PONE-D-24-02305In vivo cisplatin-resistant neuroblastoma metastatic model reveals Tumour Necrosis Factor Receptor Superfamily Member 4 (TNFRSF4) as an independent prognostic factor of survival in neuroblastomaPLOS ONE

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"O.P. received support for this project through Neuroblastoma UK, RCSI Strategic Academic Recruitment (StAR) Programme, Health Research Board - The Conor Foley Neuroblastoma Cancer Research Foundation. C.G. was funded by Irish Research Council Postgraduate Programme (GOIPG/2019/3220), R.S. - Irish Research Council - The Conor Foley Neuroblastoma Cancer Research Foundation (EPSPG/2021/95). M.S. was funded by Instituto de Salud Carlos III through the projects “PI20/000530”, “PI23/01144” and “PMP21/00073” (Co-funded by the European Regional Development Fund/European Social Fund; “A way to make Europe”/ “Investing in your future”). L.D.-J. is recipient of a Ramón y Cajal scheme (Grant No. RyC-2021-034346-I), funded by the Spanish Ministry for Science and Innovation (MCIN)"

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Additional Editor Comments:

Both reviewers agreed the manuscript to be interesting. However, they also raised concerns regarding the presentation, analysis, and interpretation of the data. They also noted a lack of data and discussion to fully support the authors' hypothesis and claims made in the manuscript. Additionally, the reviewers identified issues with some of the experimental designs, including the animal model. It is important to address each of these concerns meticulously and provide successful responses.

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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

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

Reviewer #1: Yes

Reviewer #2: No

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

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: 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: This manuscript utilizes a murine model of neuroblastoma to compare a parental cell line versus its cisplatin resistant derivative to assess a possible impact on metastasis. The authors discover a disproportional metastatic tumor load associated with cisplatin resistance and identify dysregulated genes in these tumors associated with clinical relevance. The gene TNFRSF4 was shown to correlate with poor prognostic outcomes, though not risk of recurrence. Data indicated that TNFRSF4 did not appear to be regulated by changes in MYCN expression and the knockdown of TNFRSF4 expression did not significantly affect cell viability in vitro.

Here are my comments:

Major Comments:

1) On page 19, the authors indicate the following in regard to multivariate Cox regression analysis:

“The increased likelihood of recurrence based on Low expression of TNFRSF4 was no longer significant in this analysis, but this variable remained significant at increasing the likelihood of death 1.7-fold (p=0.007). Then, TNFRSF4 Low expression was an independent prognostic factor of survival in neuroblastoma.”

If TNFRSF4 is no longer considered significant to the likelihood of recurrence, what role would you speculate TNFRSF4 is responsible for in the increased likelihood of death?

One would assume this role must be different from the roles of the remaining hazards, which all correlated with both “Risk of Recurrence” and “Risk of Death”.

2) In Figure 7 (Section 3.7, page 22), the authors indicated that siRNA knockdown of KIF11 in both Kelly and SK-N-AS neuroblastoma cells served as a positive control for cell viability and proliferation. Figure 7D corroborated this result. However, siRNA knockdown of TNFRSF4 (confirmed in Figure 7E) revealed minimal effects in the neuroblastomas. Unfortunately, that leaves the manuscript with no functional assay and therefore, no data to suggest what role TNFRSF4 plays in the predicted “increased likelihood of death” witnessed in patients. Can the authors offer any assay which helps point in the direction of a possible mechanism of action?

3) As a follow-up to Comment #2, given the volume of RNA Sequencing data you have acquired and the use of pathway analysis performed (using iDEP, for instance), could this data help to identify a relevant dysregulated pathway for a predicted function of TNFRSF4 in neuroblastoma mortality?

4) In the first paragraph of the Discussion (page 24), the authors indicate:

“Our study identified a potential relationship between the drug-resistant phenotype, which causes relapse in approximately 50% of neuroblastomas, the formation of a sentinel lymph node microenvironment during tumour metastasis and immune-related gene expression alterations”.

I apologize, but can you clarify exactly what “immune-related gene expression alterations” were covered in this manuscript so far?

Section 3.4 (page 15 – 16) indicates the title:

“Immune-Related Genes are Associated with Neuroblastoma Outcomes”

This section mentions “candidate genes” associated with “neuroblastoma aggressiveness” and assumes “a direct neuroblastoma – immune cell interaction would be facilitated by either proteins secreted or expressed on the cancer cell surface”. However, the reader has no indication exactly how these candidates are immune related. Since this section deals with “Immune-Related Genes”, shouldn’t we know how they are immune related?

5) The Discussion further mentions (bottom of page 24):

“Pathway enrichment analysis of the profiled immuno-oncology panel via iDEP and Metascape points towards some dysregulated genes that can modulate the formation of metastatic sites through cytokine signalling”.

The text then identifies the cytokine CCL2 as upregulated in cisplatin-resistant tumors and refers to a supplemental figure. Why was this new data only introduced in the Discussion? This is an experimental result. Shouldn’t it have been mentioned in Section 3.4?

6) In the Discussion (page 26), the authors identify that TNFRSF4 “upregulated in the lymph nodes, playing a key role in effector Treg cell development”, that “expression was higher in tumour infiltrating T cells than peripheral blood T cells“, and “in metastatic melanoma, sentinel lymph nodes had a significantly higher number of OX40+ lymphocytes than healthy lymph nodes expression was higher in tumour infiltrating T cells than peripheral blood T cells“.

The authors further indicate that these data “conflict with our observation of decreased TNFRSF4 expression in cisplatin-resistant lymph node metastases in neuroblastoma; however, many of these studies assessed TNFRSF4 expression in lymphocytes within tumour-positive lymph nodes rather than in tumour cells themselves, as we did in our study.“

Given this inversed proportion of expression for TNFRSF4 between lymph node lymphocytes and the tumors themselves, is this correlation necessary or advantageous and why would you propose this is occurring?

Minor Comments:

1) I am curious why the authors chose SCID Beige mice as a model system for this study. Were nude athymic mice not viable for metastatic modeling?

2) On page 4 (Section 2.2 of the Materials and Methods), the authors indicate the following:

“Fox Chase SCID Beige mice, which have defective B cells, T cells and NK cells, were injected via tail vein with a suspension of either KellyLuc (cisplatin-sensitive, n=10) or KellyCis83Luc (cisplatin-resistant, n=10) cells.”

Could you indicate how many cells were introduced, the volume, and the characteristics of the “suspension” solution?

3) Figure 3 (associated with Section 3.2, page 15) appears to have been incorrectly labeled as Figure 1. It is also incorrectly labeled in the text as Figure 1A on page 14 and Figures 1B, C, D, and E on page 16. The actual Figure 1 is identified in Section 3.1 (page 12).

4) On page 21 (Section 3.6), the authors state:

“Owing to the significant inverse correlation shown between MYCN and TNFRSF4 in multiple neuroblastoma datasets (Figure 7A and Supplementary Figure S7)…”

I believe you are referring to Supplementary Figure S6, titled “Correlation between MYCN and TNFRSF4 expression…” which indicates the comparison of the Westermann cohort versus the the Fischer cohort of neuroblastomas.

Supplementary Figure S7 is titled: “Expression of TNFRSF4 and MYCN in a panel of neuroblastoma cell lines.”

As a consequence, Supplementary Figure 6 is not mentioned anywhere in the text and Supplementary Figure S7 needs to be properly introduced.

5) On page 25 (in the Discussion), Supplemental Figure S9 is indicated as associated with an experiment involving CCL2. There is no Supplemental Figure S9 in the supplementary material. This is Supplemental Figure S8.

6) In the Discussion (page 25), the following statement should probably have a citation to support it:

“The CCL2-CCR2 pair has a long track record of multiple protumorigenic roles, including the promotion of tumour growth and metastasis in many cancer types.”

Taken together, this manuscript offers a solid set of experiments, but requires a few gaps and corrections be addressed.

Reviewer #2: This manuscript focuses on further characterization of an established cisplastin-resistant neuroblastoma model (KellyCis83), in the context of metastasis, an important consequence and deadly outcome for many patients with high-risk NB. Given that metastatic NB is highly drug resistant, the in vivo RNA sequencing dataset and assessments made regarding TNFRSF4 would provide a valuable resource to the research community.

One concern is the conclusion that TNFRSF4 disregulation is specific to NB tumor cells. Bulk tumor was dissected, stained and profiled using RNA sequencing. Why is it assumed that leukocytes and/or other stromal cells are not perhaps an additional and/or functional the source of TNFRSF4 in the in vivo model? Please clarify with tissue specific expression profiling from the met biopsies.

Related to the previous comment regarding the potential for TNFRSF4 expression in stromal tissues, scRNAseq data from NB patients (Wienke et al. Cancer Cell 2024) show TNFRSF4 expression in NB-associated Tregs and potentially an immune suppressive activity by this cell type. This recently published data might lead one to guess that increased stromal TNFRSF4 expression associates with worsened outcomes, which contradicts the data shown in Fig 4. It may be a consideration to discuss this published finding as it relates to the current manuscript, KellyCis83 and TNFRSF4.

Even though characterization of these Kelly cell lines was previously published, it would be beneficial to comment in section 3.1 or elsewhere on differences between Kelly and KellyCis83Luc with respect to growth dynamics and genomic changes (chromosomal gains, losses, rearrangements). For example, are any changes consistent with chromosomal loss discussed in patients on pg. 18?

Is there an explanation for why the RT-PCR data (pg. 15, Fig. 1 C and E) are inconsistent with the RNA sequencing. Is it that these genes differentially expressed in stromal cells? This could be further explored on tumor section and/or clarified in the manuscript.

In the discussion, it is mentioned that the tumor size of KellyCis83 mets are increased. It would be interesting to present this data in Fig 1 to compliment met number.

It does not look like the stiffness data presented in Fig.1F is significantly different in Kelly versus KellyCis83?. While this may be a possible explanation for the observed phenotypic changes, without significant changes, this reviewer does not fully understand the value of presenting and discussing this data in the manuscript. A p-value in Fig.1F may help clarify this point.

Minor comments:

-"Figure 1: Functional enrichment analysis..." (pg.15) figure legend and associated references in the text appear mislabelled and based on the presented sequence should be Figure 3 vs Figure 1?

-A legend for "dot sizes correspond to adjusted p values" in Fig1 (maybe Fig.3, pg 15) could be helpful

-Reference the source of "large cohort of 498 neuroblastomas" on page 16, line 2.

-Raw data from the RNA sequencing do not appear to be deposited in a publically available database. Sharing of these data would facilitate their use in other studies.

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Reviewer #1: No

Reviewer #2: No

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In vivo cisplatin-resistant neuroblastoma metastatic model reveals Tumour Necrosis Factor Receptor Superfamily Member 4 (TNFRSF4) as an independent prognostic factor of survival in neuroblastoma

PONE-D-24-02305R1

Dear Dr. Piskareva,

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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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

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: 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

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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: This manuscript identified a gene which correlates with poor outcome for high-risk neuroblastoma patients based upon a murine metastasis study. The material presented is sound and the analysis does not exaggerate its impact or conclusions. Therefore, I recommend its acceptance for publication.

Reviewer #2: Thank you to the authors for their thoughtful response and updated manuscript.

All of my questions and comments have been addressed.

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Reviewer #1: No

Reviewer #2: No

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