Assessing Radiation Effects on Chemo-Treated BT20 and 4T1 Breast Cancer, and Neuroblastoma Cell Lines: A Study of Single and Multiple-Cell Ionization via Infrared Laser Trapping

Round 1

Reviewer 1 Report

The major purpose of the manuscript presented by Goangul et al. was to explore radiation sensitivity of a breast carcinoma line (BT20), treated with the compound 2 DMDD and a laser trapping technology. In more detail, authors report on a radiation sensitization of the BT20 cells following DMDD application in line with a decreased threshold radiation dose (TRD), with increasing cell mass and multiple cell ionization, attributed to ionizing radiation field's chain effect and water molecule absorption at 1064 nm.

Although the study, in principle, is of potential interest to the radiobiological community, there are several major experimental concerns that prohibit the reviewer´s recommendation for publication in Radiation in its current form. Major points of criticism are given below.

Major points of criticism:

1.     Introduction section. The first paragraph (lines 48-56) covers instructions for authors  and does not reflect an introduction. These sentences should be omitted. Moreover, the introduction section is weak in performance and does not adequately introduce to  infrared laser trapping, the major experimental technique used in the manuscript.

2.     Material and methods section, Mechanistic framework. This paragraph does not cover a description of the method but mainly covers a discussion of underlying mechanisms, that is not appropriate in the material and methods section.

3.     Authors stated (line 181-182): “The amount of the radiation energy of the laser incident absorbed by the cell determines the threshold ionization energy needed for cell death…”. However, analyses of cell diameter and cell mass are considered not to be appropriate parameter for cell death and DNA repair capacity. Accordingly, assays including gammaH2AX foci analyses, colorimetric assays for cell proliferation and apoptosis assays (TUNEL staining, FACS analyses) may strengthen their statements.

4.     Figure 2 covers a graphical display of the values presented in table 1 and does not contribute additional information. Thus, table 1 should be omitted.

5.     Authors provided a comparison with (published data) of N2a and 4T1 cells (section 4.3) but failed to describe these comparisons in the abstract section.

6.     Authors provided data on a combined laser trap and compound 2 DMDD treatment for 2 and 24 hours, however, did not indicate findings on either monotherapy with irradiation or 2 DMDD. How do their parameters differ following sole laser and sole DMDD treatment at 2h and 24h.

7.     In line with that, authors restricted their analyses to treatment with a single DMDD concentration (100 μM per well). Is there a dose response relationship with varying amounts of the compound applied?

Author Response

We would like to express our appreciation for the time you have taken from your busy schedule to review our manuscript. We have diligently addressed the concerns and feedback provided. Please find our response below:
Reviewer

1. Introduction section. The first paragraph (lines 48-56) covers instructions for authors and does not reflect an introduction. These sentences should be omitted. Moreover, the introduction section is weak in performance and does not adequately introduce to infrared laser trapping, the major experimental technique used in the manuscript.

Response

The following lines were removed, “The first paragraph (lines 48-56) covers instructions for authors and does not reflect an introduction. These sentences should be omitted.”

We add the following to address the concern raise by the reviewer:

Reviewer

2. Material and methods section, Mechanistic framework. This paragraph does not cover a description of the method but mainly covers a discussion of underlying mechanisms, that is not appropriate in the material and methods section.

Response

The section on Mechanistic framework was removed and to the discussion section.

 Reviewer

3. Authors stated (line 181-182): “The amount of the radiation energy of the laser incident absorbed by the cell determines the threshold ionization energy needed for cell death…”. However, analyses of cell diameter and cell mass are considered not to be appropriate parameter for cell death and DNA repair capacity. Accordingly, assays including gammaH2AX foci analyses, colorimetric assays for cell proliferation and apoptosis assays (TUNEL staining, FACS analyses) may strengthen their statements.

Response:  We understand the reviewer’s points point and agrees that suggestions of assays, including gammaH2AX foci analyses, colorimetric assays for cell proliferation, and apoptosis assays (TUNEL staining, FACS analyses), can provide both quantitative and qualitative data that help demonstrate the relationship between absorbed radiation energy and cell death. However, the point we are making is this, “While other assays provide valuable insights into the mechanisms and effects of radiation exposure, the occurrence of cell rupture serves as a definitive endpoint for assessing cell viability in our experimental setup. The amount of radiation energy incident upon the cell and absorbed by it determines the threshold ionization energy required for cell death, starting from the moment the cell enters the trap until its departure.” We add the statement in quotes the manuscripts. Furthermore, while we acknowledge that parameters such as cell diameter and mass may not directly signify cell death, they do play a role in determining the absorbed energy that is determining the cellular response to radiation. The absorbed energy is a key determinant of the cellular response, and we have focused on elucidating the mechanisms through which absorbed radiation energy affects cell viability and DNA repair capacity.

 Reviewer

4. Figure 2 covers a graphical display of the values presented in table 1 and does not contribute additional information. Thus, table 1 should be omitted.

Response: Remove Table 1

Reviewer

5. Authors provided a comparison with (published data) of N2a and 4T1 cells (section 4.3) but failed to describe these comparisons in the abstract section.

 Response

Abstract was modified to the following:

Background: Our study aimed to assess the radiation sensitivity of BT20, a human breast tumor cell line, using the laser trapping technique and compare it with N2a and 4T1 cells. Additionally, we investigated the impact of the antitumor compound 2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) on radiation sensitivity.

Methods and Materials: We employed laser trapping to calculate both the threshold ionization energy (TIE) and threshold radiation dose (TRD) for BT20, N2a, and 4T1 cells. We assessed the effect of DMDD on BT20 cells' radiosensitivity and conducted comparisons across these cell lines.

Results: Our findings reveal that DMDD significantly enhances the radiosensitivity of BT20 breast carcinoma cells. Moreover, we observed distinct trends in TIE and TRD across the three cell lines, with differences attributed to variations in cell size and composition. When multiple cell ionizations were considered, a notable reduction in TRD was observed, implicating factors such as the chain effect of ionizing radiation and the influence of DMDD. The study found that TIE increased with the number of cells in the trap while TRD consistently decrease across all three cell lines, suggesting comparable radiation sensitivity, and oligostilbene treatment further reduced TRD, presenting potential for enhancing therapeutic ratios in cancer treatment.

Conclusion: The antitumor compound DMDD enhances the radiosensitivity of BT20 breast carcinoma cells, highlighting its potential in cancer treatment. Furthermore, our study underscores the impact of cell size and multiple cell ionizations on TRD. Leveraging laser trapping techniques, biocompatible nanoparticles, and advanced optical tweezers opens promising avenues for personalized and effective cancer therapy approaches.

Reviewer

6. Authors provided data on a combined laser trap and compound 2 DMDD treatment for 2 and 24 hours, however, did not indicate findings on either monotherapy with irradiation or 2 DMDD. How do their parameters differ following sole laser and sole DMDD treatment at 2h and 24h.

 Response

            We are somewhat confused by the reviewers concern here. While it is true that there have been previous studies focusing on specific cell types such as BT20 with laser trap[https://doi.org/10.1364/BOE.3.002190] and 4T1 with DMDD [https://doi.org/10.1038/s41598-017-07162-3] sole treatments treatment, our overarching aim in this study is to elucidate the combined effects of these modalities within the context of radio-chemo treatment.

We have the following paragraph in the introduction and hoping this address reviewers concern:

“In a recent study that investigated the radiation sensitivity of 4T1 breast cancer cells, both untreated and treated with an antitumor compound from DMDD, researchers utilized a laser trapping technique. The findings revealed that both the TIE and TRD decreased with longer treatment periods, suggesting an increase in radiation sensitivity due to the treatment [22]. In a separate study, researchers explored the radiation response of untreated Na2 cells to infrared laser trap ionization, focusing on the relationship between TIE, TRD, and cell mass. The study found an inverse correlation between TRD and cell mass, with TRD decreasing as cell mass increases. This suggests a potential method for calculating TRD in vivo, offering insights for optimizing radiation dosimetry in cancer treatment [29]”.

Additionally, our research is designed to provide a holistic understanding of the radio-chemo treatment approach, considering the integrated effects of both laser trap and DMDD therapies. By incorporating insights from these studies and building upon them within the framework of our research, we aim to offer a comprehensive analysis of the radio-chemo treatment modality and its potential synergistic benefits.

Reviewer

7. In line with that, authors restricted their analyses to treatment with a single DMDD concentration (100 μM per well). Is there a dose response relationship with varying amounts of the compound applied?

Response

The dose response of DMDD was previously explored for its anti-proliferative and anti-tumor activities. A concentration of 100 uM was used to ensure the effectiveness of both activities. The publication can be found via this link:

The antidiabetic compound 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione, isolated from averrhoa carambola L., demonstrates significant antitumor potential against human breast cancer cells - PMC (nih.gov)

Reviewer 2 Report

Review of the paper entitled “Assessing Radiation Effects on Chemo-Treated BT20 and 4T1 Breast Cancer, and Neuroblastoma Cell Lines: A Study of Single and Multiple Cell Ionization via Infrared Laser Trapping”

This is a very interesting work employing infrared laser trapping to estimate (or measure (maybe)) the cell ionizations and applies an antidiabetic compound 2-Dodecyl-6-methoxycyclohexa-2, 5-diene-1, 4-dione (DMDD) in human cancer cell lines; DMDD has been reported for antitumor activities. I have quite a few comments and concerns and they mentioned below.

1.      Application of DMDD: The authors cite reported antitumor activities of DMDD.

(a)    Have they repeated the literature reported data (lines 87-90) and reproduced them? I do not find any panel “g” in Figure 1 (ii).

(b)   What do the authors mean by “decrease with increasing treatment period”.

(c)    Have the authors verified the dual action of DMD by reproducing the reported data? I have my concern here (see point a above).

2.      My main concern is also heating associated with infrared laser trapping component (see for example, Biophys J. 2003 Feb; 84(2): 1308–1316. doi: 10.1016/S0006-3495(03)74946-7). So, this method has already, in principle, a hyperthermic component. How can the authors account for that while measuring (or estimating) TIE and TRD?

3.      The standard deviations are large and hence the claim 424-427 should be carefully considered. These results do not appear to be precise and compelling.

4.      The cell ionizations cannot be totally accounted for through the membrane damage and hence the TIE and TRD should be very carefully defined.

5.      Employing Optical Tweezers to trap tumors is a good concept but more compelling evidences should be cited.

6.      The manuscript has typographical errors, e.g., “ in the title.

The manuscript cannot be accepted in the present form. The authors must show strong proof of reproducibility of data.

7.    The authors have done a poor job in referencing (see ref. 53 and onwards). The corresponding author has overlooked this.

Presentation can be improved. But, my main concern is the scientific sound ness and merit of the paper

Author Response

Dear Reviewer,

   We would like to express our appreciation for the time you have taken from your busy schedule to review our manuscript. We have diligently addressed the concerns and feedback provided. Please find our response below:

Reviewer:

This is a very interesting work employing infrared laser trapping to estimate (or measure (maybe)) the cell ionizations and applies an antidiabetic compound 2-Dodecyl-6-methoxycyclohexa-2, 5-diene-1, 4-dione (DMDD) in human cancer cell lines; DMDD has been reported for antitumor activities. I have quite a few comments and concerns and they mentioned below.

1. Application of DMDD: The authors cite reported antitumor activities of DMDD.

• Have they repeated the literature reported data (lines 87-90) and reproduced them?

Response

Yes; this support by reference 22-29

 I do not find any panel “g” in Figure 1 (ii).  Error.

Response

This was fixed.

• What do the authors mean by “decrease with increasing treatment period”.

Response

This was one finding of recently publication: Muhammed, E., Chen, L., Gao, Y., & Erenso, D. (2019). Chemo-treated 4T1 breast cancer cells radiation response measured by single and multiple cell ionization using infrared laser trap. Scientific reports, 9(1), 17547.

Both TIE and TRD decrease with increasing treatment periods" means that as the duration of the treatment extends, the amount of energy (Threshold Ionization Energy) and the dose of radiation (Threshold Radiation Dose) required to achieve a specific effect on the cells (such as ionization) are reduced. This implies that the cells become more sensitive to the treatment over time, so less energy or a lower dose is needed for the same level of impact.

Reviewer

• Have the authors verified the dual action of DMD by reproducing the reported data? I have my concern here (see point a above). We have not verified the dual action DMDD as such we will rephrase our statement to :

Response

Statement has been rephrased

Reviewer

2. My main concern is also heating associated with infrared laser trapping component (see for example, Biophys J. 2003 Feb; 84(2): 1308–1316. doi: 10.1016/S0006-3495(03)74946-7). So, this method has already, in principle, a hyperthermic component. How can the authors account for that while measuring (or estimating) TIE and TRD?

Response

The following was added in “The Material and Method”:

We are aware of the hyperthermic effects that can accompany infrared laser trapping [39]. Drawing on insights from previous research, we mitigate these effects in our evaluation of Threshold Ionization Energy (TIE) and Threshold Radiation Dose (TRD) [22-29]. This is achieved through precise monitoring and regulation of the laser's intensity and the duration of exposure, which helps in minimizing thermal buildup.”

This response demonstrates awareness of the issue and outlines steps taken to mitigate its impact on the research findings. It is acknowledged that thermal effects can cause charging in cells, and that pulsed NIR lasers may induce ionization through non-thermal pathways. The observed ejection of cells perpendicular to the laser beam in optical trapping experiments is indicative of charge presence in the cells, a phenomenon substantiated by recent research by” …Measurement of Charge and Refractive Indices in Optically Trapped and Ionized Living Cells. Tomography, 9(1), 70-88” in their study on the measurement of charge and refractive indices in optically trapped and ionized living cells.

 Further it is well established that thermal effects can lead to charging and also because Pulse NIR laser can also bring about ionization by alternative pathway.  Since the cell was ejected from the trapped perpendicular to the beam direction this is because of the cell charge. This was recently demonstrated: Muhammed, E., Erenso, D. B., Gao, Y., Chen, L., Kelley, M., Vazquez, C., ... & Crogman, H. T. (2022). Measurement of Charge and Refractive Indices in Optically Trapped and Ionized Living Cells. Tomography, 9(1), 70-88.

Reviewer

3. The standard deviations are large and hence the claim 424-427 should be carefully considered. These results do not appear to be precise and compelling.

Response

  We revise the following : “Furthermore, as our method enables very precise measurements of radiation energy, the possibility of a more effective therapeutic ratio based on the cellular level is confirmed here, which reduces tissue toxicity caused by radiation while promoting sterilization of the cancerous cells.”,

to ,

“While our method demonstrates the potential for highly precise measurements of radiation energy, leading to a promising avenue for optimizing the therapeutic ratio at the cellular level, the observed large standard deviations call for cautious interpretation. These preliminary findings suggest a reduced tissue toxicity and enhanced sterilization of cancerous cells, yet further detailed studies are necessary to corroborate these results and refine their application in clinical settings.”

Reviewer

4. The cell ionizations cannot be totally accounted for through the membrane damage and hence the TIE and TRD should be very carefully defined.

Response
"We acknowledge the complexity of cellular responses to ionization, which may extend beyond membrane damage alone. Accordingly, we are committed to refining the definitions of TIE and TRD, incorporating a broader range of cellular effects. This will ensure a more comprehensive understanding and accurate assessment of the ionization process, paving the way for future research to explore these mechanisms in greater detail."

Reviewer

5. Employing Optical Tweezers to trap tumors is a good concept but more compelling evidences should be cited.

Response

"We appreciate the suggestion to strengthen our argument with additional evidence. To address this, we will review the latest literature to identify further studies that demonstrate the efficacy and potential of Optical Tweezers in cancer research. This will include exploring diverse applications and outcomes that support our concept, ensuring a robust foundation for our claims and enhancing the credibility of our approach."

Reviewer

6. The manuscript has typographical errors, e.g., “ in the title.

Response

Typos fixed after a careful read.

Reviewer

7.    The authors have done a poor job in referencing (see ref. 53 and onwards). The corresponding author has overlooked this.

Response

Fixed

Reviewer 3 Report

This article is very interesting and describes a very useful implementation of novel methods. Nevertheless, there are some importants additions that are needed to be made. 

The radiation used to irradiate the cells is not clearly stated. The same infrared used for the trapping is also used for the irradiation? The question comes from the fact that a lot of discussion is made (in the article) for radiation therapy which is not the same as thermotherapy. So this needs to be made more clear. Also, it is important to explain why the term ionisation is used, when no ionising radiation is implemented. I think that the results of this method do not come from ionisation, but for cell membrane lysis. This needs be explained.

Specific comments:

P2L48-56: Please remove this whole paragraph....

P2L74: Use the term radiation therapy, instead of radiotherapy.

Figure 3: Please include e, f, j, h in the description.

Table 3: Please respect the use of capitals letters or not. Use the same for all similar variables. Additionally, it would help to use vertical lines to separate TIE, Mass, and TRD.

Figure 5: Quality needs be highly improved.

Author Response

We would like to express our appreciation for the time you have taken from your busy schedule to review our manuscript. We have diligently addressed the concerns and feedback provided. Please find our response below:

Reviewer 

The radiation used to irradiate the cells is not clearly stated. The same infrared used for the trapping is also used for the irradiation. The question comes from the fact that a lot of discussion is made (in the article) for radiation therapy which is not the same as thermotherapy. So this needs to be made more clear. Also, it is important to explain why the term ionisation is used, when no ionising radiation is implemented. I think that the results of this method do not come from ionisation, but for cell membrane lysis. These needs be explained.

Response

We have added the following in the method section, “We must point out here that near-infrared (NIR) lasers themselves do not directly ionize atoms or molecules in the way that ultraviolet (UV) or X-ray photons can, due to their lower photon energies and such ionization alternative pathways such as Multiphoton Absorption, Tunnel Ionization,  or Laser-Induced Breakdown are responsible for cell ionization^36-40.”

Specific comments:

Reviewer 

P2L48-56: Please remove this whole paragraph.... 

Response

It was removed.

Reviewer 

P2L74: Use the term radiation therapy, instead of radiotherapy.

Response

This change was made.

Reviewer 

Figure 3: Please include e, f, j, h in the description.

Response

This was fixed.

Reviewer 

Table 3: Please respect the use of capitals letters or not. Use the same for all similar variables. Additionally, it would help to use vertical lines to separate TIE, Mass, and TRD.

However, Table 3 is now Table 2 as another requested the removal of table 1 removal to eliminate redundancy with figure 2 .

Response

This was fixed as per your suggestion. All variables are similar.

Reviewer 

Figure 5: Quality needs be highly improved.

Response

We included a better-quality figure in the manuscript.

Round 2

Reviewer 1 Report

In the revised version of the manuscript, the authors addressed my previous concerns in an adequate manner.

Reviewer 2 Report

This is fine. Accept.

This is fine. Accept.

Reviewer 3 Report

The article has been highly improved!