PONE-D-21-09835

Guiding cell adhesion and motility by modulating crosslinking and topographic properties of microgel arrays

PLOS ONE

Dear Dr. Sechi,

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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PLOS ONE

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2. Thank you for stating in your Funding Statement:

This work was partly supported by the Center for Chemical Polymer Technology (CPT), which was supported by the EU and the federal state of North Rhine Westphalia (grant EFRE 30 00883 02). AP thanks the financial support of the Deutsche Forschungsgemeinschaft (DFG) of the Collaborative Research Center SFB 985 “Functional Microgels and Microgel Systems”. The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Additional Editor Comments:

When preparing your revised version, please pay special attention to the critical comments of reviewer 3. In particular, comment on the concerns regarding possible differences in cell adhesive protein adsorption, which could indeed affect the observed differences between arrays and controls. Also, please discuss more critically if differences between arrays are significant or if the presence of the arrayed microgels is the primary effect. This will be important for future optimization and possible applications.

In line with the PLOS data sharing policy, please also provide clear links connecting the provided supplementary data with the figures (e.g. in the figure legends). If not already done for all figures, please also provide all mean/median values used to prepare the box plots as well as all values used to perform the statistical tests (not only their outcome).

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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: No

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #2: Yes

Reviewer #3: 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: This manuscript describes the patterning of hydrogel microparticles and the response of cell spreading to the corresponding interfaces. The impact of the resulting patterns on the spreading and motility of Sertoli cells and Gas2L1 KO cells was characterised. The dynamics of focal adhesions was then studied. The authors then switch to the study of B16F1 cells for the study of the impact of microgel size on cell adhesion, motility and adhesion turnover. Finally, the impact of microgel mechanics was studied. Such phenomena and the impact of regulators of actin assembly on sensing of such topographies are interesting, but important aspects are not properly characterised or insufficiently detailed. The following should be addressed prior to considering this manuscript any further:

1. Understanding factors regulating the sensing of nanotopography is important, but it is not clear why select Gas2L1 specifically. Many other regulators of actin assembly or FA regulators could have been picked up.

2. In addition, there are other platforms accessible to regulate nanoscale geometry and topography. It is not clear why this specific microgel platform was selected and how it allows to solve unanswered questions on the role of Gas2L1. Beyond the fact that Gas2L1 regulates motility, a fact previously established, this study does not demonstrate further roles for this player in topography sensing.

3. Similarly, it is not clear why the two cell models selected are particularly appropriate to study the impact of microgel pattern topographies on cell adhesion. It is also odd that some of the experiments are carried out only with B16F1 cells when most of the study focuses on Sertoli cells. If the aim of the study is to mimic topographical features present in the matrix, why not carry out all experiments in the same cell type. The argument presented, that clearer trends could be observed breaks down since no real trend correlating motility or FA turnover with microgel topography (and size) is observed. Given that all other experiments were carried out with Sertoli cells, studying the impact of microgel size on motility and FA turnover seems important.

4. The differences observed in terms of cell motility and FA turnover are proposed to result from differences in matrix. Clearly cells sense and align to the patterns generated, but this was previously reported. However, differences observed between glass, films and arrays (patterns) could arise from differences in cell adhesive protein adsorption. I could not find details of any specific protein adsorption or biofunctionalisation with peptides, therefore I assume that this was not controlled. This aspect should be investigated for the different substrates studied (including differences in microgel diameters and stiffness).

5. In Fig 6. FA assembly seems to be differentially regulated by Gas2L1 expression when cultured on glass, but not when cultured on films or arrays. Trends (relative rates of assembly/disassembly of FAs) are overall very similar for WT and KO lines. This does not support the notion that Gas2L1 is mediating the sensing of the microgel properties. It is not clear what properties are particularly targeted either in this study (topography/size of microgels, chemistry, mechancis).

6. In Fig 7, the mobile fractions measured for the KO cells are lower than for WT, on glass. However, in Fig 6, the assembly and disassembly rates of FA are faster. The two observations seem to be contradicting.

7. In Fig 9, trends are proposed for the cell response to different microgels. E.g. "their average speed and directionality were greatly reduced on 800, 1200 and 1600 nm arrays". Considering that there is no apparent statistical trend (the averages do not even follow a clear trend when the microgel size was varied from 300 to 1600 nm), any notion that the microgel size (and associated topography?) is regulating cell adhesion and motility should be removed from this manuscript. The only clear difference observed is between glass/film and arrays. But again, this breaks down when analysing the impact of substrate topography/patterning on FA assembly/disassembly.

8. No statistical analysis results are reported in Fig 9.

9. What is the actual stiffness of the microgels used in Fig 10? This should be measured.

10. Conclusions made in the discussion, specifically (but not only) "the migration of B16F1 cells is inversely correlated with microgel array spacing" are not supported by the data presented. This needs to be carefully rewritten. Similarly "In this context, we have developed a (...) cell adhesion and migration". I do not see evidence that the system presented allow to modulate actin cytoskeleton architecture. There is no control in the phenotypes observed and no clear trends when correlating spacing and migration/FA dynamics are observed. The cytoskeleton architecture has not been systematically studied or characterised.

11. The conclusion that "the variation of microgel array topographic and mechanical features can efficiently be used for the modulation of cell adhesion and motility" is misleading. There is no real control achieved (which would be evidenced by trends with microgel size for example). The only trends observed are the response of cells to films and arrays compared to glass. But this could be the result of differences in adhesive protein adsorption promoting differences in cell adhesion.

12. Fig. 7. It would be useful to show some of the normalised fluorescence intensities prior to photobleaching in the traces presented.

13. The manuscript is sometimes vague and the language used could be more specific. For example, "Focal adhesion dynamics is also modulated by microgels". What properties of microgels? Or, "the kinetics of the focal adhesion protein zyxin is decreased". The kinetics of which phenomenon? Presumably recruitment and disassembly. Similarly, "whereas focal adhesion speed was clearly reduced". This is presumably the FA formation rate, or the FA turnover.

Reviewer #2: Riegert et al investigated how surface bound microgel arrays affect cell adhesion and migration. They controlled and varied the spacing of patterning and degree of crosslinking and showed that cell adhesion and migration are indeed affected. Greater spacing led to increased cell polarization and migration directionality. The author further showed that this is directly correlated with the focal adhesion dynamics. This is a very interesting study, and I find the article generally well written and conclusions directly supported by the observation. I have a few comments:

Fig 2 – are the second column a line cross-sectional profile or is it projected vertically? In A, the periodicity is very clear, but seems to be diminished to noise in B. Further, in C, the microgel is much narrower than the surface, but the line profile in D seems to suggest they’re about the same width. The authors should indicate where the cross-section is drawn on the first column that produced the profile on the second column.

In the current design, both the gel with and the gap width seems to be increasing at the same time. So, is it the microgel array width or their gap that has the stronger effect on their influence to cell adhesion? For instance, keeping the gels at 300nm width, but spacing hem 1600 nm apart, would the authors expect to see similar result as gel/gap width = 300/300 or 1600/1600?

What is the mechanism behind the cells’ alignment in the direction of the pattern at 300nm (Fig 8A)? Given the authors stated that the surface swelling made it behave like a connected sheet. How does the cell get the cues to polarize in this context along the patterns. While the authors stated that the molecular mechanism is outside the scope of the current study, can they provide some reasonable interpretations and what they hypothesize might be the cause?

Fig 9 C, D doesn’t seem as significant as authors stated. For instance, in Fig 9C, film, 800, 1600nm doesn’t seem to be different. Similarly, glass, 300 and 1200 do not seem that different. How many independent experiments did the authors perform, and how was the variation between experiments? Please include statistical test between the necessary groups stated in the manuscript.

Reviewer #3: In this work, Sechi and coworkers developed surface-supported microgel arrays featuring different spacing and elasticity and investigated the effect of such topographic and mechanical cues on the adhesion and migration of different cell types. Building up on previous research from their group, the authors optimized the fabrication method of the biomaterial by varying the microgel size and crosslinking degree to control the spacing between adjacent arrays as well as the materials mechanical strength. After characterization of the synthesized microgels and the printed microgel arrays, the authors used this platform to study the response of cells (B16F1 and Sertoli cells) in terms of adhesion and migration in comparison to cells cultured on glass and microgel films. Sertoli cells were found to elongate and align their morphology, actin cytoskeleton and focal adhesions in respect to the orientation of the arrays. Additionally, the rate of motility and directionality were investigated, and a more pronounced impact of microgels on the migration of Gas2L1 KO vs. wild-type Sertoli cells was found, while directionality was not significantly impacted. Microgels topography and its stiffness was as well found as effective means of modulating focal adhesion dynamism. Regarding B16F1 cells, their migration average speed was reduced on microgel topography, and showed dependence on the array spacing (with a lower limit of control found at 300 nm spacing), reflecting differences in focal adhesion dynamics. The findings of this work are useful to understand the influence of different design parameters of microgel culture platforms on cell functionality. In a future perspective, these concepts could guide the development of implantable scaffolds with controlled cell adhesion and migration, to support wound healing and tissue engineering.

This is a technically sound investigation: the aim of the work is clearly formulated, the experiments are competently performed, the analysis of different parameters on cell response is exhaustive and the results are well presented. The findings are interesting, the conclusions are supported by the data and the overall quality and clarity of this manuscript are very good. Some experimental details are missing, and some typos need correction, all aspects that can be easily implemented. I positively support this article and recommend the acceptance for publication in PLOS One after minor corrections noted (see below)

1- Experimental Section: some important missing details should be included:

- The authors mention the “microgel films” (Page 10) as one of the control substrates to be compared to the “microgel array”. However, the preparation of the microgel films is not described in the Exp. Section. Are they prepared similarly to the microgel arrays, except that the printing part is skipped? Please, complete this part for clarity.

- Page 6, line 120: “Microgels were subsequently purified by dialysis for several days”. How many days were needed typically? Please, inform average purification time.

- Page 8, formula to calculate the degree of microgel swelling: Please, define the different terms or magnitudes of the equation at first mention. In connection to that, how were the swelling degree and the volume phase transition temperature measured? I suggest mentioning it briefly, even when they are shown in the Supp. Info.

- Page 25, legend of Fig. 7, lines 554-555: “The thin lines above and below the thick curves indicate the S.E.M.” Please, define “S.E.M”.

2- There are some typos to correct in the text:

- Page 4, line 90: “regulate” instead of “regulated”

- The authors vary between “cross linking” and “crosslinking” throughout the text. Please, unify style. Same for “cross linker”, “crosslinker” and “cross-linker”.

- Supporting Information: “FTIR spectra” instead of “FITR spectra”.

- Page 25, line 565: “chose” instead of “chosen”.

- Page 31, line 715: “ratio” instead of “ration”.

3- My last comment is more a suggestion to the authors. With the investigation of design parameters (spacing and stiffness) on diverse cell responses (morphology, cytoskeleton and focal adhesion alignment, focal adhesion turnover, migration rate and directionality, etc), across different substrates (glass, microgel array, microgel films) and cell types (B16F1 cells, and wild-type vs Gas2L1 KO Sartoli cells), at some point the reader may get lost with so many data. I believe that a clear summary of the different findings at the end of the discussion section will be very appreciated by the reader and will nicely wrap up the main findings. This could be implemented, for example, in the form of a table that compares which factor has a significant influence on a given response.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-21-09835R1

Guiding cell adhesion and motility by modulating cross-linking and topographic properties of microgel arrays

PLOS ONE

Dear Dr. Sechi,

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 Aug 07 2021 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: http://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,

Kerstin G. Blank

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Reviewer 1 is now more positive about the manuscript. However, he stongly suggests to adapt and rewrite some of the conclusions. I agree with this opinion. Please ensure that the conclusions about the cellular response to the microgels are backed up by the statistical analysis. In general, I strongly recommend to include the responses to this reviewer in the manuscript or SI as the reader may have similar questions.

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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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: 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: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

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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

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #2: Yes

Reviewer #3: 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: Reviewer: The authors have addressed some of the comments raised, but left some of the most important comments regarding the validation of some of their conclusions. Despite the replies made, the associated text and discussion were not corrected appropriately. I still disagree with these conclusions and do not recommend publication of this manuscript unless these aspects are fully and appropriately addressed.

For clarity, I am attaching a pdf of my comments.

5. In Fig 6. FA assembly seems to be differentially regulated by Gas2L1 expression

when cultured on glass, but not when cultured on films or arrays. Trends (relative rates

of assembly/disassembly of FAs) are overall very similar for WT and KO lines. This

does not support the notion that Gas2L1 is mediating the sensing of the microgel

properties. It is not clear what properties are particularly targeted either in this study

(topography/size of microgels, chemistry, mechanics).

In this part of the study, we have not analysed specific microgel properties but the

impact of microgels on FA dynamics in control cells and cells lacking Gas2L1. We

clearly show that microgels are effective in regulating the motility and adhesion of

Sertoli cells. Furthermore, FA assembly and disassembly rates are both reduced in

Gas2L1 KO cells compared to control cells on microgels arrays and films (see suppl.

Table S4) indicating that Gas2L1 is somehow involved in sensing of microgel

substrata. Obviously, we do not know, at this stage, the molecular mechanisms for

this process that will be investigated in future studies.

R: I am not disputing the fact that microgels regulate the directionality of cell motility. I am disputing the proposed role of Gas2L1 on this process. In Fig 5 (A-D), cells display high speed on the array compared to glass or film substrates, whether they are WT or KO, although the speed of KO cells is overall reduced. The trend in directionality is also the same. This is also mirrored by the identical trends observed in Fig 6 (assembly and disassembly rates, and FA speed, are reduced on films compared to arrays, both for WT and KO). Therefore, I conclude that, although Gas2L1 has an impact on cell adhesion and motility, it does not regulate the sensing of the topography. This needs to be corrected (and highlighted) in the text, abstract, intro, discussion and conclusion.

Interestingly, such effect is more prominent in Sertoli cells harboring a knockout of Gas2L1, a component of the cytoskeleton that mediates the interaction between microtubules and microfilaments. Moreover, on microgel arrays, the kinetics of the focal adhesion protein zyxin is decreased in wild-type and increased in Gas2L1 KO Sertoli cells. Finally, increasing microgel cross-linking causes a stronger reduction of focal adhesion turnover in Gas2L1 KO cells.

"we further reasoned that the present microgel system could be used to preferentially modulate the migration of wild-type or Gas2L1 KO Sertoli cells. A correct hypothesis would result in a clear difference in the rate of motility between the two Sertoli cell lines with the Gas2L1 cells migrating faster than wild-type cells."

R: P21. L466. Again, I am not disputing that the WT and KO migrate at different rate, they do. But they respond in a similar way to microgels and their topography, compared to glass. Therefore, the microgel system is not preferentially modulating the migration of WT or KO cells. Gas2L1 has an impact on migration, independent of these substrates.

"A closer analysis of the data showed that the ratio between the average speeds of Gas2L1 KO and wild-type cells was higher for cells on microgel films and microgel arrays than for cells on glass coverslips (S3 Table), clearly showing a more pronounced impact of microgels on the migration of Gas2L1 KO Sertoli cells."

"Conversely, in cells on microgel arrays, focal adhesion assembly was significantly reduced only in Gas2L1 KO cells (Fig. 6A-B, D-E)."

"It is important to note that the ratio between the assembly rate of Gas2L1 KO and wild-type cells was much smaller in cells on microgel films (S4 Table). The ratios for focal adhesion disassembly rate and speed followed a similar trend (S4 Table). Similar comparisons also showed that the focal adhesion size ratio was higher for cells on microgel arrays, whereas focal adhesion life span ratio was higher for cells on microgel films (S4 Table). Collectively, these findings demonstrate that the surface-grafted microgels can be used as an effective system to modulate focal adhesion dynamics in Sertoli cells and have a larger impact on Gas2L1 KO cells."

" Furthermore, the ratio between the mobile fractions of zyxin in wild-type and Gas2L1 KO cells was increased in cells seeded on microgel arrays (S5 Table). Thus, microgel arrays preferentially modulated zyxin kinetics in Gas2L1 KO cells, thus serving as an effective tool for highlighting differences of focal adhesion behavior between genotypically diverse cell types."

R: We noted the effort to characterise ratios between migration rates, directionality, rates of assembly/disassembly etc. between KO and WT and compare their ratios, but note that, as stated above, the overall trends remain unchanged and there is no indication that the ratios reported in Tables S3-S5 demonstrate quantitative differences in the response of KO and WT cells to patterns. Considering the standard deviations, the min/max ratios (calculated from minimising/maximising the corresponding values before calculating min/max ratios) are significantly overlapping.

Overall, the role of Gas2L1 on cell migration is clear, but its role on sensing topography of the microgels studied is not. This should be corrected in the manuscript throughout and clearly stated.

6. In Fig 7, the mobile fractions measured for the KO cells are lower than for WT, on

glass. However, in Fig 6, the assembly and disassembly rates of FA are faster. The

two observations seem to be contradicting.

The mobile fraction of zyxin reflects its dynamic behaviour within focal adhesions.

Such behaviour may or may not reflect the dynamic behaviour of focal adhesions that

depends on the function of several proteins. Hence, in our opinion, the more static

behaviour of zyxin in KO cells on glass does not necessarily represent a “functional”

contradiction. In fact, it is possible that the behaviour of one single focal adhesion

protein is affected in a certain way, whereas focal adhesions as a whole behave in a

opposite way.

R: This still needs to be stated and discussed. There is no evidence for the behaviour proposed by the authors. I do not recommend suggesting it without evidence or without reference to appropriate literature.

7. In Fig 9, trends are proposed for the cell response to different microgels. E.g. "their

average speed and directionality were greatly reduced on 800, 1200 and 1600 nm

arrays". Considering that there is no apparent statistical trend (the averages do not

even follow a clear trend when the microgel size was varied from 300 to 1600 nm),

any notion that the microgel size (and associated topography?) is regulating cell

adhesion and motility should be removed from this manuscript. The only clear

difference observed is between glass/film and arrays. But again, this breaks down

when analysing the impact of substrate topography/patterning on FA

assembly/disassembly.

We thank the reviewer for the interesting comment on our interpretation of the data.

We based our interpretation of the data and statements on the statistical analysis (see

suppl. Tables S7 and S8). In our opinion there is a clear trend showing that the

average speed decreases as the microgel spacing increases.

R: I disagree. Fig 9 does not show stats, but 1600 nm is clearly above 1200 and 800 nm. Similarly, other processes quantified in Figure 9 clearly do not show trends. Some stats should be included directly in this figure, to facilitate its quantitative analysis.

Likewise, spacing greater than 300 nm consistently leads to higher directionality of cell motility. We cannot, at present, precisely explain why cells on 1600 nm microgel arrays regain part of their motility. However, we note that the 1600 nm microgel arrays form a pyramidal structure characterised by one microgel sitting on the top of two microgels. This

technical limitation arises by both the fact that we have used the largest possible

PDMS stamp to print the 1600 nm arrays, and that physical limitations do not allow to

generate larger microgels during the synthesis. Regardless of these current technical

drawbacks, it is certainly important to investigate how cell behaviour changes when

the distance between adjacent arrays increases.

R: clearly this calls for a modification of the conclusions. The microgel size does not clearly modulate migration. Only 300 nm gels do.

10. Conclusions made in the discussion, specifically (but not only) "the migration of

B16F1 cells is inversely correlated with microgel array spacing" are not supported by

the data presented. This needs to be carefully rewritten. Similarly, "In this context, we

have developed a (...) cell adhesion and migration". I do not see evidence that the

system presented allow to modulate actin cytoskeleton architecture. There is no

control in the phenotypes observed and no clear trends when correlating spacing and

migration/FA dynamics are observed. The cytoskeleton architecture has not been

systematically studied or characterised.

In our opinion, the statement “the migration of B16F1 cells is inversely correlated with

microgel array spacing” is correct and supported by Fig. 9 (with the possible exception

of the motility of cells on 1600 nm microgel arrays).

R: As noted above, there is no trend between migration speed, directionality and assembly/disassembly rates in Figure 9. According to Table S7 cells migrate faster on 1600 nm gels. The directionality is only significantly different for cells on 300 nm patterns. The assembly/disassembly rates follow a see-saw pattern, which does not constitute a trend.

11. The conclusion that "the variation of microgel array topographic and mechanical

features can efficiently be used for the modulation of cell adhesion and motility" is

misleading. There is no real control achieved (which would be evidenced by trends

with microgel size for example). The only trends observed are the response of cells to

films and arrays compared to glass. But this could be the result of differences in

adhesive protein adsorption promoting differences in cell adhesion.

In our opinion, the statement "the variation of microgel array topographic and

mechanical features can efficiently be used for the modulation of cell adhesion and

motility" highlighted by the reviewer is supported by solid experimental evidence. As

to the topography, we have not only compared cell motility and adhesion on glass to

the same biological processes on films and arrays, but also films to arrays.

Furthermore, we have changed arrays spacing (i.e., microgel topography) and clearly

show that it can modulate cell adhesion and motility. Also in this case, the comparison

was done with glass, but also with films and between pairs of different array spacings

(see statistical analysis in the suppl. data). Regarding the mechanical feature (i.e.,

content of cross-linker), we clearly show that focal adhesion turnover is clearly

modulated by this microgel feature in both WT and Gas2L1 KO cells.

R: As noted above, this still requires revising. This study presents some interesting results, but I dispute some of its conclusions and this should not be discarded.

12. Fig. 7. It would be useful to show some of the normalised fluorescence intensities

prior to photobleaching in the traces presented.

As the reviewer certainly knows, the normalised intensities prior to photobleaching are

equal to 1 and will be visualised as curves parallel to the X axis for all the conditions

(i.e., glass, films and arrays). In our opinion, the information will not add any relevant

detail to the figure but introducing it will cause the curves to be squeezed together

(because the Y axis will include a range of values up to 1 or more) thus making any

difference difficult to appreciate.

R: The value of presenting normalised fluorescence intensities directly prior to bleaching (for a few tens of s is to clearly show whether some gradual photobleaching of the systems was observed simply during imaging.

R: Statistical Analysis.

Experiments should all be carried out at least in triplicates, rather than duplicates and triplicates.

Reviewer #2: (No Response)

Reviewer #3: The authors have addressed my suggestions satisfactorily. I recommend publication since the revised manuscript now meets the journal standards.

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Reviewer #1: No

Reviewer #2: Yes: Isaac T.S. Li

Reviewer #3: No

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Attachments

Attachment

Submitted filename: PONE-D-21-09835_comments.pdf

Guiding cell adhesion and motility by modulating cross-linking and topographic properties of microgel arrays

PONE-D-21-09835R2

Dear Dr. Sechi,

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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Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: All comments have been addressed

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Reviewer #1: Partly

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

Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Most of the comments made have been addressed. Some of the issues raised could be better addressed and I would still dispute some of the conclusions, but this would require further investigation and I understand it would delay publication.

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Reviewer #1: No