PONE-D-21-39755Triple-negative breast cancer influences a mixed M1/M2 macrophage phenotype associated with tumor aggressivenessPLOS ONE

Dear Dr. Supannikar Tawinwung,

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Data obtained with THP-1 cells should be validated with PBMC-derived primary macrophages which will be helpful to the improvement of the manuscript, in terms of clarifying the methodology and the goal of this manuscript. The authors should also show if such M1-associated genes induced by conditioned THP-1 cells is accompanied by expression of classical M1 surface markers. The authors also need to address  the protein level after stimulation of THP-1 cells with CM medium through western blot. The manuscript should be checked for typos and grammar errors.

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: General comments

In this manuscript the authors attempt to characterize an in-vitro THP-1 cell -based macrophage model exposed to conditioned media from TNBC, trying to validate the results in specific patients’ datasets. Their conclusion is that the obtained TNBC conditioned THP-1 cells phenotype is mixed between the M1- and M2 -like one, suggesting the heterogeneity of TAM phenotype in breast cancer.

In my opinion the manuscript cannot be accepted and requires major revisions.

As a general comment, I find it difficult to accept that a tumor cell line (THP-1 cells) conditioned with TNBC media could be compared to TAMs. At least the results obtained with THP-1 cells should be validated with PBMC-derived primary macrophages.

First of all, conditioned THP-1 cells express M1-associated genes without reporting the indicated markers from a classically-stimulated positive control

Except for CXCL10 and TGF1B, ThP-1 conditioning does not induce a significant increase for any of the markers indicated in Figure 2, when compared to the corresponding control. So it is not clear if these conditioned cells have reached their polarization status or not. Then Figure 3 shows a relevant increase in the IL-6 , IL-10 and TNFa release by TNBC-conditioned cells, so these data should be included in Figure 2. It is also not clear in Figure 2 the media of which cancer cell type was used to condition ThP-1 cells.

Additionally, ThP-1 alternative polarization (labelled as M2, obtained with stimulation with IL-4+IL13 I believe) does not induce a significant increase of the typical associated markers (Figure 2), which is also quite strange. Since IL-4 and IL-13 produce different effects, the indicated markers should be evaluated in cells stimulated with IL-4 alone and IL-13 alone. Additionally, data regarding the expression of M1 and M2-related metabolic enzymes, such as iNOS, PKM2 or ARG1 should also be included.

To evaluate the functional features of these conditioned ThP-1 cells, some migration experiments were performed finding that TAMs and M2-polarized macrophages increased the motility of TBNC cells. In the same way, the authors should study the capacity of these conditioned cells to increase the invasive properties of TBNC cells and analyze the expression levels of classical migration/invasion markers such as N-cadherin, E-cadherin or vimentin. Also, since these cells exhibit a significant increase in the CXCL10 expression levels, the ability of these cells to induce CD8+ T cell recruitment should be reported.

Specific comments

Page 3, line 40 -42 � In this paragraph this sentence is written: “Accumulated evidence suggests that TAMs are a heterogeneous and plastic population, in which polarized TAMs can be identified as M1- and M2-like macrophages”. Please reference.

6. Line 219 � Please write the full name for the acronym BCA.

Figure legends: please specify the cell types and the treatment each result comes from.

Methods: please describe the transwell assay with more detail.

Reviewer #2: This manuscript is well written and performed experiments well, after a minor revision manuscript could be accepted Manuscript Title: "Triple-negative breast cancer influences a mixed M1/M2 macrophage phenotype associated with tumor aggressiveness."

Manuscript is written well and informative way. Data were clean and clear and followed the flow of writing.

I wonder that if authors also need to consider the protein level after stimulation of THP-1 cells with CM medium through western blotting which will tell more profound story of the manuscript.

I recommend that if authors could perform major proteins western blotting and showed the phenotypic condition in protein level that would be more readable.

After the minor revision of experiments this manuscript could be accepted in this journal

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Reviewer #1: No

Reviewer #2: Yes: Yuba Raj Pokharel

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Triple-negative breast cancer influences a mixed M1/M2 macrophage phenotype associated with tumor aggressiveness

PONE-D-21-39755R1

Dear Dr. Tawinwung,

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.

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Kind regards,

Dominique Heymann, Ph.D.

Academic Editor

PLOS ONE

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