Cancer Stem Cells and Glioblastoma: Time for Innovative Biomarkers of Radio-Resistance?

Round 1

Reviewer 1 Report

In the introduction, the authors state their intent to evaluate the role of CSCs in extracranial solid cancers other than GBM. However, there is a mention of biomarkers within the context of GBM. To enhance clarity, it is advisable to rephrase the segment "other than GBM."

The authors accurately acknowledge the contribution of glioma CSCs to radiotherapy resistance through the activation of DNA damage response pathways (Results, lines 104-107). In addition to solid tumors, it's important to briefly address the matter of chemoresistance and the corresponding mechanisms in glioma CSCs at this point.

In the methods section, the authors comprehensively describe their literature search process. While this review was not conducted as a systematic review, it might be valuable to consider including a flow chart illustrating their search strategy. Could the authors also provide information about the number of studies included in this review?

Please ensure that each column in table 1 is accompanied by an appropriate header, particularly column 3. A suggestion is to allocate group headings in the biomarker column, indicating whether the biomarkers are membrane proteins, intracellular markers, or transcription factors.

Figure 1 should be accompanied by a detailed figure legend outlining the biomarkers involved in radio-resistance, along with their abbreviations.

There are missing markers such as Nestin, Notch signaling components like Notch1 and Jagged1, HIF-1α, STAT3, and DNA repair pathways (RAD51 and BRCA1/2). These markers should be discussed and supported by pertinent references.

Lastly, it would be insightful to hear the author's opinions and thoughts on the future directions of biomarkers in GBM, along with their predictive, diagnostic, and therapeutic value. Please provide commentary on the relevance of these aspects in the field, as mentioned in the abstract.

The review is well-written. Please conduct a thorough spelling check and formatting check, as there are minor mistakes throughout the manuscript.

Author Response

We thank the editor and reviewers for valuable suggestions that improved the quality of this review.

Below are the point-by-point responses to the reviewers' suggestions and comments.

We remain at the disposal for further modifications or additions if deemed useful by the auditors.

Kind Ragrads,

We thank the reviewer for this suggestion. We have integrated the text as suggested.

In the introduction, the authors state their intent to evaluate the role of CSCs in extracranial solid cancers other than GBM. However, there is a mention of biomarkers within the context of GBM. To enhance clarity, it is advisable to rephrase the segment "other than GBM."

Thanks for the suggestion. We have made the recommended correction.

In the methods section, the authors comprehensively describe their literature search process. While this review was not conducted as a systematic review, it might be valuable to consider including a flow chart illustrating their search strategy. Could the authors also provide information about the number of studies included in this review.

We thank the reviewer for the suggestion. As we have stated before, we have integrated the materials and methods part so as not to confuse the reader (this is a descriptive review, not a systematic review).

Please ensure that each column in table 1 is accompanied by an appropriate header, particularly column 3. A suggestion is to allocate group headings in the biomarker column, indicating whether the biomarkers are membrane proteins, intracellular markers, or transcription factors.

We thank the reviewer for the suggestion.

We have made the suggested changes.

Figure 1 should be accompanied by a detailed figure legend outlining the biomarkers involved in radioresistance, along with their abbreviations.

We thank the reviewer for the suggestion.

We have made the suggested changes.

There are missing markers such as Nestin, Notch signaling components like Notch1 and Jagged1, HIF-1α, STAT3, and DNA repair pathways (RAD51 and BRCA1/2). These markers should be discussed and supported by pertinent references.

We thank the reviewer for the suggestion. We have integrated the list of biomarkers.

Lastly, it would be insightful to hear the author's opinions and thoughts on the future directions of biomarkers in GBM, along with their predictive, diagnostic, and therapeutic value. Please provide commentary on the relevance of these aspects in the field, as mentioned in the abstract.

We thank the reviewer for the suggestion. We have added this section.

Author Response File: Author Response.docx

Reviewer 2 Report

This review focuses on the latest data on glioblastoma stem cells, especially in the light of their radioresistance, that is, the presence of molecular markers of radioresistance in them. Undoubtedly, it will be of interest to a wide range of readers - researchers working in the field of oncogenesis of gliomas, drug resistance of tumors, in particular, radioresistance. After reading the review, there are several comments that in no way detract from the significance of the material presented in this review, but are necessary to improve the quality of the review.

1.      The abbreviation GBM stands for glioblastoma multiforme, this diagnosis has been abolished by the WHO classification of 2021, so it is better to abbreviate GB.

2.      L. 17-18 Please change the font.

3.      L.38-40. «Unfortunately, there have been no improvements in the treatment of this tumor in the last 20 years, and all phase 3 studies have failed in their intention to change the standard therapy» - Please, add  the references, especially when you so categorical.

4.      L. 44. Merge the references.

5.      L. 46-47. Add the references.

6.      L. 52-57. You write that the analysis of the articles followed the previously established inclusion criteria, do I understand you correctly that the only inclusion criterion is the presence of the term "cancer stem cells". I doubt that this criterion is sufficient, even taking into account paragraph 2.3. It would be nice to get closer to a meta-analysis, but consider this only as a suggestion (https://doi.org/10.1002/mar.21657).

7.      L. 90-92. It would be better to write down what specific characteristics of stem cells controlled "various characteristics of cancer by interacting with cells and the extracellular matrix (ECM) in their environment." The phrase is not entirely clear.

8.      L. 92-95. It would be better to write specifically what «soluble factors such as interleukins, cytokines, growth factors, and other metabolites into the tumor microenvironment (TME) ». If these are the ones you describe in the next sentence, then why are the links different? Also it should be better to see the whole picture / information in the review itself, and not follow the links. On the one hand, the review should not contain a lot of "water", on the other hand, it is not necessary to clutter up the review with unnecessary information.

9.      L. 104. Please correct the word – example. Also You wrote here that «through activation of DNA damage response pathways and increased DNA repair capacity»… There are several DNA repair pathways—base excision repair (BER), nucleotide excision repair (NER), non-homologous end joining (NHEJ), mismatch repair (MMR), homologous recombination (HR) - what exactly are you talking about here?

10.  L. 123 – “to uncontrolled amplification “- the phrase only applies to genes, not cells.

11.  L. 137-139. The phrase «Radiotherapy (RT) induces DNA damage, either directly or indirectly through the generation of water-derived radicals and reactive oxygen species (ROS), which interact with macromolecules such as DNA, lipids, and proteins» is redundant, because You wrote about DNA damage twice.

12.  L. 160 Please explain (write some explanations and links) what you mean by the term “high-efficiency DNA damage detection points”.

13.  L. 209-211. The key point is why authors of a glioblastoma article should list “the most important CSCs biomarkers that could play a role in radio-resistance of solid tumors other than glioblastoma”. Please rewrite / add or remove this section altogether!

14.  L. 242. Perhaps there should have been a section dedicated to CD144…

15.  L. 244. Please add the references.

16.  3.3.1. Surface markers (CD133, CD144, EpCAM, TIM-3), 3.1.2 Intracellular markers, 3.1.3. Transcription Factor as biomarker. 3.1.4. Extracellular biomarkers.

 It is necessary to describe the molecular mechanism linking these receptors/ Intracellular markers /TFs (Nanog, SOX2, OCT-3/4, EZH2)/ Extracellular biomarkers with the occurrence of radioresistance. At the same time, only those markers should be given for which statistics are actually shown for patients undergoing radiotherapy, or at least experimentally on model animals. It is written crudely.

17.  L. 323-373. Please edit the paragraph on aldehyde dehydrogenase (ALDH) - it is written very crudely, there are incidental phrases («immune сheckpoints point expression»), you can take an article as a basis -doi: 10.3389/fimmu.2021.756606.

18.  Please, when describing markers, decipher their abbreviated names, for example:  EZH2 - Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (https://www.genecards.org/cgi-bin/carddisp.pl?gene=EZH2)

19.  L. 506 and L. 511 - What are those numbers in brackets?

20.  Table 1 - In the table you write that CD133 has a biological function - Antioxidant scavenger system, CD133-induced hypoxia, however, there is not a word about this in the text above, it is necessary to coordinate the text with the table. Also, it is not correctly write “CD44 interaction with extracellular domain activates a number of signaling pathways” - This is very general, please be more specific.

21.  Figure 1 is very uninformative, needs to be redrawn.

Minor editing of English language required. Please see my comments.

Author Response

We thank the editor and reviewers for valuable suggestions that improved the quality of this review.

Attached please find our point-by-point responses to the reviewers' suggestions and comments. Thank you again.

Author Response File: Author Response.pdf

Reviewer 3 Report

In the manuscript titled "Cancer stem cells and glioblastoma. Time for innovative bi- 2 omarkers of radio-resistance?" prepared by Pasqualetti et al., the author reviewed recent advances in glioma stem cells as well as the implication in biomarker discovery. I think the article brings about an interesting question, although I have a couple of minor suggestions as follow:

1. This is a review article. So I feel the material and method section is unnecessary.

2. The author mainly discussed about the protein markers that are typically used for CSC biology study. Could the author discuss which types of clinical sample are feasible for biomarker discovery?

3. The author may also consider discuss the diversified genetic background in glioma, which may bring additional insight into the biomarker selection. For example, IDH mutated cancer stem cells exhibit distinctive biological and metabolic patterns (PMID: 35611837).

English is ok. Minor grammar check may be needed.

Author Response

We thank the editor and reviewers for valuable suggestions that improved the quality of this review.

Below are the point-by-point responses to the reviewers' suggestions and comments.

We remain at the disposal for further modifications or additions if deemed useful by the auditors.

Kind Ragrads,

1. 
   This is a review article. So I feel the material and method section is unnecessary.

We thank the reviewer for this suggestion. We have taken care to make this part as descriptive as possible, precisely to make the reader realise that this is a descriptive review.

2. The author mainly discussed about the protein markers that are typically used for CSC biology study. Could the author discuss which types of clinical sample are feasible for biomarker discovery?

We thank the reviewer for this suggestion. We have taken care to specify in the text how biomarkers are developed in neuro-oncology and the associated limitations, especially with regard to translational studies.

3. The author may also consider discuss the diversified genetic background in glioma, which may bring additional insight into the biomarker selection. For example, IDH mutated cancer stem cells exhibit distinctive biological and metabolic patterns (PMID: 35611837).

We thank the reviewer for this suggestion. We have integrated the text as suggested.

In the introduction, the authors state their intent to evaluate the role of CSCs in extracranial solid cancers other than GBM. However, there is a mention of biomarkers within the context of GBM. To enhance clarity, it is advisable to rephrase the segment "other than GBM."

Thanks for the suggestion. We have made the recommended correction.

In the methods section, the authors comprehensively describe their literature search process. While this review was not conducted as a systematic review, it might be valuable to consider including a flow chart illustrating their search strategy. Could the authors also provide information about the number of studies included in this review.

We thank the reviewer for the suggestion. As we have stated before, we have integrated the materials and methods part so as not to confuse the reader (this is a descriptive review, not a systematic review).

Please ensure that each column in table 1 is accompanied by an appropriate header, particularly column 3. A suggestion is to allocate group headings in the biomarker column, indicating whether the biomarkers are membrane proteins, intracellular markers, or transcription factors.

We thank the reviewer for the suggestion.

We have made the suggested changes.

Figure 1 should be accompanied by a detailed figure legend outlining the biomarkers involved in radioresistance, along with their abbreviations.

We thank the reviewer for the suggestion.

We have made the suggested changes.

There are missing markers such as Nestin, Notch signaling components like Notch1 and Jagged1, HIF-1α, STAT3, and DNA repair pathways (RAD51 and BRCA1/2). These markers should be discussed and supported by pertinent references.

We thank the reviewer for the suggestion. We have integrated the list of biomarkers.

Lastly, it would be insightful to hear the author's opinions and thoughts on the future directions of biomarkers in GBM, along with their predictive, diagnostic, and therapeutic value. Please provide commentary on the relevance of these aspects in the field, as mentioned in the abstract.

We thank the reviewer for the suggestion. We have added this section.

Round 2

Reviewer 1 Report

The authors are to be commended for their thorough and comprehensive revision. I would like to offer some minor suggestions for further improvement:

1. - Table 1: Ensure uniformity in referencing style for PMIDs, aligning them with the format used for other markers.

2.  

3. - Please review the manuscript meticulously to address grammar and formatting issues. Pay particular attention to maintaining consistency in language style, whether British or American English (tumor vs. tumour). Also, address formatting concerns in lines 486-489 and reference no. 104 in the text (X.X Kim et al.).

4.  

5. - Abstract: In scientific writing, it's important to maintain objectivity. Avoid subjective language such as 'unfortunately' in the abstract.

Thank you!

Recommend spell check.

Author Response

like to thank you and the reviewers for your support in this second round.

Thanks to your suggestions the quality of this review has been substantially improved.

Below are the point-by-point responses to reviewer 1’s suggestions.

We remain open to any further modifications or changes you consider necessary to further refine the review.

Kind regards

Mario Miniati, M.D., Ph.D.

University of Pisa, Italy

The authors are to be commended for their thorough and comprehensive revision. I would like to offer some minor suggestions for further improvement:

- Table 1: Ensure uniformity in referencing style for PMIDs, aligning them with the format used for other markers.

Thank you for your suggestion. We made the requested changes.

- Please review the manuscript meticulously to address grammar and formatting issues. Pay particular attention to maintaining consistency in language style, whether British or American English (tumor vs. tumour). Also, address formatting concerns in lines 486-489 and reference no. 104 in the text (X.X Kim et al.).

Thank you for your suggestions. We carefully re-read the paper and made the changes.

- Abstract: In scientific writing, it's important to maintain objectivity. Avoid subjective language such as 'unfortunately' in the abstract.

Thank you for your suggestion. We made the changes, avoiding subjective language.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors, thank you very much for your reports on my comments, I still have some comments on the changed text.

L. 2. Please rewrite – «radioresistance» spelled together.

L.11. I am not sure, but perhaps the passive voice should be used. – «Unfortunately, glioblastoma is characterized by a high level of resistance to radiation therapy».

L. 20-21, L. 40-41 “In light of the failures of clinical trials conducted over the past 20 years, urgent clarification of the mechanisms conferring glioblastoma (GBMGB) progression and radioresistance is required.” It is very categorical and not entirely correct phrase. Please take a look and correct with at least these literary references (https://doi.org/10.1038/s41591-022-01897-x, https://doi.org/10.1039/D2BM01996E).

L. 22- 27. Please check the scientific English. “spread of cancer”, «discuss the scientific literature» - doesn't sound scientifically correct.

L. 61. Should be added the established term “radioresistance”.

L. 53. If this is an ordinary review, and not a meta-analysis, then why single out materials and methods, results? I don't understand.

L. 75. Explain what «language suitability» means as a parameter for including an article in a review, for example, if the article has already been published in high-impact journals? It seems to me that section 2 should be removed or briefly rewritten - for what year did you take articles published in high-impact journals.

L. 98 – L.104 You write quite obvious things that can be omitted, everyone knows that the classification of gliomas is based, among other things, on mutations in the iDH gene.

L.105. You missed the word – “cells” after “cancer stem”

L. 108 Change, please, “IDH mutated Gliomas” to “IDH mutant gliomas”

L. 127. Earlier I have been written about this sentence. (L. 104. Please correct the word – example. Also You wrote here that «through activation of DNA damage response pathways and increased DNA repair capacity»…There are several DNA repair pathways—base excision repair (BER), nucleotide excision repair (NER), non-homologous end joining (NHEJ), mismatch repair (MMR), homologous recombination (HR) - what exactly are you talking about here?

Unfortunately my comment has not been corrected.

L. 140. ”CSCs constitute only a small percentage (0.05-1%) of the total tumor cell population within a tumor mass” – Plagiarism from doi: 10.1002/0471141755.ph1425s61

L.168 – L.170. – You write about the fact that radiotherapy causes DNA damage, and also causes the formation of ROS, which cause damage to proteins, etc. This is not correct, since radiotherapy causes water radiolysis, the formation of ACE, and therefore damage to DNA, lipids and proteins.

L. 236-238. It is not clear why in an article devoted to the mechanisms of the occurrence of radioresistance due to stem cells mention the carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP).

My previous comments (13. L. 209-211. The key point is why authors of a glioblastoma article should list “the most important CSCs biomarkers that could play a role in radio-resistance of solid tumors other than glioblastoma”. Please rewrite / add or remove this section altogether!) has not been corrected!

Subsection 3.3.1. «Surface markers», 3.1.2 Intracellular markers, 3.1.3. Transcription Factor as biomarker. 3.1.4. Extracellular biomarkers, should be dedicated to markers of CSCs only glioblastoma, and only those which related to radioresistance.

Also change the name of the subsection “3.3. Biomarkers associated with CSC”, since we are talking not just about stem cells, but in the context of the emergence of radioresistance.

If you write about other tumors, or markers that are not related to radioresistance, then it will be a big cumbersome, unreadable text that has nothing to do with the title of your article (Cancer stem cells and glioblastoma. Time for innovative biomarkers of radioresistance?).

Unfortunately a very small discussion, from which I did not understand about "Future Directions", it is also worth noting that the authors do not single out any particular marker, which, in their opinion, is the most promising. Written very generally and very streamlined.

The picture is not informative.

Links inserted in the form of a picture or what?

It is necessary to check English for not only spelling, but also the correct use of terms and stable scientific expressions.

Author Response

Dear Editor,

I would like to thank you and the reviewers for your support in this second round.

Thanks to your suggestions the quality of this review has been substantially improved.

Below are the point-by-point responses to reviewer 2's suggestions.

We remain open to any further modifications or changes you consider necessary to further refine the review.

Kind regards

Mario Miniati, M.D., Ph.D.

University of Pisa, Italy

2. 2. Please rewrite – «radioresistance» spelled together.

We thank the reviewer for this suggestion; we changed the term radioresistance throughout the paper.

L.11. I am not sure, but perhaps the passive voice should be used. – «Unfortunately, glioblastoma is characterized by a high level of resistance to radiation therapy».

We thank the reviewer for this suggestion. We edited the sentence as suggested.

10. 20-21, L. 40-41 “In light of the failures of clinical trials conducted over the past 20 years, urgent clarification of the mechanisms conferring glioblastoma (GBMGB) progression and radioresistance is required.” It is very categorical and not entirely correct phrase. Please take a look and correct with at least these literary references (https://doi.org/10.1038/s41591-022-01897-x, https://doi.org/10.1039/D2BM01996E).

Thanks for this suggestion. We edited the sentence as suggested. We also added the two reported references.

27. 22- 27. Please check the scientific English. “spread of cancer”, «discuss the scientific literature» - doesn't sound scientifically correct.

We thank the reviewer for this suggestion, and we edited the sentences as suggested.

61. 61. Should be added the established term “radioresistance”.

In agreement with the first suggestion, we changed the term radioresistance throughout the paper.

53. 53. If this is an ordinary review, and not a meta-analysis, then why single out materials and methods, results? I don't understand.

Thanking the reviewer for this suggestion, we modified the structure of the paper

75. 75. Explain what «language suitability» means as a parameter for including an article in a review, for example, if the article has already been published in high-impact journals? It seems to me that section 2 should be removed or briefly rewritten - for what year did you take articles published in high-impact journals.

Thanking the reviewer for this suggestion, we removed session 2

1. 98 – L.104 You write quite obvious things that can be omitted, everyone knows that the classification of gliomas is based, among other things, on mutations in the iDH gene.

Following this suggestion, we removed the section on glioblastoma biomarkers.

L.105. You missed the word – “cells” after “cancer stem”

We deleted the flagged paragraph.

1. 108 Change, please, “IDH mutated Gliomas” to “IDH mutant gliomas”

We changed the sentence as suggested.

127. 127. Earlier I have been written about this sentence. (L. 104. Please correct the word – example. Also You wrote here that «through activation of DNA damage response pathways and increased DNA repair capacity»…There are several DNA repair pathways—base excision repair (BER), nucleotide excision repair (NER), non-homologous end joining (NHEJ), mismatch repair (MMR), homologous recombination (HR) - what exactly are you talking about here? Unfortunately my comment has not been corrected.

Thanks for this suggestion. We modified the paragraph as indicated.

140. 140. ”CSCs constitute only a small percentage (0.05-1%) of the total tumor cell population within a tumor mass” – Plagiarism from doi: 10.1002/0471141755.ph1425s61

Thanks for this suggestion. We modified the sentence.

L.168 – L.170. – You write about the fact that radiotherapy causes DNA damage, and also causes the formation of ROS, which cause damage to proteins, etc. This is not correct, since radiotherapy causes water radiolysis, the formation of ACE, and therefore damage to DNA, lipids and proteins.

Following reviewer’s suggestion, we modified the sentence to make it more comprehensive and clear.

238. 236-238. It is not clear why in an article devoted to the mechanisms of the occurrence of radioresistance due to stem cells mention the carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP).

Thanks for the suggestion. We removed the corresponding paragraph.

My previous comments (13. L. 209-211. The key point is why authors of a glioblastoma article should list “the most important CSCs biomarkers that could play a role in radio-resistance of solid tumors other than glioblastoma”. Please rewrite / add or remove this section altogether!) has not been corrected!

Thanks for the suggestion. We apologize for the omission. We amended the paragraph.

Subsection 3.3.1. «Surface markers», 3.1.2 Intracellular markers, 3.1.3. Transcription Factor as biomarker. 3.1.4. Extracellular biomarkers, should be dedicated to markers of CSCs only glioblastoma, and only those which related to radioresistance.

We modified the text including only biomarkers of glioblastoma radioresistance.

Also change the name of the subsection “3.3. Biomarkers associated with CSC”, since we are talking not just about stem cells, but in the context of the emergence of radioresistance.

Thanks for the suggestion. We modified the title of the section.

If you write about other tumors, or markers that are not related to radioresistance, then it will be a big cumbersome, unreadable text that has nothing to do with the title of your article (Cancer stem cells and glioblastoma. Time for innovative biomarkers of radioresistance?).

Following the suggestion, we modified the text including only biomarkers of glioblastoma radioresistance.

Unfortunately, a very small discussion, from which I did not understand about "Future Directions", it is also worth noting that the authors do not single out any particular marker, which, in their opinion, is the most promising. Written very generally and very streamlined.

We expanded and supplemented the paragraph, and changed the title.

The picture is not informative. Links inserted in the form of a picture or what?

We modified the figure and inserted the corresponding link in the text.

Author Response File: Author Response.docx

Round 3

Reviewer 2 Report

1.      Dear authors, unfortunately, you have not corrected the comments that I asked for. In particular: «20-21, L. 40-41 “In light of the failures of clinical trials conducted over the past 20 years, urgent clarification of the mechanisms conferring glioblastoma (GBMGB) progression and radioresistance is required.” It is very categorical and not entirely correct phrase. Please take a look and correct with at least these literary references (https://doi.org/10.1038/s41591-022-01897-x, https://doi.org/10.1039/D2BM01996E). Thanks for this suggestion. We edited the sentence as suggested. We also added the two reported references».

2.      By the way, while analyzing the links, I noticed now that you very often quote - Pasqualetti F, this is unacceptable.

3.      L.70-71. «Some key points related to IDH mutations and cancer stem (22)», L. 80 «CSCs exert those skills through…» - English and scientific language, please. Also change “error-free technique” to “error-free process” – it is more correct.

4.      L.197-199. You write that in recent years several stem cell-related biomarkers have been identified in various solid and hematologic tumors, unfortunately without translational significance, primarily for the treatment of glioblastomas. If they have no translational significance, then how can they even be used to treat any cancer?

5.      L. 271-274. “TIM-3 (T-cell immunoglobulin mucin-3) is a type 1 cell-surface glycoprotein expressed on surface of leukemic stem cells in more than 90% of patients with acute myeloid leukemia (AML) but not in that of normal hematopoietic stem cell (HSC)”. It’s probably interesting how many of them are on the surface of glioblastoma cells.

6.      L. 442-L.443 – “Transcription factor regulators of stemness have been indicated as potential biomarkers in many cancers other than glioblastoma”. A strange proposal, considering that the TFs whose expression is altered in glioblastoma cells are listed below.

7.      Nanog, OCT-3/4 - these transcription factors are not associated with radioresistance.

8.      L. 538- 540. This does not apply to gliomas. https://www.proteinatlas.org/ENSG00000106462-EZH2/pathology

9.      L. 562-L.564. “Genetic materials can be delivered via exosomes in order to reciprocally regulate gene expression by protein and miRNA”. Reprint. Not exactly written.

10.  The conclusion of the review is undoubtedly improved, but it is still not entirely specific, and gives the reader an idea of which markers need to be suppressed or, on the contrary, increased, in what way the authors propose to do this, which markers will be simply prognostic and will have only diagnostic value.

11.  The drawing is also undoubtedly improved, but Biorender allows you to make a more meaningful drawing using their wonderful templates.

I have some minor editorial comments regarding the English language.

Author Response

Dear Editor,
I would like to thank you and the reviewers for your support in this fourth round. Thanks to your suggestions the quality of this review has been improved. Below are the point-by- point responses to reviewer 2's suggestions.
We remain open to any further modifications or changes you consider necessary to further refine the review.
Kind Regards
Mario Miniati, M.D., Ph.D.

Dear authors, unfortunately, you have not corrected the comments that I asked for. In particular: «20-21, L. 40-41 “In light of the failures of clinical trials conducted over the past 20 years, urgent clarification of the mechanisms conferring glioblastoma (GBMGB) progression and radio-resistance is required.” It is very categorical and not entirely correct phrase. Please take a look and correct with at least these literary references (https://doi.org/10.1038/s41591-022-01897-x, https://doi.org/10.1039/D2BM01996E).

Thanks for this suggestion. We edited the sentence as suggested. We also added the two reported references». The phrase “In light of the failures of clinical trials conducted over the past 20 years...” has been deleted.

By the way, while analyzing the links, I noticed now that you very often quote - Pasqualetti F, this is unacceptable.

We apologize for this, although pertinently, we agree that there may be a conflict of interest. We have left only two citations from the author.

L.70-71. «Some key points related to IDH mutations and cancer stem (22)», L. 80 «CSCs exert those skills through...» - English and scientific language, please. Also change “error-free technique” to “error-free process” – it is more correct.

Thanks for this suggestion. We edited the sentence as suggested.

4. L.197-199. You write that in recent years several stem cell-related biomarkers have been identified in various solid and hematologic tumors, unfortunately without translational significance, primarily for the treatment of glioblastomas. If they have no translational significance, then how can they even be used to treat any cancer? MDPI | Reply review report 20/09/23, 06)47 https://susy.mdpi.com/user/manuscripts/review/41526741?report=32312876 Pagina 4 di 5.

Thanks for this suggestion. We edited the sentence as suggested.

5. L. 271-274. “TIM-3 (T-cell immunoglobulin mucin-3) is a type 1 cell-surface glycoprotein expressed on surface of leukemic stem cells in more than 90% of patients with acute myeloid leukemia (AML) but not in that of normal hematopoietic stem cell (HSC)”. It’s probably interesting how many of them are on the surface of glioblastoma cells.

Thanks for this suggestion. We edited the sentence as suggested.

6. L. 442-L.443 – “Transcription factor regulators of stemness have been indicated as potential biomarkers in many cancers other than glioblastoma”. A strange proposal, considering that the TFs whose expression is altered in glioblastoma cells are listed below.

Thanks for this suggestion. We removed the sentence.

7. Nanog, OCT-3/4 - these transcription factors are not associated with radioresistance.

Thanks for this suggestion. We removed the paragraph.

8. L. 538- 540. This does not apply to gliomas. https://www.proteinatlas.org/ENSG000001064 62-EZH2/pathology

Thanks for this suggestion. We removed the sentence.

9. L. 562-L.564. “Genetic materials can be delivered via exosomes in order to reciprocally regulate gene expression by protein and miRNA”. Reprint. Not exactly written.

Thanks for this suggestion. We edited the sentence.

10. The conclusion of the review is undoubtedly improved, but it is still not entirely specific, and gives the reader an idea of which markers need to be suppressed or, on the contrary, increased, in what way the authors propose to do this, which markers will be simply prognostic and will have only diagnostic value.

Thank you very much for this observation. After re-reading it we agree that the last paragraph needed to be rewritten. We have changed the whole paragraph and hope that the reviewer will agree with our point of view.

11. The drawing is also undoubtedly improved, but Biorender allows you to make a more meaningful drawing using their wonderful templates.

We have changed the figure, thank you for letting us know.

Author Response File: Author Response.pdf