Microgravity as an Anti-Metastatic Agent in an In Vitro Glioma Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors presented an interesting study, determining the effect of microgravity on glioma cells, which is a relatively unexplored area of research. This originality can contribute to a better understanding of how gravity affects cancer cell behavior.

There are some issues that could be clarified, i.e., the Authors indicate the anti-metastatic impact of microgravity on glioma cells. It is difficult to justify this, as the study was performed only on one cell line. To determine anti-metastatic efficacy, a normal cell line should be included. 

Based on the obtained results, try to clarify mechanisms underlying the observed alterations in GFAP, Vinculin, and Connexin-43. Providing insights into the molecular pathways involved would improve the clarity of the study.

While the study is informative about cell culture responses, the translation of these findings to in vivo conditions is unclear. Additional clarification or prediction could bridge this gap and provide a more comprehensive understanding of glioblastoma cell behavior in microgravity.

Please determine the limitations of the study, particularly in the case of in vivo studies,  sample size, and mechanistic insights, which should be addressed in the discussion/conclusions. 

Additionally there is a lack of statistical analysis for cell death, and fig.2 has no description of Y axis. 

some minor remarks; 

line 127 - Na3 citrate should be Na3C6H5O7?

line 262- is mormogravity - should be normogravity

 

Author Response

The changes are highlighted in the text: the new sentences inserted are in green

the deleted sentences are in yellow

• Consideration of the metastatic nature of C6 cells is a prerequisite originating from numerous experimental studies that have validated the C6 cell line as the gold standard for glioblastoma research. Although these cells were developed in Wistar rats, their morphology and physiology are very similar to those observed in human glioblastoma. In particular for our purpose, it has been observed that the C6 cells have a high aggressive behaviour towards surrounding tissue. Therefore, we assumed this known ability as an experimental model without further verification. An explanatory note has been inserted at the end of Introduction section.
• A summary paragraph where an insight into molecular pathways involved underlying the observed alterations in GFAP, Vinculin, and Connexin-43, has been detailed in the final part of the Discussion section.
• The same sentence illustrating the translation of our in vivo findings has been inserted at the end of discussion section and in the limits of the study.
• The paragraph on the limitations of the study has been implemented according to the referee’s suggestions 
• Y axis description has been inserted in Figure 2. The statistical analysis of cell death has been detailed.
• Minor remarks have been corrected.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The article “Microgravity as Anti-Metastatic Agent in an in Vitro Glioma Model” by Sabbatini et al. analyzed the effect of microgravity environment on C6 glioblastoma cell survival, cytoskeletal organization and expression of some specific proteins, involved in cytoskeleton dynamics and intercellular interactions. By using a 3D-Random Positioning Machine, in order to reproduce the effect of microgravity in cell cultures, Authors observed that the microgravity exposure up to 72 h induced a reduction of cell proliferation, an increase of the number of necrotic cells and some alterations related to the expression/distribution/organization of cytoskeletal elements.

Some revisions are required for acceptance.

 

3.2. Cell Death

Analysis of apoptosis was performed based on labelling with PI as described in Riccardi and Nicoletti, Nat. Protoc. 2006. As shown in Figure 2, exposure to microgravity did not induce apoptosis; after 72 h, only a little increase of necrotic population was observed. Nevertheless, cell proliferation was clearly reduced at 48 and 72 h of microgravity exposure.

- To ensure the absence of apoptosis induction, I suggest another assay to analyze apoptotic population, as Annexin V/propidium iodide labelling and flow cytometric analysis.

 

3.3. GFAP

In this paragraph, Authors reported “Western blot analysis showed that GFAP amount is reduced after 24h of microgravity exposure,….. Instead, at 48h and 72h of microgravity exposure the content of GFAP increase largely, also if their morphological appearance is highly irregular (Fig. 3G,H)” (lines 225-230).

- Actually, the increase of GFAP is well evident only at 48h, there is no difference in GFAP expression between normo- and microgravity condition at 72h. In addition, the meaning of the sentence “also if their morphological appearance is highly irregular” is confusing, Authors should better explain: what does “their” refers to? Please, try to change the expression “appearance irregular”.

3.4. Vinculin

Regarding vinculin alterations, Authors reported “Following C6 exposure to microgravity, we have observed after 24h an increase in number of vimentin-positive focal contact (Table 1)” (lines 251-252).

- I suppose that this result refers to vinculin, not to vimentin.

- From data reported in table 1, at 24 h the number of vinculin spots in normogravity is very similar to that one in microgravity, Authors should rewrite the sentence.

 

 

Author Response

The changes are highlighted in the text: the new sentences inserted are in green,

the deleted sentences are in yellow

3.2 Cell Death

Cell death has been reanalysed by Annexin V/propidium iodide labelling and flow cytometric analysis, according to referee suggestion. The new data is shown in Figure 2 and Table 1.

3.3 GFAP

The paragraph has been corrected in the points indicated: the sentence has been rewritten and the expression changed and explained.

3.4 Vinculin

The paragraph has been corrected in the points indicated: the sentence has been rewritten and the term corrected.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors have improved their manuscript, however some parts still require corrections/revisions e.g:

 

• line 92-93 - place only reference [25] instead of [for a review see 25]. 
• Figure 1 - y axis - there is "cell numer", and should be "cell number"
• figure 2 - the upper part is not visible, the frame is out of the page, and in the description, it is "Cytofluorimeyric" and should be "Cytofluorimetric". Despite this, I suggest combining Fig 1 and 2 together; currently, the graphical representation is not representative. 
• line 454 - instead of "limit" place "limitations"

Comments on the Quality of English Language

editing of English language required

Author Response

All reviewer observations have been corrected.

Figure 1 and 2 have been combined together.

English language editing of has been performed throughout the manuscript.