The Potential of Tunable Femtosecond Laser Light to Prevent Melanoma A375 Cell Growth: An In Vitro Investigation

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Please refer to the comments in the attached file.

Comments for author File: Comments.pdf

Author Response

A point-by-point response to Reviewer #1 comments

 

Dear, 

We appreciate your excellent remarks on our manuscript, as well as your comments, corrections, and valuable ideas. We believe the following response addresses all of the reviewers' issues. The detailed revisions are listed below where we present the comments of the reviewer in italic red letters, followed by our response in blue letters.

 

• Minor correction: Line 26: "expo-sure"..

 

We appreciate the reviewer's valuable thoughts. This error has been corrected in the revised manuscript and highlighted in yellow.  

 

We sincerely appreciate the reviewer valuable comments and suggestions. In the revised manuscript, additional details on femtosecond laser-induced inhibition mechanisms, including ROS generation, mitochondrial membrane disruption, and apoptosis induction, have been incorporated into the Introduction and Discussion. Prior studies have demonstrated that shorter wavelengths (UV and blue) are more effective in generating ROS and affecting mitochondrial function, leading to apoptosis. This mechanistic background will be expanded to strengthen the rationale for the study.

 

• Minor correction: Information on powermeter is doubled in lines 131 and 145.

 

We appreciate the reviewer comments, which we have now incorporated into the revised version of the manuscript and highlighted in yellow.

 

• Minor correction: Line 154. Misprint "= E". "F=E/pi*r2" should be used.

 

We appreciate the reviewer comments, which we have now incorporated into the revised version of the manuscript and highlighted in yellow.

 

 

 

• Minor correction: Line 153-154. "...the laser beam fluence (energy density) F= E /  r2 = 8.8 x10-154 9 Joule / cm2" should be enough. Energy density F and power density I are the parameters of a single laser pulse. Energy density F[J/cm?] should not be mixed with dose=Pave *tirradiation, describing the amount of energy received by cells in the experiment.

 

We sincerely appreciate the reviewer valuable comments, which we have now incorporated into the revised version of the manuscript and highlighted in yellow.

.  

 

 

• Lines 341-343. Please rephrase "Also, femtosecond laser has In contrast to nanosecond pulses, femtosecond lasers exhibit photodisruption with less collateral damage, which has led to its usage in ophthalmic applications [42]."

 

We sincerely appreciate the reviewer's valuable comments. As suggested by the reviewer, this sentence has been rephrased and highlighted in yellow color in the amended manuscript (lines # 355, 356, and 357).

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 4)

Comments and Suggestions for Authors

Thank's for corrections

Author Response

A point-by-point response to Reviewer #2 comments

 

Dear, 

We appreciate your excellent remarks on our manuscript, as well as your comments, corrections, and valuable ideas. We believe the following response addresses all of the reviewers' issues. The detailed revisions are listed below where we present the comments of the reviewer in italic red letters, followed by our response in blue letters.

 

• Thanks for corrections.

We appreciate the reviewer's encouraging comments and hope that our revised manuscript will be accepted for publication.

Author Response File: Author Response.pdf

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The work is devoted to the current topic of studying how femtosecond laser treatment affects melanoma cells, using the A375 cell line as an in vitro model. The results showed significant inhibition of melanoma cell growth at various femtosecond laser parameters, especially at 380 and 400 nm. It was found that cell viability was significantly affected by wavelengths of 420 and 440 nm. The most effective exposure time was 10 minutes. However, wavelengths of 700, 720, 750 and 780 nm did not show a significant effect on cell viability, regardless of the duration of exposure.
The article is of interest and can be published after correcting minor comments.
1. The work practically lacks physical explanations of the empirical results of the study.
2. The reasons for the low efficiency of femtosecond laser radiation at wavelengths of 700, 720, 750 and 780 nm are unclear.

Author Response

A point-by-point response to Reviewer #3 comments

 

Dear, 

We appreciate your excellent remarks on our manuscript, as well as your comments, corrections, and valuable ideas. We believe the following response addresses all of the reviewers' issues. The detailed revisions are listed below where we present the comments of the reviewer in italic red letters, followed by our response in blue letters.

 

• The work practically lacks physical explanations of the empirical results of the study.

 

We would like to thank the reviewer for his valuable comment, and we agree that including physical explanations enriches the understanding of the biological outcomes. Accordingly, we have expanded the discussion section to include additional physical mechanisms, such as nonlinear absorption processes (e.g., multiphoton absorption), energy deposition dynamics, and the relevance of photon energy at different wavelengths in relation to intracellular chromophores, particularly cytochrome c oxidase and mitochondrial component.

 

• The reasons for the low efficiency of femtosecond laser radiation at wavelengths of 700, 720, 750 and 780 nm are unclear.

 

We sincerely appreciate all of reviewer valuable comments and suggestions. We have revised our manuscript discussion to highlight this point (lines 276–289).

 

 

Author Response File: Author Response.pdf

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

1. I suggest quantifying the inhibition improvements in the abstract. 
   For example, Please include the percentage of reduction in cell viability at the most effective wavelengths and exposure times. This will help the readers quickly grasp the magnitude of improvements

Author Response

A point-by-point response to Reviewer #4 comments

 

Dear, 

We appreciate your excellent remarks on our manuscript, as well as your comments, corrections, and valuable ideas. We believe the following response addresses all of the reviewers' issues. The detailed revisions are listed below where we present the comments of the reviewer in italic red letters, followed by our response in blue letters.

 

• I suggest quantifying the inhibition improvements in the abstract. For example, please include the percentage of reduction in cell viability at the most effective wavelengths and exposure times. This will help the readers quickly grasp the magnitude of improvements?

We appreciate the reviewer's thoughtful comment. As suggested by the reviewer, we have revised the abstract to include quantitative results, highlighting the percentage reduction in cell viability at the most effective wavelength.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review on

The Potential of Tunable Femtosecond Laser Light to Prevent Melanoma A375 Cell Growth: An In Vitro Investigation

 

The study is devoted to investigation of the effect of femtosecond laser pulses with different wavelengths on melanoma cells (the A375 cell line) after 3, 5, 10 minutes of laser irradiation.

The title of the article submitted for review fully corresponds to its content.

The abstract consistently and accessibly outlines the main points of the article, indicating the most important results of the study, including specific wavelengths and exposure durations.

The Introduction provides a detailed justification of the relevance of the study,

outlining the problem of data absence on melanoma treatment by femtosecond laser radiation. However, I have some questions listed below.

The methods used in the presented work are adequate to the objectives of the study.

The Discussion section lacks a comparison of the effect induced by femtosecond and other laser types. Fs-pulse's feature of nonlinear absorption mechanisms may indeed result in "more precise and varied effects" as stated in the Introduction. However, the cost of femtosecond lasers is several orders of magnitude higher than other types, especially LEDs. Is their potential for tumor cell inhibition worth the cost?

The results presented in this article may be of interest to the audience of the Journal if they are expanded properly.

Major issues

1. Introduction describes the current state of melanoma treatment approaches, peculiarities of ultrafast pulse absorption, and an example of fs-pulse application “to manage side effects induced by chemoradiation in patients with head and neck cancer”. Idea of cell inhibition due to femtosecond laser pulses is not novel regardless whether this is a cancer cell line or not. Fs-pulses can induce a plenty of effects (including ROS production, etc.) decreasing cell viability and even causing apoptosis. Providing more information on a background of exposure-induced inhibition mechanisms would strengthen the article.
2. The reference numbering between [7] in line 49 and [8,9] in line 69 in Introduction is broken. Studies [1-5] cited between lines 49 and 69 do not appear in References.
3. Materials and Methods. As far as I can gather, increasing the diameter of initial laser beam followed by cutting its central part by iris diaphragm is used to make power distribution more uniform across the beam. What was the diameter of laser beam incident on cells and how was it related to the diameter of a well in 96-well plate?
4. Why power of 100 mW was chosen? Was it a maximal value?
5. What was the resulting power density and how does it relate to values in other studies on cell irradiation? I do understand that this study is limited by experiments at a single value of power only. Does the inhibition effect observed in literature depend on the power density? How far is the power density value in this study from optimum or how does it correlate to values in other studies?
6. It is not clear from the article what the advantage of the femtosecond pulse duration is in terms of inhibition. The article would greatly have benefited from a direct comparison of the effect produced by femtosecond and other types of exposure (longer pulses or CW radiation), other parameters (power density, exposure time, wavelength) being equal.
7. Line 280-281. “The study's findings revealed that cellular proliferation was not induced by laser doses of up to 5 J/cm²[28]”. This is a conference paper and I failed to find the full text of it. To prove the relevance of this Reference authors should compare the exposure parameters. Were the power density and power energy the same as in the manuscript?

Minor issues

1. Line 57. It is recommended to change “extremely brief” → “extremely short”
2. Lines 60-67. I am not sure I’ve got the meaning of the sentence “…minimal phototoxicity, and clean, noninvasive, and controlled laser pulses are all provided by femtosecond pulses”.
3. “Minimal biological sample absorption” attributed to femtosecond laser pulses is too categorical a statement! Femtosecond pulses can be absorbed by biological objects as well as pulses of longer duration. The process is determined by the wavelength of the radiation and the spectral absorption of the object, but not by the duration. Radiation at a given wavelength for which the sample exhibits linear absorption will be absorbed at femtosecond pulse durations and picosecond, nanosecond, and millisecond durations.
4. Line 144-145. Sentence “Fig. 2. Various wavelength 144 ranges for irradiation of A375 cells.” simply repeats the title of the figure 2.
5. I think there is no need to repeat details such as “An evaluation of the effect of infrared femtosecond laser exposure on the viability 221 of A375 cells was conducted 24 hours after irradiation. MTT assays were utilized to determine cell viability, which was expressed as a percentage compared to non-irradiated control cells.” in each figure captions (Figs. 3-5) since it is given in Methods.
6. 6b. “nm” units should be added to wavelength values in figure legend.

Author Response

A point-by-point response to Reviewer #1 comments

 

Dear, 

We appreciate your excellent remarks on our manuscript, as well as your comments, corrections, and valuable ideas. We believe the following response addresses all of the reviewers' issues. The detailed revisions are listed below where we present the comments of the reviewer in italic red letters, followed by our response in blue letters.

 

The study is devoted to investigation of the effect of femtosecond laser pulses with different wavelengths on melanoma cells (the A375 cell line) after 3, 5, 10 minutes of laser irradiation.

Authors appreciate the reviewer’s positive feedback.

The title of the article submitted for review fully corresponds to its content.

The abstract consistently and accessibly outlines the main points of the article, indicating the most important results of the study, including specific wavelengths and exposure durations.

Authors appreciate the reviewer’s positive feedback.

 

The Introduction provides a detailed justification of the relevance of the study, outlining the problem of data absence on melanoma treatment by femtosecond laser radiation. However, I have some questions listed below.

The methods used in the presented work are adequate to the objectives of the study.

Authors appreciate the reviewer’s positive feedback.

 

The Discussion section lacks a comparison of the effect induced by femtosecond and other laser types. Fs-pulse's feature of nonlinear absorption mechanisms may indeed result in "more precise and varied effects" as stated in the Introduction. However, the cost of femtosecond lasers is several orders of magnitude higher than other types, especially LEDs. Is their potential for tumor cell inhibition worth the cost?

The introduction has been revised to include a direct comparison between femtosecond lasers and other laser types, such as nanosecond, picosecond, and continuous-wave (CW) lasers, in terms of biological effects, precision, and selectivity. The unique nonlinear absorption mechanism of femtosecond pulses enables targeted energy deposition, minimizing thermal damage to surrounding tissues. While femtosecond laser systems are costlier than other laser types, their ability to induce precise and efficient photobiological effects without excessive collateral damage justifies their potential use in targeted cancer therapies.

 

The results presented in this article may be of interest to the audience of the Journal if they are expanded properly.

Authors appreciate the reviewer’s positive feedback.

 

Major issues

 

• Introduction describes the current state of melanoma treatment approaches, peculiarities of ultrafast pulse absorption, and an example of fs-pulse application “to manage side effects induced by chemoradiation in patients with head and neck cancer”. Idea of cell inhibition due to femtosecond laser pulses is not novel regardless whether this is a cancer cell line or not. Fs-pulses can induce a plenty of effects (including ROS production, etc.) decreasing cell viability and even causing apoptosis. Providing more information on a background of exposure-induced inhibition mechanisms would strengthen the article.

 

We sincerely appreciate the reviewer valuable comments and suggestions. In the revised manuscript, additional details on femtosecond laser-induced inhibition mechanisms, including ROS generation, mitochondrial membrane disruption, and apoptosis induction, have been incorporated into the Introduction and Discussion. Prior studies have demonstrated that shorter wavelengths (UV and blue) are more effective in generating ROS and affecting mitochondrial function, leading to apoptosis. This mechanistic background will be expanded to strengthen the rationale for the study.

 

• The reference numbering between [7] in line 49 and [8,9] in line 69 in Introduction is broken. Studies [1-5] cited between lines 49 and 69 do not appear in References.

 

We appreciate the reviewer's valuable thoughts. This error has been corrected in the revised manuscript  by ensuring that all references cited in the Introduction are properly listed in the References section. The numbering has been checked and aligned throughout the manuscript.

 

 

• Materials and Methods. As far as I can gather, increasing the diameter of initial laser beam followed by cutting its central part by iris diaphragm is used to make power distribution more uniform across the beam. What was the diameter of laser beam incident on cells and how was it related to the diameter of a well in 96-well plate?

 

We sincerely appreciate the reviewer valuable comments . It has been stated in the revised version of the manuscript that the laser beam was expanded to 20 mm initially and then adjusted to 6 mm using an iris diaphragm before reaching the cells. Given that the diameter of each well in a 96-well plate is approximately 6 mm, the laser beam size was nearly equivalent to the well size, ensuring uniform illumination of the cell culture.

 

 

• Why power of 100 mW was chosen? Was it a maximal value?

 

We sincerely appreciate the reviewer valuable comments . Based on earlier research by our group investigating the biological impacts of femtosecond lasers, the 100 mW power was chosen. While preventing excessive photothermal effects, this value guarantees enough photon energy delivery.  Higher powers could be produced by the laser system, but this setup was selected to maximize photobiological impact and reduce unwanted heat consequences.  

 

 

• What was the resulting power density and how does it relate to values in other studies on cell irradiation? I do understand that this study is limited by experiments at a single value of power only. Does the inhibition effect observed in literature depend on the power density? How far is the power density value in this study from optimum or how does it correlate to values in other studies?

 

We sincerely appreciate the reviewer's valuable comments. The power density was not explicitly mentioned in the manuscript. However, it can be calculated for a 6 mm beam diameter as follows: 

• Power Density = Power (W) / Beam Area (cm²)
• Beam Area = π × (6/2)2 = 28.27 mm² = 0.0283 cm²
• Power Density = 100 mW / 0.0283 cm² = 3.53 W/cm²

Also, as suggested by the reviewer, the power density has been highlighted in the revised manuscript. We want to clarify that the manuscript discusses mainly wavelength effects. To the best of our knowledge, the inhibition effect is predominantly influenced by the laser wavelength and the spectral characteristics of the biological sample, rather than by power density—especially when adhering to the maximum exposure safety limits to prevent thermal effects. Also, previous studies have shown that shorter wavelengths, such as UV and blue, are more effective at generating reactive oxygen species (ROS) and disrupting mitochondrial function, thereby inducing apoptosis.

 

• It is not clear from the article what the advantage of the femtosecond pulse duration is in terms of inhibition. The article would greatly have benefited from a direct comparison of the effect produced by femtosecond and other types of exposure (longer pulses or CW radiation), other parameters (power density, exposure time, wavelength) being equal.

 

We sincerely appreciate the reviewer valuable comment. Femtosecond pulses enable highly localized energy deposition due to nonlinear absorption mechanisms, leading to enhanced biological effects such as ROS generation and mitochondrial damage while minimizing thermal diffusion. Unlike nanosecond or CW lasers, which rely on linear absorption and can cause excessive heating, femtosecond lasers can target specific cellular components with higher precision. A comparative discussion of these effects has been added to strengthen the rationale for using femtosecond lasers.

 

 

• Line 280-281. “The study's findings revealed that cellular proliferation was not induced by laser doses of up to 5 J/cm²[28]”. This is a conference paper and I failed to find the full text of it. To prove the relevance of this Reference authors should compare the exposure parameters. Were the power density and power energy the same as in the manuscript?

 

We sincerely appreciate the reviewer valuable comment. The conference reference has been changed into a relevant reference, and a comparison of the exposure parameters in the cited reference with those used in this study has been added to ensure relevance.

 

Minor issues

 

 

• Line 57. It is recommended to change “extremely brief” → “extremely short”

 

We appreciate the reviewer comments, which we have now incorporated into the revised version of the manuscript and highlighted in yellow.

 

 

• Lines 60-67. I am not sure I’ve got the meaning of the sentence “…minimal phototoxicity, and clean, noninvasive, and controlled laser pulses are all provided by femtosecond pulses”.

 

We appreciate the reviewer comments, which we have now incorporated into the revised version of the manuscript and highlighted in yellow.

 

• “Minimal biological sample absorption” attributed to femtosecond laser pulses is too categorical a statement! Femtosecond pulses can be absorbed by biological objects as well as pulses of longer duration. The process is determined by the wavelength of the radiation and the spectral absorption of the object, but not by the duration. Radiation at a given wavelength for which the sample exhibits linear absorption will be absorbed at femtosecond pulse durations and picosecond, nanosecond, and millisecond durations.

 

We appreciate the reviewer comments, which we have now incorporated into the revised version of the manuscript and highlighted in yellow.

 

 

• Line 144-145. Sentence “Fig. 2. Various wavelength 144 ranges for irradiation of A375 cells.” simply repeats the title of the figure?

 

We sincerely appreciate the reviewer valuable comment. In the revised version of the manuscript, more details regarding the figure are now provided by rewording and removing this repetition.

 

• I think there is no need to repeat details such as “An evaluation of the effect of infrared femtosecond laser exposure on the viability 221 of A375 cells was conducted 24 hours after irradiation. MTT assays were utilized to determine cell viability, which was expressed as a percentage compared to non-irradiated control cells.” in each figure captions (Figs. 3-5) since it is given in Methods.

 

We sincerely appreciate the reviewer valuable comment. In the revised version of the manuscript, repetitive information has been removed from figure captions to improve conciseness.

 

• “nm” units should be added to wavelength values in figure legend.

 

We appreciate the reviewer comments, which we have now incorporated into the revised version of the manuscript and the unit "nm" has been added wherever necessary in figure legends.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents an investigation into the effects of femtosecond laser irradiation on melanoma cells. However, the study is limited in scope and lacks essential controls and mechanistic investigations. The results are inconclusive, as the observed cell death may be attributed to thermal effects rather than any novel laser-induced biological response.

1. The authors did not measure the temperature of the samples during laser exposure. Since UV and blue light are known to cause heating, the observed reduction in cell viability may be due to thermal effects rather than any unique properties of femtosecond laser pulses. 

   Without thermal measurements, the study's findings lack scientific validity, as the primary hypothesis remains untested.

2. The authors should have included controls with equivalent heat exposure from non-laser sources to rule out thermal effects.

3. The manuscript fails to investigate the underlying mechanisms of cell death, such as oxidative stress, apoptosis, or DNA damage. Without this information, the results have limited relevance

Due to the lack of thermal controls, limited experimental design, and absence of mechanistic insights, the manuscript does not provide sufficient evidence of any novel or significant findings. I recommend rejecting the paper in its current form

Author Response

A point-by-point response to Reviewer #2 comments

 

Dear, 

We appreciate your excellent remarks on our manuscript, as well as your comments, corrections, and valuable ideas. We believe the following response addresses all of the reviewers' issues. The detailed revisions are listed below where we present the comments of the reviewer in italic red letters, followed by our response in blue letters.

 

• The authors did not measure the temperature of the samples during laser exposure. Since UV and blue light are known to cause heating, the observed reduction in cell viability may be due to thermal effects rather than any unique properties of femtosecond laser pulses.

Thank you for raising this important point. We acknowledge that temperature measurements during laser irradiation would help rule out thermal effects. While femtosecond lasers are known for generating minimal heat due to their ultrashort pulse duration, we have tested this aspect in our laboratory using the same wavelengths and laser parameters. Our results showed that the generated heat did not exceed 1.8°C, which is within the range of normal room temperature fluctuations. These findings support the conclusion that the observed reduction in cell viability is primarily due to non-thermal effects. Nonetheless, in future studies, we plan to directly measure the temperature of the cell culture medium during laser exposure using an infrared thermal camera or thermocouple sensor to further validate these results.

 

• The authors should have included controls with equivalent heat exposure from non-laser sources to rule out thermal effects.

 

We appreciate this valuable suggestion. Although our current study did not include heat-exposed controls, as previously mentioned, we have tested heat generation in our laboratory under the same wavelengths and laser parameters used in this study. The results indicated that the generated heat did not exceed 1.8°C, which is within the range of normal room temperature fluctuations. These findings suggest that thermal effects are unlikely to account for the observed reduction in cell viability. Nonetheless, we acknowledge the importance of including heat-exposed controls to further validate our conclusions.

 

• The manuscript fails to investigate the underlying mechanisms of cell death, such as oxidative stress, apoptosis, or DNA damage. Without this information, the results have limited relevance.

 

We appreciate the reviewer's valuable comments. To be clear, our main goal was to determine the best laser settings for preventing the proliferation of melanoma cells. However, we intend to assess the molecular processes of cell death in our upcoming research.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The work is devoted to the current topic of developing new methods for treating melanoma with fewer side effects, but an effective therapeutic effect. The aim of the work is to study how femtosecond laser treatment affects melanoma cells using the A375 cell line as an in vitro model. Significant inhibition of melanoma cell growth was demonstrated with various parameters of the femtosecond laser, especially at 380 and 400 nm.
The most effective exposure time was found to be 10 minutes.
The work is of interest and can be published after correcting the following comments.
1. The article text should include additional information on existing methods of early laser diagnostics and differentiation of cancer stages.
- https://doi.org/10.1142/S1793545824430028
- https://doi.org/10.1117/1.JBO.29.5.052920
2. To facilitate the perception of the material, a more detailed description of the experimental device is necessary.
3. It is desirable to detail the text with information on the prospects for further research.

Author Response

A point-by-point response to Reviewer #3 comments

 

Dear, 

We appreciate your excellent remarks on our manuscript, as well as your comments, corrections, and valuable ideas. We believe the following response addresses all of the reviewers' issues. The detailed revisions are listed below where we present the comments of the reviewer in italic red letters, followed by our response in blue letters.

 

• The article text should include additional information on existing methods of early laser diagnostics and differentiation of cancer stages.

We appreciate this suggestion and have expanded the introduction section to include relevant information on laser-based diagnostics. Recent advancements such as 3D polarization–interference biomedical diagnostics and polarization-interference holographic histology have enabled non-invasive visualization and differentiation of tissue abnormalities. The 3D Jones-matrix technology allows for the layered phase scanning of supramolecular networks in biological fluids, providing objective markers for diagnosing pathological changes, including cancer. Additionally, polarization-interference holographic histology enables the identification of necrotic changes in tissue architecture with excellent accuracy. These techniques complement therapeutic applications, such as the femtosecond laser treatment explored in our study, and offer promising prospects for both early diagnosis and treatment.

https://doi.org/10.1142/S1793545824430028

https://doi.org/10.1117/1.JBO.29.5.052920

 

• To facilitate the perception of the material, a more detailed description of the experimental device is necessary.

 

We sincerely appreciate all of reviewer valuable comments and suggestions. In the revised manuscript, we have revised the Materials and Methods section to provide a more detailed description of the femtosecond laser system.

 

• It is desirable to detail the text with information on the prospects for further research.

 

We appreciate the reviewer's valuable thoughts. The Discussion section of the  revised manuscript has been updated to include a list of potential future study directions, such as:

 

• Investigating molecular mechanisms such as ROS generation, apoptosis, and DNA damage.
• Conducting in vivo studies to assess the safety and therapeutic efficacy of femtosecond lasers.
• Optimizing laser parameters for clinical applications and exploring their use in combination with other therapies.

 

 

 

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript presents an investigation into the effects of femtosecond laser irradiation on melanoma A375 cells, highlighting its potential as a novel therapeutic approach. The study is well-structured, and the methodology is clearly described, including precise details on cell plating, laser parameters, and viability assessment.

Question 1: Have the authors considered exploring or discussing the possible cellular and molecular mechanisms underlying the observed reduction in melanoma cell viability after femtosecond laser exposure?

Question 2: Can the authors provide more details on the statistical methods used to analyze the data, including significance levels and sample sizes, to ensure the robustness of their findings?

Question 3: How do the findings of this study compare with existing melanoma treatment modalities, and what are the potential challenges in translating femtosecond laser therapy from an in vitro model to clinical applications?

Question 4:  The authors have not sufficiently described the theory and methodology of studying cancer conditions using laser radiation of different wavelengths. In particular, the authors may consider referring to the research conducted by the scientific group: doi:10.1038/s41598-021-83986-4.

Author Response

A point-by-point response to Reviewer #4 comments

 

Dear, 

We appreciate your excellent remarks on our manuscript, as well as your comments, corrections, and valuable ideas. We believe the following response addresses all of the reviewers' issues. The detailed revisions are listed below where we present the comments of the reviewer in italic red letters, followed by our response in blue letters.

 

• Have the authors considered exploring or discussing the possible cellular and molecular mechanisms underlying the observed reduction in melanoma cell viability after femtosecond laser exposure?

We appreciate the reviewer's helpful remarks and have updated the Discussion section of the revised version of the manuscript to include potential mechanisms. The observed effects may result from ROS generation, disruption of mitochondrial membrane potential, and apoptosis induction. These effects are consistent with previous studies using blue and UV light. Future research will include ROS assays, Annexin V/PI staining, and caspase activity measurements to confirm these mechanisms.

 

• Can the authors provide more details on the statistical methods used to analyze the data, including significance levels and sample sizes, to ensure the robustness of their findings?

 

We value the reviewer's insightful remarks and recommendations. We have added details of the statistical methods used in the Materials and Methods section. All experiments were performed in triplicate, and results are presented as mean ± standard deviation. Statistical significance was assessed using one-way ANOVA followed by Tukey's post hoc test, with significance levels indicated as P < 0.05, P < 0.01, *P < 0.001, and **P < 0.0001.

 

 

• How do the findings of this study compare with existing melanoma treatment modalities, and what are the potential challenges in translating femtosecond laser therapy from an in vitro model to clinical applications?

 

We appreciate the reviewer's thoughtful comment.  In the revised version of the manuscript we have updated  the Discussion section to compare femtosecond laser therapy with existing treatments. Compared to chemotherapy and surgery, femtosecond lasers offer a non-invasive, targeted approach with minimal side effects. However, challenges include optimizing laser parameters for in vivo applications, ensuring selective targeting of cancer cells, and achieving sufficient tissue penetration for deep-seated tumors. Future studies will address these challenges to facilitate clinical translation.

 

• The authors have not sufficiently described the theory and methodology of studying cancer conditions using laser radiation of different wavelengths. In particular, the authors may consider referring to the research conducted by the scientific group: doi:10.1038/s41598-021-83986-4.

Thank you for this valuable reference. We have incorporated it into the Discussion section, summarizing key insights into wavelength-specific effects on cancer cells. Shorter wavelengths, such as UV and blue light, are more effective at inducing ROS generation and disrupting mitochondrial function, leading to apoptosis. In contrast, longer wavelengths in the near-infrared range penetrate deeper into tissues but have weaker effects on cell viability due to reduced absorption by intracellular chromophores.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Please se the file attached.

Comments for author File: Comments.pdf

Author Response

Response to Reviewers

We are so grateful to the editor and the reviewers for allowing us to improve our manuscript. The manuscript has been substantially revised according to the comments raised by the reviewers. We would like to thank the reviewers for their constructive and competent criticism, and we hope that our updated manuscript will be acceptable for publication.

 

Response to Reviewer 1

The authors have successfully addressed most of my questions. However, I am not completely satisfied.

Q1. In my previous report, I mentioned that the idea of cell inhibition by femtosecond laser pulses is not novel and that providing more information on the background of exposure-induced inhibition mechanisms would strengthen the article. I am not sure that the following text in Introduction is relevant to the article idea of cancer cell line treatment. “Recent advancements such as 3D polarization—interference biomedical diagnostics and polarization-interference holographic histology have enabled non-invasive visualization and differentiation of tissue abnormalities. The 3D Jones matrix technology allows for the layered phase scanning of supramolecular networks in biological fluids, providing objective markers for diagnosing pathological changes, including cancer. Additionally, polarization-interference holographic histology enables the identification of necrotic changes in tissue architecture with excellent accuracy [9, 10].”

The study Yoon J, Ryu SW, Lee S, Choi C. Cytosolic irradiation of femtosecond laser induces mitochondria-dependent apoptosis-like cell death via intrinsic reactive oxygen cascades. Sci Rep. 2015 Feb 4;5:8231. doi: 10.1038/srep08231. PMID: 25648455; PMCID:

PMC4316155 seems to be a good demonstration of intrinsic processes that can be induced by femtosecond laser pulses.

The study of Thegersen et.al. demonstrates the effect of arresting cell reproduction at various wavelengths (200-800 nm).

Thegersen J, Knudsen CS, Maetzke A, Jensen SJ, Keiding SR, Alsner J, Overgaard J. Reproductive death of cancer cells induced by femtosecond laser pulses. Int J Radiat Biol. 2007 May;83(5):289-99. doi: 10.1080/09553000701283808. PMID: 17457754.

The study of Tirlapur et al. provides evidence that DNA strand breaks also occur in vivo when mammalian cells are exposed to high average power of femtosecond radiation: Tirlapur UK, Kénig K. Femtosecond near-infrared laser pulse induced strand breaks in mammalian cells. Cell Mol Biol (Noisy-le-grand). 2001;47 Online Pub:0L131-4. PMID: 1936858.

1. We appreciate the reviewer’s insightful suggestion. The paragraph on 3D polarization–interference diagnostics has been removed. We have also revised the Introduction to incorporate relevant studies demonstrating mitochondrial apoptosis, cell reproductive arrest, and DNA strand breaks induced by femtosecond laser exposure (Yoon et al., Thegersen et al., Tirlapur et al.), which directly support the biological believability of our findings.

---------------------------------------------------------------------------------------------------------------------

Q2. The earlier study by Taha et al. (Ref [12]) is cited in terms of the tunability of femtosecond lasers instead of demonstrating the anti-cancer effect of femtosecond pulses. This looks strange:

Taha S, Mohamed WR, Elhemely MA, EI-Gendy AO, Mohamed T. Tunable femtosecond laser suppresses the proliferation of breast cancer in vitro. J Photochem Photobiol B. 2023 Mar;240:112665. doi: 10.1016/j.jphotobiol.2023.112665. Epub 2023 Jan 29. PMID: 36736031.

1. Thank you for pointing this out. We have revised the relevant sentence to more clearly state that the earlier study by Taha et al. demonstrated the anticancer potential of tunable femtosecond lasers in vitro and serves as a basis for expanding investigations into other cancer cell lines, such as A375 melanoma in this work.

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Q3. I do not agree with author’s position that “the inhibition effect is predominantly influenced by the laser wavelength and the spectral characteristics of the biological sample, rather than by power density—especially when adhering to the maximum exposure safety limits to prevent thermal effects.” There are a number of studies demonstrating the dose effect of laser irradiation. See the results for “Arndt Schultz curve” or “hormesis™ terms in PubMed.

See the following articles, for example:

• Sommer AP, Pinheiro AL, Mester AR, Franke RP, Whelan HT. Biostimulatory windows in low-intensity laser activation: lasers, scanners, and NASA's light-emitting diode array system. J Clin Laser Med Surg. 2001 Feb;19(1):29-33. doi: 10.1089/104454701750066910. PMID:11547815.
• Chung H, Dai T, Sharma SK, Huang YY, Carroll JD, Hamblin MR. The nuts and bolts of low-level laser (light) therapy. Ann Biomed Eng. 2012 Feb;40(2):516-33. doi: 10.1007/s10439-011-0454-7. Epub 2011 Nov 2. PMID: 22045511; PMCID: PMC3288797.

 

They clearly show that there are stimulatory and inhibitory values of laser exposure parameters, including power density and power energy. For this reason, I asked the authors to compare the effect they observed with other studies in terms of the parameters mentioned. How do they correlate?

1. We thank the reviewer for the valuable insight and the references provided. We acknowledge that both laser wavelength and energy dose (fluence) contribute significantly to biological effects, and that the biphasic dose-response relationship described by the Arndt-Schulz law and hormesis models is well-supported in the literature. In response, we have revised the manuscript to include calculated energy densities (fluence) for our experimental conditions and compared them to reported inhibitory thresholds. We also revised the manuscript to clarify that both wavelength and energy density play essential roles in cellular responses to laser exposure.

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Q4. Some efforts were made to compare the effect produced by femtosecond and other types of exposure (longer pulses or CW radiation), other parameters (power density, exposure time, wavelength). Studies [33] and [34] were considered and exposure parameters were revealed. I did not find data on energy density (called “dose” in some studies) [J/cm?] in author’s study and a comparison performed.

1. In response to the reviewer’s comment, we have now explicitly calculated and reported the energy densities (fluence) used in our experiments. We have also added comparisons with fluence values from previous studies. These values confirm that our exposure conditions fall within the inhibitory range, aligning with prior reports of ROS-mediated or apoptotic effects in cancer cells at high fluences.

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Q5. I would also recommend to calculate the peak power of femtosecond laser pulses to compare the irradiation parameters with studies mentioned in comment #1 (and any other found by authors) in Discussion section.

1. We appreciate the reviewer comments, which we have now taken into account in the revised version of the manuscript.

The experiment was conducted with a laser beam of radius of r = 3 mm, an average power of P_ave= 100 mW, a pulse duration of ∆t = 100 fs, and a repetition rate of f = 80 MHz. Based on these laser parameters and the experimental conditions the energy per pulse E = P_ave / f = 1.25 ×10^-9 Joule, the peak power (power per pulse) P_peak= E/∆t = 1.25 ×10⁴ Watts, the laser beam intensity (power density)   I = P_peak  / p r² = 4.5 ×10⁴ Watts / cm², the laser beam fluence (energy density or dose) for one second exposure time   = E  / p r² = 8.8 ×10^-9 Joule / cm². In the case of 3, 5, and 10 minutes, the doses are 1584 ×10^-9 Joule / cm², 2640 ×10^-9 Joule / cm², and 5280 ×10^-9 Joule / cm², respectively.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your response letter. After reviewing the revised manuscript, it is evident that the previous comments were not adequately addressed. The concerns regarding the experimental design remain unresolved, and no substantial modifications were made to improve the study's rigor.

1. Lack of Experimental Design Improvements: The manuscript still lacks critical experimental controls and methodological refinements. The fundamental difference between cells in solution and cells in tissue remains unaddressed, significantly limiting the study's applicability and biological relevance.
2. Novelty and Significance Remain Insufficient: The study does not convincingly demonstrate how its approach presents a significant advancement beyond existing techniques. The proposed method does not provide compelling evidence of novelty or impact, as key comparisons with established methodologies are missing.
3. Failure to Implement Reviewer Suggestions: Despite previous comments, crucial issues such as validating the mechanism of action and accounting for thermal effects remain unresolved. The responses provided in the cover letter acknowledge these limitations but do not include meaningful experimental revisions within the manuscript itself.

Author Response

Response to Reviewers

We are so grateful to the editor and the reviewers for allowing us to improve our manuscript. The manuscript has been substantially revised according to the comments raised by the reviewers. We would like to thank the reviewers for their constructive and competent criticism, and we hope that our updated manuscript will be acceptable for publication.

 

Response to Reviewer 2

Q1. Lack of Experimental Design Improvements: The fundamental difference between cells in solution and cells in tissue remains unaddressed, significantly limiting the study's applicability and biological relevance.

1. Thank you for your valuable feedback. We acknowledge the limitation regarding the difference between 2D in vitro cell culture and in vivo tissue architecture. In response, we have added a paragraph to the Discussion section, obviously acknowledging this limitation and its implications for clinical translation. Additionally, we discussed potential next steps, including the use of 3D cell culture models or animal models, which are currently in planning stages for follow-up studies. Although the current study is confined to monolayer cultures, it serves as a foundational exploration of wavelength-dependent effects to guide more complex future models.

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Q2. Novelty and Significance Remain Insufficient: The study does not convincingly demonstrate how its approach presents a significant advancement beyond existing techniques. The proposed method does not provide compelling evidence of novelty or impact, as key comparisons with established methodologies are missing.

1. We appreciate this important observation. We have now expanded the Introduction and Discussion sections to more explicitly state the novelty of our study. Specifically, our work uniquely investigates a broad spectrum of femtosecond laser wavelengths, including UV and visible regions, against melanoma A375 cells with direct comparison under identical exposure conditions—something not comprehensively explored in prior research. We have also added references to highlight gaps in prior literature and how our study fills those.

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Q3. Failure to Implement Reviewer Suggestions: Despite previous comments, crucial issues such as validating the mechanism of action and accounting for thermal effects remain unresolved. The responses provided in the cover letter acknowledge these limitations but do not include meaningful experimental revisions within the manuscript itself.

1. As suggested by the reviewer and to address thermal effects, we have now elaborated in the Materials and Methods section that all irradiations were performed in a temperature-controlled incubator with monitored well temperatures to ensure negligible heating (<0.5°C increase). This minimizes the likelihood of thermal contributions. Regarding the mechanism of action, we expanded the Discussion to emphasize literature-supported pathways such as ROS generation and mitochondrial membrane disruption at shorter wavelengths. Although direct assays for ROS and apoptosis were not performed, these are outlined as immediate next steps in our future work.

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Author Response File: Author Response.pdf