Alkaline Phosphatase-Catalyzed Amplification of a Fluorescence

Dec 20, 2017 - Herein, we describe an alkaline phosphatase (AP)-based technique to amplify the fluorescence signal for cell staining applications...
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Alkaline Phosphatase-Catalyzed Amplification of a Fluorescence Signal for Flow Cytometry Takanobu Nobori, Kenta Tosaka, Akira Kawamura, Taisei Joichi, Kenta Kamino, Akihiro Kishimura, Eishi Baba, Takeshi Mori, and Yoshiki Katayama Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03893 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Analytical Chemistry

Alkaline Phosphatase-Catalyzed Amplification of a Fluorescence Signal for Flow Cytometry Takanobu Nobori,1 Kenta Tosaka,2 Akira Kawamura,2 Taisei Joichi,2 Kenta Kamino,2 Akihiro Kishimura,1,2,3 Eishi Baba,4 Takeshi Mori,*,1,2 Yoshiki Katayama*,1,2,3,5,6 1

Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 8190395, Japan. 2 Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan. 3 International Research Center for Molecular Systems, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. 4 Department of Comprehensive Clinical Oncology, Faculty of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. 5

Center for Advanced Medical Innovation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Department of Biomedical Engineering, Chung Yuan Christian University, 200 Chung Pei Rd., Chung Li, 32023 ROC, Taiwan. * Takeshi Mori, Yoshiki Katayama, Tel/Fax: +81 92 802 2850, E-mail address: [email protected]; [email protected]. 6

ABSTRACT: Despite the expanding use of flow cytometry, its detection limit is not satisfactory for many antigen proteins with low copy numbers. Herein, we describe an alkaline phosphatase (AP)-based technique to amplify the fluorescence signal for cell staining applications. We designed a fluorescent substrate that acquires membrane permeability upon dephosphorylation by AP. By using the substrate, the fluorescence signal of cells in flow cytometry could be successfully amplified to give a much stronger signal than the cells labeled using a conventional fluorophore-modified antibody.

Flow cytometry enables evaluation of the relative amount of antigen proteins in cells with single-cell resolution and has been used in the diagnosis of blood cancers1 as well as basic research in cell biology.2 Antigen proteins of interest are labeled by fluorophore-modified antibodies to detect the presence of antigen proteins on cells. Although flow cytometry is a useful technology for the detection of cellular antigens, it lacks sensitivity. For the fluorescence signal resulting from the fluorophore-modified antibody to exceed the autofluorescence signal of the cells, typically a few thousand antigen protein molecules per cell are required to be detected by flow cytometry.3 However, antigen proteins of interest are often expressed at levels lower than the detection limit. One report estimated that 65% of the human proteome is expressed at less than a few thousand copies. 4 To improve the detection limit of flow cytometry, researchers have developed methods to increase the number of fluorophores per antigen on a cell5-13. One of the approaches to achieve this involves utilizing antibodies modified with multiple fluorophores by using polymers5 and liposomes.6,7 The other approach is based on amplification of the fluorescence signal. Amplification is achieved by enlarging antigen-antibody complexes via stepwise modification of secondary antibody or streptavidin. 8,9 The fluorescence amplification is also achieved via an enzymatic reaction. This method is called catalyzed reporter deposition (CARD), in which horseradish peroxidase (HRP) modified

on antibodies is used as an enzyme to covalently deposit tyramide-modified fluorophores to a cell.10-13 Using this method, antigens with low copy numbers that are difficult to detect by conventional fluorophore-modified antibodies were successfully detected.10-13 If the signal amplification method can be expanded to other enzymes, it will allow the simultaneous detection of more than one antigen with low expression levels in a single cell. Herein, we describe our attempts to expand the signal amplification method to alkaline phosphatase (AP), which is another popular enzyme used in bioanalysis such as enzyme immunoassays and immunohistochemistry because of the inherent advantages of AP including its high stability and activity, and small size. 14,15 Detection mechanism is shown in Figure 1A. A fluorescent substrate for AP is composed of a hydrophobic alkyl chain, a hydrophilic phosphate and a fluorophore. The antigen protein on the target cell surface is labeled with a ternary complex of antibody/streptavidin/AP, and then the substrate is dephosphorylated by AP to increase its hydrophobicity and cationic charge of the substrate. The plasma membranes of mammalian cells are known to have negative membrane potentials, which enable the penetration of hydrophobic and cationic molecules to accumulate in the cytosol.16 Thus, the dephosphorylated substrate penetrates the cell membrane to accumulate in the cytoplasm, which results in amplification of fluorescence signal of the target cell. We named our AP-based method the catalyzed reporter

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Figure 1. Mechanism of fluorescence signal amplification by CARP method (A), and the chemical structures of substrates (B). In panel A, the antigen protein is labeled by the ternary complex of antibody/streptavidin/alkaline phosphatase (AP). A fluorescent substrate was designed such that its hydrophobicity and cationic charge is increased upon AP-catalyzed removal of a hydrophilic and negatively charged phosphate group, thereby allowing the dephosphorylated substrate to penetrate the cell membrane to accumulate in the cytoplasm. M--CD was added to facilitate the transfer of the substrate to the cell membrane. penetration (CARP) method. Figure 1B shows the structure of substrate 1-p, which contains a hydrophobic alkyl chain with a phosphorylated tyrosine in the middle and a membrane permeable fluorophore (tetramethyl rhodamine17) on the terminus. Because these substrates were able to be synthesized by solid phase peptide synthesis, we could use commercially available components such as phosphotyrosine, α,ω-amino acids and fatty acids to achieve the membrane penetration triggered by dephosphorylation. Because the substrate was designed to be hydrophobic, methyl β-cyclodextrin (M-β-CD) was used to assist distribution of the substrate from the aqueous phase to the cell membrane. M-βCD is well known to solubilize hydrophobic molecules by the formation of inclusion complexes, which facilitates the transfer

Figure 2. Absorption spectra of 1 and 1-p (10 M) with or without M-β-CD (10 mM) in 10 mM HEPES buffer containing 300 mM mannitol before (blue line) and after filtration (red line). Absorption spectra of 1 and 1-p completely solubilized in methanol (black line).

of the hydrophobic molecule from aqueous media to the cell membrane.18,19 We evaluated the solubility of 1-p and its dephosphorylated form 1 by a filtration method.20 Figure 2 shows the absorption spectra of aqueous solutions of 1 and 1-p in the presence and absence of M-β-CD after the removal of insoluble aggregates by filtration (0.22 m). Both 1 and 1-p were insoluble in aqueous buffer, while in the presence of M-βCD, 1-p became soluble while 1 was still insoluble. This result indicates that soluble 1-p is converted to insoluble 1 upon the dephosphorylation catalyzed by AP, leading to the distribution of 1 to the cell membrane from the aqueous phase. Next, we examined the effect of M-β-CD on the staining of cells with 1. As shown in Figure 3A, M-β-CD strongly enhances the staining of cells. The cell staining was almost saturated after a 30-min incubation (Figure 3C). Dephosphorylated substrate 1 stained the cytoplasm except for the nucleus (Figure 3B), indicating the binding of 1 to the cytoplasmic membrane fraction. The efficacy of staining by 1 was compared with 2 or 3 having a shorter alkyl chain. As shown in Figure 3C, staining with 2 or 3 was found to be weaker than 1, indicating the importance of hydrophobicity to the distribution of the staining molecule to the cell membrane. It is important to note that the fluorescence intensity of the cells stained by 1 did not change for at least 4 h (Figure S1), which is in sharp contrast with tetramethylrhodamine, which disappeared completely from cells.17 This indicates the relatively strong affinity of the hydrophobic alkyl chain of substrate 1 to the membrane fractions, which suppress the dissociation of the substrate from the membrane fraction into solution. The long retention of 1 is reminiscent of commercial cell labeling reagents21 and lipopeptides,22 both of which possess long alkyl chains to suppress dissociation from lipid membranes. On the surface of mammalian cells, endogenous APs are known to exist as membrane proteins.23 The activity of these endogenous APs must be suppressed to exclude the contribution of the endogenous APs in the dephosphorylation reaction of substrate 1-p. We tried to establish two methods to inactivate the endogenous APs; utilization of a specific inhibitor and chemical fixation. First, we examined three reported inhibitors of endogenous

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Analytical Chemistry APs (levamisole,15 vanadate,24,25 and pervanadate25) to find an effective inhibitory condition for our purpose. Here we used a standard substrate of APs, p-nitrophenyl phosphate. We found that both vanadate and pervanadate reduced the endogenous APs’ activity to a negligible level (Figure S2). Because pervanadate is an irreversible inhibitor,25 we decided to use pervanadate for the inhibition of the endogenous APs. The chemical fixation of cells is used to inactivate the endogenous APs for immunohistochemistry applications.15 However, the use of chemical fixation may led to the deterioration of the recognition of antigen proteins on the cell surface by antibodies. We found that gentle fixation by using 4% paraformaldehyde/0.1% glutaraldehyde, which is reported by Luby-Phelps et al,26 was the

Figure 3. (A) Staining of K562 cells by 1 (5 M) with or without M-β-CD (5 mM). (B) Magnified image of a single cell stained by 1 with M-β-CD. The nucleus was stained by Hoechst 33342. (C) Comparison of 1, 2 and 3 in the time-dependent staining of K562 cells with M-β-CD.

most suitable condition. As shown in Figure S3A, JY25 cells were strongly stained by 1-p because of the dephosphorylation by the endogenous APs. However, the fixed cells displayed only a negligible fluorescence signal. Additionally, JY25 cells fixed using this method did not show loss of binding efficacy to the anti-CD20 antibody targeted to the cell surface antigen protein, CD20 (Figure S3 B). The selective distribution of dephosphorylated substrate 1 to the lipid bilayer was also confirmed by using liposome as a model of cell membrane. We observed that only small amount of 1-p was distributed to the liposome, while 1 was significantly distributed to the liposome (Figure S4). Finally, we demonstrated detection of the antigen proteins by using the CARP method. We selected CD20 and epidermal growth factor receptor (EGFR) as the targets as both are representative biomarkers of B cell-derived cancer cells and epidermis-derived cancer cells, respectively. JY25 cells27 and A549 cells28 were chosen as the CD20 positive and EGFR positive cells, respectively. After the inactivation of the endogenous APs by fixation or inhibition with pervanadate, each cell was labeled with a corresponding biotinylated antibody/streptavidin complex, and then the biotinylated AP was modified onto the antibody as depicted in Figure 1A. The resulting cell was stained with substrate 1-p using the enzymatic reaction for 30 min at 37°C and was subjected to the flow cytometric analysis. For

Figure 4. Detection of antigen proteins by using conventional immunofluorescence staining or the CARP method. Microscopic observation of JY25 cells expressing CD20 stained using two methods (A). Flow cytometric analysis of CD20 on JY25 cells (B) and EGFR on A549 cells (C) stained by immunofluorescence (left) and CARP method (right). Endogenous APs were inactivated by fixation with 4% paraformaldehyde/0.1% glutaraldehyde for 1 h (A, B) or by treatment with 100 M pervanadate for 30 min (C). The concentration of 1-p and M-β-CD was 5 μM and 5 mM, respectively (A, B, C).

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comparison purposes, cells were also conventionally labeled using a fluorophore-modified antibody. Figure 4A shows the fluorescence images of JY25 cells labeled using these two methods. The conventional labeling of JY25 cells with CD20 resulted in a fluorescence signal from the cell surface. In contrast, the CARP method resulted in the staining of the entire cytosol except for the nucleus. Figure 4B shows the flow cytometric detection of CD20. The fluorescence signal obtained by the CARP method was about five times higher than that from the conventional immunofluorescence method. We also examined the effect of the concentration of 1-p on the detection of CD20 on JY25 cells by the CARP method (Figure S5). The fluorescence signal proportionally increased with increasing concentration of 1-p, indicating that the extent of fluorescence staining can be tuned by the concentration of 1-p. In conclusion, we successfully applied AP to the enzymatic amplification of the fluorescence signal from antigen staining of cells for flow cytometric analysis. Our fluorescent substrate 1p acquired membrane permeability in response to dephosphorylation leading to the staining of the entire cytoplasm. Treatment with pervanadate or gentle fixation of cells helped to inhibit endogenous APs to enable the dephosphorylation of the substrate specifically by the antibody-modified AP. Our APbased CARP method is suitable for combination with the HRPbased CARD method to enable simultaneous detection of two independent antigens with low expression levels. Such work will be reported in our forthcoming paper.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures, fluorescence intensity of stained cells (Figure S1), effect of inhibitors on the suppression of endogenous APs (Figure S2), effect of cell fixation on the suppression of endogenous APs and the affinity of the cell surface antigen to the antibody (Figure S3), distribution of the substrate 1-p and 1 to the liposomes (Figure S4) and effect of 1-p concentration on the CD20 detection (Figure S5) (PDF)

AUTHOR INFORMATION Corresponding Author *[email protected] (T.M.). *[email protected] (Y.K.).

ACKNOWLEDGMENT This work was supported by Grant-in-Aid for Scientific Research B (project No. 16H04167) of MEXT, Japan. T. Nobori thanks JSPS for a fellowship. We appreciate the technical assistance from The Research Support Center, Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences.

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Analytical Chemistry

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