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The authors addressed all my concerns.
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The authors have carefully addressed all the reviewers' comments and revised the manuscript accordingly. Therefore, the manuscript is acceptable for publication in this journal.
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Dear Dr. Dong,
Please revise the manuscript according to the reviewer's comments before re-submitting the text to PeerJ.
Kind regards,
Dr. Sander Bekeschus
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a rebuttal letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the rebuttal letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the rebuttal letter. Directions on how to prepare a rebuttal letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
This is a well writing manuscript with scientific sound. The article structure and figures presentation are organized in a clear and unbiased manner. The only concern from the reviewer is the gramma errors and typos in the manuscript. The authors should carefully go through the manuscript to revise the typos.
The goal of this manuscript is to identify the effects of giant cell tumor stromal cells in giant cell tumor progression. This is an interesting study and important to the field. To address this scientific question, the authors isolated GCTSCs and compared them with normal BMMSCs. In addition, they identified IL-6 and IL-10 secreted by GCTSCs in regulation of tumor macrophage polarization. The research aims and experimental designs were well defined, and results were relevant to their hypotheses.
The authors identified the role of GCTSCs in regulation of tumor macrophage polarization. In addition, IL-6 and L-10 were discovered as the potential underlying mechanisms in this regulations. However, how IL-6/IL-10 can promote M2 macrophage polarization was not fully discussed. Please discuss more detail potential mechanisms in the paper.
This is an important and novel study. However, it is not clear how to apply these findings to clinical applications. The authors should carefully discuss and link their discoveries to future clinical trials and studies.
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In the present study, the authors investigated the effects of GCT stromal cells (GCTSCs) on proliferation, apoptosis, and polarization of human macrophages. The results indicated that GCTSCs expressed a similar panel of MSC-related markers but had higher proliferation and relative lower differentiation abilities compared with BMMSCs. Meanwhile, GCT CD14+ monocytes contain an increased proportion of CD206+ M2 macrophages as compared to normal human blood CD14+ monocytes. In addition, co-culture of GCTSCs with macrophages promoted M2 macrophage polarization and increased the release of IL-6 and IL-10 from GCTSCs. These findings suggest that GCTSCs contribute to GCT pathogenesis possibly by promoting M2 macrophage polarization via increased secretion of secret IL-6 and IL-10. The reviewer has the following major points that need to be carefully addressed:
1) Lines 90-91, it is stated that “Normal MSCs were obtained from the bone marrow of C57 mice as described previously.” It is questionable to use murine BMMSCs as compared with human GCTSCs in term of their immune profiles, proliferation, and multipotent differentiation capabilities.
2) Lines 92-96, as is known, exogenous M-CSF is usually required to culture and maintain CD14+ monocytes isolated from peripheral blood. It is unclear whether this growth factor was included in the present study.
3) In Figure 2b, the expression of a panel of MSC-related makers, STRO-1, SSEA-4, CD146, CD73, and CD90 was detected in GCTSCs by immunofluorescence (IF) staining, but NOT in BMMSCs. To support the conclusion, paralleled experiment should be performed in in BMSCCs. Meanwhile, the quality of IF is concerned with a relatively high background signal in nuclei while a relatively weak signal for CD146 and CD73. Flow cytometry analysis should be more convincing as was shown in Figure 2a.
4) Figure 4a showed that CD14+ monocytes from GCT and peripheral blood contain 85.05% and 61.15% of CD206+ M2 macrophages, respectively. However, in Figure 2b, why was the percentage of CD206+ M2 macrophages among GCT CD14+ monocytes less than 10%? After co-culture with BMMSCs and three strains of GCTSCs, the proportion of CD206+ M2 macrophages in blood-derived CD14+ monocytes were 8.6%, 11.0%, 11.6%, 19.1%, respectively, which were much lower than the basal percentage (61.15%) of CD206+ M2 macrophages. How to explain such discrepancies?
5) As is known, IL-10 is one of the major anti-inflammatory cytokines secreted by M2 macrophages. It is unknown whether induced M2 macrophages also contribute to the elevated IL-10 levels in the co-cultured GCTSCs and macrophages.
6) It cannot be definitely concluded that GCTSCs contribute to M2 macrophage polarization through elevated secretion of IL-6 and IL-10 unless neutralizing antibodies for IL-6 and IL-10 were utilized in the study.
The present manuscript entitled “Giant cell tumor stromal cells: Osteoblast lineage-derived cells secrete IL-6 and IL-10 for M2 macrophages polarization” describes the role of giant cell tumor stromal cells (GCTSCs) in the macrophage polarization by releasing of IL-6 and IL-10. Authors also investigated the mesenchymal stem cells (MSCs) characters of GCTSCs and the effect of GCTSCs on macrophage proliferation and apoptosis.
The manuscript includes interesting and informative outcomes, however, this manuscript seems to be somewhat superficial and need further investigations to confirm the relation of macrophages polarization and IL-6, IL-10 released by GCTSCs. Therefore, the following revisions should be addressed to be able to publish.
1. Most of the experiments of this manuscript was based on the comparison of BMMSC and GCTSCs. However, the GCTSCs were isolated from human samples while the BMMSC were obtained from C57 mice. The results would be more reliable if the authors used human BMMSC as control.
2. The evidence that GCTSCs affect macrophages polarization by secreting IL-6, IL-10 was weak. Please do more experiments to testify this statement, for example, down-regulate IL-6, IL-10 of GCTSCs and investigate how it affects the polarization of macrophages.
What’s the purpose to test the proportion of M2 macrophage in human blood in figure 4?
Please add description or discussion.
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Comments to the Author:
The present manuscript entitled “Giant cell tumor stromal cells: Osteoblast lineage-derived cells secrete IL-6 and IL-10 for M2 macrophages polarization” describes the role of giant cell tumor
stromal cells (GCTSCs) in the macrophage polarization by releasing of IL-6 and IL-10. Authors also investigated the mesenchymal stem cells (MSCs) characters of GCTSCs and the effect of GCTSCs on macrophage proliferation and apoptosis.
The manuscript includes interesting and informative outcomes, however, this manuscript seems to be somewhat superficial and need further investigations to confirm the relation of macrophages polarization and IL-6, IL-10 released by GCTSCs. Therefore, the following revisions should be addressed to be able to publish this work on this journal.
There are some issues that the author should clarify as follows:
1. Most of the experiments of this manuscript was based on the comparison of BMMSC and GCTSCs. However, the GCTSCs were isolated from human samples while the BMMSC were obtained from C57 mice. The results would be more reliable if the authors used human BMMSC as control.
2. The evidence that GCTSCs affect macrophages polarization by secreting IL-6, IL-10 was weak. Please do more experiments to testify this statement, for example, down-regulate IL-6, IL-10 of GCTSCs and investigate how it affects the polarization of macrophages.
3. Please modify the title of the manuscript as the research didn’t provide convincing evidence of the key role of IL-6, IL-10.
4. What’s the purpose to test the proportion of M2 macrophage in human blood in figure 4?
5. The language should be revised, as there were some unclear points that was distracting and impedes the scientific presentation. Moreover, proofreading is needed since there are several grammatical errors and misspelling words in the manuscript.
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