PONE-D-20-14145

Overcoming TRAIL-resistance by sensitizing prostate cancer 3D spheroids with taxanes

PLOS ONE

Dear Dr. King,

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, the authors compare sensitivity of DU145 and PC3 prostate cancer cell lines grown in 2D vs 3D to the

Please provide additional details regarding the CAFs used (hTERT PF179T). Were these purchased or created in the authors’ lab?

Figures 1, 2: Does the ratio of prostate cancer cells to fibroblasts used to create the spheroids impact the ratio of each or the size of the final formed spheroid? This should be directed addressed.

Page 12, Lines 260-270: results from several experiments are described but this data is not shown in any of the figures and quantitative results are not provided. If these results are to be included in the manuscript, the data should be included in the figures and a quantitative measure of outcomes (cell death, sensitivity, etc) should be provided with appropriate statistics.

Page 12: Line 260 states that DU145 cells were resistant to TRAIL alone, but then Line 263-264 state that treated DU145 cells show morphological changes of apoptosis. This seems at odds. This should be clarified and the data and analysis should be provided.

Use of the word “cells” can be confusing in places because its variably used to refer to cells in 2D vs 3D. Please try to clarify; for example, could refer to 3D cultures strictly as “spheroids”, or always referring to 2D cultures as “monolayer cells”.

Page 14, Line 301: Typo…I think “DU145” should be “PC3”

Page 14, Lines ~300-310: Same issue as in preceding DU145 section. Authors state that treatment did not impact cells, but then describe morphological changes of apoptosis…this seems discrepant.?

The difference in sensitivity to the TRAIL+chemo combo in 2D vs 3D of DU145 (no difference) and PC (big difference) is perhaps the most interesting result from Figures 3-4 and this difference in behavior between the two cell lines could be emphasized.

For experiments in Figure 5, how can death of cancer cells be differentiated from death of fibroblasts following treatment? This should be explained. If the assay is not able to differentiate death of cancer cells from fibroblasts, this could impact interpretation of results.

To my knowledge, there is little available clinical data to support targeting TRAIL in prostate cancer. If such data is available, it would be worthwhile to discuss since it is otherwise not so clear why the authors chose to focus on TRAIL therapy in combination with taxanes (which do have a demonstrated role in clinical management of mCRPC).

Reviewer #2: The authors have evaluated the effects of TRAIL, cabazitaxel and docetaxel in 3D spheroids. The results of this paper show some benefits of combination therapy in vitro. The endpoints of the study should be better described. More experimental work is needed to completely support the conclusions. This reviewer has the following specific suggestions and recommendations:

1. Page 12, the differences between NAF and CAF are mentioned. What is the difference in expression of key molecules which regulate proliferation and migration between NAF and CAF? This is not clearly explained in the manuscript.

2. apoptosis is detected mostly by morphological changes. This is not sufficient. Additional tests should be considered. For example, the authors should use PARP cleavage. This is an important part of the manuscript and should be better presented.

3. In order to further support the findings on page 14, I recommend to perform the cell cycle analysis.

4. in order to ensure consistency in the manuscript, I recommend to analyze the expression of death receptors (DR) after combination therapy (page 17).

5. Importantly, the argumentation for use of only androgen receptor-negative cell lines in the paper is not complete. Key findings should be repeated with at least one androgen receptor-positive cell line.

Reviewer #3: This manuscript seeks to test the hypothesis that the combination of TRAIL + the taxanes docetaxel (DTX) and cabazitaxel (CBZ) sensitizes the prostate cancer cell lines PC3 and DU145 to cell death compared to treatment with these agents alone. While the article has several interesting points it has major issues that would require careful attention and further experimentation.

1. The authors part from the premise that 2D and 3D cultures of PC3 and DU145 cells are resistant to treatment with DTX or CBZ because no apoptosis is observed after 24 h of treatment with 0.25 uM (250 nM) of each drug. There are two major problems with this rationale:

a) Treatment for 24 h, according to the Methods and Results sections, is not sufficient time to induce PC3 or DU145 apoptotic cell death with taxanes. The reason is that for taxanes to induce apoptosis, the cells need to be first arrested in mitosis, which is then followed up by cells rounding up, detaching from the plates, and undergoing mitotic catastrophe. For significant cell death to be observed, this process needs to be followed up for approximately 48-72 h.

b) 0.25 uM, or 250 nM, is a very high concentration of taxanes, which may not induce apoptotic cell death at 24 h for the reasons stated above but may induce lysosomal or other non-apoptotic modes of cell death after 24 h. Typically PC3 and DU145 cells growing in 2D are sensitive and undergo apoptotic cell death when treated a low nM concentrations (5-20 nM) for 48 h - 72 h. In fact, DTX-resistant PC3 and DU145 cells are normally selected and maintained in culture at 10 nM concentrations. The use of very high taxane concentrations in this manuscript must be justified. The claim that these cells are resistant to high taxane concentrations after treatment for only 24 h is questionable. The cells are simply not ready to display the characteristics of apoptosis at that time point. The authors should use a dose-response, time-response experiment to compare taxane resistance between 2D or 3D PC3 and DU145 cell lines, or select the cells for taxane resistance for several weeks by incrementally increasing drug concentrations while selecting surviving cells.

2. The combination treatment of taxane plus TRAIL at 48 h does induce cell death, but the problem is that apparently the treatments with taxanes alone and TRAIL alone were conducted for 24 h. This is what the Methods and Results sections imply, even when the figure legends suggest that treatments were done at 48 h. This needs to be clarified because it is possible that the observed loss of viability could be due to induction of cell death by taxanes after 48 incubation, and not by the TRAIL-taxane combination. If the single treatments were done at 24 h and the combinatorial treatment at 48 h, then the following comparisons are needed: single and dual treatments at both 24 h and 48 h.

3. Was TRAIL used as 400 ng/ml or 100 ng/ml? Both concentrations are mentioned in the text without explanation.

4. The measurement of apoptosis using the Annexin V/PI method needs a positive control to rule out the possibility that trypsinization may have cleaved Annexin V from the cell surface, which may have resulted in underestimation of cell death. The combination of TRAIL (100 ng/ml) + Actinomycin D causes robust apoptosis between 6-12 h and could be used as a suitable positive control for this assay.

5. The flow cytometry panels in Figures 3 and 4 need better resolution. Very difficult to see the numbers.

6. Was necrotic cell death estimated (AV-/PI+) for each treatment? If so the numbers should be provided more clearly in the flow figures. It is possible that the high taxane concentrations may have induced some necrotic cell death.

7. Treatment with DTX or CBZ led to downregulation of the TRAIL receptors DR4 and DR5 in both PC3 and DU145 spheroids; however, 3D DU145 cultures were still sensitive to TRAIL + taxanes whereas PC3 cultures were more resistant, suggesting that downregulation of these receptors may not be related to the cell viability response. The authors argue that this difference may be related to the presence of more stem cells in the PC3 spheroids but the data is not presented. These contradictory results need more detailed discussion, and perhaps additional supportive data.

8. To establish the role of the DR4 and DR5 receptors in influencing sensitivity to TRAIL + taxanes in PC3 and DU145 spheroids the authors should knockdown these receptors in 2D cells and examine culture sensitivity to this combinatorial treatment.

9. The cancer cells and the the fibroblasts were stained with different Cell Tracker fluorescent dyes. How do the authors know that these dyes are specific for cancer cells or fibroblasts?

10. It is not clear from the Methods if the PCa cells were mixed with the fibroblasts and then seeded as a mixture, or if the fibroblasts and the PCa cells were seeded sequentially. Regardless, it seems that the presence of fibroblasts does not influence cellular responses to TRAIL + taxanes. Discussion of this issue needs elaboration.

11. What are the comparison groups in Fig. 5, particularly for the bars with significant values? The statistical analysis needs to be clarified for this figure.

12. There are several key statements in the Introduction and Discussion sections that need citations.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Overcoming TRAIL-resistance by sensitizing prostate cancer 3D spheroids with taxanes

PONE-D-20-14145R1

Dear Dr. King,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: No further comments necessary. The comments have been addressed.

No additional comments regarding ethics.

Reviewer #3: The authors have satisfactorily addressed all my earlier comments by including new data in the Figures and adding clarifications or explanations to the manuscript text.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No