Microbiome Dysbiosis Is Associated with Castration Resistance and Cancer Stemness in Metastatic Prostate Cancer

Round 1

Reviewer 1 Report

This manuscript proposes a significant association between the human microbiome and castration resistance in prostate cancer, highlighting the importance of considering microbial factors in understanding the aggressive and invasive nature of the disease. This subject is interesting and follows recent trends about the role of the human microbiome in cancer progression. 

However, the authors attempt to state this conclusion through multi-level correlations, which makes it challenging to prove a direct connection between castration-resistant prostate cancer and microbiome levels. It would be much better to use clinical patient diagnostic and outcome data to find the connection to microbiome levels instead of gene expression since gene expression is only an indicator of disease progress. 

In addition to this major concern, I also have the following main points: 

1.     Multiple potential pathways were considered to be related to castration-resistant mechanisms. Why do the authors hypothesize PI3K/AKT and cancer stemness? Were any other pathways looked at? Were any pathways related to immunity response that had been reported previously? 

2.     Please describe more thoroughly why studying the correlation is necessary. 

3.     Why was only the lymph node cohort plotted in Fig. 3A? What are these species, and is there a specific reason why only twenty species were included in this figure? 

4.     While individually checking each pathway, are there any genes commonly present in cancer stem cell, EMT, and pluripotency pathways? Are these genes the driver that dominates the negative correlation? 

5.     Similar to question number 3, why were only twenty species selected in Fig. 5B?

Concerns related to data presentation: 

1.     Many figures should be reorganized, including Fig. 2A, Fig. 3, and Fig. 5. The figure legend is either too small or has a display error. 

2.     The take-home message of Fig. 2A and 4A needs to be clarified. Another type of plot should be considered to show the main point.

3.     In Fig. 2B, how can the overlap between liver and bone be 11, but the common marker among three metastasis sites is 35? 

4.     For Fig. 2C, why only show the expression of these markers while comparing the levels of two species? Why not plot all common markers? How about other species?

5.     The GSEA plots, e.g., Fig. 3A and 5A, should include enrichment scores and FDR. 

6.     The results of Fig. 3B could be more apparent to the readers. What does this plot represent? Another plot type should be considered. 

Minor concerns: 

1.     The sentence starting with “Through mechanisms…” (line 38) is not accurate. Genomic alterations associated with PCa might impact AR signaling, but this doesn’t mean these genomic changes are caused by AR or AR activity. 

2.     For Fig. 1A, it is hard to differentiate between the colors of each study. 

3.     Normalization and contamination correction are not directly related to the conclusion that the authors want to emphasize in this study. Therefore, including Fig. 1 in the supplementary results would be better.

4.     When the Endocrine Resistance pathway is indicated for the first time in the 2.3 section, a reference should be mentioned.

5.     It is unclear which method was used to calculate the relative gene expression in Fig. 2C and 4C.

6.     The use of “Pluripotency” has been repetitious in the discussion section (lines 237 and 251).

7.     The version of packages used for bioinformatics should be mentioned in the method section.

8.     The description “Standard parameters were chosen” is insufficient for the method section. More details should be provided.

9.     More analysis details about cross-study normalization should be provided.

10.  Consider including the gene lists used in the pathway analyses in the supplementary results.

The discussion section was difficult to follow because the arguments were not presented clearly and cohesively. Additionally, repetitive language and ambiguous sentences made the text harder to understand. It would be helpful to revise and clarify these elements to improve the overall quality of the manuscript.

Author Response

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Reviewer 2 Report

The current manuscript explores the association between microbial dysbiosis and castration resistant, metastatic prostate cancer, as well as the cancer stemness pathways.

How did the authors select literature for cancer stemness markers? 

Did the authors consider the quality of sequencing data? Do cite the source of these datasets appropriately. 

Moderate improvements are needed for materials and methods section to ensure that the work can be replicated. 

Author Response

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Reviewer 3 Report

February 14, 2024

Ms. Ref. No.: ijms-2861827

Journal: International Journal of Molecular Sciences.

Title: Microbiome Dysbiosis Associates to Castration Resistance and Cancer Stemness in Metastatic Prostate Cancer.

Comments:

Thank you for your efforts in composing an article on such a pertinent subject. I have taken the liberty of providing you with a few observations that I believe will serve to enhance the quality of your work. Please find my feedback outlined in the following paragraphs

1-      The RNA sequencing data pertained to biopsies of bone and soft tissue from patients who had metastatic CRPC. What was the reason for selecting bone for the study?

2-      According to this article, only bone (n = 159), lymph node (n = 92), and liver (n = 39) metastases were analyzed. What was the reason for selecting these samples?

3-      Two studies, phs000915 (with a sample size of 147) and phs001141 (with a sample size of 143), were mentioned. Please explain how the sample size was determined?

4-      Is it possible that by these results, generalize the conclusion?

5-      In order to improve the clarity of the introduction, it is recommended that you include some of the following sources as references:

·         https://doi.org/10.3390/ijms25042152

·         https://doi.org/10.1007/s00210-023-02551-0

·         https://doi.org/10.3390/ijms25042054

Author Response

Please see the attachment.

Author Response File: Author Response.docx