PONE-D-25-11860

The effect of cold ischemia time on hypoxia, EMT, and apoptosis pathways in normal colon mucosa

PLOS ONE

Dear Dr. Filipowicz,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

Specifically:

After careful consideration of the reviewer comments and editorial assessment, we regret to inform you that we are unable to proceed with publication.

Your findings on how cold ischemia time (CIT) may affect gene expression in normal colon mucosa are clearly presented and technically sound. However, the study remains largely descriptive, and the conclusions regarding cancer-related transcriptional changes are not directly supported by functional validation. Moreover, the focus on normal tissue limits the broader applicability of the findings.

Given these limitations, we do not believe the manuscript meets the publication criteria for PLOS ONE in its current form. We thank you again for considering the journal and encourage you to further develop this work for submission elsewhere.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

Kind regards,

Yingkun Xu, M.D

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Thank you for submitting your manuscript to PLOS ONE. We appreciate the thoughtful study design and attention to data quality.

After careful consideration of the reviewer comments and editorial assessment, we regret to inform you that we are unable to proceed with publication.

Your findings on how cold ischemia time (CIT) may affect gene expression in normal colon mucosa are clearly presented and technically sound. However, the study remains largely descriptive, and the conclusions regarding cancer-related transcriptional changes are not directly supported by functional validation. Moreover, the focus on normal tissue limits the broader applicability of the findings.

Given these limitations, we do not believe the manuscript meets the publication criteria for PLOS ONE in its current form. We thank you again for considering the journal and encourage you to further develop this work for submission elsewhere.

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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?

Reviewer #1: Yes

Reviewer #2: Yes

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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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: accepted.

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) (Limit 200 to 20000 Characters)

Reviewer #2: The paper explains the effect of cold ischemia time (CIT) and its importance in transcriptomic studies and reproducibility. The study objective evaluating CIT effects on cancer-related genes is relevant and practical. The findings from this study suggest a CIT limit (≤45–60 min), information that is valuable for the research community.

The number of samples, patient count and CIT intervals are clearly described but whether all 54 samples were paired per patient across all time points needs further clarification.

Overall, the analysis is comprehensive. The combination of RNA-seq, functional enrichment and WGCNA strengthens the biological interpretation. Identifying DEGs and linking them to apoptosis, hypoxia, EMT and cancer-related pathways is relevant and informative.

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Reviewer #1: No

Reviewer #2: No

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Dear Dr. Filipowicz,

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.

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Kind regards,

Aniruddha Datta

Academic Editor

PLOS One

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"This study was supported by the Foundation for Polish Science (FNP) under the International Research Agendas Program to J.P.D. and A.P., co-financed by the European Union under the European Regional Development Fund (MAB/2018/6); and the Ministry of Science and Higher Education, (grant No. 2/566516/ SPUB/SP/2023) to L.K.

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Additional Editor Comments (if provided):

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

Reviewer #5: All comments have been addressed

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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

The PLOS Data policy

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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Reviewer #3: The authors have updated their manuscript with the comments and questions raised by the reviewers. I have no further feedback.

Reviewer #4: Summary

This study investigates the effect of cold ischemia time (CIT) on the transcriptomic profile of normal colonic mucosa, with specific attention to cancer-related pathways such as apoptosis, hypoxia, and epithelial–mesenchymal transition (EMT). The authors analyzed 54 normal mucosal samples from 9 colorectal cancer patients and performed RNA-seq under CIT conditions ranging from 0 to 60 minutes. The manuscript describes the experimental workflow, RNA quality assessment, differential expression analysis, WGCNA, and pathway enrichment.

In the Results, the authors report 44 significant DEGs between T0 and T5, enriched in apoptosis, hypoxia, and EMT pathways. They note minimal transcriptomic changes before T3, a small increase at T4 (6 DEGs), and a larger increase at T5. The Discussion proposes that CIT can induce tumor-like molecular signals and stresses the importance of controlling ischemia time during tissue acquisition. The authors recommend keeping CIT under 45 minutes.

Questions and Concerns

The proposed “safe CIT threshold” appears overly optimistic and contradicts multiple prior studies.

The manuscript recommends limiting CIT to 45–60 minutes to minimize molecular artifacts. However, previous literature shows that both gene and protein expression in colonic tissue can be significantly altered within minutes.

• Spruessel et al. demonstrated that a few minutes to 30 minutes of ischemia can markedly change gene and protein expression in colon samples:

https://pubmed.ncbi.nlm.nih.gov/15211754/

• von der Heyde et al. (2024, Cell Death & Disease, Nature Publishing Group) showed that proteomic dysregulation emerges above 15 minutes of ischemia:

“Specimen ischemia time above 15 min is mostly associated with a dysregulation of proteins in the immune-response pathway.”

https://www.nature.com/articles/s41419-024-07090-x

These findings suggest that the authors’ proposed CIT tolerance window may not be sufficiently conservative.

The study relies solely on mRNA measurements and lacks multi-omic validation.

No proteomic, immunohistochemical, or additional orthogonal assays are provided. This is a notable gap because proteomic alterations often occur earlier and more sensitively than transcriptomic changes.

For example, von der Heyde et al. (2024) report:

“The proteomics analysis revealed that specimen ischemia time above 15 min is mostly associated with a dysregulation of proteins in the immune-response pathway.”

https://www.nature.com/articles/s41419-024-07090-x

Without multi-omics confirmation, it is difficult to determine whether the reported mRNA changes reflect true biological responses or early degradation phenomena.

Warm ischemia time (WIT) is acknowledged but not recorded or modeled, which is inconsistent with established standards.

Although the authors state that WIT is important, they do not:

• document each patient’s WIT;

• include WIT as a covariate in statistical models (e.g., edgeR, linear models);

• analyze interactions between WIT and CIT.

This is problematic because prior work has emphasized that WIT must be explicitly accounted for. Musella et al. (2013) show that both WIT and CIT shape molecular stability in colon tissue:

“A critical time point for tissue handling in colon seems to be 60 minutes at room temperature … accounting for warm ischemia in this tumor type.”

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053406

Without controlling for WIT, the attribution of transcriptional changes solely to CIT is not well supported.

Reviewer #5: This manuscript presents a well-designed and carefully executed study examining the impact of cold ischemia time (CIT) on transcriptomic profiles of normal colon mucosa. The topic is timely and highly relevant, as preanalytical variables such as CIT remain a major source of variability and irreproducibility in transcriptomic and biobanking-based research.

The experimental design is a clear strength of the study. The use of paired samples collected from the same patients at multiple predefined time points provides strong internal control and allows for a robust assessment of time-dependent transcriptional changes. The application of RNA sequencing combined with functional enrichment analysis and WGCNA is appropriate and well justified, and the statistical analyses are performed rigorously and transparently. The clarification regarding the fully paired nature of the dataset and the use of ComBat-seq to account for patient-specific effects adequately addresses potential concerns about confounding.

The results are clearly presented and convincingly demonstrate that prolonged CIT induces transcriptional changes in pathways related to hypoxia, apoptosis, EMT, and cancer-associated signaling, even in morphologically normal tissue. The identification of a practical CIT threshold (≤45–60 minutes) is particularly valuable and provides actionable guidance for researchers involved in tissue collection, biobanking, and transcriptomic analyses.

The manuscript is generally well written, logically structured, and accessible to a broad readership. The discussion appropriately contextualizes the findings within existing literature and highlights both the strengths and limitations of the study, including sample size and the use of a targeted gene panel.

Overall, this work represents a solid and meaningful contribution to the field of preanalytical variability and transcriptomic research. The conclusions are well supported by the data, and the study should be of broad interest to researchers working with human tissues, particularly in cancer and biobanking contexts.

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Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

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Attachments

Attachment

Submitted filename: PONE-D-25-11860_R1.pdf

The effect of cold ischemia time on hypoxia, EMT, and apoptosis pathways in normal colon mucosa

PONE-D-25-11860R2

Dear Dr. Filipowicz,

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.

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Kind regards,

Aniruddha Datta

Academic Editor

PLOS One

Additional Editor Comments (optional):

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

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Reviewer #4: The authors have adequately addressed the major concerns. The recommended CIT threshold has been revised to ≤30 minutes and appropriately contextualized relative to prior literature. The conclusions are now clearly restricted to transcriptomic findings within the targeted gene panel.

Limitations regarding the lack of multi-omic validation and the absence of patient-specific WIT measurements are transparently acknowledged. While these constraints remain, they do not undermine the validity of the presented data within the defined scope.

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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.

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Reviewer #4: No

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