The Role of Oral Pathobionts’ Outer Membrane Vesicles in Cancer Pathology and Therapeutic Development

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Cells

cells-4218421, entitled “The Role of Oral Bacterial Outer Membrane Vesicles in Cancer Pathology and Therapeutic Development” has been carefully reviewed.

 

Overall Assessment

This review provides a comprehensive overview of the role of oral bacterial outer membrane vesicles (OMVs) in cancer pathology and therapeutic development, with a focus on Porphyromonas gingivalis and Fusobacterium nucleatum. The topic is highly relevant to the intersection of microbiology, inflammation, and oncology, and the manuscript synthesizes a substantial body of recent literature (2018–2025) to unravel the multifaceted mechanisms by which OMVs drive cancer initiation, progression, and metastasis. The structure is logical, covering OMV biogenesis, microbial carcinogenesis pathways, OMV-mediated cancer hallmarks, and OMV-mitochondria crosstalk, supplemented with informative figures. Overall, this review makes a valuable contribution to the field by highlighting OMVs as key mediators of host-microbe interactions in cancer and identifying potential therapeutic avenues. However, several revisions are needed to enhance clarity, address gaps, and strengthen the scientific rigor.

 

Comments

1. Mechanistic Specificity and Contrast Between Bacterial Species

While the manuscript thoroughly describes general mechanisms of OMV-induced cancer, it often conflates P. gingivalis and F. nucleatum OMV effects without clear differentiation. For example, the section on cancer initiation (Section 6) mentions both species activating TLRs but does not specify whether their OMVs target distinct TLR subtypes (e.g., P. gingivalis OMVs preferentially activating TLR2 vs. F. nucleatum OMVs activating TLR4) or induce different downstream cytokine profiles. A comparative table or subsection explicitly contrasting the unique virulence factors (e.g., gingipains in P. gingivalis OMVs, FadA/Fap2 in F. nucleatum OMVs) and their specific oncogenic pathways would greatly improve clarity. Additionally, the manuscript should address why certain OMVs (e.g., from E. coli, L. rhamnosus) do not promote cancer cell migration (mentioned in Section 8) to highlight the specificity of oral pathobiont OMVs.

 

2. Integration of Clinical Evidence

The review primarily focuses on preclinical (in vitro/in vivo) mechanisms but lacks sufficient integration of clinical data linking oral bacterial OMVs to human cancer outcomes. For instance, while it mentions associations between P. gingivalis/F. nucleatum and esophageal/colorectal cancer, it does not discuss clinical studies detecting OMVs or their cargo (e.g., LPS, gingipains) in human tumour tissues, blood, or saliva, nor their correlation with patient prognosis (e.g., survival, treatment response). Incorporating such clinical evidence—including diagnostic potential of OMV-derived biomarkers—would bridge the gap between preclinical findings and translational relevance.

 

3. Clarification of OMV-Mitochondria Crosstalk Mechanisms

Section 9 on OMV-mitochondria interactions is a strength but requires more precise mechanistic detail. The manuscript states that P. gingivalis OMVs induce DRP1-dependent mitochondrial fission, but it does not explain how OMV cargo (e.g., LPS, gingipains) directly or indirectly regulates DRP1 phosphorylation (Ser616) or Mfn2 expression. Additionally, the link between mitochondrial fragmentation and the Warburg effect is mentioned but not elaborated: Do OMV-induced mitochondrial dysfunction directly drive metabolic reprogramming (e.g., via ROS-mediated HIF-1α stabilization) or is it a secondary effect of inflammation? Clarifying these pathways would strengthen the mechanistic depth.

 

4. Discussion of Therapeutic Challenges and Future Directions

The "Therapeutic Development" section (implied but not explicitly structured) is underdeveloped. While the manuscript mentions engineered OMVs as potential immunotherapeutic platforms, it does not address key challenges such as OMV targeting to tumour cells, immunogenicity of bacterial-derived OMVs, or delivery barriers (e.g., clearance by the reticuloendothelial system). A dedicated subsection on current therapeutic strategies (e.g., anti-OMV antibodies, OMV-based vaccines, inhibition of OMV biogenesis/secretion) and their preclinical/clinical progress would enhance the review’s translational impact. Additionally, future directions should include addressing gaps like OMV heterogeneity (e.g., different OMV subpopulations with distinct oncogenic potential) and the role of host microbiota in modulating OMV effects.

 

5. Figure Annotations and Accessibility

Figures 1–4 are informative but require improved annotations: (a) In Figure 2, label specific OMV cargo (e.g., gingipains, FadA) alongside pathways they regulate; (b) In Figure 3, clarify the distinction between TLR2/TLR4 activation outcomes (pro-inflammatory vs. anti-inflammatory); (c) Ensure all abbreviations (e.g., TREM1, cGAS-STING) are defined in figure captions for reader accessibility.

 

6. Redundancy and Structural Streamlining

Several mechanisms (e.g., NF-κB activation, ROS production) are repeated across sections (e.g., inflammation, cancer initiation, progression). Streamlining these overlapping discussions—for example, by centralizing core inflammatory pathways and cross-referencing their downstream effects (DNA damage, EMT, immune evasion)—would improve readability. Additionally, Section 5 (Role of OMVs in Cancer Stages) overlaps with Sections 6–8; restructuring to clearly separate initiation, progression, and metastasis (with distinct subheadings) would enhance logical flow.

 

7. Terminology Consistency

Ensure consistent use of terminology: (a) "Pathobionts" is preferred over "pathogens" for oral bacteria (as they are part of the microbiota but become pathogenic in dysbiosis).

 

8. Citation Updates and Accuracy

Verify citations for recent studies (2024–2025) to ensure they are correctly attributed (e.g., Muñoz-Medel et al. 2024, Chen et al. 2023). Additionally, include citations for clinical studies linking OMVs to cancer (e.g., detection of F. nucleatum OMVs in colorectal cancer tissues) and therapeutic applications (e.g., engineered OMV vaccines in preclinical cancer models) to support claims.

 

Recommendation

The manuscript is of high scientific interest and has the potential to be a valuable resource for researchers in microbiology, oncology, and translational medicine. I recommend Major Revision to address the above comments, particularly enhancing mechanistic specificity, integrating clinical evidence, clarifying therapeutic directions, and streamlining structure. With these revisions, the review will more effectively bridge preclinical mechanisms and clinical relevance, providing a comprehensive and impactful analysis of oral bacterial OMVs in cancer.

Author Response

Reviewer 1. 

Overall Assessment

This review provides a comprehensive overview of the role of oral bacterial outer membrane vesicles (OMVs) in cancer pathology and therapeutic development, with a focus on Porphyromonas gingivalis and Fusobacterium nucleatum. The topic is highly relevant to the intersection of microbiology, inflammation, and oncology, and the manuscript synthesizes a substantial body of recent literature (2018–2025) to unravel the multifaceted mechanisms by which OMVs drive cancer initiation, progression, and metastasis. The structure is logical, covering OMV biogenesis, microbial carcinogenesis pathways, OMV-mediated cancer hallmarks, and OMV-mitochondria crosstalk, supplemented with informative figures. Overall, this review makes a valuable contribution to the field by highlighting OMVs as key mediators of host-microbe interactions in cancer and identifying potential therapeutic avenues. However, several revisions are needed to enhance clarity, address gaps, and strengthen the scientific rigor.

 

Comments

1. Mechanistic Specificity and Contrast Between Bacterial Species

While the manuscript thoroughly describes general mechanisms of OMV-induced cancer, it often conflates P. gingivalis and F. nucleatum OMV effects without clear differentiation. For example, the section on cancer initiation (Section 6) mentions both species activating TLRs but does not specify whether their OMVs target distinct TLR subtypes (e.g., P. gingivalis OMVs preferentially activating TLR2 vs. F. nucleatum OMVs activating TLR4) or induce different downstream cytokine profiles. A comparative table or subsection explicitly contrasting the unique virulence factors (e.g., gingipains in P. gingivalis OMVs, FadA/Fap2 in F. nucleatum OMVs) and their specific oncogenic pathways would greatly improve clarity. Additionally, the manuscript should address why certain OMVs (e.g., from E. coli, L. rhamnosus) do not promote cancer cell migration (mentioned in Section 8) to highlight the specificity of oral pathobiont OMVs.

Response:

We appreciate this important suggestion. We have revised the manuscript to clearly distinguish between P. gingivalis and F. nucleatum OMV-mediated mechanisms. Specifically:

• A new comparative table has been added (Table 1, page 22) summarizing key OMV cargo, receptor interactions (e.g., TLR2 vs TLR4), and downstream oncogenic pathways.
• The relevant sections have been revised to explicitly differentiate species-specific effects. (Page 16)
• We have also clarified why OMVs from non-pathobionts (e.g., coli, Lactobacillus rhamnosus) do not exhibit similar pro-tumorigenic effects. (Page 23).

 

2. Integration of Clinical Evidence

The review primarily focuses on preclinical (in vitro/in vivo) mechanisms but lacks sufficient integration of clinical data linking oral bacterial OMVs to human cancer outcomes. For instance, while it mentions associations between P. gingivalis/F. nucleatum and esophageal/colorectal cancer, it does not discuss clinical studies detecting OMVs or their cargo (e.g., LPS, gingipains) in human tumour tissues, blood, or saliva, nor their correlation with patient prognosis (e.g., survival, treatment response). Incorporating such clinical evidence—including diagnostic potential of OMV-derived biomarkers—would bridge the gap between preclinical findings and translational relevance.

Response:

We thank the reviewer for this valuable suggestion. In response, we have strengthened the integration of clinical evidence throughout the manuscript and introduced a dedicated subsection entitled “Clinical Evidence and Translational Relevance of OMVs”. (Page 16)

Specifically, we have incorporated evidence demonstrating the presence of oral pathobionts, including Fusobacterium nucleatum and Porphyromonas gingivalis, in human tumour tissues and biofluids. These studies highlight associations between microbial burden and clinical outcomes such as tumour stage, metastasis, chemotherapeutic response, and patient survival.

We have also expanded the discussion on the potential of microbial and OMV-associated components as diagnostic biomarkers, including their detection in saliva, tumour tissues, and circulation, supporting their relevance for non-invasive cancer diagnosis and prognosis.

Furthermore, we acknowledge that while substantial clinical evidence supports the association between oral microbiota and cancer, direct detection and characterisation of OMVs in patient-derived samples remain limited. This limitation has now been explicitly addressed in the manuscript, and we highlight it as an important direction for future translational research.

3. Clarification of OMV-Mitochondria Crosstalk Mechanisms

Section 9 on OMV-mitochondria interactions is a strength but requires more precise mechanistic detail. The manuscript states that P. gingivalis OMVs induce DRP1-dependent mitochondrial fission, but it does not explain how OMV cargo (e.g., LPS, gingipains) directly or indirectly regulates DRP1 phosphorylation (Ser616) or Mfn2 expression. Additionally, the link between mitochondrial fragmentation and the Warburg effect is mentioned but not elaborated: Do OMV-induced mitochondrial dysfunction directly drive metabolic reprogramming (e.g., via ROS-mediated HIF-1α stabilization) or is it a secondary effect of inflammation? Clarifying these pathways would strengthen the mechanistic depth.

Response:

We thank the reviewer for this insightful comment. In response, we have expanded the section on OMV-mitochondria crosstalk to provide additional mechanistic detail. Specifically, we have incorporated discussion on mitochondrial dynamics, including the potential involvement of DRP1-mediated mitochondrial fission in OMV-induced mitochondrial dysfunction. We have also clarified the role of OMV-induced reactive oxygen species (ROS) in stabilising HIF-1α and promoting metabolic reprogramming. Furthermore, we have strengthened the link between mitochondrial dysfunction and the Warburg effect to better explain how OMVs may contribute to tumour metabolic adaptation. These additions improve the mechanistic depth of this section and address the reviewer’s concern.

4. Discussion of Therapeutic Challenges and Future Directions

The "Therapeutic Development" section (implied but not explicitly structured) is underdeveloped. While the manuscript mentions engineered OMVs as potential immunotherapeutic platforms, it does not address key challenges such as OMV targeting to tumour cells, immunogenicity of bacterial-derived OMVs, or delivery barriers (e.g., clearance by the reticuloendothelial system). A dedicated subsection on current therapeutic strategies (e.g., anti-OMV antibodies, OMV-based vaccines, inhibition of OMV biogenesis/secretion) and their preclinical/clinical progress would enhance the review’s translational impact. Additionally, future directions should include addressing gaps like OMV heterogeneity (e.g., different OMV subpopulations with distinct oncogenic potential) and the role of host microbiota in modulating OMV effects.

Response:

We thank the reviewer for this valuable suggestion. In response, we have restructured and expanded the therapeutic section, now entitled “Therapeutic Targeting of Oral Bacterial OMVs: Opportunities, Challenges, and Future Directions.” (Page 46)

Specifically, we have incorporated a dedicated subsection addressing key challenges associated with OMV-based therapies, including limited tumour-specific targeting, rapid clearance by the reticuloendothelial system, and the intrinsic immunogenicity of bacterial-derived OMVs. We have also discussed current therapeutic strategies, including OMV-based vaccines, drug delivery systems, and approaches targeting OMV biogenesis and function.

Furthermore, we have expanded the discussion of future directions, highlighting important gaps such as OMV heterogeneity and the influence of host microbiota on OMV function and therapeutic outcomes. These additions improve the translational relevance of the manuscript and directly address the reviewer’s concerns.

5. Figure Annotations and Accessibility

Figures 1–4 are informative but require improved annotations: (a) In Figure 2, label specific OMV cargo (e.g., gingipains, FadA) alongside pathways they regulate; (b) In Figure 3, clarify the distinction between TLR2/TLR4 activation outcomes (pro-inflammatory vs. anti-inflammatory); (c) Ensure all abbreviations (e.g., TREM1, cGAS-STING) are defined in figure captions for reader accessibility.

Response:

We have now added, gingipains to Figure 2 and the pathways they dysregulate. We have also in Figure 3 legend indicated the red and green arrows showing pro-inflammatory and anti-inflammatory pathways induced. We have been mindful of the complexity of the figures and made them more comprehensible to the reader.

6. Redundancy and Structural Streamlining

Several mechanisms (e.g., NF-κB activation, ROS production) are repeated across sections (e.g., inflammation, cancer initiation, progression). Streamlining these overlapping discussions—for example, by centralizing core inflammatory pathways and cross-referencing their downstream effects (DNA damage, EMT, immune evasion)—would improve readability. Additionally, Section 5 (Role of OMVs in Cancer Stages) overlaps with Sections 6–8; restructuring to clearly separate initiation, progression, and metastasis (with distinct subheadings) would enhance logical flow.

Response:

We thank the reviewer for this valuable suggestion. In response, we have carefully revised the manuscript to improve clarity, reduce redundancy, and enhance structural organisation.

To address repetition of key mechanisms (e.g., NF-κB activation, ROS production), we have streamlined overlapping content by centralising core inflammatory and oxidative pathways within earlier sections of the manuscript. Subsequent sections now refer to these mechanisms with minimal repetition, focusing instead on their specific roles in cancer initiation, progression, and metastasis.

In addition, we have restructured the section on the role of OMVs in cancer to improve logical flow. The previous combined section has been revised to clearly distinguish between cancer initiation, progression, and metastasis, with dedicated subheadings for each stage. This restructuring reduces overlap between Sections 5–8 and provides a more coherent framework for presenting OMV-mediated mechanisms across different stages of tumour development.

 

7. Terminology Consistency

Ensure consistent use of terminology: (a) "Pathobionts" is preferred over "pathogens" for oral bacteria (as they are part of the microbiota but become pathogenic in dysbiosis).

Response:

We appreciate this important point. The terminology has been revised throughout the manuscript to ensure consistent use of “oral pathobionts” when referring to bacteria such as Porphyromonas gingivalis and Fusobacterium nucleatum. The term “pathogens” is now used only where appropriate (e.g., general infection context), while “pathobionts” is consistently used in the context of dysbiosis-associated disease. We have also improved consistency across related terms, including:

“Oral microbiota” vs “oral microbiome”

“Oral bacteria” vs “oral pathobionts”

This improves conceptual precision and aligns with current microbiome literature.

 

8. Citation Updates and Accuracy

Verify citations for recent studies (2024–2025) to ensure they are correctly attributed (e.g., Muñoz-Medel et al. 2024, Chen et al. 2023). Additionally, include citations for clinical studies linking OMVs to cancer (e.g., detection of F. nucleatum OMVs in colorectal cancer tissues) and therapeutic applications (e.g., engineered OMV vaccines in preclinical cancer models) to support claims.

Response:

We have included new citations throughout the manuscript and validate the references cited.

Recommendation

The manuscript is of high scientific interest and has the potential to be a valuable resource for researchers in microbiology, oncology, and translational medicine. I recommend Major Revision to address the above comments, particularly enhancing mechanistic specificity, integrating clinical evidence, clarifying therapeutic directions, and streamlining structure. With these revisions, the review will more effectively bridge preclinical mechanisms and clinical relevance, providing a comprehensive and impactful analysis of oral bacterial OMVs in cancer.

Reviewer 2 Report

Comments and Suggestions for Authors

This is a review article titled ‘The role of oral bacterial outer membrane vesicles in cancer pathology and therapeutic development’ by Hadjigol et al.,. Overall, this manuscript contains a great amount of information and cites a substantial literature base. It addresses a potentially interesting topic of the role of oral bacterial OMVs in cancer biology and possible therapeutic applications. However, there are a few issues that need to be addressed before its acceptance for publication.

 

Major comments

1. My first concern is that the manuscript is labeled a “systematic review,” but the review methodology is not presented at the standard required for a systematic review. The abstract states, “In this systematic review, PubMed, Web of Science, and Google Scholar were searched…” and lists keywords and a time emphasis. However, I could not identify a dedicated methods section describing the search strategy, date of last search, screening workflow, inclusion and exclusion criteria, study selection process, handling of duplicates, or risk-of-bias assessment. The authors should either convert the paper clearly into a narrative review or fully revise it as a systematic review with PRISMA-style methodology, study-selection flow diagram, and transparent eligibility criteria.

 

2. The manuscript appears to be unbalanced as the scope does not remain sufficiently centered on OMVs, despite the title, which is specifically about oral bacterial outer membrane vesicles in cancer pathology and therapeutic development. Sections 3 and 4 discuss general microbiota-associated carcinogenesis and the oral microbiome’s role in carcinogenesis across many mechanisms before the OMV-focused section begins later. The dedicated OMV-centered cancer section starts only at Section 5.

 

3. The manuscript frequently uses overly definitive language for a field that appears largely preclinical and associative. The Highlights begin with “This review demonstrates…” and the bullets state that OMVs contribute to initiation, progression, metastasis, immune evasion, etc. Likewise, the abstract states that oral pathobiont-derived OMVs “have been implicated in promoting” multiple malignant phenotypes. Given that much of the cited evidence appears mechanistic, in vitro, animal-based, or associative rather than clinical/causal, the claims should be softened and qualified more consistently.

 

4. The manuscript often moves from literature on P. gingivalis or F. nucleatum infection broadly into statements about OMV-mediated mechanisms. For example, the general microbiota/carcinogenesis sections discuss bacterial effects and later transition into OMV-centered claims. This may overstate what is specifically proven for OMVs. The authors should explicitly label each mechanism, and a summary table would help substantially.

 

5. Given the amount of information gathered and presented in this manuscript, it would greatly benefit from a structured evidence table. I suggest a table with columns such as “oral bacterium”; “OMV cargo/mechanism”; “model system”; “cancer type”; “phenotype observed”; “evidence strength”; “limitations”. This would be especially useful because the review blends natural-pathogen OMVs and engineered therapeutic OMVs, which are conceptually related but biologically distinct.

Minor comments

1. There is repetition in the discussion around oral microbiota/carcinogenesis. The paragraph beginning “Significant clinical evidence has established a robust correlation…” is then essentially repeated in the next sentences with very similar wording and content. This section should be consolidated.

 

2. There are many language/grammar issues throughout the manuscript. Examples include: “OMVs include those from oral bacteria are composed…”; “A second mechanism are changes…”; “This review draws on these recent findings and investigates these…”. A thorough language edit is needed.

 

3. Terminology should be standardized throughout. The authors alternate between oral microbiota, oral microbiome, oral pathobionts, periodontal organisms, etc. A clearer terminology framework would improve precision, particularly because the paper aims to focus on OMVs from specific oral Gram-negative bacteria.

Author Response

Reviewer 2

Comments and Suggestions for Authors

This is a review article titled ‘The role of oral bacterial outer membrane vesicles in cancer pathology and therapeutic development’ by Hadjigol et al.,. Overall, this manuscript contains a great amount of information and cites a substantial literature base. It addresses a potentially interesting topic of the role of oral bacterial OMVs in cancer biology and possible therapeutic applications. However, there are a few issues that need to be addressed before its acceptance for publication.

 

Major comments

1. My first concern is that the manuscript is labeled a “systematic review,” but the review methodology is not presented at the standard required for a systematic review. The abstract states, “In this systematic review, PubMed, Web of Science, and Google Scholar were searched…” and lists keywords and a time emphasis. However, I could not identify a dedicated methods section describing the search strategy, date of last search, screening workflow, inclusion and exclusion criteria, study selection process, handling of duplicates, or risk-of-bias assessment. The authors should either convert the paper clearly into a narrative review or fully revise it as a systematic review with PRISMA-style methodology, study-selection flow diagram, and transparent eligibility criteria.

Response:

We thank the reviewer for this important observation. We acknowledge that the manuscript does not fully meet PRISMA standards required for a systematic review. Therefore, the manuscript has been revised to reflect a narrative review format. Specifically, the term “systematic review” has been removed from the abstract and main text. The literature search description has been retained in a simplified form to reflect a structured narrative approach, rather than a formal systematic methodology. This revision ensures methodological clarity and aligns with the scope of the manuscript.

 

2. The manuscript appears to be unbalanced as the scope does not remain sufficiently centered on OMVs, despite the title, which is specifically about oral bacterial outer membrane vesicles in cancer pathology and therapeutic development. Sections 3 and 4 discuss general microbiota-associated carcinogenesis and the oral microbiome’s role in carcinogenesis across many mechanisms before the OMV-focused section begins later. The dedicated OMV-centered cancer section starts only at Section 5.

Response:

We agree that maintaining a clear focus on outer membrane vesicles (OMVs) is important given the scope of the review but as the topic of OMVs involvement in cancer progression is an exciting and emerging field it is critical to provide readers with a clear understanding of why OMVs have become prominent.

Thus, the early sections including sections 3 and 4 were intentionally included to provide the mechanistic and biological context necessary to interpret the emerging OMV-specific literature. As the reviewer correctly notes, the OMV-focused cancer section begins in Section 5; however, the current body of OMV research in cancer remains relatively early-stage. Consequently, much of our understanding of OMV function is inferred from well-established bacterial pathogenic mechanisms. This is biologically justified, as OMVs carry a substantial proportion of the virulence factors of their parent bacteria while possessing distinct properties as nanoscale entities, including enhanced tissue penetration, immune modulation, and systemic dissemination.

To address the reviewer’s concern, we have revised the manuscript to more clearly centre OMVs throughout these earlier sections. Specifically, we have:

• strengthened the linkage between bacterial mechanisms and OMV-mediated processes
• explicitly highlighted where OMVs are likely to underpin the described effects
• added bridging statements to guide the reader from general bacterial carcinogenesis to OMV-specific mechanisms

We have also clarified the reviews structure in the introduction (last paragraph) to ensure readers understand that the early sections establish a mechanistic framework that is subsequently resolved in OMV-specific detail in Section 5 onwards.

We believe these revisions improve the balance of the manuscript while preserving the necessary biological context required to interpret OMV-driven cancer pathology.

Added paragraph at the end of the introduction section:

Although substantial evidence links oral pathobionts to cancer, the mechanisms by which the oral microbiome drives carcinogenesis through host-microbe interactions are yet to be fully elucidated. Emerging research suggests that outer membrane vesicles (OMVs) from oral pathobionts are critical mediators of these processes. OMVs encapsulate the virulence repertoire of their parent bacteria while exhibiting distinct nanoscale properties, including enhanced tissue penetration, immune modulation, and systemic dissemination, enabling them to influence host signalling independently of whole bacterial cells. While many studies report effects of bacterial infection, direct attribution to OMVs remains limited in some contexts. Consequently, this review adopts a framework whereby established mechanisms of oral bacteria-associated carcinogenesis are interpreted through an OMV-bias, as many of these effects are likely mediated or amplified by OMV-dependent processes. The initial sections therefore establish the biological context necessary to interpret this emerging field, while subsequent sections focus explicitly on the recent OMV-driven experimental, pathological, and translational evidence, defining their role as key effectors of cancer progression and potential therapeutic targets.

 

3. The manuscript frequently uses overly definitive language for a field that appears largely preclinical and associative. The Highlights begin with “This review demonstrates…” and the bullets state that OMVs contribute to initiation, progression, metastasis, immune evasion, etc. Likewise, the abstract states that oral pathobiont-derived OMVs “have been implicated in promoting” multiple malignant phenotypes. Given that much of the cited evidence appears mechanistic, in vitro, animal-based, or associative rather than clinical/causal, the claims should be softened and qualified more consistently.

Response:

We would agree that some statements in the manuscript were expressed too definitively given the predominantly preclinical and emerging nature of the OMV-cancer literature. To address this, we have systematically revised the manuscript to adopt more appropriately qualified language throughout. Statements implying causality or generality have been replaced with more cautious phrasing where appropriate. We have also revised the Highlights and Abstract to better reflect the strength and limitations of the available evidence, ensuring that the distinction between mechanistic/preclinical findings and clinical relevance is clearly conveyed

 

4. The manuscript often moves from literature on P. gingivalis or F. nucleatum infection broadly into statements about OMV-mediated mechanisms. For example, the general microbiota/carcinogenesis sections discuss bacterial effects and later transition into OMV-centered claims. This may overstate what is specifically proven for OMVs. The authors should explicitly label each mechanism, and a summary table would help substantially.

Response:

We have now explicitly labelled each mechanism and have included a summary table (Table 1) of the features of OMVs for P. gingivalis and F. nucleatum on page 22.

5. Given the amount of information gathered and presented in this manuscript, it would greatly benefit from a structured evidence table. I suggest a table with columns such as “oral bacterium”; “OMV cargo/mechanism”; “model system”; “cancer type”; “phenotype observed”; “evidence strength”; “limitations”. This would be especially useful because the review blends natural-pathogen OMVs and engineered therapeutic OMVs, which are conceptually related but biologically distinct.

Response:

We thank the reviewer for this excellent suggestion. A structured evidence table (Table 2) has been added (Page 30), including: Oral bacterium, OMV cargo/mechanism, model system, cancer type, observed phenotype, evidence strength, limitations

This table integrates both: natural OMV studies and engineered OMV applications. This significantly improves clarity and allows readers to quickly assess the strength of evidence.

Minor comments

1. There is repetition in the discussion around oral microbiota/carcinogenesis. The paragraph beginning “Significant clinical evidence has established a robust correlation…” is then essentially repeated in the next sentences with very similar wording and content. This section should be consolidated.

Response:

We have revised the manuscript to reduce repetition so as to enhance clarity throughout.

2. There are many language/grammar issues throughout the manuscript. Examples include: “OMVs include those from oral bacteria are composed…”; “A second mechanism are changes…”; “This review draws on these recent findings and investigates these…”. A thorough language edit is needed.

Response:

We have gone through the manuscript and completed a thorough language edit.

3. Terminology should be standardized throughout. The authors alternate between oral microbiota, oral microbiome, oral pathobionts, periodontal organisms, etc. A clearer terminology framework would improve precision, particularly because the paper aims to focus on OMVs from specific oral Gram-negative bacteria.

Response:

We have revised the manuscript to have a clear terminology framework throughout.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This review provides an insightful overview of oral bacterial OMVs in cancer. With the above revisions to enhance logic, clarity, and translational relevance, the manuscript merits publication in Cells.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed most of my major scientific concerns in the revised version. In particular, they have removed the systematic-review framing, clarified the rationale for the broader background sections, and added structured summary tables that improve the distinction between general bacterial effects and OMV-specific mechanisms. These revisions substantially strengthen the manuscript. However, the revised file still contains many language, terminology, and presentation issues. I strongly recommend a thorough English editing before publication. Overall, I consider the manuscript much improved and potentially suitable for publication after minor editorial revision.