Dear Dr. Motsch,

Thank you very much for submitting your manuscript "Self-organization in brain tumors: how cell morphology and cell density influence glioma pattern formation" for consideration at PLOS Computational Biology. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. 

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, 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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Philip K Maini

Associate Editor

PLOS Computational Biology

Feilim Mac Gabhann

Editor-in-Chief

PLOS Computational Biology

***********************

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

[LINK]

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors propose a novel agent-based model (ABM) that simulates patterns of self-organization of elliptic/ellipsoid-shaped glioma cells in 2 & 3D. This is an exciting contribution and the authors are correct that studying the complex dynamics of such emergent flocks and (onco)streams – cells moving in the same/opposite directions - may potentially hold great promise for assisting in the development of innovative cytoreductive and cytotoxic therapeutic strategies, much needed for these extremely difficult to treat brain neoplasms.

Like any other model, this one has its limitations as e.g. the absence of biochemical factors reduces its levers to biomechanics, which is perfectly ok as long as one keeps this in mind when evaluating the results. For instance, the authors correctly emphasize that it is contrary to most concepts that an increase in cell density would lead to a reduction in flocks, and present this as an unexpected, noteworthy finding. Well – if cell-cell ‘avoidance’ is hard-coded in this ABM (pgs. 2 &3), wouldn’t one assume that cell ‘repulsion’ and/or change of cell directionality or ‘steering’ will become naturally more pronounced, particularly at higher N. The difficulty I am having with this is that real cancer cells are characterized by what is known as a loss of ‘contact inhibition’ … in other words, they exhibit the opposite of such ‘cell-cell avoidance’. It is precisely this feature that allows cells to form 2D-cell layer clusters in dishes and multicellular tumor spheroids in gels. In this context, the authors perhaps may find this paper an interesting read:

https://onlinelibrary.wiley.com/doi/abs/10.1046/j.1365-2184.2001.00202.x

For the sake of the argument, once the import of cell-cell avoidance is reversed and perhaps (in form of PDEs in a discrete-continuum extension of this work) one would introduce biochemical factors secreted by the agents/cells (autocrine, with paracrine impact) in response to microenvironmental triggers, cells at higher N [a non-explicit representation of either a replication capability of the agents (say depending on space-lattice availability in this biomechanical setting) or a dynamic Tumor growth term, N_t which are both missing here, say Gompertz or Logistic] would likely display more same-direction flock and less opposite-direction streaming behavior, particularly as N increases, which is realistic in a spatially confined area (3D tissue, skull); similarly, the impact of (different cell size/shape) ‘packing’ – an exciting extension of this work, correctly mentioned on pg. 13 - will likely be less pronounced in this cell-cell avoidance setting. Rather, one would think that securing cell outflow from a rapidly proliferating tumor surface is critical to manage the onset of central apoptosis and necrosis (see clinical MRIs with circular contrast enhancement) which in turn is to some degree a function of dwindling vascular support across the surface. In this 3/4D scenario, what would be interesting to study – with a model such as this one - is if directionality, steering angle & velocity/speed change as a function of the distance to the main tumor – with the hypothesis that the closer the individual cell is to the main tumor, in an area of metabolic constraint and reduced tissue carrying capacity, the more likely it is to follow others departing in the same direction (away from the tumor), with a straight angle at higher speed – while at some distance, in a nutrient-rich environment with less mechanical confinement through the growing main mass, sparsely seeded invasive cells could explore multi-directionality and angles with higher degrees of freedom to forage (– analogous to complex systems in social insects like ants). This would likely result in fractal like invasive patterns that we have seen in gels and that (probably reinforced by fractal-like neovascular architecture) have been noted for real tumor boundaries in the past (see e.g. works by Cross S.S.).

In summary, to put the claim of this work i.e. having clinical potential into perspective, I would like to encourage the authors to discuss key limitations of their current work, both in setup and results, in more detail.

Finally, as a minor item, in Figure 1B, it appears that the blue labels being used to denote ‘cells’ – but this is an H&E staining and convention is that it depicts cell ‘nuclei’ in blue, cell bodies in pink. Perhaps I misunderstood, but worth clarifying.

Reviewer #2: The authors study an important question in tumor biology in general and glioma in particular. They investigate the effects of cell shape and density on organization and direction of movement. The model is elegant and the results are interesting. The presentation and Figures are clear. I have minor suggestions/questions:

1. Did the author observe the existence of streams in experimental data? Figure 1 is static; though some cells are elongated, the direction of movement can not be determined.

2. The dynamic behavior of circular cells is not surprising as it is predetermined by the equations; see page 4, lines 106-109. I suggest mentioning this point in the discussion.

3. I suggest combining Figures 10-12 into a single Figure.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: Thomas S. Deisboeck, MD, MBA

Associate Professor of Radiology

Massachusetts General Hospital

Harvard Medical School

Reviewer #2: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit 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. For instructions see http://journals.plos.org/ploscompbiol/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Motsch,

We are pleased to inform you that your manuscript 'Self-organization in brain tumors: how cell morphology and cell density influence glioma pattern formation' has been provisionally accepted for publication in PLOS Computational Biology.

Before your manuscript can be formally accepted you will 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.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology. 

Best regards,

Philip K Maini

Associate Editor

PLOS Computational Biology

Feilim Mac Gabhann

Editor-in-Chief

PLOS Computational Biology

***********************************************************