude mice that had received MCFꢀ7 human
breast tumor xenografts, fluorescence images were dynamically monitored at various
time points. The probe IRDye800CWꢀE2 rapidly targeted tumor areas; fluorescence
signals were detected as early as 4 h, reached a peak at around 12 h, and persisted for
48 h postꢀinjection. (Fig. 6A). The tumorꢀtoꢀbackground ratios (TBR) were 1.78 ±
0.20 at 2 h, 3.34 ± 0.46 at 12 h, and 2.43 ± 0.19 at 48 h (Fig. 6C). To further determine
the specificity of IRDye800CWꢀE2 targeting to ERα, a blocking study was also
performed by the coꢀinjection of IRDye800CWꢀE2 in combination with the ERα
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ligand E2. As shown in Fig. 6B, the tumor uptake of IRDye800CWꢀE2 decreased
dramatically after blocking compared to that of the imaging group. Moreover, the
TBR in the blocking group was also lower than that in the imaging group, confirming
the specificity of IRDye800CWꢀE2 for ERα.
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After the in vivo observations, the ex vivo tumors and major organ imaging was
conducted. As shown in Fig. 6D, a higher fluorescence signal was evident within the
tumor after injection with the IRDye800CWꢀE2 probe in the imaging group than the
signal in the blocking group. The average radiant efficiency of tumor fluorescence
signals in the blocking group as 64.2% lower than that in imaging group (Fig. 6E).
The efficient blocking study further confirmed the specific binding of
IRDye800CWꢀE2 to ERα. The fluorescence signal for IRDye800CWꢀE2 was also
detected in the liver and kidney, indicating the elimination of IRDye800CWꢀE2
mainly through the kidney and, to a lesser extent, through the liver.
Figure 6. In vivo fluorescence images and the biodistribution of IRDye800CWꢀE2 in MCFꢀ7
tumorꢀbearing nude mice. (A) Representative mouse NIR fluorescence images before injection
and at 2, 4, 6, 12, 24, and 48 h after the injection of 10 nmol IRDye800CWꢀE2; (B) Representative
NIR fluorescence images of mice with excess E2 blocking; (C) Region of interest analysis of
tumor to normal tissue fluorescence signal ratios of IRDye800CWꢀE2 in the imaging and blocking
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groups; (D) Representative ex vivo organ NIR fluorescence images (Labels: T, tumor; H, heart; L,
liver; S, spleen; L, lung; K, kidney; I, intestines) at 48 h postꢀinjection of 10 nmol
IRDye800CWꢀE2 with (blocking group) or without (imaging group) 100 nmol E2; (E) Quantitative
analysis of fluorescence intensity of dissected organs. *P < 0.05.
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CONCLUSION
In the present study, we have succeeded in the design and synthesis of the novel
ERαꢀtargeted NIR fluorescent probe IRDye800CWꢀE2 for targeted ERα (+) breast
tumor imaging in vivo. This is, to the best of our knowledge, the only application of
IRDye800CWꢀE2 as a fluorescent probe for the in vivo imaging of ERα for breast
tumor detection to date. The IRDye800CWꢀE2 probe exhibited good ERα binding
affinity. For in vivo imaging studies, IRDye800CWꢀE2 was able to efficiently target
ERα with high imaging sensitivity and specificity and good tumorꢀtoꢀbackground
ratios in MCFꢀ7 xenograft tumors. Our results indicated that IRDye800CWꢀE2 shows
potential to efficiently visualize ERα levels in breast tumors and enable the early
detection and treatment of breast cancer, offering potential for clinical translation.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI:
Cartoon displays how to select adjacent normal tissue and H NMR and 13C
1
NMR spectral information for compound 4 (PDF)
AUTHOR INFORMATION
Corresponding Authors
Jie Tian, Eꢀmails: jie.tian@ia.ac.cn, tian@ieee.org. Phone.: 8610 62527995, Fax: 86
10 62527995, Website: http://www.3dmed.net;
Zhen Cheng, Eꢀmail: zcheng@stanford.edu. Phone: 650ꢀ723ꢀ7866, Fax:
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650ꢀ736ꢀ7925.
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Notes
The authors declare no conflicts of interest.
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ACKNOWLEDGEMENTS
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This paper is supported by Ministry of Science and Technology of China under Grant
No. 2017YFA0205200, 2017YFC1309100, 2016YFC0102000, the National Natural
Science Foundation of China under Grant No. 81470083, 81527805, 61231004,
81601548, 81772011, 81701771, the International Innovation Team of CAS under
Grant No. 20140491524, Beijing Municipal Science & Technology Commission No.
Z161100002616022, China Postdoctoral Science Foundation funded project
(2017M613084), Shaanxi Postdoctoral Science Foundation funded project
(2017BSHEDZZ91).
ABBREVIATIONS
ERα, estrogen receptorꢀalpha; E2, 17βꢀestradiol; NIR, nearꢀinfrared; ERαꢀLBD,
ERαꢀligand binding domain; TLC, analytical thinꢀlayer chromatography; TEA,
triethylamine; HPLC, highꢀperformance liquid chromatography; MS, mass
spectrometry; DMEM, dulbecco minimum essential medium; FBS, fetal bovine serum;
RBA, relative binding affinity; PDB, protein data bank; FITC,
fluoresceinꢀ5ꢀisothiocyanate; DAPI, 4',6ꢀdiamidinoꢀ2ꢀphenylindole; CLE, confocal
laser endomicroscopy; ROI, regions of interest.
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Molecular Pharmaceutics
Page 22 of 22
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A novel estrogen receptor αꢀtargeted nearꢀinfrared fluorescent probe
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for in vivo detection of breast tumor
Chu Tang, Yang Du, Qian Liang, Zhen Cheng, Jie Tian
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