PONE-D-22-31029Immunostaining protocol for infiltrating brain cancer spheroids for light-sheet imagingPLOS ONE

Dear Dr. Jauffred,

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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Michelle R. Dawson

Section Editor

PLOS ONE

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7. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

​

Please revise your manuscript to address the reviewers comments. Also, please review your submission carefully to eliminate any typos or grammatical errors.

I look forward to receiving your revised manuscript.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: Yes

Reviewer #2: Partly

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3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

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4. If the manuscript contains new data, have the authors made this data fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the article 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 highlight any specific errors that need correcting in the box below.

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 submitted manuscript describes a very useful technique for staining and imaging infiltrating brain cancer cells using a spheroid model. It is easy to imagine that this technique could be utilized for models of various types of infiltrating tumors. It would be useful, but may not be strictly necessary to have some quantifiable benchmarks associated with the protocol - what percentage of surface cells were stained using this method, and what percentage of those cells are expected to infiltrate the Matrigel?

The speculation that as the cells migrate from the tumor surface, the cells underneath will then repeat the process of vim expression and infiltration is an interesting one, but is this something that the authors have been able to validate in any way? Would this require another round of staining or is this something that could be easily observed in the same assay based on the penetration of the vim staining within the spheroid? An answer to this question could be rather impactful in terms of the application of this technique.

The manuscript could use some slight editing for grammar and the protocol has some additional characters (????) that don't influence the method but add some confusion.

Reviewer #2: This manuscript described a protocol for immunostaining of glioblastoma spheroid for two laser light sheet imaging and proved the utility of this protocol by showing a distinct surface staining of vimentin on spheroid and the protrusion of vimentin in migration assay. However, as a protocol article, the current version didn't provide the readers enough detail of the experiment, and there are some confusions in the protocol. I highly recommend the authors carefully organize the protocol part of this article make it really useful to the community. And my suggestions are as follows.

1. what are those question markers in section "cell culture of primary specimen"

2. Replace all the "agent" to " reagent"

3. in the fixation reagent section there is description about tips of how to wash cells. Where in the Procedure section these tips should be applied to? Is in the "Immunostaining spheroids through hydrogel"? However, there are different description in the "Immunostaining spheroids through hydrogel" fixation section. Which one the readers should follow? It is really confusing!

4. in the Cell Culture section, please specify the number of cells and size of culture dishes. Not just use the description"small culture plate"

5. In Gravitation-assisted spheroid formation what is the sentence "The spheroids can be immunofluorescence stained directly using the procedure in section Immunostaining surface-attached

cells" meaning?

6. In "Imaging spheroids in hydrogel", why the authors put plate on ice to liquefy the hydrogel matrix, isn't matrigel already dissolved in PFA? And can the authors please add a schematic cartoon to show how to set up the imaging tube?

7. In "Immunostaining surface-attached cells", why wash the cells with cold PBS, doesn't that detach the cells? And please specify the time of washes.

8. For primary antibody staining, what time and temperature of incubation the author was using for Vim1? Because in the data part, authors mentioned they have tried 1 hour and 3 hour and no difference was observed. Please make it clear in the protocol and make sure consistent with the data description.

9. For the Data part, in figure 3C, it is hard to understand the way of quantification of the intensity based on the schematic the authors provided.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-22-31029R1Immunostaining protocol for infiltrating brain cancer spheroids for light-sheet imagingPLOS ONE

Dear Dr. Jauffred,

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 Feb 25 2023 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,

Michelle R. Dawson

Section 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.

Additional Editor Comments (if provided):

Thank you for addressing the main concerns in the reviews. A few minor concerns remain. First, in the description of isolated cancer cells it is unclear whether this is human subjects research or not, if it is human subjects research an IRB approval would be required and that should be stated. Otherwise, the author should use the appropriate language to show that is not human subjects research. In addition, the reviewers had some concerns with the grammar and writing. I noticed some portions of this protocol are still very conversational. Please read it once more and correct any colloquial language since this is a scientific paper. After these small issues are addressed, I would be happy to accept your protocol.

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Reviewers' comments: N/A

[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.

Immunostaining protocol for infiltrating brain cancer spheroids for light-sheet imaging

PONE-D-22-31029R2

Dear Dr. Jauffred,

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.

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Kind regards,

Michelle R. Dawson

Section Editor

PLOS ONE