PONE-D-20-08672

The SRG rat, a novel Sprague-Dawley Rag2/Il2rg double-knockout validated for human tumor oncology studies.

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: Yes

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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?

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

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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: No

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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 paper, Noto FK et al, developed the SRG OncoRat model, a SCID rat on the Sprague-Dawley background that harbors a double knockout for the Rag2 and Il2rgamma genes. Similar to NSG mice, SRG rats showed enhanced immunodeficiency, lacking B, T, and NK cells. Combining these genetic changes in this immunodeficient rat allow the use of fewer animals through enhanced engraftment rates with more uniform tumor kinetics, and improved tumor growth profiles for both tumor cell lines and PDXs.

The paper discusses different aspects impacting the engraftment of human tumors: the comparison with NSG mice, the growth kinetics of several human cancer cell lines, the molecular characterization and histological analyses for PDXs...

Experiments are well conducted, the manuscript is clear, well written and the conclusions are supported by the data.

This reviewer only has minor points to be addressed:

- Figure legends 1 and 2: delete "252" and "278" refer to B cells and circulating mature B cells, respectively.

- Figure 5: the authors only showed a tumor growth curve from a single NSCLC PDXs established in SRG rats; please, provide growth curves for others 6 PDXs. This figure could also go to supplementary.

- To evaluate the genomic instability of the PDX model, the authors performed NGS after P1, P2 and P3, but any result is showed. Please, provide a figure or a table supporting this section.

- The authors assessed the growth of several tumor cell lines in SRG rats, including HCT-116, MIA-PaCa-2, HCC1954, and 786-O, but only the colorectal carcinoma cell line HCT-116 was also injected in NSG mice as comparison. They stated "growth kinetics of these human cancer cell lines have been tested by others in mouse xenograft models [14]", but any of these cell lines (MIA-PaCa-2, HCC1954, and 786-O) are mentioned in ref. 14. The authors should provide others refs. to demonstrate the higher engraftment rate of these cell lines in SRG rat in comparison to NSG mice.

Reviewer #2: In “The SRG rat, a novel Sprague-Dawley Rag2/Il2rg double-knockout validated for human tumor oncology studies” (PONE-D-20-08672), Noto et al generated SRG rat model carrying mutations in Rag2 and Il2rg. SRG rats had severe immunodificient phenotypes, i.e., lack of mature T/B cells and reduced NK cells. Then the authors demonstrated the advantages of using SRG rats in both cell-line-derived xenograft (CDX) and patient-derived xenograft (PDX) models. In CDX models, the engraftment rate (>90%) of VCaP cells was much higher than that of NSG mice. In PDX models, the authors achieved 7 PDX out of 9 samples from patients with lung adenocarcinoma. In both CDX and PDX models, SRG rats supported higher tumor growth-rate and easier sample collection, which makes them an alternative oncology model to mice.

This work is very interesting and adds important knowledge of immunodeficient rat models in oncology studies. However, the quality of this manuscript needs to be substantially improved before it’s accepted by Plos One.

The major issues:

1. Although the authors used “novel” in the title, similar models have already been characterized previously. These models include FSG (Prkdc/Il2rg; Mashimo et al. Cell Rep. 2012.), SD-RG (Rag1/Rag2/Il2rg; He et al. FASEB J. 2019) and RRG (Rag1/Il2rg; Ménoret et al. Transplantation. 2018). I highly recommend the authors to change the title and acknowledge the previous research in the manuscript.

2. The mouse strain NSG (NOD/LtSz-Scid Il2rg−/−) does not equal Nod-Scid. However, the authors may make the readers confused in the figure legends “Figure 4. VCaP xenograft model in NSG mouse and SRG OncoRat. NOD-Scid mice and SRG rats were inoculated with 5x106 and 10x106 VCaP cells, respectively, subcutaneously in the hind flank.” and “Supplemental Figure 2: Growth curve of VCaP in NOD-Scid mice. Mean weight in mg with SEM. Molecular analysis of VCaP tumors confirmed expression of the androgen receptor (AR) in both the NSG (NOD-scid) mouse and SRG rat models (Figure 4B,C).” To avoid misleading, I hope the authors to clarify which mouse strain was used in their experiments.

3. The statistical analysis and presentation should be improved. In the first paragraph of Results section, for example, the authors wrote “The SRG rat spleen also has slightly lower NK cells compared to the wild-type rat spleen (2.81% vs. 3.96%, respectively; Figure 1G -I).” 2.81% or 3.96% is just one representative result of the triplicate experiments. The authors should use the “Mean±SD” as shown in Figure 1I to describe this difference. The authors claimed that “Not only did the tumors grow faster in SRG rats, their individual growth kinetics were more uniform, leading to consistent tumor volumes throughout their growth.” However, I could not find any statistical analysis to verify the “uniform” growth kinetics.

4. In the last part of Results section, the authors did NGS to evaluate the genomic instability of their PDX models. However, there are no detailed results (figures or tables) or access number of the sequencing data. So I recommend the authors to provide the missing details in both Methods and Results sections.

The minor issues:

1. The authors verified the deletions in Rag2 and Il2rg respectively by Sanger sequencing. Their results would be solidified if they could examine the expression of these two genes at mRNA and protein levels.

2. Unlike Fig3A, Fig3BCD did not have NSG data as control. Although the authors cited previous results as “Growth kinetics of these human cancer cell lines have been tested by others in mouse xenograft models [14].”, I don’t think they are good controls because the experimental settings varied between different labs.

3. I highly recommend the authors to revise the manuscript carefully to avoid typographical or grammatical errors.

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Reviewer #1: No

Reviewer #2: No

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The SRG rat, a Sprague-Dawley Rag2/Il2rg double-knockout validated for human tumor oncology studies.

PONE-D-20-08672R1

Dear Dr. Noto,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Francesco Bertolini, MD, PhD

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

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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

**********

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

Reviewer #1: Yes

Reviewer #2: 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: 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: The authors have addressed all of my comments and concerns through their response and edits, and the manuscript is definitely improved. I have no further suggestions or corrections.

Reviewer #2: I carefully read the revised manuscript “The SRG rat, a Sprague-Dawley Rag2/Il2rg double-knockout validated for human tumor oncology studies” (PONE-D-20-08672R1). Noto et al made substantial improvement in revising the manuscript. I was satisfied with their responses to my previous comments. However, I hope the authors to be aware of the issues below.

1. I noticed that the authors used SCID/NCr mice as control for VCaP PDX. SCID/NCr mice lack T and B cells, but still have NK cells. SRG rats lack not only T and B cells due to Rag2 KO but also NK cells due to Il2rg KO. So the high growth-rate of PDX in SRG rats may be alternatively explained by this difference. I recommend the authors to address this in the text.

2. To benefit the field in rat PDX models, I highly recommend the authors to deposit their NGS data to the public server such as NCBI.

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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