NK Cells Can Preferentially Target Prostate Cancer Stem-like Cells via the TRAIL/DR5 Signaling Pathway
Round 1
Reviewer 1 Report
In their paper, Seki et al. focused on the selective action of NK cells prostate cancer stem(-like; S-L) cells. Using an approach based on the 3D spheroid-based separation of S-L cells from the population androgen-dependent LNCaP populations and on S-L cultivation in the presence of NK cells, they found a selective cytostatic effect against S-L cells that they related to the TRAIL/DR5 pathway. This a convincing and nicely presented study, definitely worth of publishing in Biomolecules after several points have been addressed:
- The whole story is based on the assumption that only S-L cells have been propagated in 3D. It may be true but it would also be reasonable to expect more information on CSC-specific markers in the analysed S-L cells FACS, immunofluorescence);
- it would also be interesting to see the photos of the cells in culture and in co-cultures;
- The legend to Fig.1B is unclear. Please describe explicitly the meaning of filled vs. open bars.
- The WST-8 test is broadly used in such assays, however it is sensitive to the differences in cell metabolic activity. As such, it can give misleading results when performed alone. Growth curves/annexinV tests would add to the message of the story;
Author Response
Please see the attachment explaining my response to your comments.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript by Seki et al. sorted and analyzed a subset of DR5 positive LNCaP subpopulation for cytotoxic effects by natural killer cells. Authors revealed that NK cells preferentially reduce cell viability of DR5-expressing LNCaP cells. An important key message of authors is that NK cells reduce viability of prostate cancer stem-like cells. However, this point has not been addressed.
This manuscript lacks important controls to support the statements.
Major points:
- Authors claim that DR5 positive cell population of a human prostate cancer cell line (LNCaP) consists of stem-like cells. However, the stem-like characteristics of prostate cancer cells must be verified. It is unclear whether this DR5-positive subpopulation has indeed stemness characteristics. Authors must verify the activity as stem-like cells functionally e.g. by tumorsphere assays. Also, authors must provide additional stem cell markers.
- Authors must show the underlying molecular basis of lower cell viability. Is it apoptosis? Several markers of apoptosis, necrosis, necroptosis and cell cycle inhibitors must be analyzed.
Author Response
Please see the attachment explaining my response to your comments.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
I have no more comments
Author Response
Dear Sir,
Thank you for your kind comment.
Yours sincerly
Tomohiro Yano PhD
Toyo University
Reviewer 2 Report
Thank you for the response.
It would be better for the general reader that authors confirm on page 2 which stemness markers exactly were detected and that the analyzed stemness markers are increased in the stem-like LNCaP population. (CD44 alone is not sufficient to characterize prostate cancer stemness).
I guess authors analyzed besides CD44, the markers CD24, CD133, SOX2 and Oct3/4, which should be mentioned as described in their earlier paper.
Author Response
Dear Sir
Thank you for your helpful comment.
In reply to the comment on cancer stem markers, we added the additional explanation on cancer stem cell markers in this revised version (please see line 90-line 91).
We hope that this revised version of our paper is now suitable for publication in Special Issue of Biomolecules, "Molecular Signaling in Prostate Development and Prostate Cancer” edited by Dr Wen-Yang Hu, as a Communication.
Yours sincerely
Tomohiro Yano PhD
Toyo Univerity
