Obtaining More Accurate Signals: Spatiotemporal Imaging of Cancer

Subscriber access provided by Northern Illinois University

Article

Obtaining More Accurate Signals: Spatiotemporal Imaging of Cancer Sites Enabled by a Photoactivatable Aptamer-Based Strategy Heng Xiao, Yuqi Chen, Erfeng Yuan, Wei Li, Zhuoran Jiang, Lai Wei, Haomiao Su, Weiwu Zeng, Yunjiu Gan, Zijing Wang, Bifeng Yuan, Shanshan Qin, Xiaohua Leng, Xin Zhou, Songmei Liu, and Xiang Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07450 • Publication Date (Web): 23 Aug 2016 Downloaded from http://pubs.acs.org on August 24, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Obtaining More Accurate Signals: Spatiotemporal Imaging of Cancer Sites Enabled by a Photoactivatable Aptamer-Based Strategy Heng Xiao, ,§,¶ Yuqi Chen, ,¶ Erfeng Yuan,‡,¶ Wei Li, Zhuoran Jiang, Lai Wei, Haomiao Su, † † † † † Weiwu Zeng, Yunjiu Gan, Zijing Wang, Bifeng Yuan, Shanshan Qin, Xiaohua Leng,‡ Xin † Zhou,‡ Songmei Liu*,‡ and Xiang Zhou*, †

†

†

†

†

†

†

College of Chemistry and Molecular Sciences and Institute of Advanced Studies, Wuhan University, Wuhan, Hubei 430072, P. R. China ‡

Zhongnan Hospital, Wuhan University, Wuhan, Hubei 430072, P. R. China

§

Research Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China

Supporting Information Placeholder ABSTRACT: Early cancer diagnosis is of great significance to relative cancer prevention and clinical therapy, and it is crucial to efficiently recognize cancerous tumor sites at the molecular level. Herein, we proposed a versatile and efficient strategy based on aptamer recognition and photoactivation imaging for cancer diagnosis. This is the first time that a visible light-controlled photoactivatable aptamer-based platform has been applied for cancer diagnosis. The photoactivatable aptamer-based strategy can accurately detect nucleolin-overexpressed tumor cells and can be used for highly selective cancer cell screening and tissue imaging. This strategy is available for both formalin-fixed paraffin-embedded (FFPE) tissue specimens and frozen sections. Moreover, the photoactivation techniques showed a great progress in more accurate and persistent imaging to the use of traditional fluorophores. Significantly, the application of this strategy can produce the same accurate results in tissue specimen analysis as with classical hematoxylin-eosin (H&E) staining and immunohistochemical (IHC) technology.

KEYWORDS: aptamer, photoactivation, cancer site, fluorescent imaging, nucleolin, breast cancer.

10-16

INTRODUCTION The development of novel molecular-level differentiation methods has contributed to early diagnosis and prompted 1 therapy. Cancer biomarkers are often measured and evaluated for early cancer diagnosis by means of immunoenzymebased immunohistochemical (IHC) methods, a process that detects antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. However, antibody-based methods are affected by the limitations of antibodies. For example, there are no guidelines or standard methods to determine the va2 lidity of these reagents. In recent years, aptamer-based biomarker-target cancer diagnosis strategies have gained popularity. Aptamers have been widely studied for their use in cancer diagnostic applications due to their unique ability to recognize certain mo3-5 lecular characteristics. Due to many distinct advantages, such as good reproducibility, ease of synthesis, excellent biocompatibility, high thermal and environmental stability, and relatively small size and molecular mass, which allow for fast 6-7 tissue penetration and clearance from blood, aptamers provide superior alternatives to antibodies as cell-specific 8agents and show great potential for use in cancer diagnosis. 9 Several recent advances in aptamer-based detection ap-

proach have been established that could provide various detection signals, but they still suffer from autofluorescence interference and nonspecific adsorption; Additionally, they are time consuming, expensive or require specific equipment. Aptamer-based fluorescent imaging shows great promise because of a wide range of benefits fluorescent imaging has, derived from its combination of high spatiotemporal resolu17 tion, high detection sensitivity, wide signal dynamic range and linear relationships for biomarker quantification. However, traditional fluorescent imaging techniques suffer from background interference due to biological autofluorescence; this autofluorescence can severely limit the signal to back18-19 ground ratio (SBR). Development of quick, economical and sensitive cancer diagnoses methods to overcome biological autofluorescence is of great significance to cancer diagnosis. An attractive alternative approach to solve this problem is to use fluorescence photoactivation, a key approach that triggers the fluorescence of fluorescent probes by appropriate 20-21 excited wavelength. Fluorescence photoactivation can produce a more favorable SBR for accurate imaging by removing background-the auto-fluorescent signal-via subtract20 ing the original caged image signal from the uncaged one. In principle, this can avoid the problem of generating local oxidative damage that is inherent to photobleaching, produc-

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ing a more accurate and persistent image signal. Thus, this method has

Figure 1. Mechanism of spatiotemporal imaging of cancer sites enabled by a photoactivatable aptamer-based strategy (A) Schematic illustration of the photoactivatable fluorescent aptamer-based strategy for in situ spatiotemporal imaging of tumor cells. (B) Schematic illustration of traditional fluorescent imaging strategy and photoactivatable fluorescent imaging strategy for cancer site imaging. emerged as a very promising strategy for visualizing and tracking molecular and cellular dynamics with high temporal 22-27 However, and spatial resolution in biological systems. until now, this method hasn’t been applied to tumor biomarker imaging and cancer diagnosis. Herein, we first applied this strategy to tumor biomarker imaging and cancer diagnosis by combining a photoactivatable fluorophore with an aptamer probe to achieve photoactivatable aptamer-based cancer site imaging in order to produce more accurate results (Figure 1A). In the presence of target molecule in the cancerous cells, the aptamer probe can selectively recognizes the cancer site. Then the cancer site can be detected at a high spatiotemporal resolution in a noninvasive manner by controlling both the time and application site of the excitation light. In traditional fluorescent imaging strategy, the signals of cancer sites were usually used to contrast with the signals of adjacent cancer sites, which revealed an ex-situ comparison. However, in photoactivatable fluorescent imaging strategy, we can get a more favorable SBR for accurate imaging by removing background signal via subtracting the nonirradiated signal from the in situ irradiated one (Figure 1B).

Page 2 of 8

MATERIALS AND METHODS Materials and Instruments. The following solvent, compounds and reagents were commercially available: All chemicals were of analytical grade were bought from Sigma-Aldrich. Oligonucleotides were synthesized by Sangon Biotech (Shanghai, China). 3’ azide-modified AS1411 ssDNA is 5’GGTGGTGGTGGTTGTGGTGGTGGTGG-3’-N3. 3’ FAMmodified AS1411 ssDNA is 5’GGTGGTGGTGGTTGTGGTGGTGGTGG-3’-FAM. 3’ azide-modified random ssDNA is 5’1 13 GAGTGGTTTCACATAAAATGTACCAA-3’-N3. H and C NMR spectra were recorded on Varian Mercury 300 spectrometers, respectively. HRMS were recorded on Thermo Fisher LTO Orbitrap XL. API-ES of compounds and LC-MS were recorded on Agilent LC/MSD. For mass-spectrometric analysis of oligonucleotide, an LCQ Deca XP Plus Ion-Trap mass spectrometer equipped with a ESI interface (ThermoFisher Scientific, Waltham, MA, USA) was used in line with the HPLC system. Gel Imaging is collected in Pharos FX Molecular imager (Bio-Rad, USA) and portable UV lamp (GL-9406, JiangSu). UV absorption spectra were collected on SHIMADZU UV-2550. Fluorescent emission spectra were collected on PerkinElmer LS 55. Quartz cuvettes with 300 μL volume were used for emission measurements. Unless otherwise specified, all spectra were taken at an ambient temperature. Preparation of caged fluorescent oligonucleotides. To 200 μL 0.5 M potassium phosphate buffer (pH=7.0), 200 μL 1 mM CuSO4/ 6 mM BTTAA (2-(4-((bis((1-tert-butyl-1H1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1yl)acetic acid) aqueous solution, 200 μL 10 mM freshly prepared sodium ascorbate aqueous solution and 200 μL 1 mM alkyne caged TokyoGreen (ACTG) in DMSO/tBuOH (3:1) were added sequentially. To the above solution, 200 μL 0.1 mM azide oligonucleotice aqueous solution was added, and the solution was stored at room temperature for 12 h. The reaction was purified by means of reverse-phase HPLC (MeCN/50 mM trimethylaminecanbonic acid buffer=10/90→50/50 over 30 min, flow: 1.0 mL/min, the excitation and emission wavelength of the fluorescence detector was 488 and 520 nm, respectively). Cell culture. MCF-7 Human breast cancer cells and CHO Chinese hamster ovary cells (CCTCC, China) were cultured in DMEM (Hyclone, China) supplemented with 10% FBS (Hangzhou Sijiqing Biological engineering Materials Corporation, China). Cells were maintained in a humidified atmosphere of 5/95 (v/ v) CO2/air at 37 °C. Photoactivatable fluorescence imaging in living cells. The MCF-7 human breast cancer cells and CHO Chinese hamster ovary cells (CCTCC, China) were cultured in DMEM (Hyclone, China) supplemented with 10% FBS (Hangzhou Sijiqing Biological engineering Materials Corporation, China). Cells were maintained in a humidified atmosphere of 5/95 (v/ v) CO2/air at 37 °C. For confocal microscopy imaging, MCF-7 and CHO cells were plated in a 35-mm confocal dish (Nest, China) for 24 h and grown to around 30% confluence prior to experiments. Then cells were washed three times with PBS and


Page 3 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


incubated with 300 nM CTG-AS1411 probe or random probe at 37 °C in 5% CO2 for 40 min. Cells were then washed three times with PBS to remove the excess probes and immersed in 1 mL PBS. Then, confocal imaging was acquired on Nikon Confocal Laser Scanning Microscope (TE2000, Japan) with an objective lens (×60). The fluorescent images were taken with green filter (excitation: 488 nm) at 15.0 % power. Cells were activated with scans from the 488 nm laser at 15.0 % power or 408 nm laser at 1.0 % power for 20 s one time. Images and merges were obtained with EZ-C1 software. Fluorescence intensity was analyzed by image J software. Flow Cytometry Assay. Generally, living cells were scraped from culture dishes, then CTG-AS1411 probe or 5 random probe (300 nM) was incubated with 2 × 10 cells o at 37 C for 40 min in PBS. After centrifugation at 2000 rpm for 2 min, the cells were collected and washed with PBS 3 times. The cells were then resuspended in 100 μL PBS, samples need irradiation were irradiated under 365 nm UV light for 30 min and then subjected to flow cytometry. Flow cytometry analysis was conducted on BD Accuri™ C6 Flow Cytometer (BD Biosciences, USA) by counting 2000 events, and the data were analyzed using Cflow Plus software.

Photoactivatable fluorescence imaging in FFPE tissue sections. Formalin-fixed paraffin-embedded human breast cancer tumor tissue and human benign breast tissue speciemens (Zhongnan Hospital of Wuhan University) were subjected to a 4 μm thick serial sectioning for immunohistochemical staining. After xylene dewaxing, the slides were de-benzened using conventional gradient ethanol and immersed in water. After incubation for 10 min with 3% H2O2, the slides were washed thoroughly using PBS and placed in the phosphate-EDTA buffer solution (50%, pH 9.0). Antigens were retrieved in the 121 °C high-pressure cooker for 5 min, followed by cooling down of the slides to room temperature. Approximately, 200 μL of the CTG-AS1411 probe (300 nM in PBS) were added to each slide. The slides were incubated with probes for 30 min at room temperature and washed with PBS. The slides were then washed 3 times with PBS. The slides were then washed with distilled water and sealed with neutral resin. The CTG-AS1411 probe staining slides were directly detected after incubation. The photoactivation procedure was consistent with the procedure described in living cells confocal imaging section. Synthesis of ACTG. 1-(2-nitrophenyl) ethyl TokyoGreen (TG-NPE) was synthesized by following a literature pro26 cedure. TG-NPE 52.6 mg (0.1 mmol), EDCI 20 mg (0.1 mmol), HOAT 7 mg (0.05 mmol) were suspended in DMF (5mL) and propargylamine 11 mg (0.2 mmol) in DMF (1 mL) was added with stirring. The reaction mixture was stirred for 12 hours at room temperature under an Ar atmosphere. After the vacuum evaporation of the solvent, the residue was purified on a silica gel column (CH2Cl2/MeOH = 97/3) to afford compound ACTG 39 1 mg (0.07 mmol, 70%, orange solid). H-NMR (300 MHz, CDCl3) δ: 1.75 (s, 3H, J = 4.2 Hz), 2.02 (d, 3H), 2.29 (s, 1H), 4.19 (d, 2H, J = 2.1 Hz), 4.58 (s, 1H), 6.18 (q, 1H, J = 6 Hz), 6.40 (s, 1H), 6.53 (d, 1H, J = 9.9 Hz), 6.71 (t, 1H, J = 8.1 Hz), 6.80-6.96 (m, 4H), 7.07 (d, 1H, J = 8.4 Hz), 7.26 (s, 1H),

7.48 (t, 1H, J = 7.5 Hz), 7.63 (t, 1H, J = 6 Hz), 7.71 (d, 1H, J 13 = 6.9 Hz), 8.06 (d, 1H, J = 8.1 Hz) ppm. C-NMR (75 MHz, CDCl3) δ: 19.9, 23.3, 28.7, 67.2, 71.7, 71.8, 72.3, 78.9, 102.5, 102.7, 105.7, 105.7, 112.2, 113.4, 113.6, 114.8, 114.9, 116.7, 118.8, 125.0, 126.1, 127.0, 128.8, 129.4, 130.3, 134.2, 137.6, 138.4, 147.2, 148.5, 154.0, 154.2, 157.7, 158.6, 161.7, 167.4, 185.7 + ppm. HRMS (ESI ): m/z calcd for C33H26N2O7 (M+H) 563.18128; detected 563.18228.

RESULTS AND DISCUSSION Fabrication of CTG-AS1411. To demonstrate the effectiveness of our approach, we report here a new photoactivatable 26 dye, ACTG. Caged TokyoGreen, a caged fluorescein derivative, was used as the photoactivatable imaging component. The cage strategy removes caged groups (photoremovable protecting groups) by appropriate light illumination, resulting in original bioactive molecule release. TG-NPE was used as the photoremovable protecting group and a derivative alkyne group as the oligonucleotide attachment point (Figure S1). This design ensures the greatest activation of fluorescence upon light irradiation and the successful modification of caged TokyoGreen (CTG) to the oligonucleotide probe via copper-catalysed azide-alkyne cycloaddition (Figure S2). Then the oligonucleotide conjugation and photoactivatable experiment were investigated. To achieve the specific targeting, we used AS1411, a G-rich oligonucleotide that can function as an aptamer to nucleolin (NCL), a bcl-2 mRNA binding protein that is overexpressed on the plasma membrane of breast cancer cells, but has a very low level of ex30 pression on normal cells. Considering that NCL overexpression on the plasma membrane has been linked to various 31-32 human diseases such as breast cancer, NCL has been regarded as a cancer biomarker and the NCL-specific binding oligonucleotide AS1411 is regarded as a promising targeting 33-35 ligand for breast cancer. Furthermore, the G-quadruplex oligonucleotide shows distinct nuclease resistance relative to single-stranded nucleotides in serum and cellular environ36-37 ments. Thus, the G-quadruplex aptamer-based strategy can provide a robust imaging signal in biological analysis and clinical diagnosis. Azide-modified oligonucleotide (azideI AS1411) was incubated with ACTG under Cu and BTTAA ligand catalysis to provide the caged fluorescent oligonucleotide (caged TokyoGreen-modified AS1411, CTG-AS1411) after HPLC (Figure S2). The probe structure was confirmed by ESI mass spectrometry (Figure S7). Optical properties of CTG-AS1411. As a derivative of caged TokyoGreen, ACTG also showed remarkable fluorescence activation upon UV irradiation (Figure S3, S4, S5 and S6). Afterwards, the optical properties were detected after successfully preparing the CTG-AS1411 probe. The caged fluorescent oligonucleotide was almost nonfluorescent in a buffered aqueous solution due to the quenching of the singlet excited state via the photoinduced electron-transfer (PeT) process, and a dramatic fluorescence increase was observed by means of 365 nm UV irradiation (Figure 2A, 2B). By removing the caging group, the photoproduct was also identified by ESI mass spectrometry (Figure S7). We also found that the fluorescence intensity of the caged fluorescent oligonucleotide polyacrylamide gel electrophoresis (PAGE) band in-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

creased in an irradiation time-dependent manner (Figure 2C and S9). Furthermore, we unexpectedly found that this caged fluorescent probe was

Page 4 of 8

causing little damage to the organism. These results suggest that the caged fluorescent oligonucleotide probe CTG-AS1411 can serve as a useful marker for photoactivatable imaging experiments.

Photoactivatable fluorescence detection for cancer cells.

Figure 2. Photoactivation experiments in photoactivatable fluorescent aptamer-based strategy. (A) Uncaging scheme and structure of CTG-AS1411. (B) Fluorescence spectral change of CTG-AS1411 (100 nM) upon UV irradiation (365 nm). (C) Fluorescence image of a native PAGE gel after irradiation. Marker was a FAM-labelled oligonucleotide with same sequence. The solutions of caged fluorescent oligonucleotides were irradiated with UV light (365 nm) for various periods of time (0, 5, 10, 15, 20, 25, 30 min). (D) Confocal laser scanning microscopy of MCF-7 cells (top row) and CHO cells (bottom row) incubated with CTG-AS1411 probe. Scale bar = 20 μm. DIC: bright-field images. also activated by 488 nm or 408 nm visible light under appropriate conditions (Figure S10), which had not been 26 reported previously. Based on this inspiring phenomenon, biological samples can be irradiated by visible light,

To determine the feasibility of the caged fluorescent aptamer probe for the purpose of cancer-selective imaging, we carried out a photoactivatable reaction and traced the resulting fluorescence signal in living MCF-7 human breast cancer cells and normal healthy CHO cells. Before observation, the cells were incubated with a CTGAS1411 probe or random sequence. In CHO cells, there was non-specific fluorescence background staining (Figure 2D, S11a), which because of the non-specific absorption, stability and low background fluorescence noise of our probe even in a physiologic environment. Probes conjugated within cells were then activated with a 488 nm or 408 nm laser for 20 s, one time. After light irradiation treatment, an increase in bright fluorescence was observed on the cell membranes of the MCF-7 breast cancer cells at single-cell resolution with a high signalto-noise ratio (Figure 2D, S11a and S12), while no obvious fluorescence growth was observed from MCF-7 cells and CHO cells treated with random probe (Figure S12). Upon light irradiation, the fluorescence intensity from the sample incubated with the CTG-AS1411 probe was over 20 times that of the non-irradiated cells, while other samples produced a relatively low signal with/without light illumination (Figure 2D and S11A). Moreover, the flow cytometry results (Figure S13) were consistent with those of confocal microscopy. All of these results demonstrated that the caged fluorescent aptamer probe can selectively recognize MCF-7 breast cancer cells and effectively image the target cells versus normal cells via photoactivation with a high SBR. In fact, clinical samples which are used to cancer diagnosis are much more complicated than working buffer in the laboratory, and a practical diagnostic approach should possess strong selectivity, sensitivity and antiinterference ability. To test the practicality of our method, we also deployed a CTG-AS1411 probe directly into unmodified healthy human serum and human whole blood, a realistically complex and contaminant-ridden clinical material (Figure S11B and S11C). The mimic physiologic detection environments did not interfere with the detection selectivity and sensitivity of our probe. Furthermore, the CTG-AS1411 probe did not target the numerous leukocytes appearing in the blood, reflecting the low expression of NCL in leukocytes and indicating the prominent detection specificity for cancer cells in our method. Moreover, the nuclease resistance ability of the aptamer probe was also verified by gel-electrophoresis (Figure S14).

Photoactivatable fluorescence detection for tissue specimens. Herein, we applied our caged fluorescent aptamer probe as a novel means for cancer diagnosis to image breast cancer tissues versus benign breast tissue. (Figure 3A and S16) Two cases of formalin-fixed paraffinembedded (FFPE) cancer tissue sections and three cases of FFPE benign tissue sections were taken as experimental samples. After incubating with the CTG-AS1411


Page 5 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


probe, the breast cancer tissue showed a bright green fluorescence signal after being irradiated with 488 nm and 408 nm light, with negligible fluorescence signal observed before light illumination. However, benign breast tissue under

Figure 3. Experiment of FFPE tumor tissue sections and frozen tissue sections in photoactivatable fluorescent aptamerbased strategy. (A) Spatiotemporally controlled imaging of FFPE tumor tissue sections: DIC (panel 1), time-lapsed fluorescence (panels 2-9), and merged DIC/fluorescence (panel 10) images of breast cancer tumor sections (top row) and benign hyperplastic breast tumor sections (bottom row) upon 488 nm illumination and subsequently 408 nm. Scale bar = 40 μm. (B) Spatiotemporally controlled imaging of frozen tissue sections: DIC (panel 1), time-lapsed fluorescence (panels 2-7), and merged DIC/fluorescence (panel 8) images of breast cancer tissue sections (top row) and benign breast tissue sections (bottom row) upon 488 nm illumination and subsequently 408 nm. All tumour frozen sections were stained by 300 nM CTG-AS1411 probe. A 20 s 488 nm or 408 nm laser was applied to the selected area. Scale bar = 20 μm. DIC: bright-field images. the same treatment conditions exhibited a very weak fluorescence signal, likely due to background noise before light irradiation, while there was no distinct fluorescence increase after the same irradiation conditions were applied. These results clearly illustrate that the caged fluorescent aptamer probe can specifically recognize the corresponding breast cancer tissue and can be observed by photoactivating the caged fluorescent aptamer probe. In addition, more accurate fluorescence intensity signals of tissue sections can be obtained by subtracting the original untreated image signal from the 17 light-activated signal. We also analysed FFPE tissue sections of tumor and normal tissue obtained from the 4T1 tumor model nude mice. The AS1411 aptamer binds with high affinity and selectivity to nucleolin, which is also overexpressed on the cell membrane of 4T1 breast cancer cells (Figure S15). Our photoactivatable aptamerbased strategy also shows obvious distinction between tumor and normal tissue sections in 4T1 tumor model nude mice. At present, one of the most prevalent cancer diagnostic methods is FFPE pathological section analysis via immunoenzyme-based immunohistochemical (IHC) methods. Patients can receive diagnostic results in 3-7 days from these methods, which are time-consuming due to their complexity. Therefore, we sought to apply this technology to breast cancer diagnosis in rapid fro-

zen tissue section analysis with the CTG-AS1411 probe. The frozen tissue sections stained with CTG-AS1411 probe were immediately analysed by fluorescence confocal imaging. As shown in Figure 3B, we found a similar fluorescence increase phenomenon upon photoactivation in

Figure 4. Comparison of photoactivatable fluorescent probe with traditional fluorescent dyes. (A) Spatiotemporally controlled imaging of FFPE tumor tissue sections: DIC (panel 1), time-lapsed fluorescence (panels 2-7), and merged DIC/fluorescence (panel 8) images of CTG-AS1411 stained sections (top row) and FAM-AS1411 stained sections (bottom row) upon 488 nm illumination and subsequently 408 nm. (B) and (C) Quantification of FI in CTG-AS1411 (B) and FAMAS1411 (C) stained sections. Fluorescence density analysis by imageJ software, data are presented as mean ± SEM (n= 3). DIC: bright-field images. breast cancer tissue sections, while the fluorescence intensities of benign sections were almost unchanged. The application of photoactivatable aptamer-based techniques is promising for accurate and timely diagnosis of breast cancer. Comparison with traditional fluorescent dyes. To further understand the benefits of the photoactivatable fluorescent strategy we proposed, probe and the traditional fluorescent probe to do the same photoactivation experiments of FFPE breast cancer tissue sections (Figure 4, S17 and S18). The tissue sections which were incubated with CTG-AS1411 reflected obvious fluorescence increase after photoactivation. However, the sections which were incubated with FAM-AS1411, the same aptamer sequence labelled with FAM, showed gradual fluorescence decay upon photo-illumination. The fluorescence intensity of CTG-AS1411 incubated tissue sections showed about 8.6-fold increase and FAM-AS1411 incubated one showed 30% fluorescence decrease (Figure 4B and 4C). These experiments validated that the fluorescence photoactivation strategy can not only obtain more accurate imaging signals, but also offer more persistent fluorescent signal relative to traditional uncaged fluorophores. Comparison with classic diagnostic methods. To demonstrate the feasibility of the photoactivatable aptamerbased strategy, we also compared this approach with traditional IHC and H&E staining methods using FFPE pathological sections of breast cancer tissue and benign tissue from the same site (Figure 5). Tissues from the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

same site were chosen to minimize location-based differences in cell morphology and expression levels of cancer-related proteins, such as HER2 and nucleolin. For the novel photoactivatable aptamer-based method (Figure 5A), CTG-AS1411-stained breast cancer tissue sections exhibited a remarkable fluorescence increase after photoactivation, while

Figure 5. Comparison of photoactivatable aptamer-based strategy with IHC technology and H&E staining. (A) Fluorescent images of CTG-AS1411 probe stained breast cancer tissue and benign tissue. (B) Expression of HER2 in the breast cancer tissue and benign tissue as estimated by IHC. (C) H&E histology of breast cancer tissues and benign tissue. DIC: bright-field images. no obvious fluorescent signals were observed in benign tissue. Compared with classical H&E staining and IHC technology (Figure 5B and 5C), these results also reveal excellent breast cancer diagnostic capability of the aptamer strategy. It is worth mentioning that compared to IHC, this method is more convenient and time saving, which is very important for patient diagnosis.

CONCLUSION

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed information on the experimental methods; Figure S1-S2: Molecular design, oligonucleotide conjugation and photoactivation; Figure S3-6: Optical spectra of ACTG; Figure S7: The ESI-TOF results of azide-AS1411, CTG-AS1411 before and after UV irradiation; Figure S8: Absorption spectra of CTG-AS1411; Figure S9: PAGE analysis of CTG-AS1411 upon UV light irradiation; Figure S10: Relative fluorescence intensity of ACTG upon 408 nm or 488 nm light irradiation; Figure S11: Spatiotemporally controlled imaging in intact living cells; Figure S12: Spatiotemporally controlled imaging of MCF-7 cells after CTG-AS1411 or CTG-random sequence incubation; Figure S13: Flow Cytometric Analysis; Figure S14: PAGE analysis of the nuclease resistance ability of the aptamer probe; Figure S15: Photoactivatable fluorescent detection in FFPE 4T1 model nude mice tissue specimens using CTG-AS1411 probe; Figure S16: Spatiotemporally controlled imaging of FFPE tissue sections; Figure S17-S18: Spatiotemporally controlled imaging of FFPE breast cancer tumour tissue sections using CTG-AS1411 and FAM-AS1411 probe; Scheme S1: Synthesis of ACTG; Synthesis process of ACTG. Figure S19-21. 1 13 The H NMR, C NMR, and high-resolution mass spectra of ACTG (PDF).

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

Author Contributions

In summary, we have developed a general and simple photoactivatable aptamer-based strategy with high selectivity and accurate signal-output by combining the AS1411 aptamer sequence with a caged photoactivatable fluorophore. This is the first study to use a photoactivatable technique for aptamer-based cancer cell recognition and light-controlled cancer site imaging. By means of controlling visible light, which has deeper penetration capability, we can selectively image cancer cells of interest with high temporal and spatial resolution. More importantly, in contrast to traditional fluorophore, which suffer severe SBR and photo-bleaching, the photoactivation strategy in tissue sections diagnosis showed more accurate and persistent signals. Compared with classical IHC technology and H&E staining, our method also revealed excellent breast cancer diagnostic capability, and this photoactivatable fluorescence recovery technique may be well-suited for accurate biomarker imaging and related cancer diagnosis in a complex clinical sample. The ability to discriminate the cancer site in tissue specimens can increase diagnostic accuracy and timeliness of therapeutic decisions when used in combination with other diagnostic goldstandard methods. Custom-made aptamer sequences coupled with the use of photoacivatable fluorophores could be a simple but powerful tool for cancer site imaging and cancerrelated early diagnosis.

ASSOCIATED CONTENT

Page 6 of 8

¶

These authors contributed equally (H.X., Y.C. and E.Y.).

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank the 973 Program (2012CB720600, 2012CB720603, and 2012CB720605), the National Science Foundation of China (21432008, 91413109, 81373256 and 91213302).

REFERENCES (1) Tan, W.; Donovan, M. J.; Jiang, J. Aptamers From Cell-based Selection For Bioanalytical Applications. Chem. Rev. 2013, 113, 28422862. (2) Bordeaux, J.; Welsh, A.; Agarwal, S.; Killiam, E.; Baquero, M.; Hanna, J.; Anagnostou, V.; Rimm, D. Antibody Validation. Biotechniques 2010, 48, 197-209. (3) Yang, C. J.; Jockusch, S.; Vicens, M.; Turro, N. J.; Tan, W. Lightswitching Excimer Probes for Rapid Protein Monitoring in Complex Biological Fluids. Proc. Natl. Acad. Sci. U S A 2005, 102, 17278-17283. (4) Navani N. K.; Li Y. Nucleic Acid Aptamers and Enzymes as Sensors. Curr. Opin. Chem. Biol., 2006, 10, 272-281. (5) Liu, J.; Cao, Z.; Lu, Y. Functional Nucleic Acid Sensors. Chem. Rev. 2009, 109, 1948-1998. (6) Tavitian, B.; Terrazzino, S.; Kuhnast, B.; Marzabal, S.; Stettler, O.; Dolle, F.; Deverre, J. R.; Jobert, A.; Hinnen, F.; Bendriem, B.; Crouzel,


Page 7 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


C.; Di Giamberardino, L. In Vivo Imaging of Oligonucleotides with Positron Emission Tomography. Nat. Med. 1998, 4, 467-471. (7) Zhao, W.; Schafer, S.; Choi, J.; Yamanaka, Y. J.; Lombardi, M. L.; Bose, S.; Carlson, A. L.; Phillips, J. A.; Teo, W.; Droujinine, I. A.; Cui, C. H.; Jain, R. K.; Lammerding, J.; Love, J. C.; Lin, C. P.; Sarkar, D.; Karnik, R.; Karp, J. M. Cell-surface Sensors for Real-time Probing of Cellular Environments. Nat. Nanotechnol. 2011, 6, 524-531. (8) Liang, H.; Zhang, X. B.; Lv, Y.; Gong, L.; Wang, R.; Zhu, X.; Yang, R.; Tan, W. Functional DNA-containing Nanomaterials: Cellular Applications in Biosensing, Imaging, and Targeted Therapy. Acc. Chem. Res. 2014, 47, 1891-1901. (9) Li L.; Tong R.; Chu H.; Wang W.; Langer R.; Kohane D. S. Aptamer Photoregulation in Vivo. Proc. Natl. Acad. Sci. U S A, 2014, 111, 17099-17103. (10) Yuan Q.; Wu Y.; Wang J.; Lu D.; Zhao Z.; Liu T.; Zhang X.; Tan W. Targeted Bioimaging and Photodynamic Therapy Nanoplatform Using an Aptamer-Guided G-Quadruplex DNA Carrier and Near‐ Infrared Light. Angew. Chem. Int. Ed., 2013, 52, 13965-13969. (11) Wang, J.; Wei, T.; Li, X.; Zhang, B.; Wang, J.; Huang, C.; Yuan, Q. Near-Infrared-Light-Mediated Imaging of Latent Fingerprints based on Molecular Recognition. Angew. Chem., Int. Ed. 2014, 53, 1616-1620. (12) Liu, J.; Lee, J. H.; Lu, Y. Quantum Dot Encoding of Aptamerlinked Nanostructures for One-pot Simultaneous Detection of Multiple Analytes. Anal. Chem. 2007, 79, 4120-4125. (13) Phillips J. A.; Xu Y.; Xia Z.; Fan Z. H.; Tan W. Enrichment of Cancer Cells Using Aptamers Immobilized on a Microfluidic Channel. Anal. Chem., 2009, 81, 1033-1039. (14) Li W.; Wang J.; Ren J.; Qu X. Endogenous Signalling Control of Cell Adhesion by Using Aptamer Functionalized Biocompatible Hydrogel. Chem. Sci., 2015, 6, 6762-6768. (15) You M.; Chen Y.; Peng L.; Han D.; Yin B.; Ye B.; Tan W. Engineering DNA Aptamers for Novel Analytical and Biomedical Applications. Chem. Sci., 2011, 2, 1003-1010. (16) Li, F.; Zhang, H. Q.; Wang, Z. X.; Newbigging, A. M.; Reid, M. S.; Li, X. F.; Le, X. C. Aptamers Facilitate Amplified Detection of Biomolecules. Anal. Chem. 2015, 87, 274-292. (17) Hicke, B. J.; Stephens, A. W.; Gould, T.; Chang, Y. F.; Lynott, C. K.; Heil, J.; Borkowski, S.; Hilger, C. S.; Cook, G.; Warren, S.; Schmidt, P. G. Tumor Targeting by an Aptamer. J. Nucl. Med. 2006, 47, 668-678. (18) Choi, H. S.; Gibbs, S. L.; Lee, J. H.; Kim, S. H.; Ashitate, Y.; Liu, F.; Hyun, H.; Park, G.; Xie, Y.; Bae, S.; Henary, M.; Frangioni, J. V. Targeted Zwitterionic Near-infrared Fluorophores for Improved Optical Imaging. Nat. Biotechnol. 2013, 31, 148-153. (19) Frangioni, J. V. In Vivo Near-infrared Fluorescence Imaging. Curr. Opin. Chem. Biol. 2003, 7, 626-634. (20) Adams, S. R.; Tsien, R. Y. Controlling Cell Chemistry with Caged Compounds. Annu. Rev. Physiol. 1993, 55, 755-784. (21) Mitchison, T. J.; Sawin, K. E.; Theriot, J. A.; Gee, K.; Mallavarapu, A. Caged Fluorescent Probes. Methods Enzymol. 1998, 291, 63-78.

(22) Politz, J. C. Use of Caged Fluorochromes to Track Macromolecular Movement in Living Cells. Trends Cell Biol. 1999, 9, 284-287. (23) Politz, J. C.; Tuft, R. A.; Pederson, T.; Singer, R. H. Movement of Nuclear Poly (A) RNA Throughout the Interchromatin Space in Living Cells. Curr. Biol. 1999, 9, 285-291. (24) Lim, R. K.; Lin, Q. Photoinducible Bioorthogonal Chemistry: a Spatiotemporally Controllable Tool to Visualize and Perturb Proteins in Live Cells. Acc. Chem. Res. 2011, 44, 828-839. (25) Puliti D.; Warther D.; Orange C.; Specht A.; Goeldner M. Small Photoactivatable Molecules for Controlled Fluorescence Activation in Living Cells. Bioorg. Med. Chem., 2011, 19, 1023-1029. (26) Kobayashi, T.; Urano, Y.; Kamiya, M.; Ueno, T.; Kojima, H.; Nagano, T. Highly Activatable and Rapidly Releasable Caged Fluorescein Derivatives. J. Am. Chem. Soc. 2007, 129, 6696-6697. (27) Kobayashi, T.; Komatsu, T.; Kamiya, M.; Campos, C.; GonzalezGaitan, M.; Terai, T.; Hanaoka, K.; Nagano, T.; Urano, Y. Highly Activatable and Environment-insensitive Optical Highlighters for Selective Spatiotemporal Imaging of Target Proteins. J. Am. Chem. Soc. 2012, 134, 11153-11160. (28) Dakin, K.; Zhao, Y.; Li, W. H. LAMP, a New Imaging Assay of Gap Junctional Communication Unveils That Ca2+ Influx Inhibits Cell Coupling. Nat. Methods 2005, 2, 55-62. (29) Jing J.; Chen J.; Hai Y.; Zhan J.; Xu P.; Zhang J. Rational Design of ZnSalen as a Single and Two Photon Activatable Fluorophore in Living Cells. Chem. Sci., 2012, 3, 3315-3320. (30) Bates P. J.; Laber D. A.; Miller D. M.; Thomas S. D.; Trent J. O. Discovery and Development of The G-rich Oligonucleotide AS1411 as a Novel Treatment for Cancer. Exp. Mol. Pathol., 2009, 86, 151-164. (31) Ginisty, H.; Sicard, H.; Roger, B.; Bouvet, P. Structure and Functions of Nucleolin. J. Cell Sci. 1999, 112, 761-772. (32) Srivastava, M.; Pollard, H. B. Molecular Dissection of Nucleolin's role in Growth and Cell Proliferation: New Insights. FASEB J. 1999, 13, 1911-1922. (33) Derenzini M.; Sirri V.; Trere D.; Ochs R. L. The Quantity of Nucleolar Proteins Nucleolin and Protein B23 is Related to Cell Doubling Time in Human Cancer Cells. Lab. Invest., 1995, 73, 497-502. (34) Otake, Y.; Soundararajan, S.; Sengupta, T. K.; Kio, E. A.; Smith, J. C.; Pineda-Roman, M.; Stuart, R. K.; Spicer, E. K.; Fernandes, D. J. Overexpression of Nucleolin in Chronic Lymphocytic Leukemia Cells Induces Stabilization of bcl2 mRNA. Blood 2007, 109, 3069-3075. (35) Xu K.; Luduena R. F. Characterization of Nuclear βII-tubulin in Tumor Cells: A Possible Novel Target for Taxol. Cell Motil. Cytoskeleton, 2002, 53, 39-52. (36) Bishop, J. S.; Guy-Caffey, J. K.; Ojwang, J. O.; Smith, S. R.; Hogan, M. E.; Cossum, P. A.; Rando, R. F.; Chaudhary, N. Intramolecular G-quartet Motifs Confer Nuclease Resistance to a Potent anti-HIV Oligonucleotide. J. Biol. Chem. 1996, 271, 5698-5703. (37) Cao, Z.; Huang, C. C.; Tan, W. Nuclease Resistance of Telomerelike Oligonucleotides Monitored in Live Cells by Fluorescence Anisotropy Imaging. Anal. Chem. 2006, 78, 1478-1484.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 8

Table of contents graphic


8

Obtaining More Accurate Signals: Spatiotemporal Imaging of Cancer

Recommend Documents