Simple Summary: Cancer cells often have aberrant sialic acid expression. We used molecularly imprinted polymers in this study as novel tools for analyzing sialic acid expression as a biomarker on cancer cells. The sialic acid imprinted polymer shell was synthesized on a polystyrene core, providing low-density support for improving the suspension stability and scattering properties of the molecularly imprinted particles compared to previous core-shell formats. Our results show that these particles have an increased ability to bind to cancer cells. The binding of these particles may be inhibited by two different pentavalent sialic acid conjugates, pointing to the specificity of the sialic acid imprinted particles. Sialic acid (SA) is a monosaccharide usually linked to the terminus of glycan chains on the cell surface. It plays a crucial role in many biological processes, and hypersialylation is a common feature in cancer. Lectins are widely used to analyze the cell surface expression of SA. However, these protein molecules are usually expensive and easily denatured, which calls for the development of alternative glycan-specific receptors and cell imaging technologies. In this study, SA-imprinted fluorescent core-shell molecularly imprinted polymer particles (SA-MIPs) were employed to recognize SA on the cell surface of cancer cell lines. The SA-MIPs improved suspensibility and scattering properties compared with previously used core-shell SA-MIPs. Although SA-imprinting was performed using SA without preference for the α2,3- and α2,6-SA forms, we screened the cancer cell lines analyzed using the lectins Maackia Amurensis Lectin I (MAL I, α2,3-SA) and Sambucus Nigra Lectin (SNA, α2,6-SA). Our results show that the selected cancer cell lines in this study presented a varied binding behavior with the SA-MIPs. The binding pattern of the lectins was also demonstrated. Moreover, two different pentavalent SA conjugates were used to inhibit the binding of the SA-MIPs to breast, skin, and lung cancer cell lines, demonstrating the specificity of the SA-MIPs in both flow cytometry and confocal fluorescence microscopy. We concluded that the synthesized SA-MIPs might be a powerful future tool in the diagnostic analysis of various cancer cells.
Keywords: cancer; imprinting; molecularly imprinted polymers; SA conjugates; sialic acid
Sialic acid (SA) is a nine-carbon monosaccharide located at the terminal end of cell surface proteins, lipids, or secreted proteins [[
Moreover, the level of SA expression in cancer has been shown to result in the cancer cell's increased metastatic and invasive potential [[
Boronic-acid-based semi-covalent imprinting is widely used to recognize glycoproteins since they bind reversibly with cis-diol groups of the saccharide units [[
We recently reported these particles' synthesis and binding properties in solution and cell labeling assays with two cancer cell lines, the epidermal carcinoma cell line A431 and pulmonary epithelial carcinoma cell line A549 [[
In addition, the specificity of the SA-MIPs was validated using two pentavalent SA conjugates, ME0752 and ME0976 [[
The biotin-labeled lectins MAL I and SNA were purchased from Vector Laboratories (Burlingame, CA, USA). The streptavidin-fluorescein isothiocyanate (FITC) was obtained from Agilent Technologies (Santa Clara, CA, USA). The Falcon multi-chamber culture glass slides were purchased from Corning (Glendale, AZ, USA). The mounting medium ProlongQR Gold antifade reagent, phosphate buffered saline (PBS), vinylbenzene boronic acid (VBBA), and 4′,6-diamino-2-phenylindole (DAPI) were bought from Thermo Fisher Scientific (Waltham, MA, USA). Triton 100X, methacrylamide (MAAm), ethylene glycol dimethacrylate (EGDMA), formaldehyde, and rhodamine phalloidin were purchased from Sigma–Aldrich (Taufkirchen, Germany). The 2,2′-Azobis(2,4-dimethylvaleronitril) (ABDV) initiator was obtained from Wako Chemicals (Neuss, Germany). Human cell lines including MDA-MB-468, MDA-MB-231, CAMA-1, T-47D, MCF7, SK-BR-3, Hs 578T, A549, A-431, PC-3, THP-1, and Jurkat were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). Hek-n cells are primary human epidermal keratinocytes isolated from neonatal foreskin and were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The following cells were cultured in a cell culture medium purchased from Thermo Fisher Scientific (Waltham, MA, USA): MDA-MB-231 and MDA-MB-468 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). MCF7, Jurkat, THP-1, and T-47D cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 50 µg/mL gentamycin. Hs-578T cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin (PEST), and 10 µg/mL insulin. CAMA-1 was cultured in RPMI-1640 medium supplemented with 10% FBS, 1% PEST, and 1% sodium pyruvate. A549 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% PEST. SK-BR-3 and PC-3 were cultured in DMEM supplemented with 10% FBS, 1% GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA), and 1% PEST. Hek-n cells were maintained in EpiLife growth medium with 60 mM calcium chloride supplemented with 1% human keratinocyte growth supplement (HKGS) and 0.2% gentamycin/amphotericin. A-431 cells were cultured in Eagle's Minimum Essential Medium (EMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS, 1% L-glutamine and 1% non-essential amino acids (Thermo Fisher Scientific, Waltham, MA, USA). All the cell lines were cultured at 37 °C with 5% CO
The synthesis of SA-MIPs was performed as we recently reported [[
Briefly, the cells were first washed twice with 2 mL PBS. Next, the cells were divided into several flow cytometry tubes (5 × 10
A total of 1 × 10
In this assay, the SA-MIPs were pre-treated with pentavalent SA conjugates (SA conjugates) before being used in a MIP staining assay according to the experimental procedure described above. Pre-treatment was conducted by pre-incubating the MIP in PBS with different concentrations (20 and 200 µM) of the SA conjugates ME0976 [[
For the confocal fluorescence microscopy analysis, 1 × 10
In this study, two different lectins, MAL I (α2,3-SA) and SNA (α2,6-SA) were used to analyze the expression of SA on the surface of thirteen human cancer cell lines by flow cytometry: SK-BR-3, MDA-MB-468, PC-3, THP-1, Jurkat, A-431, MCF7, MDA-MB-231, A549, Hek-n, CAMA-1, T-47D, and Hs 578T cells. The α2,3-SA and α2,6-SA lectin staining results are presented in histograms (Figure 1). The MFI of unstained control cells as the background is also shown for each cell line in Figure 1 (black line). The breast cancer cell line CAMA-1 shows the lowest expression of α2,3-SA. For α2,6-SA expression, the breast cancer cell line MCF7 and prostate cell line PC-3 show the least pronounced expression. High expression levels of α2,3-SA were determined in the breast cancer cell lines Hs 578T and MDA-MB-231 and the lung carcinoma cell line A549. The experiment has been repeated twice with minor deviations. Therefore, one representative experiment out of two performed is shown.
The binding properties of the SA-MIPs were analyzed using flow cytometry. The MFI of SA-MIP binding to the cell lines SK-BR-3, MDA-MB-468, PC-3, THP-1, Jurkat, A-431, MCF7, MDA-MB-231, A549, Hek-n, CAMA-1, T-47D, and Hs 578T are shown in Figure 2. The SA-MIP binding properties are displayed in histograms (Figure 2A) and bar diagrams showing the MFI (Figure 2B). The order of the cell lines is based on the binding capacity of the SA-MIPs.
Three cell lines with different binding properties to SA-MIPs, A549 lung carcinoma cells (high binding), MCF7 breast cell line (average binding), and A-431 skin carcinoma cells (low binding), were selected for further analysis. The specificity of the SA-MIP binding was assessed using flow cytometry. The SA-MIPs were pre-incubated with SA-conjugates ME0752 or ME0976 at a concentration of either 20 µM or 200 µM, respectively, and then applied to cell-binding assays. For all three cell lines, a reduction in MFI was observed for SA-MIP binding to cells after incubation with both concentrations of SA-conjugates (Figure 3A–C). The highest reduction occurred in the skin carcinoma cell line A-431 (Figure 3B).
To visualize the binding of the SA-MIPs to the cells and characterize the fluorescence properties of the SA-MIPs, the three selected cancer cell lines, A549, MCF7, and A-431 were analyzed using confocal fluorescence microscopy. In addition to staining with SA-MIPs, all cell lines were stained with DAPI and phalloidin for nuclei and cytoskeleton visualization, respectively. The binding pattern and distribution of the SA-MIPs were different in the three cell lines (Figure 4A–L).
The A549 cells showed a uniform distribution of SA-MIPs (Figure 4B). In contrast, the A-431 cells showed a very low binding of the SA-MIPs (Figure 4F), whereas MCF7 cells showed a heterogenous SA-MIP binding pattern (Figure 4J).
The SA-MIP binding pattern changed after pre-treatment with 200 µM of SA conjugate ME0752. The A549 cells showed less binding of SA-MIPs (Figure 4C). In contrast, A-431 cells showed that several SA-MIPs were bound as small particles (Figure 4G). MCF7 cells showed similar amounts of bound particles (Figure 4K).
After pre-treatment with 200 µM of SA conjugate ME0976, the SA-MIP binding pattern for A549 cells (Figure 4D) and A-431 cells (Figure 4H) remained similar to pre-treatment with ME0752. The MCF7 cells showed fewer bound particles than those pre-treated using ME0752 (Figure 4L).
We previously reported about developing and using core-shell SA-imprinted particles for determining cell surface glycans [[
All thirteen cancer cell lines were stained with the improved SA-MIP batch and analyzed by flow cytometry. Our results revealed a different staining pattern for the cancer cell lines, which cannot be compared with the α2,3-SA, and α2,6-SA expression since the SA-MIPs were imprinted with SA. In addition, the weak nature of glycan-mediated interactions may affect the MAL I and SNA binding properties, as well as specificity [[
We further developed the use of pentavalent SA conjugates to validate the binding of the SA-MIPs to three different cell types, A549 lung carcinoma, MCF7 breast cancer, and A-431 skin carcinoma cells [[
The staining patterns of the SA-MIPs were visualized on the selected cancer cell lines, A549, MCF7, and A-431, using confocal fluorescence microscopy. The binding pattern to the lung carcinoma A549 cells revealed a uniform distribution of SA-MIPs. Most interestingly, the addition of pentavalent SA conjugates changed the SA-MIP staining pattern of the cells by diminishing the SA-MIP binding. For all three cell types analyzed, the bound MIPs were more diluted on the cell surface after adding the SA conjugates. A549 cells showed fewer bound particles, whereas MCF7 showed similar numbers. In contrast, the number of SA-MIPs bound to A-431 cells was increased. The preincubation of MIP particles with the SA conjugates prior to cell binding results in the presence of the spacer molecules in the polymer layer of the particles, which facilitates solubilization of the MIP particles in the assay suspension.
In our flow cytometry results, α2,3-SA was expressed at comparable levels in A-431 and MCF7 cells. Confocal microscopy showed very few SA-MIP particles bound to the cell surface of A-431 cells. The different distribution of the SA-MIPs on A549 observed in the confocal microscopy images can be explained by the high expression of both α2,3-SA and α2,6-SA seen in flow cytometry. The MFI values for MAL I staining were high on all cell lines, whereas A549 expressed α2,6-SA to a greater extent, as seen in the SNA staining results.
The SA-MIPs were imprinted without selectivity for the two forms α2,3-SA or α2,6-SA and, therefore, did not reveal clear specificity compared to cell staining using MAL and SNA lectins. Moreover, the SA-MIPs are significantly larger than lectins and can be expected to display multivalent interactions with the cell surface. The fact that each cell line has distinct characteristics and morphology may also influence the binding behavior of the larger MIP particles.
We analyzed the SA expression in thirteen different cancer cell lines using SA-MIPs together with MAL I and SNA. The results show that the varying expression of α2,3- and α2,6-SA results in different binding capacities for SA-MIPs. Preincubation of the SA-MIPs with pentavalent SA conjugates reduced the overall binding of the MIPs, pointing to the specificity of the MIPs to bind SA. In conclusion, synthesized SA-MIPs may be applied as effective tools to analyze the potential biomarker SA expressed on the surface of cancer cells..
Graph: Figure 1 MAL I (α2,3-SA) and SNA (α2,6-SA) lectin binding to the thirteen cancer cell lines. Both lectins were used at a concentration of 5 µg/mL. The flow cytometry histograms show the mean fluorescence intensity (MFI) of unstained control cells (black lines) and lectin-stained cells (blue lines for MAL I and red lines for SNA). One representative experiment out of two performed is shown.
Graph: Figure 2 SA-MIP binding to the thirteen cancer cell lines as shown by MFI. The concentration of SA-MIPs is 0.1 mg/mL. (A) The flow cytometry histograms show the MFI of unstained control cells (black lines) and SA-MIP-stained cells (red lines); (B) the bar diagrams show the MFI of SA-MIP-stained cells. One representative experiment out of two performed is shown.
Graph: Figure 3 SA-MIPs were pre-incubated with different concentrations of SA conjugates and analyzed with flow cytometry. The reduction in binding compared to SA-MIP binding alone is shown. The SA conjugates ME0752 and ME0976 were added to the SA-MIPs at 20 µM and 200 µM, respectively, and the particles were used thereafter to stain (A) A549, (B) A-431, and (C) MCF7 cells. The chemical structures for ME0752 and ME0976 are shown in (D). One representative experiment out of two performed is shown.
Graph: Figure 4 Confocal fluorescence microscopy images of SA-MIPs staining for three different cancer cell lines. A549 (A–D), A-431 (E–H), and MCF7 (I–L) were stained with SA-MIPs (B,F,J) in green, rhodamine-phalloidin (actin filaments) in red and DAPI (nuclei) in blue. The two columns on the right show staining with SA-MIPs after pre-treatment with the 200 µM of SA conjugates ME0752 (C,G,K) and ME0976 (D,H,L). Scale bar: 20 µm.
S.B., Y.Z., M.M.S., Z.E.-S. and A.G.W. conceived and designed the study. S.B., Z.E.-S., Y.Z., A.V., T.W., L.S. and Y.Z. conducted the cell-based studies and performed experiments; M.K., K.G. and K.R. designed and synthesized chemical compounds; Z.E.-S., S.B., Y.Z., J.L.P., L.O., M.M.S. and A.G.W. analyzed the data; K.G., L.O., M.M.S., P.H. and J.L.P. provided advice and technical assistance; E.J., M.E. and R.C. synthesized the pentavalent SA conjugates; S.B., Z.E.-S., Y.Z., P.H., M.M.S. and A.G.W. wrote the manuscript with contributions from all authors. All authors have read and agreed to the published version of the manuscript.
This study was funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement number 721297, the Swedish Knowledge Foundation grant number 20160165, the Malmö Cancer Center, Malmö, Sweden, Biofilms Research Center for Biointerfaces and Malmö University, Sweden.
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The data presented in this study are available in this article.
The authors declare no conflict of interest.
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