Dear Dr Xiong,

Thank you for submitting your manuscript entitled "Cell2Spatial: precisely deciphering spatial transcriptomic spots at single-cell granularity" for consideration as a Methods and Resources Article by PLOS Biology. Please accept my sincere apologies for the delay in getting back to you as we consulted with an academic editor about your submission.

Your manuscript has now been evaluated by the PLOS Biology editorial staff, as well as by an academic editor with relevant expertise, and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. After your manuscript has passed the checks it will be sent out for review. To provide the metadata for your submission, please Login to Editorial Manager (https://www.editorialmanager.com/pbiology) within two working days, i.e. by Jun 06 2025 11:59PM.

If your manuscript has been previously peer-reviewed at another journal, PLOS Biology is willing to work with those reviews in order to avoid re-starting the process. Submission of the previous reviews is entirely optional and our ability to use them effectively will depend on the willingness of the previous journal to confirm the content of the reports and share the reviewer identities. Please note that we reserve the right to invite additional reviewers if we consider that additional/independent reviewers are needed, although we aim to avoid this as far as possible. In our experience, working with previous reviews does save time.

If you would like us to consider previous reviewer reports, please edit your cover letter to let us know and include the name of the journal where the work was previously considered and the manuscript ID it was given. In addition, please upload a response to the reviews as a 'Prior Peer Review' file type, which should include the reports in full and a point-by-point reply detailing how you have or plan to address the reviewers' concerns.

During the process of completing your manuscript submission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Richard

Richard Hodge, PhD

Senior Editor, PLOS Biology

rhodge@plos.org

PLOS

Empowering researchers to transform science

Carlyle House, Carlyle Road, Cambridge, CB4 3DN, United Kingdom

California (U.S.) corporation #C2354500, based in San Francisco

Dear Qing,

Thank you for your patience while we considered your revised manuscript "Cell2Spatial: precisely deciphering spatial transcriptomic spots at single-cell granularity" for publication as a Methods and Resources article at PLOS Biology. Please accept my sincere apologies for the long delays that you have experienced during the peer review process. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and the three of the original reviewers at the previous journal.

Based on the reviews, I am pleased to say that we are likely to accept this manuscript for publication, provided you satisfactorily address the remaining points raised by the reviewers. In addition, please also make sure to address the following data and other policy-related requests that I have provided below (A-G):

(A) We routinely suggest changes to titles to ensure maximum accessibility for a broad, non-specialist readership. In this case, we would suggest a minor edit to the title, as follows. Please ensure you change both the manuscript file and the online submission system, as they need to match for final acceptance:

“Cell2Spatial is a computational framework that maps single cells to spatial transcriptomic spots to reconstruct tissue architecture”

(B) We note that your financial disclosure statement in the online submission form says that this study was not supported by any specific funding. Please provide additional details about any funding received to conduct the study, including the names of the funding agencies and grant numbers.

(C) You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

-Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

-Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:

Figure 2D-E, 2G-H, 3A-F, 3I-L, 4D, 4G, 4I, 5C, 5G, 6C, 6F-H, 7B, S1B-C, S2E-H, S3D, S4G-H, S5D, S5F, S6B, S7E-H, S9A-F

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

(D) Please also ensure that each of the relevant figure legends in your manuscript include information on *WHERE THE UNDERLYING DATA CAN BE FOUND*, and ensure your supplemental data file/s has a legend.

(E) Thank you for depositing the full analysis code in Github (https://github.com/lihuamei/Cell2Spatial.Reproduce). However, please note that we cannot accept sole deposition of code in GitHub, as this could be changed after publication. However, you can archive this version of your publicly available GitHub code to Zenodo. Once you do this, it will generate a DOI number, which you will need to provide in the Data Accessibility Statement (you are welcome to also provide the GitHub access information). See the process for doing this here: https://docs.github.com/en/repositories/archiving-a-github-repository/referencing-and-citing-content

(F) Please ensure that you are using best practice for statistical reporting and data presentation. These are our guidelines https://journals.plos.org/plosbiology/s/best-practices-in-research-reporting#loc-statistical-reporting and a useful resource on data presentation https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002128

- If you are reporting experiments where n ≤ 5, please plot each individual data point.

(G) Please ensure that your Data Statement in the submission system accurately describes where your data can be found and is in final format, as it will be published as written there.

------------------------------------------------------------------------

As you address these items, please take this last chance to 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 cover letter that accompanies your revised manuscript.

In addition to these revisions, you may need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests shortly. If you do not receive a separate email within a few days, please assume that checks have been completed, and no additional changes are required.

We expect to receive your revised manuscript within 1 month.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable, if not applicable please do not delete your existing 'Response to Reviewers' file.)

- a track-changes file indicating any changes that you have made to the manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://plos.org/published-peer-review-history/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. 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

Please do not hesitate to contact me should you have any questions.

Best regards,

Richard

Richard Hodge, Ph.D.

Senior Editor, PLOS Biology

rhodge@plos.org

------------------------------------------------------------------------

Reviewer remarks:

Reviewer #1: The authors have successfully addressed all of my concerns. I have no other comments.

Reviewer #2: The revised manuscript is a significant improvement, and the authors have addressed my major concerns. I have some minor comments:

L23: "Several methods have been publicly disclosed. One common approach, like Seurat [18]"

Seurat is an R package with lots of methods. Can you be more specific?

L36: "In the case of low-resolution ST data (such as Spatial-based data)"

Isn't all ST spatial-based?

L68: "Spatial context is incorporated using Seurat's canonical correlation analysis (CCA) [18], which produces a Euclidean distance matrix from the Uniform Manifold Approximation and Projection (UMAP)"

Doesn't it use the PCA, not the UMAP?

"For high-resolution ST data, where spots typically capture single cells, it assigns a cell count of 1 per spot."

Both STOMICs and VisiumHD are sub-cellular resolution. Are the spots being binned to be approx. single cell sized? I would also be interested to know what the future development plans are for sub-cellular resolution datasets.

There are still some improvements that could be made when asserting claims about cell2spatial's performance compared to other tools in the results section of the manuscript. For instance the addition of a % performance difference/increase between tools, or quoting the Cell2Spatial metric and the range in the other tools (as the authors have done in the response to reviewers) could suffice.

L110, L128 (Only Jaccard index is quoted for Figure 2?), L153, L164-6 (how compares with the other tools?), L175-7 (other tools?), L194 (other tools?) etc.

L798: Dropbox link? Is there a better repository for this?

Reviewer #3: I am pleased to see that the authors have addressed most of the previous concerns. The additional analysis provided by the author strengthens the paper's claims. Overall, I have a positive view of the paper and support its publication. However, I suggest a few minor revisions are needed before the manuscript is finalized.

1. To address the previous comment from Reviewer 1. the authors have incorporated a comprehensive benchmarking study in their revised manuscript, citing Li et al., 2022. They have used their own simulated spatial transcriptomics (ST) datasets to evaluate the performance of Cell2Spatial and other tools. While this is a valuable exercise, the Li et al. paper provides its own set of benchmark datasets (already published). To further validate their claims of superior performance and provide a more direct comparison to the published literature, it would be highly informative to simply apply Cell2Spatial on those datasets and see how it performs on the publicly available benchmark datasets from the Li et al. study. I would recommend that the authors run their tool on these datasets and include the results in the manuscript.

2. Figure 2H, Consistency Result: The box plot shows the Jaccard index for single-cell mapping accuracy at different noise levels. Firstly, it is counterintuitive that for some tools, like CellTrek, the consistency seems to increase as the noise level increases. This could suggest that the consistency results are also subject to a high degree of randomness and using only one dataset is not enough for a consistent comparison. Additionally, the Jaccard index values for all tools are quite low, with the highest mean index being less than 0.20. The author should provide some comments/discussions on these phenomenon.

3. Figure 3H, Lower-Left Panel: The lower-left panel of Figure 3H, which displays the cell counts in a whole mouse brain ST slice using Cell2Spatial, shows a large contiguous area with the same cell count. The authors should provide an explanation for this phenomenon and exclude those spots from downstream analysis if appropriate.

4. Typo in Figure 1: In the upper-right portion of Figure 1, within the "Single-cell RNA-seq (SC) data" section, there is a typo in the legend. The "Cell Type" list shows "Type4" twice. This should be corrected for clarity.

I am confident that addressing these points will further enhance the quality of the manuscript and its contribution to the field.

Dear Qing,

On behalf of my colleagues and the Academic Editor, Selene Fernandez-Valverde, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

IMPORTANT: We noted during our routine checks that the Data Availability Statement states that the structured spatial and corresponding single-cell data is deposited in a Google Drive (https://drive.google.com/file/d/13D9k1rb47XA7MBbpEcTgwxAlCXNEvdn-/view?usp=drive_link). Please note that we cannot accept deposition of data in a Google Drive and we ask that this data is deposited in the Zenodo database or similar. Due to the upcoming grant application, we have decided to move forward to editorial acceptance on your study, but I would be grateful if you could please move this data to an official data repository during the production process. Please update the Data Availability Statement in the online submission form and the manuscript file to reflect this.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Best wishes, 

Richard

Richard Hodge, PhD

Senior Editor, PLOS Biology

rhodge@plos.org

PLOS

Empowering researchers to transform science

Carlyle House, Carlyle Road, Cambridge, CB4 3DN, United Kingdom

California (U.S.) corporation #C2354500, based in San Francisco