Anticancer Effects of Combined Blue Light and Ionizing Irradiation
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis study proposes a promising strategy combining blue light irradiation with radiotherapy. Preliminary data support its synergistic antitumor effects and a mechanism mediated through the ERK signaling pathway. Although the work has limitations in mechanistic depth and model diversity, it holds value as an exploratory study and is worthy of publication, provided the authors address the following major points (Major Revision recommended).
1. Mechanistic investigation is preliminary:
Only the phosphorylation status of ERK1/2 was examined, without validation of upstream regulators (e.g., Ras/Raf) or downstream effectors (e.g., c-Myc, Cyclin D1). It is recommended to include additional analyses of key nodes within the ERK pathway or perform functional rescue experiments using ERK inhibitors or activators to establish a causal relationship.
2. Limited cell model:
The study relies solely on the SAS HNSCC cell line, which may not adequately represent the heterogeneity of head and neck squamous cell carcinoma. To strengthen the generalizability of the conclusions, at least one additional HNSCC cell line (e.g., CAL27 or FaDu) should be included.
3. Lack of dose–response and time-course analyses:
Critical parameters—such as blue light intensity (mW/cm²), irradiation duration, and X-ray dose—are not sufficiently detailed, and no data are presented on how varying combinations of these parameters affect outcomes. Dose-titration experiments are recommended to optimize the combined treatment regimen.
4. Absence of normal cell toxicity assessment:
It remains unclear whether blue light alone or in combination with radiation exhibits selective toxicity toward cancer cells versus normal epithelial cells—a key consideration for therapeutic safety. The inclusion of a normal oral keratinocyte cell line (e.g., HaCaT) as a control is strongly advised.
no
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsEvaluation of Originality/Novelty
The manuscript “Anticancer Effects of Combined Blue Light and Ionizing Irradiation” addresses an interesting and potentially innovative topic. The study explores, for the first time, the anticancer effects of blue light in combination with ionizing radiation, particularly in HNSCC cell lines. Although blue light has been widely studied in various biomedical contexts, including its antiproliferative effects and synergistic action with antitumor agents, no prior studies have evaluated its combination with radiotherapy. This represents a meaningful novel contribution.
The manuscript also provides original insights into the underlying mechanisms, especially regarding the modulation of ROS and ERK1/2 signaling. The finding that blue light combined with 2 Gy yields antiproliferative effects comparable to 6 Gy alone is noteworthy and suggests potential clinical relevance through radiation dose reduction. The differential mechanistic response observed between SAS and Ca9-22 cells adds value by highlighting cell-line–specific heterogeneity. Additionally, the negative results, such as the lack of increased apoptosis and absence of changes in radiosensitivity, are appropriately presented and reinforce the conclusion that the observed effects do not stem from classical pathways.
However, some aspects temper the overall level of novelty. The antiproliferative action of blue light alone is well documented, and the study essentially confirms these effects in HNSCC. The experimental methods employed are standard and do not introduce methodological innovation. Moreover, the work is entirely in vitro, which limits the translational impact at this stage.
Overall, the study offers moderate originality. Its main strength lies in the unprecedented combination of blue light with ionizing radiation and the identification of a possible mechanism involving ROS–ERK1/2 modulation. These contributions are scientifically relevant and justify consideration for publication, although the findings do not constitute a disruptive advance in radiation-based cancer therapy.
Significance of Content
The manuscript presents clear scientific significance, primarily due to its novel proposal of combining blue light with ionizing radiation and its investigation of the associated molecular mechanisms. This combined approach is original and adds value to the current literature. However, the overall impact of the study remains moderate, mainly because all experiments were conducted exclusively in vitro, without translational validation or additional experimental approaches that could strengthen the mechanistic conclusions. Expanding the experimental depth in future studies would substantially enhance the significance and applicability of the findings.
Quality of Presentation
The manuscript is well structured, clear, and logically organized. The overall presentation is appropriate for a biomedical article; however, there are elements that could be improved to enhance fluency, visual impact, and narrative clarity. I highlight these points below:
Results (Lines 149–257)
Currently, the results are presented in a continuous structure spanning from page 7 to the end, covering the effects of blue light, its combination with radiation, apoptosis, ERK1/2, and ROS. I suggest reorganizing this section to improve clarity and segmentation, as follows:
- Effects of Blue Light Irradiation Alone
- Combined Effects of Blue Light and Ionizing Radiation
- Effects on Reactive Oxygen Species (ROS)
- Regulation of ERK1/2 Phosphorylation
- Effects on Apoptosis
- Effects on Clonogenic Survival
Although the authors already use subsections to delineate the results, I believe that the new sequence and grouping proposed above strengthen the scientific narrative. The Isolated Effect (section 3.1) establishes the baseline; the Combined Effect (section 3.2) presents the main finding; the Functional Effect (Clonogenic assay – section 3.3) validates the main finding using a gold-standard assay (better grouping); and the Mechanistic Sections (3.4, 3.5, and 3.6) explore the underlying mechanisms in a logical and sequential manner.
Additional observations:
- Line 159: Special attention should be given to Figures 2B and 2C, which show horizontal distortions.
- Line 179: Special attention should be given to Figure 3B, which shows horizontal distortions.
- Line 207: Special attention should be given to Figure 4A, which shows horizontal distortions.
- Line 233: Special attention should be given to Figure 6A, which shows horizontal distortions.
Scientific Soundness
Overall, the study is scientifically sound and addresses a relevant and understudied topic. The hypotheses are clearly stated and logically aligned with existing literature. The experimental design is appropriate, and the methods employed, cell viability assays, apoptosis analysis, Western blotting, ROS measurement, and clonogenic assays, are well-established and suitable for the research objectives.
However, several points require attention to strengthen the scientific rigor of the manuscript:
- Mechanistic evidence is suggestive but not demonstrated
Although the results support an association between blue light exposure, reduced ROS levels, and decreased ERK1/2 phosphorylation, the study lacks functional experiments to establish causality. The use of ERK pathway inhibitors or ROS modulators would substantially reinforce the proposed mechanism. - ROS analysis is limited to SAS cells
Because the study highlights the heterogeneity between SAS and Ca9-22 cells, it would be scientifically more robust to assess ROS levels in both cell lines. The current analysis limits the strength of the mechanistic interpretation. - All experiments are in vitro
While acceptable for preliminary investigation, the lack of in vivo or ex vivo validation restricts the translational impact. This limitation should be more explicitly acknowledged in the Discussion. - Temperature control during blue light irradiation
The manuscript does not specify whether the temperature was monitored during blue light exposure. As LED sources may cause localized heating, clarification is needed to ensure that the effects observed are attributable to light and not thermal artifacts. - Statistical details require clarification
The manuscript would benefit from reporting the number of biological replicates for each experiment and confirming whether assumptions of normality were met for the statistical tests used. - Interpretation of “Effective Dose Reduction” should be tempered
Although the finding that 30 min of blue light combined with 2 Gy mimics the antiproliferative effect of 6 Gy is noteworthy, statements suggesting dose reduction in radiotherapy should be phrased more cautiously. No assays evaluating DNA damage, repair kinetics, or checkpoint activation were performed.
Despite these points, the data are internally consistent, the results are interpreted appropriately, and the conclusions are generally well supported by the experiments presented. Addressing the issues above would significantly improve the scientific robustness of the manuscript.
Interest to Readers
The manuscript addresses a relevant and timely topic that is likely to attract the interest of readers working in radiation biology, cancer research, photobiology, and molecular signaling. The proposed combination of blue light with ionizing radiation is novel and conceptually appealing, particularly because it involves a simple, low-cost physical modality that may complement established radiotherapeutic strategies. This innovative angle increases the manuscript’s relevance to researchers exploring radiosensitization and multimodal anticancer approaches.
The study also aligns well with the scope of journals focused on molecular mechanisms and experimental oncology, given its emphasis on ROS modulation, ERK1/2 signaling, and cellular stress responses. Readers interested in the cellular consequences of combined physical stimuli or in non-pharmacological therapeutic strategies may find the work particularly engaging.
However, the overall interest is somewhat limited by the entirely in vitro nature of the experiments and by the preliminary stage of the mechanistic insights. The absence of translational components—such as in vivo models, 3D cultures, or functional validation of the proposed pathways—may reduce the appeal for readers seeking clinically oriented or application-driven studies. In addition, the heterogeneity of responses between cell lines, although scientifically relevant, may restrict the generalizability of the findings.
Despite these limitations, the manuscript provides a novel conceptual framework and raises questions that could stimulate further investigation, thereby maintaining moderate to high interest for the intended readership.
Overall Merit
The overall merit of the manuscript is moderate. The study presents a novel and conceptually interesting approach by combining blue light with ionizing radiation, and it provides relevant preliminary insights into ROS modulation and ERK1/2 signaling. These contributions add value to the field and justify consideration for publication.
However, the impact of the work is limited by several factors: (i) all experiments were performed exclusively in vitro, which restricts the translational relevance; (ii) the mechanistic findings, although promising, remain preliminary and would benefit from functional validation; and (iii) some results confirm known effects of blue light rather than expanding the field substantively. Taken together, the manuscript offers meaningful but not high-impact contributions.
With further mechanistic reinforcement and potential expansion into more advanced models, the work could achieve higher significance. As currently presented, its overall merit aligns with moderate relevance within the journal’s scope.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
Thank you for submitting the revised version of your manuscript entitled “Anticancer Effects of Combined Blue Light and Ionizing Irradiation”, along with the detailed response to the reviewers’ comments.
I have carefully evaluated the revised manuscript and your point-by-point responses. The revisions adequately address all concerns raised during the review process. In particular, the explicit acknowledgment of the exclusively in vitro nature of the study, the clarification of the mechanistic limitations, and the tempered interpretation of translational implications significantly strengthen the scientific rigor and transparency of the work.
The reorganization of the Results section, improvement of figure quality, inclusion of additional ROS data, clarification of statistical analyses, and discussion of potential thermal effects further improved the clarity and overall quality of the manuscript. The study now presents a balanced and well-contextualized contribution, highlighting its novelty while appropriately delineating its limitations.
Based on the revisions made, the manuscript is suitable for publication.
