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In situ recognition of cell-surface glycans and targeted imaging of cancer cells.

Xu XD, Cheng H, Chen WH, Cheng SX, Zhuo RX, Zhang XZ - Sci Rep (2013)

Bottom Line: Fluorescent sensors capable of recognizing cancer-associated glycans, such as sialyl Lewis X (sLe(x)) tetrasaccharide, have great potential for cancer diagnosis and therapy.Here we report boronic acid-functionalized peptide-based fluorescent sensors (BPFSs) for in situ recognition and differentiation of cancer-associated glycans, as well as targeted imaging of cancer cells.The newly developed peptide-based sensor will find great potential as a fluorescent probe for cancer diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China.

ABSTRACT
Fluorescent sensors capable of recognizing cancer-associated glycans, such as sialyl Lewis X (sLe(x)) tetrasaccharide, have great potential for cancer diagnosis and therapy. Studies on water-soluble and biocompatible sensors for in situ recognition of cancer-associated glycans in live cells and targeted imaging of cancer cells are very limited at present. Here we report boronic acid-functionalized peptide-based fluorescent sensors (BPFSs) for in situ recognition and differentiation of cancer-associated glycans, as well as targeted imaging of cancer cells. By screening BPFSs with different structures, it was demonstrated that BPFS₁ with a FRGDF peptide could recognize cell-surface glycan of sLe(x) with high specificity and thereafter fluorescently label and discriminate cancer cells through the cooperation with the specific recognition between RGD and integrins. The newly developed peptide-based sensor will find great potential as a fluorescent probe for cancer diagnosis.

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Related in: MedlinePlus

Flow cytometry profiles (A) and quantification of the cellular fluorescence shown via MFI (B) of HepG2 cells incubated with BPFS1 (40 μM) for 5 min (BPFS1), incubated with neuraminidase for 30 min and then further incubated with BPFS1 (40 μM) for 5 min (Neuraminidase), incubated with fucosidase for 30 min and then further incubated with BPFS1 (40 μM) for 5 min (Fucosidase), and incubated with BPFS1 (40 μM) for 5 min and then further incubated with fructose (400 μM) for 30 min (Fructose).Control represents the cells incubated without BPFS1.
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f7: Flow cytometry profiles (A) and quantification of the cellular fluorescence shown via MFI (B) of HepG2 cells incubated with BPFS1 (40 μM) for 5 min (BPFS1), incubated with neuraminidase for 30 min and then further incubated with BPFS1 (40 μM) for 5 min (Neuraminidase), incubated with fucosidase for 30 min and then further incubated with BPFS1 (40 μM) for 5 min (Fucosidase), and incubated with BPFS1 (40 μM) for 5 min and then further incubated with fructose (400 μM) for 30 min (Fructose).Control represents the cells incubated without BPFS1.

Mentions: To determine the binding sites between BPFS1 and cell-surface sLex, neuraminidase specifically catalyzing the hydrolysis of α(2,3) sialic acid linkages and fucosidase specifically catalyzing the hydrolysis of α(1,3)- as well as (1,4)-linked fucose were respectively used to incubate with HepG2 cells for 4 h. Subsequently, BPFS1 (40 μM) was added and the cells were further incubated for another 5 min. From the flow cytometry profile presented in Figure 7, in comparison with the cells only incubated with BPFS1 (40 μM, 5 min), the addition of neuraminidase results in around 72% decrease in MFI while the addition of fucosidase induces around 78% decrease in MFI, indicating that both sialic acid and fucose residues of cell-surface sLex are the binding sites for BPFS1. To prove this statement, fructose (400 μM), one of the strongest 1:1 boronic acid binders454647, was employed to incubate with HepG2 cells labelled by BPFS1. It is noteworthy that the effective competition can be only observed when fructose with a high concentration (at least 10-fold excess compared to BPFS1 concentration) is added. The possible reason is that the monosaccharide of fructose poorly competes with the multi-saccharides of cell-surface sLex since the multivalent interactions are nearly stronger than the sum of monovalent interactions3348. As shown in Figure 7B, because the competitive reaction between fructose and sLex with BPFS1 leads to the shedding of the cell bound BPFS1 molecules, there is around 85% decrease in MFI after 30 min incubation. With respect to the residual cellular fluorescence, it mainly corresponds to the cell bound BPFS1 molecules via the interaction between RGD segments and the receptor of integrins, which is in agreement with the CLSM observation in Figures 4F1 and 4G1. The results of enzymatic hydrolysis and fructose competition not only provide the information for the binding sites of BPFS1 with cell-surface sLex, but quantitatively indicate the influence of RGD sequence on the binding affinity of BPFS1 with sLex. From the Figure 7B, with addition of neuraminidase to hydrolyze sialic acid linkage of sLex into Lex, the cellular fluorescence shows a 3.6-fold decrease, which is lower than decreased value (7.7-fold) revealed in Figure 2B, implying that the interaction between RGD sequence and its receptors could improve the binding affinity of phenylboronic acid group with cell-surface sLex and thus strengthen the labelling and targeting ability of BPFS1. This interesting finding provides a valuable chance to use BPFS1 as probe for clinical cancer diagnosis since the dual-targeting functions of BPFS1 can facilitate each other and simultaneously recognize two types of cancer-biomarkers.


In situ recognition of cell-surface glycans and targeted imaging of cancer cells.

Xu XD, Cheng H, Chen WH, Cheng SX, Zhuo RX, Zhang XZ - Sci Rep (2013)

Flow cytometry profiles (A) and quantification of the cellular fluorescence shown via MFI (B) of HepG2 cells incubated with BPFS1 (40 μM) for 5 min (BPFS1), incubated with neuraminidase for 30 min and then further incubated with BPFS1 (40 μM) for 5 min (Neuraminidase), incubated with fucosidase for 30 min and then further incubated with BPFS1 (40 μM) for 5 min (Fucosidase), and incubated with BPFS1 (40 μM) for 5 min and then further incubated with fructose (400 μM) for 30 min (Fructose).Control represents the cells incubated without BPFS1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3775094&req=5

f7: Flow cytometry profiles (A) and quantification of the cellular fluorescence shown via MFI (B) of HepG2 cells incubated with BPFS1 (40 μM) for 5 min (BPFS1), incubated with neuraminidase for 30 min and then further incubated with BPFS1 (40 μM) for 5 min (Neuraminidase), incubated with fucosidase for 30 min and then further incubated with BPFS1 (40 μM) for 5 min (Fucosidase), and incubated with BPFS1 (40 μM) for 5 min and then further incubated with fructose (400 μM) for 30 min (Fructose).Control represents the cells incubated without BPFS1.
Mentions: To determine the binding sites between BPFS1 and cell-surface sLex, neuraminidase specifically catalyzing the hydrolysis of α(2,3) sialic acid linkages and fucosidase specifically catalyzing the hydrolysis of α(1,3)- as well as (1,4)-linked fucose were respectively used to incubate with HepG2 cells for 4 h. Subsequently, BPFS1 (40 μM) was added and the cells were further incubated for another 5 min. From the flow cytometry profile presented in Figure 7, in comparison with the cells only incubated with BPFS1 (40 μM, 5 min), the addition of neuraminidase results in around 72% decrease in MFI while the addition of fucosidase induces around 78% decrease in MFI, indicating that both sialic acid and fucose residues of cell-surface sLex are the binding sites for BPFS1. To prove this statement, fructose (400 μM), one of the strongest 1:1 boronic acid binders454647, was employed to incubate with HepG2 cells labelled by BPFS1. It is noteworthy that the effective competition can be only observed when fructose with a high concentration (at least 10-fold excess compared to BPFS1 concentration) is added. The possible reason is that the monosaccharide of fructose poorly competes with the multi-saccharides of cell-surface sLex since the multivalent interactions are nearly stronger than the sum of monovalent interactions3348. As shown in Figure 7B, because the competitive reaction between fructose and sLex with BPFS1 leads to the shedding of the cell bound BPFS1 molecules, there is around 85% decrease in MFI after 30 min incubation. With respect to the residual cellular fluorescence, it mainly corresponds to the cell bound BPFS1 molecules via the interaction between RGD segments and the receptor of integrins, which is in agreement with the CLSM observation in Figures 4F1 and 4G1. The results of enzymatic hydrolysis and fructose competition not only provide the information for the binding sites of BPFS1 with cell-surface sLex, but quantitatively indicate the influence of RGD sequence on the binding affinity of BPFS1 with sLex. From the Figure 7B, with addition of neuraminidase to hydrolyze sialic acid linkage of sLex into Lex, the cellular fluorescence shows a 3.6-fold decrease, which is lower than decreased value (7.7-fold) revealed in Figure 2B, implying that the interaction between RGD sequence and its receptors could improve the binding affinity of phenylboronic acid group with cell-surface sLex and thus strengthen the labelling and targeting ability of BPFS1. This interesting finding provides a valuable chance to use BPFS1 as probe for clinical cancer diagnosis since the dual-targeting functions of BPFS1 can facilitate each other and simultaneously recognize two types of cancer-biomarkers.

Bottom Line: Fluorescent sensors capable of recognizing cancer-associated glycans, such as sialyl Lewis X (sLe(x)) tetrasaccharide, have great potential for cancer diagnosis and therapy.Here we report boronic acid-functionalized peptide-based fluorescent sensors (BPFSs) for in situ recognition and differentiation of cancer-associated glycans, as well as targeted imaging of cancer cells.The newly developed peptide-based sensor will find great potential as a fluorescent probe for cancer diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China.

ABSTRACT
Fluorescent sensors capable of recognizing cancer-associated glycans, such as sialyl Lewis X (sLe(x)) tetrasaccharide, have great potential for cancer diagnosis and therapy. Studies on water-soluble and biocompatible sensors for in situ recognition of cancer-associated glycans in live cells and targeted imaging of cancer cells are very limited at present. Here we report boronic acid-functionalized peptide-based fluorescent sensors (BPFSs) for in situ recognition and differentiation of cancer-associated glycans, as well as targeted imaging of cancer cells. By screening BPFSs with different structures, it was demonstrated that BPFS₁ with a FRGDF peptide could recognize cell-surface glycan of sLe(x) with high specificity and thereafter fluorescently label and discriminate cancer cells through the cooperation with the specific recognition between RGD and integrins. The newly developed peptide-based sensor will find great potential as a fluorescent probe for cancer diagnosis.

Show MeSH
Related in: MedlinePlus