Ultrastructural Evidence of Interactions Between Eosinophils and Mast Cells in Gastric Cancer: Considerations in AllergoOncology Research

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper describes an ultrastructural study on the interaction between degranulating mast cells and cytotoxic eosinophils in gastric cancer. The main findings of this study are as follows: In gastric cancer tumor tissues, atypical accumulations of mast cells and eosinophils (TATEM) were observed. TATEM was identified in 9 out of 72 cases (12.5%). Ultrastructural analysis using electron microscopy revealed that mast cells and eosinophils closely interact within the tumor microenvironment, suggesting an allergy-like immune response. Mast cells exhibited both piecemeal degranulation and anaphylactic degranulation. Some mast cells showed signs of recovery from anaphylactic degranulation. Eosinophils demonstrated cytolysis accompanied by degranulation, and in some cases, ETosis (extracellular trap cell death) was observed. During ETosis, nuclear chromatin and specific granules were released extracellularly. The simultaneous activation of mast cells and eosinophils suggests the release of cytotoxic substances, such as TNF-α and eosinophil-specific granules, into the tumor microenvironment, which may exert antitumor effects. TATEM tended to be associated with early-stage (stage I–II) gastric cancer, although this did not reach statistical significance. This study suggests that allergy-like immune responses may influence the tumor microenvironment and supports the concept of AllergoOncology. These findings indicate that the interaction between mast cells and eosinophils may represent an essential immune response in the tumor microenvironment of gastric cancer.

This is a fascinating study, but several concerns should be noted:

1. In the structure of the paper, the Methods section is placed after the Discussion. Please correct the order.

2. Please illustrate the method of selecting study samples.

3. In the Discussion, please address the limitations of this study. For example, the study subjects were limited to surgical specimens collected between 1998 and 2005, and patient backgrounds and treatments may differ from current clinical practice. The cases in which TATEM was observed accounted for only 12.5% (9 cases) of the total, which limits the generalizability of the results. Larger studies are needed. The specific mechanisms underlying the association of TATEM with early-stage gastric cancer are not presented. It is unclear whether the observed association is causal or merely correlational. Detailed molecular-level analysis of the specific role of the immune response indicated by TATEM and its impact on the tumor microenvironment is lacking.

Comments on the Quality of English Language

Minor English editing is needed. 

Author Response

Please refer to the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study present the results obtained during  examination  of a 72 resected cancer specimen, regarding interaction and degranulation patterns of tumour-associated tissue eosinophils and mast cells (TATEM) in a series of gastric carcinoma cases.

The data obtained  by authors reveal a close association between degranulating mast cells and cytolytic eosinophils, with or without ETosis, in two cases of gastric cancer, indicate that mast cells may be involved in the induction of eosinophil cytolysis.
The presence of cytolytic eosinophils and mast cell anaphylactic degranulation  it suggests the release of cytotoxic mediators that may exert antitumour effects. Bioactive compounds, such as TNF-alpha  present in mast cell granules  may have significant anticancer properties. The Fisher’s  test indicated  an association between TATEM and low-stage gastric cancer (stages I–II), suggesting that this immune response may be more prominent in early-stage tumours or less active in advanced disease.

The manuscript is well written, the results are presented concisely, and the conclusions are well supported by the findings. This study, for the first time, provides evidence of a close association between degranulating mast cells and cytolytic eosinophils, with or without ETosis, in the tumour tissue of gastric carcinomas. This represents the novelty of the study, and for this reason, I recommend its publication.

Minor revisions:

1.The manuscript contains one or two sentences that begin with an abbreviation. These sentences should be revised, as a sentence should not begin with an abbreviation.
2. Subchapter 4.4, which currently appears in the Methods section, should be moved to the Discussion chapter.

Author Response

Please refer to the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Heterotypic Aggregation Between Degranulating Mast Cells and Cytolytic Eosinophils, With or Without ETosis, in Gastric Cancer: An Ultrastructural Study Within the AllergoOncology Paradigm

 

In this manuscript Rigoli et al., present a detailed and original ultrastructural investigation of tumor-associated mast cells and eosinophils in gastric cancer. The authors provide a compelling TEM evidence of heterotypic cellular interactions suggestive of allergy-like immune responses in the TME, with possible anti-tumoral effects. This study is grounded in the emerging field of AllergoOncology and offers valuable morphological insight. Despite these merits there are several flaws in the manuscript. I believe several concerns around the manuscript's clarity, interpretation of significance, and broader translational impact must be addressed before it can be considered for publication.

For the starters, please consider shortening the title it’s mouthful. 

Use short forms only once when it appears for the first time. Avoid repetitions. 

Intro needs refinement as there’s an abrupt change of topic as opposed to smoother transition. 

Would it be possible to provide clear, high definition or high quality images and please improve the figure legend to reflect its content. 

I’d restructure the entire manuscript so as to explain each figure in a sequence and avoid back and forth while referring the figures. I’d go in a sequence. High resolution images are necessary and so is the figure legend for each figure. Maybe add graphical overlays or annotated arrows. Maybe add schematic diagrams to complement the EM images and help the reader understand degranulation stages and interactions more intuitively.

There’s no consistency in the figure, font, font size etc. I’d keep everything consistent. 

The discussion assumes cytotoxic or anti-tumoral roles for the mast cell/eosinophil interactions, but these remain speculative. The authors should more clearly frame these interpretations as hypotheses and discuss alternative explanations whether these interactions may also contribute to tumor progression, angiogenesis, or fibrosis in certain contexts.

 Only 9 of 72 cases exhibited TATEM (12.5%). The observed association with low-stage disease (P = 0.06) is interesting but not statistically significant. The authors should temper claims of potential clinical relevance and clearly state that larger studies are needed to validate these findings.

The manuscript often relies on overly technical language without accessible framing, limiting its readability to non-specialists. I’d consider reworking the abstract and intro to more clearly articulate the core question, significance, and translational relevance. Define key terms like “ETosis” earlier and more clearly.

There are no functional assays or biomarker data to complement the ultrastructural observations. I’d add a limitations section which should explicitly acknowledge that these findings are morphological only, and that functional confirmation such as in vitro co-culture, cytokine profiling, gene expression will be necessary to validate mechanisms.

I think the manuscript would benefit from a light language edit to improve clarity and reduce repetition.

Last but not the least, please verify consistency in formatting references including volume and page numbers. Also some DOI formats appear non-uniform.

All in all, this paper has strong potential for publication but after careful revision. The topic is novel, and the methodology is solid, but interpretation and framing need refinement.

Best of luck and keep up the good work,

Cheers!

Comments on the Quality of English Language

Heterotypic Aggregation Between Degranulating Mast Cells and Cytolytic Eosinophils, With or Without ETosis, in Gastric Cancer: An Ultrastructural Study Within the AllergoOncology Paradigm

 

In this manuscript Rigoli et al., present a detailed and original ultrastructural investigation of tumor-associated mast cells and eosinophils in gastric cancer. The authors provide a compelling TEM evidence of heterotypic cellular interactions suggestive of allergy-like immune responses in the TME, with possible anti-tumoral effects. This study is grounded in the emerging field of AllergoOncology and offers valuable morphological insight. Despite these merits there are several flaws in the manuscript. I believe several concerns around the manuscript's clarity, interpretation of significance, and broader translational impact must be addressed before it can be considered for publication.

For the starters, please consider shortening the title it’s mouthful. 

Use short forms only once when it appears for the first time. Avoid repetitions. 

Intro needs refinement as there’s an abrupt change of topic as opposed to smoother transition. 

Would it be possible to provide clear, high definition or high quality images and please improve the figure legend to reflect its content. 

I’d restructure the entire manuscript so as to explain each figure in a sequence and avoid back and forth while referring the figures. I’d go in a sequence. High resolution images are necessary and so is the figure legend for each figure. Maybe add graphical overlays or annotated arrows. Maybe add schematic diagrams to complement the EM images and help the reader understand degranulation stages and interactions more intuitively.

There’s no consistency in the figure, font, font size etc. I’d keep everything consistent. 

The discussion assumes cytotoxic or anti-tumoral roles for the mast cell/eosinophil interactions, but these remain speculative. The authors should more clearly frame these interpretations as hypotheses and discuss alternative explanations whether these interactions may also contribute to tumor progression, angiogenesis, or fibrosis in certain contexts.

 Only 9 of 72 cases exhibited TATEM (12.5%). The observed association with low-stage disease (P = 0.06) is interesting but not statistically significant. The authors should temper claims of potential clinical relevance and clearly state that larger studies are needed to validate these findings.

The manuscript often relies on overly technical language without accessible framing, limiting its readability to non-specialists. I’d consider reworking the abstract and intro to more clearly articulate the core question, significance, and translational relevance. Define key terms like “ETosis” earlier and more clearly.

There are no functional assays or biomarker data to complement the ultrastructural observations. I’d add a limitations section which should explicitly acknowledge that these findings are morphological only, and that functional confirmation such as in vitro co-culture, cytokine profiling, gene expression will be necessary to validate mechanisms.

I think the manuscript would benefit from a light language edit to improve clarity and reduce repetition.

Last but not the least, please verify consistency in formatting references including volume and page numbers. Also some DOI formats appear non-uniform.

All in all, this paper has strong potential for publication but after careful revision. The topic is novel, and the methodology is solid, but interpretation and framing need refinement.

Best of luck and keep up the good work,

Cheers!

Author Response

Please refer to the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have revised the manuscript following the reviewer's comments. No further comments are available. 

Comments on the Quality of English Language

Minor English editing is required. 

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you very much for addressing all the concerns and comments. I believe authors have done a great job refining the manuscript and thus it will help in disseminate and furthering our understanding in pushing the boundaries in this area of research. 
Best of luck and keep up the good work,

Cheers!