Feasibility of rabbit auricular VX2 tumor model as an experimental model for intra-arterial embolization

Woosun Choi; Byung Kook Kwak; Jisung Jung; Serah Park; Soon Auck Hong; Sang Lim Choi

doi:10.1371/journal.pone.0316282

Peer Review History

Original SubmissionJuly 7, 2024
29 Aug 2024 Decision Letter - Zhengwei Huang, Editor PONE-D-24-24136Feasibility of rabbit auricular VX2 tumor model as an experimental model for intra-arterial embolizationPLOS ONE Dear Dr. Kwak, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. According to our reviewers' comments, a Major Revision was needed for this manuscript. Please carefully revise it. Please submit your revised manuscript by Oct 13 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: rabbit auricular VX2 tumor model is much easier to establish, comparing with hepatic vx2 tumor model, however, rabbit auricular VX2 tumor model cannot simulate the dual blood supply system of the human, moreover the author should add the regular hepatic vx2 tumor model for the whole comparison. eventually less than half (11/24) models are successful, the feasibility is very low. Reviewer #2: Feasibility of rabbit auricular VX2 tumor model as an experimental model for intraarterial embolization The authors provide a well written manuscript describing a small study characterizing VX2 tumor development and characterization in rabbit auricles. The authors characterize growth and characteristics through thermography, ultrasonography, angiography and histopathology, which the authors report has not been previously done for rabbit auricular VX2 tumor research. The majority of the paper is sound. There are some minor deficiencies in the histopathology images and descriptions supplied for this manuscript. I am recommending minor edits which are below. Thank you to the authors for your curiosity and hard work in generating this manuscript. Line 51: remove the word ‘height’ Line 102: Within parentheses, change ‘two-four’ to ‘2 to 4’ to be consistent with the authors’ chosen enumeration rather than spelling of single digits. Same with spelled numbers on lines 159, 216, 222, 224 Figure 5 legend: A. remove the word ‘clearly’ C. Remove the word ‘clearly’, remove the phrase ‘abundant mitosis’ and replace with ‘mitotic activity’. Mitoses are most definitely not abundant in the image supplied. D. I am not convinced that the image depicts lymphovascular invasion. First, image quality is poor. Second, the surrounding fibrovascular tissue somewhat conforms to the purported embolus, and it’s margins around the embolus are fragmented, suggesting artifactual separation from the neoplastic cells. Third, endothelium cannot be seen lining the colorless space. I suspect this is simple tumoral extension with artifactual separation of the underlying stroma. Please prove me wrong by submitting a higher quality image for figure 5D that shows an endothelial lining, or provide an image of an immunohistochemical stain or other marking examination that highlights the endothelial cells and proves the neoplastic cells are in a vessel. Reviewer #3: The author implanted VX2 tumors on the auricle of rabbits, and the study has certain significance. However, this manuscript needs to solve the following problems: 1.H&E figure needs to add ruler; 2.Previous studies have reported that VX2 tumors were implanted on the liver (PMID:35667125、36510170). 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Revision 1
7 Nov 2024 Author Response Dear Editor: We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. We have answered all the questions below to the best of our ability. Reviewer Comments to Author: Reviewer #1 rabbit auricular VX2 tumor model is much easier to establish, comparing with hepatic vx2 tumor model, however, rabbit auricular VX2 tumor model cannot simulate the dual blood supply system of the human, moreover the author should add the regular hepatic vx2 tumor model for the whole comparison. eventually less than half (11/24) models are successful, the feasibility is very low. -Thank you for your advice. The VX2 tumor is an anaplastic squamous cell carcinoma and is not of hepatocyte origin; hence, it receives only arterial supply and not portal supply when implanted in the liver. This has been described in reference number 4 in our manuscript. The hepatic VX2 model is similar to hepatocellular carcinoma in that the blood supply to the tumor comes from the arterial system; thus, it is a hypervascular tumor and is richly revascularized from the arterial system after treatment. These characteristics are also present in the rabbit auricular VX2 tumor model. However, since we agree that it is important to compare the rabbit liver VX2 model with the auricle VX2 model, we have added a paragraph about the comparison in the discussion section. Main text Compared to the rabbit hepatic VX2 tumor model, which is commonly used as an arterial embolization model, the rabbit auricular VX2 tumor model has several advantages. First, when preparing the model, the tumor can be more easily accessed by inoculating it into the rabbit ear via a syringe; thus, it is more convenient to prepare this model than preparing the hepatic VX2 tumor model, which generally requires laparotomy; moreover, there is a reduced chance of death of experimental animals due to complications of laparotomy. In addition, the VX2 tumor implanted in the liver requires additional imaging studies, such as CT or US, to determine whether the tumor growth is appropriate for conducting additional experiments such as arterial interventional studies. However, for the auricular VX2 tumor model, the optimal timing for additional experiments can be easily determined because the growth of the tumor can be directly confirmed with the naked eye. Unlike the liver model, the auricular VX2 tumor model can be visually inspected without additional imaging tests to observe the tumor response after experiments such as intra-arterial embolization or ablation. The VX2 tumor implanted in the liver is commonly used as an intra-arterial embolization model because the blood supply to the tumor from the hepatic artery is very similar to that observed in hepatocellular carcinoma. In particular, the hepatic VX2 tumor model is a hypervascular tumor and the blood supply of the tumor originates in the arterial system [3, 4]. However, although the VX2 implanted in the auricle is supplied from the auricular artery, not the hepatic artery, the tumor originates in the arterial system and has the characteristics of a hypervascular tumor. Therefore, compared to the rabbit hepatic VX2 model, the auricular model is appropriate to use as an arterial embolization model. However, since the surrounding tissues are different, such as the liver and auricle, their characteristics as an experimental model may be different when conducting an ablative study. The low feasibility may be due to a calculation error in the title of Table 1 of the main text, which should actually be “19 auricles” but was incorrectly written as “24 rabbits.” We have corrected this in the revised manuscript. In our experiment, the model succeeded in 19 of 24 auricles, except 5, thus indicating a success rate of 79.2%. Reviewer #2 Feasibility of rabbit auricular VX2 tumor model as an experimental model for intraarterial embolization The authors provide a well written manuscript describing a small study characterizing VX2 tumor development and characterization in rabbit auricles. The authors characterize growth and characteristics through thermography, ultrasonography, angiography and histopathology, which the authors report has not been previously done for rabbit auricular VX2 tumor research. The majority of the paper is sound. There are some minor deficiencies in the histopathology images and descriptions supplied for this manuscript. I am recommending minor edits which are below. Thank you to the authors for your curiosity and hard work in generating this manuscript. Line 51: remove the word ‘height’ Line 102: Within parentheses, change ‘two-four’ to ‘2 to 4’ to be consistent with the authors’ chosen enumeration rather than spelling of single digits. Same with spelled numbers on lines 159, 216, 222, 224 - We greatly appreciate your comment. We have revised the manuscript according to your comment. Figure 5 legend: A. remove the word ‘clearly’ C. Remove the word ‘clearly’, remove the phrase ‘abundant mitosis’ and replace with ‘mitotic activity’. Mitoses are most definitely not abundant in the image supplied. - In accordance with your recommendation, we have removed the word “clearly” and revised the phrases in the main text and figure legends. Fig 5. Microscopy of VX2 tumor in the auricle (A) The tumor is located in the subepidermal space and exhibits nodular growth (original magnification, 2×; scale bar, 1000 μm). (B) Areas of necrosis are present (indicated by 'n') (original magnification, 100×; scale bar, 200 μm). (C) High-power magnification reveals malignant tumor cells with pleomorphic, enlarged nuclei and mitotic activity (original magnification, 400×; scale bar, 50 μm). (D) Lymphovascular invasion is suspected (indicated by the arrow) (original magnification, 400×; scale bar, 50 μm). Main text A total of 6 rabbits were sacrificed. However, the tumor did not grow in one auricle in two rabbits; thus, histologic features were evaluated in 10 auricles (Fig 5). All tumors were located in the subepidermal space and characterized by nodular growth (Fig 5A). Histologically, necrotic areas were observed in all 10 tumors, with an average proportion of 27% (range, 5–50%) (Fig 5B). Tumor cells demonstrated malignant histologic features of pleomorphism and large nuclei, prominent nucleoli, and mitotic activity (Fig 5C). Epidermal damage, manifested as erosion and ulcer, was observed in three auricles (30%). Cartilage damage was observed in one auricle, and the tumor was observed in both the dorsal and ventral aspects of the cartilage. Lymphovascular invasion was suspected in 5 of the 10 auricles (50%) (Fig 5D). Figure 5 legend: D. I am not convinced that the image depicts lymphovascular invasion. First, image quality is poor. Second, the surrounding fibrovascular tissue somewhat conforms to the purported embolus, and it’s margins around the embolus are fragmented, suggesting artifactual separation from the neoplastic cells. Third, endothelium cannot be seen lining the colorless space. I suspect this is simple tumoral extension with artifactual separation of the underlying stroma. Please prove me wrong by submitting a higher quality image for figure 5D that shows an endothelial lining, or provide an image of an immunohistochemical stain or other marking examination that highlights the endothelial cells and proves the neoplastic cells are in a vessel. - As discussed with a board-certified pathologist (S.A.H.), in our study, lymphovascular invasion was reanalyzed, and D2-40, CD31, and CD34 immunostainings were performed to confirm lymphovascular invasion. However, while these immunohistochemical stainings were conducted, the relevant area could not be fully evaluated due to its removal during additional sectioning. Although the findings are not definitive, the pathologist suggested a potential lymphovascular invasion based on the following observations: first, the suspected area of lymphovascular invasion was distant from the main tumor; second, there was no surrounding fibrosis; and third, a region suspected to contain endothelial cells was identified. Accordingly, we have revised the main text and figure legend to include the term "suspected." Furthermore, Figure 5d has been updated to display a new image showing the endothelial lining. Fig 5. Microscopy of VX2 tumor in the auricle (A) The tumor is located in the subepidermal space and exhibits nodular growth (original magnification, 2×; scale bar, 1000 μm). (B) Areas of necrosis are present (indicated by 'n') (original magnification, 100×; scale bar, 200 μm). (C) High-power magnification reveals malignant tumor cells with pleomorphic, enlarged nuclei and mitotic activity (original magnification, 400×; scale bar, 50 μm). (D) Lymphovascular invasion is suspected (indicated by the arrow) (original magnification, 400×; scale bar, 50 μm). Main text A total of 6 rabbits were sacrificed. However, the tumor did not grow in one auricle in two rabbits; thus, histologic features were evaluated in 10 auricles (Fig 5). All tumors were located in the subepidermal space and characterized by nodular growth (Fig 5A). Histologically, necrotic areas were observed in all 10 tumors, with an average proportion of 27% (range, 5–50%) (Fig 5B). Tumor cells demonstrated malignant histologic features of pleomorphism and large nuclei, prominent nucleoli, and mitotic activity (Fig 5C). Epidermal damage, manifested as erosion and ulcer, was observed in three auricles (30%). Cartilage damage was observed in one auricle, and the tumor was observed in both the dorsal and ventral aspects of the cartilage. Lymphovascular invasion was suspected in 5 of the 10 auricles (50%) (Fig 5D). Reviewer #3 The author implanted VX2 tumors on the auricle of rabbits, and the study has certain significance. However, this manuscript needs to solve the following problems: 1.H&E figure needs to add ruler; - We greatly appreciate your comment. According to your comment, we have added a ruler to the H&E figure. 2.Previous studies have reported that VX2 tumors were implanted on the liver (PMID:35667125、36510170). What are the advantages of auricle tumor compared with VX2 tumor implantation in liver? - Thank you for your comment. The rabbit auricular VX2 tumor model has the following advantages over the rabbit hepatic VX2 tumor model: the tumor can be more easily accessed by inoculating it into the rabbit ear via a syringe, whereas the liver model generally requires laparotomy. In addition, in the auricular VX2 tumor model, the timing for additional experiments could be easily determined because the growth of the tumor could be directly confirmed with the naked eye. Furthermore, the tumor response can be visually confirmed without additional imaging tests after experiments such as intra-arterial embolization or ablation. We have added a paragraph about the comparison in the discussion section. Main text Compared to the rabbit hepatic VX2 tumor model, which is commonly used as an arterial embolization model, the rabbit auricular VX2 tumor model has several advantages. First, when preparing the model, the tumor can be more easily accessed by inoculating it into the rabbit ear via a syringe; thus, it is more convenient to prepare this model than preparing the hepatic VX2 tumor model, which generally requires laparotomy; moreover, there is a reduced chance of death of experimental animals due to complications of laparotomy. In addition, the VX2 tumor implanted in the liver requires additional imaging studies, such as CT or US, to determine whether the tumor growth is appropriate for conducting additional experiments such as arterial interventional studies. However, for the auricular VX2 tumor model, the optimal timing for additional experiments can be easily determined because the growth of the tumor can be directly confirmed with the naked eye. Unlike the liver model, the auricular VX2 tumor model can be visually inspected without additional imaging tests to observe the tumor response after experiments such as intra-arterial embolization or ablation. The VX2 tumor implanted in the liver is commonly used as an intra-arterial embolization model because the blood supply to the tumor from the hepatic artery is very similar to that observed in hepatocellular carcinoma. In particular, the hepatic VX2 tumor model is a hypervascular tumor and the blood supply of the tumor originates in the arterial system [3, 4]. However, although the VX2 implanted in the auricle is supplied from the auricular artery, not the hepatic artery, the tumor originates in the arterial system and has the characteristics of a hypervascular tumor. Therefore, compared to the rabbit hepatic VX2 model, the auricular model is appropriate to use as an arterial embolization model. However, since the surrounding tissues are different, such as the liver and auricle, their characteristics as an experimental model may be different when conducting an ablative study. 3. The P in statistical analysis is recommended to be capitalized italics. - We greatly appreciate your comment. According to your comment, we have revised the term to capital italics. Once again, we express our gratitude for your insightful comments. Attachments Attachment Submitted filename: Response_to_reviewers_(PONE)-v1.0.docx https://doi.org/10.1371/journal.pone.0316282.r002
9 Dec 2024 Decision Letter - Zhengwei Huang, Editor Feasibility of rabbit auricular VX2 tumor model as an experimental model for intra-arterial embolization PONE-D-24-24136R1 Dear Dr. Byung Kook Kwak, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Zhengwei Huang Academic Editor PLOS ONE Additional Editor Comments (optional): The manuscript is acceptable. 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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 current manuscript is acceptable as all the issues from the reviwers have been well addressed after revision. ****** 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: Yes: Qun Tang ******** https://doi.org/10.1371/journal.pone.0316282.r003
Formally Accepted
13 Dec 2024 Acceptance Letter - Zhengwei Huang, Editor PONE-D-24-24136R1 PLOS ONE Dear Dr. Kwak, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Zhengwei Huang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0316282.r004

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