Dear Dr. Morshneva,

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 submit your revised manuscript by Jul 06 2025 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.

• 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: https://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,

Li Yang, M.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. 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. Thank you for stating the following financial disclosure:

“This study was supported by the Russian Science Foundation (project No. 24-25-20164, https://rscf.ru/project/24-25-20164/) and the St. Petersburg Science Foundation (Russia).”

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files.

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

4. 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.

[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?

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

**********

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

Reviewer #1: Dr. Morshneva et al. submitted the manuscript entitled: An in vitro tumor recurrence model based on platinum-resistant colon cancer cells as a research tool for studying cancer cell dormancy, in which the authors reported the establishment of platinum-resistant tumor recurrence model, as well as detailed validation and exploration of the drug-resistant cell line. The authors checked expression level of essential proteins and RNAs as well as ROS level. Generally speaking, this is a meaningful work, and the topic will be of interest to the potential readers of Plos One. However, as characterization and validation are fundamental for reports on a new cell model, I have some major comments for authors to further improvement.

1. The authors did not directly compare drug-resistance cells with parental cells, such as IC50 shifts, survival curves or dose response study.

2. The authors did not perform short-term stability studies of the new cells, such as colony formation study and apoptosis studies when applied to platinum drugs.

3. The authors did not perform long-term stability studies of the new cells, such as passage-stability testing.

4. I’m wondering if the authors observed cross drug-resistance on HCT116 cspl-R or HCT116 oxpl-R? Can these cell lines resist other platinum drugs?

5. The language used occasionally feels a bit choppy, with ideas jumping abruptly from one paragraph to the next.

Considering the importance of this work, but lacking results from some essential experiments, I recommend rejecting the submission and offering the opportunity to resubmit after substantial revisions.

Reviewer #2: This manuscript presents a well-structured and detailed study of a novel in vitro model of tumor recurrence using platinum-resistant HCT116 colon cancer cells. The model aims to recapitulate cellular dormancy and recurrence following oxaliplatin/cisplatin treatment. The authors comprehensively characterize dormancy-associated features, including cell cycle arrest, reduced ROS, increased autophagy, EMT marker expression, and upregulation of stemness-related genes.

The study is methodologically sound and offers a potentially valuable tool for investigating cancer cell dormancy and recurrence. However, several areas would benefit from clarification, more rigorous analysis, and contextualization within the broader literature:

1. The authors state: "To date, no dormancy models utilizing drug-resistant cells have been reported in the literature." (Lines 439–441)

While this claim may be valid, it would benefit from clearer positioning against existing models of dormancy. For example, the recent review by Kamat et al. (Molecular Cancer, 2025, Vol. 24) discusses a range of drug-tolerant and dormancy-related systems. The authors should acknowledge these works and better articulate the specific novelty of their approach.

2. The use of FUCCI labeling is appropriate and informative. However, additional validation—such as Ki-67 immunostaining—would help more definitively distinguish true G0 quiescence from G1 arrest.

3. The manuscript reports dynamic changes in dormancy-related markers (e.g., FoxO1/3, LC3, Beclin, Twist1), but does not explore their causality. Are these markers drivers of dormancy, or merely consequences of treatment-induced stress?

4. The manuscript requires thorough language editing for grammar, clarity, and consistency.

Reviewer #3: General Assessment

In this manuscript (PONE-D-25-19582), Morshneva et al. present a well-conceived and methodologically rigorous in vitro model that simulates tumor recurrence and cancer cell dormancy using platinum-resistant HCT116 colon cancer cells. By exploiting the chemoresistant phenotype, the authors recreate a clinically relevant scenario in which residual, therapy-surviving tumor cells enter a non-proliferative state before eventually repopulating, a key feature of tumor relapse. This approach addresses a fundamental challenge in dormancy research: the limited availability of residual cells post-chemotherapy in traditional models. The model’s high reproducibility and scalability enhance its potential utility in mechanistic studies and preclinical screening of dormancy-targeting therapies.

A key strength of this work lies in the comprehensive and multi-dimensional characterization of the quiescent/dormant state. Through the integration of FUCCI-based cell cycle imaging, qPCR, immunoblotting, ROS quantification, and viability assays, the authors convincingly demonstrate that treated cells enter a reversible growth-arrested state marked by G0/G1 accumulation, reduced ROS levels, increased autophagy, drug resistance, and elevated expression of stemness and EMT markers. The flexibility of the model, demonstrated by manipulating drug type, dose, and exposure time, further enhances its applicability. However, despite the convincing phenotypic profiling, the manuscript would benefit from additional functional validation experiments, clearer handling of interpretive inconsistencies (particularly regarding EMT and treatment comparisons), and improvements in data presentation. Addressing the following concerns is essential for strengthening the manuscript’s scientific rigor and suitability for publication.

Major Comments

1. Inconsistent Use of Resistant Cell Lines: Although both cisplatin- and oxaliplatin-resistant cell lines are described, the authors alternate between them across experiments despite noting that they exhibit different repopulation kinetics (lines 453–458). This inconsistency may raise concerns about selective data presentation. To enhance the model’s generalizability and scientific transparency, key findings should be validated in both cell lines.

2. Lack of Functional Validation of Dormancy Pathways: While dormancy-associated features are well-characterized, functional testing of core pathways (e.g., p38 MAPK, autophagy, ROS detoxification) is lacking. Perturbing these pathways using small-molecule inhibitors (e.g., SB203580 for p38, 3-MA for autophagy) or gene knockdown approaches (e.g., FOXO3 or Beclin1) would significantly strengthen the mechanistic conclusions.

3. Inconsistencies in EMT Marker Expression: The simultaneous upregulation of E-cadherin and N-cadherin is acknowledged but insufficiently addressed. This contradiction could reflect epithelial-mesenchymal plasticity or cellular heterogeneity. Follow-up experiments such as single-cell transcriptomics or immunofluorescence staining are recommended to clarify the phenotype

4. Discussion of Model Limitations: The current discussion does not adequately address the model’s limitations, particularly the absence of tumor microenvironmental components (immune, stromal, and hypoxic factors), which are critical regulators of dormancy in vivo. This gap should be more thoroughly considered in the context of the model’s translational relevance.

5. Data Presentation and Clarity: Several figures and legends are overly dense or lack clarity. Normalization methods, time points, and statistical annotations (including p-values and sample size) should be consistently reported. Graphs would benefit from cleaner labeling and clearer presentation of fold-changes relative to key states (e.g., peak quiescence or baseline proliferation).

6. Figure Legends and Abbreviations: Figure legends should be more comprehensive and include all abbreviations and treatment details referenced in the figure. This will improve interpretability without needing to refer back to the main text.

7. Discrepancy Between Imaging and Graphs in Figure 2: There is a mismatch between the days shown in fluorescent images (days 2 and 4) and the time points in the graph (days 3, 4, 5). This should be clarified or aligned for consistency.

8. Drug Treatment Justification: In Materials and Methods (line 92), the rationale for selecting 25–50 μM cisplatin and 50–150 μM oxaliplatin with exposure times of 6/24 hours should be explicitly stated or supported by literature references.

9. Drug Concentration Justification (Line 276): Drug concentrations for viability testing (e.g., etoposide, irinotecan, 5-FU) are listed without justification. Appropriate references or pilot data should be provided to validate the chosen doses.

10. Inconsistency in Figure 5 Treatments: The rationale for using cisplatin in Figure 5a and oxaliplatin in Figures 5b/c is unclear. Uniform treatment or explanation of the differential use is necessary.

11. Unjustified Reagent Choice (Line 225): The detailed 0–33-day time-course is performed using cisplatin, not oxaliplatin. A justification for this choice is needed, or results should be presented for both agents to support the model’s robustness.

12. Statistical Reporting: The authors report SEM instead of SD without justification. Given the small number of replicates (n=3), reporting SD may more accurately reflect biological variability unless multiple independent experiments were conducted.

13. Misinterpretation of FUCCI Data (Line 224): The claim that cell cycle distribution “returns to its initial state” by day 13 is not fully supported by the data. mKO2-Cdt1-positive cells still represent ~35% versus ~10% at day 0. This needs rephrasing or additional justification.

14. Missing p27 mRNA Data: In Figure 3, the accumulation of p27 is shown by western blot, but not confirmed at the transcript level. Including qPCR results would provide a more complete assessment of cell cycle regulation.

15. Overstated Persistence of Beclin (Line 304): The text claims Beclin accumulation persists through repopulation, but Figure 5 suggests otherwise. This conclusion should be revised or supported with additional quantification.

16. Need for Quantification of Immunoblots (Line 307): The claim that survivin accumulation corresponds with LC3/Beclin decline is not visually convincing. Quantitative densitometry should be provided to support this interpretation.

Minor Comments

• A brief comparison with other drug-induced dormancy models (e.g., breast or lung cancer models treated with doxorubicin or docetaxel) would help contextualize the significance of this work within the field.

• Minor typographical and formatting issues (e.g., inconsistent italicization of gene names, figure label placement) should be corrected throughout the manuscript.

• In Figure 1b, visual cues such as arrows should be added to highlight key features (e.g., vacuolization, multinucleation) for clarity.

Reviewer #4: Overall, this is a clear, concise, and well-written manuscript. The introduction is relevant and research based. Sufficient information about the previous study findings is presented for readers to follow the present study rationale and procedures. The authors make a systematic contribution to the research literature in this area of investigation.

Specific comments follow

p. 4, lines 74: The term "present" suggests that there should be a "presented."

Please provide original full resolution images of Fig 1b

**********

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

Reviewer #3: No

Reviewer #4: Yes:  Umar Farooq

**********

[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.

Dear Dr. Morshneva,

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 submit your revised manuscript by Oct 02 2025 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.

• 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 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: https://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,

Li Yang, M.D.

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

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.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: Thank you for carefully addressing my comments and suggestions. The revised manuscript presents the significance of your findings more clearly and effectively. All my previous comments have been addressed, and I appreciate the descriptive and detailed responses provided by the authors, as well as the thoughtful revisions made throughout the manuscript.

Reviewer #4: When applied to platinum medicines, the scientists did not conduct short-term stability investigations of the new cells, such as colony formation and apoptosis studies.

The novel cells were not subjected to long-term stability experiments by the authors, including passage-stability testing.

The figures must be clearly and meaningfully prepared at high quality. Both by themselves and with the typography, the manuscript's images lacked visual appeal. It was suggested that they be changed and modified.

Occasionally, the wording feels a little chaotic, with concepts leaping from one paragraph to the next without explanation.

The article does not investigate the causal relationship between the dynamic changes in dormancy-related markers (such as FoxO1/3, LC3, Beclin, and Twist1) that are reported in the study.

Do these indicators indicate reasons for dormancy, or are they just effects of stress brought on by treatment?

Absence of Functional Validation of Dormancy Pathways: Although characteristics linked to dormancy have been thoroughly described, there is a dearth of functional testing for key pathways (such as p38 MAPK, autophagy, and ROS detoxification). The mechanistic results would be much strengthened if these pathways were perturbed using gene knockdown techniques (e.g., FOXO3 or Beclin1) or smallmolecule inhibitors (e.g., SB203580 for p38, 3-MA for autophagy).

Limitation is missing in the study

Disparities in EMT Marker Expression: Ecadherin and N-cadherin's concurrent overexpression is recognized but not adequately addressed

**********

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

Reviewer #3: No

Reviewer #4: Yes:  Dr Umar Farooq

**********

[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

An in vitro tumor recurrence model based on platinum-resistant colon cancer cells as a research tool for studying cancer cell dormancy

PONE-D-25-19582R2

Dear Dr. Morshneva,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .

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,

Li Yang, M.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thanks for the authors' efforts to comprehensively improve your manuscript according to editor's and reviewers' comments. I am pleased to inform you that your paper can be accepted for publication now. Thanks for the chance to assess your work. Additionally, many thanks for all the reviewers' precious inputs.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #4: Yes

**********

Reviewer #4: Dear Author(s),

I am pleased to inform you that your manuscript has been accepted for publication. The paper makes a valuable contribution to the field of Pharmacy, and the research is well-structured, clearly written, and supported with appropriate methodology and references.

Strengths of the Paper:

The study addresses an important and relevant research problem.

The objectives are clearly stated, and the methodology is sound.

The results are presented effectively with logical interpretation.

The discussion is insightful and supported by recent and relevant literature.

The overall presentation of the manuscript is clear, concise, and professionally written.

I commend the authors for their efforts and the quality of work presented. No further revisions are required at this stage.

Congratulations on your acceptance, and I wish you continued success in your research endeavors.

**********

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 #4: Yes:  Dr. Umar Farooq

**********