Multicolor Imaging of Cancer Cells with Fluorophore-Tagged

Jul 23, 2014 - The discrimination of the type of cancer cells remains challenging due to the subtle differences in their expression of membrane recept...
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Multicolor Imaging of Cancer Cells with Fluorophore-Tagged Aptamers for Single Cell Typing Song Wang, Hao Kong, Xiaoyun Gong, Sichun Zhang,* and Xinrong Zhang Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing, 100084, P. R. China S Supporting Information *

ABSTRACT: The discrimination of the type of cancer cells remains challenging due to the subtle differences in their expression of membrane receptors. In this work, we developed a multicolor cell imaging method for distinguishing the type of cancer cells with fluorophore-tagged aptamers. We found that the interaction between aptamers and cancer cells was affected by both of the sequence of aptamers and the labeled dyes. As the coownership of biomarkers for different cancer cell lines, the fluorophore-tagged aptamers interacted with different cancer cell lines in different degree, resulting in a distinct color to discriminate the type of cancer cells at single cell level. Taking advantage of the cross-reactive ability of the fluorophore-tagged aptamers, we could not only distinguish the cancerous cells quickly from large quantities of noncancerous cells, but also identify the type of the cancerous cells. This work has potential application for cancer diagnostic and therapy in the future.

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ensemble aptamers (ENSaptamers, i.e., rationally designed, nonspecific DNA sequences) for molecular identify and cellular targets discrimination.22 In our previous work, aptamer-AuNPs sensor array was developed and cell discrimination was realized by using reported protein aptamers as nonspecific receptors.23 These methods deal with a large amount of cells and obtain recognition signals from different set of cells, making them not suitable for single cell detection. In this study, we proposed a multicolor cell imaging method for cell discrimination by using fluorophore-tagged aptamers (Scheme 1). By labeling different fluorophores to aptamers, more than one target could be visualized simultaneously on the same cell, and we can get more information for cell discrimination. Combining simultaneously multicolor imaging with cross-reactive sensing strategies, we could recognize the types of cells intuitively cell by cell. Different from the random designed DNA, the aptamers from cell-SELEX showed little binding properties with the normal cell, which was important for the detection of cancer cells. Therefore, we could not only detect the cancer cells from the large amount of normal cells but also distinguish the type of a specific cell.

dentifying cancer at the single cell level holds great promise for cancer diagnosis, prognosis, and therapy.1−3 There are more than 200 distinct types of cancer cells, affecting over 60 human organs.4 Cancer cell types are characterized by different kinds and/or amounts of specific biomarkers, and therefore methods that could identify the subtle differences between different cells are highly desired.5 Many methods have been proposed for cellular typing based on the specific intra/ extracellular biomarkers, such as imaging of specific biomarkers by confocal microscopy,6−9 immunotyping by flow cytometry,10,11 and specific biomarkers detection by microarrays.12,13 However, those methods need prior knowledge of the specific markers and corresponding antibodies for different cancers, which is not the case with many cancers.14 Pattern recognition is an alternative strategy for cell subtyping. Materials that show different interactions with cells, such as fluorescent polymers,15,16 nanomaterials,17−21 and DNAs,22,23 are also used for discriminating the cell types. Aptamers are single-stranded oligonucleotides that have specificity and affinity to a wide range of targets that vary from small molecules to cancer cells.24−26 Through cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) procedure, a series of aptamers showing high affinity to cancer cells have been selected and applied for cancer cell detection.27−32 As the co-ownership of biomarkers for different cancer cell lines and the nonspecific interactions, many cell-SELEX aptamers are semi specific to a given cell line.33 To circumvent this, Tan et al. used aptamer-conjugated magnetic nanoparticles (ACMNPs) for pattern recognition of cancer cells in various complex biological media.21 Fan et al. used © 2014 American Chemical Society

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EXPERIMENTAL SECTION Materials. Unless otherwise specified, chemicals were of reagent grade and used as received. All materials were

Received: May 5, 2014 Accepted: July 23, 2014 Published: July 23, 2014 8261

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Scheme 1. Schematic Presentation of the Multicolor Imaging for Single Cell Typing with Fluorophore-Tagged Aptamers

Figure 1. Cell imaging with single aptamer. a−d represent L-02, Hela, MCF-7, and A549 interacted with Cy3 labeled TC01 aptamer respectively, e− h represent L-02, Hela, MCF-7, and A549 interacted with Cy3 labeled TC02 aptamer respectively, i−l represent L-02, Hela, MCF-7, and A549 interacted with Cy3 labeled TD05 aptamer, respectively. The concentration of the aptamer was 10 nM.

Cells. Cell lines listed in Table S2 were obtained from Cell Resource Center, IBMS, CAMS/PUMC. MCF-7, HepG2, Hela, and A549 cells were cultured in DMEM medium. L-02 cells were cultured in RPMI 1640 medium. All medium for cancer cells was supplemented with 10% fetal bovine serum (FBS) and 100 IU/mL penicillin-streptomycin. Cells were grown at 37 °C in a humidified atmosphere containing 5%

purchased from Corning (NY, USA), unless otherwise noted. Fluorescein 5-isothiocyanate (FITC) was purchased from Sigma-Aldrich (St. Louis, USA). Cy3 and Cy5 were purchased from Abgent (San Diego, USA). Aptamers were synthesized by Sangon, Shanghai. The sequences of them were obtained from the literature9,27 and shown in Table S1. 8262

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Figure 2. Cell imaging with three aptamers simultaneously. a−d represent L-02, Hela, MCF-7, and A549 interacted with three fluorophore-tagged aptamers (Apt-FITC, TD05-Cy3, and TE13-Cy5) respectively. The concentration of the aptamer was 10 nM.

CO2. Cells were passaged by trypsinization with 0.1% trypsin (0.02% EDTA, 0.05% dextrose, and 0.1% trypsin) in phosphine-buffered saline (PBS; pH 7.2). The cell density was determined using a hemocytometer before any experiments. Gel Electrophoresis Investigation. In the gel electrophoresis investigation, 2.5 μL of 50 uM Cy5 labeled TD09 aptamer was added to 22.5 μL of different cell medium, after incubation at 37 °C for different period of time, the sample was applied to a polyacrylamide (PAGE) gel (15% acrylamide, 19:1 acrylamide/bis(acrylamide)) to separate the cleaved products from the substrate. The electrophoresis was carried in 1× trisborateEDTA (TBE) buffer (90 mM Tris, 90 mM boric acid, and 10 mM EDTA, pH 8.0) at 300 V for 1.0 h. Cell-Sensing Studies. Cells suspension (150 μL, 10000 cells) was added to a glass-bottom dish, then 150 μL medium containing fluorophore-tagged aptamers (20 nM respectively) was added. After culturing for 6 h, the medium was removed and the cells were washed three times using Dulbecco’s PBS (DPBS, 1 mL each time), then added 100 μL DPBS for fluorescence imaging. All cellular fluorescent images were collected under the same conditions using an Olympus IX81 confocal microscope in sequential mode with a 40× objective. The average fluorescent intensity of a single cell was recorded and then processed using classical linear discriminant analysis (LDA) in SYSTAT (version 12.0).

Figure 3. Jackknifed classification matrix obtained using LDA for nine aptamers for the four cancer cell lines Hela, MCF-7, A549, and HepG2.

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the dyes and the tested cells. Then we labeled Cy3 on TC01 aptamer, which is chosen from previous literature, and incubated it with the four cell lines, clear fluorescent images was observed for three types of cancer cells (Figure S2 f−h). As control, normal cells (noncancerous cells) did not give fluorescent images (Figure S2e). When we labeled fluorescein isothiocyanate (FITC) on the same aptamer, the fluorescent responses were quite different with Cy3 (Figure S2 i−l). The results indicate that the dyes modified on aptamers may affect the interaction between the cells and the apatmers due to the structure difference of cyanine and the fluorescein. Therefore, it is concluded that the differential interactions between fluorophore-tagged aptamers and cancer cells not only come from the different expression level of specific biomarkers but also are affected by the dyes labeled on the aptamers. The stability of the aptamers under the incubate condition has also been investigated using polyacrylamide gel electrophoresis. Taking TD09 aptamer as the example, it did not digest in cell culture medium without FBS (Figure S3A, lane 8 and 9). In cell culture medium contain FBS, the aptamer digests slightly over time (Figure S3A, lane 7−1). When we incubated the aptamer with Hela cells and recorded the average fluorescence intensity of the cells, we could found that the intensity after 6 h was the highest (Figure S3B). This indicated that the TD09 aptamer continuing into cell within 6 h, the

RESULTS AND DISCUSSION Cancer Cell Discrimination with a Specific Aptamer. At the first of our research, we tested the interaction between reported aptamers and cell lines. We incubated four cell lines (L-02, Hela, MCF-7 and A549) with 10 nM Cy3-labeled aptamers for 6 h in cell medium and then took confocal microscopy fluorescent images of the cells. Fluorescent images were observed for three types of cancer cells (Figure 1b−d, f− h, j−l). As control, normal cells (noncancerous cells) did not give fluorescent images (Figure 1a,e,i). Additionally, a random DNA was used as a control sequence and no fluorescence was observed for either cancerous or normal cells (Figure S1). The results indicate that a single cell-SELEX aptamer is a great candidate for cell discrimination between the normal and cancerous cells, but not very suitable for cell discrimination between different cancer cell lines. To make sure of the reliability of our experiment, we also studied the interaction between the dyes and the cell lines. We incubated the above four cell lines with 10 nM Cy3 for 6 h in cell medium. The fluorescent images of the cells were taken and no fluorescent signal was observed (Figure S2a−d). Similar results were observed when we used other dyes, Cy5 or FITC, instead of Cy3, proving that there was no interaction between 8263

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Figure 4. (a) Confocal images of five cell lines after incubated with three fluorophore-tagged aptamers simultaneously for 6 h. The concentration of aptamers was 10 nM. (b) Fluorescence responses of four cancer cell lines (Hela, MCF7, HepG2, and A549) using fluorophore-tagged aptamers. Each value is the average of ten cells. (c) Canonical score plot for two factors of fluorescence response patterns obtained with the aptamer arrays.

slightly digest of aptamers has little effect on the uptake of aptamers. Cancer Cell Discrimination with Multiple Aptamers Simultaneously. When we look into the results in Figure 1, we could find that the three fluorophore-tagged aptamers interact with cancerous cells, but in different degree (Figure 1b, f, and j; c, g, and k; d, h, and l). These results imply that aptamers generated from cell-SELEX procedure could be

candidates of a cross-reactive sensing strategy for cell typing. As a “proof-of-concept” study, we labeled three aptamers (Apt, TD05, and TE13) with FITC, Cy3, and Cy5 and incubated them with different cell lines, respectively. A significant difference of the merge colors was observed for different cancer cell lines (Figure 2b−d). With an appropriate set of aptamers, we could get more subtle information for cell discrimination. Stepwise analysis 8264

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Figure 5. Confocal images of the cell mixture incubated with three fluorophore-tagged aptamers simultaneously for 6 h. (a) The fluorescent image of normal and cancerous liver cells. (b) The bright field images of a. (c) The merge image of a and b. (d) The fluorescent image of cancerous Cervix cells and cancerous liver cells. (e) The bright field images of c. (f) The merge image of d and e. The concentration of aptamers was 10 nM.

Cell Typing of Cell Mixtures. The multicolor imaging method could be applied for single cell typing in cell mixtures. We incubated the cell mixture (L-02 and small amount of HepG2) with the three fluorophore-tagged aptamers simultaneously, and the cancerous cells were clearly observed among normal cells (Figure 5a,c), proving that our multicolor imaging array could effectively detect cancer cells sensitively. When it comes to cancerous cell mixtures, Hela cells and HepG2 cells as example, after incubating with the three fluorophore-tagged aptamers simultaneously, two distinct merge colors could be observed (Figure 5d,f), indicating that our multicolor imaging method works well for cancer cell mixtures.

with different aptamer set(s) was used to determine the best aptamer set for cell typing. First, we incubated nine aptamers with four cancer cells individually. Then the fluorescence intensity of individual cell was recorded with ten cells for each cell line. Subsequently, the data of intensities was subjected to LDA, and the Jackknifed classification matrix using a different combination of aptamers is shown in Figure 3. We observed the maximum differentiation grouping using three aptamers (TC02, TD09, and TC01) in the present study. Multicolor Imaging and Cell Typing with Three Fluorophore-Tagged Aptamers. We labeled TC01, TC02, and TD09 aptamers with FITC, Cy3, and Cy5, respectively, and incubated them with normal cell L-02 and cancer cell HeLa, MCF7, HepG2, and A549 simultaneously. All of the cancer cells give obvious fluorescent signals and differ from each other by colors and intensities while the normal cell L-02 gives little fluorescent signal (Figure 4a). The colors represent the different interactions between the cell lines and the fluorophore-tagged aptamers, therefore we could recognize the cancer cell lines visually from the merge colors of each cell. In order to achieve precise single cell typing, we constructed a training matrix (3 fluorophore-tagged aptamers × 4 cancer cell lines × 10 cells). First, we recorded the intensity of the three channels of a single cell, ten cells for each cell line. Figure 4b presented the average fluorescence intensity of the four cancer cell lines after incubation with three fluorophore-tagged aptamers. Next, we subjected the data of intensities to LDA for discrimination. LDA converted the patterns of the training matrix to canonical scores, the different cell types were separated into four respective groups with 100% accuracy, indicating the ability of this set of aptamers to differentiate between the four cancer cell lines at single cell level. The three canonical factors are 79.0%, 19.0%, and 2.0%, and the plot of the first two factors is presented in Figure 4c. We then tested the system against unknown types of cells, and the discrimination accuracy of unknown cells was 88.9%% (24 out of 27, Table S3).

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CONCLUSIONS

In summary, we found that the interactions between fluorophore-tagged aptamers and target cells are cross-reactive. On the basis of this phenomenon, we have developed a multicolor cell imaging method using fluorophore-tagged aptamers to detect cancer cells from the large amount of normal cells and distinguish cancer cell lines. Compared with traditional cell typing methods, there are two main advantages of this method: first, it allows cancerous cells to be easily picked out from a larger number of normal cells; and second, the type of the cancerous cells can be identified at single cell level. In the present study, we select only three aptamers due to the limitation of the instrument (there are three fluorescence channels in a commercial confocal microscopy). If we labeled aptamers with different fluorescent dyes whose emission spectra are not overlapping, and the sensing platform could record more than three fluorescence signals simultaneously, the discriminate abilities of the present method could be further improved. We believe that this single cell multicolor imaging has potential application in cancer diagnostic and therapy. 8265

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details and additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interests.

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ACKNOWLEDGMENTS This work is supported by the Ministry of Science and Technology of China (Nos. 2013CB933800 and 2012YQ12006003) and the National Natural Science Foundation of China (Nos. 21390410 and 21125525).

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