Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS)

Xiaoling Leng; Guofu Huang; Siyi Li; Miaomiao Yao; Jianbing Ding; Fucheng Ma

doi:10.1371/journal.pone.0261138

Peer Review History

Original SubmissionJune 29, 2020
16 Nov 2020 Decision Letter - Hanna Landenmark, Editor PONE-D-20-18825 Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS) PLOS ONE Dear Dr. Ding, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please see the reviewer reports below. The reviewers have requested further information on the aims and motivations, as well as further details in the methodology to help ensure that the study is reproducible by another researcher (PLOS ONE publication criterion #3). We also ask that you ensure that your manuscript is copyedited for English grammar and use. 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Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. In the Methods section, please provide the source, product number and any lot numbers of the primary antibodies used for the immunohistochemical analysis conducted for your study. 3. In your Methods section, please provide additional information about the participant recruitment method and the demographic details of your participants. 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Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: I Don't Know ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: No ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have demonstrated that the correlation between the parameters of less invasive CEUS and the expression level of CSC/EMT markers in breast cancer by IHC and RT-PCR. However, the experimental results are not sufficient to state their conclusions. The authors must evaluate IHC either in the same cell (double staining) or by the staining using serial sections. RT-PCR should use RNA extracted from cancer cells or normal cells recovered by laser microdissection, rather than RNA extracted from whole tissues. Furthermore, it is necessary to use the TaqMan probe method, which has a high quantitative yield, instead of the cybr green method, which is a semi-quantitative method. Minor points: 1) Please insert scale bars in Fig.1. 2)The information of antibodies of IHC should be written down in Materials and Methods. 3)Please describe the detailed statistical analysis methods in Materials and Methods. 4)There are inappropriate citations, such as quoting a lung cancer paper even though it is a breast cancer citation. Please review the selection of cited papers. 5) There are many misspellings. Please check the spelling. Reviewer #2: Contrast-enhanced ultrasound (CEUS) procedure carried out with new medical equipment and new-generation micro-bubble contrast reagents has been suggested to increase the accuracy in providing information on the microcirculation. The overall aim of the research described in this manuscript is to assess whether CEUS-based features may provide prognostic type of information. In particular, CEUS-based microcirculation features are examined for correlation with cancer-stem cell (CSC)-like subpopulations including those undergoing epithelial-mesenchymal transition (EMT). The rationale for focusing on tumor cells with CSC and EMT-like characteristics relates to potential roles in tumor heterogeneity and aggressiveness. The manuscript including the background, methods, and results would benefit from better language use and inclusion of information in terms of providing a clearer understanding of the rationale and the objectives of the study, the methodologies used, data interpretation including implications. Specific Comments- 1. Some of the information presented in the discussion should be considered to be moved to the background to help provide a basis for the study. 2. Several published studies (eg Ji et al, 2017. Clin Hemorheol Microcirc, Zhao et al, 2017 Onco Targets Ther, Cao et al, 2014. Ultrasound Med Biol, Wang et al, 2017, PLoS One, and Vraka et al, 2918 In Vivo) have assessed the correlation between CEUS-based parameters and prognostic markers in breast cancer should be alluded to and referenced in the background and/or discussion. 3. The reason for use of 2D US prior to the CEUS should be included in the method. 4. A figure that depicts the various parameters obtained related to the CEUS describe on page 6 in the methods should be included (see ). The functional implications of these parameters in terms of circulation/perfusion should be clearly stated to help understand their significance when described in the result sections. Use of full names (and not abbreviations) is recommended throughout the manuscript. 5. A rationale should be provided for the criteria used to follow the various biomarkers described in the IHC section of the method. The nature of the antibodies used what they recognize on target proteins should be included. What is the rationale for looking for cytoplasmic localization of CD24? β-catenin localization in cytoplasmic/nucleus can relate to WNT-pathway activation and potentially EMT, given this was this aspect inspected? How epithelial/carcinoma vs stromal cells distinguished? Where antibodies for cytokeratins used? 6. EMT induction requires the expression of transcription factors. Thus, to better show the relationship of the biomarkers used, it will be important to assess the expression (correlation) of some of EMT-TF with the biomarkers and the CEUS-features tested in the current study. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0261138.r001
Revision 1
11 May 2021 Author Response Dear editor, On behalf of all co-authors, I would like to thank you for your letter concerning our manuscript. We greatly appreciate you and the reviewers for the critical reading of our manuscript and giving us the favorable comments and instructive suggestions. We have carefully proof-read and revised the manuscript according to the reviewers’ comments and the editorial notes. Here we submit the revised manuscript. In the following pages, the responses to the comments are described point-by-point. Once again, we want to extend our appreciation to you and the reviewers for the valuable and helpful comments. We sincerely hope that the manuscript has been revised to your satisfaction, and we would be grateful if the manuscript could be considered for publication. In addition, we would like to update our Funding Statement as follows: This work was supported by Natural Science Foundation of Xinjiang Uygur Autonomous Region (2018D01C276). Sincerely yours, Jianbing Ding Basic Medical College, Xinjiang Medical University, No. 393 Xinyi Road, Urumqi 830011, Xinjiang, China. Tel: 86-18999230065. Email: dingjb1234@ aliyun.com & 1601379937@qq.com The reviewer’s comments and the authors’ responses: Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have demonstrated that the correlation between the parameters of less invasive CEUS and the expression level of CSC/EMT markers in breast cancer by IHC and RT-PCR. However, the experimental results are not sufficient to state their conclusions. Response: To validate our conclusions, we have further performed Western blot analysis of CSC-related and EMT-related markers in tumor and tumor adjacent tissues. The results showed that compared with normal tissues adjacent to cancer, the breast cancer tissues had significantly up-regulated protein expression levels of CD44, N-cadherin, and β-catenin, but significantly down-regulated protein expression levels of E-cadherin and CD24 (Fig. 3A and 3B). No significant difference was observed in the Vimentin protein expression levels between the cancer and adjacent tissues. We have added this result to the revised Fig. 3 and modified the revised manuscript accordingly. However, further studies are still needed to validate our results. The authors must evaluate IHC either in the same cell (double staining) or by the staining using serial sections. Response: In immunohistochemistry, the staining color position and depth indicated the expression levels and patterns, and it was difficult to perform double staining on the very one section. Therefore, these indicators were subjected to IHC staining on serial sections. However, the reviewer raised a very good point. We will do as suggested in future studies. RT-PCR should use RNA extracted from cancer cells or normal cells recovered by laser microdissection, rather than RNA extracted from whole tissues. Response: The tumor samples were obtained under the guidance of B-ultrasound. The hyperplastic gland tissues were used as control. Due to limited funding, we did not recover the cells by laser microdissection. However, the reviewer raised a very good point. We will do as suggested in future studies. Furthermore, it is necessary to use the TaqMan probe method, which has a high quantitative yield, instead of the cybr green method, which is a semi-quantitative method. Response: The quantitative real-time PCR was performed to detect the mRNA expression levels of the target genes. However, the reviewer raised a very good point. We will do as suggested in future studies. Minor points: 1) Please insert scale bars in Fig.1. Response: In fact, the figures were taken at the magnification of 200×, which has been added in the figure legends. 2) The information of antibodies of IHC should be written down in Materials and Methods. Response: According to the comment, we have added the information of IHC antibodies in Materials and Methods section of the revised manuscript. 3) Please describe the detailed statistical analysis methods in Materials and Methods. Response: If the data conformed to the normal distribution, the single-factor analysis of variance was performed. Multiple comparisons were performed with the sidak method for the data with homogeneity of variance, while the Tamhane method was used for the data with the heterogeneity of variance. The data without normal distribution were first subjected to the logarithmical transform for normalization, and then the above-mentioned one-way analysis of variance method was performed for statistical analysis, or the Wilcoxon rank sum test was used. Correlation analysis of the expression index was performed with the Pearson correlation. 4) There are inappropriate citations, such as quoting a lung cancer paper even though it is a breast cancer citation. Please review the selection of cited papers. Response: Sorry for the inappropriate citations. According to the comment, corresponding changes have been made herein. 5) There are many misspellings. Please check the spelling. Response: According to the comment, we have further gotten editorial help to improve the English writing of the revised manuscript. Please check! Reviewer #2: Contrast-enhanced ultrasound (CEUS) procedure carried out with new medical equipment and new-generation micro-bubble contrast reagents has been suggested to increase the accuracy in providing information on the microcirculation. The overall aim of the research described in this manuscript is to assess whether CEUS-based features may provide prognostic type of information. In particular, CEUS-based microcirculation features are examined for correlation with cancer-stem cell (CSC)-like subpopulations including those undergoing epithelial-mesenchymal transition (EMT). The rationale for focusing on tumor cells with CSC and EMT-like characteristics relates to potential roles in tumor heterogeneity and aggressiveness. The manuscript including the background, methods, and results would benefit from better language use and inclusion of information in terms of providing a clearer understanding of the rationale and the objectives of the study, the methodologies used, data interpretation including implications. Specific Comments- 1. Some of the information presented in the discussion should be considered to be moved to the background to help provide a basis for the study. Response: According to the comment, corresponding changes have been made herein. 2. Several published studies (eg Ji et al, 2017. Clin Hemorheol Microcirc, Zhao et al, 2017 Onco Targets Ther, Cao et al, 2014. Ultrasound Med Biol, Wang et al, 2017, PLoS One, and Vraka et al, 2918 In Vivo) have assessed the correlation between CEUS-based parameters and prognostic markers in breast cancer should be alluded to and referenced in the background and/or discussion. Response: According to the comment, we have added the following reference to the Discussion section. Please check! Reference: Vraka I, Panourgias E, Sifakis E, Koureas A, Galanis P, Dellaportas D, Gouliamos A, Antoniou A. In Vivo.Correlation Between Contrast-enhanced Ultrasound Characteristics (Qualitative and Quantitative) and Pathological Prognostic Factors in Breast Cancer. 2018;32(4):945-954. Epub 2018/07/30. doi: 10.21873/invivo.11333. PubMed PMID: 29936484;PubMed Central PMCID: PMC6117754. 3. The reason for use of 2D US prior to the CEUS should be included in the method. Response: In fact, for the CEUS, the contrast mode should be conducted on the basis of a clear two-dimensional ultrasound image. Although the results needed not to be analyzed for the two-dimensional ultrasound, the operation method would be necessary, which represented the diagnostic rule for contrast ultrasound. However, these methodological information needed not to be explained in the study. 4. A figure that depicts the various parameters obtained related to the CEUS describe on page 6 in the methods should be included (see ). The functional implications of these parameters in terms of circulation/perfusion should be clearly stated to help understand their significance when described in the result sections. Use of full names (and not abbreviations) is recommended throughout the manuscript. Response: According to the comment, corresponding information has been added herein. For example, we have used the full names instead of abbreviations according to the suggestion. 5. A rationale should be provided for the criteria used to follow the various biomarkers described in the IHC section of the method. The nature of the antibodies used what they recognize on target proteins should be included. What is the rationale for looking for cytoplasmic localization of CD24? β-catenin localization in cytoplasmic/nucleus can relate to WNT-pathway activation and potentially EMT, given this was this aspect inspected? How epithelial/carcinoma vs stromal cells distinguished? Where antibodies for cytokeratins used? Response: According to the comment, corresponding information concerning the primary antibodies and the criteria for expression evaluation have been added in the revised manuscript. Moreover, based on the Human-Protein-Atlas database, CD24 is located in the cytoplasm. The activation of the WNT pathway would cause the translocation of β-catenin from the cytoplasm to the nucleus [1]. In the nucleus, β-catenin combines with the LEF/TCF transcription factor to replace the auxiliary inhibitor, and this combination recruits the additional auxiliary activators of the Wnt target genes to promote the process of EMT [2]. There have been articles [3] reporting that CD24 is the target of Wnt in colorectal cancers. Therefore, from database predictions and previous literature, the WNT pathway activation and potential EMT would affect the CD24 cytoplasmic localization. In addition, through information retrieving, the keratin (epithelial cells) and vimentin (stromal cells) are used to distinguish the epithelial cells from the stromal cells. References: [1] Borgal L, Habbig S, Hatzold J, Liebau MC, Dafinger C, Sacarea I, Hammerschmidt M, Benzing T, Schermer B. The ciliary protein nephrocystin-4 translocates the canonical Wnt regulator Jade-1 to the nucleus to negatively regulate β-catenin signaling. J Biol Chem. 2012 Jul 20;287(30):25370-80. [2] Kobayashi W, Ozawa M. The epithelial-mesenchymal transition induced by transcription factor LEF-1 is independent of β-catenin. Biochem Biophys Rep. 2018 Jun 12;15:13-18. [3] Chen YC, Lee TH, Tzeng SL. Reduced DAXX Expression Is Associated with Reduced CD24 Expression in Colorectal Cancer. Cells. 2019 Oct 12;8(10):1242. 6. EMT induction requires the expression of transcription factors. Thus, to better show the relationship of the biomarkers used, it will be important to assess the expression (correlation) of some of EMT-TF with the biomarkers and the CEUS-features tested in the current study. Response: Based on the PROMO database, Genecards database and UCSC database, the transcription factors related to EMT indicators were predicted. The common transcription factors included the SP1, YY-1, etc. Through literature searching, the transcription factors were all related to EMT, which regulated the process of EMT through modulating the gene transcription. It has been reported that the prepared nanobubbles showed high affinity and specificity to breast cancer cells and tumors with the neuropeptide YY 1 receptors, with minimal toxicity and damages to the organs [1]. Biodegradable photoluminescent nanobubbles were used as ultrasound contrast agents for targeted breast cancer imaging. The transcription factor prediction results were obtained as attached files. Reference: [1] Li J, Tian Y, Shan D, Gong A, Zeng L, Ren W, Xiang L, Gerhard E, Zhao J, Yang J, Wu A. Neuropeptide Y Y1 receptor-mediated biodegradable photoluminescent nanobubbles as ultrasound contrast agents for targeted breast cancer imaging. Biomaterials. 2017 Feb;116:106-117. Attachments Attachment Submitted filename: 2021 04 30 Response letter revised.docx https://doi.org/10.1371/journal.pone.0261138.r002
12 Jul 2021 Decision Letter - Stella E. Tsirka, Editor PONE-D-20-18825R1 Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS) PLOS ONE Dear Dr. Ding, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. As evident from the Reviewer's comments, it is critical to re-address the images in Figure 1. It is very hard to see how the images provided come from serial sections, or how they correlate with each other, so that one can extract meaningful conclusions. Please provide a revised Figure 1 with appropriate images, and/or low magnifications of the images to allow for evaluation of location of panels and potential co-localizations, as well as the assessment of the same tumor. Please submit your revised manuscript by Aug 26 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Stella E. Tsirka Academic Editor PLOS ONE Additional Editor Comments (if provided): As evident from the Reviewer's comments, it is critical to re-address the images in Figure 1. It is very hard to see how the images provided come from serial sections, or how they correlate with each other, so that one can extract meaningful conclusions. Please provide a revised Figure 1 with appropriate images, and/or low magnifications of the images to [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: They have not essentially revised for my major concerns. My biggest concerns are below. Although authors described that they use the serial section for IHC, but as far as I can see in the picture in Figure 1, I can not think that they have observed the same tumor or normal site in the continuous section. The photos should be shown the same tumor or normal site in the serial section. In figure4, the amounts of N-cadherin, Beta-catenin, and Vimentin proteins in these representative photos are not consistent with these quantitative graphs. Thus, I think that their experimental results are inadequate to draw their conclusions. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0261138.r003
Revision 2
25 Aug 2021 Author Response Dear editor, We are resubmitting the Manuscript ID PONE-D-20-18825R1 entitled Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS) to PLOS ONE. Our responses to the comments by the reviewers are outlined below. Please also see the revised manuscript for details. For easily reading, we use the TRACK function of MICROSOFT WORD. Please simply select “Accept changes” to get rid of the TRACK markers if you do not like the tracks. In addition, we would like to update our Funding Statement as follows: This work was supported by the Supporting Project from Xinjiang Science and Technology Department (No.2020E0269). Additional Editor Comments: As evident from the Reviewer's comments, it is critical to re-address the images in Figure 1. It is very hard to see how the images provided come from serial sections, or how they correlate with each other, so that one can extract meaningful conclusions. Please provide a revised Figure 1 with appropriate images, and/or low magnifications of the images to allow for evaluation of location of panels and potential co-localizations, as well as the assessment of the same tumor. Response: As suggested, we have further provided a revised Figure 2 (the original Figure 1) with high and low magnifications of the same tumor or normal sites. We have also made corresponding changes in the revised manuscript. Please check! Reviewers' comments: Reviewer #1: They have not essentially revised for my major concerns. My biggest concerns are below. Although authors described that they use the serial section for IHC, but as far as I can see in the picture in Figure 1, I can not think that they have observed the same tumor or normal site in the continuous section. The photos should be shown the same tumor or normal site in the serial section. Response: As suggested, we have further provided a revised Figure 2 (the original Figure 1) with high and low magnifications of the same tumor or normal sites. We have also made corresponding changes in the revised manuscript. Please check! In figure4, the amounts of N-cadherin, Beta-catenin, and Vimentin proteins in these representative photos are not consistent with these quantitative graphs. Response: As suggested, we have modified the representative and quantitative Western blot results in the revised Figure 4 to ensure that the amounts of N-cadherin, Beta-catenin, and Vimentin proteins in these representative photos were consistent with these quantitative graphs. We have also made corresponding changes in the revised manuscript. Please check! Thus, I think that their experimental results are inadequate to draw their conclusions. Response: As suggested, we have modified some results and some conclusion to ensure that the conclusions were supported by the experimental results. For example, the overstated conclusions have been modified. Please check the revised manuscript for details. Attachments Attachment Submitted filename: 2021 08 25 Response letter revised.docx.docx https://doi.org/10.1371/journal.pone.0261138.r004
18 Oct 2021 Decision Letter - Stella E. Tsirka, Editor PONE-D-20-18825R2Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS)PLOS ONE Dear Dr. Ding, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please fix the figure legends as indicated by the reviewers. Please submit your revised manuscript by Dec 02 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. 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The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: I think it has improved. The IHC analysis result, which was a major concern, seems to be a mixture of those that can be compared at the same point in the continuous section and those that are not. However, since the tumor sample is very small, I understand that it is difficult to analyze. No more peer-reviewed rounds are needed. Minor points. 1) The square part of the low-magnification photo of CD24 and the square part of the magnifying magnification are 90 degrees out of alignment. Please correct it as it is confusing. 2) The word Breast is missing in the graph on the upper right of Figure 4B. 3) There is a misspelling in the upper left corner of Table 1. The term of "cancer" is "caner". 4) The value of N-cadherin in Table 5 is 0.000. Please enter an appropriate value. Reviewer #2: Figure legends are non-existence or very brief. Keys of various symbols and color codes should be explained in the legends to allow a better appreciation of the results and hence the conclusions. The relationship between figures 1 and 2 needs to clearly stated. It will be helpful to indicate n values for various analyses when possible. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0261138.r005
Revision 3
22 Nov 2021 Author Response Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Response: We confirm that the reference list is complete and correct. No further changes to the reference list are needed. We did not cite any retracted papers. Reviewers' comments: Reviewer's Responses to Questions Reviewer #1: I think it has improved. The IHC analysis result, which was a major concern, seems to be a mixture of those that can be compared at the same point in the continuous section and those that are not. However, since the tumor sample is very small, I understand that it is difficult to analyze. No more peer-reviewed rounds are needed. Minor points. 1) The square part of the low-magnification photo of CD24 and the square part of the magnifying magnification are 90 degrees out of alignment. Please correct it as it is confusing. Response: Sorry for the confusion. We have corrected the low-magnification photo of CD24 in the revised Figure 2. Please check! 2) The word Breast is missing in the graph on the upper right of Figure 4B. Response: Sorry for the typo. We have corrected this in the revised Figure 4. Please check! 3) There is a misspelling in the upper left corner of Table 1. The term of "cancer" is "caner". Response: Sorry for the typo. We have corrected "caner" into "cancer" in the revised Table 1. Please check! 4) The value of N-cadherin in Table 5 is 0.000. Please enter an appropriate value. Response: To be more clear, we have changed P=0.000 into P<0.001. Please check the revised Table 5 for details. Reviewer #2: Figure legends are non-existence or very brief. Keys of various symbols and color codes should be explained in the legends to allow a better appreciation of the results and hence the conclusions. The relationship between figures 1 and 2 needs to clearly stated. It will be helpful to indicate n values for various analyses when possible. Response: As suggested, we have further provided more detailed information to the figure legends. Figure 1 shows the time intensity curve analysis of contrast-enhanced ultrasound (CEUS). The representative sections of CEUS were marked before surgery. Then, the tissue specimens were collected from these marked sections and subjected to immunohistochemical analysis of CSCs and EMT proteins (Figure 2). We have further clarified this in the revised manuscript. Please check! Additionally, the n values have been indicated in the figure legends and table titles. Please check! Attachments Attachment Submitted filename: 2021 11 19 response to reviewer letter revised.docx https://doi.org/10.1371/journal.pone.0261138.r006
25 Nov 2021 Decision Letter - Stella E. Tsirka, Editor Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS) PONE-D-20-18825R3 Dear Dr. Ding, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Stella E. Tsirka Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0261138.r007
Formally Accepted
13 Dec 2021 Acceptance Letter - Stella E. Tsirka, Editor PONE-D-20-18825R3 Correlation of breast cancer microcirculation construction with tumor stem cells (CSCs) and epithelial-mesenchymal transition (EMT) based on contrast-enhanced ultrasound (CEUS) Dear Dr. Ding: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Stella E. Tsirka Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0261138.r008

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