Comprehensive Analysis of Innate Immunophenotyping Based on Immune Score Predicting Immune Alterations and Prognosis in Breast Cancer Patients

Round 1

Reviewer 1 Report

In this manuscript, Liu and colleagues did a comprehensive analysis of the public breast cancer datasets and pointed out that CXCL9 may serve as a key innate immune checkpoint for breast cancer immunotherapy, then they also predicted three small compounds targeting CXCL9. However, the authors need do some in vitro experiments to confirm their findings.

Author Response

Dear Editor and Reviewers:

Thank you for taking time out of your busy schedule to review this manuscript. Now we have carefully corrected and replied the manuscript for this revision. The revision instructions are as follows:

Point 1: In this manuscript, Liu and colleagues did a comprehensive analysis of the public breast cancer datasets and pointed out that CXCL9 may serve as a key innate immune checkpoint for breast cancer immunotherapy, then they also predicted three small compounds targeting CXCL9. However, the authors need do some in vitro experiments to confirm their findings.

Response 1: We totally agree with the reviewer and understand that vitro experiments may better and directly confirmed the function of CXCL9 in breast cancer. However, in the present study, we mainly focused on screened and identified the key innate immune checkpoint for breast cancer therapy and prediction of breast cancer prognosis. In addition, we do not have clinical tissue samples from breast cancer patients. Thus, I'm afraid I can't supplement the relevant confirmatory experiments for now. But, we would perform the vitro experiments and nude mouse model in future research for confirmation the function and mechanism of the key checkpoint CXCL9 in breast cancer therapy. Finally, We  deeply grateful for your valuable advice in this manuscript and great helpful for our work.

Reviewer 2 Report

In this manuscript, Weiguang Liu et al. performed a series of comprehensive analysis for public transcriptome data in breast cancer patients, constructed the model of risk prognostic based on the innate-cluster-immune (ICI) score, and identified the candidate innate immune response signatures, which may affect innate immune checkpoint for breast cancer therapy, they . Overall, this manuscript systematically summarized these bioinformatic data and seems to be complete and solid, but most of these data are lack of experimental confirmation. Additionally, to be honest, the manuscript is not well-written, grammatical errors could be observed in many places, it will be great to find someone to polish the English. The authors, however, should consider the following specific comments to further strengthen the manuscript.

1\ In Figure 1A, the authors mentioned that they found that 20 out of the differentially expressed innate immune-related genes could be mapped to the prognostic signatures in breast cancer patients, but only 19 genes were shown. Additionally, are the CNV frequency data (Gain or Loss) in Figure 1C-D consistent with the gene expression difference of the same genes?  the author should provide the number of breast cancer patients in high or low gene expression groups.

2\ In Figure 2B, the survival difference between cluster A and cluster B&C seems to be not that much, can the authors provide the details of statistical method?  Please add the labels of scale bar in Figure 2C.

3\ The font size of labels in most of the figures are too small, which make it very hard to get the details for readers, the author should make some adjustment.

4\ The authors should check the protein level of CXCL9 in breast cancer tissue and normal tissue samples to confirm the mRNA level difference, can perform western blot in several breast cancer patients.

5\ The authors should further separate the breast cancer patients to ER+(Luminal A and B), HER2+, and triple-negative groups based on IHC information, and check if CXCL9 play different roles in specific groups?

6\ Too many main figures in this manuscript, the authors should move some of the main figures to supplementary data.

Author Response

Dear Editor and Reviewers:

Thank you for taking time out of your busy schedule to review this manuscript. Now we have carefully corrected and replied the manuscript for this revision. The revision instructions are as follows:

Point 1: In Figure 1A, the authors mentioned that they found that 20 out of the differentially expressed innate immune-related genes could be mapped to the prognostic signatures in breast cancer patients, but only 19 genes were shown. Additionally, are the CNV frequency data (Gain or Loss) in Figure 1C-D consistent with the gene expression difference of the same genes?  the author should provide the number of breast cancer patients in high or low gene expression groups

Response 1: Firstly, we great thankful for your comments. We have corrected the results. According to the CNV data of breast cancer, we identified 17 out of these 20 selected innate immune signatures and shown in result 1. And  the number of breast cancer patients in high or low gene expression groups also exhibited in overall survival plot for breast cancer patients.

Point 2:  In Figure 2B, the survival difference between cluster A and cluster B&C seems to be not that much, can the authors provide the details of statistical method?  Please add the labels of scale bar in Figure 2C.

Response 2: In figure 2B, the survival probability difference among cluster A, cluster B and cluster C were used chi-square test via R version 4.0.3. We performed GSVA analysis through deferentially expressed genes between cluster A and cluster B&C were shown in Figure 2C. And all of these KEGG enriched pathway were selected via FDR<0.01 and |logFold Change|>1.

Point 3: The font size of labels in most of the figures are too small, which make it very hard to get the details for readers, the author should make some adjustment.

Response 3: we have adjust the font size in all figures.

Point 4: The authors should check the protein level of CXCL9 in breast cancer tissue and normal tissue samples to confirm the mRNA level difference, can perform western blot in several breast cancer patients.

Response 4: We understanding that the in vitro assay for confirmed the the protein level of CXCL9 in breast cancer tissue and normal tissue samples may better support these results. However, in the present research, we mainly focused on the identification of the key innate immune checkpoint for prediction of breast cancer prognosis and the effective of breast cancer therapy. Besides, we performed molecular docking analysis to identified small drug for CXCL9. We totally agree with your comment, but we don’t have breast cancer tissues samples for now. Thus, We can’t add relevant experimental results right now. But, we would performed the in vitro cell experiment and in vivo nude mouse model experiment for further confirmed the function of CXCL9 in breast cancer progress.

Point 5: The authors should further separate the breast cancer patients to ER+(Luminal A and B), HER2+, and triple-negative groups based on IHC information, and check if CXCL9 play different roles in specific groups?

Response 5: To further confirmed the function of CXCL9 in breast cancer, we detected the expression level of CXCL9 in the subtype breast cancer patients, including ER+(Luminal A and B), HER2+, and triple-negative groups.

Point 6: Too many main figures in this manuscript, the authors should move some of the main figures to supplementary data.

Response 6: Indeed, we have re-adjusted these results in this manuscript and some of figures were moved to the supplementary materials.