Immunological Effects of Cold Atmospheric Plasma-Treated Cells in Comparison with Those of Cells Treated with Lactaptin-Based Anticancer Drugs
, Diana Novak 1,2, Mikhail Varlamov 1, Mikhail Biryukov 1,2, Anna Nushtaeva 1, Galina Kochneva 3, Dmitriy Zakrevsky 4,5, Irina Schweigert 6, Vladimir Richter 1 and Olga Koval 1,2,*Round 1
Reviewer 1 Report
This manuscript describes experimental work concerning the possible immunological effects of MX-7 cellas treated with Cold Atmospheric Plasma (CAP) compared to lactaptin-based anticancer drugs.
The results are interesting, and the paper is overall well written. Therefore, I recommend its publication on Biophysica, after a few minor points have been addressed. I list them below:
1) When describing the CAP source, please give also the average power transferred to the plasma. This is useful for comparison between different devices.
2) The Materials and Methods section should include a subsection about how the CAP treatment is performed, so as to allow other scholars to properly repeat the experiments. Issues such that the distance between the plasma source and the cells (including the length of the plasma plume), the kind of support in which the cells are hosted, the volume of medium in which the cells are immersed during the treatment, if the medium is changed afterwards, ecc., should be detailed.
3) The Conclusions section should not start with "Thus", which implies a dependence from a previous sentence. Also, I suggest to expand this section a little bit, shortly summarizing the objectives of the work, and then giving the conclusions that have been obtained.
Author Response
General Comment: This manuscript describes experimental work concerning the possible immunological effects of MX-7 cells treated with Cold Atmospheric Plasma (CAP) compared to lactaptin-based anticancer drugs. The results are interesting, and the paper is overall well written. Therefore, I recommend its publication on Biophysica, after a few minor points have been addressed. I list them below:
Response: We thank the reviewer for nice words about our work and a very detailed analysis of our manuscript.
Point 1: When describing the CAP source, please give also the average power transferred to the plasma. This is useful for comparison between different devices.
Response 1: This data are presented in the current version Materials and Methods, Line 143.
Point 2: The Materials and Methods section should include a subsection about how the CAP treatment is performed, so as to allow other scholars to properly repeat the experiments. Issues such that the distance between the plasma source and the cells (including the length of the plasma plume), the kind of support in which the cells are hosted, the volume of medium in which the cells are immersed during the treatment, if the medium is changed afterwards, ecc., should be detailed.
Response 2: These data are presented in the current version Materials and Methods, Lines 144-148.
Point 3: The Conclusions section should not start with "Thus", which implies a dependence from a previous sentence. Also, I suggest to expand this section a little bit, shortly summarizing the objectives of the work, and then giving the conclusions that have been obtained.
Response 3: We thank the reviewer for this valuable suggestion. Please, see the new parts of the text (Lines 448-458):
The main purpose of the current study was to compare the immunological effects of MX7 rhabdomyosarcoma cells treated with various potential inducers of ICD: cold atmospheric plasma jet, recombinant vaccinia virus and cytotoxic protein RL2. Among the studied inductors, RL2 caused the most significant changes in ICD-related signs which agrees well with the observed in vivo effect of vaccination produced by the RL2-treated tumor cells. CAP-treated cells were not capable of activating anti-tumor immune response under the irradiation regimen used: they demonstrated poor IFNα release, had a weak potency to be untaken by DCs, and were low-efficient in support of maturation of DCs. It is possible that the conditions of the treatment cells by CAP, that stimulate ICD in vitro, are not optimal for activation of the immune system in vivo, and further investigation of CAP regimens to enhance the activation of the immune system is required.
Reviewer 2 Report
The enhancement of immunogenic cell death (ICD) may enhance the therapeutic effects in various cancers, including metastatizing or unresctable malignancies. In this manuscript, Troiskaya O et al demonstrated that cold atmospheric plasma (CAP) increased maturation and phagocytosis weakly in dendritic cells. Unfortunately, the induction of ICD by CAP was lower than other compounds; however, it may be nice to publish your data to show the biological effects of CAP.
Major points
#1. Did MX-7 completely die after CAP treatment?
#2. It may be nice to show the ratio of engulfed MX-7 cells in Figure 3(b). CAP treatment group, 5.7 % was phagocytosed, while 40.2 % was not. In RL-2 treated MX-7, 30.3 % was phagocytosed, while 25.4 % was not. Please show the proportion of phagocytosed MX-7 and examine the significant differences.
Author Response
General Comment: The enhancement of immunogenic cell death (ICD) may enhance the therapeutic effects in various cancers, including metastatizing or unresctable malignancies. In this manuscript, Troiskaya O et al demonstrated that cold atmospheric plasma (CAP) increased maturation and phagocytosis weakly in dendritic cells. Unfortunately, the induction of ICD by CAP was lower than other compounds; however, it may be nice to publish your data to show the biological effects of CAP.
Response: We thank the reviewer for nice words about our work.
Point 1: Did MX-7 completely die after CAP treatment?
Response 1: We added this information in the text. Please, see Lines 252-254: Since tumor cells treated with the ICD inducer must emit DAMPs, these may not be cells in the final stage of death but have passed the point of no return.
Point 2: It may be nice to show the ratio of engulfed MX-7 cells in Figure 3(b). CAP treatment group, 5.7 % was phagocytosed, while 40.2 % was not. In RL-2 treated MX-7, 30.3 % was phagocytosed, while 25.4 % was not. Please show the proportion of phagocytosed MX-7 and examine the significant differences.
Response 2: We thank the reviewer for this valuable suggestion. We added such histogram on the Fig. 3c. We have also described these data in the main text (Lines 305-308): Looking at the ratio of engulfed MX-7 cells to the total MX-7 been added to DCs, more than 50% of cells were taken up in the case of RL2, while only 13% of cells were taken up in the case of CAP-treated cells (Figure 3c).
