Effects of stanniocalcin-1 overexpressing hepatocellular carcinoma cells on macrophage migration

Cherry C. T. Leung; Chris K. C. Wong

doi:10.1371/journal.pone.0241932

Peer Review History

Original SubmissionJuly 23, 2020
7 Sep 2020 Decision Letter - Joe W. Ramos, Editor PONE-D-20-22832 Effects of Stanniocalcin-1 Overexpressing Hepatocellular Carcinoma Cells on Macrophage Migration PLOS ONE Dear Dr. Wong, 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. There are many technical issues with the manuscript as described below by the reviewers. Importantly additional methodological details are needed, improved blotting controls, validation of TNF-α and IL-6 levels in the culture media. Evaluation of the effect of Rho inhibitors in the assay system to independently confirm the conclusion that Rho is responsible would strengthen the work. There is also need to address data presentation in some figures (eg Bar Graphs). 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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. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines: (1) Please state the source and number of mice used in the study (2) Please provide details of animal welfare (e.g., shelter, food, water, environmental enrichment) (3) Please describe any steps taken to minimize animal suffering and distress, such as by administering anaesthesia (4) Please include the method of euthanasia (5) Please describe the post-operative care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being. Thank you for your attention to these requests. 3. Please provide additional information about each of the cell lines used in this work, including the source and any quality control testing procedures (authentication, characterisation, and mycoplasma testing). For more information, please see http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines. 4. In the Methods section, please provide the source, product number and any lot numbers of the primary antibodies used for the Western blot analysis in your study. 5. At this time, we ask that you please provide scale bars on the microscopy images presented in Figure 1 and 6 and refer to the scale bar in the corresponding Figure legend. 6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [Note: HTML markup is below. 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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: No 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: Yes Reviewer #2: Yes ****** 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: Comments of Reviewer Human stanniocalcin-1 (STC1) is a glycoprotein known to participate in inflammation and tumor progression. However, its role in cancer-macrophage interaction at the tumor environment is not known. In this study, the co-culture of the human metastatic hepatocellular carcinoma cell line (MHCC97L) stably transfected with a control vector (MHCC97L/P), or STC1-overexpressing vector (MHCC97L/S1) with human leukemia monocytic cell line (THP-1) was conducted. Authors found that STC1-overexpressing hepatocellular carcinoma (MHCCp7L) suppressed the migratory activity of THP-1, indicating the inhibitory effect of STC1-overexpressing cancer cells on macrophage migration/infiltration in the relationship between tumor STC1 level and macrophage infiltration. Major point. In Boyden chamber-based co-culture, relative TNF-α and IL-6 mRNAs expression were increased in human leukemia monocytic cells (THP-1) by stimulating with PMA, an activator of protein kinase C. Productions of TNF-α and IL-6 in THP-1 may enhanced in THP-1 cells and these factors impact MHCC97L/P or S1 cells. NF-kB signaling may be activated by increased TNF-α and IL-6. Overexpression of STC-1 may suppress activation of NF-kB signaling. This may impact cellular events linked to migration. Authors should be shown these results with additional experiment. Author should determin the levels of TNF-α and IL-6 in condition medium. These experiments may be very important in this study. Minor point 1. Experimental methods are very poor. This is important to keep reproducibility in experiments by other researchers. Especially, authors should be described cell number used in the experiments of western blot and co-culture. 2. Authors should describe the weight of tumor tissues obtained from mice in immunohistochemical staining. Reviewer #2: This work reports the role of TME on immune cell infiltration using MHCC97L and THP-1 cell lines. The authors demonstrated that STC-1 overexpressing MHCC97L cells can inhibit transwell migration of THP-1 and identified various changes in cytokines and gene expression in STC-1 overexpressing cells that may be the cause of this inhibitory action. Interestingly, RhoA signaling pathway is implicated as a plausible mechanism of action of STC1. Although definitive proof and explicit mechanism behind STC1 effects is still missing. These results continues to demonstrate the importance of the TME and establishes a baseline by which STC1 overexpressing cancer cells can affect macrophage infiltration. Comments – 1. Should include scale bar in figures with representative images (Fig 1, Fig 6). 2. Please include information on how many trials/replicates were performed or analyzed for the migration assay (Fig 1, Fig 2, Fig 3). From how many tumor sections was data in Fig 6 generated and is the data quantified from all fields in a tumor section or from select sections only? 3. I agree from your images that it does appear that 97L/S1 co-culture appears to reduce THP-1 transwell migration, however, I wonder how accurate the data is. Your methods describe that cells were counted by light microscopy. From the images, it looks like there are large masses of cells that migrated through, how were those counted? Also, are these quantification per microscopy field or did you image the entire bottom of the transwell and count all cells? How were the pathways selected in Fig 3A? 4.Fig 3B top panel has no error bars. Was only one replicate analyzed for this bar graph? I interpreted the method section as having 4 replicates run for the transcriptome analysis. Similarly for Fig 5A. 5. In Fig 5C, is actin the representative loading control for the blot? Since RhoA pathway affects actin dynamics, it may be better to use a different loading control to compare your groups (maybe GAPDH). 6. Since RhoA pathway was proposed, it would be nice to do a confirmatory assay perhaps using an inhibitor of RhoA and demonstrating that THP-1 migration is still inhibited. 7. There are some grammatical errors in the writing and formatting inconsistencies that should be addressed before publication. 8. As a general comment, it would be better to show dots when possible on your bar graph (or use alternative means for data display) to indicate what the values were from each of your analyzed experiments used to generate the mean and SD for the bar graphs. This improves the transparency of the data. ****** 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.0241932.r001
Revision 1
3 Oct 2020 Author Response Editorial 1. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines: (a) Please state the source and number of mice used in the study Response: Ten 6-week-old male BALB-c nude mice were used in each group. (b) Please provide details of animal welfare (e.g., shelter, food, water, environmental enrichment) Response: In the study, the mice were maintained in a pathogen-free room, under controlled temperature (23 ± 1ºC ambient temperature), a 12-h light/dark cycle, and food and water ad libitum. (c) Please describe any steps taken to minimize animal suffering and distress, such as by administering anaesthesia Response: The mice were sacrificed by cervical dislocation to minimize the suffering and distress. (d) Please include the method of euthanasia Response: As abovementioned, the animals were sacrificed by cervical dislocation. (e) Please describe the post-operative care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being. Responses: Body weight and tumor volume was measured twice a week to monitor their health and tumor growth. 3. Please provide additional information about each of the cell lines used in this work, including the source and any quality control testing procedures (authentication, characterisation, and mycoplasma testing). For more information, please see http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines. 4. In the Methods section, please provide the source, product number and any lot numbers of the primary antibodies used for the Western blot analysis in your study. Response: The information is listed in Table 2. 5. At this time, we ask that you please provide scale bars on the microscopy images presented in Figure 1 and 6 and refer to the scale bar in the corresponding Figure legend. Response: Added as per your comment. 6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Reviewer #1: Human stanniocalcin-1 (STC1) is a glycoprotein known to participate in inflammation and tumor progression. However, its role in cancer-macrophage interaction at the tumor environment is not known. In this study, the co-culture of the human metastatic hepatocellular carcinoma cell line (MHCC97L) stably transfected with a control vector (MHCC97L/P), or STC1-overexpressing vector (MHCC97L/S1) with human leukemia monocytic cell line (THP-1) was conducted. Authors found that STC1-overexpressing hepatocellular carcinoma (MHCCp7L) suppressed the migratory activity of THP-1, indicating the inhibitory effect of STC1-overexpressing cancer cells on macrophage migration/infiltration in the relationship between tumor STC1 level and macrophage infiltration. Major point. In Boyden chamber-based co-culture, relative TNF-α and IL-6 mRNAs expression were increased in human leukemia monocytic cells (THP-1) by stimulating with PMA, an activator of protein kinase C. Productions of TNF-α and IL-6 in THP-1 may enhanced in THP-1 cells and these factors impact MHCC97L/P or S1 cells. NF-kB signaling may be activated by increased TNF-α and IL-6. Overexpression of STC-1 may suppress activation of NF-kB signaling. This may impact cellular events linked to migration. Authors should be shown these results with additional experiment. Author should determine the levels of TNF-α and IL-6 in condition medium. These experiments may be very important in this study. Response: Agree with your comments. As indicated in the antibody cytokine arrays (Fig 4), we do not see significant changes in the levels of TNF-α and IL-6 in the conditioned media of the MHCC97L/P or S1 cells. Minor point. 1. Experimental methods are very poor. This is important to keep reproducibility in experiments by other researchers. Especially, authors should be described cell number used in the experiments of western blot and co-culture. Response: Sorry for the unclear information. We have added the details accordingly. 2. Authors should describe the weight of tumor tissues obtained from mice in immunohistochemical staining. Response: The description has been added in the revised manuscript. Reviewer #2: This work reports the role of TME on immune cell infiltration using MHCC97L and THP-1 cell lines. The authors demonstrated that STC-1 overexpressing MHCC97L cells can inhibit transwell migration of THP-1 and identified various changes in cytokines and gene expression in STC-1 overexpressing cells that may be the cause of this inhibitory action. Interestingly, RhoA signaling pathway is implicated as a plausible mechanism of action of STC1. Although definitive proof and explicit mechanism behind STC1 effects is still missing. These results continues to demonstrate the importance of the TME and establishes a baseline by which STC1 overexpressing cancer cells can affect macrophage infiltration. Comments 1. Should include scale bar in figures with representative images (Fig 1, Fig 6). Response: Scale bars have been added in the revised manuscript. 2. Please include information on how many trials/replicates were performed or analyzed for the migration assay (Fig 1, Fig 2, Fig 3). From how many tumor sections was data in Fig 6 generated and is the data quantified from all fields in a tumor section or from select sections only? Response: Sorry for the unclear information. Migration assays were performed and analyzed in triplicates. For tumor sections, fluorescent signals of 3 pairs of tumors, 3 sections per tumor, had been analyzed, all showing higher fluorescent count in 97L/P tumors. The representative data from Fig 6 was generated from one of the pair sets. Fluorescent count was quantified from the whole tumor sections. The information has been added in the revised manuscript. 3. I agree from your images that it does appear that 97L/S1 co-culture appears to reduce THP-1 transwell migration, however, I wonder how accurate the data is. Your methods describe that cells were counted by light microscopy. From the images, it looks like there are large masses of cells that migrated through, how were those counted? Also, are these quantification per microscopy field or did you image the entire bottom of the transwell and count all cells? Response: Data of migrated cells from Fig 1A and 1B (24 hr) were quantified from 4-5 random fields within the inserts. Sometimes, large masses of cells were observed which made it difficult to count. We estimated the number of migrated cells per unit area of a single cell. For figure 1B, the graph was from the 24hr-experiment. How were the pathways selected in Fig 3A? Response: As mentioned in the Discussion, RhoA pathway was selected according to the transcriptomic data since it was associated with monocyte/macrophage motility. The 2 pathways (MYPT1/MLC2 and LIMK/cofilin) are two common RhoA downstream pathways related to cell motility. 4.Fig 3B top panel has no error bars. Was only one replicate analyzed for this bar graph? I interpreted the method section as having 4 replicates run for the transcriptome analysis. Similarly for Fig 5A. Response: We originally took the means of the bioinformatics data to plot the graph and indicated the significance, as the p values are shown in the supplementary Table 1-DEG.xlsx . We did not add the standard deviations in our previous version. As per your comment, we have added the error bars in the revised figures. 5. In Fig 5C, is actin the representative loading control for the blot? Since RhoA pathway affects actin dynamics, it may be better to use a different loading control to compare your groups (maybe GAPDH). Response: The samples have been used to run for GAPDH and the data are shown in the revised manuscript. 6. Since RhoA pathway was proposed, it would be nice to do a confirmatory assay perhaps using an inhibitor of RhoA and demonstrating that THP-1 migration is still inhibited. Response: As per your suggestion, we used the ROCK inhibitor (Y27632) in the THP-1 migration assay. A reduction in ThP-1 cell migration is observed. The data are presented in the supplementary Figure 1. 7. There are some grammatical errors in the writing and formatting inconsistencies that should be addressed before publication. Response: The manuscript has been edited. Attachments Attachment Submitted filename: Reponses to Reviewers.docx https://doi.org/10.1371/journal.pone.0241932.r002
23 Oct 2020 Decision Letter - Joe W. Ramos, Editor Effects of Stanniocalcin-1 Overexpressing Hepatocellular Carcinoma Cells on Macrophage Migration PONE-D-20-22832R1 Dear Dr. Wong, 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, Joe W. Ramos, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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 Reviewer #2: 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: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 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: Yes ****** 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: This reviewer recommends that the revised manuscript was perfectly revised and this reviewer will be suitable for publication in PLOS ONE. This manuscript does not have concerns about dual publication, research ethics, or publication ethics. Reviewer #2: (No Response) ****** 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: Yes:** Masayoshi Yamaguchi Reviewer #2: No https://doi.org/10.1371/journal.pone.0241932.r003
Formally Accepted
27 Oct 2020 Acceptance Letter - Joe W. Ramos, Editor PONE-D-20-22832R1 Effects of Stanniocalcin-1 Overexpressing Hepatocellular Carcinoma Cells on Macrophage Migration Dear Dr. Wong: 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. Joe W. Ramos Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0241932.r004

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