Comparison of urine proteomes from tumor-bearing mice with those from tumor-resected mice

  1. Heng Ziqi
  2. Zhao Chenyang
  3. Gao Youhe

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Abstract

[Objective]

This study focuses on the most important concern of surgeons - whether they resected all of the tumor. Urine can reflect early changes associated with physiological or pathophysiological processes. Based on the above ideas, we conducted experiments to explore changes in the urine proteome between tumor-bearing mice and tumor-resected mice.

[Method]

The tumor-bearing mouse model was established with MC38 mouse colon cancer cells, and the mice were divided into the healthy control group, complete resection group, and nonresection group. Urine was collected 7 days and 30 days after resection. Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) was used to identify the urine proteome and then analyze differentially expressed proteins and biological pathways.

[Results]

(1) Seven days after tumor resection, there were 20 differentially expressed proteins that could distinguish between the complete resection group and the nonresection group. The biological process includes circadian rhythm, Notch signaling pathway, leukocyte cell–cell adhesion, and heterophilic cell–cell adhesion via plasma membrane cell adhesion molecules. (2) Thirty days after tumor resection, there were 33 differentially expressed proteins that could distinguish between the complete resection group and the nonresection group. The biological process includes cell adhesion, complement activation, the alternative pathway, the immune system process, and angiogenesis. (3) There was no significant difference between the two groups at 30 days after tumor resection between the complete resection group and the healthy control group.

[Conclusion]

Changes in the urine proteome can reflect tumors with or without complete resection.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7711832.

    Comparison of urine proteomes from tumor-bearing mice with those from tumor-resected mice

    Heng Ziqi et al have developed a diagnostic tool using urine biomarker analysis for post tumor resection. The disappearance of protein biomarkers after resection helps in determining the success of the surgical procedure. For the purpose of the study, 10 mice were injected with the MC38 cell line into the right hind limb, and 5 mice served as healthy controls. They had similar surgical procedures to ensure that the changes due to wound healing do not overlap with the tumor resection process. There are some points that I would like to highlight in the study.

    Major Issues regarding the study:

    1. For chronic conditions (like cancer), the serum biomarker will not diminish immediately after treatment/surgery. If we consider appropriate time points, we can highlight different aspects of the pathophysiology condition of the subject. Hence we cannot discount the importance of blood biomarker discovery.

    2. In order to test the reproducibility of the study, the consistency of biomarkers among different cell lines should be tested.

    3. In order to eliminate the chances of false association of suggested biomarkers with the changes post-resection, multiple time points are required. If the changes in the tumor microenvironment hamper the wound healing response compared to healthy wildtype, then there is a chance that the biomarkers reported could be due to slow healing of the tissue.

    4. If there would have been partial resection of the tumor, then we might get a few more biomarkers as the effect of tumor regrowth and the metastasis response by the body would have been more realistic scenario.

    5. The changes in the urine proteome depend on various factors. If it is considered urine as a sink for waste disposal in case of chronic conditions, then the changes could be rapid. In such cases, the proteome should be monitored continuously to validate if the changes are really due to treatment or amelioration of the chronic condition.

    6. The process of change in the urine proteome biomarker could be further elucidated from the data. Linking the results with the processes involved in the tissue damage repair mechanism and withdrawal of immune response can provide insight into the difference between complete resection and the condition between primary tumor disappearance and secondary tumor appearance.

    Minor issues which could have been addressed in this article:

    1. The cancer progression in different tissue should be considered while developing this diagnostic tool.

    2. If the changes in the serum biomarker would have been considered, then this would have been a more comprehensive study.

    3. In human cancer patients, the changes in urine biomarkers are largely affected by ongoing therapy, diet, and multiple surgical procedures. In that case, how does the author suggest that we should plan out urine collection?

    4. With the MC38 cell lines the author has established the time period for this study. This ensures that the biomarker clears out in case of complete resection. But this does not consider the cases where cancer cell dormancy occurs.

    Competing interests

    The author declares that they have no competing interests.

  2. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6781847.

    Heng Ziqi et al have asked a basic question about differentiating between the individuals with completely resected tumors and ones without complete tumor resection by comparing their urine proteomes at two different time points. The experiments were conducted on fifteen rats, which were divided into three groups of five rats each. The authors carried out a reasonable scramble analysis to show the significance of their findings. However, I have several major concerns with their work, which I have described below:

    Major Comments

    1. While the authors satisfactorily show that upon complete resection, the urine proteome of the tumor-bearing rats reverts to a state similar to that of the healthy rats, the reverse causality relation is not sufficiently convincing. The authors have not compared their results to a case where the tumor is partially resected. For the completeness of the study, I would suggest the authors to include tumor-bearing rats from a "partial tumor resection" category with partially resected tumor and check if their proteomes show any differences. Also, I would suggest the authors to discuss the assumptions of the study explicitly while describing their conclusions towards the end of results section or in Discussions section.
    2. The authors have cited a few studies which indicate that the position of tumor implantation dictates changes in urine proteome. In addition, several other factors also lead to changes in urine proteome.
    3. Also, the effect of using another cancer cell line on urine proteome is currently not discussed. I would suggest the authors to check if their results hold when they conduct this study using another cell line.

     

    Minor Comments

    1. In the second paragraph of Introduction section, the authors have included phrases like "stable and balanced" and disturbing factors, which lack specifics. I would suggest avoid these.
    2. In the third paragraph, the authors have cited some previous studies conducted with Walker-256 tumor bearing rats, which show that the location of the tumor implantation dictates the changes in the urine proteome. It would be helpful if this is mentioned explicitly in the text.
    3. In the last paragraph of Introduction, a more complete explanation of the experimental procedure is needed. Also, concluding the Introduction with a summary of results and the implications of the findings of the study will provide the reader with a complete picture of the background and that of the implications of this study.
    4. The authors have done a few preparatory steps before they established the tumor bearing models. It would make the figure 1 more complete if the authors include these steps in the figure. Also, I would suggest the authors to specify more details instead of simply mentioning "Bioinformatics analysis" in this figure.
    5. In figure 2 for the Non-resection group, the authors have used the word recurrence incorrectly. It is misleading to call it a case of recurrence as the original tumor was never resected.
    6. In 3.2 section of the Results, there are sentences that begin with "In the cellular component category…" which are confusing. I failed to understand what the authors are trying to convey. I suggest to reword these.
    7. Figures 4 and 5 need a more detailed legend as it is difficult to understand the take home messages from sub-figures b and c for both of these figures.
    8. In first paragraph of section 3.2.3 of Results, saying "level of resection of the tumor" is misleading and needs rewording.

     

  3. Version published to 10.1101/2022.06.08.495253 on bioRxiv