Unravelling the metastasis-preventing effect of miR-200c in vitro and in vivo

  1. Bianca Köhler
  2. Emily Brieger
  3. Tom Brandstätter
  4. Elisa Hörterer
  5. Ulrich Wilk
  6. Jana Pöhmerer
  7. Anna Jötten
  8. Philipp Paulitschke
  9. Chase P Broedersz
  10. Stefan Zahler
  11. Joachim O Rädler
  12. Ernst Wagner
  13. Andreas Roidl

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Abstract

Advanced breast cancer as well as insufficient treatment can lead to the dissemination of malignant cells from the primary tumor to distant organs. Recent research has shown that miR-200c can hamper certain steps of the invasion-metastasis cascade. However, it is still unclear, whether sole miR-200c expression is sufficient to prevent breast cancer cells from metastasis formation. Hence, we performed a xenograft mouse experiment with inducible miR-200c expression in MDA-MB 231 cells. The ex vivo analysis of metastatic sites in a multitude of organs including lung, liver, brain, and spleen has revealed a dramatically reduced metastatic burden of mice with miR-200c expressing tumors. A fundamental prerequisite for metastasis formation is the motility of cancer cells and, therefore, their migration. Consequently, we analyzed the effect of miR-200c on collective and single cell migration in vitro , utilizing MDA-MB 231 and MCF7 cell systems with genetically modified miR-200c expression. Analysis of collective cell migration has resulted in confluence dependent motility of cells with altered miR-200c expression. Additionally, scratch assays have shown enhanced predisposition of miR-200c negative cells to leave cell clusters. The in-between stage of collective and single cell migration was validated using transwell assays, which have displayed reduced migration of miR-200c positive cells. Finally, to measure migration on single cell level, a novel assay on dumbbell shaped micropatterns was performed, which revealed that miR-200c critically determines confined cell motility. All of these results demonstrate that exclusive expression of miR-200c impedes metastasis formation in vivo and migration in vitro and highlight miR-200c as metastatic suppressor in breast cancer.

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    Referee #3

    Evidence, reproducibility and clarity

    Bianca Köhler and colleagues investigate the influence of miR-200c on cancer cell migration through a series of in vitro and in vivo experiments. After inducing miR-200c overexpression in MDA-MB 231 cells with doxycycline treatment, the researchers observe a reduction in metastases when these cells are implanted into mice. Using live imaging analysis, the authors found that MCF7 cells lacking miR-200c display increased mobility compared to wildtype cells, particularly at low cell density. Conversely, MDA-MB 231 cells with miR-200c overexpression show decreased mobility, irrespective of cell density in culture. Scratch assays show a diminished invasive capacity of MDA-MB 231 cells with heightened miR-200c expression. This finding aligns with results from transwell assays, where wildtype MCF7 cells exhibit reduced migration compared to MCF7 cells lacking miR-200c. In examining the impact of miR-200c on single-cell migration, a micropattern assay with two square islands connected by a thin bridge reveals a decrease in both transition frequency and transition speed of migrating MDA-MB-231 cells upon miR-200c expression.

    In summary, this study provides a comprehensive exploration of the effects of miR-200c on cancer cell migration in various experimental contexts, offering insights into potential therapeutic implications.

    The manuscript exhibits clear and articulate writing, coupled with well-explained experiments. To enhance its value, a more thorough characterization of miR-200c's mechanism of action, validation of its targets, and a more detailed analysis of in vivo metastases would be beneficial.

    The manuscript's novelty appears limited, as the role of miR-200c as a tumor suppressor and its association with decreased metastatic potential in breast cancer cells have been previously documented (Klicka et al., Front Oncol 2022; Ljepoja et al., Plos One 2019). Highlighting unique contributions or contextualizing findings within existing literature would strengthen the manuscript's distinctiveness.

    Comments to the authors: I recommend integrating these suggestions into the manuscript to enhance its scientific rigor and relevance.

    Metastases Characterization:

    Consider providing histological images illustrating the distribution of cancer cells in metastatic organs. This visual representation could offer readers valuable insights into the nature and characteristics of metastases arising from MDA-MB 231 cells.

    Tumor Growth Impact:

    Address the potential impact of tumor growth on metastatic dissemination by correcting for variations in primary tumor size when quantifying metastases in vivo. Accounting for this variable will strengthen the reliability and interpretation of the results.

    Control Experiments:

    Strengthen the experimental design by including a scrambled miRNA sequence as a control. This addition will contribute to a more robust comparison, ensuring observed effects are specifically attributed to miR-200c.

    Target Validation for Mechanistic Insights:

    Improve the understanding of miR-200c's mechanism of action by validating some of its natural targets. This step will provide a more solid foundation for interpreting experimental outcomes and unraveling the intricacies of miR-200c function.

    Clinical Correlation:

    Explore the possibility of correlating miR-200c expression with the progression of specific tumor diseases in patients. This potential correlation could contribute valuable clinical insights to the manuscript.

    Translational Potential:

    Once natural targets of miR-200c are validated, explore the translational potential by investigating whether these targets can be targeted by available drugs. Testing these drugs in tumor mouse models would further assess their efficacy and potential clinical applications.

    Significance

    In summary, this study provides a comprehensive exploration of the effects of miR-200c on cancer cell migration in various experimental contexts, offering insights into potential therapeutic implications.

    The manuscript exhibits clear and articulate writing, coupled with well-explained experiments. To enhance its value, a more thorough characterization of miR-200c's mechanism of action, validation of its targets, and a more detailed analysis of in vivo metastases would be beneficial.

    The manuscript's novelty appears limited, as the role of miR-200c as a tumor suppressor and its association with decreased metastatic potential in breast cancer cells have been previously documented (Klicka et al., Front Oncol 2022; Ljepoja et al., Plos One 2019). Highlighting unique contributions or contextualizing findings within existing literature would strengthen the manuscript's distinctiveness.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Kohler et al analyze the impact of miR200c on cell motility in vitro and breast cancer metastasis in mouse models. The they show that miR200c represses metastasis to several different organs and propose that reduced motility is a significant cause of this. The experiments are generally sound and well performed. However, the insight gained with the study does not go much beyond what is already known about miR200c function in breast cancer. The experimental tools used in the study could provide the opportunity to reveal novel insights into the role of miR200c in metastasis. However, the investigators did not take full advantage of this and thus we are left with findings that are rather predictable based on the current literature. Details below.

    Major points:

    1. The primary weakness of this study is limited novelty. miR200c has been shown to regulate migration and invasion of breast cancer cells in several previous studies, and this includes analysis using the same breast cancer cell lines that Kohler et al use in the current study, MCF7 and MDA-MB-231 (Jurmeister et al Mol Cell Bio 2012; Zhang et al Genet Mol Res 2017) and a study by the same group (Ljepoja et al Plos One 2019). Moreover, previous studies have also shown that miR200c represses metastasis in two different claudin low triple negative breast cancer models, MDA-MB-231 and genetically-engineered p53 null transplantable model (Simpson et al Genes 2022, Knezevic et al Oncogene 2016). Of note, Kohler et al do analyze metastases not only in lungs, but also in liver, brain and spleen and this could be a source of novel insights depending on the scientific questions. Is the miR200c mediated repression of metastasis caused by the same mechanisms in all these organs, or is it context dependent? What about molecular mediators downstream of miR200c?
    2. The authors focus primarily on migration issues as the potential cause of miR200c mediated repression of metastasis. However, there is significant literature on the role of miR200c in cancer progression. miR200c has been associated with multiple cellular functions, including regulation of epithelial mesenchymal transition (EMT) by repressing key EMT transcription factors ZEB1 and ZEB2. EMT regulation of course may suggest an effect on cell motility, but also several other functions, such as stem cell activity, plasticity, survival under stress and many more. Indeed, in a clinical setting some may question the importance of migration, considering that breast cancer cells disseminate from the primary tumor early in the process and upon diagnosis the cells are likely already lodged in secondary organs. Therefore, it is probable that cell functions such as survival under stress, proliferation and plasticity would be of even higher importance compared to cell motility. I would think that miR200c functional studies need to go beyond cell motility to generate additional insights into its role in metastasis and reveal potentially actionable targets.
    3. The investigators use a dox inducible system to express miR200c in MDA-MB-231 mammary tumors in mice. The mice were treated with dox to induce miR200c when the tumors reached 200 mm3 in size. This is a rather early induction of miR200c and may not address the ability of miR200c to repress actively growing metastatic lesions. I think these experiments should also be done by waiting longer before miR200c induction. What happens if the tumors are allowed to grow to 500 mm3 or 750 mm3? This would really test the ability of miR200c to inhibit overt metastasis.

    Minor points:

    1. Although in some figures the plots/graphs show individual data points, this is not always the case. All box plots and bar graphs should show individual data points (biological replicates).
    2. Representative histological examples of the metastases in Figure 1C-1D should be shown.
    3. Presentation of the data in Figure 2C-2F is confusing. Statistics are also missing.

    Significance

    Although the study is technically sound, it suffers from limited novelty. Overall conclusions are predictable from previous studies. Of note, this study does provide somewhat more detailed analysis of migratory regulation by miR200c in cancer cells compared to previous reports. However, the study's advance is still quite modest.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary: in this manuscript, Kohler and coworkers describe the role of miR-200c in preventing breast cancer cell migration in vitro and metastasis in vivo (using sub cutaneous injections of human breast cancer cell lines in nude mice). The novelty of this manuscript resides in the in vivo work, as the role of miR-200c in preventing cell migration and EMT in vitro is widely established (and recognised in this manuscript).

    Major comments:

    • the authors need to measure miR-200c expression in their experimental systems. Here they described a DOX-inducible system to express miR-200c in MDA-MB-231 cells and they used KO MCF7 cells, but the levels of miR-200c are not reported at all in the manuscript. It is essential to show that DOX treatment induces miR-200c expression both in vitro and in vivo.
    • the experiments presented in figure 4 do not contain the appropriate controls. In all the other figures, inducible MDA-MB-231 cells are presented, in the presence and absence of DOX. However, in this figure WT MDA-MB-231 cells are compared with the inducible variant in the presence of DOX. All the experiments in this figures need to be repeated with the inducible cells in the absence of DOX
    • in the would healing experiments (figure 3), both KO and induction of miR-200c result in increased migration (which is not consistent with the rest of the data shown in the paper). This point should be explained more clearly in the discussion. In addition, the behaviour of the cells in the absence of DOX (figure 3G) seems very different in the control vs miR-200c cells (figure 3E) - this issue needs to be addressed in the discussion, as it could suggest that other factors independent of miR-200c expression might contribute to the difference between the 2 cell lines.
    • in some instances, the authors draw conclusions from data that are not statistically significant, as in supplementary figure S2A and B, in relation to which the authors state 'both analysis were additionally validated... by crystal violet staining', but the quantifications show no significant differences
    • all the migration experiments in vitro are in 2D. This should be highlighted as a limitation of this study. In addition, it is not appropriate to describe migrating cells as 'invasive', when this was not assessed in the experiment.

    Minor comments:

    • it is not clear what the difference between figure 4 A and B is
    • it would be good to better clarify the rational behind and the physiological relevance of the confined cell motility experiment
    • the authors measured differences in tumour volume it vivo, therefore it would be useful to assess cell proliferation in vitro as well. This is also important as proliferation can impact the cell migration assays used in this study.
    • MCF7 cell migration is minimal, making it difficult to draw meaningful conclusions from these experiments. Longer migration times might be helpful here
    • I was not able to open the supplementary videos, so I cannot comment on them.

    Significance

    General assessment: the strongest aspect of the study is the characterisation of the role of miR-200c expression in metastasis formation. However, the study lacks several controls. In my opinion, the in vivo work should be expanded, as the in vitro is mostly a confirmation of previous work. The data seem to hint to potential effect in organo-tropism, which warrant further investigation.

    Advance: the in vivo work is novel, extending the knowledge of miR200c role in metastasis, while most of the in vitro work is incremental or confirmatory.

    Audience: cancer biology researchers will mostly be interested in this work. There is potential for translational implications, but this needs to be strengthen.

    I am a cancer cell biologist, expert in cancer cell migration and invasion. Most of my expertise is in 2D and 3D in vitro models, but I am also very familiar with mouse breast cancer models. I don't have sufficient expertise to comment on the analysis of the confined cell motility assay.

  5. Version published to 10.1101/2023.11.14.566527v1 on bioRxiv