Dear Dr. Dyson,

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.

==============================

Dear Dr. Paul J. Dyson,

Following the review of your Research Article titled " High-throughput screening of drug libraries identifies a new synergistic drug combination for the treatment of retinoblastoma", I recommend that it should be revised taking into account the changes requested by the reviewers. Since the requested changes are major, the revised manuscript will undergo a second round of review by the same reviewers.

==============================

Please submit your revised manuscript by Feb 09 2025 11:59PM of the date of this decision. 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Mounir Tilaoui, Ph.D

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

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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: Partly

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

Reviewer #5: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: 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: No

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. 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 #5: Yes

**********

Reviewer #1: Page 12, line 267 – For the 21 drugs advanced that showed enhancement with thermotherapy, why did the authors choose only drugs that were not active at 37oC? Wouldn’t it be more advantageous to have a drug that both works at normal body temperature and then is further enhanced with thermotherapy?

Page 15, line 326 – I would recommend adding the avg values for carboplatin-etoposide and carboplatin-vincristine in the text as well.

Page 15, Line 341 – authors state that the combination of carboplatin (3.3 uM) and gemcitabine (2.5 uM) only resulted in 60% cell inhibition. Yet, the IC50 of gemcitabine alone is listed as 0.045 uM. Similarly, the dose-response curve in Figure 1 for gemcitabine shows ~60% inhibition at concentrations of 0.1 uM. If the curve in Figure 1 is to be believed, gemcitabine at 2500 nM (Log ~3.4) would be expected to be close to 100% inhibition on its own. Why the big discrepancy in cell viability/% inhibition values between Figure 1 and Figure 2?

Page 15, Line 342 – Quantitating synergistic effects is not trivial to perform and even more difficult to describe. For example, the line “This corresponds to a 15-fold reduction in the concentration of carboplatin required to produce such an effect when compared to carboplatin alone”, yet according to Figure 2, this same % inhibition (~60%) is observed even when 0 uM of carboplatin is used. It is also unclear where the 15-fold reduction number comes from as 15 fold of 3.3 uM is ~50uM and yet 50uM in Figure 2a with no Gemcitabine only inhibited ~11%.

Page 16, line 368 – Authors state “At these doses, carboplatin-gemcitabine resulted in 60% cell viability inhibition in Y79 cells compared to 20% in RPE-1 cells, indicating a promising therapeutic window.” However, the numbers they quote do not appear to be what is depicted in Figure 2c, carboplatin-gemcitabine in Y79 cells looks to be ~60% inhibition as they state, but in RPE-1 cells, the inhibition looks to be even higher inhibition ~65%. No ideas where authors get 20% cell inhibition value from. The figure in 2c makes it appear that there is no therapeutic window using any of the chemotherapeutics as almost all Cell inhibition is similar between Y79 and RPE-1 cells.

Similarly, the other cell inhibition percentages for the other drugs do not appear to be correct either. Authors state 25% and 18% inhibition for carboplatin-irinotecan in Y79 and RPE-1 cells, respectively. However, the graph in Figure 2c clearly shows a greater amount of cell inhibition for the RPE-1 cells.

Page 18, line 397 – Authors state “Furthermore, drug combinations induced greater levels of apoptosis than single 398 drugs as determined by flow cytometry (Fig. 3a,b).” Yet, the flow graphs in Figure 3a do not back up this statement. In fact, Gemcitabine alone had 54.2% in the early apoptotic region and 19.2% in the late apoptotic/dead cell region. Yet the carboplatin + gemcitabine only had 46.9% in early and 12.4% in late. Similarly, etoposide alone appeared to do better in apoptotic induction as compared to the carboplatin + etoposide.

For the quantitation in Figure 2b, were the graphs in figure 2a not used for the analysis? I assume the dots on the bar chart are the individual data points. So if we take etoposide as an example, why is there no dot at 66% (or 76% if you are combining early and late apoptosis regions)?

Reviewer #2: This manuscript present the high-throughput screening of a drug library to identify candidates for synergistic drug combinations for the treatment of retinoblastoma. The study is straightforward and clearly described and the manuscript is well written. However, I believe there are some (minor) issues that need to be addressed by the authors.

Aim of library screening

In the introduction, the authors clearly describe the goal of the high-throughput screening, to identify candidates that are both active for RB (cytotoxic to Y79 cells) and selective (non-toxic to RPE-1) with and without thermotherapy. However, after identifying candidates through the high-throughput screen, candidates are tested in drug combinations and the focus shifts towards multi-drug treatments and away from thermotherapy. If the goal is to identify candidates for multi-drug treatments, why not include combinations with carboplatin in the initial screen? And why use the delta HTS parameter for score if the downstream combination with carboplatin are performed at 37C? Also, authors introduce the drug library as a general drug library of approved drugs. But during hit selection, pharmacologic targets (line 305) are used as a criteria. So, why not only screen drugs with relevant pharmacologic targets? Or why exclude hits with no relevant target but identified through the screen? And it was little disappointing to found out at the end of the discussion that gemcitabine and platinum-based drug combinations are already used as effective treatments. The authors should address these issues and explain in more detail why certain choices were made. Furthermore, the authors should explain what the third round of screening entailed and why it was performed. And the scheme

Minor issues

- Line 170-171: Sentence repeated

- Line 237: Approved by who? Add to text

- Line 241-242: Please explain the scoring the main text. It is explained in the methods section, but this is important for the results section.

- Line 325: Are the authors comparing the observed in vitro screening results with other in vitro results? If so, please add reference. If there refer to actual clinical data, the authors should not compare in vitro screening data to clinical treatments.

- Line 533-549: A lot of speculation. The authors should tone down this section.

- I'm missing a conclusion section or conclusion paragraph at the end of the manuscript

Figures

- Figure 1b: Not informative, as selections can be stated as text

- Figure 3. FACS scatter plots look all very similar and numbers are not readable. Overall not informative. Edu images are counter to the bar plot, as it looks like, there is more fluorescent signal in the gemcitabine treated cells

Reviewer #3: Tseng et al. describe the results of a high-throughput screening campaign in retinoblastoma cells (Y79) for the purpose of identifying synergistic drug combinations and synergy with thermotherapy. The combination the authors focused on post-screening was carboplatin with gemcitabine. This combination demonstrated a favorable therapeutic window, reducing the required carboplatin dose by 15-fold while maintaining cytotoxicity in Y79 cells and sparing RPE-1 cells. The authors also perform mechanistic studies. In vivo studies using an orthotopic xenograft model of RB showed that intravitreal gemcitabine, alone or with systemic carboplatin, delayed tumor progression compared to vehicle with potentially less tumor infiltration. These findings highlight the potential of carboplatin-gemcitabine as a promising chemotherapeutic combination for RB patients.

Retinoblastoma is a challenging disease. I congratulate the group on their efforts to help push the knowledge of RB treatment forward. I think this study has merit but some experimental and statistical issues currently prevent a recommendation for publication without addressing the following points:

Major points:

1. The two cell lines were grown in different conditions (20% vs 10% FBS, and RPMI vs DMEM). It is unclear how much of the discrepancy in response is due to this. One recommended experiment is to condition both lines to the opposite condition to validate the hits in a 2x2 matrix. That these are suspension/spheroidical vs adherent cells should also be made clear and discussed as potential confounder.

2. RPE-1 are hTERT-immortalized cells. This wasn’t made clear in the paper and can have implications in the interpretation of the study as this line was used as reference to which Y79 data were compared. Importantly, describing such a line as “normal” is problematic; “non-cancerous” or “non-transformed” would be more appropriate.

3. The study bases all conclusions on only the Y79 line in both in vitro and in vivo studies. The authors should discuss this limitation in the paper.

4. Another HTS synergistic screen was conducted using Y79 cells previously by Mahida et al. (https://doi.org/10.1371/journal.pone.0059156). I could not find a mention of this work in this study. How does this prior study compare with the current study? were there consistent and inconsistent observations?

5. There seems to be no data presented on drug treatment and thermotherapy in vivo. Given the premise of the manuscript, how do these drugs or their combination perform as a chemothermotherapy in vivo?

6. Bar graphs in Fig2D, 2E, 3B, and 3D are difficult to read as the dots were made the same color as the bars, so it is difficult to see the distribution. A box plot may be more appropriate and easier to visualize here or change the dots into circles of a different color (e.g. black).

7. Most of the above mentioned bar graphs do not show p-values or statistical comparisons, making the figure difficult to interpret.

8. The study should evaluate the statistical significance of synergy in Fig2 analyses (It appears the paper is using SynergyFinder 1.0, but the p-value calculations are likely offered in newer versions: https://www.bioconductor.org/packages/release/bioc/vignettes/synergyfinder/inst/doc/User_tutorual_of_the_SynergyFinder_plus.html).

9. Line 422: This effect is not statistically significant from Fig 3D and should be stated clearly that there was no effect of the combination on EdU incorporation. Moreover, it is odd that the p-value is > 0.1 between DMSO and gemcitabine alone in Fig 3D. It appears this is an effect of only one outlier in the DMSO group. The location of the dots in gemcitabine also appear higher than what the bar graph is indicating (the dots are difficult to see). Taken together, conclusions based on the current results are problematic and require additional replicates given the variability. It’s also unclear what each dot represents (different fields of view in the same well? different wells? different experiment dates?).

10. In Fig 4, the optic nerve appears to be double the number of animals used likely because two optic nerves are being counted as independent experiments/samples from the same animal. This breaks the assumption of the Fisher exact test that observations must be independent. One solution would be to count the number of animals with any positive tumor infiltration in the optic nerve, or increase the number of animals which will allow a better study of brain infiltration as well.

11. In Fig 4, how does carboplatin alone compare to gemcitabine or the combination? This is a key control.

12. Line 528-531: The statement is somewhat misleading. The in vivo data shows that there was no statistical difference in survival between standard of care melphalan and/or gemcitabine with the combination of gemcitabine+carboplatin. The authors can make this clearer by breaking the discussion on survival and tumor infiltration into separate statements.

Minor edits:

1. Line 221: Spelling: cone-rod homeobox RNA

2. Figure 1: Spelling: Under ‘Second screened out’ box, the word ‘Highly’ is misspelled.

3. Figure 4: Clarity: The colors of the lines are very similar to one another. Perhaps different colors or dashed lines could be used instead.

Reviewer #4: Manuscript Number: PONE-D-24-22008

PLOS ONE

Research Article

“High-throughput screening of drug libraries identifies a new synergistic drug combination for the treatment of retinoblastoma”

The authors identify gemcitabine, a nucleoside analogue inhibitor of DNA synthesis, as a potential re-purposed retinoblastoma (RB) therapy when applied in synergistic combinations with thermotherapy and/or cytotoxic agent carboplatin. Gemcitabine was identified through HTS on a set of FDA-approved compounds and some 80 additional anti-cancer agents. Compounds were tested for efficacy on target RB (Y79 cell line) and toxicity in RPE-1 (normal retinal pigment epithelial) as a counterscreen. Follow-up mechanistic assays and efficacy testing in orthotopic xenograft mice models of RB add high value to this work. The paper is well-written, experimental design and reporting is sound, and figures/results are clear and effective. Great work! I recommend publication with no revisions.

Reviewer #5: - Fig 3A & B: It would be better if authors add the data for the negative control/ vehicle control used in these experiments

- Fig 3C & D: To be clear on the whole experiment, it is advised to add the carboplatin alone data to figure 3C and 3D

- It is advised to discuss about potential toxicity studies to evaluate any side effects and overall safety of the gemcitabine and carboplatin combination, particularly focusing on pediatric models to ensure relevance to the target patient population.

- While the potential benefits of combining carboplatin and gemcitabine with thermotherapy were suggested, detailed experimental data on the efficacy and safety of this combined approach in vivo were not provided. Are there any in vivo experimental models authors would like to test their suggested treatment options (chemotherapy+ thermotherapy) or would like to include in the discussion as a future direction of this study?

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: Markus de Raad

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

**********

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Dear Dr. Dyson,

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.

==============================

Following the review of your manuscript titled "High-throughput screening of drug libraries identifies a new synergistic drug combination for the treatment of retinoblastoma" I recommend that it should be revised taking into account the changes requested by the reviewer. Since the requested changes are minor, the revised manuscript will undergo a second round of review by the same reviewer.

==============================

Please submit your revised manuscript by Aug 24 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,

Mounir Tilaoui, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

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.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

Reviewer #5: All comments have been addressed

**********

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

Reviewer #2: Yes

Reviewer #5: Yes

**********

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

Reviewer #2: I Don't Know

Reviewer #5: Yes

**********

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

The PLOS Data policy

Reviewer #2: No

Reviewer #5: Yes

**********

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

Reviewer #2: Yes

Reviewer #5: Yes

**********

Reviewer #2: I would like to thank the authors for the responses to my questions and concerns. The authors made substantial changes to the manucript which improved the overall quality of the presented work.

However, there are still some minor issues that need to be addressed.

Line 279-281: "Out of the 38 identified drugs, the 11 which scored highest in the Y79 cell line in a third round of screening, with an HTS score 280 of over 0.9, were chosen for further investigation". The authors need to include the data for this third round of screening and should describe this third round of screening in the manuscript. Since the selections were based on the all 3 rounds, the data, results and outcomes of this third round of screening should be presented as findings in the manuscript. The given statement is not enough.

Reviewer #5: (No Response)

**********

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 #2: Yes: Markus de Raad

Reviewer #5: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org

Dear Dr. Dyson,

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. The manuscript had been significantly improved after two revisions, however there are still a few minor issues that have to be addressed. 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 Nov 21 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,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

1. 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.

2. 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.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #6: (No Response)

Reviewer #7: (No Response)

Reviewer #8: (No Response)

**********

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

Reviewer #2: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

**********

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

Reviewer #2: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

**********

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

The PLOS Data policy

Reviewer #2: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

**********

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

Reviewer #2: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

**********

Reviewer #2: (No Response)

Reviewer #6: Thank you for the opportunity to review your article on drug discovery in retinoblastoma and the candidacy of gemcitabine-carboplatin. I find that the methods and statistical analysis are strong and adequate to test your hypothesis. At this time I believe that there remains some revisions before this article is acceptable for publication. Please see my comments below.

Intro:

- 81-82) “carboplatin with etoposide with or without vincristine” what are the routes of administration (ROA) of these medications? Unclear if standard of care is systemic or intravitreal based on text.

- 90-101) no indication of ROA being intravitreal. Please make more clear what the in vivo experiments are testing and make sure it is reflected elsewhere in the article including abstract.

Methods:

- How was eye survival determined? Difficult to ascertain significance of this metric without this defined.

- This was addressed in discussion, but why only use one RB cell line for identification of active compounds in vitro? Does this not restrict generalizability?

- 135) 42 °C in the first hour and then transferred to incubator at 37 °C. — this is enough to mimic thermotherapy? Citation for this proof of concept?

Results:

362) carboplatin-vincristine ( avg of -2.98) clinically used but < 0 relates to an antagonistic effect and 417) carboplatin-vincristine demonstrated a strong antagonistic effect ( < -10).

- This was briefly explored in discussion but please elaborate. Does this finding contrast to prior literature? What implication does this finding have on your results if any? What are some explanations behind this?

Discussion:

633-635) grade 3/4 neutropenia and thrombocytopenia “are generally manageable”. “Manageable” does not seem to capture the gravity of this type of adverse event. Consider rephrasing and including a clinical citation.

Thank you again for the chance to review this compelling manuscript!

Reviewer #7: The article describes the process and results of screening a large number of chemotherapeutic agents to identify a drug that is at the same time cytotoxic to the tumour and gentle on RPE cells. I also find from the previous comments and clarifications given that the authors deliberately aimed at finding drugs that are inactive at normal temperature and become active only on thermotherapy. As a clinician I have the following comments to make.

1. In thermo-chemotherapy, thermotherapy is an addendum to the chemotherapy. The ability of the laser that is used to perform TTT (transpupillary thermotherapy) (to penetrate the tumour depth is limited, and hence large tumours cannot be heated with TTT throughout the extent of the tumour while chemotherapy can circulate to the entire extent of the tumour. Hence it is important that a chemo therapeutic agent should be active at body temperature as well as at higher temperature attained by thermotherapy.

In this respect the authors have been self-contradictory in their replies to previous reviewers.

Eg. The following is the explanatory note for reviewer 1 comment 1. ‘Since retinoblastoma patients are typically very young children and higher dose of systemic chemotherapy results in serious associated toxicity, we were looking for drugs that are inactive at body temperature and can be selectively activated at the tumor site.’ – Seems to clearly state that the intent of this experiment is to identify only drugs that are inactive at body temperature.

However, in the same version while replying to comments of reviewer 2, comment 1- the authors state ‘“Our overarching goal was to identify candidate drugs that could enhance or complement current chemotherapy regimens, either as monotherapies or in rationally designed combinations, with or without thermotherapy.”

Hence the stress by the authors should be only of identifying potential new chemotherapeutic drugs for retinoblastoma with or without added efficacy enhancement with thermotherapy.

Otherwise I have no specific comments on methodology, results and interpretation which have been extensively reviewed and corrected already.

Reviewer #8: This manuscript by Tseng et al. presents an HTS and validation of compounds synergistic for selective retinoblastoma toxicity. This is a revised manuscript, but this reviewer is seeing it for the first time. Overall it a really well written and well conceived manuscript. There are some minor technical issues that do not take away from the overall conclusions drawn from this work, but addressing these will further aid a reader’s overall understanding and reproducibility of the work described here.

1. how were the concentration ranges chosen for dose response assays (lines 158-163)?

2. does seeding RPE-1 cells with compounds already in the growth media affect attachment/morphology (line 138)?

3. was the switch from a 72 hr viability assay to a 5 day with the combination therapy necessitated by the switch from 384 to 96 well?

4. what was the seeding density in the 96 well assay (line 176), and why did your dosing strategy change with the combination from initial screening (line 178 vs 136)?

5. how were the Y79 cells growing that they could be stained and imaged in a 96 well plate (lines 200-208)? They can grow semi-attached but usually don’t stay that way through washes. It might be helpful to include arrows pointing to the EdU incorporated cells.

6. Does the lack of inhibition by clinically used carboplatin-etoposide combination seen in this synergy experiment point to any issues with the other conclusions drawn from this in vitro work? (line 414)

7. Figure legends: please include statistical test, n, and indications of what asterisks mean

8. Figure 3 title is an overstatement – the EdU analysis does not show “mechanism of action”. Please rephrase.

9. The authors use a variable slope model to analyse their dose response curves. How many data points did they have between 0.78 to 100uM (how many are needed to use variable slope vs standard slope?)

10. Please mention the post hoc test used for one way ANOVA (line 250)

11. It would be helpful if it were possible to include tumor/eye images for the in vivo study

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Reviewer #2: Yes: Markus de Raad

Reviewer #6: No

Reviewer #7: No

Reviewer #8: No

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<p>High-throughput screening of drug libraries identifies a new synergistic drug combination for the treatment of retinoblastoma

PONE-D-24-22008R3

Dear Dr. Dyson,

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. The authors perfectly addressed all reviewers' comments.

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #6: All comments have been addressed

Reviewer #7: All comments have been addressed

Reviewer #8: All comments have been addressed

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

Reviewer #6: (No Response)

Reviewer #7: Yes

Reviewer #8: Yes

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

Reviewer #6: (No Response)

Reviewer #7: Yes

Reviewer #8: Yes

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

The PLOS Data policy

Reviewer #6: (No Response)

Reviewer #7: Yes

Reviewer #8: Yes

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

Reviewer #6: (No Response)

Reviewer #7: Yes

Reviewer #8: Yes

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Reviewer #6: (No Response)

Reviewer #7: The article has already been extensively reviewed by multiple reviewers. The authors seem to have addressed all the queries raised. i believe the paper is ready for publication

Reviewer #8: The authors have effectively addressed all reviewer concerns, although the eye survival method details requested by reviewer 6 do not appear to be in the methods and should be added, as the authors mention in their Response to Reviewers.

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Reviewer #6: No

Reviewer #7: No

Reviewer #8: No

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