Laminin 511-E8 Fragment Offers Superior Adhesion Properties for Gastric Cancer Cells Compared with Full-Length Laminin 511
Round 1
Reviewer 1 Report
In this study, authors indicate that the laminin 511-E8 fragment had impact on the adhesion, morphology, and proliferation of gastric cancer cells. Overall, the method is solid, and the result is reasonable.
Minor comments
- Please justify why authors picked up laminin in GC in this study.
- If authors can, please discuss laminin-E8 expressions is a certain type of GC molecular subtype (https://aacrjournals.org/clincancerres/article/23/15/4441/257504/Clinical-Significance-of-Four-Molecular-Subtypes) and Lauren histologic subtype. In other words, in Figure 7 and discussion section, authors implicated (not clearly) that laminin-E8 might be associated with RhoA and Cdc42. RhoA has been believed to be associated with Lauren diffuse subtype (https://pubmed.ncbi.nlm.nih.gov/27364501/ and more).
- Please justify why authors selected the cell lines. The cell lines are associated with Lauren histology subtypes or any phenotype relevance?
Author Response
COMMENTS FOR THE AUTHOR
Reviewer #1
Comments and Suggestions for Authors
In this study, authors indicate that the laminin 511-E8 fragment had impact on the adhesion, morphology, and proliferation of gastric cancer cells. Overall, the method is solid, and the result is reasonable.
Minor comments
1. Please justify why authors picked up laminin in GC in this study.
Response: Thank you for taking the time reviewing our manuscript. I have revised and added several sentences in the Introduction section to describe why we selected laminin for gastric cancer cells in the present study.
2. If authors can, please discuss laminin-E8 expressions is a certain type of GC molecular subtype (https://aacrjournals.org/clincancerres/article/23/15/4441/257504/Clinical-Significance-of-Four-Molecular-Subtypes) and Lauren histologic subtype. In other words, in Figure 7 and discussion section, authors implicated (not clearly) that laminin-E8 might be associated with RhoA and Cdc42. RhoA has been believed to be associated with Lauren diffuse subtype (https://pubmed.ncbi.nlm.nih.gov/27364501/ and more).
Response: Thank you for your suggestion, we have added one paragraph in the Discussion section to address the relationship between RhoA activity and diffuse type gastric cancers.
3. Please justify why authors selected the cell lines. The cell lines are associated with Lauren histology subtypes or any phenotype relevance?
Response: SH-10-TC cells were established from gastric mucinous adenocarcinoma, MKN74 cells from moderately differentiated tubular adenocarcinoma, and SC-6-JCK and MKN45 cells from poorly differentiated adenocarcinoma. Thus, MKN74 was classified as intestinal type, whereas SH-10-TC, SC-6-JCK, and MKN45 cells were classified as diffuse type according to the Lauren classification.
We have added this information to the Methods section.
Reviewer 2 Report
This study showed that Ln511-E8 fragment had a greater impact on the adhesion, morphology, and proliferation of gastric cancer cells than full-length laminin 511. However, some concerns as following should be addressed.
- The Introduction section did not clear mention and define the Ln511-E8 fragment and laminin (Ln) 111, Ln121, Ln211, Ln221, Ln411, Ln421, Ln511, and Ln521. The numbers did not indicate specific genes of laminins. The readers cannot understand what they are. The authors should provide clear backgrounds on these commercial products. It is also suggested that the mentions for the fragments in the first paragraph of the discussion section should be moved to the Introduction section.
- In the cell proliferation experiments (Figure 2), it is not clear whether the end point (48h) results might be affected by cell size or cell area (shown in Figure 4). It is suggested that the cell proliferation should be analyzed by time course or directly count the cell numbers.
- In Figure 6, it is not clear whether the data are presented from one experiment. No standard deviation was shown. Is there any statistically significant difference among groups?
- In the experiments of Figure 7, the concentrations of treated Cdc42 GTPase inhibitor are so high (100 microM). The effect of high concentration of the inhibitor could not be specific for Cdc42 GTPase activity. The issue for the specificity of this inhibitor should be addressed.
Author Response
COMMENTS FOR THE AUTHOR
Reviewer #2
This study showed that Ln511-E8 fragment had a greater impact on the adhesion, morphology, and proliferation of gastric cancer cells than full-length laminin 511. However, some concerns as following should be addressed.
1. The Introduction section did not clear mention and define the Ln511-E8 fragment and laminin (Ln) 111, Ln121, Ln211, Ln221, Ln411, Ln421, Ln511, and Ln521. The numbers did not indicate specific genes of laminins. The readers cannot understand what they are. The authors should provide clear backgrounds on these commercial products. It is also suggested that the mentions for the fragments in the first paragraph of the discussion section should be moved to the Introduction section.
Response: We appreciate the reviewer’s valuable suggestion. We have revised several sentences in the Introduction.
2. In the cell proliferation experiments (Figure 2), it is not clear whether the end point (48h) results might be affected by cell size or cell area (shown in Figure 4). It is suggested that the cell proliferation should be analyzed by time course or directly count the cell numbers.
Response: We performed additional experiments to explore the issues raised by the reviewers. We directly counted the number of cells using an automated cell counter and added the results as shown in Fig. 3.
3. In Figure 6, it is not clear whether the data are presented from one experiment. No standard deviation was shown. Is there any statistically significant difference among groups?
Response: We obtained data from a single experiment using gene expression array plates (i.e., a single plate per sample, three plates in total). We added a sentence to the legend of Fig. 7 to explain this issue.
4. In the experiments of Figure 7, the concentrations of treated Cdc42 GTPase inhibitor are so high (100 microM). The effect of high concentration of the inhibitor could not be specific for Cdc42 GTPase activity. The issue for the specificity of this inhibitor should be addressed.
Response: We agree with the reviewer’s comment.
We performed additional experiments using Rac1 and Rho inhibitors and have revised Fig. 8 accordingly. In addition, we added several sentences to the Discussion section to describe this issue.
Round 2
Reviewer 1 Report
Authors addressed the comments. No further comments.
Author Response
Reviewer #1
Authors addressed the comments. No further comments.
Response: Thank you for taking the time reviewing our manuscript.
Reviewer 2 Report
The authors have made efforts to revise the manuscript according to the reviewer’s suggestion. One minor issue is that all figures need to be added ticks in the y-axis.
Author Response
Reviewer #2
The authors have made efforts to revise the manuscript according to the reviewer’s suggestion. One minor issue is that all figures need to be added ticks in the y-axis.
Response: We have revised the Figures accordingly.
