Expression of Tenascin-C Is Upregulated in the Early Stages of Radiation Pneumonitis/Fibrosis in a Novel Mouse Model

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

As I pointed out last time, securing a minimum number of samples and performing statistical analysis are essential, and papers that do not meet this requirement cannot be evaluated. Shouldn't the authors change their paper to focus only on the creation of the RILD mouse model? After that, they should increase the number of samples and report on radiation pneumonitis markers.

Author Response

Comment 1

As I pointed out last time, securing a minimum number of samples and performing statistical analysis are essential, and papers that do not meet this requirement cannot be evaluated. Shouldn't the authors change their paper to focus only on the creation of the RILD mouse model? After that, they should increase the number of samples and report on radiation pneumonitis markers.

 

Response 1

Thank you for your suggestion.

As the reviewer pointed out, the principal objective of this paper is to demonstrate the creation of a novel mouse model of RILD and to provide a comprehensive description in the sequential change of the nature of the lung lesions observed through a comparative analysis of CT images and histological findings. An example of the characterization of the pathological changes, we focused on tenascin-C a well-known spatiotemporally limited marker of inflammation, because we already had preliminary data that TNC was up-reregulated in a RILD patient. Although the expression pattern of TNC in the lung lesion may suggest the molecule could be an early marker of RILD, evaluating the utility and feasibility of TNC as a biomarker is the next issue, which will indeed require increasing sample sizes and careful statistical analysis.

To clarify this point, we have modified the title by adding the word ‘novel’　in front of ‘mouse model’, rephrased．

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Title: 

Accurate and specific.

Consistent use of capitalization.

 

Abstract

Clearly describes the research focus and findings.

Some sentences are complex and could be simplified for better readability.

A bit verbose; can be more concise.

 

Introduction

Provides a thorough background on RILD and TNC.

Smooth transition from general RT information to specific study details.

Long sentences could be split for clarity. Example: "RP typically presents 1–6 months after RT and transitions to RF months to years later, and sometimes RILD becomes lethal."

Good coverage of mechanisms and prior studies. Some references to the literature are not smoothly integrated.

 

Results

Sequential CT Image Changes

Describes the experimental setup and findings accurately.

Some sentences are overly detailed. Example: "Exposure time was 17 seconds, and images were reconstructed at 400-μm thickness..."

Ensure consistent past tense usage. Example: "No obvious change was observed" (passive voice, consider active voice for clarity).

 

Comparison of CT Image Findings with Pathological Findings

Provides a clear comparison of CT and histological findings.

Descriptions of histological changes are sometimes too detailed for the main text and could be simplified or moved to a methods section.

Some sentences could be split for clarity. Example: "However, at 16 weeks, small inflammatory lesions, accompanied by abundant foam cells in the alveolar spaces, were observed."

 

Discussion

Discusses findings in the context of existing literature accurately.

Good, but some sections could be more concise.

Ensure clarity in complex sentences. Example: "The 'linear shadowing, consolidation, and loss of lung volume' at a later stage could correspond to histologic findings such as the decrease in inflammatory cells..."

 

Conclusion

Summarizes the study's findings accurately.

Clear and concise.

Correct, but ensure consistency in verb tenses and avoid passive voice where possible.

Comments on the Quality of English Language

Minor editing of the English language required

Author Response

Comment 1

Title: 

Accurate and specific.

Consistent use of capitalization.

 

Response 1

Thank you for your suggestion.

We have corrected the capitalization in the title.

Title: Expression of Tenascin-C is Upregulated in the Early Stages of Radiation Pneumonitis/Fibrosis in a Novel Mouse Model

 

Comment 2

Abstract

Clearly describes the research focus and findings.

Some sentences are complex and could be simplified for better readability.

A bit verbose; can be more concise.

 

Response 2

Thank you for your suggestion.

Complex or long sentences were corrected to be shorter.

 

Comment 3

Introduction

Provides a thorough background on RILD and TNC.

Smooth transition from general RT information to specific study details.

Long sentences could be split for clarity. Example: "RP typically presents 1–6 months after RT and transitions to RF months to years later, and sometimes RILD becomes lethal."　

 

 

Response 3

Thank you for your suggestion. Revised the sentence as follows.

 

RP typically presents 1 to 6 months after RT. It advances to RF over several months to years. In some cases, RILD can become fatal

 

Comment 4

Good coverage of mechanisms and prior studies. Some references to the literature are not smoothly integrated.

 

Response 4

Thank you for your suggestion.

We have revised it to smoothly integrate references to the literature.

 

Comment 5

Results

Sequential CT Image Changes

Describes the experimental setup and findings accurately.

Some sentences are overly detailed. Example: "Exposure time was 17 seconds, and images were reconstructed at 400-μm thickness..."

Ensure consistent past tense usage. Example: "No obvious change was observed" (passive voice, consider active voice for clarity).

 

Response 5

Thank you for your suggestion. Revised the sentence as follows.

 

"Exposure time was 17 seconds, and images were reconstructed at 400-μm thickness...

⇒The images were reconstructed by obtaining end-expiratory images using a respiratory gating technique.

 

"No obvious change was observed" 

⇒We could not observe any obvious changes in the mice until 16 weeks after irradiation.

 

Comment 6

Comparison of CT Image Findings with Pathological Findings

Provides a clear comparison of CT and histological findings.

Descriptions of histological changes are sometimes too detailed for the main text and could be simplified or moved to a methods section.

Some sentences could be split for clarity. Example: "However, at 16 weeks, small inflammatory lesions, accompanied by abundant foam cells in the alveolar spaces, were observed."

 

Response 6

Thank you for your suggestion. Revised the sentence as follows.

 

However, at 16 weeks, small inflammatory lesions were observed. Deposition of TNC began to be observed in the thickened alveolar septa near the inflammatory foci (Figure 3 A, B). At 20 weeks, when clear consolidation was confirmed in the CT images, the histology of the lesions showed features of organizing pneumonia consisting of fibroblasts, macrophages, and lymphocytes, loose-collagen fibers (Figure 3 C).

 

Comment 7

Discussion

Discusses findings in the context of existing literature accurately.

Good, but some sections could be more concise.

Ensure clarity in complex sentences. Example: "The 'linear shadowing, consolidation, and loss of lung volume' at a later stage could correspond to histologic findings such as the decrease in inflammatory cells..."

 

Response 7

Thank you for your suggestion. Revised the sentence as follows.

 

The "linear shadowing, consolidation, and loss of lung volume" at a later stage, indicating a progression from RP to RF. These changes could correspond to histologic findings such as the decrease in inflammatory cells, formation of mature collagen fibers, and progression of destruction and atrophy. These pathological findings were similar to those of RF in humans ^{6, 28}.

 

 

Comment 8

Conclusion

Summarizes the study's findings accurately.

Clear and concise.

Correct, but ensure consistency in verb tenses and avoid passive voice where possible.

 

Response 8

Thank you for your suggestion. Revised the sentence as follows.

 

I revised the text to ensure consistency in verb tenses and to avoid passive voice whenever possible.

 

Reviewer 3 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Dear Authors,

thank you for all the provided explanations. I recommend the article for publication in that form.

Author Response

Comment 1

Dear Authors,

thank you for all the provided explanations. I recommend the article for publication in that form.

 

Response 1

Thank you for your comments.

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

General Comments:

The manuscript presents an interesting study on radiation-induced lung damage (RILD) using a mouse model. The work is timely and relevant, especially with the increased use of stereotactic body radiotherapy (SBRT) in lung cancer treatment. The study is well-conceived, with a focus on comparing CT imaging findings with pathological changes and evaluating the role of Tenascin-C (TNC) as a potential biomarker for RILD.

However, the manuscript could benefit from some revisions to improve clarity, structure, and depth of analysis. Below are detailed comments on specific sections of the manuscript.

Title:

Consider revising the title to better reflect the study's focus on the use of TNC as a potential biomarker for RILD. 

Introduction:

·       The introduction provides a good background on RILD and the clinical relevance of the study. However, it could benefit from a clearer statement of the research hypothesis or specific objectives of the study.

·       The transition between the general discussion of RILD and the specific focus on TNC could be smoother. Consider adding a sentence to explicitly link TNC to the progression of RILD, setting the stage for its investigation in the study.

Materials and Methods:

·       The description of the Pb shielding device and irradiation setup is thorough, but it would be helpful to include a rationale for the chosen radiation dose (30 Gy/1fr) and its relevance to human SBRT.

·       The method for quantifying TNC-expressing regions is well described, but additional details on the validation of this method and any potential sources of error would strengthen the section.

Results:

·       The results are presented clearly, with appropriate use of figures to illustrate key findings. However, the text could be more concise, particularly in the descriptions of CT findings, to avoid redundancy.

·       In the section discussing the comparison of CT findings with histological findings, consider summarizing the key differences between the two methods in a table for better readability.

·       The results of TNC quantification are interesting but could be further analyzed. For example, statistical analysis of the differences in TNC expression over time would add rigor to the conclusions.

Figures:

·       Figure 1: The diagrams of the Pb shielding device and study design are helpful, but the figure legend could be more descriptive, explaining each panel in more detail.

·       Figure 2: The sequential CT images are essential for illustrating the progression of RILD but consider labeling key features directly on the images for clarity.

·       Figure 3: The histological comparison is crucial, but the figure could be enhanced by including higher magnification insets to highlight specific cellular changes.

·       Figure 5: The line graph of TNC positive areas is informative, but the figure legend should clarify whether error bars represent standard deviation or standard error.

Discussion:

·       The discussion effectively interprets the findings in the context of existing literature. However, it could be improved by explicitly addressing the limitations of the study, such as the small sample size and potential differences between the mouse model and human RILD.

·       Consider expanding on the implications of TNC as a biomarker for RILD, including potential clinical applications and the need for further research to validate these findings in human studies.

·       The discussion on the partial lung irradiation model could be more concise, focusing on its relevance to SBRT and potential for future studies.

Conclusion:

·       The conclusion is appropriate but could be strengthened by clearly summarizing the main findings and their significance for the field of radiation oncology.

 

Author Response

Comment 1

Title:

Consider revising the title to better reflect the study's focus on the use of TNC as a potential biomarker for RILD. 

 

Response 1

We also appreciate your point that we should focus on the relationship between TNC and RILD. On the other hand, another reviewer pointed out that there is not enough evidence to consider TNC as a biomarker in terms of sample size and statistical analysis. In light of this, I have revised the title slightly as follows.

Title: Expression of Tenascin-C is Upregulated in the Early Stages of Radiation Pneumonitis/Fibrosis in a Novel Mouse Model

 

Comment 2

Introduction:

• The introduction provides a good background on RILD and the clinical relevance of the study. However, it could benefit from a clearer statement of the research hypothesis or specific objectives of the study.
• The transition between the general discussion of RILD and the specific focus on TNC could be smoother. Consider adding a sentence to explicitly link TNC to the progression of RILD, setting the stage for its investigation in the study.

Response 2

Thank you for your suggestion. We have added the following sentences in the Introduction section.

 

After irradiation of the lungs, inflammation occurs in the lung tissue, and various factors related to tissue repair are activated. TNC might be one of these factors. Therefore, we hypothesized that TNC contributes to the progression of inflammation and tissue remodeling during the development of RILD.

 

Comment 3

Materials and Methods:

• The description of the Pb shielding device and irradiation setup is thorough, but it would be helpful to include a rationale for the chosen radiation dose (30 Gy/1fr) and its relevance to human SBRT.

 

Response 3

Thank you for your suggestion.

Dose fractions of 30Gy/1fr have been used clinically in SBRT for lung cancer patients. The following text and reference have been included in the Discussion section (Partial lung irradiation model for mice).

 

Our study, fractionated irradiation is difficult due to system constraints, so we used single-dose irradiation. In human studies, single-dose irradiation to the lungs is performed at 30-34 Gy. Based on this human study data, we adopted a dose of 30 Gy in 1 fraction.

 

 

Comment 4

The method for quantifying TNC-expressing regions is well described, but additional details on the validation of this method and any potential sources of error would strengthen the section.

 

Response 4

Thank you for your suggestion. The following text has been included in the Discussion section (TNC is upregulated at the early stage of radiation pneumonitis).

 

“In this study, the ratio of TNC-positive area to entire lung section area was quantified. Several other quantification methods were considered. One method was to measure the TNC-positive rate in a randomly selected field of view under a microscope. In this method, there was a risk of underestimating the TNC positivity rate if lung tissue outside the irradiated area was selected. The other method of selecting regions with strong TNC expression might result in bias.”

 

Comment 5

Results:

The results are presented clearly, with appropriate use of figures to illustrate key findings. However, the text could be more concise, particularly in the descriptions of CT findings, to avoid redundancy.

 

Response 5

Thank you for your suggestion. The statement in the Result section (Sequential CT image changes of the focally irradiated lung of a mouse with 30 Gy/1fr) is corrected as follows.

 

At 20 weeks, we observed patchy ground-glass opacity in the irradiated right lung. By 24 and 28 weeks, this opacity had expanded and became denser. At 32 weeks, we noted linear shadows, consolidation with lung volume loss, and compensated expansion in the non-irradiated left lung.

 

Comment 6

In the section discussing the comparison of CT findings with histological findings, consider summarizing the key differences between the two methods in a table for better readability.

 

Response 6

Thank you for your suggestion.

We have added the Table 1, which summarized the CT and histological findings at each period.

 

Comment 7

The results of TNC quantification are interesting but could be further analyzed. For example, statistical analysis of the differences in TNC expression over time would add rigor to the conclusions

 

Response 7

Thank you for your suggestion. In conducting this research, we have determined that it would be challenging to observe statistically significant differences. Therefore, we did not set a sample size and calculate the power for the study. In our future study, with the aim of investigating differences based on strain or differences related to knockout mice, we will consider the importance of setting the power and determining the sample size.

Considering the limitations of quantitative evaluation, we attempted to measure blood concentrations using ELISA with the aim of applying it as a biomarker, but it was unsuccessful. We are currently attempting to quantify the RNA and protein levels in lung tissue to analyze the molecular function of TNC.

 

Comment 8

Figures:

Figure 1: The diagrams of the Pb shielding device and study design are helpful, but the figure legend could be more descriptive, explaining each panel in more detail.

 

Response 8

Thank you for your suggestion. We have added the following sentences in Figure 1.

In Group 1, sequential computed tomography (CT) scans were obtained for the same mice at 12, 16, 20, 24, 28, and 32 weeks after irradiation. In this group, to eliminate individual differences in observations, we will perform sedation and CT evaluation of RILD on the same mouse each time. In Group 2, the CT images and pathological findings were compared at 12, 16, 20, 24, 28, 36, and 44 weeks after irradiation. In this group, to confirm CT findings of RILD pathologically, we perform autopsies at each time point.

 

Comment 9

• Figure 2: The sequential CT images are essential for illustrating the progression of RILD but consider labeling key features directly on the images for clarity.

 

Response 9

Thank you for your suggestion.

We have added labels for the key features in Figure 2.

 

Comment 10

• Figure 3: The histological comparison is crucial, but the figure could be enhanced by including higher magnification insets to highlight specific cellular changes.

 

Response 10

Thank you for your suggestion. We have added higher magnification insets in Figure 3C.

 

Comment 11

• Figure 5: The line graph of TNC positive areas is informative, but the figure legend should clarify whether error bars represent standard deviation or standard error.

 

Response 11

Thank you for your suggestion.

The dots in figure 5 represented actual scores for TNC positivity rates, not the standard deviation or standard error. We have modified the legend in Figure 5.

 

Comment 12

Discussion:

• The discussion effectively interprets the findings in the context of existing literature. However, it could be improved by explicitly addressing the limitations of the study, such as the small sample size and potential differences between the mouse model and human RILD.

 

Response 12

Thank you for your suggestion. We have added the following sentences in the limitation section.

 

Third, while this study has the potential to contribute to understanding the mechanisms of RILD, there is a possibility that findings in mice may differ from those in humans.

 

Comment 13

• Consider expanding on the implications of TNC as a biomarker for RILD, including potential clinical applications and the need for further research to validate these findings in human studies.

 

Response 13

Thank you for your suggestion. We have added the following sentences in the Discussion section.

 

If a biomarker for RILD is identified, attempting to measure blood concentrations using ELISA could potentially be applied to evaluate RILD in humans. The key benefit of discovering a biomarker for RILD is identifying severe cases early, potentially improving treatment outcomes.

 

Comment 14

• The discussion on the partial lung irradiation model could be more concise, focusing on its relevance to SBRT and potential for future studies.

 

Response 14

Thank you for your suggestion. We have added the following sentences in the Discussion section.

 

This model has the potential to contribute to the optimization of treatment parameters such as radiation dose, frequency, and irradiation area in SBRT, as well as to the elucidation of the mechanisms of RILD.

 

Comment 15

Conclusion:

• The conclusion is appropriate but could be strengthened by clearly summarizing the main findings and their significance for the field of radiation oncology.

 

Response 15

Thank you for your suggestion. We have added the following sentences in the Conclusion section.

 

It might be useful for identifying patients at high risk of developing RILD and providing appropriate care at an early stage.

Round 2

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

As I pointed out last time, it is essential to secure a minimum sample size and perform statistical analysis, and papers that do not meet this requirement cannot be evaluated. In other words, the data can be used to suit the story. Either delete that data or fill in the required N number before including it in your paper.

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

Dear Authors,

In general article is interesting but the results do not support major scientific claims. 

Your research explores the role of tenascin-C, in the early stages of radiation-induced lung damage. You proposed a mouse model with localized lung irradiation and tracked the development of radiation pneumonitis and radiation fibrosis through sequential CT scans and histological analysis. 

Despite all interesting results, the major findings require curation: 

1. I can't find any information about the control group - at least a single mouse should be observed (Ideally 3 of them) as a control group. How can we exclude other factors from the experiments (Like CT scans themselves?)

2. TNC positive area should be also presented in the table (ideally as a value for every sample - there are not many of them). There is one thing I observed that makes the results foggy - did you assess all 3 mice at every point? For some samples, I observed just two points and I can assume that the results were identical but I am not sure about that.

3. TNC level at 20W is for sure biased by a single very high reading? Please check again this sample if it is correct - I would even suggest removing it or making an additional description with an explanation in the article.

Reviewer 2 Report

Comments and Suggestions for Authors

1. Introduction:

1. Clarification is needed on the abbreviation SBRT (stereotactic body radiotherapy) upon first mention.
2. Specify the timeframe for the development of RP and RF after RT, as mentioned in references 1-3.
3. Include a brief explanation of how TNC could serve as a potential biomarker or therapeutic target for RILD.
4. The transition from RP to RF could be elaborated upon, highlighting the progression of lung damage.
5. Consider citing recent studies (if available) that provide insights into the molecular mechanisms underlying RILD.
6. Ensure consistency in the use of terminology (e.g., radiation-induced lung damage vs. RILD).
7. Check for parallelism in sentence structures to enhance readability.

 

2. Materials and Methods:

1. Provide justification for choosing BALB/c mice for the study.
2. Include details about the source and housing conditions of the mice for reproducibility.
3. Clarify the dimensions and specifications of the Pb shielding device to ensure its replicability.
4. Expand on the anesthesia protocol, including the rationale for choosing specific drugs and doses.
5. Include information about any potential adverse effects of anesthesia on the study outcomes.
6. Clarify the rationale for the selected radiation dose and parameters.
7. Specify the criteria used for categorizing mice into Groups 1 and 2.
8. Provide a rationale for the selected time points for sequential CT scans and histopathological analysis.
9. Describe the methodology for lung tissue processing and sectioning in more detail.
10. Provide a brief explanation of why hematoxylin and eosin (H&E) and Elastica-Sirius Red (SR) staining were chosen for histopathological analysis.
11. Mention any potential limitations or challenges encountered during the histopathological analysis.
12. Specify the concentration and source of the anti-TNC antibodies used for immunohistochemical staining.
13. Describe the antigen retrieval process in more detail, including its importance and rationale.
14. Clarify the method used for quantifying TNC-expressing regions and justify the chosen approach.
15. Provide details on how potential confounding factors were controlled during image analysis.
16. Mention any statistical methods employed for data analysis and interpretation.
17. Include information on how the study adhered to ethical guidelines for animal experimentation.
18. Maintain consistency in tense usage throughout the section.
19. Use clear and concise language to describe experimental procedures and methodologies.
20. Check for grammatical errors and typos, particularly in complex sentences.
21. Consider breaking down lengthy paragraphs into smaller, more digestible chunks for clarity.

 

3. Results:

1. Sequential CT image changes:

1. Provide details on the method used for CT image acquisition, such as slice thickness and reconstruction parameters.
2. Specify any potential reasons for the mouse's death during the CT scan at 20 weeks, if known.
3. Clarify if any external factors, such as environmental changes or interventions, were controlled during the study period.

 

2. Comparison of CT images with pathological findings:

1. Ensure clarity in the description of the histological findings, particularly regarding the appearance of specific cell types and tissue structures.
2. Consider providing a brief explanation of how Elastica-Sirius Red staining works to enhance understanding for readers unfamiliar with the technique.
3. Specify the criteria used to define the stages of RP and RF based on histopathological features.

 

4. Discussion:

1. Include a brief rationale for selecting BALB/c mice for the study, considering their relevance to lung pathology and irradiation models.
2. Provide more context on the significance of differences in RILD outcomes between sexes, as mentioned in reference 18.
3. Clarify whether the observed differences in fibrosis-related gene expression between different radiation doses (20 Gy vs. 90 Gy) have implications for clinical practice or mechanistic understanding.
4. Discuss the potential translational implications of the findings, particularly regarding the relevance of the mouse model to human RILD.
5. Consider addressing potential limitations of using TNC as an early marker for RILD, such as specificity and sensitivity issues.
6. Elaborate on the implications of TNC as a biomarker for RILD in clinical practice, including its potential role in guiding therapeutic decisions.
7. Maintain a logical flow of ideas and arguments to enhance readability and comprehension.
8. Check for grammatical errors and typos to ensure the clarity of the discussion points.

Comments on the Quality of English Language

Moderate editing of English language required

Reviewer 3 Report

Comments and Suggestions for Authors

The core of this paper is the evaluation of the relationship between TNC expression and RILD using this mouse model. It is said that the peak was reached at 20 weeks after irradiation, but it is not possible to know whether the peak has been reached until statistical analysis is performed. Furthermore, due to the small number of mice studied, some individuals show a TNC-positive rate of 0%. The authors have responded that they will consider this in the future, but I think this is an issue that should at least be resolved in this paper. Unfortunately, unless we increase the N number in Fig. 4 and perform statistical analysis, we cannot demonstrate that TNC is a useful inflammatory marker that precedes pulmonary fibrosis, as stated in the conclusion of this paper.