PONE-D-19-18985

Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

PLOS ONE

Dear Dr. Goueli,

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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ACADEMIC EDITOR:

Please refer to reviwers' comments below with particular reference to including increased emphasis on how this approach is superior to currently established protocols e.g. potential to use with live cells. Both reviewers have also commented on the length and number of figures. Please try to more succinctly present the data.

As suggested by reviewer 1, an introduction that includes a wider characterisation of the role of CD73 and CD39 in human physiology/ pathology is warranted.

==============================

We would appreciate receiving your revised manuscript by Sep 21 2019 11:59PM. When you are 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.

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Please include the following items when submitting your revised manuscript:

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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.

We look forward to receiving your revised manuscript.

Kind regards,

Paul A Beavis

Academic Editor

PLOS ONE

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The authors acknowledge the financial support by Promega Corp for carrying out this research.

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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

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: N/A

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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

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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: No

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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: Reviewer comments

Due to the increasing interest in targeting the CD73/CD39 and adenosine receptor axis in the context of immunotherapy to treat cancer, and promising outcomes both in pre-clinical and clinical settings, there is a need to develop new and effective therapeutic agents targeting these receptors. The authors have developed and clearly demonstrated a sensitive assay platform utilizing Promega’s own AMP-GLO or ADP-GLO kits to test the efficacy and specificity of small molecule inhibitors and antibodies for inhibiting the activity of CD39 and CD73. There are however several issues that need to be addressed as outlined below.

Major

A potential limitation of using a luciferase-based detection assay is inhibition of luciferase by inhibitors used. POM1 was mentioned in prior literature to inhibit luciferase in a different enzyme activity kit (doi: 10.4049/jimmunol.1003884). Have the authors confirmed their assay using orthogonal methods, such as alternative enzyme assay or testing their inhibitors at varying concentrations in the absence of CD73 or CD39? It may be important for future screening to include in the protocol a test for potential inhibitors interfering with the output of the AMP-GLO or ADP-GLO kits.

Protein concentration used in western blots for CD79 or CD39 in cells should be shown using either a housekeeping control, or the total protein concentration shown if other methods were used. Alternatively, a FACS based assay could be performed to show expression of cell surface CD79 or CD39.

One of the advantages of this assay as described by the authors is that a homologous detection protocol could be adapted to broad range of kinases and proteins that metabolise ATP. This is great for identifying cross reactivity, which is an advantage over other assays. However, it is difficult to decipher between groups in Figure 17A and 17B. It does appear that at high concentrations of inhibitor POM1, there is inhibition of enzyme activity in each group, but POM1 could be specific at lower doses. Showing the X-axis as a Log10[inhibitor] will make these figures easier to interpret.

Include statistics and p-values.

State competing interests, such as funding from Promega.

Minor

The large number of figures is unnecessary and some streamlining of the figures can make the paper easier to read. Joining Fig 3+4+5, figure 6+7, Figures 8+9+10, figures 12+13, figures 14+15+16 into their respective figures for example. The Figure legends will need to be modified in each case.

Error bars in several figures are hidden behind the point. It may be relevant to show these in front for consistency.

Higher resolution figures should also be provided for most of the figures, this is especially relevant for figure 11.

Including several examples from literature on the advantages of using membrane bound proteins rather than purified proteins in screening for inhibitors, could enhance the rationale of using this approach on live cells. Is it possible to use this assay for primary mouse or human cells, and if so, could this lead to other applications for this assay, such as regulation of enzyme function on cells in response to stimuli, or confirming active inhibition of receptors in vivo after therapy?

Optimization of cell numbers for adherent or non-adherent cells in the assay may be of interest to readers and could be provided as supplementary.

Sufficient references used, covering several studies targeting the CD39/CD73 and adenosine axis in combination with checkpoint inhibitors to treat tumors. Identified its importance in tumor escape, regulation of anti-tumor immunity (both myeloid and lymphoid populations) in both preclinical and clinical studies. Increased tumor CD73 and CD39 expression correlate with poorer patient outcomes. It may be prudent to include J.Stagg 2010 PNAS paper, which is the original paper that utilized anti CD73 against cancer. More recent papers have also linked CD39 expression as a marker of tumor-reactive T cells in tumor infiltrating CD8 populations in both primary and metastatic tumors. Citing these in the text can be relevant.

Relating to the methods for preparation of adherent cells for the assay, it was not clearly mentioned if these cells need to be re-trypsinized after culture overnight in clear bottom plate to be transferred into 96 well plate?

Few spelling errors throughout the paper to be corrected.

Reviewer #2: The manuscript highlights a system to accurately assess the activity of CD73 and CD39 in vitro, an important assay as CD73 and CD39 inhibitors and antibodies are of increasing interest clinically. However, the manuscript requires a number of changes to improve and increase its readability for a greater audience. Many of the sections could be condensed to highlight further the main message of the manuscript succinctly.

Major points:

The introduction is long and highlights much detail about the combinatorial potential of targeting the adenosinergic pathway alongside other therapeutic modalities in cancer, the authors should consider whether their may be a more appropriate introduction to provide a better understanding to their results and the assay they have developed.

Methods section provides numerous details about the reagents used, however, this could be more strategically integrated within the methods.

Please highlight the clones of antibodies being used in addition to their catalogue number. Ab71322 is no longer available from the company and the authors should reconsider use of this data.

In scheme 1 – please include in part (a) adenylate kinase as mentioned in the legend for this section. In (b) please keep the names of the reagents or symbols used to identify them consistent to that in (a).

The number of figures is excessive, many could be combined to make a multiple figure panel, increasing the flow of the paper. Within the figure legends it describes that these figures are performed in triplicate, is that biological or technical triplicates?

In Figure 1 – the inset graph in panel A is not well defined.

Figure 2 – the legend references schematic 1A, it is unclear how this section of the schema relates to the figure, should this be a reference to Scheme 1B where CD73 activity is performed? Figure 2 is also not referenced by number in the body of the text (see line 415 “as shown in figure”).

Figure 3 – uses the same concentration of AMP, in line 433 in reference to this figure the text states “…using different concentrations of AMP”. Please keep units consistent within the manuscript ie in this figure the units used are ng whereas pg has been used in the text. Similarly, line 466 includes mU which are not utilized elsewhere.

Figure 11 – please highlight which antibody number relates to which antibody and the specific clone (this may be most practical to do in the methods section). Some antibodies within the methods section highlight the same clone from different companies being used in this assay, please detail their reproducibility. In addition, many of the CD73 antibodies did not inhibit with CD73 enzymatic activity, please discuss whether this is in keeping with what is known about that antibodies specificity. Line 543 preceding this figure also states that “control antibodies showed minimal effect…” it is unclear within the figure which control antibodies are used and whether any in addition to CD39 are apparent.

Figure 13 – Anti-CD39 was also used in this figure as a negative control for CD73 activity – it would be of interest to look at this antibodies ability to impede CD39 enzymatic activity alongside POM1 and ARL67156.

In Figure 17, POM1 is shown to lack specificity to CD39 and instead inhibits multiple enzymes, it would be of value to comment on the use of POM1 to delineate CD39-mediated anti-tumor efficacy.

In general, the figure legends contain a large amount of information that belongs in the material and methods, please look to make these more succinct.

It would be of interest to utilize this system to measure CD73 and CD39 activity, or the inhibition of enzymatic activity from samples ex vivo rather than in vitro. Can this system be used to measure the presence and activity of CD73 and CD39 inhibitors in circulation? The presence of increased soluble CD73/CD39 or exosome-derived CD73/CD39? Or the enzymatic activity in the tumor microenvironment ex vivo? Please comment or display the utility of this assay in these settings.

Please comment on the advantages/disadvantages of this system compared to those currently utilized. In addition, very limited discussion of the impact of the results is provided throughout and a greater emphasis should be placed towards ensuring this is clear to the reader.

Minor points:

Please check the manuscript for consistency regarding CD73, CD39 and the Farage cell line as well as other nomenclature.

Please provide some reference to the differences in temperature used within the assays. For example, figure 2 at 23 degrees C while Figure 12 is 37 degrees.

Please see some small errors to correct:

Line 19 – increased to increase

Line 20 /65– immunosuppressant, generally refers to a drug type reconsider use of this word throughout.

Line 22/25 – CD39 while upstream of CD73 and the conversion of AMP to adenosine, should not be referres to as a major source for adenosine.

Line 34 – specify the what novel inhibitors you refer too for cancer therapy.

Line 36 – simplify “immune-checkpoint blockers drugs”

Line 70/71 – Rephrase sentence

Line 92 – Replace effector with CD8+ T cells

Line 104/105 – Please explain low cross reactivity with other nucleotides? Do you mean ectonucleotidases?

Line 115 – Fc should have the gamma symbol not that currently shown.

Line 116/118 – refer as A2AR inhibitor or antagonist, not anti A2AR.

Line 121 – AZD4635’s remove the ‘s

Line 124/126 – rephrase sentence

Line 133 – why refer to them as tentative therapeutic approaches?

Line 136 – should read “as well as the tumor itself”

Line 137 – should read monitors

Line 157 – EMEM?

Line 166 – rephrase kinases kits.

Line 272 – per what well?

Line 284 – A.1.?

Line 354 – should read “a very potent immunosuppressive”

Line 354 – 365 – redundant or better included in the introduction please revise.

Line 369/370 – relevance for the discussion of the bacterial enzyme within this paper?

Line 425 – delete i.e.,

Line 444 – should read “Fig. 4, CD73..”

Line 534 – Delete three.

Line 540/541 – refers to CD39 antibodies should read antibody as only one is used and the use of control antibodies which are not clearly shown within the figure should be clarified.

Line 564 – should read “while the other monitors”

Line 608 – remove the use of totally.

Line 656 – should read panel not penal.

Figure 13/17 legends please check for incorrectly formatted symbols.

Line 659 replace that with to.

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Reviewer #1: No

Reviewer #2: No

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-19-18985R1

Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

PLOS ONE

Dear Dr. Goueli,

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.

==============================

ACADEMIC EDITOR:

There are some minor points that require a bit more attention to make the manuscript acceptable for publication. Please refer to the comments of reviewer 2 with regards to Figure 7 in particular.

==============================

We would appreciate receiving your revised manuscript by Nov 09 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled '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.

We look forward to receiving your revised manuscript.

Kind regards,

Paul A Beavis

Academic Editor

PLOS ONE

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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: All comments have been addressed

Reviewer #2: (No Response)

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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

Reviewer #2: Partly

**********

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

Reviewer #1: N/A

Reviewer #2: N/A

**********

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

**********

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

**********

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: It will be prudent to go over the text and legends again before final submission as I have spotted several grammar and spelling errors that haven't been corrected. Figure 1A legend needs clarification, as it is unclear if the figures shown are performed in the presence or absence of 100uM ATP? Aside from these, the paper is much more readable and most comments have been sufficiently addressed.

Reviewer #2: Some points were addressed but there remains a major need to provide appropriate level of detail for the interpretation of figures.

Major points:

- Figure 7 – antibody details must be provided within – it is not sufficient to label with just Ab1, Ab2 etc without detailing which antibody clone it relates to. It is unclear which of the bars represents the control (non-CD73/CD39 antibodies), these are essential details in the interpretation of the graph. In addition, clones for all antibodies should be detailed within methods, not just catalog info.

- The figure quality is very poor and must be improved for publication. Labels are difficult to read as they are in many cases blurry.

Minor points:

Authors should check for spelling and grammatical errors throughout the manuscript see below for some identified.

- Correct the misspelling of Farage cell line, often referred to as Farag as well as Jurkat referred to as Jurkate.

- Define A2AR at the first time it is abbreviated.

- Line 34 – delete (,)

- Line 51 – should read “leading candidate in…”

- Line 57 – Ref 8 is strange – primary article by Bastid, Cancer Immunology Research, 2015 would serve this statement better and broader across multiple cancers.

- Line 70 – delete ‘That…’

- Line 119 – should read checkpoint

- Line 115 and 120 – delete “anti” in relation to A2AR – misleading suggestive that it’s an antibody, please ensure this is removed throughout the text.

- Line 123 – should read A2AR

- Line 142 – delete (s)

- Line 221 – 96-well is repeated

- Line 259 – should read ‘or for a certain time period’

- Line 267 – aberrant )

- Line 272 – should read “known CD39 inhibitor…”

- Line 334 – should read 80-90%

- Line 355 – should read “, 10 min”

- Line 390 – should read “remaining”

- Line 621 – should read “antibody”

- Line 711 – should read from not form.

- Line 757 - should read ‘a diverse set’

- Line 763 - should read ‘that because POM1’

- Line 789 – should read ‘and is not easily’

- Line 805 – should read ‘not only can it’

- Reference 22 and 29 is listed as the same

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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: No

Reviewer #2: No

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

Monitoring and characterizing soluble and membrane-bound ectonucleotidases CD73 and CD39

PONE-D-19-18985R2

Dear Dr. Goueli,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Paul A Beavis

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for making the remaining changes to the manuscript.

Reviewers' comments:

N/A.