Claisened Hexafluoro Inhibits Metastatic Spreading of Amoeboid Melanoma Cells

Round 1

Reviewer 1 Report

In the manuscript, Leo and co-workers examined whether Claisened Hexafluoro as a novel inhibitor of amoeboid motility of melanoma cells by modulating mitochondrial activity and activating the AMPK signaling. Usually metastatic melanoma cells can migrate different procedures depend upon the microenvironment including rounded/amoeboid-type motility and the elongated/mesenchymal-type motility. Importantly, the dissemination of highly invasive melanoma cells usually move the amoeboid motility as escaping route and metastasize and developing a complex clinical challenge. In addition, Claisened Hexafluoro is able to control with the adhesion abilities and the stemness features of melanoma cells. Hence, the authors suggest that the studies may provide the justification for future therapeutic usage of Claisened Hexafluoro for metastatic dissemination of melanoma. Overall the studies proposed a strong translation values for an unmet clinical need.

Comments:

1. There are some small editorial issues in this manuscript such as, in the Fig 1 legend, the “L” was not missing. The H, I and L of Fig 1 didn’t state what cells were used. Also, there several grammatical issues which need editorial attention.
2. Fig 1B, Rho GTP bands are overexpressed. They can use lower exposures of the RhoGTP bands.
3. Fig 2, the figure itself did not have any level properly. Also, number 3 panel, the confocal picture is not clear.
4. Fig 6 was the key experiments of the manuscript. However, the authors need to show the overall lung health which is not clear in the representative Fig 6B. Need more deeper molecular pathology to validate their results.

Author Response

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Reviewer 2 Report

In this manuscript, Leo et al reported the utility of a chemical analog of Honokiol, Claisened Hexafluoro, as a potential inhibitors of ameboid motility process in invasive melanoma. The authors presented a good set of in vitro experiments covering multiple aspects of cancer cell invasion followed with an in vivo lung colonization experiment. The authors showed that the potential MOA of this compound is through the dysregulation of mitochondrial output; the authors used seahorse to measure metabolic activities and showed AMPK signaling activation to show CH-induced decreased ATP production. The results presented were quite comprehensive, promising and pointed the utility of CH in melanoma.

However, my concern is that this is a single cell line study. It is hard to say whether all this good effects of CH is applicable to multiple ameboid melanoma lines or is it just A375M6? The comparison with the mesenchymal cell line HS294T were also done only one time (Figure 1). As such, additional validation experiments on other melanoma cell lines will be needed to show the potential generality of the proposed use of CH. Below are my specific comments:

1) Most of the experiments is based on A375M6 cell line. The authors should replicate important experiments (not all) to show that the CH compound can similarly prevent the invasive phenotype of another ameboid melanoma cells. The only other melanoma line tested was a mesenchymal HS294T line which were used as a "negative" control.

2) In figure 1, the authors seem to propose that the decrease in Rho-GTP and EPHA2 are indicative of an ameboid melanoma line (the decrease in the western blot seems quite weak because the loading control also showed similar slight decrease). Would HS294T line not show these decrease upon CH treatment?

3) In the same line, would a ROCK and/or EPHA2 inhibitors be able to mimic the effect of CH? If yes, then the authors can be convinced that the Rho-ROCK pathway is the main target of CH. 

4) The effect of 10uM of CH was tested in a 24 and 48hr time window. Has the author tested the survival of the melanoma cells over a longer period? It is possible that the CH is inducing cell death over a longer time period and the decreased motility is just an early phenomenon preceding cell death.

5) As the authors correctly pointed out, many of the MAPK inhibitor resistant lines are dependent on OXPHOS and some were shown to be sensitive to ROCK inhibitors. The authors should test the efficacy of CH on one or two of those lines.

Minor comments:
1) Figure 2 does not have the panel labels 
2) There are quite a few grammatical & lexical errors in the paper. English copy-editing is recommended

Author Response

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Reviewer 3 Report

In the manuscript entitled “Claisened Hexafluoro inhibits metastatic spreading of amoeboid melanoma cells” Leo et al . demonstrated the inhibition of ameboid metastatic dissemination of melanoma cells following the administration of the Claisened Hexafluoro (CH) compound. The rational of the study and the experimental setting are well presented. Overall the manuscript shows interesting results clearly organized for a straightforward read by a broad audience of readers.

However, minor adjustments could improve the study:

- In order to better define the stem-like phenotype of melanoma cells, authors should assess the expression of ABCB5 (PMID: 18202660) and CD271 markers (PMID: 20596026) on A375M6 cells.

- Authors should report mice tumor weight and liver and kidney morphology in order to exclude toxicity phenomena on organs following CH in vivo treatment.

- Figure 1L legend is missing.

- In Figure 2, panel letters are missing.

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Reviewer 4 Report

This article although only vitro compile a lot of adaptable methods to validate the conclusion propose. The article is really pleasant to read and very clear to follow one conclusion to another. In the conclusion I would have appreciate to find that all these results are only vitro and need to be confirm clinically with an appropriate targeting to get to the cancer cells 

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Reviewer 5 Report

The aim of the work by Leo and colleagues is to demonstrate that Claisened Hexafluoro (CH), a chemical analog of Honokiol, blocks melanoma metastatic dissemination by inhibiting amoeboid motility. The long-term goal is to propose CH for melanoma treatment.

The authors have investigated the effect of CH on two melanoma cell lines, A375M6 (amoeboid phenotype) or Hs294T (mesenchymal phenotype), showing that CH inhibits cell invasion only in A375M6. They have first characterized the toxicity level of CH onto the cells by using different concentrations, but the chosen concentration is then partially reducing cell survival (Fig 1A): why not choose 5 μM (100% cell survival at 24 and 48 hrs)? Is there a change in cell morphology during CH treatment? To prove that CH inhibits ameboid motility, the time-lapse experiment should be better investigated. Only 1 cell is shown in the video, and the video of the CH-treated cell is very rapid (it’s much shorter than the control) and it is not clear whether the cell moves away from the focus of the camera or simply dies. No quantification of the experiment nor indication of how many times the experiment has been done is included. The experiment should be done on Hs294T as well, to verify that CH treatment does not impact the type of motility in these cells in a context where cell motility can be better appreciated.

The authors suggest that the ameboid phenotype is associated with stem-like features, but very few references are indicated. Stemness in melanoma is still a controversial field, where specific markers have not been identified, or if tentatively identified, they have been disputed. As an example, a recent systematic review and meta-analysis (Madjd et al, DOI: 10.5301/jbm.5000209) shows that CD133 is not an appropriate biomarker to identify melanoma stem cells. That said, the markers are evaluated on adherent cells (Figure 3), known to show a reduced number of stem-like cells in culture when compared to melanosphere (Perego et al, https://doi.org/10.1038/jid.2010.69). The authors performed a melanosphere assay in vitro, where they claim that CH treatment reduces the number (and the size?) of the spheres. No quantification is provided, only 2 spheroids are shown in the figure! The experiment should be better addressed to draw solid conclusions: melanospheres should be counted and size annotated. Serial replating should be performed to evaluate self-renewal potential of control and CH-treated spheroids.

Metastatic dissemination is a multistep process involving different phases: cells have to leave the primary tumor, intravasate, survive in the body fluids, extravasate, and seed at distant sites (also shown in the graphical abstract). Lung colonization does not recapitulate the majority of these phases. As CH treatment decreases the adhesion of A375M6 cells to the endothelium it would be desirable to verify that metastases are reduced upon CH treatment in a spontaneous model of metastasis (A375M6 cells transplanted subcute, tumor resected, and metastases analysed at lymph nodes and distant sites).

Minor points:

• Few typos along the text should be corrected
• 1L lacks the legend
• Font size of Fig. 2 legend should be reduced
• Cells in the images of Fig. 2 cannot be seen
• The different morphology of mitochondria in Fig. 4 cannot be appreciated
• how metastases are calculated should be indicated (all lungs are paraffin-embedded? How many sections of how many lungs are examined? How many mice/group were analysed?)

Author Response

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Reviewer 6 Report

This is undoubtedly an experimentally sound and scientifically concrete manuscript that deserves to be published in CANCERS, providing that the authors will improve it as suggested in the following lines:

1. Figure 1L must be renamed as 1J, throughout the whole manuscript, including Figure legends.
2. A quantification graph for Fig. 3E is advised to be included in the revised manuscript version.
3. Fig. 4I does not illustrate mitochondria morphology, but their distribution in the cell; please, re-phrase accordingly.
4. Replace "NANOg" with "NANOG".
5. A protein of reference as control (e.g. Actin, or Tubulin) must be added in Fig. 5C.
6. Besides stemness, did authors examine the EMT activation program, which is known to significantly contribute to Invasion-Metastasis Cascade?? If not, western blotting and immunofluorescence profiling of Vimentin and E-Cadherin expression is strongly suggested to be examined.
7. Did authors examine the effect of CH on other kinase activities, such as Akt, p38, Erk1/2 and Junk1/2/3?? It is important to examine the response of other critical kinases, besides AMPK, to CH exposure.
8. Fig. 2 demonstrates that CH inhibits adhesion. This is contrary to its ability to block metastasis and metastatic spreading. Lower adhesion means higher detachment and elevated migration levels. Authors have to explain how the reduction in adhesion forces help melanoma cells to compromise and attenuate their metastatic capacities to migrate. This is an issue of major importance for the manuscript.
9. Fig. 1A shows that CH cannot affect melanoma-cell survival. However, as described in Figs. 4 and 5, cells exposed to CH are subjected to an energetic crisis, compromise in their mitochondrial functions and production of ROS species. How can cells retain their survival and growth capacities unaffected in the presence of CH, since CH can induce oxidative and energetic stress??? Authors must discuss this discrepancy in mechanistic detail. It is a very important issue for the clinical exploitation of CH in metastatic-melanoma biology.          

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Round 2

Reviewer 2 Report

The authors have updated and improved the manuscripts as suggested. The reviewer has no additional comments.

Reviewer 5 Report

The authors duly addressed all my concerns. They did not carry out one of the proposed experiments, but I do understand the reason and I hope it will be part of a follow-up manuscript.

Reviewer 6 Report

Accept.