Combining Vascular Targeting Agents with Radiation: An Effective Anti-Tumor Treatment but Associated with Radiation-Induced Systemic Toxicity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the current manuscript, the authors analyzed the effects of combination therapies using angiogenesis inhibitors, vascular disrupting agents, and radiation in vivo models. The results are interesting and the manuscript is almost well written. Here are my comments that would improve the manuscript.

1.           What dose “π” means in the figure legends 1 and 2?  I assume that it indicates the combination groups in both figures.

2.           It is unclear how the authors selected the analyzed cytokines. Also, the SE levels were remarkably high in some of the analysis (e.g. IL-6 at 72 hours after RT, and IL-10 at 1 hour after RT). It is unclear how the authors regarded the results from the manuscript.

3.           If the authors wanted to show the feasibility of these therapies, the authors need to check the blood cell numbers at least because it might be one of the side effects.

4.          Discussion section seems long, and the authors need to try to summarize the points to be discussed.

Author Response

1. What dose “π” means in the figure legends 1 and 2?  I assume that it indicates the combination groups in both figures.

Response: This was strange. When we uploaded the original version of our manuscript the combination groups were clearly indicated by solid triangles in the figure legend. However, in the versions sent to the reviewers it was changed to ”ττ”! Why this happened is unknown. In the re-submitted version the symbols are correct and we hope that they remain unchanged when it is uploaded.

2. It is unclear how the authors selected the analyzed cytokines. Also, the SE levels were remarkably high in some of the analysis (e.g. IL-6 at 72 hours after RT, and IL-10 at 1 hour after RT). It is unclear how the authors regarded the results from the manuscript.

Response: We selected a broad spectrum of cytokines that were available in commercial kits and had previously been evaluated in pre-clinical radiation studies in our laboratory. Comments explaining this have now been added to section 2.5. As to the remarkably high SE values, then the SE values have a tendency to be large when analyzing cytokines due to high noise levels even though the study was done in duplicate and we have 14 measurements per treatment. We now refer to this point (with a relevant publication) in lines 317-318.  Finally, our study was not designed to elucidate mechanisms, but rather to demonstrate the fact that when investigating potential anti-tumor effects when combining radiation and VTAs there is a potential negative effect of induced systemic toxicity and any discussion of the mechanisms would be pure speculation and goes beyond our study.

3. If the authors wanted to show the feasibility of these therapies, the authors need to check the blood cell numbers at least because it might be one of the side effects.

Response: What side effects are causing the drop in body weight is unclear. When we planned this study, we were simply investigating the potential of combining two vascular targeting agents that have different modes of action (i.e., an angiogenesis inhibitor and a vascular disrupting agent) and see how they could be used to enhance response to radiation therapy. Based on our previous studies and those of others we did not expect any systemic toxicity effects, so were surprised by the data of figure 4. This seemed to be a radiation mediated effect and when researching the literature it became obvious that radiation could increase various cytokines and cause an inflammatory response that perhaps could then influence the toxicity profile of the angiogenesis inhibitor. Hence our additional measurements shown in table 4. Clearly, we do not know exactly what is causing the increase in toxicity and a more detailed study investigating a number of factors including blood cell numbers, various enzyme levels, and histological assessment of a range of normal tissues, is required. However, that would involve a large, more comprehensive study that goes beyond the scope of the current study. Nevertheless, we have now added some additional comments related to this specific issue at the end of the discussion (see lines 330-334).

4. Discussion section seems long, and the authors need to try to summarize the points to be discussed.

Response: The reviewer is correct that the discussion is long. However, we believe this length was necessary to cover all the relevant aspects of our study.

Reviewer 2 Report

Comments and Suggestions for Authors

The study explores the combination of vascular targeting agents (VTAs) with radiation for enhanced anti-tumor effects. While the combination of TNP-470 and combretastatin A-4 phosphate (CA4P) with radiation showed promising tumor growth inhibition, significant systemic toxicity was observed, especially with TNP-470. The study lacks a detailed exploration of the mechanisms behind this toxicity and the role of cytokines beyond IL-6. Additionally, the long-term impact of this treatment, optimization of dosing, and broader applicability across different cancer models need further investigation. Overall, the paper provides valuable insights but requires further clarification on several critical points.

 

Comment for authors

1. The paper's title is too wordy, consider revising it.

2. The introduction section lacks sufficient background information and requires significant improvements.

3. The introduction should include a discussion on how radiation impacts biological systems to provide readers with a comprehensive understanding of its effects. Incorporating following article (https://doi.org/10.3390/ijms23169288) in the introduction section will be helpful for readers to understand how radiations affect biological systems.

4. For Figures 1–4, it is recommended to visually represent the solid and hollow symbols directly on the graphs, rather than describing them in the figure captions.

5. The paper identifies an increase in systemic toxicity when combining TNP-470 with radiation, but the exact mechanism behind this toxicity is not fully explained, explain possibilities beyond IL-6 in the discussion section.

6. The study focuses on short-term systemic toxicity, such as weight loss. What about the long-term impact of this combination therapy, and could chronic effects arise that were not observed in the short-term assessments?

7. The concept of vascular normalization as a potential mechanism to enhance radiation response is mentioned but not deeply explored.

8. The paper mentions that VDAs may induce hypoxia in the tumor core, potentially reducing the efficacy of radiation. Did the authors measure tumor oxygenation during their experiments, and how might this hypoxia have influenced their results?

9. The paper uses specific doses of TNP-470, CA4P, and radiation. How they optimize the treatment dose? There is no data provided about this.

10. The study measures systemic toxicity using weight loss as a proxy, but how does the combination therapy affect normal tissues beyond weight changes? Are there other indicators of damage to normal tissues that could have been evaluated, such as histopathology or specific organ function tests?

 

11. The paper contained typos and grammatical errors. Double-check and correct them in the revised version.

Comments on the Quality of English Language

The paper contained typos and grammatical errors. Double-check and correct them in the revised version.

Author Response

1. The paper's title is too wordy, consider revising it.

Response: The reviewer is correct and we have significantly shortened the title.

2. The introduction section lacks sufficient background information and requires significant improvements.

Response: We have modified the introduction to improve the understanding of areas that were unclear, especially concerning TNP-470 (see lines 48-59). We also added details regarding radiation as requested along with additional references (see lines 80-83).

3. The introduction should include a discussion on how radiation impacts biological systems to provide readers with a comprehensive understanding of its effects. Incorporating following article (https://doi.org/10.3390/ijms23169288) in the introduction section will be helpful for readers to understand how radiations affect biological systems.

Response: Additional comments regarding radiation have been added as requested (see point 2). We also thank the reviewer for making us aware of the excellent review article on microwave radiation. However, we did not include it in the reference list because the focus of that review was microwave radiation which has a different mechanism of action to X-ray radiation as used in the current study and we felt it might confuse those not so familiar with the different types of radiation.

4. For Figures 1–4, it is recommended to visually represent the solid and hollow symbols directly on the graphs, rather than describing them in the figure captions.

Response: We agree with the reviewer that figures are easier to understand if the legends are actually in the figure. Thus, in line with the reviewer’s suggestion we have now added the relevant details to the figures themselves. However, we still kept the information in the legends to avoid confusion regarding the additional details concerning the doses, scheduling, and routes of administration used for each treatment.

5. The paper identifies an increase in systemic toxicity when combining TNP-470 with radiation, but the exact mechanism behind this toxicity is not fully explained, explain possibilities beyond IL-6 in the discussion section.

Response: Our study was not designed to elucidate possible mechanisms for the increase in toxicity when combining TNP-470 and radiation and any discussion beyond what we have already included would be pure speculation and we are not comfortable with that.

6. The study focuses on short-term systemic toxicity, such as weight loss. What about the long-term impact of this combination therapy, and could chronic effects arise that were not observed in the short-term assessments?

Response: This is a good point. Unfortunately, all the mice were tumor bearing and according to our animal license, when tumors reached a maximum size of 1000 mm³ we were required to euthanize them and this was long before any chronic toxicity affects would occur.

7. The concept of vascular normalization as a potential mechanism to enhance radiation response is mentioned but not deeply explored.

Response: Additional comments regarding normalization have been added (see lines 270-274).

8. The paper mentions that VDAs may induce hypoxia in the tumor core, potentially reducing the efficacy of radiation. Did the authors measure tumor oxygenation during their experiments, and how might this hypoxia have influenced their results?

Response: We did not measure hypoxia in this specific study, but do have previously published data showing that after treatment with CA4P there was a significant increase in hypoxia. This was mentioned in the discussion. We have no data on what happens when mice are treated with TNP-470. Any effect on hypoxia would not have influenced the radiation response since the various drug treatments were started after radiation had been given. Of course, we do not know whether an induction of hypoxia would have affected the cytokine levels and are not aware of any studies addressing this issue, so any discussion of this aspect would be pure speculation on our part.

9. The paper uses specific doses of TNP-470, CA4P, and radiation. How they optimize the treatment dose? There is no data provided about this.

Response: The specific doses of CA4P and TNP-470 selected were based on previous studies by us and others. This is now stated in the Materials and Methods along with relevant references (see section 2.2). Interestingly, the data of Figure 1 clearly show that the effects of CA4P and TNP-470 on tumor growth inhibition were identical, thus confirming that the doses we selected were optimal. The use of 10 Gy radiation in figures 3 and 4 was simply because it was the maximum dose used in the combination studies shown in figure 2 and is now stated in the text (see lines 318-321).

10. The study measures systemic toxicity using weight loss as a proxy, but how does the combination therapy affect normal tissues beyond weight changes? Are there other indicators of damage to normal tissues that could have been evaluated, such as histopathology or specific organ function tests?

Response: What side effects are causing the drop in body weight is unclear. When we planned this study, we were simply investigating the potential of combining two vascular targeting agents that have different modes of action (i.e., an angiogenesis inhibitor and a vascular disrupting agent) and see how they could be used to enhance response to radiation therapy. Based on our previous studies and those of others we did not expect any systemic toxicity effects, so were surprised by the data of figure 4. This seemed to be a radiation mediated effect and when researching the literature it became obvious that radiation could increase various cytokines and cause an inflammatory response that perhaps could then influence the toxicity profile of the angiogenesis inhibitor. Hence our additional measurements shown in table 4. Clearly, we do not know exactly what is causing the increase in toxicity and a more detailed study investigating a number of factors including blood cell numbers, various enzyme levels, and histological assessment of a range of normal tissues, is required. However, that would involve a large, comprehensive study that goes beyond the scope of the current study. Nevertheless, we have now added some additional comments related to this specific issue at the end of the discussion (see lines 330-334).

11. The paper contained typos and grammatical errors. Double-check and correct them in the revised version.

Response: Typos and grammatical errors were seen and corrected.

Reviewer 3 Report

Comments and Suggestions for Authors

Your manuscript exploring the combination of AIs, vVDAs, and radiation in treating C3H mammary carcinomas presents an intriguing approach. However, several major concerns need addressing to enhance the clarity and robustness of your study. 1. The manuscript requires substantial revisions for language clarity. Many sentences are currently difficult to understand. 2. The graphical representation in the figures appears outdated. I recommend using modern graphing software, such as GraphPad, for clearer and more contemporary visuals. 3. It is claimed that there is a significant difference in body weight between the groups shown in Figure 4, yet there appears to be no statistical analysis provided. Including p-values or other statistical tests will substantiate your claims. 4. Converting Table 1 into graphical curves could significantly enhance readability and comprehension of the data presented. 5. The results pertaining to cytokines do not seem to correlate with the body weight data in Figure 4. Clarifying this relationship, or providing a rationale for the observed discrepancy, would be beneficial. 6. A deeper analysis of the toxicity associated with the combination therapies is crucial. This should include a pathological evaluation of various organs to better understand potential side effects.

Comments on the Quality of English Language

Substantial revisions are required.

Author Response

1. The manuscript requires substantial revisions for language clarity. Many sentences are currently difficult to understand.

Response: We have re-checked the manuscript and made changes to sentences that were not clear.

2. The graphical representation in the figures appears outdated. I recommend using modern graphing software, such as GraphPad, for clearer and more contemporary visuals.

Response: This is the first time ever that someone has raised concerns about the way the figures in my publications look. To be perfectly honest, I do not believe that using modern graphing software will actually improve the figure quality beyond which is currently seen. 

3. It is claimed that there is a significant difference in body weight between the groups shown in Figure 4, yet there appears to be no statistical analysis provided. Including p-values or other statistical tests will substantiate your claims.

Response: The reviewer is correct and we have now modified our comments to indicate this. The problem was that the statistical comparison was made between the mouse body weight after radiation with the values obtained with radiation + TNP-470 or radiation + CA4P + TNP-470. As one can see from figure 4, the radiation only curve stops at 14 days. This is because the tumors had reached the maximal size allowed under our animal license and consequently the animals were euthanized. Thus, we could only do the statistical analysis up to day 14 and it would seem odd to indicate the significant difference only on the data up to this point, when it is obvious that values after day 14 are probably also “significantly” lower. To address this comment we have, therefore, modified the text as shown in lines 190-191.

4. Converting Table 1 into graphical curves could significantly enhance readability and comprehension of the data presented.

Response: We agree with the reviewer that graphical curves generally are better than tables. However, we originally represented this data in figure form, but decided to go with a table because it made the data clearer and easier to understand.

5. The results pertaining to cytokines do not seem to correlate with the body weight data in Figure 4. Clarifying this relationship, or providing a rationale for the observed discrepancy, would be beneficial.

Response: We agree with the reviewer, but cannot explain the lack of correlation between the cytokine and body weight data. Hence, our comments on lines 328-330.

6. A deeper analysis of the toxicity associated with the combination therapies is crucial. This should include a pathological evaluation of various organs to better understand potential side effects.

Response: What side effects are causing the drop in body weight is unclear. When we planned this study, we were simply investigating the potential of combining two vascular targeting agents that have different modes of action (i.e., an angiogenesis inhibitor and a vascular disrupting agent) and see how they could be used to enhance response to radiation therapy. Based on our previous studies and those of others we did not expect any systemic toxicity effects, so were surprised by the data of figure 4. This seemed to be a radiation mediated effect and when researching the literature it became obvious that radiation could increase various cytokines and cause an inflammatory response that perhaps could then influence the toxicity profile of the angiogenesis inhibitor. Hence our additional measurements shown in table 4. Clearly, we do not know exactly what is causing the increase in toxicity and a more detailed study investigating a number of factors including blood cell numbers, various enzyme levels, and histological assessment of a range of normal tissues, is required. However, that would involve a large, comprehensive study that goes beyond the scope of the current study. Nevertheless, we have now added some additional comments related to this specific issue at the end of the discussion (see lines 330-334).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Accept in present form