Review Reports - KRT6A Is a Biomarker of PAS Progression and Enhances the Invasive Ability of Trophoblast Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigates blood samples from patients diagnosed with Placenta Accreta Spectrum (PAS) disorders at 30 weeks of gestation and just before birth for protein biomarkers of disease. The authors identify KRT6 as a potential candidate. They go on to over-expression KRT6 in BeWo and HTR-8 cells and show that the expression of this particular keratin enhances cell migration and invasion. Bioinformatics analyses further show that KRT6 is often associated with the MYC pathway and increased cellular "aggressiveness".

Overall, this is an interesting study but several points need to be addressed. The most important points are highlighted with an asterisk:

It would appear that the authors have previously conducted similar proteomic surveys of PAS and control patients. KRT6 was already identified in these previous studies. Please make that clear in the text.
* What is the abundance profile of KRT6 in control patients at 30 wks of gestation and just before birth? This data should be included.
It is not clear how the integration of previous data accounted for the possibility that some proteins are up-regulated only because of the progression of pregnancy. Please explain better how proteins that are up-regulated with gestation were ruled out. Looking at the IHC stainings, it would appear that KRT6 is up-regulated as pregnancy advances even in control patients.
The overlap in Fig. 2A is unclear. Is KRT6 the only shared protein? Which list is from which study?
* Fig. 2B would indicate the KRT6 is only up-regulated in 3/5 patients. Even if this is statistically significant, please elaborate on this point and discuss the value of KRT6 as biomarker. Looking at Fig. 2B, normal KRT6 levels are not indicative of low risk.
Following on from this comment, can the authors please look back at the clinical records to determine what else is different in the 3 patients that had elevated KRT6 levels. Can they also please spell out in precisely how many follow-up samples that were used for histology they observed the up-regulation of KRT6, in in how many this was not observed?
Fig. 5, the phase contrast images in A and B are invisible. In general, BeWo cells are a poor model for trophoblast invasion.
Fig. 6, please improve the layout of this figure - make the plots the same size, align them to each other etc
Line 25. please correct the sentence
Line 95, please correct the sentence
Line 293, "BeWo" - or leave these cells out entirely, as they are a poor model of migration and invasion

Author Response

Dear Reviewer,

Thank you very much for your thorough and insightful comments on our manuscript. Your professional guidance has been extremely valuable, and I have learned a great deal from this review process.

We have carefully considered each of your suggestions and have revised the manuscript accordingly. A point-by-point response to all comments has been prepared and is attached to this letter.

We greatly appreciate the time and effort you have dedicated to reviewing our work. Your input has significantly improved the quality of our manuscript.

Thank you again for your thoughtful review.

Sincerely,

Zhirong Guo
On behalf of all authors

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this paper the authors investigate the plasma profile of 5 placenta accreta patients in an aim to identifying a potential causative factor and biomarker of PAS progression.

This is a retrospective study enrolling PAS patients.

PAS diagnosis is well described and clinically defined, Ultrasound score: the ultrasound scoring system for PAS developed by Peking University First Hospital was adopted, and scoring was performed by two experienced sonographers. Blood samples were collected at 2 time points, one sample before 30 weeks of gestation (glucose challenge test) and another 2 weeks prior to delivery.

Plasma proteins were analysed by mass spectrometry (methods well described) and data is shown nicely in figure 1. The clustering analysis clearly shows separation between the 2 time points and functional enrichment analysis, including Gene Ontology and Gene Set Enrichment identified that as PAS progresses, compared to the 30th week of gestation, abnormal biological processes in PAS before delivery include cell keratinization, keratinocyte differentiation, epidermal cell differentiation, and cornification of keratinocytes

C1) Please ensure that all text in the figures can be read at 100% zoom (This applies to all figures in the manuscript)

26 proteins were identified and to identify those associated with PAS the authors performed an intersection analysis using another publication of a proteomic data set from term PAS patients described in ref 29. This analysis identified KRT6A to be the hub protein between to two datasets. KRT6a increased with gestation in the study subjects, localized to the invasive trophoblast in a UMAP plot of the ref29 dataset and was increased above a control group

C2) Why is KRT6 the only common protein detected between the 2 studies ? The other study identifies a number of keratins? Please can the authors discuss.

C3) What is the control group in this figure and fig 3. There is no description in the paper or tables of a healthy control group, please correct this.

The authors then move on to functional analysis starting with immunohistochemistry of the placenta. They show that KRT6A strongly labels the choriotrophoblast and the invasive EVT at the maternal fetal interface.

C4) Fig 3F The 15 week control sample is not decidua it is a blood clot, please remove

They then move on 2 cell lines 1)HTR8SVneo is a first trimester invasive cell line and BeWo is a term choriocarcinoma line not routinely used for invasive studies but more as a model of cell fusion.

C5) Why was Bewo used as it is not invasive?

They establish stable lentiviral transfected lines over expressing KRT6A and perform cell proliferation, apoptosis and invasion assays, They show that KRT6A only affected cell invasion leading to an increase in numbers of migrated cells in the KRT6 overexpressing lines .

C6) It is impossible to see the cells in the wound healing images please provide phase contrast images.

The authors lastly perform a gene set enrichment study utilizing a single cell sequencing study of PAS placentas Ref(27) and showed that that MYC_TAGRETS_V1 signal pathway was activated in the KRT6A+ trophoblast cells.

C7) Please can the authors add more details about the other studies that they have used in their analysis in the results section to provide more context on why they were used i.e. what did ref 29 and 27 actually show?

Finally in equating the overt invasion of EVT in PAS with cancer the authors perform a pan-cancer analysis of KRT6A expression and its correlation with MYC signaling using data from the TCGA and GTEx databases and show a strong correlation between KRT6 and numerous cancer types.

The discussion is well written and limitations are addressed appropriately.

Author Response

Dear Reviewer,

Thank you very much for your thorough and insightful comments on our manuscript. Your professional guidance has been extremely valuable, and I have learned a great deal from this review process.

We have carefully considered each of your suggestions and have revised the manuscript accordingly. A point-by-point response to all comments has been prepared and is attached to this letter.

We greatly appreciate the time and effort you have dedicated to reviewing our work. Your input has significantly improved the quality of our manuscript.

Thank you again for your thoughtful review.

Sincerely,

Zhirong Guo
On behalf of all authors

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript by Zhirong Guo et al entitled “KRT6A is a biomarker of PAS progression and enhances the invasive ability of trophoblast cells”, the authors aim to highlight the potential of a newly characterise KRT6 gene has potential biomarker for Placenta Accreta Spectrum. This protein is initially identified as part of a bioinformatics/ proteomic analysis across 10 human samples (5 of whom have been diagnosed with Placenta Accreta Spectrum). Further characterisation of the actual overexpression of the protein is performed further either in human placenta sections or using human placental cell lines. In the latter, this is achieved by overexpression of the gene via plasmid transfection. Experiments are then conducted to correlate the changes in expression with overall changes is motility and invasions, cell proliferation and apoptosis.

The work is of interest and provides a new target to consider as biomarker and potential regulator of Placenta Accreta Spectrum. The experiments provided are somewhat conclusive of a potential regulatory role, even if preliminary. The main drawback to the study is the lack of information about any of the experiments carried out when related to the gain of function analysis. This makes it impossible for readers, including this reviewer, to make any informed judgment on the work presented.

Here are a few points that the authors need to consider to bolster their observations as listed below:

Vey little information is provided to explain how the cell lines were engineered to express KRT6A. This would need to be fully documented. Is it a stable clonal expression? If so, authors should provide evidence that their observations are not clone specific.

Figure 4 present some Western blots data. Again, no information is given to understand what has been done. It seems that the gene expressed is actually Flag-KRT6. Is this correct? Why not use the wild type version of the protein. If indeed a fusion protein is the expressed protein, then what is the Western blot work probed against? Why not use KRT6 antibody given that those were used earlier.

The use of a Flag-KRT6 protein, when expressed, would also lead to questions related to the potential artefacts of the fusion version rather than the full lengths KRT6 as regulator of the different phenotypes measured and this would need to be considered.

Figure 5 appears to relate to some invasion data for some of the panels. Panel B seems to show some scratch assay analysis but is titled as cell invasion. It is not clear how the authors can justify this as being the correct experiment to reach such conclusions ?

Analysis of KRT6 expression in human placenta is shown by immunofluorescence /immunohistochemistry in Figure 3. Authors refer to difference in expression levels but no quantification is shown to allow to make this assessment. Some analysis should be carried out to offer statistical evaluations of the changes if any.

The final work aims to shed new light into the potential pathways where KRT6 involvement could play a role. Again, the information available to make sense of what is shown is scares at best. It seems cancer samples were used for some of the panels presented. It is unclear as to why those were selected, given the focus of this study. If there is not enough data available to relate this to Placenta Accreta Spectrum, then at least, authors should make some efforts to show that the identified pathways appear to be affected in their samples, either placental or cell lines to support their findings.

Author Response

Dear Reviewer,

Thank you very much for your thorough and insightful comments on our manuscript. Your professional guidance has been extremely valuable, and I have learned a great deal from this review process.

We have carefully considered each of your suggestions and have revised the manuscript accordingly. A point-by-point response to all comments has been prepared and is attached to this letter.

We greatly appreciate the time and effort you have dedicated to reviewing our work. Your input has significantly improved the quality of our manuscript.

Thank you again for your thoughtful review.

Sincerely,

Zhirong Guo
On behalf of all authors

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have done an appropriate job in this revision and responded well to all points raised. Although the findings are somewhat marginal, they seem interesting and at least partially novel.

Author Response

Dear Reviewer,

We sincerely thank you for the positive and encouraging feedback on our revision. We truly appreciate the acknowledgment that our findings are interesting and partially novel. This encouragement means a great deal to us and further motivates us to continue exploring the role of KRT6A in PAS progression in our future studies. Thank you again for your time and expertise.

Sincerely,

Zhirong Guo
On behalf of all authors

Reviewer 2 Report

Comments and Suggestions for Authors

Many thanks to the authors for their extensive revisions to the manuscript. I am very happy to say that these changes have greatly improved the paper from start to finish and congratulations to all involved. This is an impressive body of work.

Author Response

Dear Reviewer,

We are deeply grateful for your generous and encouraging words. Your positive feedback means a great deal to us and is a strong affirmation of the effort we have put into this revision. We are truly delighted to know that you find the manuscript greatly improved from start to finish.

We also sincerely appreciate your recognition of this work as an impressive body of work. Your constructive and thoughtful guidance throughout the review process has been invaluable in helping us strengthen the quality of our study.

Thank you once again for your time, expertise, and encouragement.

Sincerely,

Zhirong Guo
On behalf of all authors

Reviewer 3 Report

Comments and Suggestions for Authors

In this resubmission of their manuscript by Zhirong Guo et al entitled “KRT6A is a biomarker of PAS progression and enhances the invasive ability of trophoblast cells”, the authors have provided some text changes to somewhat improve the reading of their work.

It is however not sufficient and much more would need to be done to make the current study suitable for readers to understand the work.

Taking the previous comments (some of which have just been copied and pasted) as examples (more could be offered but is beyond the scope of this review), it is clear that much more work is needed to allow people to be able to appreciate the work that has been carried out.

Figure 4: the method does not state that KRT6 has been expressed as a fusion protein in the form of Flag-KRT6. Is this correct? If this is the case, where is the Flag peptide in the control system as this will be the control to use throughout the work presented. As before, points raised related to use the wild type version of the protein. If indeed a fusion protein is the expressed protein Why not use KRT6 antibody given that those were used earlier.

Figure 5 appears to relate to some invasion data for some of the panels. Panel B seems to show some scratch assay analysis but is titled as cell invasion. Some information is given to explain how they have poured Matrigel onto the cells to transformed this into an invasion assay. Whilst this may be a possible way of analysis this, it would need to be validated as this is a very unusual way to do so. Please note also that Fig 5C seem to show some transwell analysis but as before no information is provided to explain this assay. Furthermore Fig 5D, according to the legend relates to Bewo cells whilst everything in the panel is labelled as HTR8

Author Response

Please see the attachment

Author Response File: Author Response.pdf