Specific Position of the Pulmonary Hilar Node in Cancer Immunity: Immunohistochemical and Morphometrical Study Using Lung Regional Nodes Obtained from Non-Small Cell Cancer Patients Without Metastasis

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Areas for improvement:

1. Limited discussion of lung cancer lymphatic drainage patterns: the introduction mentions that the hilar node is considered the most proximal node along the lymphatic drainage route in lung cancer, but doesn´t fully explain the normal lymphatic drainage patterns of the lung and how they differ between lobes. This context would help readers understand the subsequent findings about different immune responses in upper vs. lower lobe cancers.
2. Inadequate explanation of the "skip metastasis" phenomenon: The authors briefly mention that hilar nodes are sometimes free from metastasis when paratracheal nodes are involved, but don´t elaborate on this important concept or cite key references about skip metastasis in lung cancer.
3. Limited background on the sentinel node concept: Since the authors compare their findings to previous work on sentinel nodes in gastric cancer, a brief explanation of the sentinel node concept would be beneficial.
4. Minimal discussion of monocyte-derived vs. self-renewal macrophages: While the authors discuss this distinction in the discussion section, introducing it earlier would help frame the research question.
5. References could be more comprehensive: While the paper includes 44 references, the introduction primarily focuses on the immunosuppressive role of macrophages and CD169-positive macrophages role in cross-presentation. Additional references on lung-specific immune responses and regional differences in thoracic lymph nodes would strengthen the background.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

In the present work Aoki et al. attempted to present a immunohistochemical and morphometrical study using lung regional nodes from cancer patients. Their work is partially interesting since there are some important that should be addressed.

First of all, the whole scope and question of this work is unclear. The authors should clearly state their rationale and scope in the “Introduction” section. It is unclear, overall, what the authors are trying to achieve and what question they are trying to answer.

Concerning the “Materials and Methods” section, it appears that the authors have performed the mcroscopic analyses manually. There are many computational tools to quantify or semi-quantify histological images, and I don’t understand why the authors have not tried to standardize their work with a computational workflow. Their methodology, if followed by another scientist it would probably lead to different results, which makes imperative the use of standard computational methods for quantification. For example (as they also mention), the use of ImageJ or other open source software would greatly improve their methodology.

The authors should summarize their patient cohort, as well as the anthropometric characteristics, in a table in the “Materials and Methods” section. They should not refer to a table placed in the “Results” section in the “Materials and Methods” section. They should also summarize the lymph nodes inspected per patient in a table in the “Materials and Methods” section. As I have understood it, each patient had multiple nodes inspected, which should be summarized in a separate table.

In the “Results” section the authors refer to percentages of “cluster” node areas; what does that mean? They should explain the meaning of their measurements in detail. Especially, if they want for other researchers and clinicians to use or repeat their work.

Explain in detail what “overlapped clusters” mean.

The authors should highlight their findings. Explain how their approach could be useful in the clinical practice. How can it be used for the diagnosis (?) or prognosis (?) or lung cancer. This was also my first comment concerning the scope of their work. Explain why this specific approach was used. Why is it important to perform an immunohistochemical and morphometrical inspection of peripheral lymph nodes in lung cancer patients (this goes beyond the obvious) and most importantly they should present a method that can be used in a standard and measurable way (not by hand or empirically).

Comments on the Quality of English Language

Minor proof-reading and editing

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

The introduction provides a good overview of the role of nodal dendritic cells and macrophages in cancer immunity. However, it could benefit from a clearer statement of the research gap and the specific objectives of the study.

Consider adding a brief explanation of why the hilar node is of particular interest compared to other regional nodes.

The results are comprehensive but somewhat dense. The presentation could be improved by summarizing key findings in a more concise manner, possibly using subheadings to guide the reader.

The figures are informative, but the legends could be expanded to include more details about the staining methods and significance of the highlighted areas.

The discussion effectively interprets the results in the context of existing literature. However, it would be helpful to explicitly address the limitations of the study (e.g., the use of "candidate DCs" without definitive confirmation of their identity).

The conclusion could be strengthened by suggesting potential clinical implications or future research directions based on the findings.

The methods are well-described, but the morphometric analysis section could be clarified further. For example, provide more details on how the "clusters" were defined and traced, and how reproducibility was ensured.

The references are relevant and up-to-date. However, a few recent studies on the role of CD169+ macrophages in cancer immunity could be added to bolster the background.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The authors have addressed my previous comments and have significantly improved their manuscript.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled “Specific position of the pulmonary hilar node in cancer immunity: An immunohistochemical and morphometrical study using lung regional nodes obtained from non-small cell cancer patients without metastasis” presents a detailed study on the distribution of macrophages and dendritic cells in the pulmonary lymph nodes of patients with non-small cell lung cancer without metastasis. However, the authors should clarify the following aspects:

1. How did they ensure that the observed differences in the distribution of macrophages and dendritic cells were not due to individual variations in the patients' immune systems?
2. Were the immunohistochemistry methods used in the study validated with appropriate positive and negative controls?
3. What criteria were used to define and quantify the overlap between CD169-positive cells and candidate dendritic cells?
4. How did they rule out the possibility that the correlation between the anatomical location of the lymph nodes and the observed immune activity was not a methodological artefact?
5. Were other relevant immune markers considered that could influence antigen presentation activity in pulmonary lymph nodes?
6. Are there any previous studies that support or contradict these findings regarding the differential distribution of macrophages and dendritic cells in regional lymph nodes?
7. How are these results interpreted in the context of lung cancer progression and their potential impact on the anti-tumour immune response?
8. Was the potential influence of chronic pulmonary diseases, such as COPD or pulmonary fibrosis, on the cellular composition of the lymph nodes analysed?
9. Could a functional analysis of these cells, such as in vitro immune activation studies, provide further evidence of their role in the anti-tumour response?
10. Would a longitudinal analysis allow for an assessment of whether the cellular composition of the lymph nodes changes over time during cancer progression?
11. Would it be feasible to integrate transcriptomic or proteomic analyses to better characterise the immune profiles of the different lymph nodes?
12. Has the functionality of CD169-positive cells and their actual capacity for antigen cross-presentation been considered in experimental models?
13. How could these findings contribute to the development or improvement of immunotherapeutic strategies for lung cancer patients?
14. Would a comparative analysis with lymph nodes from other types of cancer provide additional insights into the specificity of these findings in lung cancer?

Reviewer 2 Report

Comments and Suggestions for Authors

1. The authors performed immunohistochemical and morphometric analyses to show differences in the distributions of DC-SIGN-, CD68-, and CD169-positive cells in the paratracheal, subcarinal, and hilar nodes from 25 non-small cell lung cancer patients without metastasis. Please elaborate the innovative contributions of the manuscript in detail.
2. In the table 2, overlapped clusters and evaluation of anticancer activity in patients with lower lobe cancer, should be demonstrated in detail.
3. In the table 4, primary monoclonal antibodies, their dilution and specific treatment, should be demonstrated in detail.

Comments on the Quality of English Language

Please thoroughly revise the language before your submission.

Reviewer 3 Report

Comments and Suggestions for Authors

Nodal dendritic cells (DCs) and CD169-positive macrophages, possibly cross-present cancer antigens earlier in the proximal node than in the distal node. In this study, the authors found that CD169-positive and DC-positive cells were colocalized in the subcapsular and paracortical sinuses, whereas CD68-positive, self-renewal alveolar macrophages were present in the medullary sinus. This complementary distribution was more evident in nodes other than hilar nodes. Monocyte-derived cells occupied a significantly larger area in the hilar nodes of patients with upper lobe cancer than in patients with lower lobe cancer. They concluded that the specific site occupying the lung hilum with collateral vessels seemed to determine the hilar node enlargement. In general, the experiments were well-performed and the manuscript was well-written.

1) In the last sentence of Abstract, "hilar node morphology" is inaccurate, since no size or morphological index was measured. So "hilar node enlargement" may be better.

2) Not all abbreviations have been introduced with full names, for example, DC-SIGN in Abstract, "M" and "F" in Figures and tables.

3) Scale bars for Figure 1BCEF, Figure 2BCEF and Figure 3BCEF are missing.

4) Through analyzing your data, what are the clinical implications to the non-small cell cancer treatment?