Exploring the Microbiome in Breast Cancer: The Role of Fusobacterium nucleatum as an Onco-Immune Modulator

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review discusses microbiome in breast inflammatory and cancerous conditions: The role of Fusobacterium nucleatum as Onco-Immune modulator. Overall, it addresses an important topic, which is the role of the microbiome in breast cancer, specifically addressing the onco-immune modulatory role of F. nucleatum, intending to aid in the development of personalized anticancer therapy. However, several areas need major improvement before publication.

 Major Comments

The manuscript does not clearly define the specific knowledge gap it aims to fill. A more focused introduction is needed to explain what has been covered in recent reviews and how this review differs or adds new value. Without this context, the novelty and contribution of the paper remain unclear. The authors should also critically discuss recent literature to position their review within the current landscape.
Needs more critical analysis on the onco-immunomodulatory role of F. nucleatum. In the abstract, the manuscript stated a “deeper understanding of nucleatum biology and its interactions with host cells and co-existing symbiotic microbiota could aid in the development of personalized anticancer therapy”, which is also partly the aim of this review. However, the manuscript hasn’t included evidence from patients or animal model studies that support the potential role F. nucleatum in cancer immunotherapy, radiotherapy resistance, chemotherapy resistance and, overall effect on tumour response to treatment. Thus, I suggest including recent studies that support the objective of this review.
Add a figure about the Onco-Immunomodulatory role of breast cancer, considering the ideas stated in the review, like how it induces the proinflammatory microenvironment, manipulates antitumor immunity and the range of chemokines and cytokines involved. You can create a figure using BioRender to present a good summary of the Onco-Immunomodulatory role of nucleatum in breast cancer.

You should refer to the recent reviews about the microbiome and cancer to rewrite the contents discussed in the review. Other updated reviews should be included so you can increase the value of your review by filling the gaps that are not discussed in these papers or incorporating updated information.

Lack of flow: For example, 1) on page 2, the paragraph about the diversity of microbiome in breast cancer should come before the paragraph that discuss about the determined bacteria in breast tissues, 2) dedicate a paragraph about the overview of the bacterium, nucleatum, before introducing its onco-Immunomodulatory role.
No strong evidence of direct DNA damage in host cells for some bacteria listed. So, specifically list the bacteria that cause direct or indirect DNA damage in line 487, paragraph 7, page 11.
Lack of source: add to some very informative paragraphs, like paragraph 4 on page 7.
The evidence you included to support how the microbiome affects the molecular alterations in breast cancer is not enough. Add more paragraphs to this section.

Minor Comments

The word "investigating" implies original research, which is usually not the primary aim of a review paper. So instead of starting with “investigating,” use more declarative or descriptive language to modify your title.
It's recommended to spell out "breast cancer" on first mention, and if using an abbreviation afterward, use BC with a clear definition in parentheses.
nucleatum is not italized in many places
Fusobacterium nucleatum (F. nucleatum)- this abbreviation should be indicated/used when you start to use it for the first time in the manuscript on page 2, not page 12
Make sure to write the whole manuscript in the same font size

 

Author Response

Response to the reviewer 1

This review discusses microbiome in breast inflammatory and cancerous conditions: The role of Fusobacterium nucleatum as Onco-Immune modulator. Overall, it addresses an important topic, which is the role of the microbiome in breast cancer, specifically addressing the onco-immune modulatory role of F. nucleatum, intending to aid in the development of personalized anticancer therapy. However, several areas need major improvement before publication.

 We thank the reviewer for the valuable suggestions.

Major Comments

1. The manuscript does not clearly define the specific knowledge gap it aims to fill. A more focused introduction is needed to explain what has been covered in recent reviews and how this review differs or adds new value. Without this context, the novelty and contribution of the paper remain unclear. The authors should also critically discuss recent literature to position their review within the current landscape.
   Needs more critical analysis on the onco-immunomodulatory role of F. nucleatum. In the abstract, the manuscript stated a “deeper understanding of nucleatum biology and its interactions with host cells and co-existing symbiotic microbiota could aid in the development of personalized anticancer therapy”, which is also partly the aim of this review. However, the manuscript hasn’t included evidence from patients or animal model studies that support the potential role F. nucleatum in cancer immunotherapy, radiotherapy resistance, chemotherapy resistance and, overall effect on tumour response to treatment. Thus, I suggest including recent studies that support the objective of this review.

We modified the introduction to address the dual role of Fusobacterium nucleatum as an oncogenic bacterium but also an immunomodulatory one (highlighted in red).

According to reviewer suggestion, we have added recent literature on the role of Fusobacterium nucleatum in breast cancer to that already present in the manuscript. We also added in introduction the sentence “Recent papers reported the role of F.nucleatum in promoting breast cancer progression in patients and in animal models “

2. 
   Add a figure about the Onco-Immunomodulatory role of breast cancer, considering the ideas stated in the review, like how it induces the proinflammatory microenvironment, manipulates antitumor immunity and the range of chemokines and cytokines involved. You can create a figure using BioRender to present a good summary of the Onco-Immunomodulatory role of nucleatum in breast cancer.

 

We added a figure (Figure 3) about the onco-Immunomodulatory role of Fusobacterium nucleatum

You should refer to the recent reviews about the microbiome and cancer to rewrite the contents discussed in the review. Other updated reviews should be included so you can increase the value of your review by filling the gaps that are not discussed in these papers or incorporating updated information. 

We have updated the introduction.

3. Lack of flow: For example, 1) on page 2, the paragraph about the diversity of microbiome in breast cancer should come before the paragraph that discuss about the determined bacteria in breast tissues 2) dedicate a paragraph about the overview of the bacterium, nucleatum, before introducing its onco-Immunomodulatory role

1)The structure of the manuscript is the following: the review begins with the microbiome of healthy breast and milk, then analyses the microbiome perturbations in breast inflammation, such as mastitis, and in breast cancer.

2)A brief introduction to Fusobacterium nucleatum was given at the beginning of chapter 5.

4. 
   No strong evidence of direct DNA damage in host cells for some bacteria listed. So, specifically list the bacteria that cause direct or indirect DNA damage in line 487, paragraph 7, page 11.
   Lack of source: add to some very informative paragraphs, like paragraph 4 on page 7.
   The evidence you included to support how the microbiome affects the molecular alterations in breast cancer is not enough. Add more paragraphs to this section.

We added a paragraph to the section.

Minor Comments

The word "investigating" implies original research, which is usually not the primary aim of a review paper. So instead of starting with “investigating,” use more declarative or descriptive language to modify your title.

According to reviewer suggestion, we changed the title of the manuscript in:Exploring the Microbiome in breast cancer: the role of Fusobacterium nucleatum as onco-immune modulator.

It's recommended to spell out "breast cancer" on first mention, and if using an abbreviation afterward, use BC with a clear definition in parentheses.
nucleatum is not italized in many places
Fusobacterium nucleatum (F. nucleatum)- this abbreviation should be indicated/used when you start to use it for the first time in the manuscript on page 2, not page 12
Make sure to write the whole manuscript in the same font size

We checked.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript contains the comprehensive review of the microbiome in breast cancer and emphasizes the specific role of Fusobacterium nucleatum as the onco-immune modulator. All the references are reliable and review is very interesting. However, it may be accepted for publication after major and minor corrections.

Major revisions:

1. The title does not reflect the manuscript content. First, it is not an investigation but review. Second, the term "Breast Inflammatory Conditions" is too extensive and has not been reviewed comprehensively in this manuscript. The title limited by microbiome, breast cancer and Fusobacterium nucleatum  seems to be more appropriate.
2. Part 2 "Microbiota in Breast Feeding in Healthy and Inflammatory Conditions" is too long due to some excessive parts, for example role of breast milk for newborns and their health. This gives nothing to main idea of the manuscript. So, part 2 is recommended to be shortened substantially.
3. Part 4 describes a specific role of Fusobacterium nucleatum as the onco-immunomodulatory agent in breast cancer. However, in Part 3 this bacterium or higher taxa (phylum  Fusobacteriota, class Fusobacteriia, order Fusobacteriales, family Fusobacteriaceae) has not been mentioned at all as a component of breast microbiome in health or disease. Thus, all such findings should be revealed in the cited references and given in Part 3.

Minor revisions:

1. Lines 18, 19, 30, 38 and the whole text, tables and figures. According to the International Code of Nomenclature of Prokaryotes all Latin names of bacteria from species to phylum should be written with Italic font.
2. Line 20. "The human milk microbiota (HMM) is a complex biological fluid..." is not correct, because microbiota is not a fluid.
3. Lines 22, 31. The "BC" and "MYC" abbreviations should be deciphered in Abstract just after theirthe first appearance.
4. Lines 24-26. The sentence is incorrect, because estrobolome is not a process, it is a set of bacterial genes and metabolites.
5. Line 49. "Lactobacillusiners and Prevotella" should be exchanged by "Lactobacillus iners and Prevotella".
6. Line 60. The statement "the breast hosts a large ecosystem" is incorrect, because the classical definition of ecosystem term in ecology includes both biota and biotope. Here the term "community" seems to be more appropriate.
7. Line 64. The Figure caption is too short and unclear. It should be corrected.
8. Lines 65-71 contains a lot of facts, but absolutely without references. It is improper, and every fact should be supported by a reference.
9. Line 73. The "growth experiments" is unclear and is better to be exchanged by "bacterial cultivation"
10. Lines 77-78. Term "relative presence" is incorrect and should be exchanged by "relative abundance".
11. Line 91. Term "a large" is not proper here and should be exchanged by "a diverse".
12. Line 93-94. The statement "The diversity of breast microbiota is comparable to that of the gut microbiome but significantly higher than that observed in the vaginal microbiome [1]." is absolutely incorrect. Moreover, the cited article does not contain this information. The statement is better to be removed.
13. Lines 112-113. The statement "The most prevalent bacteria belong to facultative anaerobic or strictly aerobic groups..." is absolutely incorrect. The bacteria "Bifidobacterium, and Bacteroidetes (including Prevotella)" (line 116) are strict obligate anaerobes.
14. Lines 127-136 contains only description of Figure 2 and should be shortened and transferred to the figure caption.
15. Lines 145-148. Incorrect and unclear statement "A growing body of evidence supports the concept of a “gut-lung axis,” a bidirectional communication pathway between the gastrointestinal and respiratory microbiomes, further emphasizing the immunomodulatory potential of human milk in neonatal health [18]." What is the relation between gut-lung axis and breast milk taking into account absence of common blood supply routes between lungs and breast? In common this phrase is strange and incorrect and recommended to be removed.
16. Lines 157-159. These facts should be supported by references.
17. Lines 195-197. Again, these facts should be supported by references. 

Author Response

Response to the reviewer 2

The manuscript contains the comprehensive review of the microbiome in breast cancer and emphasizes the specific role of Fusobacterium nucleatum as the onco-immune modulator. All the references are reliable and review is very interesting. However, it may be accepted for publication after major and minor corrections.

 We thank the reviewer for the valuable suggestions.

Major revisions:

The title does not reflect the manuscript content. First, it is not an investigation but review. Second, the term "Breast Inflammatory Conditions" is too extensive and has not been reviewed comprehensively in this manuscript. The title limited by microbiome, breast cancer and Fusobacterium nucleatum seems to be more appropriate.

According to reviewer suggestion, we changed the title of the manuscript in:

Exploring the Microbiome in breast cancer: the role of Fusobacterium nucleatum as onco-immune modulator.

Part 2 "Microbiota in Breast Feeding in Healthy and Inflammatory Conditions" is too long due to some excessive parts, for example role of breast milk for newborns and their health. This gives nothing to main idea of the manuscript. So, part 2 is recommended to be shortened substantially

According to reviewer suggestion, we have shortened Part 2 substantially.

Part 4 describes a specific role of Fusobacterium nucleatum as the onco-immunomodulatory agent in breast cancer. However, in Part 3 this bacterium or higher taxa (phylum  Fusobacteriota, class Fusobacteriia, order Fusobacteriales, family Fusobacteriaceae) has not been mentioned at all as a component of breast microbiome in health or disease. Thus, all such findings should be revealed in the cited references and given in Part 3.

We mentioned Fusobacteriaceae in Part 4 (The microbiota in Breast Cancer) and we also added a sentence about Fusobacterium nucleatun at the end of Part 4.

Minor revisions:

1. Lines 18, 19, 30, 38 and the whole text, tables and figures. According to the International Code of Nomenclature of Prokaryotes all Latin names of bacteria from species to phylum should be written with Italic font. We checked.
2. Line 20. "The human milk microbiota (HMM) is a complex biological fluid..." is not correct, because microbiota is not a fluid. We corrected.
3. Lines 22, 31. The "BC" and "MYC" abbreviations should be deciphered in Abstract just after theirthe first appearance. We checked
4. Lines 24-26. The sentence is incorrect, because estrobolome is not a process, it is a set of bacterial genes and metabolites. We corrected.
5. Line 49. "Lactobacillusiners and Prevotella" should be exchanged by "Lactobacillus iners and Prevotella". We corrected.
6. Line 60. The statement "the breast hosts a large ecosystem" is incorrect, because the classical definition of ecosystem term in ecology includes both biota and biotope. Here the term "community" seems to be more appropriate. We corrected.
7. Line 64. The Figure caption is too short and unclear. It should be corrected.

The captions for Figure 1 (lines 67-73) and Figure 2 (lines 129-138) were mistakenly included in the main body by the Microorganisms editorial team. We repositioned them near the corresponding figures.

1. Lines 65-71 contains a lot of facts, but absolutely without references. It is improper, and every fact should be supported by a reference. We added new references
2. Line 73. The "growth experiments" is unclear and is better to be exchanged by "bacterial cultivation". We modified.
3. Lines 77-78. Term "relative presence" is incorrect and should be exchanged by "relative abundance". We modified.
4. Line 91. Term "a large" is not proper here and should be exchanged by "a diverse". We modified.
5. Line 93-94. The statement "The diversity of breast microbiota is comparable to that of the gut microbiome but significantly higher than that observed in the vaginal microbiome [1]." is absolutely incorrect. Moreover, the cited article does not contain this information. The statement is better to be removed. We corrected the reference.
6. Lines 112-113. The statement "The most prevalent bacteria belong to facultative anaerobic or strictly aerobic groups..." is absolutely incorrect. The bacteria "Bifidobacterium, and Bacteroidetes (including Prevotella)" (line 116) are strict obligate anaerobes.

We removed the statement.

1. Lines 127-136 contains only description of Figure 2 and should be shortened and transferred to the figure caption.

The captions for Figure 1 (lines 67-73) and Figure 2 (lines 129-138) were mistakenly included in the main body by the Microorganisms editorial team. We repositioned them near the corresponding figures

1. Lines 145-148. Incorrect and unclear statement "A growing body of evidence supports the concept of a “gut-lung axis,” a bidirectional communication pathway between the gastrointestinal and respiratory microbiomes, further emphasizing the immunomodulatory potential of human milk in neonatal health [18]." What is the relation between gut-lung axis and breast milk taking into account absence of common blood supply routes between lungs and breast? In common this phrase is strange and incorrect and recommended to be removed.

We modified the statement and added a reference.

1. Lines 157-159. These facts should be supported by references We added a reference.
2. Lines 195-197. Again, these facts should be supported by references. We added a reference.

Reviewer 3 Report

Comments and Suggestions for Authors

The review discussed the microbiota of breast and the important bacteria in different situation of breast, which is very interesting and meaningful, but there are some confused places to be checked. Also, the structure of the manuscript was chaotic. I don’t think it meet the standard of publication.

1. It was unclear in the abstract that the microbiota in the manuscript was belong to which part of the breast? The milk? Or the tissue? This is very important. Also, the abstract was of logical mess, please modify.
2. L270, The number of the subtitle was wrong, please check.
3. L42, The introduction part shouldn’t repeat the words in the abstract. Please revise.
4. L49, wrong name of the Lactobacillusiners? Please check.
5. 1, Lactobacillus should be totally italic, also an abundant dot should be deleted.
6. The introduction part was also confused, for I think the microbiota in other tissue needn’t be mentioned.
7. L81, Thanks to....too colloquial...
8. L131, the format of the referene was wrong, please check.

 

Author Response

Response to the reviewer 3

The review discussed the microbiota of breast and the important bacteria in different situation of breast, which is very interesting and meaningful, but there are some confused places to be checked. Also, the structure of the manuscript was chaotic. I don’t think it meet the standard of publication.

 We thank the reviewer for the valuable suggestions.

 

1. It was unclear in the abstract that the microbiota in the manuscript was belong to which part of the breast? The milk? Or the tissue? This is very important. Also, the abstract was of logical mess, please modify.

We modified the abstract to define better the structure, which is the same as the manuscript. The review begins with the microbiome of healthy breast and milk, then analyses the microbiome perturbations in breast inflammation, such as mastitis, and in breast cancer.

1. L270, The number of the subtitle was wrong, please check.

The numbering of the chapters, from 3 onwards, was wrong by the Microorganisms editorial team. We have renumbered them correctly

1. L42, The introduction part shouldn’t repeat the words in the abstract. Please revise. We modified
2. L49, wrong name of the Lactobacillusiners? Please check. We corrected.
3. 1, Lactobacillus should be totally italic, also an abundant dot should be deleted. We modified
4. The introduction part was also confused, for I think the microbiota in other tissue needn’t be mentioned. We modified the introduction
5. L81, Thanks to....too colloquial... We corrected.
6. L131, the format of the referene was wrong, please check. We corrected the format of the 2 references

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, the authors address many of the comments. However, some areas need improvement before publication.

 Major improvement

• They added a new figure to enhance the presentation about the role of nucleatum as onco-immune modulator
• They included evidence of direct DNA damage in host cells for listed bacteria
• Added requested information
• Corrected many minor comments

Major Comments to be revised

• The manuscript does not clearly define the specific knowledge gap it aims to fill. A more focused introduction is needed to explain what has been covered in recent reviews, Little et al 2023 (onco-modulatory role of nucleatum) microbiome and breast cancer, and how this review differs or adds new value. Without this context, the novelty and contribution of the paper remain unclear. The authors should also critically discuss recent literature to position their review within the current landscape.

 

• Lack of flow: For example, the last 2 paragraphs of the introduction, on page 4, need revision
• Lack of source: add source to some very informative paragraphs, like paragraph 5 on page 7.

Minor Comments to be revised

• Words like nucleatum, invitro and invivo are not etalized
• Make sure to write the whole manuscript in the same font size
• Formatting errors, for example, writing the figure title of fig-2 and-3 on top and bottom. The bottom one only is enough.

Author Response

Response to

Second round Reviewer 2

 

Overall, the authors address many of the comments. However, some areas need improvement before publication.

 Major improvement

• They added a new figure to enhance the presentation about the role of nucleatum as onco-immune modulator
• They included evidence of direct DNA damage in host cells for listed bacteria
• Added requested information
• Corrected many minor comments

Major Comments to be revised

• The manuscript does not clearly define the specific knowledge gap it aims to fill. A more focused introduction is needed to explain what has been covered in recent reviews, Little et al 2023 (onco-modulatory role of nucleatum) microbiome and breast cancer, and how this review differs or adds new value. Without this context, the novelty and contribution of the paper remain unclear. The authors should also critically discuss recent literature to position their review within the current landscape.

 We thank the reviewer for the valuable suggestions. We improved the introduction, emphasizing the main points and novelty of the review. We also discussed the most recent literature in part 5 of the manuscript.

 

• Lack of flow: For example, the last 2 paragraphs of the introduction, on page 4, need revision
• Lack of source: add source to some very informative paragraphs, like paragraph 5 on page 7.

We modified and added further references.

Minor Comments to be revised

• Words like nucleatum, invitro and invivo are not etalized
• Make sure to write the whole manuscript in the same font size
• Formatting errors, for example, writing the figure title of fig-2 and-3 on top and bottom. The bottom one only is enough.

We corrected

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript contains the comprehensive review of the microbiome in breast cancer and emphasizes the specific role of Fusobacterium nucleatum as the onco-immune modulator. All the references are reliable and review is very interesting. All unclarities have been successfully removed during revision. The manuscript is recommended to be published.

Reviewer 3 Report

Comments and Suggestions for Authors

Thanks for your modification. This manuscript supplied a new idea about microbiome in breast inflammatory and cancerous conditions, which may be important to breast cancer studies in future.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, The manuscript has been improved as the authors have addressed nearly all of the comments:

• Added a new figure illustrating the role of F. nucleatum as an onco-immune modulator.
• Included evidence of direct DNA damage in host cells for the listed bacteria.
• Included the requested additional information.
• Rewritten the introduction to incorporate recent literature
• Corrected many minor issues.

I believe that the manuscript is ready for publication.