■ GENERAL COMMENTS TO THE EDITORS AND THE REVEIWERS:

We would like to extend our gratitude to you and the reviewers of the “PLOS One” for taking the time and efforts to review our manuscript. Many of the valuable and constructive points you raised truly inspired the authors. After considering the reviewers’ comments, we revised the manuscript and have indicated the corrections and changes made with yellow highlights in the manuscript.

The revision, based on the review team’s collective input, includes a number of positive changes. Based on your guidance, we:

• Enhanced clarity through general revision of the manuscript

• Added new descriptions and figures

• Revised the title

We now wish to submit the revised manuscript. The specific revisions and corrections made in response to the reviewers’ comments are as follows:

We have uploaded the marked file.

Please include the following items when submitting your revised manuscript:

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Answer:

As recommended, we revised manuscript format.

2. 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.

Answer:

Answer: We attached the immunoblotting raw data file. Please find the attached.

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.

3. In the ethics statement in the manuscript and in the online submission form, please provide additional information about the patient records/samples used in your retrospective study, including: a) whether all data were fully anonymized before you accessed them; b) the date range (month and year) during which patients' medical records/samples were accessed.

Answer:

As recommended, we added the sentence in “materials and methods” section as follows:

“The patients' medical records and samples were fully anonymized before we accessed them in September 2018.”

4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

Answer:

As recommended, the ethics statement appear in the Methods section

5. Please provide additional information about each of the cell lines used in this work, including 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.

Answer:

The paper included the purchase information about the cell lines we used. We purchased the cells from the “Korean Cell Line Bank,” a research institute that acquired the status of the International Depository Authority and the institute sent the cells after quality control. For more information on cell lines, please refer to the following sites:

https://cellbank.snu.ac.kr/main/tmpl/sub_main.php?m_cd=6&m_id=0201&sp=2&c_id=558

6. Please provide accession numbers and/or URLs for the datasets obtained from the TCGA and GDSC databases.

Answer:

As recommended, we added new URLs in materials and methods as follows:

TCGA data: https://portal.gdc.cancer.gov/

GDSC data: https://www.cancerrxgene.org/celllines

7. At this time, we ask that you please provide scale bars on the microscopy images presented in Figures 1 and 5, and refer to the scale bar in the corresponding Figure legend.

Answer:

As recommended, scar bars were added in figures 1, 3 and 5

8. Thank you for stating the following in the Financial Disclosure section:

"YES

This study was supported by Daewon Pharmaceutical Co., Ltd. in 2018. (To Byoung Kwan Son) ".

We note that one or more of the authors have an affiliation to the commercial funders of this research study : [Daewon Pharmaceutical Co., Ltd].

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

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If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

Answer:

The funding organization did not play any role in our study and provided only financial support. We have reviewed the author roles in the Author Contribution section and confirm the previous statements are correctly stated.

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Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

Answer:

As recommended, we added a new sentence in the “Competing Interests Statement” section as follows.

“This does not alter our adherence to PLOS ONE policies on sharing data and materials.”

9. 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.

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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: No

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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

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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:

• In my opinion the title is not comprehensible, please improve it.

Answer:

We revised the title as follows:

High SLC2A1 expression with retinoic acid-induced inhibition promoted cancer survival by suppressing CD8 T cells and B cells in gastric cancer ->

High SLC2A1 expression associated with suppressing CD8 T cells and B cells promoted cancer survival in gastric cancer

• SLC2A1 is an acronym, and as such the first time it is mentioned it should be made explicit, do the same for all the others throughout the text.

Answer:

As recommended, we added the full name of SLC2A1 as “solute carrier family 2 member 1 (SLC2A1)”

• Retinoic acid, tretinoin: please uniform the names for ATRA.

Answer:

As the reviewer well pointed out, we revised as ATRA.

• What is the purpose of Supplementary table 1? Please include all information for all the patients, also as an average values between groups.

Answer:

To avoid confusion among readers, we removed the supplementary table 1

• Mat&met Supplementary information, 1µM retinoic acid: this is too generic definition, please include what kind of retinoic acid is (ATRA? 9-cis? 13-cis?) and the producer.

Answer:

Of the many kinds of retinotic acid, we used all-trans-retinoic acid (ATRA). The relevant Information, place of purchase, and product number was inserted in the Materials and methods section as follows: 1 μM retinoic acid (ATRA, all-trans-retinoic acid, R2625, Sigma)

• Why for RT-PCR GAPDH was used, and instead for immunoblotting beta-actin? Please explain or justify as limitation.

Answer:

Genes such as GAPDH, beta-actin, and alpha-tubulin are all widely used in experiments as housekeeping genes. Unless there are special conditions (such as separating cytosol and nucleus, experiments that can change actin, etc..), it is safe to use any housekeeping gene. As many scholars prefer bata-actin in immunoblotting and GAPDH in PCR (including realtime PCR), we have used it accordingly. I have attached two papers that used actin in immunoblotting and GAPDH in PCR for more information. They also use two housekeeping genes in a paper.

Figure 4 and 5 of J Biol Chem. 2015 Jul 3;290(27):17029-40.

Figure 1D and 1E of PLoS One. 2015; 10(8): e0128943.

• “MKN-45 cells were seeded at 3,400 cells per well” please add the type of plate.

Answer:

Although the experiment was conducted at 96 well plate, we have not specified the information in the script. Thanks to the sharp point by the reviewer, we added the information as follows: MKN-45 cells were seeded at 3,400 cells per well in 96 well plate.

• Results, “189 normal mucosa, 279 primary cancer and 58 metastatic cancer samples […] primary tumour or metastatic cancer samples” in light of this it is necessary to give some more and correct information about included patients and type of isolated tissue from them, because only 279 were depicted above, if the samples are 279 primary cancer ‘or’ 58 metastatic samples, patients should be more. It should be indicated by how many patients all three types, or only two, or only one samples were taken, justify why the healthy samples are less than the tumors, and so on.

Answer:

Metastatic cancer, primary cancer, and normal tissue were collected from one patient. In the process of making tissue microarray, there were many cases of missing normal tissues.

We revised the original sentence in the “Clinical manifestations of SLC2A1” section as follows:

“We have analyzed 189 normal and 58 metastatic tumor samples from a total 279 primary cancer samples.”

• Table 2: please define all the acronyms throughout the text, for example here CI and HR.

Answer:

We added the explanation as “HR, hazard ratio; CI, confidence interval” in the table 2.

• Figure 1: please define all the elements in the figure in the captions, here for example the numbers of low and high.

Answer:

We appreciate your insight.

We added “low <0.5” and “high >0.5” in EHC.

We added “low <10.2” and “high >10.2” in TCGA.

• Fig.1b: please add statistic direction

Answer:

We added the p value in the Fig 1b.

• “In the EHC, high SLC2A1 expression was significantly associated with advanced T stage, advanced N stage, large tumor size, diffuse type, high histological grade, lymphatic invasion, high PD-L1 expression, low PNI, and chemoresistance (p = 0.001, 0.001, 0.003, 0.002, 0.001, 0.001, 0.028, 0.048 and 0.002, respectively) (Table 1).” Please explain among which groups the comparison was made.

Answer:

We added “compared with low SLC2A1 expression” in the sentence.

• “SLC2A1 expression in relation to mutation, copy number alteration, and methylation status” for this paragraph some things are not explained. For example, which sample group does this analysis belong to? It is not clear why for wild type SLC2A1 expression value is 11, and so on. Add information in mat&met and in captions.

Answer:

This is an analysis based on the TCGA data for SLC2A1. It means that the mRNA value of SLC2A1 for wild type is 11.

Reviewer #2: The authors study SLC2A1 and possible chemotherapy agents targeting SLC2A1 in gastric cancer.

The manuscript is in general well written and complies with ethical standards of research on human tissue specimen. I feel a few minor changes throughout the manuscript would improve the quality of the manuscript and make it easier understandable for the readers.

Major suggestions:

Study setting:

The headline states that “High SLC2A1 expression with retinoic acid-induced inhibition promoted cancer survival by suppressing CD8 T cells and B cells in gastric cancer”, this is a confusing headline. In the manuscript the authors state that high SLC2A1 expression indicates poor prognosis in gastric cancer patients. Although tretinoin reduced the growth of cancer cell lines with high SLC2A1 expression, it is quite a bold statement to say that this promoted cancer survival, when no survival benefit was, nor could have been, shown in this study setting.

Answer:

We revised the title as follows:

High SLC2A1 expression with retinoic acid-induced inhibition promoted cancer survival by suppressing CD8 T cells and B cells in gastric cancer ->

High SLC2A1 expression associated with suppressing CD8 T cells and B cells promoted cancer survival in gastric cancer

As stated in the abstract, the aim of this study was to analyze the survival data but also genetic changes and immune profiles in gastric cancer patients with high SLC2A1 expression and to provide treatment strategies. The conclusion of the study states that strategies making use of SLC2A1 could contribute to better clinical management/research for patients with gastric cancer. The conclusion should clearly answer the aim of the study, which I feel it didn’t.

Answer:

We revised the conclusion as follows:

“Treatment involving the use of SLC2A1 could contribute to better clinical management/research for patients with gastric cancer.”

We revised the aim as follows: (treatment strategies -> treatment)

The aim of this study was to analyze the survival and genetic changes/immune profiles in patients with gastric cancer with high SLC2A1 expression and to provide treatment for improving prognosis.

Results:

The results on CD8+ cells are confusing. In the EHC, CD8+ cells are elevated in patients with high SLC2A1 expression. However, in TCGA, high SLC2A1 expression was associated with decreased CD8 T cells. This is not discussed in the paper. Why do you think this is?

Answer:

In the two EHC and TCGA cohorts, the high SLC2A1 expression group had a decrease in CD8+ T cells.

Minor suggestions:

Abstract:

A short description of methods in the abstract section of the manuscript would be good.

Introduction:

One whole paragraph of the introduction goes over results of previous studies comparing GLUT1 and 18f-FDG-PET-CT. There are plenty of studies on GLUT1 and SLC2A1 already published. Since the current study does not investigate PET imaging, this paragraph could be left out or replaced by a short one meaning statement on PET imaging and glucose metabolism in cancer.

Answer:

As recommended, we removed the contents.

The next to last paragraph of introduction states that TCGA divides gastric cancer into five molecular subtypes, I believe four subtypes have been described.

Answer:

As you pointed out, we revised as “four”.

Methods:

Does the Eulji hospital cohort consist of selected or consecutive patients? Further in the results section the authors mention normal mucosa, primary cancer and metastatis cancer samples. Are the normal mucosa samples from the same patients? If so, why only 189 samples when the cohort was 279 patients? Should be clarified in the methods section of the manuscript.

Answer:

Metastatic cancer, primary cancer, and normal tissue were collected from one patient. In the process of making tissue microarray, there were many cases of missing normal tissue.

We revised the sentence in the “Clinical manifestations of SLC2A1” section as follows:

“We have analyzed 189 normal and 58 metastatic tumor samples from a total 279 primary cancer samples.”

As neoadjuvant chemotherapy is nowadays standard of care in the treatment of gastric cancer, could this affect the results of this study?

Answer:

no

We focused mainly on stomach cancer with high SLC2A1 expression.

The authors have done a great job in defining all abbreviations in the manuscript. However, I cannot find the definition of GSEA (fourth paragraph of results).

Answer:

The definition is provided in the 1st paragraph in the materials and method section.

-> gene set enrichment analysis (GSEA version 4.3)

Figures are very nice and well representing the results on their own. In figure 1D, would it be possible to represent time as months instead of days as in figure 1C?

Answer:

We revised as months in the Fig 1D.

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Reviewer #1: No

Reviewer #2: No

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