Epithelial–Mesenchymal Transition in Osteosarcoma as a Key Driver of Pulmonary Metastasis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

A scoping review focusing on EMT in OS and how it affects the ability of the tumor to metastasize. The manuscript presents the relevant literature in a comprehensive manner and in general has a correct layout. The language is good. There are some points I suggest be revised:

1. The simple summary should provide the reader with what the authors think is the general conclusion of the research presented.
2. Section 3, EMT-Related Signaling Pathways in Cancer , is too long, should be shortened. 
3. Section 5, Regulation of EMT in OS: Key Signaling Pathways: There should be a figure in the same manner as Section 3, showing the key pathology in OS. Furthermore, this section should be rewritten so that the reader understands which data come from in vitro experiments (cell lines etc) or clinical series (they have different relevance and impact)
4. I believe the focus on miRNAs is too strong and Table 1 should be omitted
5. The authors should generally comment on EMT transition in cases of sarcomas/OS: they already are mesenchymal tumors 

Author Response

Comment 1: The simple summary should provide the reader with what the authors think is the general conclusion of the research presented.
Response 1: Thank you for this valuable suggestion. We have revised the Simple Summary to include a clear overall conclusion. Specifically, we added the sentence: “Overall, this review shows that EMT plays an important role in lung metastasis of osteosarcoma, and targeting this process may help improve treatment and patient survival.” (Page 1, Line 16–18).

 

Comment 2: Section 3, EMT-Related Signaling Pathways in Cancer, is too long and should be shortened.
Response 2: We agree with the reviewer. Section 3 has been substantially revised and shortened throughout Pages 5–9 (Lines 110–280). Specifically, we removed redundant and overly detailed step-by-step descriptions of multiple signaling pathways (TGF-β, MAPK, Notch, PI3K/Akt, STAT3, Wnt/β-catenin, etc.) and replaced them with concise summary statements.

 

Comment 3: Section 5, Regulation of EMT in OS: Key Signaling Pathways: There should be a figure in the same manner as Section 3, showing the key pathology in OS. Furthermore, this section should be rewritten so that the reader understands which data come from in vitro experiments (cell lines etc) or clinical series (they have different relevance and impact).

Response 3: We thank the reviewer for this constructive suggestion. In the revised manuscript, we have added a new figure in Section 4 (EMT in OS, Page 10, Figure 4) that illustrates EMT-mediated mechanisms of metastasis and therapy resistance in osteosarcoma. In addition, Section 5 has been revised to clarify which results are derived from in vitro experiments (e.g., cell lines) and which are based on clinical samples, emphasizing their different relevance and impact.

 

Comment 4: I believe the focus on miRNAs is too strong and Table 1 should be omitted.

Response 4: Thank you for this comment. We have revised the manuscript to reduce the emphasis on miRNAs. Specifically, Tables 1–3 in the main text have been streamlined to include only representative examples (Pages 15–24). The complete versions of these tables are now provided in the Supplementary Material as Supplementary Tables S1–S3.

 

Comment 5: The authors should generally comment on EMT transition in cases of sarcomas/OS: they already are mesenchymal tumors.

Response 5: We thank the reviewer for this helpful comment. To address this point, we have added a clarification in Section 4 (EMT in OS, Page 9, Lines 283–285). The revised text states: “Although osteosarcoma is of mesenchymal origin, many studies still use the term ‘EMT’ to describe the activation of transcription factors and signaling pathways that enhance invasion, metastasis, and therapy resistance in OS.” This note clarifies the terminology and avoids potential confusion regarding the use of EMT in the context of osteosarcoma.

Reviewer 2 Report

Comments and Suggestions for Authors

This  review comprehensively addresses the current aspects of EMT in the development of osteosarcoma lung metastases.

Here are a few minor suggestions for improvement.

The extensive ncRNA tables currently lack a systematic assignment of literature citations. Utilising the PMID Number for defining the literature citation is somewhat cumbersome. A more common practice is to directly assign the specific articles in the literature list to the corresponding ncRNAs in the tables by the corresponding literature list number. This should be added to enhance clarity and consistency.

The demonstration of the fundamental mechanisms in three relatively simple figures is currently inadequate. Additional figures with more graphic information, particularly those focussing on the function of the most important ncRNAs, should be included.

 

 

 

Author Response

Comment 1: The extensive ncRNA tables currently lack a systematic assignment of literature citations. PMID Number is somewhat cumbersome. A more common practice is to directly assign the specific articles in the literature list by their number.
Response 1: We appreciate this suggestion. In the revised manuscript (Pages 15–24), we have changed the citation format in the ncRNA tables: instead of listing the PMID numbers, we now use the reference list numbers (e.g., [23], [45]) to indicate the source articles. In addition, we shortened the tables by presenting only representative ncRNAs in the main text, while providing the complete versions in the Supplementary Material. These revisions enhance clarity, readability, and consistency.

 

Comment 2: The demonstration of the fundamental mechanisms in three relatively simple figures is currently inadequate. Additional figures with more graphic information, particularly those focusing on the function of the most important ncRNAs, should be included.

Response 2: We thank the reviewer for this helpful suggestion. While we did not include additional figures dedicated solely to ncRNAs, in the revised manuscript we added a new figure in Section 4 (EMT in OS) that illustrates EMT-mediated mechanisms of metastasis and therapy resistance in osteosarcoma. This new figure provides more graphic information to complement the existing ones and improves the overall clarity of the review.

Reviewer 3 Report

Comments and Suggestions for Authors

The article provides a broad review of the role of EMT (epithelial-mesenchymal transition) in the metastatic progression of osteosarcoma, with particular reference to pulmonary colonization. Particularly relevant is the description of cellular plasticity, which is not limited to the epithelial/mesenchymal dichotomy, but includes hybrid states capable of combining invasiveness, drug resistance and stem potential.

The complex network of signals that converge in the activation of EMT-related transcription factors (Snail, Slug, Twist, ZEB1), mediated by key pathways such as TGF-β/Smad, MAPK, PI3K/Akt, STAT3 and Wnt/β-catenin, is highlighted. Also of interest is the evidence of multilevel regulation by ncRNAs (miRNAs, lncRNAs, circRNAs), which modulate both EMT induction and therapeutic response, opening up prospects for targeted RNA-based therapy interventions.

The in-depth study of the interaction between EMT and the tumor microenvironment is interesting: hypoxia, tumor-associated fibroblasts (CAFs), M2 macrophages and inflammatory signaling pathways (COX-2/STAT3, HMGB1-RAGE) create a favorable context for metastatic progression and chemoresistance.

The review highlights the possibility of combining conventional drugs with EMT inhibitors or specific pathway modulators (e.g. TGF-β, Wnt/β-catenin, STAT3), as well as with natural molecules already tested in preclinical models (oridonin, pectolinarigenin, ginsenosides, flavonoids), approaches that could enhance sensitivity to chemotherapy and reduce the formation of lung metastases, which remain the main negative prognostic factor.

In my opinion, the article can be published.

Author Response

Comment: In my opinion, the article can be published.

Response: We sincerely thank the reviewer for the positive evaluation and supportive comments. We are encouraged that the manuscript was considered comprehensive and suitable for publication.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In the revised version of the manuscript, the authors have addressed almost all necessary points. The text is more concise and the presentation more clear. I still believe the ext should further elaborate more on the key concept of epithelial to mesenchymal transition in an already mesenchymal tumour. The authors have introduced a short comment in lines 284-286. I suggest that any information on possible morphological changes in OS cells during the molecular processes that fall into the category of EMT is included in the manuscript. If no changes are observed, it should be made clear to the reader that the findings are only molecular (consider a new title). In Figure 4, the epithelial morphology depicted should thus be removed. 

Author Response

Comment :
I still believe the text should further elaborate on the key concept of epithelial-to-mesenchymal transition in an already mesenchymal tumour. I suggest that any information on possible morphological changes in OS cells during the molecular processes that fall into the category of EMT is included in the manuscript. If no changes are observed, it should be made clear to the reader that the findings are only molecular (consider a new title). In Figure 4, the epithelial morphology depicted should thus be removed.

Response :
Thank you very much for this valuable comment. Reports of “EMT in osteosarcoma” have so far been defined mainly on the basis of molecular and functional indicators, such as decreased expression of E-cadherin, increased expression of mesenchymal markers including N-cadherin and vimentin, and activation of EMT-related transcription factors such as Snail, ZEB1, and Twist. In other words, EMT in OS is not understood as a phenomenon accompanied by morphological changes, but rather as one characterized by alterations in molecular markers and cellular properties, including invasiveness, motility, and therapy resistance.

We have clarified this internationally recognized understanding in the manuscript, revising the text in Section 4 (“EMT in OS”, page 9, lines 281–286) to avoid any misinterpretation regarding the definition of EMT. In addition, the epithelial morphology previously illustrated in Figure 4 has been removed, and the figure has been revised to focus on the signaling pathways and functional implications of EMT. The figure legend has also been updated accordingly to emphasize that EMT-like changes in OS are molecular and functional rather than morphological.

With these revisions, we believe that the concept of EMT in osteosarcoma, as well as its research significance, is now presented more accurately and clearly.