Anticancer Efficacy of Long-Term Stored Plasma-Activated Medium
Round 1
Reviewer 1 Report
The authors present clear data on a very important subject of plasma medicine. Stability of frozen plasma-activated medium over long time is an important issue for future applications. The authors measure essential parameters, such as induction of apoptosis, cell viability and the concentrations of hydrogen peroxide as well as nitrite. The experiments are carefully performed, well-controlled and clearly presented. I suggest several modifications to further improve the significance of this paper.
1) Quantitative aspect: The tests have been performed with undiluted plasma-activated medium and it has been shown that the biological effects were unchanged after six months storage of frozen plasma-activated medium. This is important and the central aspect of this paper. However, a quantitative analysis seems to be required. This is to exclude that undiluted activated medium contained the essential compounds in excess and thererfore any decrease in their content over time was not detectable. I suggest to perform titration of fresh activated medium and stored activated medium and to determine the biological effects in a quantitative way.
2) The authors utilize the Griess reaction to determine "production of RNS" (line 91). In Figure 6 b and in line 188 they speak of "increase in NO". Correction is needed and it should be stated that nitrite concentration was determined. It might then discussed that nitrite might have derived from NO. However, it is not likely that there is still NO in the stored medium.
3) The authors use the term "liquid plasma" in their manuscript. This is linguistically and scientifically unprecise, as this term does not indicate that the activated medium contains compounds that originate from the interaction between plasma compounds from the gas phase with the liquid. I suggest to use the term "plasma-activated medium" , as this term is used by the majority of researchers in this field.
4) Introduction, line 50: " we adopted liquid treatment..": The authors should refer to previous reports on the biological effects of plasma-activted medium. Central papers are:
H. Tanaka, K. Ishikawa, K. Nakamura, H. Kajiyama, H. Komo, T. Kikkawa and M. Hori, “Plasma-activated medium selectively kills glioblastoma brain tumor cells by down-regulating a survival signaling molecule, AKT kinase”, Plasma Med, vol. 1, pp. 265-277; doi: 10.1615/PlasmaMed.2012006275, 2011.
T. Adachi, H. Tanaka, S. Nonomura, H. Hara, S.-I. Kondo and M. Hori, “Plasma-activated medium induces A459 cell injury via a spiral apoptotic cascade involving the mitochondrial-nuclear network”, Free Radic Biol Med, vol. 79, pp. 28-44, 2015.
Kurake, H. Tanaka, K. Ishikawa, T. Kondo, M. Sekine, K. Nakamura, H. Kajiyama, F. Kikkawa, M. Mizuno and M. Hori, “Cell survival of glioblastoma grown in medium containing hydrogen peroxide and/or nitrite, or in plasma-activated medium”, Arch Biochem Biophys, , vol. 605, pp. 102-108, 2016.
P.-M. Girard, A. Arbabian, M. Fleury, G. Bauville, V. Puech, M. Dutreix and J. S. Sousa, “Synergistic effect of H2O2 and NO2─ in cell death induced by cold atmospheric He plasma”, Sci Rep, vol. 6, pp. 29098; doi: 10.1038/srep29098, 2016.
Canal C, Fontelo R, Hamouda I, Guillem-Marti J, Cvelbar U, Ginebra M-P. Plasma-induced selectivity in bone cancer cell death. Free Radic Biol & Medicine 110: 72-80, 2017
Sato Y, Yamada S, Takeda S, Hattori N, Nakamura K, Tanaka H, Mizuno M, Hori M, Kodera Y. Effect of plasma-activated lactated Ringer`s solution on pancreatic cancer cells in vitro and in vivo. Annnals of Surcical Oncology 25: 299-307, 2018
D. Yan, A. Talbot, N. Nourmokammadi, X. Cheng, J. Canady, J. Sherman and M. Keidar, “Principles of using cold atmospheric plasma stimulated media for cancer treatment”, Sci Rep, vol. 5, pp. 1833901- 18339017; doi: 10.1038/srep18339, 2015.
S. Mohades, M. Laroussi, J. Sears, N. Barekzi and H. Razavi, “Evaluation of the effects of a plasma-activated medium on cancer cells”, Phys Plasmas, vol. 22, pp. 122001, 2015.
5) line 30: "ability of plasma to selectively eliminate" : though most researchers in the field support the concept of selective antitumor activity of plasma and plasma-activated medium, a few reports disagree (for example: A. M. Hirst, M. S. Simms, V. M. Mann, N. J. Maitland, D. O`Connell D and F. M. Frame, “Low temperature plasma treatment induces DNA damage leading to necrotic cell death in primary prostate epithelial cells”, Br J Cancer, vol. 112, pp. 1536-1545, 2015. DOI: 10.1038/bjc.2025.113). The reason for nonselective action may be related to a dose problem, i. e. plasma and plasma-activated medium are probably selective in a defined dose range and not above.
6) The authors are encouraged to discuss their findings on the contents of the activated medium with the results obtained by Kurake et al, 2016 and Girard et al., 2016 (see above).
7) Introduction, line 25: " ....and various radicals": The description of the compounds found in plasma should be corrected/extended, as plasma (or its liquid phase) also contains nonradical reactive oxygen species (like H2O2, singlet oxygen, ozone, hypochloride) and nonradical reactive nitrogen species such as nitrite, nitrate, peroxynitrite, peroxynitrate ) in addition to radical species.
Author Response
Response to Reviewer 1 Comments
The authors present clear data on a very important subject of plasma medicine. Stability of frozen plasma-activated medium over long time is an important issue for future applications. The authors measure essential parameters, such as induction of apoptosis, cell viability and the concentrations of hydrogen peroxide as well as nitrite. The experiments are carefully performed, well-controlled and clearly presented. I suggest several modifications to further improve the significance of this paper.
1) Quantitative aspect: The tests have been performed with undiluted plasma-activated medium and it has been shown that the biological effects were unchanged after six months storage of frozen plasma-activated medium. This is important and the central aspect of this paper. However, a quantitative analysis seems to be required. This is to exclude that undiluted activated medium contained the essential compounds in excess and therefore any decrease in their content over time was not detectable. I suggest to perform titration of fresh activated medium and stored activated medium and to determine the biological effects in a quantitative way.
Response 1): According to your suggestion, we have diluted the fresh and stored plasma-activated medium and evaluated the anticancer effects of the diluted activated medium to examine whether essential components in the plasma-activated medium are decreased over time. The anticancer effects fresh and stored plasma-activated medium (PAM) diluted by 1/2 and 1/4 were comparable to those of non-diluted PAM (Figure 6). At greater dilutions, 10-fold diluted fresh and stored medium exhibited the anticancer effects, indicating that its anticancer effects range from 1/10 to 1/4 of fresh and stored PAM. Taken together, these observations indicate that the components essential for the anticancer effects in the stored PAM do not decrease over time.
2) The authors utilize the Griess reaction to determine "production of RNS" (line 91). In Figure 6 b and in line 188 they speak of "increase in NO". Correction is needed and it should be stated that nitrite concentration was determined. It might then discussed that nitrite might have derived from NO. However, it is not likely that there is still NO in the stored medium.
Response 2): We have corrected NO to nitrite and nitrate in Figure 6b and the text in the revised manuscript.
3) The authors use the term "liquid plasma" in their manuscript. This is linguistically and scientifically unprecise, as this term does not indicate that the activated medium contains compounds that originate from the interaction between plasma compounds from the gas phase with the liquid. I suggest to use the term "plasma-activated medium" , as this term is used by the majority of researchers in this field.
Response 3): We have modified the term "liquid plasma” to "plasma-activated medium" throughout the manuscript. As mentioned above, all modifications including this term are indicated in red font.
4) Introduction, line 50: " we adopted liquid treatment..": The authors should refer to previous reports on the biological effects of plasma-activted medium. Central papers are:
Response 4): We refer to 4 additional current reports (references 20–23) of plasma-activated medium based on your recommendations.
5) line 30: "ability of plasma to selectively eliminate" : though most researchers in the field support the concept of selective antitumor activity of plasma and plasma-activated medium, a few reports disagree (for example: A. M. Hirst, M. S. Simms, V. M. Mann, N. J. Maitland, D. O`Connell D and F. M. Frame, “Low temperature plasma treatment induces DNA damage leading to necrotic cell death in primary prostate epithelial cells”, Br J Cancer, vol. 112, pp. 1536-1545, 2015. DOI: 10.1038/bjc.2025.113). The reason for nonselective action may be related to a dose problem, i. e. plasma and plasma-activated medium are probably selective in a defined dose range and not above.
Response 5): We agree with your comments. Thus, we have added this point including reference 11.
6) The authors are encouraged to discuss their findings on the contents of the activated medium with the results obtained by Kurake et al, 2016 and Girard et al., 2016 (see above).
Response 6): We have discussed the possible dependency of plasma-activated medium-mediated anticancer effect on H2O2 and nitrite/nitrate including the previous studies as references.
7) Introduction, line 25: " ....and various radicals": The description of the compounds found in plasma should be corrected/extended, as plasma (or its liquid phase) also contains nonradical reactive oxygen species (like H2O2, singlet oxygen, ozone, hypochloride) and nonradical reactive nitrogen species such as nitrite, nitrate, peroxynitrite, peroxynitrate ) in addition to radical species.
Response 7): We appreciate your comments and have added the term “non-radicals”.
Reviewer 2 Report
Author must revise their manuscript majorly according to comments
Comment 1:
In this manuscript, the authors study the anticancer capability of plasma treated liquid after 6 months of storage in frozen condition. The results showed that even after a long storage period, the concentration of RONS in plasma treated liquid still maintained. It explained why the anticancer efficiency remained.
Comment 2:
The mechanism and effect of plasma treated liquid on Hela cervical cell has been reported before, please check the related article “Scientific Report 2016, 7, 45781”.
Comment 3:
The originality of this research is investigating how the anticancer effect of plasma treated liquid maintains after a long storage period. Hence, the title should be revised to enhance the comprehensiveness of the study.
Comment 4:
The term “liquid plasma” mentioned in the text should be substituted by a more precise definition “Plasma-activated Media” (or PAM), which is widely used in the field of plasma medicine.
Author Response
Response to Reviewer 2 Comments
Author must revise their manuscript majorly according to comments
Comment 1:
In this manuscript, the authors study the anticancer capability of plasma treated liquid after 6 months of storage in frozen condition. The results showed that even after a long storage period, the concentration of RONS in plasma treated liquid still maintained. It explained why the anticancer efficiency remained.
Response 1: Our results demonstrate the anticancer effect and its mechanism of stock plasma-activated medium.
Comment 2:
The mechanism and effect of plasma treated liquid on Hela cervical cell has been reported before, please check the related article “Scientific Report 2017, 7, 45781”.
Response 2: We appreciate your comments and have referred to this report.
Comment 3:
The originality of this research is investigating how the anticancer effect of plasma treated liquid maintains after a long storage period. Hence, the title should be revised to enhance the comprehensiveness of the study.
Response 3: We have modified the title as follows: “Anticancer efficacy of long-term stored plasma-activated medium” to better describe the originality and enhance the comprehensiveness of the study description.
Comment 4:
The term “liquid plasma” mentioned in the text should be substituted by a more precise definition “Plasma-activated Media” (or PAM), which is widely used in the field of plasma medicine.
Response 4: We changed the term “liquid plasma” to “plasma-activated medium” throughout the manuscript.
Reviewer 3 Report
I read the paper titled Biomedical applications of liquid plasma etc. with great interest. I have some concerns and several suggestions for improving this paper.
CONCERNS:
Liquid exposed to plasma is NOT liquid plasma. Please remove that term.
The referenced literature only goes to 2015. There have been a large number of publications regarding plasma, cancers and liquids since then. New concepts have been introduced. Even studies with frozen liquids are published. Please review current literature and remove the obsolete references.
This study documents efficacy of plasma exposed DMEM. DMEM is NOT a physiological liquid for potential human (or animal) translation. It is well documented in literature that different liquids will have different RONS compositions and storage outcomes. Why was DMEM selected?
Stability of RONS in liquids begs the question - why do we need the very expensive plasma technology? Hydrogen peroxide costs pennies. Why can't we buy that and prepare appropriate concentrations of solutions in physiological liquids? What does the remote plasma add to this scenario?
SUGGESTIONS:
The introduction is rather haphazard and does not have a logical flow. e.g. how does multi-therapy resistance to available treatments justify further studies on NTP? Please streamline it and use more current references.
Provide more details on fluorescence staining of cells. Was it done in wells? What color are dead cells? All that is seen in these images are live cells? How do you know they were just not detached and removed during washes?
Provide more details on flow cytometry graphs. What does each axis indicate and what quadrant shows what set of cells.
There is no comparison with storage at room temperature or refrigeration. There is no justification for freezing. Please address this issue.
The conclusion part of the discussion needs to be softer.
Author Response
Please find the attached file.
Author Response File: 
Author Response.pdf
Reviewer 4 Report
Specific comments:
For the preparation of liquid plasma and subsequent treatment of cells DMEM cell culture medium was used and this medium was treated with plasma. In 2.4 the authors did not mention any supplementation of the medium. Whereas in subheading 2.6 FCS-supplement was described. This is important since the cells come in contact with it. Please specify the components of the cell culture medium DMEM.
It is not necessary to mention all suppliers once again in the diagram (results section). In the subheading 2.1 the author claim to have used the AmplexRed Kit and later on in the manscript they used AmplexUlraRed. Which assay was used for H2O2 detection, AmplexRed or AmplexUltraRed?
The method used of counting the cell viability is not explained. There are many cells very close to each other which I recon makes it impossible to count them under a microscope.
The result in Fig 3 shows a spectrum of OES. Was this taken with a liquid target involved in the system or without? Does the spectrum change if a liquid is present? In your setup this could give some important hints to the species emerging from the plasma. I suggest the authors give some more explanation to the spectrum and its implications for the current study to better relate it to the current investigation.
Line 125/126 reads ‘The dissolution of plasma indicates that the plasma‐jet system is useful for various biomedical applications [21]. ‘This conclusion is very far-fetched. OES shows the presence of ROS and RNS but to my knowledge allows no conclusion to biomedical applications in general.
Why do author differentiate between live/dead staining and annexin/PI? The Hela cells are normally attached to the substrate and can be stained as the images show (Fig 4). Authors describe the staining for living cells using EthD-1. It would be more convincing to present these images as well.
Is O3 still detectable after 6 months of storage? In the stored plasma treated liquid the authors measured H2O2 as ROS. It is not clear and was not discussed why they used these two different species O3 and H2O2 (Fig 6). I suggest to measure both species at both time points or at least the same species at both time points. Further the O3 concentration looks more like 0,25 ppm at saturation level (Fig 3b).
In Fig 5b it is not clear what the authors intended to show in the diagram /results from the flow cytometry and how it transfers to the bar graph next to it. The quadrants are labelled but there is no further explanation or reference to it.
In line 187 authors gave H2O2 concentration in µm. This should be corrected.
In Fig 6B it is not necessary to give numbers for concentrations of NO with 7 digits after comma.
Figure 5. ‘Efficacy of frozen liquid plasma (LP). LP was kept in a freezer (20C) for indicated periods
(1 and 6 months), then thawed and added to the medium of HeLa cells to evaluate its efficacy’ Was the liquid plasma added or did it replace the old medium? This should be described specifically.
I strongly recommend a native English speaking person to revise the manuscript and check for spelling and grammar.
Author Response
Response to Reviewer 4 Comments
Comments and Suggestions for Authors
Specific comments:
Comment 1: For the preparation of liquid plasma and subsequent treatment of cells DMEM cell culture medium was used and this medium was treated with plasma. In 2.4 the authors did not mention any supplementation of the medium. Whereas in subheading 2.6 FCS-supplement was described. This is important since the cells come in contact with it. Please specify the components of the cell culture medium DMEM.
Response 1: We used DMEM supplemented with serum to generate plasma-activated medium. Thus, we have corrected the descriptions in section 2.4.
Comment 2: It is not necessary to mention all suppliers once again in the diagram (results section). In the subheading 2.1 the author claim to have used the AmplexRed Kit and later on in the manscript they used AmplexUlraRed. Which assay was used for H2O2 detection, AmplexRed or AmplexUltraRed?
Response 2: We used an Amplex UltraRed kit. We have deleted Amplex UltraRed from the Results section.
Comment 3: The method used of counting the cell viability is not explained. There are many cells very close to each other which I recon makes it impossible to count them under a microscope.
Response 3: Cell viability was assessed qualitatively and quantitatively. For the qualitative assay, we used a live and dead assay kit. For the quantitative viability assay, we conducted an MTT assay.
Comment 4: The result in Fig 3 shows a spectrum of OES. Was this taken with a liquid target involved in the system or without? Does the spectrum change if a liquid is present? In your setup this could give some important hints to the species emerging from the plasma. I suggest the authors give some more explanation to the spectrum and its implications for the current study to better relate it to the current investigation.
Response 4: Figure 3 is the spectrum obtained from the plasma jet in the atmosphere. Unfortunately, we do not have an analyzer for liquid at present. We agree with your suggestion. We are planning to obtain the OES of the plasma-activated medium in the near future and report the results in subsequent research papers.
Comment 5: Why do author differentiate between live/dead staining and annexin/PI? The Hela cells are normally attached to the substrate and can be stained as the images show (Fig 4). Authors describe the staining for living cells using EthD-1. It would be more convincing to present these images as well.
Response 5: To obtain more convincing results, we used two methods to evaluate the anticancer effects of plasma-activated medium. As described above, for the qualitative assay, we conducted a live/dead staining assay, while annexin/PI staining and MTT assays were conducted for quantitative analysis. Additionally, an annexin/PI staining assay was conducted to determine whether PAM induces cell death involving apoptosis, as observed for direct plasma.
Comment 6: Is O3 still detectable after 6 months of storage? In the stored plasma treated liquid the authors measured H2O2 as ROS. It is not clear and was not discussed why they used these two different species O3 and H2O2 (Fig 6). I suggest to measure both species at both time points or at least the same species at both time points. Further the O3 concentration looks more like 0,25 ppm at saturation level (Fig 3b).
Response 6: We did not measure the concentration of dissolved ozone in the medium after 6 months. We do not currently have plasma-activated medium stored for 6 months. As mentioned in the Discussion section, we measured the ozone concentration as one index of various reactive oxygen species. According to the measurement results in other plasma-activated liquid samples stored for one or two months, the concentration of dissolved ozone decreased by an order of magnitude, which is not presented in this manuscript. We inferred that other ROS/RNS rather than dissolved ozone were effective for causing apoptosis, as mentioned in the Discussion. As you suggested, we plan to determine the concentration of ozone and the OES of plasma-activated medium stored for several months in the near future and report the results in subsequent research papers.
Comment 7: In Fig 5b it is not clear what the authors intended to show in the diagram /results from the flow cytometry and how it transfers to the bar graph next to it. The quadrants are labelled but there is no further explanation or reference to it.
Response 7: We added labeled quadrants to show how the dot-plot images were transferred to the bar graph next to them. We have also added detailed explanations to the figure legend.
Comment 8: In line 187 authors gave H2O2 concentration in µm. This should be corrected.
Response 8: The H2O2 concentration was in the range of tens of μM.
Comment 9: In Fig 6B it is not necessary to give numbers for concentrations of NO with 7 digits after comma.
Response 9: We have deleted the numbers.
Comment 10: Figure 5. ‘Efficacy of frozen liquid plasma (LP). LP was kept in a freezer (-20°C) for indicated periods (1 and 6 months), then thawed and added to the medium of HeLa cells to evaluate its efficacy’ Was the liquid plasma added or did it replace the old medium? This should be described specifically.
Response 10: We have replaced the old medium with fresh or stock (frozen and thawed) plasma-activated. Thus, we have modified the sentence by deleting “the medium of”.
Comment 11: I strongly recommend a native English speaking person to revise the manuscript and check for spelling and grammar.
Response 11: The revised manuscript has been checked and corrected by a native English speaking person.
Round 2
Reviewer 2 Report
I recommend to accept this manuscript, as author have revised manuscript.
Author Response
We appreciate your recommendation.
Reviewer 3 Report
Thank you for addressing some of the concerns. However, my suggestions were not to get personal answers but to include them in the paper. After reading your responses, additional concerns have arisen.
The references do not cite any work done by the Keidar group. Danyun Yan has published on plasma treated liquid, storage, and effect on cancer cells for many years. Are you unaware of the literature? That is a serious concern for me.And if are aware, then why is it not cited?
If you already have data on room temperature and refrigeration storage, why is it not included here? Freezing expends a lot of energy and is not always feasible everywhere. Don't you think it is better if your liquid retains efficacy at room temperature?
If PBS PAM has same effects why was DMEM used? PBS is more physiological and could be used in preclinical and clinical models. DMEM cannot be. It just does not make sense to not use something that has better translation possibility.
Author Response
Comment 1: The references do not cite any work done by the Keidar group. Danyun Yan has published on plasma treated liquid, storage, and effect on cancer cells for many years. Are you unaware of the literature? That is a serious concern for me.And if are aware, then why is it not cited?
Response 1: We appreciate your suggestion and have added works of the Keidar group (Ref. 24 & 25).
Comment 2: If you already have data on room temperature and refrigeration storage, why is it not included here? Freezing expends a lot of energy and is not always feasible everywhere. Don't you think it is better if your liquid retains efficacy at room temperature?
Response 2: Unfortunately, the lab temperature is not constantly maintained in our building. In summer, the lab temperature often stays above 27 degrees. By contrast, the winter night temperature may fall below 10 degrees. Thus, just in case, we use refrigeration or freezer storage for reproducibility.
Comment 3: If PBS PAM has same effects why was DMEM used? PBS is more physiological and could be used in preclinical and clinical models. DMEM cannot be. It just does not make sense to not use something that has better translation possibility.
Response 3: When PBS PAM has been used for in vivo (mouse) experiments, we find that it is effective. However, when PBS was used as a negative control for cell culture, control cells were not well-grown compared with DMEM. Thus, we are concerned that PBS PAM effects may be exaggerated in cell cultures (in vitro).
Reviewer 4 Report
The manuscript has greatly improved. I appreciate that the authors use the term plasma-activated medium consistently throughout the manuscript.
Specific comments:
There are still some points that need discussion or revision.
Is O3 still detectable after 6 months of storage? In the fresh plasma activated medium the authors detected ozon and hydrogen peroxide, in the stored plasma activated medium the authors measured only H2O2 as ROS. It is not clear and was not discussed why they used those two different species, i.e. O3 and H2O2 (Fig 3 and 7). How is this comparable?
The result in Fig 3 shows a spectrum of OES. Was this taken with a liquid target involved in the system or without? Does the spectrum change if a liquid is present? In your setup this could give some important hints to the species emerging from the plasma. I suggest the authors give some more explanation to the spectrum and its implications for the current study to better relate it to the current investigation.
Author Response
Point 1: Is O3 still detectable after 6 months of storage? In the fresh plasma activated medium the authors detected ozon and hydrogen peroxide, in the stored plasma activated medium the authors measured only H2O2 as ROS. It is not clear and was not discussed why they used those two different species, i.e. O3 and H2O2 (Fig 3 and 7). How is this comparable?
Response 1: As mentioned in the last Response 6 to your comment, we do not know if O3 is still detectable after 6 months of storage, since we do not currently have the PAM stored for 6 months. We did not intended to infer that ozone is associated with the anticancer effect, but we just adopted dissolved ozone among various reactive oxygen species in order to find an appropriate plasma-treatment time. Since the concentration saturates in 5 minutes as shown in Fig. 3(b), we set the plasma-treatment time to 5 minutes in the preparation of PAM. On the other hand, we mentioned the dependence of the anticancer effect on H2O2.
Point 2: The result in Fig 3 shows a spectrum of OES. Was this taken with a liquid target involved in the system or without? Does the spectrum change if a liquid is present? In your setup this could give some important hints to the species emerging from the plasma. I suggest the authors give some more explanation to the spectrum and its implications for the current study to better relate it to the current investigation.
Response 2: Figure 3 is the spectrum obtained from the plasma jet in the atmosphere 1 cm apart from the nozzle end without a liquid target. The spectrum does not change even if a liquid is present, since the liquid surface is 2 cm apart from the nozzle end. We agree with your suggestion. However, we guess that the species in PAM are more important and directly associated with the apoptosis than those in the plasma jet. We are planning to detect some other species in PAM in the near future if possible, and report the results in subsequent research papers.
