The Molecular Network of Neutrophil Extracellular Traps in Hepatocellular Carcinoma: Biogenesis, Function, and Therapeutic Implications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review summarizes the knowledge accumulated to date about neutrophil-derived NETs (neutrophil extracellular traps) present in the tumor microenvironment, their formation mechanism, the mechanism of NETosis induction, and their biological function in the progression of liver cancer. The potential clinical application of NETs as novel diagnostic biomarkers and therapeutic targets is also thoroughly described.

The content is well organized and will be useful to many readers. However, the final section, "6. Conclusion and Future Perspectives" is a bit vague and lacking in content. In the abstract, the authors state, "This paper provides theoretical guidance for the development of the next generation of precision medicine strategy." However, it is unlikely that the authors' objectives have been achieved based on the content of Chapter 6. This reviewer would like the authors to send out more meaningful messages to the readers.

Below are some errors or concerns that the reviewer noticed. Please correct them accordingly.

1. Which NOX is mentioned on line 127? There are multiple NOXs that form complexes with p47phox.
2. In Figure 3, the "r" in "Extracellular" is written separately on the third line. Please correct this to make it one word.
3. Please increase the resolution of the figures a bit. The small text in the figures is hard to read.
4. On line 55, the author cites Reference 10 and states, "Researchers discovered a new function of neutrophils called extracellular neutrophil traps (NETs)." However, since Reference 10 is a review, the author should cite the original paper in which the function was first discovered. Reference 11 is the original paper on NET, so this reviewer thinks it would be best to cite it and then reference 10 as a review summarizing a series of recent reports.
5. Figure 3 does not accurately reflect the content of Chapter 3. This reviewer thinks it would be easier to understand if the authors created separate diagrams for each subsection, as in Figure 2.
6. 5.1 on line 564 should be 5.2. Please correct it.

Comments on the Quality of English Language

Did the authors check the text again to ensure that it could be understood without misunderstanding when translated back into their native language? I noticed some unnatural aspects, including the logical development. If the text has already been proofread by a native speaker, that's fine, but I would recommend checking it again in its entirety.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In the manuscript entitled “The Molecular Network of Neutrophil Extracellular Traps in Hepatocellular Carcinoma: Biogenesis, Function, and Therapeutic Implications” the authors review the current knowledge on the molecular regulatory network of neutrophil extracellular traps (NETs) in hepatocellular carcinoma microenvironment.

The review is of potential interest. Even if the precise mechanisms by which neutrophil extracellular traps contribute to tumor development have not been clearly established yet, the authors provide and unbiased overview of current understanding in the context of HCC.

I would like to suggest some modifications to further improve the potential impact of the manuscript:

1. Figure 1. “Schematic illustration of the molecular network”. No molecular network is shown in the figure.
2. Figure 2. “Molecular pathways of NETs formation through different NETosis modes”. To facilitate rapid comprehension, major abbreviations should be included in the figure legend, even if they are exhaustively detailed in the abbreviation list at the end of the manuscript. The same applies for Figure 3.
3. Figure 4 Panels (a) and (b) contain typos that need editing.
4. Figure 4 Panel (c): as the figure should focus on roles of NETs in HCC, the picture representing hydrogel mediated delivery of DNAse is out of context.
5. Figure 4 Panel (d): explain the selection of a silhouette that seems to suggest obesity.
6. Figure 5 Panel (a): resolution of the plots needs to be enhanced to improve readability.
7. Figure 5 Panel (b): Define “touch”.
8. Line 200-221. “Hepatocellular carcinoma (HCC) possesses a highly complex tumor microenvironment (TME), which consists of cells and the extracellular matrix (non-cellular components). Recently, the focus has shifted from the concept of Tumor Microenvironment (TME) to the more specific Tumor Immune Microenvironment (TIME) to emphasize the significant clinical impact that the density and composition of immune cells have at the tumor site.
9. Some references, including #6, 42, 49, 74, 89, 100 are incomplete.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Liu et al have prepared an in-depth review article on the molecular mechanisms that underly the formation of Neutrophil Extracellular Traps (NETs) in the context of the tumor microenvironment (TME) of Hepatocellular Carcinoma (HCC). The review begins with the pathways that cause NETosis in neutrophils, the basic components of the expelled chromatin that generates extracellular networks, and perhaps most importantly, and how those networks can contribute to not only immune resistance within the TME but also other pro-tumor effects including HCC metastasis.  Beyond commonly analyzed stimuli of NETosis, the authors also cover other contributing factors that modulate NET formation, again primarily focused on the TME. The authors then shift to how NETs contribute to HCC tumorigenesis and progression, how a vicious feedback loop can form between neutrophils and tumor cells mediated by NETs, and effects on postoperative recurrence and other complications. Finally, the authors do a thorough review of how NETs are detected with biomarkers that might be used as prognostic indicators, and important targets for therapeutics that might interfere with NET formation both within and outside the TME. Overall, the review is thorough, the text is well-written, and the format is logical and informative. The figures complement the descriptions in the text, and the Tables are very helpful to the reader in surveying roles of NETs, diagnostic markers, and therapeutic strategies. There are some minor issues to consider, but none of those below require more than minor editing or grammatical corrections.

Identified issues:

Lines 67-68, the authors might expand a bit on the roles that NETs play in disrupting anti-tumor functions of CD8+ T cells, as mentioned here and throughout the manuscript.

Lines 123-124, the authors might divide the description of suicidal NETosis into those that occur in vivo, vs. chemical stimulants such as PMA.

Line 125, the authors might add "activation" before protein kinase C "is initiated", in other words, the activities of PKC are initiated.

Lines 139-140, the authors should add at least one reference (or if included, refer to it here) to this sentence regarding MPO-deficient patients or how MPO inhibitors can impair NET formation.

Line 190, the authors might simply add "i.e., priming" after "pre-excitation", to match the image in Figure 2 depicted with GM-CSF simulation.

Lines 194-196, to be complete, the authors should add a reference that presents results indicating mtNETosis is deficient in patients with CGD.

Line 202, Staph aureus should be in italics.

Line 257, mitochondrial ROS has already been defined, so perhaps just write "mt ROS". Note that additional acronym definitions are repeated throughout the manuscript. While this reader appreciates the reminder of definitions especially in a new section, but some are redundant and not needed (e.g., HCC).

Line 304, TME is already defined, see note above.

Line 320, again the important role of CD8+ T cells is mentioned, but how these impact the TME and NETosis has yet to be defined, so the authors might insert that description earlier and simply indicate that this was previously described.

Line 338, yet another mention of CD8pos T cells, see above.

Line 389, "Differ" might be changed to "Differing from viral mechanisms,....." to be grammatically correct.

Line 631, "nets" should be capitalized to indicate the acronym, e.g., “NETs”.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf