Hyperbaric Oxygen Therapy Adjuvant Chemotherapy and Radiotherapy through Inhibiting Stemness in Glioblastoma

Round 1

Reviewer 1 Report

The manuscript “Hyperbaric oxygen therapy adjuvant chemotherapy and radiotherapy through inhibiting stemness in glioblastoma” represents an original experimental study of glioblastoma in cell cultures and animal models. Considering the dismal prognosis of glioblastoma, the topic undoubtedly is timely and important, and the research on certain aspects of tumour treatment in association with the stemness can be considered innovative. The contents of the manuscript correspond to the scope of the journal. Authors have investigated the impact of hyperbaric oxygen therapy on in vitro and in vivo growth of glioblastoma cell lines, as well as effect of hyperbaric oxygen therapy in combination with temozolomide and radiotherapy. The study design is sound; the results are detailed and illustrated.

There are few minor notes for potential improvements:

1) Immunohistochemistry for CD133 has been performed. The choice of a particular cancer stem cell marker should be justified, as CD133 is the first reported but not the only stem cell marker in glioblastoma.

2) Please, provide immunohistochemical images of higher quality.

3) The survival of glioblastoma can be significantly less than 12 – 15 months. See, please, Jakovlevs et al., 2019 (PMID: 32146793); among others.

4) HIF-1alpha is not the only driver of stemness in glioblastoma. Please, see Biserova et al., 2021 (PMID: 33799798) for molecular mechanisms in glioblastoma stem cells. As HIF-1alpha levels have not been evaluated in the given study, it would be appropriate to discuss it in association with the other molecular mechanisms of stemness as these could limit the efficacy of hyperbaric oxygenation by inducing stem cell differentiation via alternative pathways.

5) In glioblastoma, several molecular subtypes have been identified both by gene expression and immunohistochemistry. These subtypes were shown to predict the efficacy of the treatment. See, please, Verhaak et al., 2010 (PMID: 20129251) and Jakovlevs et al., 2019 (PMID: 32146793) for gene expression-based and immunohistochemical subtyping, respectively. Would you expect equal efficacy of hyperbaric oxygenation in all molecular subtypes of glioblastoma?

6) Although most of the abbreviations have been spelled out, some still need explanation (e.g., MTT in the Abstract (line 44) and main text (line 96); FBS (line 103); among others).

7) Check the formatting of references, please. It should be in accordance with the “Instructions for Authors”.

8) A significant fraction of references is older than 10 years, e.g., Ref. 4, 5, 6, 7, 24, 25, 27, 31, and 40. Please, consider whether you could implement some more recent references, as the concepts on aetiology and molecular pathology of gliomas have changed over the years, new epidemiological data have been acquired etc.

Finally, I would like to thank the authors for their work input and dedication. It was a pleasure and a true honour to review this manuscript.

The level of English language is generally good. Nevertheless, minor language editing might be useful to remove the few minor misprints. The title should be rephrased to improve its comprehensibility.

Author Response

The manuscript “Hyperbaric oxygen therapy adjuvant chemotherapy and radiotherapy through inhibiting stemness in glioblastoma” represents an original experimental study of glioblastoma in cell cultures and animal models. Considering the dismal prognosis of glioblastoma, the topic undoubtedly is timely and important, and the research on certain aspects of tumour treatment in association with the stemness can be considered innovative. The contents of the manuscript correspond to the scope of the journal. Authors have investigated the impact of hyperbaric oxygen therapy on in vitro and in vivo growth of glioblastoma cell lines, as well as effect of hyperbaric oxygen therapy in combination with temozolomide and radiotherapy. The study design is sound; the results are detailed and illustrated.

There are few minor notes for potential improvements:

• Immunohistochemistry for CD133 has been performed. The choice of a particular cancer stem cell marker should be justified, as CD133 is the first reported but not the only stem cell marker in glioblastoma.

Certainly, we appreciate your suggestion. In addition to the immunohistochemistry for CD133, we have conducted further experiments using Western blot to detect the expression levels of other stem cell markers, including SOX-2 and OCT4. These additional markers provide a more comprehensive evaluation of the stem cell phenotype within glioblastoma.

• Please, provide immunohistochemical images of higher quality.

Thank you for your feedback. We have now provided higher-quality immunohistochemical images in the study. These images have improved resolution and quality. If you would like to review these updated images or have any other questions or requests, please feel free to let us know.

• The survival of glioblastoma can be significantly less than 12 – 15 months. See, please, Jakovlevs et al., 2019(PMID: 32146793); among others.

Thank you for your observation. The statement has been revised accordingly to reflect the variable survival times in glioblastoma, which can be significantly less than 12-15 months, as supported by the reference to Jakovlevs et al., 2019 (PMID: 32146793).

4) HIF-1alpha is not the only driver of stemness in glioblastoma. Please, see Biserova et al., 2021 (PMID: 33799798) for molecular mechanisms in glioblastoma stem cells. As HIF-1alpha levels have not been evaluated in the given study, it would be appropriate to discuss it in association with the other molecular mechanisms of stemness as these could limit the efficacy of hyperbaric oxygenation by inducing stem cell differentiation via alternative pathways.

Certainly, we appreciate your input. You're correct that HIF-1alpha is not the sole driver of stemness in glioblastoma, and there are multiple molecular mechanisms at play in glioblastoma stem cells (GSCs). In our study, we focused on assessing the expression of CD133, SOX2, and OCT4 as indicators of stem cell characteristics. HIF-1alpha, in our context, was considered as a factor induced by hypoxia, enhancing GSC properties. Hyperbaric oxygen therapy was explored as a means to increase oxygen levels within the tumor, which, in turn, could suppress GSC properties.

We acknowledge the complexity of GSC biology and the involvement of various molecular mechanisms. In future research, it would indeed be valuable to delve deeper into the interplay of these mechanisms and their potential impact on hyperbaric oxygenation therapy.

5) In glioblastoma, several molecular subtypes have been identified both by gene expression and immunohistochemistry. These subtypes were shown to predict the efficacy of the treatment. See, please, Verhaak et al., 2010 (PMID: 20129251) and Jakovlevs et al., 2019 (PMID: 32146793) for gene expression-based and immunohistochemical subtyping, respectively. Would you expect equal efficacy of hyperbaric oxygenation in all molecular subtypes of glioblastoma?

Thank you for pointing out the importance of molecular subtypes in glioblastoma (GBM) and their potential influence on the efficacy of treatments like hyperbaric oxygenation. Indeed, GBM is a heterogeneous disease with various molecular subtypes, and the response to treatments can differ among these subtypes.

In our current study, we have chosen to work with GBM8401 (sensitive to TMZ and RT) and T98G (resistant to TMZ and RT) cell lines to validate the effects of hyperbaric oxygen therapy. These two cell lines represent different responses to conventional treatments and allow us to explore the impact of hyperbaric oxygen in distinct contexts.

In the future, we aim to establish a platform using patient-derived cells to conduct experiments. This approach will enable us to assess whether individual patients with specific molecular subtypes of GBM may benefit from adjunctive hyperbaric oxygen therapy. By tailoring treatments to the molecular characteristics of each patient's tumor, we hope to enhance the effectiveness of therapies and improve outcomes.

We appreciate your insights into the relevance of molecular subtyping in GBM treatment, and we are committed to further investigating its implications in our research

6) Although most of the abbreviations have been spelled out, some still need explanation (e.g., MTT in the Abstract (line 44) and main text (line 96); FBS (line 103); among others).

Thank you for bringing this to our attention. We have reviewed the manuscript and made the necessary revisions to spell out and explain the abbreviations.

7) Check the formatting of references, please. It should be in accordance with the “Instructions for Authors”.

Thank you for your diligence in reviewing our manuscript, and we appreciate your feedback. We have carefully checked the formatting of our references to ensure they comply with the "Instructions for Authors." We believe that our revised manuscript now adheres to the required formatting standards.

8) A significant fraction of references is older than 10 years, e.g., Ref. 4, 5, 6, 7, 24, 25, 27, 31, and 40. Please, consider whether you could implement some more recent references, as the concepts on aetiology and molecular pathology of gliomas have changed over the years, new epidemiological data have been acquired etc.

Thank you very much for your thorough review of our research and for providing valuable suggestions. We greatly appreciate your feedback. Regarding your point about updating references, we take this matter very seriously. We have reviewed the references and ensured that we have incorporated recent references from the last ten years into our study to ensure that our research reflects the latest knowledge and developments in the field.

Finally, I would like to thank the authors for their work input and dedication. It was a pleasure and a true honour to review this manuscript.

Reviewer 2 Report

In this preclinical study, the authors evaluated the impact of hyperbaric oxygen therapy alone or in combination with chemoradiation on the stemness state of glioblastoma. The authors used both in vitro assays and in vivotumor models to conduct these analyses. Based on the generated readouts, the authors conclude that hyperbaric oxygen therapy enhances the response of GBM cells to chemoradiation by imparing the ability of these tumor cells to adopt a stem cell phenotype, and presumably circumvent the resistance of GBM cells to standard therapy. While the general hypothesis behind this study is a sound one and well-worth exploring, the methodology employed by the authors has several flaws and needs to be revisited. In this regard, I have a number of comments for the authors as follows: 

1.     TMZ requires longer incubation times (i.e., longer than 72 hrs) to unfold the full killing potential of this alkylating agent. This is because the methylation of guanine nucleobases at the O6 position by TMZ requires several cycles of futile DNA repair before apoptosis is initiated by the MMR system. In my experience, for an MTT assay conducted with TMZ, at least 5 days of incubation with the drug are required for GBM cells to be killed by TMZ at clinically relevant concentrations (i.e., less than 20 μM). Although the drug itself breaks down in less than 24 hrs, it is the lingering effect of the drug on DNA (in the absence of MGMT) that requires such a long incubation for the cells to be killed. Therefore, this is especially relevant in the case MGMT-negative GBM cells lines such as GBM8401. I recommend the authors to increase the incubation time of their cells with TMZ in the MTT assay while significantly reducing the concentration of the alkylating agent. The main reason for doing this is to better understand the impact of hyperbaric oxygen therapy administered along with physiologically relevant concentrations of TMZ for humans in order to mimic a clinically relevant scenario that is translatable. While still informative, the effects reported by the authors were generated with TMZ concentrations that cannot be achieved in humans with this alkylating agent. 

2.   The neurosphere assay employed by the authors is incomplete and no conclusion could be drawn from their current results. In order to test their hypothesis on the impact of hyperbaric oxygen on neurosphere formation, the authors need to do a secondary sphere formation assay. Accordingly, GBM cells grown in neurostem cell medium need to be incubated in the presents of treatments and, at the end of the incubation time, the spheres need to be dissociated to single cells which are then transferred to fresh neurostem cell medium and observed for secondary sphere formation. If these single cells fail to form secondary spheres as a result of treatment exposure, then the authors could conclude that their treatments do indeed negatively impact the stem cell phenotype/functionality of these GBM cells.    

3.     The stem cell marker used as a histological readout by the authors to assess the impact of hyperbaric therapy on the stemness phenotype is insufficient. The stemness phenotype cannot be assayed by using only one putative stem cell biomarker in isolation. For such an assessment to be convincing, a combination of at least of few biomarkers from a much larger panel (i.e., CD133, CD44, CD15, CD70, S100A4, ALDH1A3, Nanog, OCT-4, SOX-2, Nestin, etc.) need to be included in these immunostaining analyses. Importantly, none of the above putative biomarkers is specific enough for stemness to be used in isolation for such an analysis. 

4.     The authors need to generate a Kaplan-Meier survival plot for their in vivo efficacy study conducted with hyperbaric oxygen with or without chemoradiation. First off, it would make a lot more sense to compare hyperbaric oxygen therapy alone versus hyperbaric oxygen therapy plus chemoradiation (i.e., TMZ + RT), because in a clinically relevant scenario hyperbaric oxygen therapy would make sense to be added to the Stupp protocol in the concurrent chemoradiation phase. Nonetheless, studying the effects of hyperbaric oxygen therapy in conjunction with either TMZ or RT is still acceptable provided that Kaplan-Meier plots are also generated. The photon flux data provided by the authors cannot be interpreted in isolation in the absence of such survival curves. 

Minor comment: the line 192 “To evaluate the therapeutic effect of HBO therapy alone or combined with radiotherapy…” should read “To evaluate the therapeutic effect of radiotherapy alone or combined with HBO…”. 

Author Response

In this preclinical study, the authors evaluated the impact of hyperbaric oxygen therapy alone or in combination with chemoradiation on the stemness state of glioblastoma. The authors used both in vitro assays and in vivotumor models to conduct these analyses. Based on the generated readouts, the authors conclude that hyperbaric oxygen therapy enhances the response of GBM cells to chemoradiation by imparing the ability of these tumor cells to adopt a stem cell phenotype, and presumably circumvent the resistance of GBM cells to standard therapy. While the general hypothesis behind this study is a sound one and well-worth exploring, the methodology employed by the authors has several flaws and needs to be revisited. In this regard, I have a number of comments for the authors as follows:

1. TMZ requires longer incubation times (i.e., longer than 72 hrs) to unfold the full killing potential of this alkylating agent. This is because the methylation of guanine nucleobases at the O6 position by TMZ requires several cycles of futile DNA repair before apoptosis is initiated by the MMR system. In my experience, for an MTT assay conducted with TMZ, at least 5 days of incubation with the drug are required for GBM cells to be killed by TMZ at clinically relevant concentrations (i.e., less than 20 μM). Although the drug itself breaks down in less than 24 hrs, it is the lingering effect of the drug on DNA (in the absence of MGMT) that requires such a long incubation for the cells to be killed. Therefore, this is especially relevant in the case MGMT-negative GBM cells lines such as GBM8401. I recommend the authors to increase the incubation time of their cells with TMZ in the MTT assay while significantly reducing the concentration of the alkylating agent. The main reason for doing this is to better understand the impact of hyperbaric oxygen therapy administered along with physiologically relevant concentrations of TMZ for humans in order to mimic a clinically relevant scenario that is translatable. While still informative, the effects reported by the authors were generated with TMZ concentrations that cannot be achieved in humans with this alkylating agent.

Thank you for your valuable input regarding the incubation time and concentration of TMZ in our MTT assay. We appreciate your insights into the kinetics of TMZ and its effects on GBM cells. To address the concern of achieving physiologically relevant concentrations and longer exposure times, we have decided to conduct a colony formation assay as you suggested. This approach will allow us to better understand the impact of hyperbaric oxygen therapy in combination with TMZ at concentrations and incubation times that are more clinically relevant.

By using the colony formation assay, we aim to provide a more comprehensive assessment of the long-term effects of hyperbaric oxygen therapy in conjunction with TMZ on GBM cells. This will help us simulate a scenario that better mimics clinical conditions and has greater translational relevance.

2. The neurosphere assay employed by the authors is incomplete and no conclusion could be drawn from their current results. In order to test their hypothesis on the impact of hyperbaric oxygen on neurosphere formation, the authors need to do a secondary sphere formation assay. Accordingly, GBM cells grown in neurostem cell medium need to be incubated in the presents of treatments and, at the end of the incubation time, the spheres need to be dissociated to single cells which are then transferred to fresh neurostem cell medium and observed for secondary sphere formation. If these single cells fail to form secondary spheres as a result of treatment exposure, then the authors could conclude that their treatments do indeed negatively impact the stem cell phenotype/functionality of these GBM cells.

Thank you for adopting the suggested approach to confirm the impact of hyperbaric oxygen therapy on neurosphere formation. It's important to ensure the completeness of your experimental design, and the observation that both the first and second sphere formation was inhibited in the groups subjected to HBO therapy provides valuable evidence of its effect on the stem cell phenotype/functionality of GBM cells.

3. The stem cell marker used as a histological readout by the authors to assess the impact of hyperbaric therapy on the stemness phenotype is insufficient. The stemness phenotype cannot be assayed by using only one putative stem cell biomarker in isolation. For such an assessment to be convincing, a combination of at least of few biomarkers from a much larger panel (i.e., CD133, CD44, CD15, CD70, S100A4, ALDH1A3, Nanog, OCT-4, SOX-2, Nestin, etc.) need to be included in these immunostaining analyses. Importantly, none of the above putative biomarkers is specific enough for stemness to be used in isolation for such an analysis.

Certainly, we appreciate your suggestion. In addition to the immunohistochemistry for CD133, we have conducted further experiments using Western blot to detect the expression levels of other stem cell markers, including CD44, SOX-2, and OCT4. These additional markers provide a more comprehensive evaluation of the stem cell phenotype within glioblastoma.

4. The authors need to generate a Kaplan-Meier survival plot for their in vivo efficacy study conducted with hyperbaric oxygen with or without chemoradiation. First off, it would make a lot more sense to compare hyperbaric oxygen therapy alone versus hyperbaric oxygen therapy plus chemoradiation (i.e., TMZ + RT), because in a clinically relevant scenario hyperbaric oxygen therapy would make sense to be added to the Stupp protocol in the concurrent chemoradiation phase. Nonetheless, studying the effects of hyperbaric oxygen therapy in conjunction with either TMZ or RT is still acceptable provided that Kaplan-Meier plots are also generated. The photon flux data provided by the authors cannot be interpreted in isolation in the absence of such survival curves.

Thank you for your suggestion regarding generating Kaplan-Meier survival plots for our in vivo efficacy study involving hyperbaric oxygen therapy with or without chemoradiation. We understand the importance of such survival curves in interpreting the data comprehensively.

In our study, due to the ethical guidelines and regulations set by the IACUC (Institutional Animal Care and Use Committee), we are required to adhere to specific criteria for the humane care and ethical treatment of the animals. These guidelines mandate that animals should be euthanized when their body weight falls below a certain threshold, typically 20% below their original weight. Therefore, in our research, we have used the time of euthanasia as a surrogate for the survival time of the mice since it aligns with the ethical standards and regulations governing animal research.

While generating Kaplan-Meier survival plots using these endpoints may not fully mirror the natural survival of the mice, it provides valuable information about the impact of our treatments on their overall well-being and longevity within the constraints of ethical animal research practices.

Minor comment: the line 192 “To evaluate the therapeutic effect of HBO therapy alone or combined with radiotherapy…” should read “To evaluate the therapeutic effect of radiotherapy alone or combined with HBO…”.

Thank you for making the correction. Your attention to detail is much appreciated, and it ensures the accuracy of the manuscript.

Round 2

Reviewer 2 Report

I thank the authors for accepting and implementing my suggestions and for conducting additional experiments. In my opinion, their manuscript is now significantly more complete, with conclusions strongly supported by the experimental data. I expect all these improvements to greatly benefit the potential reader. I therefore congratulate the authors for their diligent work and I recommend the current version of their manuscript for publication.