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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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Flow cytometry profiles (A), (B) and quantification of the cellular fluorescence shown via MFI (C), (D) of HepG2 cells respectively incubated with BPFS1 (20 μM) for different time (A), (C) and BPFS1 at different concentration for 5 min (B), (D).
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f5: Flow cytometry profiles (A), (B) and quantification of the cellular fluorescence shown via MFI (C), (D) of HepG2 cells respectively incubated with BPFS1 (20 μM) for different time (A), (C) and BPFS1 at different concentration for 5 min (B), (D).

Mentions: After the determination of the expression of cancer-associated glycans and integrin, BPFS1 was incubated with HepG2 cells to evaluate its ability to in situ recognize cell-surface sLex and integrins for fluorescent imaging of the target cells. The concentration of BPFS1 used here was 20 μM because more than 85% cells were live according to the cytotoxicity assay (Supplementary Fig. S15). Figure 4 shows the confocal laser scanning microscopy (CLSM) images of the cells incubated with BPFS1 for different time intervals. No fluorescence can be found for the cells incubated without BPFS1 (Figure 4A1). After incubation with BPFS1 for 1 min, besides few of internalized molecules, most of the BPFS1 molecules are bound on the cell surface to form a uniform ring-shaped fluorescence pattern (Figure 4B1). Increasing the incubation time to 3 min, the cellular fluorescence is enhanced and the vast majority of BPFS1 molecules still remain on the cell surface (Figure 4C1). However, after incubation for 5 min, most of BPFS1 molecules have been internalized to form clusters with bright fluorescence (Figure 4D1). Further elongating the incubation time to 10 min does not change the distribution pattern of BPFS1 molecules (Figure 4E1). The results of flow cytometry quantitative analysis in Figures 5A and 5C are consistent with the above CLSM observation. The mean fluorescence intensity (MFI) of the cells rapidly increases from around 480 to 1430 as the incubation time increasing from 1 to 5 min. However, there is only a slight increase in the MFI as the incubation time elongating to 10 min (MFI, around 1540). Both the CLSM observation and flow cytometry analysis indicate that BPFS1 can rapidly bind with HepG2 cells and then traffic into cells, presenting the fluorescently labelling behavior. To demonstrate that the labelling behavior of BPFS1 is built on the in situ recognition of cell-surface sLex and integrins, the antibody of CSLEX-1 was first incubated with HepG2 cells for 15 min and BPFS1 (20 μM) was then added. As shown in Figure 4F1, although the recognition between CSLEX-1 and cell-surface sLex inhibits the binding of phenylboronic acid with sLex, BPFS1 can still label HepG2 cells to form a ring-shaped fluorescence pattern through the recognition between RGD and its receptors. However, owing to the relative low expression level of integrin β3 subunit compared to sLex as shown in Figure 3, the cellular fluorescence is rather weak (Figure 5C). In addition, the analogue of BPFS1 without phenylboronic acid groups (Supplementary Fig. S16) was also incubated with HepG2 cells. From Figure 4G1, without the phenylboronic acid groups to recognize cell-surface sLex, BPFS1 can also bind with cells with weak cellular fluorescence (Figure 5C). If replacing the RGD sequence of BPFS1 with AGD (BPFS4), the cellular fluorescence slightly decreases as displayed in Figures 4H1 and 5C. All these results strongly demonstrate that the specific recognition between phenylboronic acid groups and cell-surface sLex dominates the fluorescently labelling behavior of BPFS1 while the RGD sequence could cooperate with phenylboronic acid groups to strengthen the labelling ability of BPFS1.


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), (B) and quantification of the cellular fluorescence shown via MFI (C), (D) of HepG2 cells respectively incubated with BPFS1 (20 μM) for different time (A), (C) and BPFS1 at different concentration for 5 min (B), (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Flow cytometry profiles (A), (B) and quantification of the cellular fluorescence shown via MFI (C), (D) of HepG2 cells respectively incubated with BPFS1 (20 μM) for different time (A), (C) and BPFS1 at different concentration for 5 min (B), (D).
Mentions: After the determination of the expression of cancer-associated glycans and integrin, BPFS1 was incubated with HepG2 cells to evaluate its ability to in situ recognize cell-surface sLex and integrins for fluorescent imaging of the target cells. The concentration of BPFS1 used here was 20 μM because more than 85% cells were live according to the cytotoxicity assay (Supplementary Fig. S15). Figure 4 shows the confocal laser scanning microscopy (CLSM) images of the cells incubated with BPFS1 for different time intervals. No fluorescence can be found for the cells incubated without BPFS1 (Figure 4A1). After incubation with BPFS1 for 1 min, besides few of internalized molecules, most of the BPFS1 molecules are bound on the cell surface to form a uniform ring-shaped fluorescence pattern (Figure 4B1). Increasing the incubation time to 3 min, the cellular fluorescence is enhanced and the vast majority of BPFS1 molecules still remain on the cell surface (Figure 4C1). However, after incubation for 5 min, most of BPFS1 molecules have been internalized to form clusters with bright fluorescence (Figure 4D1). Further elongating the incubation time to 10 min does not change the distribution pattern of BPFS1 molecules (Figure 4E1). The results of flow cytometry quantitative analysis in Figures 5A and 5C are consistent with the above CLSM observation. The mean fluorescence intensity (MFI) of the cells rapidly increases from around 480 to 1430 as the incubation time increasing from 1 to 5 min. However, there is only a slight increase in the MFI as the incubation time elongating to 10 min (MFI, around 1540). Both the CLSM observation and flow cytometry analysis indicate that BPFS1 can rapidly bind with HepG2 cells and then traffic into cells, presenting the fluorescently labelling behavior. To demonstrate that the labelling behavior of BPFS1 is built on the in situ recognition of cell-surface sLex and integrins, the antibody of CSLEX-1 was first incubated with HepG2 cells for 15 min and BPFS1 (20 μM) was then added. As shown in Figure 4F1, although the recognition between CSLEX-1 and cell-surface sLex inhibits the binding of phenylboronic acid with sLex, BPFS1 can still label HepG2 cells to form a ring-shaped fluorescence pattern through the recognition between RGD and its receptors. However, owing to the relative low expression level of integrin β3 subunit compared to sLex as shown in Figure 3, the cellular fluorescence is rather weak (Figure 5C). In addition, the analogue of BPFS1 without phenylboronic acid groups (Supplementary Fig. S16) was also incubated with HepG2 cells. From Figure 4G1, without the phenylboronic acid groups to recognize cell-surface sLex, BPFS1 can also bind with cells with weak cellular fluorescence (Figure 5C). If replacing the RGD sequence of BPFS1 with AGD (BPFS4), the cellular fluorescence slightly decreases as displayed in Figures 4H1 and 5C. All these results strongly demonstrate that the specific recognition between phenylboronic acid groups and cell-surface sLex dominates the fluorescently labelling behavior of BPFS1 while the RGD sequence could cooperate with phenylboronic acid groups to strengthen the labelling ability of BPFS1.

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