PONE-D-20-19549

Antigen-agnostic microfluidics-based circulating tumor cell enrichment and downstream molecular characterization

PLOS ONE

Dear Dr. Cohen,

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.

Normally I require at least two reviews; however, I respect this review and they have very good points.  Please attempt to address them.

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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

Reviewer #1: I Don't Know

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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

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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

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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: Development of an antibody-independent platform for CTCs is highly desirable and this reviewer commends the authors and company.

Since the work is intended to be incorporated into a clinical lab workflow, some comments should be made on the integrity of the PCR assays over time due to amplicon containment requirements.

Other issues from the analytical and operational aspects of the platform itself should be commented on, specifically:

1. deformation and capillary flow required by the platform should be expected to result in lysis of some patient CTCs. How are these phenomena represented in the recovery data? is this the source of spiked cell loss?

2. please expand on the concept of gap size. why is there a decreased recovery with increased gap size?

3. for the hybridization data, I read the manuscript as saying 4 spiked specimens assayed in duplicate for each cell line to generate the data. is that correct?b Can the recovery data for all experiments be pooled to generate a recovery regression model and equation related to spike level?

related to this, figure 1b shows that recovery is cell line dependent. is recovery cell size dependent?

4. In figure 3, are the spiked cell numbers per 5 mL or 1 mL of blood, please state. Also please specify the elapsed time between harvest, spiking and purification.

5. after purification, what is the optimal method of cell preservation prior to qRT PCR analysis?

6. in figure 3C, the SKBR3 10 cell spike recovery level varied over 6 CT values. please comment.

7. This manuscript should include data on performance of this method compared to the cell search on a small panel of human specimens.

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Reviewer #1: No

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Antigen-agnostic microfluidics-based circulating tumor cell enrichment and downstream molecular characterization

PONE-D-20-19549R1

Dear Dr. Cohen,

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,

Jeffrey Chalmers, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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

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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

**********

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

Reviewer #1: 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

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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

**********

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: Thankyou for addressing the points raised in the original review.

Two points to consider going forward:

1. At present, it is not clear exactly what a patient specimen report might look like and what role, if any, quantitation might play in either patient diagnosis or management during therapy. It will be interesting going forward to see if this method overcomes the limitations frequently associated with ctDNA analyais, specifically the fraction of specimens in which there is adequate analyte for statistically significant analysis.

2. Application in monitoring to response to therapy can use at least two different approaches: characterization of changes in CTC phenotypic distribution (EMT status) and how that might be quantified, vs monitoring tumor burden itself as a way to determine therapeutic success. It is not yet clear which approach this method is best suited to.

However, this study lays the groundwork for addressing those important questions.

The major reservation this reviewer sees is determining the minimum cell number in patient specimens that can be analyzed and reported with statistical significance, a problem common to all CTC platforms.

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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: Robert J. Kinders