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by
  • Yoshihiro Morita 1,
  • Macall Leslie 1 and
  • Takemi Tanaka 1,5,*
  • et al.

Reviewer 1: Anonymous Reviewer 2: Anonymous Reviewer 3: Anonymous Reviewer 4: Anonymous

Round 1

Reviewer 1 Report

Overall comments:

 

The authors addressed the question weather functional blockade of E-selectin changes the anti-tumoral effect of Doxorubicin in breast cancer. For that they used both human tissue and mice models. The research question is clinically and scientifically relevant, and the methodology was well conducted. Some revisions are needed in order to consider this article for publication.

 

Minor Revisions:

 

1. Title: Please add the study designs/populations to the title, as well as cancer type

2. Results: Please indicate more clearly in the text weather you refer to human or mice specimens.

3. Discussion: Please include a paragraph describing strengths and limitations of the present manuscript.

4. Materials and Methods, Statistical Analyses: Why did you choose a significance level of 0.10 for the in vivo analyses? Also, no statistical tests are described in this session. Please expand session describing specifically which kinds of statistical tests were used and when.

5. Figure 1, right upper box, what does the legends 1, 2 and 3 stand for? Please insert parenthesis around “c” box indication

6. Figure 2: please improve the quality of box (a) figure and of box (b) text. Also, please adjust the sizes of the figure boxes to a more homogeneous distribution.

7. References: please insert a blank space before each reference number in the text.

8. Authors contributions: please describe it.

Author Response

I appreciate reviewer’s suggestions and comments, which are highly valuable to our manuscript to be more succinct. All modifications were track-changed and I hope all suggestions were adequately addressed.

Reviewer 1

  1. Title: Please add the study designs/populations to the title, as well as cancer type

In response to reviewer’s suggestion, title was changed to “Functional Blockade of E-selectin in Tumor-associated Vessels Enhances Anti-tumor Effect of Doxorubicin in Breast Cancer”

  1. Results: Please indicate more clearly in the text weather you refer to human or mice specimens.

Species differences are clarified in results corresponding to S. Figure 1, 2, Figure 1,2, and 5 that display both human and mouse data (line 77, 82, 112-113, 226).

  1. Discussion: Please include a paragraph describing strengths and limitations of the present manuscript.

Both strengths and limitations are discussed in line 257 to 274 and 291 to 297.

  1. Materials and Methods, Statistical Analyses: Why did you choose a significance level of 0.10 for the in vivoanalyses? Also, no statistical tests are described in this session. Please expand session describing specifically which kinds of statistical tests were used and when.

All analyses and power level used in experiments in this study were 95%, 0.05; thus statistical analysis section was corrected. As suggested, we included type of analysis in each data in Figure Legends.

  1. Figure 1, right upper box, what does the legends 1, 2 and 3 stand for? Please insert parenthesis around “c” box indication

The legends are scoring index of CD45 and E-selectin. We added the sentence in line 98 and insert parenthesis around “c”.

  1. Figure 2: please improve the quality of box (a) figure and of box (b) text. Also, please adjust the sizes of the figure boxes to a more homogeneous distribution.

Image quality of Figure 2 was improved.

  1. References: please insert a blank space before each reference number in the text.

A blank space was inserted before reference number.

  1. Authors contributions: please describe it.

All contributions were added to line 400 to 408.

Reviewer 2 Report

This manuscript by Morita et al described the suppression of immune cell infiltration in doxorubicin-treated tumor by administration of anti-E-selectin aptamer. Blocking E-selectin reduced tumor metastasis was predictable. However, it is very impressive that blockade of E-selectin notably decreased tumor growth rate when combined with doxorubicin treatment. This study provided a new idea to improve anti-cancer therapy by blocking chemotherapy-induced immune cell infiltration. The conclusion in this manuscript was fully supported by the data presented.

A few questions:

  1. What is the advantage of anti-E-selectin apatmer comparing with E-selectin antibody?
  2. Was there any change in the death of tumor cells when administrated DOX/ESTA combination?

Author Response

I appreciate reviewer’s suggestions and comments, which are highly valuable to our manuscript to be more succinct. All modifications were track-changed and I hope all suggestions were adequately addressed.

Reviewer 2
1. What is the advantage of anti-E-selectin apatmer comparing with E-selectin antibody?


Since therapeutic E-selectin antibody was known to be non-specific, and currently there is no therapeutic E-selectin monoclonal antibody. Therefore, we instead discussed a general advantage of aptamer over antibody or small molecule drug was added to line 291 to 292.


2. Was there any change in the death of tumor cells when administrated DOX/ESTA combination?


We included a new data for Ki67 immunohistochemistry along with quantitative comparison among DOX/ESTA, DOX, and saline control in Figure 5.

 

Reviewer 3 Report

Morita et al  suggestsed that DOX treatment instigates de novo intratumoral infiltration of immune cells through E-selectin, and functional blockade of E-selectin may reduce residual tumor burden as well as metastasis through suppression of TH2 shift.

The manuscript is well-written and organized with solid methodology and clear conclusion.

The manuscript could be acceptable for publication after addressing the following minor comment.

More comprehensive details about DOX toxicity and potential protective agents as well as role on cardiac stem cells is highly recommended. The following reference may help. PMID: 31258475 , PMID: 28785995 ,PMID: 28460429.

 

Author Response

I appreciate reviewer’s suggestions and comments, which are highly valuable to our manuscript to be more succinct. All modifications were track-changed and I hope all suggestions were adequately addressed.

Reviewer 3

  1. More comprehensive details about DOX toxicity and potential protective agents as well as role on cardiac stem cells is highly recommended. The following reference may help. PMID: 31258475, PMID: 28785995, PMID: 28460429.

I appreciate reviewer’s comments to expand our discussion while condensing possible strengths of ESTA therapy.  As suggested current problem of DOX therapy were added to line 266.

Reviewer 4 Report

The manuscript titled "Functional Blockade of E-selectin Enhances  Anti-tumor Effect of Doxorubicin" is a very interesting paper with high translational potential in the field on immuno-oncology. The manuscript is well written and describes in an appropriate manner the methodologies followed.  The results are also clearly presented. However, i suggest to improve the translational impact of the manuscript through a description of the pharmakokinetic profiles of aptamers and their possible use in clinical trial. What about their targets in the body after i.v administration? possible interactions with albumin or lipid particles? I suggest to describe these properties in the discussion of the manuscript. Moreover, the authors have to describe the role of anti-E-selectin aptamer (ESTA) in cancer microenvironment, considering that interleukins are of crucial interest in DOXO induced cardiotoxicity ( follow Quagliariello V, Vecchione R et al., 2018 nutrients )as well as in cancer therapy (follow Zhu, J. et al, (2011) Biomedicine and Pharmacotherapy ; Ridker PM et al N Engl J Med. 2017)  ; we suggest to describe these possible effects in the discussion part of the manuscrit. After these improvements, i will consider the manuscript acceptable for publication in this journal. 

Author Response

I appreciate reviewer’s suggestions and comments, which are highly valuable to our manuscript to be more succinct. All modifications were track-changed and I hope all suggestions were adequately addressed.

Reviewer 4

  1. The results are also clearly presented. However, i suggest to improve the translational impact of the manuscript through a description of the pharmakokinetic profiles of aptamers and their possible use in clinical trial. What about their targets in the body after i.v administration? possible interactions with albumin or lipid particles? I suggest to describe these properties in the discussion of the manuscript.

We included aptamer’s strengths and weaknesses in the revised manuscript. The weaknesses include pk/pd issues with appropriate citations from line 291 to 298 and with possible solutions to overcome such obstacles in line 298 to 300.

  1. Moreover, the authors have to describe the role of anti-E-selectin aptamer (ESTA) in cancer microenvironment, considering that interleukins are of crucial interest in DOXO induced cardiotoxicity ( follow Quagliariello V, Vecchione R et al., 2018 nutrients )as well as in cancer therapy (follow Zhu, J. et al, (2011) Biomedicine and Pharmacotherapy ; Ridker PM et al N Engl J Med. 2017)  

Comments raised by the reviewer 4 is crucial as ESTA/DOX effect as well as the outcome of TH2 shift may differ in different organs. This critical point is now discussed in line 266 to 274 in the discussion.