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I have confirmed that the authors have met all the requirements suggested by the reviewers and the manuscript is ready for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
Please address minor issues marked by reviewers, which requires changes in text, figure labeling etc. It will be suitable for publication thereafter.
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response 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 response 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 response letter. Directions on how to prepare a response letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/. #]
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The authors have attempted to address majority of the suggestions provided by the reviewers. The manuscript has thus improved in quality. At the same time, there is still need to refine the text in various places in the manuscript for sentence length, punctuation, connectivity etc. As an example, I suggest the following (with edits incorporated) in some portion of the Abstract.
Melanoma is a common skin tumor that causes a high rate of mortality, especially in Europe, North America and Oceania. Immunosuppressants such as anti-PD-1 have been used in the treatment of malignant melanoma, however, nearly 60% of patients do not respond to these treatments. Sema4D, also called CD100, is expressed in T cells and tumor tissues. Sema4D and its receptor, Plexin-B1, play crucial roles in the process of immune regulation, angiogenesis, and tumor progression. Role of Sema4D in melanoma with anti-PD-1 resistance is poorly understood. Through a combination of molecular biology techniques and in silico analysis, the role of Sema4D in improving anti-PD-L1 sensitivity in melanoma was explored. The results showed that the expression of Sema4D, Plexin-B1 and PD-L1 was significantly increased in B16-F10R cells.....
Some minor edits needed in other sections:
Line No 66-67 (Word file): the word ‘treatment; is written twice, please correct.
Western blot: … ‘We investigated the expression of PD-L1, Sema4D, Plexin-B1, PI3K, p-PI3K, AKT, p-AKT, these antibodies were purchased from’….
Please edit as … We investigated the expression of PD-L1, Sema4D, Plexin-B1, PI3K, p-PI3K, AKT and p-AKT. Antibodies against these proteins were purchased from….
Figure 1 legend: … ‘Repeat the measurement three times’….It is likely these represent values obtained from three different Western blots, and not repeat measurements of the same Western blot. Please edit appropriately.
Figure 3 legend: Scale marking – please mention as ‘Major marks represent cm on the scale’.. this should suffice.
Please put 'P' in P values in a consistent way throughout the manuscript.
Supplementary data Excel file: Please provide equivalent terms in English for Sheets ‘MTT’, ‘Cell apoptosis’ and ‘Cell invasion and migration’.
Please see above
Please see above
As you can see the reviewers still find several deficiencies in the manuscript, which need to be fulfilled. These are now very specifically listed by the two reviewers. Please go through all these and rectify these issues.
Overall, the manuscript is improved. The authors addressed some of the points that have been raised except a few listed below.
From the body of data that has been added, it seems that nivolumab crossed react with murine PD-1 although authors did not provide binding assay to confirm the affinity of this antibody.
Author still haven’t investigated the source of Plexin-B1 that interacts with Sema4D on the tumor cells, mediating PD-1 resistance effect.
Line 49-50: precise what type of treatment
Line 62: what type of cancer?
These two paragraphs should be combined together
<<Sema4D and Plexin-B1 overexpression in nivolumab resistance B16-F10 cells
To investigate the mRNA and protein expression of Sema4D and Plexin-B1 in B16-F10R and B16-F10 groups, Sema4D and Plexin-B1 were detected. Compared with B16-F10 group, mRNA and protein expression of Sema4D and Plexin-B1 were significantly overexpressed in B16-F10R group (Figure 1A-E).
PD-L1 overexpression in nivolumab resistance B16-F10 cells
mRNA and protein expression of PD-L1 in B16-F10R and B16-F10 cells were detected. Compared with B16-F10 group, mRNA and protein expression of PD-L1 were significantly overexpressed in B16-F10R group (Figure 1A, F-G).>>
Line 251: Please explain what is the method and what is the source of database?
Figure 3: What technique and markers used to detect apoptosis? what is the X axis (Alexa674) of the dot plot?
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The authors have attempted to revise the manuscript entitled ‘Sema4D silencing increase the sensitivity of nivolumab to B16-F10 resistant melanoma via inhibiting the PI3K/AKT signaling pathway’, to address queries and suggestions provided by reviewers in first round of the review process. However, overall, the manuscript still needs to be edited adequately in order to achieve a publication-standard language, and address certain experimental points raised.
In the title of the manuscript ‘Sema4D silencing increase the sensitivity of nivolumab….’ Should be ‘Sema 4D silencing increases the sensitivity of nivolumab…..’.
Some sentences in the abstract still need to be rephrased/reworded to clearly convey the findings, and minor grammatical errors and punctuations be corrected.
Similarly, in the Introduction section (particularly in paragraphs 2, but also in 3, 4 and 5), the language and punctuations need to be corrected to make a clear understanding of the text and facts presented.
Many portions in the manuscript, as noted by the initial reviewers, still need complete rephrasing to make the correct meaning/interpretation. The methods section should include sufficient detailing of the procedures. The figure legends should adequately describe the experiment and data. The language and punctuation need necessary corrections throughout the manuscript.
Reviewer observations and comments in response to authors’ revised manuscript:
Please find below observations based on the initial reviewers’ suggestions and comments and their responses provided by the authors:
(Reviewer 1)
Reviewer query regarding interaction between Sema4D and Plexin-B1:
Yes, it is important to describe in the first place where Plexin-B1, in context of a receptor for Sema4D, is expressed, and the interaction between the two molecules.
Plexin-B1 knock down and use of more than one cell line:
The response from authors did not address the query raised by the Reviewer. Yes, at least an additional cell line would have strengthened the validity of the findings.
Figure legends - Methods, experiment design, and the types of data which are presented:
The methods and experimental designs need to be further elaborated, particularly for legends of Figures 1, 2, 4 and 5. In all figures legends where data is presented from replicate experiments (such as bar graphs), and a P value is derived and shown, please include the number of replicates (n) and the statistical test used to derive the P value.
Figure 1:
PD-L1 expression is data is added in Figure 1.
Point No 1 for Figure 1: Title of Figure is corrected.
Point No 2 for Figure 1: Suggested correction is made.
Point No 3 for Figure 1: Suggested correction is made.
Silencing Plexin-B1 and its effect on PD-L1 expression:
Author response is not given for reviewer query related to silencing Plexin-B1 and its effect on PD-L1 expression. Reviewer suggestion related to ‘Sema4D silencing’ in place of ‘Sema4D inhibit’…is not amended. It is still ..'inhibit' in the Results section and Figure 2 legend.
Figure 3 title:
Suggested title ‘Sema4D deficiency renders B16-F10R cells sensitive to PD-1 treatment’ has been made. But in the Results section, the title of this experiment is not changed. Please amend.
Figure 4C.
Authors can briefly describe how the invasion chambers were prepared for the assay.
Also, in Methods section, ‘B16-F10R cells invasion and migration was analyzed by Matrigel-coated invasion chambers and scratch assay’ please amend as:
‘B16-F10R cells invasion and migration was analyzed by Matrigel-coated invasion chambers and scratch assays respectively’.
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(Reviewer 2)
Basic reporting Point 1:
It appears that these sections, in particular, methods, results and figure legends still need further editing, corrections and details.
Experimental design Point 1:
Agree, that IgG would be a suitable control for PD-1 but is not included in the respective experiments.
Validity of findings Figure 3:
Untreated B16-F10 and B16-F10R groups are included by authors.
Figure 3F:
It is advisable to take the images from a suitable distance that can reveal relative differences in tumor sizes in mice, and when isolated from mice. Also, it is important to show a similar range of scale used (such as 0-15 cm) for tumor sizes from the two groups.
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(Reviewer 3)
Experimental design:
Point 2: Regarding knockdown of Sema4D in both sensitive and resistant cell lines:
The response from the authors ‘Therefore, the effect of nivolumab in sensitive cells was found to be consistent with that in insensitive cell lines through overexpression of sema4d’ is not clear /interpretable in the context of the suggestion provided by the previous reviewer.
Point 3: Western blot images of Sema4D:
Western blots of Sema4D are included.
Point 4: Regarding Sema4D knockdown and efficacy by PD-L1 regulation:
Please use correct sentence /wording (not vocalization).
Point 5: Regarding use of appropriate controls:
Authors have included untreated controls in this figure (Untreated B16-F10 and B16-F10R groups).
Point 6: Figure 3F - Regarding proper magnification to capture the tumor images:
As mentioned above, a suitable magnification can reveal relative differences in tumor sizes in mice and when isolated from mice. Also, it is advised to show tumors from both the groups on a similar scale, for example 0-15 cm scale, which would show differences in tumor sizes more clearly. The image in 3(H) (which is not marked as H), Sema4D-NC group image appears to be stretched vertically, which may be distorting tumor shapes/length in this group to some extent.
Point 7: Figure 4 - Controls and tumor microenvironment related:
Authors need to expand on response provided.
Minor revisions: Point 2
Yes, Plexin-B1 is a receptor for Sema4D.
Please see above section.
Additional observations after reviewing the revised manuscript:
Materials and Methods sections:
Western blotting: It is likely that the antibodies against the mentioned proteins were purchased….not the proteins…please correct. Please detail what antibodies were used and at what dilutions or concentrations to detect respective proteins.
RT-qPCR: DNase free Dnase set should be Rnase free Dnase set. Maintain consistent abbreviation …RT-qPCR in the section.
Sema4D overexpression: It is not mentioned how the overexpression of Sema4D was assessed.
MTT: Please mention full form of MTT when used for the first time in the manuscript.
Cell apoptosis analysis: Please include composition of binding buffer. Also mention how the cells were treated, or if a kit reagent was used, then refer to ‘performed as per manufacturer's instructions'.
Animal and tumorigenesis: Units for tumor volume should be mentioned.
All experimental details in Materials and Methods section and all procedures in Figure legends should be mentioned in the 'past tense'.
Results section:
In general, the figure legends should provide more details of the procedure and data description.
Sema4D and Plexin-B1 overexpression in nivolumab resistance in B16-F10:
Figure reference in the results section should be (Figure 1A, B, C, E and F), (not Figure 1A-E).
Also, it should be mentioned how the ratios of overexpressed proteins with GAPDH were obtained, such as by densitometry of protein bands in western blot images.
PD-L1 overexpression in nivolumab resistance B16-F10 cells:
Figure reference should be (Figure 1A, D and G), (not Fig 1A, F-G).
Preferably, in the results section, the above two sections can be combined into one as both these refer to data in Figure 1.
Sema4D ‘deficiency’ may better be mentioned as Sema4D repression or downregulation.
Figure 3: Please mark (H) for images of tumors with scale.
In all experiments, please mention what are the replicates, that is, replicate experiments, replicate samples from cultures, etc., and what do the error bars represent? If it is SD (as given in Supp Excel file) please mention as SD. What statistical test was used to obtain P values?
In ‘Sema4D deficiency potentiates the inhibitory effect of anti-PD-1 on cell invasion and migration’
…. cell migration of Sema4D-shRNA group was wider than those of Sema4D-NC group cells…
May be written as …..’the gap (or cell-free area) between migrating cells from Sema4D-shRNA group was wider than the gap between cells of the Sema4D-NC group….
It is correctly mentioned in Figure 4 legend where this data is presented.
In supplemental data Excel file, all titles should be given in equivalent English terms.
Discussion section:
Nivolumab concentration is mentioned as 50 milligram for obtaining resistant cell lines. Elsewhere in the manuscript it is mentioned 50 nanogram/ml. Please check in the discussion section.
As you can see the reviewers are of the opinion that substantial changes are needed, which will improve this manuscript to a great degree. All authors noted that it's a relevant and interesting topic. As noted by all the reviewers, please include appropriate controls in the experiments related to the use of Nivolumab as this ab is against human PD1 but used in mouse syngeneic tumor models. It will be important to show that this can block PD1 on mouse T cells.
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. #]
Review report
Thank you for the opportunity to review “Sema4D/Plexin-B1 is involved in anti-PD-1 resistance 1 in melanoma via PI3K/AKT signaling pathway” by Zhang et al. The authors attempt to study the role of Sema4D/Plexin-B1 interaction in regulating PD-1 resistance in melanoma.
Even though, the biology question addressed in this article appears to be relevant in the field, this article should be rewritten. Overall, the sentence structures are too long and the vocabularies are poorly used to communicate clear messages. There are many grammatical and spelling mistakes. The result section is not sufficiently described. No rational for the experiments was stated and experiment designs were absent or poorly explained. The discussion section seems to be more of the result section since it only contains the interpretation of the data.
The main finding of this article is that PD-1 resistance in B16-F10R can be the result of Seme4D upregulation and knock down this protein can rescue the sensitivity of these cells to PD-1 treatment. Thus, the title is not accurate. Author did not show the interaction between Sema4D and Plexin-B1. Author did not investigate the source of Plexin-B1 that interacts with Sema4D on the tumor cells, mediating PD-1 resistance effect. It is intriguing that both Sema4D and Plexin-B1 are upregulated in B16-F10R taking into account that it is the interaction between these two molecules that involves in cancer progression. Seme4D is more described to be expressed on immune cells. Can author explain the decision to only investigate Sema4D and not Plexin-B1? Did author look at the effect of Plexin-B1 knock down? The whole study was conducted with only one cell line, authors should validate this finding at least partially using one more cell line.
In all the figure legends, author should briefly describe the method, experiment design, and the types of data which are presented.
Figure 1: Can author show PDL-1 expression in B16-F10 and in B16-F10R? And, the responsiveness of both cell lines to anti-PD-1 treatment evaluated by at least cell viability or cell death.
In the figure title, author used terms that I think is not accurate:
1. “Sema4D/Plexin-B1 expression in cell” should be “Sema4D and Plexin-B1 expression in melanoma cell lines”
2. “divided” should be “derived”
3. “Sema4D/Plexin-B1” should be “Sema4D and Plexin-B1”
Figure 2: Can author show how silencing Plexin-B1 will affect PD-L1 expression?
Author used terms that I think is not accurate:
“Sema4D deficiency inhibit PD-L1 expression” should be “Sema4D silencing downregulates PD-L1 expression in B16-F10R”
Figure 3: Can author overexpress Sema4D in B16-F10 and see if that could reverse the responsiveness of this line to PD-1 treatment. In 3B, what is the X axis of the dot plot? Author has to be consistent with the way he or she presents the datasets in the graph. In 3C. the Sema4D-NC group is in grey, not black as shown in 3A and 3E.
In the figure title, author used terms that I think is not accurate:
1. “Sema4D deficiency potentiates anti-PD-1 treatment efficacy” should be “Sema4D deficiency renders B16-F10R cells sensitive to PD-1 treatment”
Figure 4: What is the experiment design? Were the cells treated with anti-PD-1? The title is not reflecting the finding in that figure. Author did not show that PD-1 treatment inhibited cell invasion and migration, but knock down Sema4D decreases the ability of the cells to migrate.
What is the experiment conducted in 4C?
Can be improved but sufficient to address the biological questions.
Should be improved.
1. Introduction, methods, results, and figure legends are not appropriately described.
1. Appropriate controls are missing. Authors should use IgG control in all the experiments. Comparisons with untreated groups are missing.
1. Figure 3: results for untreated groups are missing. Authors should include a comparison with untreated groups.
2. What is the rationale for using Balb/c mice for the animal experiment? B16-F10 cell line was origeneted from C57BL/6.
3. Figure 3F: mouse and tumor images should be captured from a distance.
The authors have described the role of Sema4D/Plexin-B1 in the anti-PD-1 treatment resistance in melanoma cells. Authors have identified PI3K/Akt pathway as a downstream target of Sema4D/Plexin-B1 in the resistance development to anti-PD1 therapy.
Major revision:
1. Authors have used nivolumab antibody which is an FDA-approved human specific PD1 blocking antibody. The B16F10 cell line model used in the study is syngeneic mouse cells. Authors should show the use of nivolumab to develop resistance is an appropriate and best model considering their different background/origin. Authors can show that nivolumab is causing cell death upon treatment.
2. The knockdown of Sema4D should have done in both sensitive and resistant cells and then check the cell viability and apoptosis to understand the effects of knockdown between these cells.
3. Authors have not shown the western blot of Sema4D in the cells.
4. Figure 2 shows that Sema4D knockdown results in the downregulation of PD-L1 expression. In the figure 3, authors show that Sema4D knockdown improves PD-1 treatment efficacy. Is the Sema4D knockdown improves the efficacy by PD-L1 regulation or it is the combination of both these effects?
5. Throughout the manuscript, appropriate control(s) were not used in the study. Using appropriate/suitable controls would have strengthened the outcome. Figure 3 authors have shown that anti-PD1 treatment efficacy was improved with Sema4D knockdown but they failed to show what was the response in Sema4D-NC and Sema4D-shRNA only groups. Please include the untreated control groups throughout the manuscript.
6. Figure 3F, please use proper magnification to capture the tumor images.
7. In the Figure 4, controls are missing. Could you please explain what would be the response in tumor cells upon anti-PD1 treatment in the absence of tumor microenvironment? Should cancer cells respond to anti-PD1 in the absence of immune and microenvironment partners?
8. Authors have shown that PI3K/Akt pathway is affected by Sema4D however, western blot does not show much of difference. Have you looked at other identified pathways, such as Jak-STAT, P53 or KRAS signaling, etc?
Minor revisions:
1. Authors are requested to appropriately write the manuscript sections.
2. Is Plexin-B1 is a ligand of Sema4D or it is vice versa?
The authors should have used proper controls in each experiment to better understand and explain the outcomes.
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