Microgravity as a Tool to Investigate Cancer Induction in Pleura Mesothelial Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In my opinion, authors should evaluate the expression of some of the adhesion factors and the cytoskeletal organization markers they found modified in their in vitro system, in human mesothelioma samples. They could use IHC and they could use statistical analyses to analyze the expression levels they will find in relation to histopathological and clinical data of the mesothelioma samples.

Comments on the Quality of English Language

English text needs only some revision for typos

Author Response

REF.: In my opinion, authors should evaluate the expression of some of the adhesion factors and the cytoskeletal organization markers they found modified in their in vitro system, in human mesothelioma samples. They could use IHC and they could use statistical analyses to analyze the expression levels they will find in relation to histopathological and clinical data of the mesothelioma samples.

RESPONSE:

Dear reviewer, thank you for your suggestion, the analysis of the expression of the adhesion factors and the cytoskeletal organization markers on different human mesothelioma samples is part of a separate work, which has as a starter point the results of the present work.

Following the tensegrity and mechano-transduction hypothesis, in the present work we intend to analyse the change occurring in cells following the loss of the physiological behaviour of the cytoskeleton and adhesion factors using microgravity as tool to induce these alterations.

The english languages has been revised

Reviewer 2 Report

Comments and Suggestions for Authors

Title: The title is short, concise, and adequate.

Abstract and keywords:

The abstract should be better organized and the division between Background + Methods + Results + Conclusion should be clear.

The keywords are appropriate.

 Introduction: The introduction is well written. However, I recommend that the authors make some suggestions and make some corrections to improve the clarity and precision of the work.

=>The opening sentence of lines 29 to 33 is very long and discusses several important subjects. To make it easier to understand, I recommend dividing the sentences into two or three.

For example:

“Cell cancer transformation originates from both exogenous and intrinsic factors inducing permanent DNA alterations. [this phrase needs a reference] According to established hallmarks, transformed cells exhibit recovered or increased prolificity, up-regulation of metabolism, and loss of the contact inhibition pathways. [this phrase needs a reference] These changes enable cells to invade adjacent tissues and even migrate at a distance, defining their invasive/metastatic behavior [1]”.

            =>In line 41 "complex cytoskeletal alterations". Could you be more specific and give examples of dynamic changes that occur?

=>In line 40 “anoikis”. The sentence should be improved to better explain the term anoikis, as there may be readers who are not familiar with the term.

=>In line 40 until 43: Consider restructuring for brevity the sentence.

=>In line 47: What are the main genetic modifications observed in mesothelioma cells that promote tumor transformation?

How do these specific genetic modifications contribute to the persistence of pro-tumorigenic conditions during the long latency period of mesothelioma?

=>In line 47, the term “might” sounds a little uncertain. If there is substantial evidence, consider using "are likely to be" or "are potential”.

=>In lines 48 until 51, the sentence can be rewritten for clarity as "This slow and late onset of the disease represents a significant clinical challenge for therapeutic intervention [10]”.

=>When the authors transition to the topic of microgravity, the connection to the previous topic could be more direct. For example, something like this: “Given the role of cytoskeletal alterations in mesothelioma, studying the effects of microgravity, a condition known to disrupt cytoskeletal organization, can provide valuable insights”.

What are the exact molecular mechanisms by which microgravity affects cytoskeletal integrity in mesothelial cells?

How can microgravity specifically influence the formation and stability of cell adhesion complexes in mesothelioma cells?

To what extent are microgravity-induced cytoskeletal changes necessary and sufficient to initiate malignant transformation in non-cancerous cells?

I would like to see these three questions answered briefly in a short paragraph incorporated into the introduction. It would be very useful to improve the introduction.

=>In my opinion, the sentence that reflects the objective of the work should be reformulated, to better impact the focus of the work. Something like this: "This study aims to broaden our understanding of cancer cell biology by investigating the role of microgravity in cytoskeletal disruption and its contribution to early cancer transformation".

 Materials and methods:

About Cell culture and simulated microgravity exposure in line 72

Why was the MeT-5A cell line specifically chosen for this study, and how does it compare to other mesothelioma cell lines in terms of biological relevance for microgravity simulation?

Is the BR95 cell line, derived from a patient with malignant pleural mesothelioma, sufficiently representative of other mesothelioma subtypes? What are the potential limitations of using this single-cell line?

When the authors establish the microgravity simulation protocol used (Random Positioning Machine), does it faithfully reproduce microgravity conditions? What are the possible limitations of this technique?

Has the formation of cell clusters in the supernatant been quantified or characterized in any way? Could this provide additional information about cytoskeletal changes?

 About Immunocytochemistry (ICC) and Confocal Laser Scanning Microscopy (CLSM) in line 91

Have the antibodies used by the authors been validated for the species and antigens in question?

Why use the Triton X-100 and not the Tween-20?

 About Western Blot analysis in line 109

Why did the authors use α-tubulin?

  Results: They are presented and described. The results are supported by figures.

In line 172 you must not start a sentence with an abbreviation, you must always write the abbreviation in full when starting a sentence.

What criteria are used to define cells as "CLUMP cells"?

Why were the actin, vinculin, connexin-43, CD44, fibulin-3, p27, and NANOG genes chosen for analysis? What is their relationship with the biological processes investigated?

The p-value must always appear in italics. (p<0.05) 

Discussion: The Results are discussed in this section. However, some issues can be improved.

How do the observed changes in the cytoskeleton and cell adhesion relate to tumor progression and metastasis?

Can the observed changes be used as biomarkers for disease diagnosis or prognosis?

Conclusion: The conclusions are adequate and supported by the Results. However, the conclusions could be more specific, mentioning the proteins and molecular mechanisms involved in the observed changes. And I would add a sentence about the applicability of this essay.  

References: The list of references is adequate. 

Author Response

Abstract and keywords:

REF.: The abstract should be better organized and the division between Background + Methods + Results + Conclusion should be clear.

RESP.: The abstract has been organized as suggested by reviewer.

 

 Introduction: The introduction is well written. However, I recommend that the authors make some suggestions and make some corrections to improve the clarity and precision of the work.

REF:: The opening sentence of lines 29 to 33 is very long and discusses several important subjects. To make it easier to understand, I recommend dividing the sentences into two or three. For example:“Cell cancer transformation originates from both exogenous and intrinsic factors inducing permanent DNA alterations. [this phrase needs a reference] According to established hallmarks, transformed cells exhibit recovered or increased prolificity, up-regulation of metabolism, and loss of the contact inhibition pathways. [this phrase needs a reference] These changes enable cells to invade adjacent tissues and even migrate at a distance, defining their invasive/metastatic behavior [1]”.

RESP.: The sentence has been re-organized as suggested by reviewer. References has been added where requested.

            REF.: In line 41 "complex cytoskeletal alterations". Could you be more specific and give examples of dynamic changes that occur?

RESP.: The main pathways determining cytoskeletal alteration has been declared (lines 44-46)

REF.:  In line 40 “anoikis”. The sentence should be improved to better explain the term anoikis, as there may be readers who are not familiar with the term.

RESP.: The term anoikis has been explained (lines 40-41) as suggested by reviewer.

REF.:  In line 40 until 43: Consider restructuring for brevity the sentence.

RESP.: The sentence has been restructuring for brevity as suggested by reviewer (lines 42-44)

REF.:  In line 47: What are the main genetic modifications observed in mesothelioma cells that promote tumor transformation? 

RESP.: The main genetic modifications observed in mesothelioma have been declared (lines 52-53 )

REF.:   How do these specific genetic modifications contribute to the persistence of pro-tumorigenic conditions during the long latency period of mesothelioma?

RESP.: An explicative sentence has been added (Lines 55-58)

REF.:   In line 47, the term “might” sounds a little uncertain. If there is substantial evidence, consider using "are likely to be" or "are potential”. 

RESP.:  The change has been executed as suggested by reviewer

REF.:   In lines 48 until 51, the sentence can be rewritten for clarity as "This slow and late onset of the disease represents a significant clinical challenge for therapeutic intervention [10]”.

RESP.: The sentence has been rewritten as suggested by reviewer

REF.:   When the authors transition to the topic of microgravity, the connection to the previous topic could be more direct. For example, something like this: “Given the role of cytoskeletal alterations in mesothelioma, studying the effects of microgravity, a condition known to disrupt cytoskeletal organization, can provide valuable insights”.

RESP.: The sentence has been added to the text as suggested by the reviewer (lines 69-71)

REF.:   

1. What are the exact molecular mechanisms by which microgravity affects cytoskeletal integrity in mesothelial cells?
2. How can microgravity specifically influence the formation and stability of cell adhesion complexes in mesothelioma cells?
3. To what extent are microgravity-induced cytoskeletal changes necessary and sufficient to initiate malignant transformation in non-cancerous cells?

I would like to see these three questions answered briefly in a short paragraph incorporated into the introduction. It would be very useful to improve the introduction.

RESP.: The sentence has been added to the text as suggested by the reviewerThe following paragraph was added to introduction (lines 71-78)

According with Tensegrity hypothesis, the cytoskeleton has been indicated as the structure involved in mediating the transfer of mechanical signals from the cell surface to the nucleus and consequence activation of biochemical pathways. Microgravity has been observed altering the mechanical-tensile forces consequently altering the physiological polymerization of cytoskeleton and the expression of adhesion molecular pattern. In our work we have explored if these dynamics can induce immortalized mesothelial cells to cancerous transformation and at what extent this change may initiate a malignant transformation.

REF.:   In my opinion, the sentence that reflects the objective of the work should be reformulated, to better impact the focus of the work. Something like this: "This study aims to broaden our understanding of cancer cell biology by investigating the role of microgravity in cytoskeletal disruption and its contribution to early cancer transformation".

RESP.:  The change has been made as suggestion of reviewer (Lines 90-92)

 

Materials and methods:

REF.:   About Cell culture and simulated microgravity exposure in line 72

Why was the MeT-5A cell line specifically chosen for this study, and how does it compare to other mesothelioma cell lines in terms of biological relevance for microgravity simulation?

RESP.:  The following sentence has been added to text (lines 99-103)

As a very large used cellular model of non-cancerous mesothelial cell line the  MeT-5A cell line was specifically chosen for the present study. These cell line has been immortalized and can guaranteed the resistance and proliferation during the mechanical experimental procedure that could damage other cellular lines.

REF.:   Is the BR95 cell line, derived from a patient with malignant pleural mesothelioma, sufficiently representative of other mesothelioma subtypes?

RESP.:  The following sentence has been added to text (lines 107-109)

 The BR95 cell line was chosen as malignant pleural mesothelioma cells due to their epithelioid nature, that make them morphologically comparable to MeT-5A.

Furthermore in the chapter Limitation and Perspective a sentence has been added on the representative limits of BR)% cells (lines 497-499)

 

REF.:   What are the potential limitations of using this single-cell line?

RESP.:  The following sentence has been added to Limitation and Perspective chapter

The use of a single cell line MeT-5A as experimental group and BR95 as malignant counterpart suffer of the primary limitation that our data can be ascribed to these cell model, but not extensively assured to other similar cells and therefore confined the result to response of defined cells only. In particular the use of MeT-5A cell flattened in their morphology and the BR95 mesothelioma  cells epithelioid in nature address our investigation mainly to epithelioid type of meningioma.

 

REF.:   When the authors establish the microgravity simulation protocol used (Random Positioning Machine), does it faithfully reproduce microgravity conditions? What are the possible limitations of this technique?

RESP.:  The reliability of RPM is discussed in Limitation and Perspective chapter in Lines (498-509)

 

REF.:   Has the formation of cell clusters in the supernatant been quantified or characterized in any way? Could this provide additional information about cytoskeletal changes?

RESP.:  The formation of cell cluster by MeT-5A was not expected, but is not surprising, because other cell lines, following microgravity exposition, have formed cell cluster. A specific characterization of cell cluster was considered don’t be a focus of the present work.

We have interpreted the formation of cell cluster as a peculiar consequence of cytoskeletal alteration and loss of adhesion profile factors. A comment on this point is reported in lines 397-408

 REF.:   About Immunocytochemistry (ICC) and Confocal Laser Scanning Microscopy (CLSM) in line 91

Have the antibodies used by the authors been validated for the species and antigens in question?

RESP.:  The antibodies used has been consider validated by the selling company, the use of the same inother experiments performed in our laboratory, have consolidate the use of the antibodies for our purpose.

 

REF.:   Why use the Triton X-100 and not the Tween-20?

RESP.:  Our experience with the protocol we have adopted has not shown any significative differences between the two compounds. My personal experience indicates that Triton X-100 is easier to manage in terms of concentration and performance.

 

 About Western Blot analysis in line 109

REF.:   Why did the authors use α-tubulin?

RESP.:  The α-tubulin was used as most stable signal in microgravity experiments. A comment on this point is reported in lines 167-170

 

  Results: They are presented and described. The results are supported by figures.

REF.:   In line 172 you must not start a sentence with an abbreviation, you must always write the abbreviation in full when starting a sentence.

RESP.:  The correction has been performed

REF.:   What criteria are used to define cells as "CLUMP cells"?

RESP.:  Clump cells was identified as floating group of five or more cells collecting cell. The indication has been added in line 204

 

REF.:   Why were the actin, vinculin, connexin-43, CD44, fibulin-3, p27, and NANOG genes chosen for

RESP.:  Actin, vinculin, connexin-43  are factors widely explored in experiments where the cytoskeleton integrity and the adhesion properties of cell are the focus of the investigation.

Fibulin-3, p27, were investigate as tumor marker. Nanog and other gene were investigated as gene involved in the Epithelial-mesenchymal transition. Specific indications on the importance of each specific factor investigated have been written at the beginning of the presentation paragraph in Results section

- actin (Line 235-236 )

- vinculin (Line  276-278)

- connexin-43 (Line  294-296)

- fibulin-3  (Line  337-340)

- P27  (Line 340-342)

- NANOG (Line 362-370)

Other informations have been reported in Discussion section also (for example fibulin-3: line 453-458)

REF.:   The p-value must always appear in italics. (p<0.05) 

RESP.: The correction has been performed

 

Discussion: The Results are discussed in this section. However, some issues can be improved.

REF.:   How do the observed changes in the cytoskeleton and cell adhesion relate to tumor progression and metastasis?

RESP.: In Discussion section we have presented our comments by discussing separately the individual factors and marker investigated in the present work in relation to tumor progression and metastasis. This may have probably caused a confusion about the collective action of the same parameters in tumor progression and metastasis. A conclusive resuming paragraph has been added (Lines 491-498)

 

REF.:   Can the observed changes be used as biomarkers for disease diagnosis or prognosis?

RESP.:  A sentence has been added on this point (Line 499-505)

 

Conclusion: The conclusions are adequate and supported by the Results.

REF.:   However, the conclusions could be more specific, mentioning the proteins and molecular mechanisms involved in the observed changes.

RESP.:  A sentence to comment this point has been added (Line 570-580)

 

REF.:   And I would add a sentence about the applicability of this essay.  

RESP.: The applications and future perspectives of the assay presented in this work have been discussed and presented in Limitations and Perspectives section. Now the paragraph has been moved at the end of Conclusion section

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In my opinion, the absence of in vivo data, even preliminary, greatly reduces the scientific value of the article. I suggest that the authors generate at least some in vivo data to support their in vitro observations.

Author Response

Referee: In my opinion, the absence of in vivo data, even preliminary, greatly reduces the scientific value of the article. I suggest that the authors generate at least some in vivo data to support their in vitro observations.

Response:  

Dear reviewer,

thank you for your interesting comment, but, as I explained to a previous reviewer, in vivo experiments will be organized for a future work. Applying microgravity protocols to cells and injecting them into an experimental animal model to observe growth takes time, and currently it cannot be performed in our laboratory. The present work is intended to be only a preliminary explorative work based on in vitro experiments, offering indications on which parameters can be explored for further in vitro functional and in vivo experiments

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

I understand the authors response to the comments. Please, include at least a paragraph in the Discussion section to describe possible in vivo experiments to perform in order to make the reader more easy to understand the potential clinical value of the data presented.

Comments on the Quality of English Language

Check the text for few typos.

Author Response

REF.: I understand the authors response to the comments. Please, include at least a paragraph in the Discussion section to describe possible in vivo experiments to perform in order to make the reader more easy to understand the potential clinical value of the data presented.

Resp.:  Following the indication of the referee suggestion, one paragraph in the discussion section has been added, to describe possible in vivo experiments (pag. 15, lines 508-525).

In lines 574-587,  a further develop of clinical potentiality of the present research was previously indicated

English language has been revised