Development of Highly Selective Fluorescent Probe Enabling Flow

Communication pubs.acs.org/bc

Development of Highly Selective Fluorescent Probe Enabling FlowCytometric Isolation of ALDH3A1-Positive Viable Cells Atsushi Yagishita,†,‡,§ Tasuku Ueno,∥,∇ Hiroyasu Esumi,○ Hideyuki Saya,§ Kazuhiro Kaneko,‡ Katsuya Tsuchihara,† and Yasuteru Urano*,∥,⊥,∇ †

Division of Translational Research, Exploratory Oncology Research and Clinical Trial Center, and ‡Department of Gastroenterology, Endoscopy Division, National Cancer Center, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan § Division of Gene Regulation, Institute for Advanced Medical Research, Graduate School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 Japan ∥ Graduate School of Pharmaceutical Sciences and ⊥Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ∇ CREST, Japan Science and Technology Agency, 7 Gobancho, Chiyoda-ku, Tokyo 102-0076, Japan ○ Research Institute for Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan S Supporting Information *

ABSTRACT: Aldehyde dehydrogenase (ALDH) is overexpressed in some subpopulations of stem cells and cancer cells. We have designed and synthesized the first selective fluorescent probe for class 3 ALDH (ALDH3A1). This probe enabled the visualization of ALDH3A1-positive cells by fluorescence microscopy as well as flow-cytometric isolation of ALDH3A1-positive viable cells from a human Caucasian esophageal squamous cell line (OE21) that heterogeneously expresses ALDH3A1.

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inside ALDH-positive cells. The design of our ALDH probe, which consists of a reactive moiety, a linker, and a fluorophore, is illustrated in Figure 1a. We focused on benzaldehyde derivatives as a reactive moiety because ALDH3A1 is known to prefer medium-chain lipid and aromatic aldehydes, such as hexanal and benzaldehyde, as substrates.10−12 Of note is the fact that benzaldehyde is known as a universally used authentic substrate for ALDH3A1. In addition, diethylaminobenzaldehyde (DEAB), an inhibitor of ALDH, was recently shown to be an excellent substrate for ALDH3A1.13 These reactive moieties were linked to BODIPY FL,14 a bright green-fluorescent dye (Figure 1b). We then tested our candidate probes with recombinant human ALDH3A1. Except for Probe 2, ALDH3A1 metabolized the probe molecules to the corresponding carboxylates without generating byproducts. Next, we examined the reactivity of the probes with other ALDH isoforms (human ALDH1A1 and 1A3) to establish their specificity. Surprisingly, Probes 1 and 2, which contain simple benzaldehyde as the reactive moiety, were metabolized by class 1 ALDH, even though benzaldehyde itself is considered a specific substrate for ALDH3A1 (Table 1). Moreover,

mong 19 known isoforms of human aldehyde dehydrogenase (ALDH), class 1 ALDHs (ALDH1A1 and ALDH1A3) are well-known as non-neoplastic and neoplastic stem cell markers, and ALDH-positivity is associated with treatment-resistant and recurrent cancer.1,2 A fluorescent substrate, ALDEFLUOR, is currently used to assay ALDH1A1 and ALDH1A3 activity3,4 and for visualizing ALDH-positive cells by fluorescence microscopy and flow cytometry.5,6 However, the biological function of class 3 ALDH (ALDH3A1) has been less-studied. ALDH3A1 plays a role in cellular oxidative stress related processes,7 and interestingly, down-regulation of ALDH3A1 causes decreased proliferation of A549 nonsmall cell lung cancer (NSCLC) cells.8 Furthermore, high levels of ALDH3A1 activity may be a feature of some kinds of stem cells.9 Therefore, there is growing interest in noninvasive approaches for analyzing and isolating ALDH3A1positive viable cells.4 ALDEFLUOR cannot be used for this purpose, as it is not metabolized by ALDH3A1 (Figure S1). To design a fluorescent substrate for ALDH3A1, we adopted the basic ALDEFLUOR assay strategy (Figure 1a). Briefly, the probe is freely permeable across intact cell membranes and is converted from the aldehyde form to the corresponding carboxylate form by intracellular ALDH; the increased negative charge of the product prevents diffusion across the plasma membrane so that metabolized probe molecules are trapped © XXXX American Chemical Society

Received: October 26, 2016 Revised: November 22, 2016

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DOI: 10.1021/acs.bioconjchem.6b00618 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry

Figure 1. Design of novel ALDH3A1 probes. (a) Strategy for detecting ALDH-expressing cells by controlling the intracellular retention of the enzyme reaction product. (b) Chemical structures of ALDEFLUOR and our Probes 1, 2, and 3.

Table 1. ALDH Isoform Specificity of Probesa

However, in cellulo imaging of ALDH3A1 was unsuccessful. However, the carboxylate form of the probe was detected in the medium after incubation with ALDH3A1-positive cells (Figure S3−4). These results indicated that probe molecules diffused into the cells and were metabolized to the corresponding carboxylic acid by ALDH3A1, but the product readily leaked out of the cells. To address this issue, we next focused on the hydrophobicity of the probe molecule. The reactive moiety of ALDEFLUOR is −CONHCH2CHO, a short-chain aliphatic aldehyde, while that of our probes is −CONH−X−Ph−CHO. Then, we found that the retention times of the carboxylate form of our probes were closer to that of the aldehyde form of ALDEFLUOR than to that of the carboxylate form of ALDEFLUOR, suggesting that generation of a carboxylate group may not be enough to disallow our probe to diffuse out from cells (Figure S5 and Table S1). Therefore, to minimize leakage of the metabolized probe molecule, its hydrophilicity should be increased. For this purpose, we decided to change the fluorophore, and we selected TokyoGreen (TG), a fluorescein analog that we previously developed,15 in which carboxylate is replaced with a

ALDH isoforms probes

ALDH1A1

ALDH1A3

ALDH3A1

Probe 1 Probe 2 Probe 3 ALDEFLUOR

+ + + +

+ −b −b +

+ −b + −b

a

Determined by high-performance liquid chromatography analysis of enzymatic reaction products. Recombinant human ALDHs were used for this study. Each enzymatic reaction was performed at 37 °C. bNo enzymatic reaction was observed.

ALDH3A1 did not metabolize Probe 2, suggesting that the structure of the linker moiety may influence substrate recognition. Overall, DEAB seemed to be the best reactive moiety for ALDH3A1 detection. We next examined in cellulo imaging assay with human Caucasian esophageal squamous cell line OE21 cells, which highly express ALDH3A1 (Figure S2). Little or no expression of class 1 ALDHs was detected in these cells (Figure S2). B

DOI: 10.1021/acs.bioconjchem.6b00618 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry

Figure 2. Detection of ALDH3A1 activity of OE21 cells. (a) Chemical structures of Probes 4 and 5. (b) Representative confocal fluorescence image of cells treated with Probe 5. OE21 cells were incubated with culture medium containing Probe 5 (40 μM) for 1.5 h at 37 °C. The inhibitor CB7 (10 μM) was added to the negative controls. Scale bar: 100 μm. (c) Representative flow cytogram (SSC vs relative fluorescence intensity of probe). Cells were prepared under the same conditions as in (b). Gates for the Probe 5 positive population were set based upon the CB7 control (i.e., higher fluorescence intensity area than the control). Experiments were run in triplicate, and the positive rates were about 10 to 15%. Negative populations are shown in yellow, and the positive populations are shown in green.

methyl group to improve the cell membrane permeability.16 Probes 4 and 5 were designed with different linker lengths (Figure 2a) and with DEAB as the reactive moiety because it provided the highest specificity for ALDH3A1 among Probes 1−3. As expected, the overall molecular hydrophobicity of the probes was improved (Figure S5 and Table S1). However, Probe 4 was not metabolized to the corresponding carboxylate, presumably because the linker was too short to avoid steric hindrance or electrostatic repulsion between TG and enzyme. However, Probe 5 was effectively metabolized to the corresponding carboxylate by ALDH3A1(Table 2). It is

metabolized probe molecules occurs primarily through energy-dependent mechanisms. In the case of ALDEFLUOR assay, inhibitors of active transport are recommended to be added to the assay buffer to suppress leakage of the metabolized product of ALDEFLUOR. Therefore, we tested several transporter inhibitors. MK-571 (a specific inhibitor of multidrug resistance-associated proteins; MRPs)18 considerably improved the retention of fluorescence (Figure S9). Flow cytometry of MK-571-treated cells revealed a much-higher percentage of positive population (∼90%) than was observed for MK-571-untreated cells (10−15%, Figure 3). The

Table 2. ALDH Isoform Specificity of Probesa ALDH isoforms probes

ALDH1A1

ALDH1A3

ALDH3A1

Probe 4 Probe 5

−b +

−b −b

−b +

a

Determined by high-performance liquid chromatography analysis of enzymatic reaction products. Recombinant human ALDHs were used for this study. Each enzymatic reaction was performed at 37 °C. bNo enzymatic reaction was observed. Figure 3. Effect of active efflux inhibitor on ALDH3A1 assay with Probe 5. Flow cytometry of OE21 cells using Probe 5 (50 μM) in combination with active efflux inhibitor MK-571 (200 μM) was far more effective in distinguishing ALDH3A1-active cells compared with results from the absence of the inhibitor. Experiments were run in triplicate, and the positive rates were about 85−95%.

interesting to note that the carboxylate form of Probe 5 was brighter than the aldehyde form; however, there was little difference in the absorption and emission spectra (Figure S6). As shown in Table S2, the kcat value of ALDH3A1 for Probe 5 was approximately 6-fold higher than that of ALDH1A1, and reverse-phase high-performance liquid chromatography (RPHPLC) analysis indicated that the carboxylate form of the probe would have lower membrane permeability than the metabolite of ALDEFLUOR. Indeed, OE21 cells were successfully imaged with Probe 5 (Figure 2b), and the fluorescence was significantly decreased in the presence of an ALDH3A1-specific inhibitor (CB7).17 Similar results were obtained with another ALDH3A1-positive cell line, TE11 (Figure S7). We also confirmed that Probe 5 is suitable for use in flow cytometry (Figures 2c and S8). During imaging studies, we noticed that the intracellular fluorescence was not well-maintained at a high level (Figure S9). The decrease of fluorescence was reduced when cells were incubated at below 4 °C, suggesting that efflux of the

percentage of positive population was dramatically reduced by the knockdown of ALDH3A1 with siRNA (Figure S10). These results clearly show that inhibition of active efflux of the metabolized probe is critical to avoid underestimation of ALDH3A1 activity. Cell viability testing with SYTOX Red dead-cell stain showed no significant difference between cells treated with Probe 5, MK-571, or both and untreated cells under these experimental conditions (Figure S11), and therefore, it should be feasible to isolate viable ALDH3A1positive cells in a cell sorter with the aid of this probe. To confirm this, we examined the isolation of the ALDH3A1-positive population from a mixture of ALDH3A1C

DOI: 10.1021/acs.bioconjchem.6b00618 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry

simple and sensitive detection and isolation of ALDH3A1positive viable cells by means of flow cytometry. It should be useful for studies of the maturational lineage biology of stem cells and progenitor cells and may also have clinical relevance in establishing the characteristics of ALDH3A1-positive cancer cells. Our design strategy could be extended to the design of probes for other classes of ALDHs by appropriately modifying the reactive moiety.

positive and -negative cells. We established an OE21 cell line in which ALDH3A1 was stably knocked down with short hairpin RNA (shRNA) as an ALDH3A1-negative cell line. Immunoblot analysis of whole-cell lysates demonstrated that ALDH3A1 was almost undetectable (“KD” in Figure S12) in the resulting cell line in comparison to the negative control (lysate of OE21 cells transfected with nontarget shRNA; “ctrl” in Figure 4a). Flow

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00618. Details of the synthesis of ALDH probes, reaction analysis procedures and analysis results, and properties of experimental materials. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 81-35814850. Fax: 81-358414855. ORCID

Atsushi Yagishita: 0000-0001-7057-7647 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Cancer Center Research and Development Fund (28-A-9 to K.T.)

Figure 4. Cell sorting of ALDH3A1-positive cells. (a) Representative flow cytogram (SSC vs relative fluorescence intensity of Probe 5) of MK-571- and shRNA-treated cells. Flow cytometry was performed in the presence of both Probe 5 (50 μM) and MK-571 (200 μM). ALDH3A1 stable knockdown in OE21 cells was achieved by using shRNA against ALDH3A1 (KD); for the control, we used nontarget shRNA (Ctrl). The ALDH3A1-positive population was significantly reduced by knockdown. (b) Representative flow cytogram of a mixedcell sample (ctrl/KD = 1:1). Cells were well-separated into two populations. (c) Relative amounts of the ALDH3A1-positive population in sorted cells. Cells in the top 25% fluorescence intensity level and in the bottom 25% fluorescence intensity level were isolated with a cell sorter and subjected to immunoblot analysis.

ABBREVIATIONS ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; TG, Tokyo Green; MRP, multidrug-resistanceassociated protein; shRNA, short hairpin RNA; Ctrl, control; KD, knock down

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REFERENCES

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DOI: 10.1021/acs.bioconjchem.6b00618 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

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DOI: 10.1021/acs.bioconjchem.6b00618 Bioconjugate Chem. XXXX, XXX, XXX−XXX