PONE-D-24-55645Targeting Tumor-Associated Genes, Immune Response, and Circulating Tumor Cells in Intrahepatic Cholangiocarcinoma: Therapeutic Potential of Atractylodes lancea (Thunb.) DC.PLOS ONE

Dear Dr. Na-Bangchang,

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National Research Council of Thailand.

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This research project was supported by the Thailand Science Research and Innovation Fundamental Fund Fiscal Year 2023 and Thammasat University (Chulabhorn International College of Medicine, Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma). Kesara Na-Bangchang is funded by the National Research Council of Thailand (NRCT), Contract number N42A671041.

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National Research Council of Thailand.

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Reviewers' comments:

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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: No

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

Reviewer #1: Yes

Reviewer #2: No

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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:  Voici la reformulation en anglais :

The presented work is very interesting and deserves to be published. However, certain improvements are necessary to enhance the clarity and rigor of the article. We recommend the following adjustments:

Revision of the quality of the graphs: Improving the resolution and presentation of the graphs would enhance readability and interpretation of the results.

Specification of the extract used: It is essential to detail the chemical composition of the extract studied and specify the location of sample collection. This information will help contextualize the results and ensure scientific reproducibility.

Reviewer #2:  Reviewer's Summary: In this exploratory companion study to a phase 2A clinical trial of Atractyloides Lancea in palliative stage Cholangiocarcinoma, Martviset et al. analyze the expression of an array of mRNAs with putative cancer relevance. It is nebulous why the authors chose exactly these genes over other tumor-associated or inflammatory genes. This study has limited use to generate hypotheses, though a non-targeted approach like RNA-seq would've been preferred. However, this study does not sufficiently support the main conclusion, namely that A. lancea reduces circulating tumor cells and inflammation. The methodology is unidimensional and the statistics are questionable in the context of a clinical, i.e., definitive study.

Reviewer's Comments:

Abstract: The abstract is mostly adequate, except for the overdrawn conclusion, as mentioned above. The authors may consider stating more explicitely that all patients were beyond curative treatment intent and in palliative care. It is not immediately apparent that RNA was isolated from all samples and not just from "the non-CCA subjects (n=16) on Day 1..." and this sentence may benefit from splitting in two. In addition, the abstract would benefit from stating the blood fraction which the RNA was isolated from (i.e., the buffy coat).

Competing Interests: Spelling, "The".

Data availability: The declaration and statement on data availability don't fit together. The former states that not all data were available, the latter that all data were included in the manuscript. The latter is false; the authors did not include all raw data. The authors should use this section to explain which data is available and which data is not available (and why). All data that can be made available should be uploaded to a public repository (e.g., JBEI EBB) or included as well-tagged supplementary table. All omissions must be justified.

Line 31: "Blood specimen" could be more specific, e.g., "Heparinized venous whole blood (20ml)" or "central venous whole blood (EDTA, 5ml)".

Line 39 and below: Some gene names are italicized and others are not. The authors may also consider discussing, why they did not analyse protein concentrations of at least some of these markers by multiplex assays like Luminex.

Line 89: I believe the authors are referring to "Marker of proliferation Ki-67" and not "monoclonal antibody Ki-67", given that they measure via PCR. This is a recurring issue throughout the manuscript.

Line 108-109: Baseline clinical and laboratory characteristics of patients in the 4 groups need to be summarized in a table in this clinical trial adjacent study. The superficial summary presented in the text is not sufficient.

Line 122 vs. Line 141: Was informed consent from patients not given in writing? Line 141 specifies "written informed consent" of the control group but only "informed consent" (line 122) from cancer patients. While not necessarily unethical, this may not be lege artis.

Line 143: Are the authors certain they centrifuged at 4000-6000 xG and not at 4000 - 6000 rpm? Such a high acceleration will rupture cells (monocytes and macrophages, for example, start rupturing at >500 xG). Accordingly, this may impair cellular RNA measurements. Buffy coats are usually isolated after 200-500 xG centrifugation over a density separation media, like Ficoll, for 30-45min without break, to isolate this fine cell layer with high purity.

Line 161: CTLA4 not CTLP4 is standard nomenclature. This is a recurring issue throughout the manuscript including in figures.

Line 173 and below, Data analysis: To keep the number of study participants undergoing a non-approved treatment low, clinical studies are regularly definitive. Definitive studies require a priori case number planning. Was a formal case number planning performed? Did the authors account for the expected loss of power due to patient death and loss to follow-up? In addition, the testing of multiple markers (considered independent) likely constitutes multiple testing. The authors do not specify any methods to reduce alpha accumulation in this context, neither for multiple testing between groups (i.e., ANOVA post tests) nor multiple markers (i.e., performing the same tests for IL10, IL6, CTLA4, etc, in the hope that at least one is positive). Normality can almost certainly not be assumed for such small case numbers, independent of any non-normality testing (which is also not specified). Given the 4 group design with two timepoints with missing values, a mixed model approach would probably be most appropriate but will likely require significantly higher case numbers to perform at adequate power. Thus, this companion study must be understood as highly exploratory, and the authors should consider introducing it as such.

Line 178 - 179: "One-way ANOVA..." or "A one-way ANOVA..." may be more appropriate than "The one-way ANOVA...". The same goes for "The paired t-test..." in the next sentence. The authors need to specify which post-test they used after the F-statistic.

Line 190 - 191: "patients [...] discontinued the study [...] due to [...] death." is a quite morbid way of phrasing this sentence, and the authors may consider more sensitive wording.

192: "before study completion."

Line 281: The authors performed RNA extraction on all nucleated blood cells and did not select for CTCs. Established methods include matching DNA mutations to known mutations from the primary tumor or from metastasis biopsies and flow cytometry for tumor cell surface markers, both of which have not been performed. The authors can thus crucially not claim that any of the observed changes in gene expression were due to circulating immune cells. To salvage this, the authors may consider spike-in controls using primary CCA tumor cells or cell lines, to generate a standard curve and estimate CTC counts in their patient samples. A similar overstatement without providing convincing data or linking to their companion study is found in line 308.

Lines 346 - 349: These sentences appear contradictory without further discussion; first the authors note reduced inflammation after AL treatment, then they suggest an improved immune response against he cancer. In addition, the latter is a highly questionable interpretation of upregulated CTLA4, an immunosuppressive molecule. Lastly, as also stated in my comments about the introduction, upregulated RNA can vastly differ from protein expression, which has unfortunately not been analyzed.

Lines 359 - 370: This whole paragraph is almost a word by word repetition of the previous paragraph.

Line 387: Circulating tumor cells have not been directly observed. For such a bold statement, one would expect confirmation by established methods and by at least two methods. Stating that inflammation was reduced would require at a minimum protein measurements, preferably functional tests, like LPS stimulated chemotaxis and ROS production.

Figure legends: It may improve reading flow to also include the chosen statistical test in the figure legends.

Fig. 2: What is presented here? Median or Mean? SEM, SD, 95% CI, IQR? Given the relatively low sample number, Median and Quartiles or Tukey boxplots would probably be most appropriate, while Mean / SD or Mean / 95% CI would also be acceptable due to the additional single point presentation.

Figure 3: I'm not sure if this figure adds enough additional information to warrant a unique figure. The authors may consider omitting this figure entirely, merging it with the previous figure, or moving it to the supplement.

Figure 4: Consider mixed models, as outlined in the comments regarding Data analysis. It is not apparent why the authors chose single point clouds for Fig 2 and violin plots for Fig 4. Violin plots have received a lot of critique lately, mostly pertaining to the regular non-disclosure of basket-size and smoothing function; the presentation in Fig. 2 - given the authors add a disclosure of the measure of central tendency and variance - is thus preferable.

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Reviewer #1: Yes:  mohamed abdelkarim

Reviewer #2: Yes:  Willem Buys

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PONE-D-24-55645R1Targeting Tumor-Associated Genes, Immune Response, and Circulating Tumor Cells in Intrahepatic Cholangiocarcinoma: Therapeutic Potential of Atractylodes lancea (Thunb.) DC.PLOS ONE

Dear Dr. Na-Bangchang,

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 May 14 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.

Please include the following items when submitting your revised manuscript:

• 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,

Keun-Yeong Jeong

Academic Editor

PLOS ONE

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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

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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

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

Reviewer #1: Yes

Reviewer #2: No

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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

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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

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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: The reviewers have adequately addressed my comments on the manuscript 'Targeting Tumor-Associated Genes, Immune Response, and Circulating Tumor Cells in Intrahepatic Cholangiocarcinoma: Therapeutic Potential of Atractylodes lancea (Thunb.) DC.' and I propose the acceptance of the article.

Reviewer #2: The authors have significiantly reworked the manuscript. The conclusion has been adequately toned down to fit the results. The limitations and further research requirements have been outlined well. It is unfortunate, that a study with such an interesting premise is so strongly impaired by a high loss to follow up from an already small sample size - yet, this is hard to avoid in terminal cancer studies.

I believe that publication is warranted, particularly as this is clinical data, after addressing two major concerns:

- Line 171 - 172: No correction for multiple comparison is detailed here. The local alpha niveau is thus likely inflated to values far higher than 0.05 due to alpha accumulation. Please detail 1) whether your workflow already contained correction for multiple testing, as is the standard in many statistics programs including PRISM, 2) whether you performed multiple testing corrections yourself, such as the Bonferroni-Holm method, or or possibly limited the false discovery rate, or 3) that the study did not correct for alpha accumulation and why.

- Figure 4: I struggle to understand, how samples were selected for this figure, as opposed to all patients in figure 2. As I understand this Figure, only samples were considered, for which data was available on day 1 and 90. However, this does not explain, how the 3 healthy subjects were selected for figure 4, as the methods section specifies they were only sampled once. Figure 4 is central to this study and would thus especially benefit from a clearer description both in the legends, as well as the results section.

Minor comments:

- Line 78-79: This sentence sounds like a citation is missing.

- Line 115-116: "is a capsule formation of"

- Line 131: "enrollment".

- Line 132 - 133: The conversion of RPM to G differs substantially by centrifuge design, i.e., the radius of the rotor. It would be highly beneficial for anyone attempting to reproduce this study, if the acceleration (in xG) was also provided. Alternatively, the authors could provide the exact model of the centrifuge and rotor, so that others can calculate the G-force.

- Line 169: "are summarized as number (%)" I'm not perfectly sure what the authors mean by this, but I assume the authors refer to the number and percent of the population fulfilling or not fulfilling a qualitative criteria.

- "Patients sampled" or "Patients available" may be more appropriate than formulating "Patients used".

- Line 177: The abstract details collection of 59 samples, this sentence mentions 64 samples.

- Table 1: It's great that the authors added this table. For quick visual assessment, the authors may consider also adding "(n,%)" over the first row of data of that table (e.g., second cell, left column). I also believe that they're likely referring to the Karnofsky (not "Kamafsky") Performence Index.

- Line 297-298: The authors don't seem to use this system (Methods), so this sentence my be irrelevant.

- Line 356: Was this Group treated with Placebo? The methods section introduces it as "best supportive care" group.

- 371 - 372: This statement is also made in the sentence prior. As the authors do not analyse immune function as part of this study, this conclusion is likely overdrawn, except if linked to previous work showing functional differences of the immune response.

- Line 375- 376: I appreciate the authors' well written and comprehensive limitation statement. The sentence ending in "...cannot be concluded." is understandable, but doesn't read well. Consider something like "Therefore, it is unclear, whether any observed changes [...] translate to differences in protein expression or cell function."

Additional suggestions:

- Line 282 - 306: The general merit of liquid biopsies and CTC could be explained with much more brevity, to make room for a more thorough discussion of the actual results, as they relate to other studies in the field.

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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.

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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:  Mohamed Abdelkarim

Reviewer #2: Yes:  Willem Buys

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[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.]

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Targeting Tumor-Associated Genes, Immune Response, and Circulating Tumor Cells in Intrahepatic Cholangiocarcinoma: Therapeutic Potential of Atractylodes lancea (Thunb.) DC.

PONE-D-24-55645R2

Dear Dr. Na-Bangchang,

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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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,

Keun-Yeong Jeong

Academic Editor

PLOS ONE

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

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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

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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: The reviewers have adequately addressed my comments on the manuscript 'Targeting

Tumor-Associated Genes, Immune Response, and Circulating Tumor Cells in Intrahepatic

Cholangiocarcinoma: Therapeutic Potential of Atractylodes lancea (Thunb.) DC.' and I propose the

acceptance of the article.

Reviewer #2: The authors have significantly improved this manuscript since the last submission. All my concerns have been addressed and I recommend publication.

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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:  mohamed abdelkarim

Reviewer #2: Yes:  Willem Buys

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