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Article
Peer-Review Record

BRAFV600E Expression in Thyrocytes Causes Recruitment of Immunosuppressive STABILIN-1 Macrophages

Cancers 2022, 14(19), 4687; https://doi.org/10.3390/cancers14194687
by Catherine Spourquet 1, Ophélie Delcorte 1, Pascale Lemoine 1, Nicolas Dauguet 2, Axelle Loriot 3, Younes Achouri 4, Maija Hollmén 5, Sirpa Jalkanen 5, François Huaux 6, Sophie Lucas 7,8, Pierre Van Meerkeeck 7, Jeffrey A. Knauf 9, James A. Fagin 10, Chantal Dessy 11, Michel Mourad 12, Patrick Henriet 1, Donatienne Tyteca 1, Etienne Marbaix 1 and Christophe E. Pierreux 1,*
Reviewer 1:
Reviewer 3: Anonymous
Reviewer 4:
Cancers 2022, 14(19), 4687; https://doi.org/10.3390/cancers14194687
Submission received: 27 July 2022 / Revised: 18 September 2022 / Accepted: 21 September 2022 / Published: 26 September 2022
(This article belongs to the Section Tumor Microenvironment)

Round 1

Reviewer 1 Report


The authors Spourquet et al. describe very well changes in immune signaling pathways induced by BRAFV600E mutation.

Nevertheless, I see some open questions for the reader and in some places I miss essential details that would be desirable for understanding and reconstructing the results presented.

- missing information about total RNA-Seq: #reads sequenced, strategie, library, etc

- seq. data should be publically available, also table of DE analysis should be provided to the reader

- how was the sequencing raw data analyzed in detail, quality, mapping, annotation, genome and annotation version, which tools and parameters other from default used?

- how was DE performed, tools, parameters other from default

- MouseMine parameter

- in general, all tools should be mentioned as well as parameters used

- can mouse model be validated in some way, by RNA-seq, Immunohistofluorescence,  etc, .. cannot see the expression changes of BRAF in the presented data

- where BRAF V600E mutation checked in RNA-Seq? is the mutation visible in all samples? would be good to know, see BRAF expression above


- what is non of leakyness of the system, anything known by the authors?

- could the reader be provided a schematic representation of the mouse model transgene?

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

Papillary thyroid carcinoma (PTC) is a histological subtype of thyroid cancer that carries BRAFV600E, an activating mutation. BRAFV600E is associated with malignant transformation, therapy resistance, and tumor progression. Spourquet and collaborators used a doxycycline-inducible model of BRAFV600E that only expresses BRAFV600E in thyrocytes to study the molecular determinants of PTC. The authors used histological, transcriptomics, and immunofluorescence assays and described a STABILIN-1 population of macrophages recruited to the neoplasm. The following statements should be clarified.

 

-RT qPCR methodology must be by the MIQE guidelines

Stephen A Bustin, Vladimir Benes, Jeremy A Garson, Jan Hellemans, Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W Pfaffl, Gregory L Shipley, Jo Vandesompele, Carl T Wittwer, The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Clinical Chemistry, Volume 55, Issue 4, Apr 1 2009, Pages 611–622, https://doi.org/10.1373/clinchem.2008.112797

 

 

In the Western Blotting section, specify the pore of the PVDF membrane. 

 

In line 476 authors state, “Altogether, our data indicate that the absence 476 of STABILIN-1 did not affect macrophage recruitment triggered by BRAFV600E expression, but the increased trend in plasma TNF concentration might suggest an increased pro-inflammatory response in the absence of STABILIN-1”. Nevertheless, the authors shouldn't imply that the trend in TNF might suggest an increased pro-inflammatory response. Authors must present data from other pro-inflammatory cytokines with statistical differences, and from the mRNA sequencing analysis, please describe the expression of pro and anti-inflammatory cytokines.

In line 69, the authors described that TC incidence is higher in women than in men. In this regard, is data sex-paired? If that is the case, please specify; if not, please do the statistical analysis considering sex. Authors should consider that the inflammatory response shows sexual dimorphism in mice:

Villapol S, Loane DJ, Burns MP. Sexual dimorphism in the inflammatory response to traumatic brain injury. Glia. 2017 Sep;65(9):1423-1438. DOI: 10.1002/glia.23171. Epub 2017 Jun 13. PMID: 28608978; PMCID: PMC5609840.

 

Du XJ. Gender modulates cardiac phenotype development in genetically modified mice. Cardiovasc Res. 2004 Aug 15;63(3):510-9. DOI: 10.1016/j.cardiores.2004.03.027. PMID: 15276476.

The authors should discuss in detail if another member of the STABILIN family was compensating for STABILIN-1 deletion.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report


Comments for author File: Comments.docx

Author Response

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Author Response File: Author Response.docx

Reviewer 4 Report

The article is up to date and following the new era.

Abstract: Line 43 is unclear to the reader. Immunosuppressive in which domain. Line 125 "ratio" may be added.

Introduction: lines 115-117 needs reference.

Methodology: Why these particular genes Line 207 were selected. Can you support by reference?

Results: I ran single cell RNAseq analysis on human samples to have a more confirmation for STAB1 gene expression at the cellular level. The gene was mostly expressed in 78% to 99% of macrophages, T lymphocytes, and epithelium in primary tumors and nodal and subcutaneous metastasis (GSE184362).

Line 310: Is there a reference or notice of authors?

Suggest checking the final gene panel in TCGA and cell lines to explain the role of the final selected genes.

 

 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Spourquet and collaborators used a doxycycline-inducible model of BRAFV600E to study the molecular determinants of PTC. They improved their manuscript according to the suggestions.

The methodology section is more straightforward.

The discussion is well-structured.

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