Sialic Acid as a Biomarker Studied in Breast Cancer Cell Lines In Vitro Using Fluorescent Molecularly Imprinted Polymers

Round 1

Reviewer 1 Report

The manuscript by Zahra El-Schich et al. uses molecularly imprinted polymers (MIPs), or “plastic antibodies” previously designed by them as nanoprobes to stain sialic acid (SA) in different cell lines of breast cancer.

The manuscript has not enough results to be presented as an original article. Previously, the authors developed these MIPs and now they use them to stain cell lines. It should be completed with additional results. Moreover, the manuscript needs to be improved as at the moment the message is confusing.

There are details that indicate that the writing has not been performed very carefully. For instance, in Figure 4. Figure Legend says that it shows the MFI but in the figure it says % of positive cells. Also, Legend says A and B but in the figure it appears only B.

 

Lines 217-220: there is a sentence saying: This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn. What does it mean? it looks like some previous comments during the preparation of the manuscript that have not been removed from there.

 

In addition, it is not clear through the manuscript the difference between the staining with MAL and SNA and then SA-MIPs. It must be better explained.

 

Confocal figures could be improved by adding in the Picture what does indicate each color?

Author Response

REVIEWER 1

The manuscript by Zahra El-Schich et al. uses molecularly imprinted polymers (MIPs), or “plastic antibodies” previously designed by them as nanoprobes to stain sialic acid (SA) in different cell lines of breast cancer.

The manuscript has not enough results to be presented as an original article. Previously, the authors developed these MIPs and now they use them to stain cell lines. It should be completed with additional results. Moreover, the manuscript needs to be improved as at the moment the message is confusing.

There are details that indicate that the writing has not been performed very carefully. For instance, in Figure 4. Figure Legend says that it shows the MFI but in the figure it says % of positive cells. Also, Legend says A and B but in the figure it appears only B.

REPLY: We agree that it was confusing that the figure 4 legend indicated MFI instead of % positive cells. Figure 4A was by mistake missing. This has now been changed and is correct.

Lines 217-220: there is a sentence saying: This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn. What does it mean? it looks like some previous comments during the preparation of the manuscript that have not been removed from there.

REPLY: This sentence was left from the template and is now removed.

In addition, it is not clear through the manuscript the difference between the staining with MAL and SNA and then SA-MIPs. It must be better explained.

REPLY: We have now clarified this on page 2 in the introduction. The following sentence is revised:

For detection of α-2,3 SA, the lectin Maackia amurensis I (MAL I) is commonly used, and for detection of α-2,6 SA, the lectin Sambucus nigra (SNA) is used. α-2,6-SA can be found on the basolateral cell membrane, whereas α-,3-SA seems to be more prominent on the apical cell membrane (Ulloa et al 2001).

We also clarify that the SA-MIPs are imprinted against combined α-2,3 and α-2,6 SA. The following sentence is revised:

Based on a ternary complex imprinting procedure we recently reported on fluorescently reporting SA imprinted nanoprobes for combined detection of both α-2,3 and α-2,6 SA on cancer cells.

Confocal figures could be improved by adding in the Picture what does indicate each color?

REPLY: There are two different confocal images in the manuscript.

Figure 2 showing MAL I or SNA using FITC (in green), actin filament using rhodamine-phalloidin (in red) and DAPI nuclear staining (in blue), and analyzed with fluorescence confocal microscopy.

Figure 5 shows SA-MIPs using NBD in green, rhodamine-phalloidin (actin filaments) in red and DAPI (nuclei) in blue, and analyzed with fluorescence confocal microscopy.

This has now been clarified in the manuscript, both in the Materials and methods section and in the Figure legends.

Reviewer 2 Report

The manuscript is well-written and describes specificity of novel nanorobes binding to rarely-detectable sialylated proteins or lipids relevant also in cancer progression.

 

Minor issues:

Authors should consider to change the title. As this one: "Sialic acid as a biomarker studied in breast cancer cells using 2fluorescent molecularly imprinted polymers" would rather suggest the evaluation of SA in the large cohort of patients. Here, it is only validation of the tool which might be used for such studies.

Authors should describe briefly the nanoprobes they use.

CD44 is not mesenchymal cell marker, please, correct it (line 85/86)

Line 160: "Hs-578T and MDAMB231 expressed EpCAM at low levels compared to MDAMB468..." should rather describe no then low expression of EpCAM on Hs-578T and MDAMB231?

Please, add description for A and B in Figure 6.

Author Response

The manuscript is well-written and describes specificity of novel nanorobes binding to rarely-detectable sialylated proteins or lipids relevant also in cancer progression.

Minor issues:

Authors should consider to change the title. As this one: "Sialic acid as a biomarker studied in breast cancer cells using fluorescent molecularly imprinted polymers" would rather suggest the evaluation of SA in the large cohort of patients. Here, it is only validation of the tool which might be used for such studies.

REPLY: We agree that the title could changed for avoiding misunderstanding.

The title is now changed to: "Sialic acid as a biomarker studied in breast cancer cell lines in vitro using fluorescent molecularly imprinted polymers"

Authors should describe briefly the nanoprobes they use.

REPLY: We agree that the description was sparse. We have now added a description of the nanoprobes.

CD44 is not mesenchymal cell marker, please, correct it (line 85/86)

REPLY: this has now been changed to ”CD44 is overexpressed in several cell types including cancer stem cells.

Line 160: "Hs-578T and MDAMB231 expressed EpCAM at low levels compared to MDAMB468..."

should rather describe no then low expression of EpCAM on Hs-578T and MDAMB231?

REPLY: : this has now been changed to ”no EpCAM” compared to…

Please, add description for A and B in Figure 6.

REPLY: This has now been clarified in the manuscript, in the Figure legends.

Figure 6. SA-MIPs were pre-incubated with different concentrations of the SA-derivatives and analyzed with flow cytometry. The reduction of binding compared to SA-MIP binding alone is shown. A. The SA-derivative ME1057 was added at 20 uM and 200 uM, respectively. B. ME0970 was added at 20 uM and 200 uM, respectively.

Reviewer 3 Report

Sialic acid as a biomarker studied in breast cancer cells using fluorescent molecularly imprinted polymers by  El-Schich  et al is an interesting study. However the authors used immunofluoresence and flow cytometry
to evaluate the EPCAM and CD44 expression. It will be interesting to see the EPCAM and CD44 mRNA and protein analysis. Additionally CD24 analysis also required  to improve the quality of the manuscript.

Author Response

Sialic acid as a biomarker studied in breast cancer cells using fluorescent molecularly imprinted polymers by  El-Schich  et al is an interesting study. However the authors used immunofluoresence and flow cytometry
to evaluate the EPCAM and CD44 expression. It will be interesting to see the EPCAM and CD44 mRNA and protein analysis. Additionally CD24 analysis also required  to improve the quality of the manuscript.

REPLY: We agree that it could be interesting to look at other markers, but we open up for this in the discussion instead and hope that next study could include CD24 as well as mRNA analysis. Indeed, we realize that CD24 is a highly glycosylated mucin-like antigen, which has recently emerged as a novel oncogene and metastasis promoter and is thereby a potential biomarker for prognosis in breast carcinoma (Jing et al, 2018).

Since the EpCAM and CD44 proteins are analyzed here to classify the breast cancer cell types, we do not see the urgent need to do mRNA analysis in this study.

 

Round 2

Reviewer 1 Report

The manuscript is well-written but unfortunately, the manuscript has not enough results to be presented as an original article. Previously the authors had already developed these MIPs. This manuscript is only the use of these MIPs to stain cell lines. This information is not enough to be presented as an original article. The article should be completed with additional results.

Author Response

We have clarified the novelty of this manuscript as suggested by the reviewers. This is the first time SA-MIPs are presented as a possible diagnostic tool for EpCAM-positive breast cancer cells. Here, we also show the novel use for SA-derivatives by pre-treating the SA-MIPs with these SA-derivatives as inhibitors. We continue the work regarding cancer biomarkers by developing the MIPs to target glycan epitopes such as Tn and sTN, as well as using fluorophores that are optimized to use for in vivo studies.  

Reviewer 3 Report

None

Author Response

We have improved the conclusions supported by the results.