Fluorescence-Enhanced Assessments for Human Breast Cancer Cell Characterizations
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript “Fluorescence-enhanced photodynamic assessments for human breast cancer cell characterizations” is devoted to the experimental study of fluorescence of cancer and normal cells samples.
Unfortunately, objectives, hypothesis, and conclusions are not clearly stated in this manuscript. As far as I understood, the ultimate goal was to use the fluorescence-based technique in diagnostics to differentiate between samples containing normal and cancerous cells. If so, a diagnostic procedure should be proposed and its validity should be evaluated. If the goal of the paper is different, it should be clearly stated in the abstract and introduction, and the corresponding conclusions should be made. While the manuscript in its current state reports several interesting experiments, their design and meaning are obscure.
The usage of porphyrins and 5-ALA for cancer cell visualization is long-standing topic in the literature. The authors should clearly describe and highlight the novelty of their results. For instance, describe what is new in this manuscript compared to the following papers:
“Enlarging the Scope of 5-Aminolevulinic Acid-Mediated Photodiagnosis towards Breast Cancers”, https://doi.org/10.3390%2Fijms232314900
“5-ALA induced fluorescence imaging for margin status identification during breast conserving surgery”, https://doi.org/10.1016/j.ejso.2023.03.019
“Intraoperative fluorescence imaging with aminolevulinic acid detects grossly occult breast cancer: a phase II randomized controlled trial”, https://doi.org/10.1186%2Fs13058-021-01442-7
Minor points.
The authors used “a sensitive homemade spectrometer”. I believe that the word homemade is not suitable here; probably “custom” or “specially designed” is better. It is also unclear how the integration time of 100µs to 10s is implemented with continuous laser?
The word “photodynamic” usually refers to a photodynamic effect which is a formation of reactive oxygen species and not fluorescence. Therefore, I recommend to remove this word from the title.
Figure 1: there are two rows of tubes, top row (colorless) and bottom row (pink). What do they correspond to? Also, I suggest to remove λ_exc because it refers to porphyrin, not the 5-ALA itself.
Figure 2: too many significant digits are given for minima/maxima locations. Could the authors also plot the fluorescence spectra of clean medium (no cells)?
Table 1: the meaning of data should be specified in the caption and probably in the main text.
Figure 6: why the spectra are different in shape for cells without 5-ALA in different samples?
“After incubation, the medium was removed and replaced with a fresh FBS.” Why FBS was used for resuspension?
" … have been cultured with six different numbers of cells of 50,000, 75,000, 100,000, 112 150,000, 200,000, and 300,000” – the counting method should be indicated. How many HFFs was in these samples?
Comments on the Quality of English Language
There are a lot of sentences which sound incomplete or grammatically incorrect. Some words are not appropriate. The manuscript should be carefully read and corrected.
There are a lot of undefined abbreviations. They should be defined prior to first usage.
Author Response
General comment: [The manuscript “Fluorescence-enhanced photodynamic assessments for human breast cancer cell characterizations” is devoted to the experimental study of fluorescence of cancer and normal cell samples.]
Comment 1: [Unfortunately, objectives, hypothesis, and conclusions are not clearly stated in this manuscript. As far as I understood, the ultimate goal was to use the fluorescence-based technique in diagnostics to differentiate between samples containing normal and cancerous cells. If so, a diagnostic procedure should be proposed and its validity should be evaluated. If the goal of the paper is different, it should be clearly stated in the abstract and introduction, and the corresponding conclusions should be made. While the manuscript in its current state reports several interesting experiments, their design and meaning are obscure.]
Response 1: [Thank you for pointing this out.
Regarding not addressing as clearly and accurately as possible objectives, hypotheses, and conclusions and not clearly stating those, we apologize. To eliminate these points and complete the explanations given in the article, the following sentences have been revised and completed.
Actually, in our study, the primary focus was on cellular characterization and monitoring using a fluorescence-based approach while diagnostics to differentiate between samples containing normal and cancerous cells especially at early stages, as you truly mentioned. To this, studying different cell numbers was to understand the relationship between cell concentration and fluorescence emission. We could investigate how the fluorescence signal changes with increasing or decreasing cell concentration by varying the number of cells from 50,000 to 300,000. This information can be valuable in determining the optimal cell concentration for diagnostic purposes or assessing the sensitivity of the fluorescence-based technique. Additionally, studying different cell numbers can provide insights into the behavior of cancerous cells at various stages of the disease. For example, comparing the fluorescence characteristics of breast cancer cells (MDAs) at primary (lower cell numbers) and metastatic (higher cell numbers) stages may help in understanding the progression of the disease and evaluating the effectiveness of diagnostic approaches.
In this work, we aimed to demonstrate the potential of this approach in distinguishing between cancerous and noncancerous breast cells based on spectral shifts, fluorescence intensity differences, and metabolic changes associated with tumor initiation and progression. Our objective was to assess the overall characteristic and detection capability of the fluorescence-based technique in characterizing breast cancer cells. Also, the evaluation of fluorescence technique for breast cancer cell characterizations using photodynamic assessments, to assess the impact of 5-ALA prodrug on cancer cell characterization and Investigation of the metabolic changes and viability of breast cancer cells could be considered among our goals.
In the abstract, it has been mentioned that "The primary and metastatic stages of BC consider different cancerous cell lines (MDAs), meaning the highest number of cells in this research (up to 300,000) represents the metastatic stages of BC, and 50,000 represents the primary level of development have been studied based on fluorescence-enhanced photodynamic characterizations.”. In continuation, it is stated in the last part of the introduction that “MDA’s different number of cells (50,000-300,000) varies fluorescence emission according to their metastatic potential." again means that 300,000 highest number of cells as the main factor causes a stronger fluorescence emission. In contrast, 50,000 cells represent a weak emission.
To categorize the content more precisely, it should be said that studying fluorescence-based approaches in photodynamic assessments for breast cancer cell characterizations as well as utilizing 5-aminolevulinic acid (5-ALA) to characterize biomarkers and metabolic changes are considered under the hypotheses of this study. Also, considering the importance of fluorescence techniques in Breast Cancer (BC) research, the efficacy of sodium fluorescein in enhancing spectral visualization, the potential of fluorescence imaging for tumor detection, and advancements in photodynamic therapy for Breast Cancer (BC) are primary conclusions which are discussed in detail in the article.
Once more we appreciate your insight and would like to clarify further objectives, hypothesis, and conclusions of our manuscript by addressing the statement “The primary objectives of this research highlight the high potential of fluorescence-based approaches for accurate discrimination and characterizations between cancerous and noncancerous cells based on biological changes associated with tumor initiation and progression. “added to the beginning of the "conclusion" section. Also, the phrase “It was believed that fluorescence techniques hold promise for timely breast cancer detection, with lipid metabolism enzyme changes correlating to MDA cell viability. Both 5-ALA and FS impact breast cancer cell characterization and detection.” has been added and revised as a finalization to the last part of the conclusion.
We apologize for any lack of clarity in the manuscript. About evaluating this approach's validity by comparing results from fluorescence-based assessments with established diagnostic methods, it should be said that you are right, but since up to this stage the time and the budget allocated to this project is almost over and the possibility of comparing the results with other researches such as UV-Visible spectroscopy or other methods will be definitely from our main future goals. Therefore, this issue will be studied and investigated by our team in the future, and it will be one of the important future goals for our research team, which also will be addressed more carefully in the future.]
Comment 2: [The usage of porphyrins and 5-ALA for cancer cell visualization is long-standing topic in the literature. The authors should clearly describe and highlight the novelty of their results. For instance, describe what is new in this manuscript compared to the following papers:
*“Enlarging the Scope of 5-Aminolevulinic Acid-Mediated Photo-diagnosis towards Breast Cancers”, https://doi.org/10.3390%2Fijms232314900
*“5-ALA induced fluorescence imaging for margin status identification during breast conserving surgery”, https://doi.org/10.1016/j.ejso.2023.03.019
*“Intraoperative fluorescence imaging with aminolevulinic acid detects grossly occult breast cancer: a phase II randomized controlled trial”, https://doi.org/10.1186%2Fs13058-021-01442-7]
Response 2: [Thank you for your comment. We tried to correct all your mentioned points, properly.
We acknowledge the key novelties of our scientific research entitled “Fluorescence-enhanced photodynamic assessments for human breast cancer cell characterizations” have been mainly focused on;
- Improving diagnostic techniques for breast cancer aims to reduce mortality rates and minimize early and late side effects, especially in the early stages of the disease.
- It explores the use of fluorescence-based techniques to study breast cancer cells, highlighting the practical and technical aspects, pathophysiological mechanisms, and limitations of this approach.
- The study utilizes a fluorescence-based technique involving sodium fluorescein as a fluorescent dye to visualize spectra variability in breast cancer cells, demonstrating the potential of this method for further implications.
- By developing early detection methods using fluorescence-based techniques, the research aims to provide non-invasive approaches for high-resolution imaging, molecular analysis, and detection of spectral differences in breast tissue.
Also, to answer the question of " what is new in our reported manuscript compared to the mentioned papers " first of all, thank you for sharing such valuable resources. Unfortunately, since I did not have access to one of the articles entitled “5-ALA induced fluorescence imaging for margin status identification during breast-conserving surgery”, it was not possible for me to check its concepts and make a comparison with this study while a bout those two others it has to be considered that, as we mentioned before while our manuscript explores the use of fluorescence-based photodynamic assessments to characterize human breast cancer cells, focusing on spectral shifts, fluorescence intensity differences, and metabolic changes for cell characterization, the "Intraoperative fluorescence imaging with aminolevulinic acid detects grossly occult breast cancer" article also delves into the impact of 5-ALA on cancer cell characterization, photodynamic processes, viability, and morphology, providing detailed insights into breast cancer physiology. In contrast to the study "Enlarging the Scope of 5-Aminolevulinic Acid-Mediated Photodiagnosis towards Breast Cancers", the manuscript specifically investigates the technique's potential in distinguishing between cancerous and noncancerous cells based on spectral shifts, fluorescence intensity differences, and metabolic changes, offering a unique perspective on oncology research.
It should be noted that to complete the results of our research article, both articles “Enlarging the Scope of 5-Aminolevulinic Acid-Mediated Photo-diagnosis towards Breast Cancers” and “Intraoperative Fluorescence Imaging with aminolevulinic acid detects grossly occult breast cancer: a phase II randomized controlled trial” have been used correctly and are cited in the references section, by addressing the statement of “Current fluorescence-based approaches, particularly using probes like 5-aminolevulinic acid (5-ALA), are crucial for accurately characterizing cancerous and noncancerous cells in breast cancer research, providing insights into metabolic changes and cancer progression. Fluorescence considerations with 5-ALA have shown high accuracy in surveying cancer cells, even in early stages, highlighting the importance of sensitivity in distinguishing between cancerous and normal cells. Using fluorescence imaging with specific probes like 5-ALA enhances the visualization of tumors contrasted against normal tissue autofluorescence, emphasizing the significance of sensitivity in these diagnostic approaches” to the ending part if the “Multivariate cell lines analysis” subsection, properly.]
Comment 3: [The authors used “a sensitive homemade spectrometer”. I believe that the word homemade is not suitable here; probably “custom” or “specially designed” is better. It is also unclear how the integration time of 100µs to 10s is implemented with continuous laser?]
Response 3: [Thank you for your valuable comment. We tried to rewrite the sentence and used the phrase “custom-designed” instead of " homemade ", as you truly mentioned.
Also considering another valuable point of view “how the integration time of 100µs to 10s is implemented with continuous laser?” better to say that, the integration time of 100µs to 10s with continuous laser is achieved by adjusting the exposure time of the detector to capture the fluorescence signal over varying durations. This allows for capturing fluorescence signals at different time intervals during continuous laser excitation, providing insights into the dynamic changes in fluorescence intensity. The implementation involves setting the detector to record fluorescence signals at specific intervals within the 100µs to 10s range while the continuous laser excitation is maintained. This approach enables the assessment of fluorescence characteristics over a range of integration times, contributing to a comprehensive analysis of the fluorescence-enhanced photodynamic assessments. By modulating the detector's exposure time during continuous laser excitation, the integration time range of 100µs to 10s can be effectively utilized to capture fluorescence data at different time points, allowing for a detailed examination of fluorescence properties in human breast cancer cell characterizations.]
Comment 4: [The word “photodynamic” usually refers to a photodynamic effect which is a formation of reactive oxygen species and not fluorescence. Therefore, I recommend to remove this word from the title.]
Response 4: [Thank you for your comment. The title has been revised accordingly as you mentioned.]
Comment 5: [Figure 1: there are two rows of tubes, top row (colorless) and bottom row (pink). What do they correspond to? Also, I suggest to remove λ_exc because it refers to porphyrin, not the 5-ALA itself.]
Response 5: [Thank you for pointing this out.
The top Eppendorf microtubes which are colorless refer to MDAs and HFF cells that have been cultured to inhibit the proliferation, metastasis, and invasion of breast cell lines as well as normal cells (HFF) as have been described through the manuscript. The bottom Eppendorf tubes (pink) show PBSs. As you mentioned, it has been labeled the two rows of micro tubes in the caption of the image as you commented, properly.
Also, regarding your mentioned point of λ_exc, while you are right, it was removed.]
Comment 6: [Figure 2: too many significant digits are given for minima/maxima locations. Could the authors also plot the fluorescence spectra of clean medium (no cells)?]
Response 6: [Since the results related to the clean medium were only related to the remnants of low-radiation lights that entered the environment, which we removed using the necessary subtractions via the spectrophotometer, and it did not contain any special information for us, unfortunately, there is no data storage. We apologize for saying that we did not have any data regarding the empty medium!]
Comment 7: [Table 1: the meaning of data should be specified in the caption and probably in the main text.]
Response 7: [Thank you for bringing that to my attention. You are right. As mentioned, the sentence is revised and stated correctly both in the main text and caption as below;
“The data presented in Table 1 are obtained from Fig. 2.”]
Comment 8: [Figure 6: why the spectra are different in shape for cells without 5-ALA in different samples?]
Response 8: [These differences could be due to variations in fluorescence intensity, which can be influenced by factors such as cell concentration and metabolic activity. The differences in spectral shape also could be attributed to variations in cellular morphology among the samples without 5-ALA, affecting the fluorescence characteristics observed in the spectra. There is also another reason that cells without 5-ALA lack the accumulation of protoporphyrin IX (PpIX), which is a key photosensitizer that contributes to the fluorescence emission observed in the spectra of samples treated with 5-ALA, which may also be influenced by the excitation wavelength used, as different wavelengths can interact differently with the cellular components, leading to variations in the fluorescence spectra observed. ]
Comment 9: [“After incubation, the medium was removed and replaced with a fresh FBS.” Why FBS was used for resuspension?]
Response 9: [The use of FBS for resuspension post-incubation could be considered as a practical choice based on its traditional role in cell culture (also based on the opinions of our biology expert colleague based on the studies and information she took from the technical reports).]
Comment 10: [" … have been cultured with six different numbers of cells of 50,000, 75,000, 100,000, 112 150,000, 200,000, and 300,000” – the counting method should be indicated. How many HFFs was in these samples?]
Response 10: [According to our biologist colleague's statements, the method they typically use for cell counting involves a Hemocytometer, which manually counts cells using light microscopy. The number of HFF cells matches those of 50,000, 315, 75,000, 100,000, 150,000, 200,000, and 300,000 cell numbers that have been employed for MDAs, during a similar culture process.]
Comment 11: [There are a lot of sentences which sound incomplete or grammatically incorrect. Some words are not appropriate. The manuscript should be carefully read and corrected. There are a lot of undefined abbreviations. They should be defined prior to first usage.]
Response 11: [We went through the entire manuscript to eliminate grammatical mistakes and undefined abbreviations, as mentioned.]
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors1- Please include in the text more details about the used CW 405 nm diode laser source. Also, please specify why the 405 nm laser was used as excitation light source.
2- Please include in the text a brief sketch that describes the home made spectrometer.
3- Please add to the text the used laser power for the excitation.
4- Please discuss in the text the sensitivity of the present fluorescence-based approaches.
5- The authors should explain the novelty of this work and its differences from earlier studies.
6- Nothing is mentioned in the text about the uncertainty in the experimental results, so please discuss this issue in the text.
7- In addition to the References list, other fluorescence-based approaches on the are proposed to be reviewed and included if they are beneficial:
Ø Sci Rep 11, 16864 (2021). https://doi.org/10.1038/s41598-021-96531-0
Ø Opt Quant Electron 56, 977 (2024). https://doi.org/10.1007/s11082-024-06781-1
Ø Spectrochimica acta. Part A, Molecular and Biomolecular Spectroscopy. 2024 Jan;305:123491. DOI: 10.1016/j.saa.2023.123491
Ø
Ø Journal of Photochemistry and photobiology. B, Biology. 2023 Mar;240:112665. DOI: 10.1016/j.jphotobiol.2023.112665.
Comments on the Quality of English Language
Moderate editing of English language required
Author Response
Comment 1: [Please include in the text more details about the used CW 405 nm diode laser source. Also, please specify why the 405 nm laser was used as excitation light source.]
Response 1: [Thank you for your comments and feedback on our work.
Regarding to add more details about the used CW 405 nm diode laser, we tried to reconsider that by addressing the statement that “The samples were exposed at the 405 nm continuous wavelength of the CNI laser diode with a power density of 15 W/cm2 (excitation power), less than 0.5 mm square, offering almost a high efficiency and lifetime compared to lamp sources”, in the main body text.
To specify why the 405 nm laser was used as an excitation light source, based on the research papers provided, a 405 nm continuous wave (CW) laser source is efficient for characterizing breast cancerous cells due to its ability to excite predominant native fluorophores like NADH and FAD, which show significant spectral discrepancies in the 420-530 nm range, aiding in maximizing cancer discrimination.
Furthermore, fluorescence-based optical techniques, particularly utilizing a 405 nm laser, show high sensitivity and good repeatability in detecting breast cancer cells, offering a cost-effective and reliable method for timely cancer detection and characterization [3]. Overall, the 405 nm laser technology proves to be instrumental in advancing the understanding and diagnosis of breast cancer through its precision, sensitivity, and cost-effectiveness(1–3).
Also, in some other reports it has been mentioned that;
The advantages of using a continuous wave (CW) 405 nm laser for cancer cell characterizations compared to other wavelengths lie in its effectiveness in photodynamic treatments and cell analysis. Research by Etcheverry et al. demonstrates that a 405 nm laser is significantly more effective in inducing cell death in cancer cells compared to red light. Additionally, Gong et al. introduce hybrid liquid crystal microlaser resonators for label-free laser emission imaging of secreted molecules associated with cell-environment interactions, showcasing the potential of laser technologies in biological studies. Furthermore, Starikova highlights the importance of optical spectroscopic imaging in medical diagnosis, emphasizing the need for techniques like resonance Raman spectroscopy for clear and effective spectral discrimination between healthy and malignant tissues, which can aid in cancer identification and real-time tissue discrimination during surgery(4,5). These findings collectively underscore the advantages of utilizing a CW 405 nm laser for precise and efficient cancer cell characterizations.]
Comment 2: [Please include in the text a brief sketch that describes the home-made spectrometer.]
Response 2: [Since this spectrometer was purchased and prepared by one of the domestic companies in the country, unfortunately, we do not have any specifications or schematics of its structure, however, the initial images of this spectrophotometer are provided along with the company's link of https://phystec.ir/product/%d8%b7%db%8c%d9%81-%d8%b3%d9%86%d8%ac-uv-vis-nir/]
Comment 3: [Please add to the text the used laser power for the excitation.]
Response 3: [Thank you very much for pointing this out.
The excited laser power at 405 nm wavelength light was measured around a power density of 15 W/cm2 (excitation power) for 10 min which has been mentioned in the main text body, as you mentioned.]
Comment 4: [Please discuss in the text the sensitivity of the present fluorescence-based approaches.]
Response 4: [Thank you for your comment.
As we discussed in our research article, the sensitivity of the current fluorescence-based approach is crucial due to its ability to accurately discriminate and characterize cancerous and noncancerous cells, particularly in breast cancer research. By utilizing probes like 5-aminolevulinic acid (5-ALA), this approach can effectively characterize metabolic changes and provide insights into functional and morphological alterations associated with cancer progression, as we mentioned in our paper. Fluorescence imaging, especially with the use of 5-ALA, has shown high accuracy in detecting cancer cells, even in early investigations, showcasing the importance of sensitivity in distinguishing between cancerous and normal cells. Additionally, the use of fluorescence imaging with specific probes like 5-ALA has demonstrated improved visualization of tumors contrasted against normal tissue autofluorescence, further emphasizing the significance of sensitivity in these approaches (6).
We added the term of “Current fluorescence-based approaches, particularly using probes like 5-aminolevulinic acid (5-ALA), are crucial for accurately characterizing cancerous and noncancerous cells in breast cancer research, providing insights into metabolic changes and cancer progression. Fluorescence considerations with 5-ALA has shown high accuracy in surveying cancer cells, even in early stages, highlighting the importance of sensitivity in distinguishing between cancerous and normal cells. The use of fluorescence imaging with specific probes like 5-ALA enhances visualization of tumor contrasted against normal tissue autofluorescence, emphasizing the significance of sensitivity in these diagnostic approaches.” in the explanation of the mentioned comment to the finishing part of the Results and discussion section.]
Comment 5: [The authors should explain the novelty of this work and its differences from earlier studies. ]
Response 5: [Thank you for bringing that to our attention.
Considering the novelty of this work comparing differences in earlier studies shows that;
The current research stands out from earlier studies by employing probes like 5-aminolevulinic acid (5-ALA) to enhance fluorescence-based approaches, enabling precise characterization of cancerous and noncancerous cells in breast cancer research. In contrast to previous studies, the current work emphasizes the importance of sensitivity in fluorescence-based techniques for the timely detection of breast cancer, especially before it spreads to axillary lymph nodes. This also focuses on utilizing fluorescence-based techniques with specific probes to achieve clear separation between different cell types, such as HFFs and MDAs, indicating reproducibility and distinct spectral characteristics. The current research highlights the significance of rapid measurements to prevent cell destruction, ensuring reliable data collection with minimal errors, a practical consideration not extensively addressed in earlier studies.
Therefore, as you mentioned the term “Recent studies have introduced innovative fluorescence imaging techniques for analyzing breast cancer cells, enabling enhanced visualization and characterization of cellular features. Advancements in fluorescence spectroscopy methods have revolutionized oncology research, offering high sensitivity and specificity in detecting cancer biomarkers and metabolic changes in cells” This has been added to the main body of the text, almost ending part of the “introduction” section. ]
Comment 6: [Nothing is mentioned in the text about the uncertainty in the experimental results, so please discuss this issue in the text.]
Response 6: [Thank you for pointing this out. As you mentioned the statement “Experimental results in fluorescence-based studies may exhibit uncertainty due to factors like sample preparation variations, environmental conditions, and spectral variability. The pH sensitivity of fluorescent proteins and reproducibility of fluorescence spectra can also contribute to measurement uncertainty. Standardized data collection protocols are crucial to minimize errors and enhance reliability.” has been added to the “Multivariate cell lines analysis “subsection part, properly.]
Comment 7: [In addition to the References list, other fluorescence-based approaches on the are proposed to be reviewed and included if they are beneficial;
- Sci Rep 11, 16864 (2021). https://doi.org/10.1038/s41598-021-96531-0
- Opt Quant Electron 56, 977 (2024). https://doi.org/10.1007/s11082-024-06781-1
- Spectrochimica acta. Part A, Molecular and Biomolecular Spectroscopy. 2024 Jan;305:123491. DOI: 10.1016/j.saa.2023.123491
- Journal of Photochemistry and photobiology. B, Biology. 2023 Mar;240:112665. DOI: 10.1016/j.jphotobiol.2023.112665.]
Response 7: [Thank you for your valuable comments.
I would also like to thank you for suggesting these titles to improve the quality of our manuscript.
We used the article titled” Real-time cancer diagnosis of breast cancer using fluorescence lifetime endoscopy based on the pH by Lee et.al,” because of the valuable explanations it provided in the field “the idea of a novel technique discriminating between tumor and normal tissues using fluorescence lifetime endoscopy (FLE)” in our work.
We also mentioned the other reports such as” Femtosecond laser-induced fluorescence spectroscopy for the rapid detection of pathogenic bacteria” by Ezzat et, al. due to its laser-induced fluorescence spectroscopy rapid detection considerations, as well as “Femtosecond laser-induced fluorescence for rapid monitoring of cardiac troponin 1 as a cardiovascular disease biomarker” again for its rapid detection considerations, in our scientific reports.]
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsAfter reviewing the paper titled "Fluorescence-enhanced photodynamic assessments for human breast cancer cell characterizations," I have several comments and questions for the authors.
1. It would be beneficial to include more recent references to highlight the latest advancements in this field.
2. What was the rationale behind selecting 50,000 and 300,000 cells for the fluorescence measurements? Were these numbers chosen based on preliminary experiments or literature references?
3. Can you provide more details on how the 100 µM concentration of 5-ALA and the 24-hour incubation time were determined? Were other concentrations and incubation times tested?
4. How does the sensitivity of the fluorescence-based technique compare with other established methods for early breast cancer detection? Have there been any direct comparisons?
5. The study mentions significant changes in cellular morphology after 5-ALA treatment. Could you provide more specific examples or images illustrating these morphological changes?
6. What are the potential clinical applications of your findings? How could this fluorescence-based approach be integrated into existing diagnostic workflows for breast cancer?
7. Overall, I suggest the author can check the manuscript for grammatical and writing issues.
Comments on the Quality of English LanguageThere manuscript owned grammatical issue and some weak explanations and might creat ambiguity to the readers.
Author Response
General comment: [After reviewing the paper titled "Fluorescence-enhanced photodynamic assessments for human breast cancer cell characterizations," I have several comments and questions for the authors.]
Response: [Thank you for your comments and feedback on our work. We appreciate your time and effort in reviewing the paper.]
Comment 1: [It would be beneficial to include more recent references to highlight the latest advancements in this field.]
Response 1: [Thank you for bringing that to my attention.
The statement “Recent studies have introduced innovative fluorescence imaging techniques for analyzing breast cancer cells, enabling enhanced visualization and characterization of cellular features. Advancements in fluorescence spectroscopy methods have revolutionized oncology research, offering high sensitivity and specificity in detecting cancer biomarkers and metabolic changes in cells.” has been added to the introduction part. ]
Comment 2: [What was the rationale behind selecting 50,000 and 300,000 cells for the fluorescence measurements? Were these numbers chosen based on preliminary experiments or literature references?]
Response 2: [Thank you for raising this question.
We want to clarify that the selection of these specific cell numbers was primarily based on the cultivation situation and the recommendations of our collaborating biologists. It is important to note that the determination of cell numbers for in vitro experiments is often arbitrary to some extent, and it is not uncommon to choose values within a range supposedly appropriate for studying the desired biological phenomena.
While there may not be a direct scientific record justifying the exact values of 50,000 and 300,000 cells, it is common practice to select cell numbers representing different stages of cancer progression is standard practice. These values were considered to encompass the primary and metastatic stages of breast cancer based on existing knowledge about the growth patterns and cellular characteristics of breast cancer cells.
The significance of comparing the outcomes between 50,000 and 300,000 cells lies in assessing the diagnostic capability of the fluorescence technique across different stages of breast cancer. Although the specific cell numbers were chosen arbitrarily, the comparison allows us to evaluate the potential of the fluorescence-based approach in detecting and distinguishing between cancerous cells at early and advanced stages.
It has been stated that "The specific cell numbers of 50,000 and 300,000 were chosen arbitrarily based on the cultivation situation and recommendations from collaborating biologists by common biological limitations. Comparing the outcomes between 50,000 and 300,000 cells allows for evaluating the fluorescence technique's diagnostic capability across various stages of breast cancer. This assessment provides valuable insights into the effectiveness of the fluorescence-based approach in detecting and distinguishing between cancerous cells at different stages of the disease." for more clarification to the Abstract of the main body of the text, for more considerations.]
Comment 3: [Can you provide more details on how the 100 µM concentration of 5-ALA and the 24-hour incubation time were determined? Were other concentrations and incubation times tested?]
Response 3: [There was a research article entitled “Small molecule additive enhances cell uptake of 5-aminolevulinic acid and conversion to protoporphyrin IX” by Harmatys et.al, 2016. Photochem Photobiol Sci. 2016 November 2; 15(11): 1408–1416. doi:10.1039/c6pp00151c, which has described the details of these preparations and the results achieved. Also, according to the consultations we had with our biology expert colleague, we used this article as a model, while our colleague added some points according to the laboratory conditions and requirements. There are also some other reports that if needed I could share those as well.
In reply to the mentioned points of considering the other concentrations and incubation times, I should say “yes” of course, for example, we faced some issues due to not enough incubation times about 6, 8, or even 12 h or the inconsistency of different concentrations, until finally, we got better results by studying this particular article “doi:10.1039/c6pp00151c” and operationalizing it.]
Comment 4: [How does the sensitivity of the fluorescence-based technique compare with other established methods for early breast cancer detection? Have there been any direct comparisons?]
Response 4: [Unfortunately, due to the limitation of some resources and time, also since still we were not sure enough about the supplementary methods provided in the published articles and reports, we have not yet succeeded in validating the results (sensitivity of our proposed approach) using other proposed methods, and in the meantime, we only rely on the results of similar articles and technical reports. Certainly, in the future, the main part of our goals will include validation of the sensitivity of the presented approach based on other standard methods.]
Comment 5: [The study mentions significant changes in cellular morphology after 5-ALA treatment. Could you provide more specific examples or images illustrating these morphological changes?]
Response 5: [In fact, it should be noted that cultured cells pose challenges due to their limited lifespan, poor stability, and viability. These limitations make it difficult for us to access these cells for our final tests and investigations, preventing us from obtaining a large number of images.]
Comment 6: [What are the potential clinical applications of your findings? How could this fluorescence-based approach be integrated into existing diagnostic workflows for breast cancer?]
Response 6: [Thank you for pointing this out.
It should be mentioned that “Incorporating fluorescence-based considerations significantly enhances clinical examinations, particularly when conducted by specialized clinicians. These techniques effectively characterize breast cancer cells, facilitating pre- and post-clinical assessments. During surgical procedures, fluorescence imaging provides real-time visualization of tumor margins and lymph nodes due to its inherent sensitivity. It guides precise tissue sampling and monitors therapeutic interventions. The ability to discriminate between cancerous and normal cells, coupled with minimal photodamage and rapid response, underscores the clinical advantages of fluorescence-based approaches.]
Comment 7: [Overall, I suggest the author can check the manuscript for grammatical and writing issues.]
Response 7: [We went through the entire manuscript to eliminate any grammatical and syntax mistakes.]
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors answered my questions to some extent.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have satisfactorily revised the manuscript in response to my earlier remarks and concerns. In my opinion, the manuscript contains now all the information and is suitable for publication in Photonics.
Comments on the Quality of English LanguageModerate editing of English language required
