PONE-D-20-14837

APC mutations in human colon lead to decreased maturation of ALDH+ stem cells along the neuroendocrine lineage: Discovery of a link between WNT and retinoic acid signalling in colon cancer development

PLOS ONE

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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: I Don't Know

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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: 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: In the manuscript by Zhang et al, the authors investigate the relationship between APC mutations and stem cell maturation in colon cancer. They use patient samples, mathematical modeling, and in vitro studies to show that APC mutation delays stem cell maturation and to connect Wnt and retinoic acid (RA) signaling. The principles of the current manuscript are important and add to the scientific understanding of colon cancer progression; however, concerns outlined below dampen the strength of these findings.

Major concerns:

1. The authors state, in the abstract and introduction, that they are investigating a correlation between Wnt signaling and RA signaling. The data shown in this manuscript have not proven this interaction.

2. The staining for ALDH1 and CGA in Figure 1 are described as having a similar pattern (1A and B – normal). However, the images shown do not depict this.

3. The authors state that “the proportion of SSTR+ cells tended to be greater than that of GLP-2R+ cells” in the colon cancer cell lines. The data shown in Figure 5 depict that one of the cell lines (COLO320) has some differences between SSTR1 and GLP-2R, but the cell lines chosen for future studies (SW480 and HT29) show no difference in the percentage of positive cells for SSTR1 and GLP-2R. The in vitro studies are lacking error and significance designations (Figure 5B-H), making the data difficult to interpret. In addition, controls are lacking in Figure 5B. The authors state that GLP-2 decreases the ALDH+ cell population, but that SST does not. Without showing the control cells, it is impossible to discern the effect of SST and GLP-2.

4. The results describe data of CGA/ALDH double staining in FAP tissues, but FAP staining isn’t shown (Figure 2). It would be helpful to show the IF staining for normal, FAP, adenoma, and carcinoma, since those are shown in panel “F.” In addition, it would be helpful to show the crypt level for all four conditions.

Minor concerns:

1. Layout of the results is difficult to follow. It would be helpful if the results and figures were in the same order.

2. In general, the figures are of poor quality. In addition, the IHC and IF images are lacking scale bars and would benefit from arrows to depict areas of interest. The text in most figures needs to be improved, and this is particularly evident in Figure 4E and F, where the green text is illegible. Similarly, the panels in Figure 5D don’t show anything.

3. It would be helpful to have the crypt levels shown on one example IHC and IF image to help orient the reader.

4. The y-axes in Figure 2 need to be labeled. As it is now, C-E show the crypt level and then there is a switch to % of the cell population. This is not labeled and left up to reader interpretation.

5. In the results and figure legend, Figure 3 is said to have multiple specific panels; however, the image only shows A and B (B is never described).

Reviewer #2: This paper by Zhang et al. describes the relationship between APC mutations, ALDH+ stem cells (SCs) and maturation along the neuroendocrine lineage in colonic crypts from FAP patients. The authors major conclusion is that progression from normal colon to adenomatous colon in APC mutant FAP patients is driven by a failure of ALDH+ SCs to differentiate into mature neuroendocrine cells (NECs). Immature intermediate progenitor NECs self-renew and proliferate causing overpopulation of the SC niche, leading to adenoma formation. The authors use IHC and IF on colon tissue from FAP tissue to make observations of NEC and SC marker positivity. Confirmatory and mechanistic experiments are performed in vitro using colon cancer cell lines. Mechanistically, the authors conclude that APC-mutant driven WNT signaling drives retinoic acid (RA) signaling to prevent maturation of colonic SCs into NECs. Overall, the studies are well conducted. However, some major and minor deficiencies prevent the paper from being suitable for publication in its current form.

MAJOR:

1. The studies are largely observational and descriptive in nature. The title indicates a link between RA and WNT signaling, but this is not mechanistically tested in this paper. Instead, the authors cite a number of previously studies to support their conclusions.

2. The mechanistic studies focus on GLP-2 and SST signaling, which are well conducted and support the major conclusions of the paper. The title should mention these mechanistic observations.

3. Many of the conclusions are based on amalgamation of previously published work. The conclusions should be tempered and based on data presented in the current paper.

4. The model in Figure 4 refers to a feedback mechanism involving the regulation of progenitor SC proliferation by mature NECs. It’s not clear that this model is fully supported by the data presented.

MINOR:

1. Page 5: Reference to 1/2 or 1/20 individuals who develop adenomatous polyps or colon cancer, respectively. Do these numbers refer to the general population? Please clarify.

2. Page 7: 5-HT is referred to as a neuro-peptide. This is incorrect. It’s a catecholamine.

3. Figure 1C, D and E: curve axes should have the same scale to enable more meaningful comparison.

4. Figure 2B: ALDH staining is much weaker in colon cancer specimens compared to normal crypts. What’s the explanation?

5. Quantitation of data in Figure 3 should be shown, similar to Figure 2.

6. Figure 3 panels are mislabeled as A and B. The text refers to panels A through J.

7. Raw flow cytometry data should be included for plots in Figure 5. The raw data could be added as supplemental material.

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Reviewer #1: No

Reviewer #2: No

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APC mutations in human colon lead to decreased neuroendocrine maturation of ALDH+ stem cells that alters GLP-2 and SST feedback signaling: Clue to a link between WNT and retinoic acid signaling in colon cancer development

PONE-D-20-14837R1

Dear Dr. Boman,

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. Please also address the minor issues highlighted by Reviewer #1. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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: 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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have done a nice job addressing previous comments and concerns.

In the revised Figure 5, it is unclear which cells are being used in the experiments for panels D-H. In addition, while the authors cite the findings here, there are not errors and statistical information on panels E-H.

Reviewer #2: The authors have adequately addressed the major and minor concerns. The paper is suitable for publication.

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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