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Metabolism-Based Click-Mediated Platform for Specific Imaging and Quantification of Cell Surface Sialic Acids Yong Liang,†,∥ Xin Jiang,‡,∥ Rong Yuan,†,∥ Yang Zhou,† Caixia Ji,† Limin Yang,† Haifeng Chen,*,‡ and Qiuquan Wang*,†,§ †

Department of Chemistry & the MOE Key Lab of Spectrochemical Analysis and Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China ‡ School of Pharmaceutical Sciences, Xiamen University, Xiamen 361005, China § State Key Lab of Marine Environmental Science, Xiamen University, Xiamen 361005, China S Supporting Information *

ABSTRACT: Although we believe that the cell surface sialic acids (Sias) are playing an important role in cell−cell interactions and related tumor metastasis processes, acquisition of their quantitative information has yet been a challenge to date. Here, we reported the construction of a new analytical platform for Sias-specific imaging and quantification. We used N-azidoacetyl-mannosamine tetraacylated as a metabolic sugar substrate to bioassemble azido-Sias on the surface of cells via the metabolic pathway of Sias de novo synthesis. These azidoSias allow us to perform a duplex Sias-specific analysis with various fluorescent and elemental reporters such as DIBOAlexa Fluor 647, DBCO-DOTA-Eu, and DBCO-PEG4-BODIPY, which can be easily labeled and/or tagged through an effective copper-free bioorthogonal click reaction. Compared to the previous reported strategies, we quantified the cell surface Sias with the LODs (3σ) down to 8.9 fmol and 0.24 pmol using 153Eu- and 10B-species unspecific isotope dilution ICPMS, in addition to their red- and green-CLSM profiling. Such a platform enables us to evaluate Sias regulation under the administration of paclitaxel, finding that 1 μM paclitaxel induced a significant Sias decrease of 67% on the surface of hepatic tumor cell SMMC-7721, while had no obvious adverse effect to that of para-carcinomatous liver cell LO2. Besides Sias, we believe that this metabolism-based click-mediated platform will provide opportunities to study other monosaccharides and their corresponding biological roles when more corresponding chemically modified sugar substrates and specific bioorthogonal reactions are developed.

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lectins family.12−15 Moreover, exact stoichiometry between the siglecs and the terminal Sias, regardless of Sias-linkage and/or sialylated-glycan structure, have not been definitely pointed out so far. In addition to the siglecs based strategies, the interaction between sophisticated modified boronic acid and the cisdihydroxyl on saccharides was also utilized. Clearly, because a myriad of similar dihydroxyl-containing molecules exist in a real biological sample, this boronic acid-based recognition method is lack of sufficient specificity toward Sias. More seriously, the stability of these boronic acid-saccharide conjugates (stability constant log K = 5−11.5)16−18 that formed by a pair of reversible covalent bonds is subtle to pH during sample preparation. In comparison, current MALDI/ESI-based mass spectrometry is the popular tool for glycan structure analysis and their relative quantification, when combining with various chemical and/or enzymatic separation and enrichment procedures.19,20 Focusing on Sias, we encountered two

tudy of cell surface sialic acids (Sias) has been one of the most intriguing while yet challenging research areas. Among the family of nine kinds of monosaccharides and countless glycan chains, sialic acid has an unusual nine-carbon backbone and always attached/disattached on the termini of glycan chains under the catalysis of sialytransferase and sialidase.1−3 The extraordinary multitude of complicated structural linkages as monomers or polymers render sialylated glycoproteins and glycolipids as the most versatile function modulators of physiological and pathological processes, such as immune cell−cell interactions and related tumor metastasis.4−8 It has been widely acknowledged that qualitative and quantitative information on the cell surface Sias are of critical importance in human health and disease studies.9−11 One of the most frequently employed methods for highly sensitive and throughput profiling of sialylated glycans was the siglecs (sialic acid-binding immunoglobulin-type lectins) based method, recognizing specific Sias linkages. However, available siglecs are restricted to discern structure-known sialylated glycans by the relative fragile noncovalent interactions. They sometimes suffer from overlapped-binding of the glycans that have no terminal Sias due to their similar domain-sequence among the © XXXX American Chemical Society

Received: October 23, 2016 Accepted: December 9, 2016 Published: December 9, 2016 A

DOI: 10.1021/acs.analchem.6b04141 Anal. Chem. XXXX, XXX, XXX−XXX

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Scheme 1. Azido-Sias on the Cell Surface through Ac4ManNAz-Based Metabolism Bioassembly and Copper-Free Click Chemistry Mediated DIBO-Alexa Fluor 647, DBCO-DOTA-Eu, and DBCO-PEG4-BODIPY Labeling/Tagging for Specific Imaging and Quantification of the Cell Surface Sias

Figure 1. Specificity of the cell surface Sias imaging of SMMC-7721 and LO2 labeled by DIBO-Alexa Fluor 647 under CLSM and Eu-signals determined using 153Eu-SUID ICPMS after the cells were tagged with DBCO-DOTA-Eu.

found to undertake the same de novo biosynthesis machinery, bioassembling Sias at the end of glycan chains on the cell surface with an intrinsic efficiency of the biotransformation, providing new possibilities for probing intracellular interactions.24−27 The azide in the bioassembled Sias on the cell surface could be labeled and/or tagged specifically by an alkynecontaining reporting-molecule via the so-called bioorthogonal click chemistry.28,29 We thus proposed to construct a metabolism-based and click-mediated duplex analytical platform (Scheme 1) for Sias-specific imaging and quantification, in

problems: (1) the negatively charged Sias has a relative low ionization efficiency compared to other neutral N-acetylationglycans in the positive ion mode during CID-fragmentation; and (2) the labile nine-carbon structure of Sias at the α-2,3/ 2,6/2,8 linkages easily loses from glycan chains before reaching the MS detector. In order to solve these problems, chemical derivatization is necessary to increase the sensitivity and stability before measurement.21−23 Fortunately, mannosamine and its derivative such as Nazidoacetyl-mannosamine tetraacylated (Ac4ManNAz) were B

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Figure 2. ESI-Q-TOF-MS spectra of Ac4ManNAz-DBCO-DOTA-Eu (a) and Ac4ManNAz-DBCO-PEG4-BODIPY (b) with 1:1 binding stoichiometry between azide and DBCO group. Calibration curves of 153Eu ICPMS intensity against cell surface Sias number and their host cells (c).

which dibenzocyclooctynol-Alexa Fluorescent 647 dye (DIBOAlexa Fluor 647) was used for imaging of the cell surface Sias under a confocal laser scan microscope (CLSM); europium complex with 10-dibenzocyclooctyne 1,4,7,10-tetraazacyclodecane-1,4,7-Tris acetic acid (DBCO−DOTA-Eu) for Siasspecific quantification using inductively coupled plasma mass spectrometry (ICPMS); and dibenzocyclooctynol (polyethy-

lene glycol)4 boron-dipyrromethene (DBCO-PEG4-BODIPY) for both imaging and quantification of the cell surface Sias. Here, hepatic tumor cell SMMC-7721 and para-carcinomatous liver cell LO2 were selected to demonstrate our ideas. First, the SMMC-7721 and LO2 cells were cultured under the presence of Ac4ManNAz donors to assemble terminal azido-Sias metabolically on the cell surface. After washing to C

DOI: 10.1021/acs.analchem.6b04141 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 3. (a) CLSM imaging of the cell surface Sias of SMMC-7721 (a, bright field; a′, dark field) and LO2 (b, bright field; b′, dark field) labeled by DBCO-PEG4-BODIPY; B-signals determined using 10B-SUID ICPMS (a″ and b″) after the Sias were tagged with DBCO-PEG4-BODIPY. Calibration curves for specific quantification of the cell surface Sias and their host cells (c).

obtained results indicated a simple 1:1 stoichiometry between DBCO-DOTA-Eu and Ac4ManNAz (Figure 2a) with the reaction kinetics up to 3.07 M−1 s−1 under physiological conditions (Figure S2). Moreover, we investigated the biotransformation of Ac4ManNAz to azido-Sias under 12, 24, 36, 48, and 72 h cell culture time. The results obtained suggested that the cell surface was saturated with azido-Sias after 48 h cultivation (see Supporting Information, Figure S3). We could thus directly quantify the Sias via 153Eu speciesunspecific isotope dilution ICPMS (153Eu-SUID ICPMS) without any other cell lysis and separation procedures after washing out the excess DBCO-DOTA-Eu (Figure 1 and Figures S4 and S5). With the assistance of an automated cell counter for cell number counting, 1.37 × 1011 Sias (2.27 × 10−13 mol) on one SMMC-7721 cell with RSD = 3.7% (n = 5) and 4.65 × 108 Sias (7.72 × 10−16 mol) on one LO2 cell with RSD = 3.9% (n = 5) were quantified. The dynamic range of the Sia number against 153Eu ICPMS intensity was from 10−14 to 10−8 mol Sia (R = 0.9997, RSD = 3% at 10−10 mol level, n = 5)

remove the excess Ac4ManNAz, the cells were labeled with DIBO-Alexa Fluor 647 via the bioorthogonal copper-free click reaction between the dibenzocyclooctyne in DIBO-Alexa Fluor 647 and the azide in the Sias. In this way, the cell surface Sias were red-profiled under CLSM (Figure 1), suggesting that we achieved a selective and efficient metabolic incorporation and click-mediated labeling of the azido-Sias, as the previous reports using other bioorthogonal fluorescent reagents.30−32 Unfortunately, CLSM imaging could hardly give quantitative information on the cell surface Sias, although relative difference in the Sias abundance on SMMC-7721 and LO2 could be distinguished. In order to obtain quantitative information on the cell surface Sias, we synthesized DBCO-DOTA-Eu (Figure S1) and used it to tag the Sias on SMMC-7721 and LO2. Before quantifying the Sias using ICPMS, we investigated the tagging efficiency of DBCO-DOTA-Eu toward Ac4ManNAz using ESI-Q-TOF-MS, considering that the bioorthogonal click reaction is independent of the chemical form to which azide linked.33,34 The D

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Figure 4. Cell surface Sias regulation of SMMC-7721 and LO2 under administration of paclitaxel, which were determined via153Eu- and 10B-SUID ICPMS.

4 and Supporting Information). Interestingly, compared to SMMC-7721, we found that paclitaxel has no obvious adverse effect to the cell surface Sias content of LO2. The determined 7.18 × 10−16 mol Sias/cell (153Eu-SUID ICPMS, RSD = 4.5%, n = 5) and 7.29 × 10−16 mol Sias/cell (10B-SUID ICPMS, RSD = 6.0%, n = 5) after paclitaxel treatment were almost unnoticeable lower than 7.72 × 10−16 mol Sias/cell of the control. These results implied that paclitaxel selectively attacked the tumor SMMC-7721 cells causing significant down-regulation of Sias, while it was almost inert to that of the normal LO2 cells at the concentration of 1 μM. Although it has been recognized that paclitaxel targets the microtubulin of tumor cells and block the cell division,36,37 the remarkable Sias down-regulation of SMMC-7721 suggested that paclitaxel can also interfere the bioassembly of Sias probably via disturbing the de novo Sias biosynthesis pathway. The selective Sias down-regulation of SMMC-7721 certainly weakens the adhesion of tumor cells with the perivascular epithelioid cells, and thus decreases the risk of tumor metastasis.38 Undoubtedly, more in-depth study of the mechanisms behind will benefit the corresponding pharmacological research and clinical practice of paclitaxel. In conclusion, the established metabolism-based clickmediated platform allows us to perform specific CLSM imaging and ICPMS quantification of the cell surface Sias as exemplified by the tumor cell SMMC-7721 and para-carcinomatous cell LO2 using DIBO-Alexa Flour 647, DBCO-DOTA-Eu, and DBCO-PEG4-BODIPY. Besides, we could count the host cells with significant self-signal amplification via the huge number of the cell surface Sias. For instance, single SMMC-7721 cell (0.15 SMMC-7721 cell as calculated) and 28 LO2 cells could be counted using ICPMS together with DBCO-DOTA-Eu, considering 1.37 × 1011 Sias on one SMMC-7721 cell and 4.65 × 108 Sias per LO2. Moreover, on this platform, we could investigate the cell surface Sias regulation under the administration of a cancer drug, providing new insights regarding the drug’s functions and the biological role of the cell surface Sias in the cell−cell interaction related tumor metastasis processes. Such deep understanding obtained will be very helpful for reconsidering the used drugs in cancer treatment and developing more drugs that are efficacious

with the limit of detection (LOD, 3σ) of Eu-tagged Sias down to 8.9 fmol (Figure 2c). Furthermore, this established metabolism-based click-mediated platform offers a possibility to use a trifunctional molecule, DBCO-PEG4-BODIPY (Scheme 1), which could be tagged to the cell surface azido-Sias with 1:1 stoichiometry between the azide and the DBCO group (Figure 2b). It permits us to perform one-dimensional targeting toward the cell surface azido-Sias and two-dimensional measurements, as evidenced by the green-profiling under CLSM and 10B-SUID ICPMS quantification of the Sias (Figure 3). In this way, the cell surface Sias of SMMC-7721 and LO2 as well as the intercellular interaction visualized vividly under CLSM (Figure 3a,b); after washing out the excess DBCO-PEG4-BODIPY (Figure S6), we directly injected the cells into ICPMS for quantifying the Sias via the determination of B tagged. In total, 1.41 × 1011 Sias (2.35 × 10−13 mol) per SMMC-7721cell (RSD = 5.9%, n = 5) and 4.71 × 108 Sias (7.81 × 10−16 mol) per LO2 cell (RSD = 6.0%, n = 5) were quantified with 0.24 pmol LOD (3σ) of Btagged Sias, which were well in agreement with those using DBCO-DOTA-Eu, although the LOD (3σ) was almost 27times higher. This LOD might be improved using a lanthanidecontaining optical and mass signals affordable molecule, for example, Eu-BCTOT for fluorescent imaging and ICPMS quantification because of the luminescent property of the Eucomplex.35 The dynamic range of the Sias number against 10B ICPMS intensity was from 10−13 to 10−8 mol Sia (R = 0.9990, RSD = 5.5% at 10−9 level, n = 5) (Figure 3c). It is well-known that the cell surface Sias participate in the cell−cell communications that related to tumor metastasis processes.1−3,8 The established Sias-specific analytical platform here provides us a novel way to further investigate Sias regulation on the cell surface and understand its corresponding role under the administration of a cancer drug. Paclitaxel was used here as an example. After the administration of paclitaxel (1 μM), the cell surface Sias of SMMC-7721 were quantified as 7.69 × 10−14 mol/cell with RSD = 4.1% (n = 5) using 153EuSUID ICPMS and 7.76 × 10−14 mol/cell RSD = 5.9% (n = 5) using 10B-SUID ICPMS, indicating that paclitaxel treatment resulted in 67% Sias decrease compared to the control (Figure E

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toward the tumor cell surface Sias. Not limited to Sias, we believe that such a strategy should find applications to other monosaccharides quantification and their host cell counting as well as study of the corresponding biological roles when more corresponding chemically modified sugar substrates and specific bioorthogonal reactions are developed.

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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.analchem.6b04141. Materials and instruments, synthesis and reaction kinetics of DBCO-DOTA-Eu, cell surface Sias imaging and quantification by DIBO-Alexa Fluor 647, DBCO-DOTAEu and DBCO-PEG4-BODIPY using CLSM and ICPMS, and the cell surface Sias regulation under treatment of paclitaxel (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +86 (0)592 2187400. *E-mail: [email protected]. ORCID

Qiuquan Wang: 0000-0002-5166-4048 Author Contributions ∥

Y.L., X.J., and R.Y. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grants 21535007, 21475108, 21275120) and the National Basic Research 973 Program (Grant 2014CB932004) as well as the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant 21521004), Fundamental Research Funds for the Central Universities (Grant 20720150128), and Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, Grant IRT13036).

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DOI: 10.1021/acs.analchem.6b04141 Anal. Chem. XXXX, XXX, XXX−XXX