Development of an Artificial 3D Liver Phantom for Analysis of Radiotherapeutic Effects In Vitro

Round 1

Reviewer 1 Report

Dear authors,

I had the opportunity to review your interesting manuscript.

Only two remarks concerning the section "limitations". I think you should discuss the limitation that hepatoblastoma cells were used -and not HCC cells- in much more detail. Second, could you discuss/explain in more detail, that the filling by agarose mixture during irradiation really is "tissue-equivalent". I´m not quite convinced about that.

Kind regards

Author Response

We thank the reviewers for their careful consideration, comments, and time. Please find our point-by-point responses below.

Only two remarks concerning the section "limitations". I think you should discuss the limitation that hepatoblastoma cells were used -and not HCC cells- in much more detail.

Answer: Thank you for pointing out these two important points. Using a single tumor cell line to study the impact of a specific therapy always limits the broader application of the data, because only one specific case is studied. The choice to use HepG2 was made because it is a widely used liver tumor cell line, displays many properties of transformed cancer cells and still has retained many hepatocyte specific features including high metabolic rates when grown in microfluidic chips (MCs). We’ve clarified this in the manuscript as well.

Second, could you discuss/explain in more detail, that the filling by agarose mixture during irradiation really is "tissue-equivalent". I´m not quite convinced about that.

Answer: The agarose mixture is used to fill the phantom “body” and mimic the properties of the aqueous environment of the human body during radiation. Several phantoms nowadays are filled with agarose and we have adapted the discussion regarding this. Additionally, we’ve changed our wording from “tissue-equivalent” to “tissue-mimicking” for clarity and accuracy.

Reviewer 2 Report

The primary therapeutic treatment of hepatocellular carcinoma (HCC) is surgical resection of the tumor from the liver tissue. However, other less invasive techniques, such as stereotactic body radiotherapy (SBRT) have garnered increasing support for use in the clinic. This study presents a novel anthropomorphic in vitro liver phantom, which simulated the relevant HCC tumor microenvironment and spatial organization, and utilized it to compare radiotherapy beam scattering effects against 2D models. The liver phantom was modeled after a 3D liver scan and designed to fit ten microfluidic chips (MCs), in which HepG2 cells were seeded. 

The paper is quite good, well-structured. Just please make Fig. 4 D-G a little bit brighter.

Thanks.

Author Response

We thank the reviewers for their careful consideration, comments, and time. Please find our point-by-point responses below.

The paper is quite good, well-structured. Just please make Fig. 4 D-G a little bit brighter.

Answer: Thank you for your kind words and suggestion. We have changed the settings of Fig. 4 to make the images better visible and hope it is ok now.

Reviewer 3 Report

In this paper, by constructing a 3D liver model, an anthropomorphic tumor environment was successfully simulated for a better understanding of HCC response to radiotherapy in vitro. Overall, this is a good research paper, however, it also has a few questions and major/minor points,

Q1: In the abstract, no results are presented. Please ensure the integrity of the abstract structure.

Q2: The expression of “microfluidic chamber” is mentioned in the introduction, and it is suggested that the “microfluidic chip” should be used as unified as possible.

Q3: In 2.1, please check the abbreviations of “microfluidic chips (MC)".

Q4: In 2.4, did this paper consider the effect of the process of changing the media on hypoxic environment? Whether this step has a significant impact on the simulated hypoxic environment？

Q5: In 2.4 and 2.5, RT should not be abbreviated directly to avoid unnecessary confusion with radiotherapy.

Q6: Please clarify how the placement of the 10 MCs was determined

Q7: In 2.5，according to "the MCs # 1-5 were located in the anterior half of the liver and the MCs 119 # 6-10 were positioned in the posterior half of the liver", I wonder if the radiation is consistent with the penetrativity of the 3D model and the actual liver. If not, what is the significance of designing the backside group?

Q8: In 3.1, according to “A dose-dependent effect was also observed in the case of hypoxic conditions.” I want to know that under the hypoxic conditions, did the γH2AX response also be prolonged? If the prolonged time was longer compared with control group, is it reasonable to choose the 30min time in figure H?

Major/minor points:

● The title is not match the main point of the conclusion, please revise.

● I suggest you rewrite the abstract. It's too controversial and verbose. Please add some results and shorten it by focusing on the main points.

● Detailed instructions are missing in the 3D printed liver model section, please supplement them in detail.

● In the results, the narratives are too messy, please keep them organized.

● Please insert a section on the implications of the study. Who benefits with it? What problem can the study help to solve?

● In the conclusions, novelty issue has not been sufficiently highlighted in the current version.

● Some figures are missing on important points, please supplement them.

● Please provide the contributions of this study more specific.

Author Response

We thank the reviewers for their careful consideration, comments, and time. Please find our point-by-point responses below.

Q1: In the abstract, no results are presented. Please ensure the integrity of the abstract structure.

Answer: Thank you for your suggestion. We have rewritten the abstract to include the results and major findings of our work and also adjusted the abstract structure.

Q2: The expression of “microfluidic chamber” is mentioned in the introduction, and it is suggested that the “microfluidic chip” should be used as unified as possible.

Answer: We now use microfluidic chip (MC) throughout the manuscript to avoid confusion and use the expression “chamber” only for the compartment inside the MC in which the cells are actually grown.

Q3: In 2.1, please check the abbreviations of “microfluidic chips (MC)".

Answer: We use the abbreviations MC and MCs for either microfluidic chip or microfluidic chips

Q4: In 2.4, did this paper consider the effect of the process of changing the media on hypoxic environment? Whether this step has a significant impact on the simulated hypoxic environment？

Answer: In the experimental set up, the cells were left in the MC for 4 h to achieve hypoxic conditions. Within these 4 hours media was not changed to ensure depletion of oxygen due to the oxygen consumption of the cells. We apologize if this was not clear and have updated the methods section to reflect this.

Q5: In 2.4 and 2.5, RT should not be abbreviated directly to avoid unnecessary confusion with radiotherapy.

Answer: We have removed “RT” as an abbreviation from the manuscript and refer to both “room temperature” and “radiotherapy” fully.

Q6: Please clarify how the placement of the 10 MCs was determined

Answer: We wanted a distribution of different positions relative to the radiation target site covering the whole liver. We changed the text to make this clearer.

Q7: In 2.5，according to "the MCs # 1-5 were located in the anterior half of the liver and the MCs 119 # 6-10 were positioned in the posterior half of the liver", I wonder if the radiation is consistent with the penetrativity of the 3D model and the actual liver. If not, what is the significance of designing the backside group?

Answer: Please see Q6. The radiation absorption and scattering should resemble the condition in the body. Therefore, cells need to also be positioned in the posterior part of the liver to analyze cells further from the radiation target. To analyze effects on different parts of the liver we positioned the MCs based on our modeling. We have now also added a supplementary figure showing the modeling during experimental planning.

Q8: In 3.1, according to “A dose-dependent effect was also observed in the case of hypoxic conditions.” I want to know that under the hypoxic conditions, did the γH2AX response also be prolonged? If the prolonged time was longer compared with control group, is it reasonable to choose the 30min time in figure H?

Answer: We wanted similar conditions for fixation of MCs after irradiation. As 5 Gy irradiation in normoxic conditions resulted in a peak of γH2AX fluorescence after 30 min and already significantly reduced γH2AX fluorescence after 1 h, this time was determined to be optimal and, therefore, MCs for all doses were fixed 30 min after irradiation.  

Major/minor points:

• The title is not matching the main point of the conclusion, please revise.

Answer: Thank you. We have revised as suggested.

• I suggest you rewrite the abstract. It's too controversial and verbose. Please add some results and shorten it by focusing on the main points.

Answer: Thank you. We have rewritten the abstract.

• Detailed instructions are missing in the 3D printed liver model section, please supplement them in detail.

Answer: We have improved the description on building the phantom with positions for the MCs and included images of the 3D printed parts in a supplementary figure for clarity.

• In the results, the narratives are too messy, please keep them organized.

Answer: We have reorganized the results section and introduced another subtitle to improve the structure.

• Please insert a section on the implications of the study. Who benefits with it? What problem can the study help to solve?

Answer: We have added these points in the discussion and conclusions.

• In the conclusions, novelty issue has not been sufficiently highlighted in the current version.

Answer: We have introduced additional emphasis on the novelty of the current study in our conclusion and discussion.

• Some figures are missing on important points, please supplement them.

Answer: We have added a supplementary figure on our modeling and some additional text for the figures.

• Please provide the contributions of this study more specific.

Answer: We have adjusted the contribution section with additional details. Thank you.  

Round 2

Reviewer 3 Report

I don not have any more questions or/and suggestions, accept.