Analysis of Cancer Stem Cell Markers in Various Histological Subtypes of Adrenocortical Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript CIMB-3333329

„Analysis of cancer stem cell markers in various histological subtypes of adrenocortical cancer” for Current Issues in Molecular Biology

 

Comments:

1. Please shorten the introduction, emphasizing the most important elements related to the work. Please pay more attention to indicate the significance of the receptors analyzed in the work in the context of ACC.

2. At the moment, the aim of the work is not fully defined. Please clarify it and clearly indicate what was the exact aim of the work.

3. Materials and methods: 2.1. Please confirm in the text that patients selected for the analyses, had not undergone previously any form of neoadjuvant treatment.

4. The value of the scale bar in the images is poorly visible. Please add information about the size of the scale bar in the figure caption.

5. Please add in the results section an information on the expression of selected receptors in the context of the advancement stage of the disease. Despite the small research group, such information may be interesting.

6. Discussion. Please elaborate on the significance of your own results. What is the significance of the expression of specific receptors for predicting, for example, 5-year survival, treatment efficacy, etc.? Do the obtained results indicate a significant importance of the tested receptors, or have their expression been only demonstrated without any specific correlations with other oncologically significant parameters?

Author Response

We appreciate the reviewer’s thoughtful and constructive feedback. Below, we provide detailed responses to each comment and describe the corresponding revisions made to the manuscript.

1.⁠ ⁠Please shorten the introduction, emphasizing the most important elements related to the work. Please pay more attention to indicate the significance of the receptors analyzed in the work in the context of ACC.

Thank you for this valuable suggestion. We have revised the introduction to focus on the most critical aspects related to the study. Specifically, we have condensed the background information and expanded the discussion on the significance of the analyzed receptors in the context of adrenal cortex. The updated introduction now provides a more concise and relevant overview.

2.⁠ ⁠At the moment, the aim of the work is not fully defined. Please clarify it and clearly indicate what was the exact aim of the work.

 

We appreciate your comment regarding the clarity of the study's aim. We have revised the manuscript to explicitly define the research objectives. The clarified aim emphasizes that the objective of this study was confirmation the presence and localization of the aforementioned markers of multipotency in the normal human adrenal gland tissue, as well as to identify and characterize them in various types of ACC.  This refinement ensures that the purpose of the study is clear and directly connected to the results presented.

 

3.⁠ ⁠Materials and methods: 2.1. Please confirm in the text that patients selected for the analyses, had not undergone previously any form of neoadjuvant treatment.

 

Thank you for pointing out this important detail. We have added a statement in the "Materials and Methods" section to confirm that none of the patients included in the analyses had undergone any form of neoadjuvant treatment prior to surgery. This addition ensures clarity and eliminates potential concerns about the patient selection process.

 

4.⁠ ⁠The value of the scale bar in the images is poorly visible. Please add information about the size of the scale bar in the figure caption.

 

Thank you for pointing this out. We have revised the figure captions to explicitly include the size of the scale bar for each image. This ensures that the information is clear and easily accessible to readers, addressing the visibility issues raised.

 

5.⁠ ⁠Please add in the results section an information on the expression of selected receptors in the context of the advancement stage of the disease. Despite the small research group, such information may be interesting.

 

Thank you for this valuable suggestion. We have added a subsection in the results section analyzing the expression of selected receptors (CD90 and LGR5) in the context of disease stage according to the ENSAT classification. Although no statistically significant differences were observed, we agree that presenting these findings provides a more comprehensive perspective. This information has been included in Section 3.1 and 3.2 of the revised manuscript.

 

6.⁠ ⁠Discussion. Please elaborate on the significance of your own results. What is the significance of the expression of specific receptors for predicting, for example, 5-year survival, treatment efficacy, etc.? Do the obtained results indicate a significant importance of the tested receptors, or have their expression been only demonstrated without any specific correlations with other oncologically significant parameters?

 

We appreciate this insightful comment. In the revised discussion, we have expanded on the significance of our findings. Specifically, we discuss the potential role of CD90 and LGR5 as prognostic markers in ACC. While CD90 expression was associated with disease-free survival, no correlations with other parameters were identified in this study. We acknowledge the need for further research in larger cohorts to assess their potential predictive value for 5-year survival and treatment efficacy. This expansion can be found in Section 4 of the revised discussion.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors did a good job revealing the presence of LGR5- and CD90-positive tumor cells in ACC. However, I have a few comments that might improve the manuscript.

1) The introduction section is very long with irrelevant information to the manuscript's topic. It can be shortened greatly to maintain the reader's attention.

2) In the material and method section: Histological evaluation not imaging (there are no images of the H&E stained slides)

3) How were the CD90+ cells counted? Visually or computerized? It is kind of hard to do it visually from these images. I recommend using a computerized system like Imagej

4) Please write the statistical analysis test under each table

5) Why you are using two different magnifications?

6) A reference is needed for the first paragraph of the discussion

7) The discussion has a lot of irrelevant information to the manuscript. At the same time not enough correlation between your findings and the previous studies on other organs

Author Response

We appreciate the reviewer’s thoughtful and constructive feedback. Below, we provide detailed responses to each comment and describe the corresponding revisions made to the manuscript.

1) The introduction section is very long with irrelevant information to the manuscript's topic. It can be shortened greatly to maintain the reader's attention.

 

Thank you for this valuable suggestion. We have revised the introduction to focus on the most critical aspects related to the study. Specifically, we have condensed the background information and expanded the discussion on the significance of the analyzed receptors in the context of adrenal cortex. The updated introduction now provides a more concise and relevant overview.

 

2) In the material and method section: Histological evaluation not imaging (there are no images of the H&E stained slides)

 

Thank you so much for your comment. We have corrected the subsections in the materials and methods in the article to make it more correct.

 

3) How were the CD90+ cells counted? Visually or computerized? It is kind of hard to do it visually from these images. I recommend using a computerized system like Imagej.

 

Thank you for your valuable comments and for drawing our attention to the incomplete presentation of the research method of our study. In this study, cell detection and quantification were performed using the open-source software QuPath (version: 0.5.1). The counting of CD90-positive cells was performed in five fields of view, each measuring 0.05 mm² (total area of 0.25 mm²), separated into areas for the tumor parenchyma and the subcapsular region. The most representative fields of view were selected based on morphological criteria.

A computerized approach was applied for cell counting within QuPath. CD90-positive cells were identified and quantified using an automated algorithm calibrated for staining intensity and cellular morphology. To ensure accuracy, the algorithm's parameters were adjusted according to the specific staining characteristics of the samples. Results were visually reviewed and validated by experienced pathologists to confirm the reliability of automated quantification.

Additionally, a semi-quantitative approach was used to assess LGR5 expression. Samples were scored on a scale from 0 to 3 in ascending order: 0 for negative samples or those with less than 10% stained tumor tissue; 1 for samples with 10 to 39% stained tumor tissue; 2 for samples with 40 to 79% stained tumor tissue; and 3 for tumors with 80% or morestained tumor tissue. Scores were determined independently by three pathologists who were blinded to the clinicopathological information.

 

4) Please write the statistical analysis test under each table.

 

We appreciate this suggestion for improving the clarity of the results. We have added details of the statistical tests used under each table in the manuscript. For example, we specified that the Kruskal–Wallis test was employed for comparisons between groups, and Spearman’s correlation analysis was used for assessing associations. These additions make the tables more informative and self-explanatory.

 

5) Why you are using two different magnifications?

 

We appreciate this question. Two different magnifications were used to enhance the visualization of the immunohistochemical findings. Lower magnification provided an overview of the tissue architecture and receptor distribution, while higher magnification allowed us to focus on the finer details, such as staining intensity and precise localization of positive cells. This approach ensured the reliability and reproducibility of our analysis.

 

6) A reference is needed for the first paragraph of the discussion

 

Thank you for bringing this to our attention. We have added appropriate references to support the statements in the first paragraph of the discussion. These references include recent studies on the incidence, prognosis, and therapeutic challenges associated with adrenocortical carcinoma (ACC). This ensures that the paragraph is well-supported and aligns with the standards of the journal.

 

7) The discussion has a lot of irrelevant information to the manuscript. At the same time not enough correlation between your findings and the previous studies on other organs

 

Thank you for pointing this out. We have thoroughly revised the discussion to remove irrelevant information and added comparisons of our findings with studies of CD90 and LGR5 expression in cancers of other organs, such as colorectal and gastric cancers. This provides context for the potential relevance of these markers in ACC and aligns our findings with broader oncological research. The revised discussion is now more focused and aligned with the manuscript's scope.

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, the authors investigated the expression and distribution of two markers, LGR5 and CD90, in different ACC types and compared the impact of their expression on patient survival. The authors had good intentions to inform the diagnosis and treatment of patients with ACC. However, there are several problems in this study: 1. Since CD90 and LGR5 are not recognized cancer stem cell markers, why did the authors only examine these two markers and not examine the expression and distribution of other recognized stem cell markers such as CD44 and CD133? 2. The authors pointed out that the expression of CD90 was correlated with the expression of KI67, but the authors did not show the staining results of KI67, nor did they provide the correlation analysis chart, suggesting an increase. 3. Cancer stem cells have high proliferation activity. Could the author try CD90, LGR5 and KI67 multi-index fluorescence immunohistochemical staining for comparison?

Author Response

We appreciate the reviewer’s thoughtful and constructive feedback. Below, we provide detailed responses to each comment and describe the corresponding revisions made to the manuscript.

1. Since CD90 and LGR5 are not recognized cancer stem cell markers, why did the authors only examine these two markers and not examine the expression and distribution of other recognized stem cell markers such as CD44 and CD133?

 

Thank you for your valuable comments and for highlighting the choice of markers in our study.
Many surface markers highly expressed in stem cells are also found in cancer cells, including TRA-1-60, SSEA-1, EpCam, ALDH1A1, LGR5, CD13, CD19, CD20, CD24, CD26, CD27, CD34, CD38, CD44, CD45, CD47, CD49f, CD66c, CD90, CD166, TNFRSF16, CD105, CD133, CD117/c-kit, CD138, CD151, and CD166 [10.4103/ctm.ctm_69_16]. Among these, CD44 and CD133 are indeed the most frequently used markers in CSC research and are also therapeutic targets in oncology.

We selected LGR5 and CD90 based on prior studies that demonstrated their presence and potential significance in various tumors. For example, LGR5 has been identified in head and neck, colon, and gastric tumors [10.1038/nature06196; 10.1371/journal.pone.0035486]. CD90 has been detected in high-grade gliomas [10.1074/mcp.M111.010744; 10.1227/01.neu.0000413227.80467.92], breast cancer [https://pmc.ncbi.nlm.nih.gov/articles/PMC4503077/pdf/ijcep0008-5105.pdf], as well as liver [10.1016/j.ccr.2008.01.013] and lung tumors [10.1309/AJCPM2Z4NGIIPBGE; 10.3892/or.2013.2784].

At the same time, we did not find immunohistochemical studies confirming the presence of stemness-related markers LGR5 and CD90 in normal human adrenal gland tissue or different types of adrenocortical carcinoma (ACC). Recognizing the importance of widely studied markers such as CD44 and CD133, we chose to focus on the less-studied markers LGR5 and CD90 to explore their potential diagnostic and prognostic value.

In future research, we plan to include CD44 and CD133 for a more comprehensive evaluation.

 

2. The authors pointed out that the expression of CD90 was correlated with the expression of KI67, but the authors did not show the staining results of KI67, nor did they provide the correlation analysis chart, suggesting an increase.

 

We thank the reviewer for this insightful comment and for carefully examining our work. However, we would like to clarify that, as stated in the manuscript, we did not observe a correlation between CD90 expression and Ki67 expression in our analyses. For this reason, we considered it unnecessary to include the staining results for Ki67 or a corresponding correlation analysis chart.

 

Nonetheless, we understand the importance of transparency and completeness in presenting data. If the reviewer believes it would add value, we are prepared to provide the staining results for Ki67 and the detailed statistical analysis in the revised manuscript. Please let us know if this would be helpful.

 

Thank you once again for your valuable feedback.

 

3. Cancer stem cells have high proliferation activity. Could the author try CD90, LGR5 and KI67 multi-index fluorescence immunohistochemical staining for comparison?

 

Thank you for your valuable feedback and recommendation for improving our manuscript. We greatly appreciate your attention to detail and the idea you have suggested. Unfortunately, within the scope of the current study, we are unable to perform multi-index fluorescent immunohistochemical staining for the markers CD90, LGR5, and KI67 due to the lack of necessary technical equipment.

Nonetheless, we understand the importance of comparing these markers for a deeper analysis of the proliferative activity of cancer stem cells and will certainly consider the possibility of simultaneous detection of CD90, LGR5, and KI67 in future studies.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is sufficiently improved, according to the comments.

Author Response

Спасибо за ваш позитивный и обнадеживающий отзыв о нашей рукописи. Мы высоко ценим время и усилия, вложенные в рассмотрение нашей работы, и ваши вдумчивые комментарии.

Reviewer 2 Report

Comments and Suggestions for Authors

Great job, thank you 

Author Response

Thank you for your positive and encouraging feedback on our manuscript. We greatly appreciate the time and effort on invested in reviewing our work and your thoughtful comments.

Reviewer 3 Report

Comments and Suggestions for Authors

"In lines 211-212, it is mentioned, 'A correlation analysis was conducted between the Ki67 proliferative index and the number of CD90+ cells beneath the tumor capsule and in the tumor parenchyma.' If the authors did not assess Ki67 expression, how did they conduct the correlation analysis?"

Author Response

Уважаемый рецензент,

Мы искренне благодарим вас за ваш подробный обзор нашей рукописи и за ценные комментарии и предложения. Ваши идеи внесли значительный вклад в улучшение качества и ясности нашей работы.

В ответ на ваш комментарий относительно корреляционного анализа мы уточнили методологию в пересмотренной рукописи. В частности, мы включили дополнительную информацию в раздел «Материалы и методы», чтобы объяснить, как индекс пролиферации Ki67 оценивался и включался в наш анализ. Кроме того, мы создали новый рисунок для визуального представления результатов статистического анализа, что, по нашему мнению, повышает прозрачность и понятность наших результатов.

Мы благодарны за возможность ответить на ваши вопросы и более уверены, что эти изменения усилили нашу рукопись. Еще раз спасибо за ваш вдумчивый обзор и за вклад в улучшение нашего исследования.

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you to the authors for their hard work.