Applications of Organoids and Spheroids in Anaplastic and Papillary Thyroid Cancer Research: A Comprehensive Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thanks for the opportunity to review this manuscript I think it is well-written and clearly reviews the field of organoids and spheroids in the more aggressive thyroid malignancies.  The diagrams and the logical flow of the narrative add value to the article.  The comments I would make are aimed at further improving the value and impact of the review.

1) The English is excellent, however the manuscript could be made more concise if some repetitive sections were revised, i.e. the benefit of 3D systems over 2D is mentioned several times. 

2) Abbreviations only need to be given in full on first use, after this the abbreviation will suffice.  Do remember however to include the abbreviations used in figures and tables in the legend.  Also a couple of abbreviations were not given in full.

3) The most important recommendation I would make is to strengthen the basis of Table 1.  The review claims to be "comprehensive", but the parameters that identified the papers included are not given.  It would be helpful if a PRISMA style approach could be added as that they the work is comprehensive within the boundaries set.  It's not that I think papers have been left out deliberately but I am sure that there are multiple studies in some areas and this body of data should be acknowledged.

4) Secondly, in respect of Table 1 it would be extremely helpful to somehow reflect the nearness of the devices to wider adoption, as the lack of standardisation is correctly highlighted as a limitation.  Most of the studies are relatively preliminary laboratory, or pilot translational studies, incorporating the robustness / number of repeats / patient samples tested would be beneficial.  This layer of detail would allow the Discussion and Conclusions to be more focused on the stat5e of the art, rather than just reviewing the individual studies; the field as a whole can be summarised.  

Author Response

• The English is excellent; however, the manuscript could be made more concise if some repetitive sections were revised, i.e. the benefit of 3D systems over 2D is mentioned several times. 

 

Answer: We thank the reviewer for their constructive feedback. In response, we have thoroughly revised the manuscript—particularly the Introduction and Section 2—to reduce redundancy related to the advantages of 3D over 2D culture systems. These benefits are now discussed in a more focused manner, ensuring that subsequent sections build upon rather than repeat this foundational information. We believe this restructuring enhances the overall conciseness and clarity of the manuscript.

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• Abbreviations only need to be given in full on first use, after this the abbreviation will suffice.  Do remember however to include the abbreviations used in figures and tables in the legend.  Also, a couple of abbreviations were not given in full.

Answer: Thank you for highlighting this important point. We have reviewed the manuscript thoroughly and ensured that all abbreviations are defined in full at first use. Specifically, we have expanded RAIRD, CSC, TIC, GPX4, and PLLA, PAX8, TSHr on their first appearance highlighted in red color.

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• The most important recommendation I would make is to strengthen the basis of Table 1.  The review claims to be "comprehensive", but the parameters that identified the papers included are not given.  It would be helpful if a PRISMA style approach could be added as that they the work is comprehensive within the boundaries set.  It's not that I think papers have been left out deliberately but I am sure that there are multiple studies in some areas and this body of data should be acknowledged.

Answer: Thank you for this constructive suggestion. While our review is designed as a representative and comprehensive within defined criteria narrative rather than a systematic review, we agree that clarity regarding study selection is essential. We have now included a brief description of the literature search strategy used to identify the studies summarized in Table 1 as a subsection entitled “4a. Study selection criteria”. This outlines the inclusion criteria and ensures transparency about how the data was compiled. Although a formal PRISMA framework was not applied, we aimed to provide a representative and balanced overview of published research using 3D models in thyroid cancer. This approach captures the breadth of recent developments while acknowledging that additional studies may exist outside our scope.

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• Secondly, in respect of Table 1 it would be extremely helpful to somehow reflect the nearness of the devices to wider adoption, as the lack of standardization is correctly highlighted as a limitation.  Most of the studies are relatively preliminary laboratory, or pilot translational studies, incorporating the robustness / number of repeats / patient samples tested would be beneficial.  This layer of detail would allow the Discussion and Conclusions to be more focused on the state of the art, rather than just reviewing the individual studies; the field as a whole can be summarised.  

Answer: Thank you for this valuable recommendation. To address your suggestion, we have added a new column to Table 1 titled “Translational Stage”, which provides a concise categorization of each study as either exploratory, preclinical, or clinically relevant. This coding reflects the study's use of patient-derived material, extent of biological validation, and proximity to clinical application. We believe this addition enhances the interpretability of the data and aligns with your suggestion to assess the translational maturity of the current body of work. We have also added brief reflections in the Future Perspectives and Clinical Implications paragraph (highlighted in red color) to summarize the current state of the field and identify areas requiring further validation and standardization.

Reviewer 2 Report

Comments and Suggestions for Authors

see attached review comments.

My ethical concern is that the way the manuscript is written it seems that each section is independently written- hence a lot of redundancy is observed. For example they keep describing the advantages to 3D cultures vs 2D cultures. In this journal is well-understood the advantages of organoid models. This makes me wonder if the manuscript was somewhat generated using Open AI or other means of combining AI generated statements.

 

Comments for author File: Comments.pdf

Author Response

• Introduction contains many redundant, generalized statements about organoids/spheroids. For example- last sentence of first paragraph. These models represent substantial advancements in our understanding of the mechanisms underlying the formation and progression of thyroid tumors, thereby facilitating the development of more precise diagnostic methods and personalized treatment strategies. 7^th sentence of third paragraph. In summary, employing organoids and spheroids in thyroid cancer research offers significant potential for deepening our understanding of the disease and for creating more effective diagnostic and therapeutic solutions.

Answer: Thank you for the valuable comments. We have substantially revised the Introduction to clearly outline the different types of thyroid cancers (DTCs, ATC, RR-PTC), their relative prevalence and clinical aggressiveness. We now describe standard treatment approaches including radioiodine therapy, and highlight the molecular alterations commonly associated with resistance, such as BRAF mutations, RET/PTC rearrangements, and NIS downregulation by adding new references (4, 7-16, 24,25 also highlighted in the reference list). This expanded clinical and molecular context better supports the need for physiologically relevant preclinical models. We have also removed the repeated generalized statements about 3D culture systems and refocused those sections to relate directly to thyroid cancer research challenges. Please find the changes in the revised manuscript marked in red.

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• Section 2- First paragraph has redundant statements from the introduction. Paragraph 2- Describes non-cancerous thyrocyte models- relevance to this review?? Section 2.2, last paragraph is redundant with previous Intro statements and should be deleted. • Section 3- First paragraph is redundant with Intro statements- delete. Subsection Organoids First paragraph is non-thyroid specific and redundant with Intro statements- delete. Subsection Spheroids- First paragraph describes historical spheroid models- delete, not necessary for this review. Second paragraph is largely redundant from earlier statements about spheroids- reduce content.

Answer: Thank you for the valuable suggestions. We agree that the first paragraph of Section 2 overlapped conceptually. Accordingly, we the revised the introduction part and removed the redundant content. The second paragraph originally aimed to explain the historical development of 3D thyroid models, including polarity correction in thyrocyte spheroids. We have revised this to clarify its relevance by linking these foundational models to the development of thyroid cancer-specific organoid platforms. Please find the changes in the revised manuscript marked in red.

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• Figure 1- Very generalized figure on 3D models- not thyroid specific and doesn’t add value to the review. It is also redundant with Figure 2 upper left corner.

Answer: We thank the reviewer for their constructive comments regarding the clarity and specificity of Figure 1. In response, we have revised the figure to clearly denote the thyroid-specific sources and outputs (e.g., “Thyroid tumor,” “Thyroid organoids/spheroids”), improving relevance to the review’s focus. We have also updated the figure legend to reflect these clarifications. We believe these adjustments improve the figure’s interpretability while retaining its conceptual purpose. Please find the changes in the revised manuscript marked in red.

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• Section 4- Redundant first paragraph from intro and previous descriptions of 3D models. Figure 2 is confusing and redundant- Disease modelling is shown in Figure 1. Drug screening is also described in Personalized Medicine section. What is the meaning of histological characterization in Drug Screening? The use of Antibody for Thyroid cancer? Are there examples of this? In disease modeling PDX goes to mouse cells?? How are embryonic tissue being used to model thyroid (disease) cancer?? Figure 2 legend- Third sentence states: “They enable gene profiling” How do 3D culture systems “enable” gene profiling? Drug testing? “creation” of biobank?

Answer: We thank the reviewer for the insightful observations regarding Figure 2.

To address these concerns, we have:

• Revised the figure legend for clarity and thyroid relevance.
• Removed or reworded ambiguous terms such as “enable gene profiling”
• Use of Antibody in Thyroid Cancer, has been replaced with the “Targeted antibody therapy” which refers to the growing interest in monoclonal antibodies and antibody-drug conjugates (ADCs) in thyroid cancer. For example, anti-PD-1 antibodies like pembrolizumab have been tested in ATC, and anti-EGFR and anti-VEGFR agents are being explored in clinical and preclinical studies.
• Regarding the "histological characterization" in the drug screening panel of Figure 2, we agree that the term may appear ambiguous. What we aimed to convey is that 3D models allow for post-treatment histological analysis—such as H&E or immunohistochemical staining—to assess tissue structure, proliferation, apoptosis, or biomarker changes in response to therapies. This provides valuable morphological and molecular insights that support drug efficacy evaluation. Therefore, we have changed the term to “morphological analysis”.
• High throughput drug screening in the personalized medicine section of the figure 2 has been replaced by “High throughput drug validation”.
• Patient derived xenografts in the disease modelling section have been replaced by the “PDX model in mice” and the mouse cells to “cancer cells” terminologies.

 

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Table 1 is very dense and hard to read- doesn’t provide added value to the review. How does the reader compare these studies, models, results from this table? Since these are described and referenced in the text its not necessary and is distracting for the reader.

Answer: We thank the reviewer for the valuable suggestion. However, we believe that, Table 1 adds significant value by offering a consolidated, comparative overview of organoid and spheroid studies in thyroid cancer research. To enhance its clarity, we have restructured the applications and effects column accordingly by shortening the descriptions, and have also add an additional column titled “Translational stage” which provides a concise categorization of each study as either exploratory, preclinical, or clinically relevant. A clarifying sentence has also been added to the introduction of Section 4 (highlighted in red color) to guide the reader on how to interpret the table effectively. Given the wide-ranging and evolving use of organoid and spheroid systems in thyroid cancer research, we believe that the table adds substantial value by offering a comprehensive, comparative snapshot of published studies, complementing the narrative in the manuscript.

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Section 4.1- Too descriptive of the references and its confusing what is important for PTC vs ATC. Why are studies on estradiol and ER being done in thyroid cancers? How is this related to the overall treatments and 3D models and how are they contributing to advancing our knowledge or changing practices??? Second paragraph should be described or stated earlier in the manuscript as it is thyroid cancer specific.

Answer: We appreciate this important suggestion. In response, we have revised Section 4.1 to clearly distinguish between findings relevant to papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma (ATC), improving thematic clarity. We also rephrased the reference to the estradiol–ER study to contextualize its significance in relation to hormone receptor profiling in PTC, particularly in light of emerging interest in sex-based differences and hormone-related signaling in thyroid cancer. While we have retained the original paragraph structure to maintain the logical flow of the applications section, we have ensured that thyroid-specific insights remain central to the discussion.

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Section 4.2- Is quite confusing as to the context of PTC and ATC. What do all these studies and findings mean? 4th paragraph- define what BI-847325 is and why/how it was used in the study.

Answer: Thank you for the constructive feedback. To improve clarity, we have revised Section 4.2 to explicitly distinguish between studies related to papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma (ATC), making the relevance and implications of each finding more apparent. Additionally, we have provided a brief explanation of BI-847325, including its mechanism of action and rationale for use in the referenced study, to enhance reader comprehension. Please find the changes in the revised manuscript marked in red.

 

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 Section 4.3- Should include more “OMICs” data that better correlate with personalized medicine besides the PDOs. Too generalized- for example, these organoid-based systems could eventually support real-time personalized treatment plans and customized regimens according to the tumor's genetic and phenotypic characteristics. How? What pathways or important markers for disease or response to treatments would be beneficial?? Speculate based on the references or knowledge from the authors opinions.

Answer: We thank the reviewer for this insightful comment. To address it, we have expanded the Future Perspectives and Clinical Implications section to include recent multi-omics studies on PTC, PDTC, and ATC that offer deep insights into the genomic, proteomic, transcriptomic, and metabolomic landscapes of thyroid cancer. These studies—although not originally performed in 3D models—identify actionable targets such as BRAF^V600E, TERT promoter mutations, RET/PTC rearrangements, immune subtypes, and metabolic signatures, which can now be functionally validated and modeled using PDOs and spheroids (cited as references from 84-88). This integration provides a strong conceptual bridge between omics-derived insights and 3D culture-based drug screening or biomarker testing. We have also added a very important study which is a single arm phase 2 study describing the role of PDOs in precision treatment of locally advanced thyroid tumor (cited as reference no 43) in the table as well as in the section 4.3

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Section 4.4- Too descriptive on the studies. How does TME, EMT and stemness play a role in thyroid cancer development, strategies of treatment, prognosis, etc.? How are these important in PTC and ATC? Last 3 paragraphs are not related to this subsection and should be separate section.

Answer: We sincerely thank the reviewer for this valuable observation. To address these concerns, we have revised Section 4.4 to explicitly clarify the functional relevance of the tumor microenvironment (TME), epithelial-mesenchymal transition (EMT), and cancer stemness in thyroid cancer development, progression, and treatment resistance—especially in relation to papillary (PTC) and anaplastic (ATC) thyroid carcinomas. Additional linking sentences have been inserted to better contextualize the importance of these biological processes in therapeutic planning and prognosis.

Furthermore, while the final three paragraphs were originally placed to illustrate advanced culture systems and biomechanical cues affecting thyroid cancer models, we have retained them in this subsection for cohesive narrative flow. Please find the changes in the revised manuscript.

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Current challenges of 3D models of thyroid cancer. Needs to be more specific to thyroid cancer models and not generalized.

Answer: We sincerely thank the reviewer for this thoughtful observation. While we agree that disease-specific insights are essential, this section was to highlight broadly applicable technical and biological limitations of 3D models that are universally relevant across cancer types. These foundational challenges—such as variability in matrix components, lack of vascularization, and difficulty incorporating immune components—directly impact thyroid cancer research, as many thyroid-specific studies are still adapting these platforms for their use, therefore we have retained the original content related to that part.

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• Section 6 (which should be section 5??)- Future perspectives and clinical implications is well written, seems to be in a slightly different style of writing, but is still relatively non-specific to thyroid cancer. Paragraph 2 is non-cancer models of thyroid and not relevant to the review.

Answer: We appreciate the reviewer's positive feedback on the "Future Perspectives and Clinical Implications" section. In response, we have revised the section by integrating thyroid cancer-specific multi-omics studies and references to emerging molecular subtypes in papillary and anaplastic thyroid cancers to enhance the translational relevance of 3D models. We retained the non-cancer thyroid models content in Paragraph 2 as it demonstrates foundational advances in thyroid-specific organ-on-a-chip and microfluidic technologies, which are increasingly adapted for thyroid cancer modelling and drug testing. We added transitional phrases to align this content with the review's goals. The refined section now provides a thyroid cancer–centered outlook while maintaining key technological insights for future directions.

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• Conclusions section is highly redundant and should be deleted.

Answer: We appreciate the reviewer’s observation. While we agree that the original conclusion had some overlap with earlier sections, we believe that a concise conclusion is essential to summarize key insights and implications of the review. In response, we have revised the conclusion to reduce redundancy, emphasize thyroid cancer–specific insights, without repeating previously detailed content. We hope this streamlined version better aligns with the expectations of the journal and reader while maintaining coherence and closure for the manuscript.

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• “Physiologically relevant” is used 9 times in the manuscript!

Answer: We thank the reviewer for pointing this out. We have carefully revised the manuscript to reduce the repetitive use of the phrase "physiologically relevant" and replaced it with suitable alternatives to improve variety and readability (highlighted in red color in the manuscript).

Reviewer 3 Report

Comments and Suggestions for Authors

The review 'Applications of Organoids and Spheroids in Anaplastic and Papillary Thyroid Cancer Research: A Comprehensive Review' gives a comprehensive overview of the use of 3D cultures in the context of Thyroid Cancers. 

In my opinion, the review is well written and relevant literature cited.

Just a minor note:

Line 39: PDTCs = please specify the acronym.

 

Author Response

The review 'Applications of Organoids and Spheroids in Anaplastic and Papillary Thyroid Cancer Research: A Comprehensive Review' gives a comprehensive overview of the use of 3D cultures in the context of Thyroid Cancers. 

In my opinion, the review is well written and relevant literature cited.

Just a minor note:

Line 39: PDTCs = please specify the acronym.

Answer: Thank you for valuable observation. We have now defined “PDTCs” as “poorly differentiated thyroid carcinomas” at its first mention in the manuscript to ensure clarity, highlighted in red color.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thanks for responding and making some substantial changes to the review.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors did an excellent job at addressing the previous review concerns. The introduction is well-written and describes the context of treatment and organoid models to thyroid cancer disease and standard care. These significant improvements allow for a greater understanding of the field and summarizes the previous 3D organoid models very well.