Shenqi Fuzheng injection reverses M2 macrophage-mediated cisplatin resistance through the PI3K pathway in breast cancer

Bin Yan; Rong Shi; Yi-yu Lu; Dong-dong Fang; Mei-na Ye; Qian-mei Zhou

doi:10.1371/journal.pone.0279752

Peer Review History

Original SubmissionJuly 15, 2022
16 Aug 2022 Decision Letter - Ming Tan, Editor PONE-D-22-20060Shenqi Fuzheng Injection Reverses M2 Macrophages-Mediated Cisplatin Resistance through the PI3K Pathway in Breast Cancer TreatmentPLOS ONE Dear Dr. zhou, 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 carefully address the reviewer's concerns, and the manuscript needs a deep editing by an English editor before resubmission. Please submit your revised manuscript by Sep 30 2022 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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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors showed the Shenqi Fuzheng injection could reverse M2 macrophages-mediated cisplatin resistance through the PI3K pathway in breast cancer treatment. There are few major flaws in the manuscript which might lead to difficulty in understanding the whole study. 1. The preparation of SQFZ is missing. How and where to obtain the SQFZ not stated. Identification of the SQFZ also not provided. These are all important information as it provides details information for others in case they interested to study further on this SQFZ. 2. Animal husbandry information missing and kindly provide animal ethic reference number in the methodology section. 3. What is DDP? The function of DDP was not mentioned in the manuscript. 4. Due to the cell MDA-MB-231 was donated by the hospital, and it is important to do the characterization and identification of the cell via flow cytometry. This was missing in the manuscript. 5. The methodology especially "Coculture procedure" somehow is very confusing and unclear. Is both cells coculture at the same time or THP-1 cell to be cultured first then only co-culture with MDA-MB-231? Is both cells using the same media with DDP? 6. Why the cells need to be stimulated twice and with difference inducers, i.e., PMA and IL-4/IL-13? 7. What is M2 macrophages cell? How to culture it? There is no information that related to M2 macrophages cell at all in the methodology. 8. Regarding the MTT assay, what type of cell going to be tested here? No information about the cells in this section. 9. According to the section results, " SQFZ significantly improves the sensitivity to cisplatin in MDA-MB-231/DDP cells and M2 macrophages", the authors stated that cytotoxicity effect detected on MDA-MB-231/DDP cells with or without M2 macrophages upon SQFZ treatment. It is kind of confusing here as the author never mention that both cells will be coculture and test for cytotoxicity effect. The main problem is how to differentiate the cell death between MDA and macrophages cell within the same well? The methodology itself seems rise a major concern here. 10. The manuscript need to send for English editing service as there were many grammatically errors and structure problem. ****** 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 ******** [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.0279752.r001
Revision 1
23 Nov 2022 Author Response Dear editor and reviewers, On behalf of my co-authors, we thank you very much for giving us an opportunity to revise our manuscript, we appreciate you and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled “Shenqi Fuzheng Injection Reverses M2 Macrophage-Mediated Cisplatin Resistance through the PI3K Pathway in Breast Cancer” (PONE-D-22-20060). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction. The main corrections in the paper and the responds to the reviewer’s comments are as flowing: Responds to the reviewer’s comments: 1. The preparation of SQFZ is missing. How and where to obtain the SQFZ not stated. Identification of the SQFZ also not provided. These are all important information as it provides details information for others in case they interested to study further on this SQFZ. Response: SQFZ (Pharmaceutical factory Batch No.180820) was obtained from Lizhu group Limin pharmaceutical Co. Ltd.( Guangdong, China). Radix Astragali and Radix Codonopsis are its main raw materials. The effective components are extracted and separated to obtain the intravenous preparation of traditional Chinese medicine, containing 160 g/L of the crude drug. These have been added in the manuscript and marked in red. 2. Animal husbandry information missing and kindly provide animal ethic reference number in the methodology section. Response: All procedures conformed to the requirements of animal welfare and were approved by the ethical committee of Shanghai Traditional Chinese Medicine (approval number PZSHUTCM210402003). The animal ethic reference number has been added. 3. What is DDP? The function of DDP was not mentioned in the manuscript. Response: DDP is cisplatin. Cisplatin (DDP) is a major chemotherapeutic agent used for the treatment of triple-negative breast cancer (TNBC). However, a risk of developing resistance to cisplatin treatment exists, and leads to treatment failure. It has been added in the manuscript. 4. Due to the cell MDA-MB-231 was donated by the hospital, and it is important to do the characterization and identification of the cell via flow cytometry. This was missing in the manuscript. Response: The hospital has identified the cells before donating them to us. The intracellular concentration of DDP was determined by HPLC in MDA-MB-231 and MDA-MB-231 / DDP. Cells were incubated with DDP for 24 h. The concentration of DDP in MDA-MB-231 cells was (47.10 ± 2.37) ng / (5 × 104) cells. However, the concentration of DDP in MDA-MB-231 / DDP cells was (6.30 ± 1.64) ng / (5 × 104) cells. There was significant difference between them. 5. The methodology especially "Coculture procedure" somehow is very confusing and unclear. Is both cells coculture at the same time or THP-1 cell to be cultured first then only co-culture with MDA-MB-231? Is both cells using the same media with DDP? Response: The THP-1 cells were seeded in the upper chamber. PMA was used to stimulate cells for 24 h, and IL-4/IL-13 was used to stimulate cells for another 24 h. The upper chamber was washed thrice with phosphate-buffered saline (PBS). MDA-MB-231/DDP cells were seeded in the lower chamber for 24 h. Subsequently, the upper chamber containing THP-1-derived macrophages were placed on the top of the lower chamer containing MDA-MB-231/DDP cells. Only the lower chamer containing MDA-MB-231/DDP cells used the media with DDP. 6. Why the cells need to be stimulated twice and with difference inducers, i.e., PMA and IL-4/IL-13? Response: THP-1 is a human monocytic leukemia cell. THP-1-derived macrophages were characterized after stimulation with phorbol-12-myristate 13-acetate (PMA). IL-4/IL-13 induced macrophages polarizing toward M2 type. Therefore, the cells need to be stimulated twice and with difference inducers. 7. What is M2 macrophages cell? How to culture it? There is no information that related to M2 macrophages cell at all in the methodology. Response: Macrophages are classified into two major phenotypes based on their functions—the M1 phenotype with proinflammatory responses and antitumor functions, whereas the M2 phenotype is anti-inflammatory and tumor-promoting. The cells were cultured in RPMI 1640 with 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37℃ in a humidified 5% CO2 atmosphere. PMA was used to stimulate cells for 24 h, and IL-4/IL-13 was used to stimulate cells for another 24 h. 8. Regarding the MTT assay, what type of cell going to be tested here? No information about the cells in this section. Response: The cocultured cells of MDA-MB-231/DDP and M2 or MDA-MB-231/DDP cells alone were tested here. It has been added in the manuscript. To determine the effects of SQFZ on M2 macrophages, we assessed the cytotoxic effects of SQFZ on MDA-MB-231/DDP cells with or without M2 macrophages. Cell survival was determined by the MTT assay. 9. According to the section results, " SQFZ significantly improves the sensitivity to cisplatin in MDA-MB-231/DDP cells and M2 macrophages", the authors stated that cytotoxicity effect detected on MDA-MB-231/DDP cells with or without M2 macrophages upon SQFZ treatment. It is kind of confusing here as the author never mention that both cells will be coculture and test for cytotoxicity effect. The main problem is how to differentiate the cell death between MDA and macrophages cell within the same well? The methodology itself seems rise a major concern here. Response: MDA-MB-231/DDP cells with or without M2 will be cocultured and tested for cytotoxicity effect. It has been added in the manuscript. The coculture systems we used were non-contact coculture. THP-1 cells in the upper chamber affected MDA-MB-231/DDP cells in the lower chamber by some secretions such as cytokines and chemokines. MDA-MB-231/DDP cells treated with SQFZ were tested for cytotoxicity effect. 10. The manuscript need to send for English editing service as there were many grammatically errors and structure problem. Response: We have worked on both language and readability and have also involved native English speakers for language corrections. We really hope that language level has been improved. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0279752.r002
14 Dec 2022 Decision Letter - Ming Tan, Editor Shenqi Fuzheng Injection Reverses M2 Macrophage-Mediated Cisplatin Resistance through the PI3K Pathway in Breast Cancer PONE-D-22-20060R1 Dear Dr. zhou, 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, Ming Tan Academic Editor PLOS ONE Additional Editor Comments (optional): The authors have addressed all the concerns of the original reviewer and the manuscript has been improved. Reviewers' comments: https://doi.org/10.1371/journal.pone.0279752.r003
Formally Accepted
4 Jan 2023 Acceptance Letter - Ming Tan, Editor PONE-D-22-20060R1 Shenqi Fuzheng Injection Reverses M2 Macrophage-Mediated Cisplatin Resistance through the PI3K Pathway in Breast Cancer Dear Dr. Zhou: 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. Ming Tan Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0279752.r004

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