Cancer cell lines show high heritability for motility but not generation time

Tumour evolution depends on heritable differences between cells in traits affecting cell survival or replication. It is well established that cancer cells are genetically and phenotypically heterogeneous; however, the extent to which this phenotypic variation is heritable is far less well explored. Here, we estimate the broad-sense heritability (H2) of two cell traits related to cancer hallmarks––cell motility and generation time––within populations of four cancer cell lines in vitro and find that motility is strongly heritable. This heritability is stable across multiple cell generations, with heritability values at the high end of those measured for a range of traits in natural populations of animals or plants. These findings confirm a central assumption of cancer evolution, provide a first quantification of the evolvability of key traits in cancer cells and indicate that there is ample raw material for experimental evolution in cancer cell lines. Generation time, a trait directly affecting cell fitness, shows substantially lower values of heritability than cell speed, consistent with its having been under directional selection removing heritable variation.


Comments to the Author(s)
The manuscript entitled "Cancer cell lines show high heritability for motility but not generation time" by Wass et al. tracked cell migration and proliferation of hundreds of cell family from multiple cells to test whether motility and proliferation rate are inheritable. The authors did a lot of works and provided interesting data. However, there are limitations in this study. The authors didn't compare what they found with relevant previous works. In addition, there are concerns regarding data processing/selection and alternative interpretation of the collected data. It is recommended that this paper can be revised before being accepted. The detailed comments are listed below: 1. There were some previous works regarding whether the cell motility can be inherited in the literature. The work from Yan et al is a short-term study more similar to the presented work. They saw low correlation between two daughter cells. The works from Chen et al are long-term heritability studies. They showed cellular motility and altered gene expression can be inherited. It is recommended that the authors should explicitly discuss what's new in the present work and also why the authors got similar or different conclusions. As the measurement methods are different, it is totally fine to get different observations. However, it would be essential to discuss relevant previous works for comparison. Three relevant papers are listed below: "Stochastic variations of migration speed between cells in clonal populations" "Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations" "Single-cell RNA-sequencing of migratory breast cancer cells: discovering genes associated with cancer metastasis" The authors claim that "22.7% of cells tracked (2048 individual cells) met the requirements to be used in the analysis." Excluding majority of the data can be biased. Other reviewers raised similar concerns as well. It is recommended that the authors should better explain how they excluded data? For example, the data were excluded for three possible reasons. x% for A reason, y% for B reason, and z% for C reason. Also, if including those excluded data, will it change the conclusion?
It is recommended that the authors can discuss the possibility that cellular motility was driven by secreted signals (e.g. secreted proteins/exosome). If secreted signals are the dominant drivers of cell motility. Neighboring cells naturally share similar motility. It may not be caused by heritability as suggested by the authors. Further data analysis maybe necessary to eliminate this concern.
Review form: Reviewer 2 Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept as is

Comments to the Author(s)
Congratulations on a very nice piece of work. I look forward to citing it.

Review form: Reviewer 3
Is the manuscript scientifically sound in its present form? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? Yes

Recommendation? Accept with minor revision (please list in comments)
Comments to the Author(s) I am satisfied with the authors' replies, bar two minor comments: 1. Could the data showing the speed of cells that move off-screen before before dividing (0 daughters) vs cells which divide on-screen (min 2 daughters) be shown? 4. Can formula used for the parent-offspring regression be shown? I presume given the reply that h is then the variance -for someone outside the field such as myself this would be useful to see.

21-Jan-2020
Dear Dr Dash, The editors assigned to your paper ("Cancer cell lines show high heritability for motility but not generation time") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Thank you for your patience while the paper was reviewed -unfortunately, we had some difficulty in soliciting suitable reviewers to assess your manuscript; however, 3 reviewers have now reported. Two are broadly in favour of publication, but the concerns raised by the first reviewer are substantial and we would ask you to respond to these effectively -the revision you submit will be sent to this reviewer for consultation.
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) The manuscript entitled "Cancer cell lines show high heritability for motility but not generation time" by Wass et al. tracked cell migration and proliferation of hundreds of cell family from multiple cells to test whether motility and proliferation rate are inheritable. The authors did a lot of works and provided interesting data. However, there are limitations in this study. The authors didn't compare what they found with relevant previous works. In addition, there are concerns regarding data processing/selection and alternative interpretation of the collected data. It is recommended that this paper can be revised before being accepted. The detailed comments are listed below: 1. There were some previous works regarding whether the cell motility can be inherited in the literature. The work from Yan et al is a short-term study more similar to the presented work. They saw low correlation between two daughter cells. The works from Chen et al are long-term heritability studies. They showed cellular motility and altered gene expression can be inherited. It is recommended that the authors should explicitly discuss what's new in the present work and also why the authors got similar or different conclusions. As the measurement methods are different, it is totally fine to get different observations. However, it would be essential to discuss relevant previous works for comparison. Three relevant papers are listed below: "Stochastic variations of migration speed between cells in clonal populations" "Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations" "Single-cell RNA-sequencing of migratory breast cancer cells: discovering genes associated with cancer metastasis" The authors claim that "22.7% of cells tracked (2048 individual cells) met the requirements to be used in the analysis." Excluding majority of the data can be biased. Other reviewers raised similar concerns as well. It is recommended that the authors should better explain how they excluded data? For example, the data were excluded for three possible reasons. x% for A reason, y% for B reason, and z% for C reason. Also, if including those excluded data, will it change the conclusion?
It is recommended that the authors can discuss the possibility that cellular motility was driven by secreted signals (e.g. secreted proteins/exosome). If secreted signals are the dominant drivers of cell motility. Neighboring cells naturally share similar motility. It may not be caused by heritability as suggested by the authors. Further data analysis maybe necessary to eliminate this concern.
Reviewer: 2 Comments to the Author(s) Congratulations on a very nice piece of work. I look forward to citing it.
Reviewer: 3 Comments to the Author(s) I am satisfied with the authors' replies, bar two minor comments: 1. Could the data showing the speed of cells that move off-screen before before dividing (0 daughters) vs cells which divide on-screen (min 2 daughters) be shown? 4. Can formula used for the parent-offspring regression be shown? I presume given the reply that h is then the variance -for someone outside the field such as myself this would be useful to see.

Author's Response to Decision Letter for (RSOS-191645.R0)
See Appendix A.

Comments to the Author(s)
The authors addressed the comments raised by the reviewers.

Recommendation? Accept as is
Comments to the Author(s) I'm satisfied with the author's revised manuscript. Congrats on a nice study.

06-Mar-2020
Dear Dr Dash, It is a pleasure to accept your manuscript entitled "Cancer cell lines show high heritability for motility but not generation time" in its current form for publication in Royal Society Open Science. The comments of the reviewer(s) who reviewed your manuscript are included at the foot of this letter.
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Response to Reviewers
We thank the reviewers and Editor for their time and their helpful suggestions.
Please find our point-by-point responses to review, and details of amendments to the manuscript, below. Editor's and reviewers' comments are in bold type.
Reviewer 1 1.1: There were some previous works regarding whether the cell motility can be inherited in the literature. The work from Yan et al is a short-term study more similar to the presented work. They saw low correlation between two daughter cells. The works from Chen et al are long-term heritability studies. They showed cellular motility and altered gene expression can be inherited. It is recommended that the authors should explicitly discuss what's new in the present work and also why the authors got similar or different conclusions. As the measurement methods are different, it is totally fine to get different observations. However, it would be essential to discuss relevant previous works for comparison. Three relevant papers are listed below: "Stochastic variations of migration speed between cells in clonal populations" "Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations" "Single-cell RNA-sequencing of migratory breast cancer cells: discovering genes associated with cancer metastasis" We now cite all three of these studies in the Introduction (page 3, lines 34-38). In the Discussion, we also briefly consider differences in methods and analysis that could explain the discrepancy between our results and those reported by Yan and Irima, who in 2014 tracked sister cell pairs and reported that their speeds were uncorrelated (page 10, lines 206-214).
The reviewer is correct that any number of differences in the experimental method could be responsible for the discrepancy. We now mention a few of these, along with a point about the difference in our datasets and analyses that might also have contributed. I reproduce Figure 4d from Yan and Irima (left). They report that they found no significant correlation between the speed of sister cells D1 and D2, but the trendline on the graph (present in the original Figure) suggests that a correlation may have been present, although it fell below their chosen threshold of significance. We include slightly more data points in all our regression analyses, allowing for greater sensitivity.
The experimental and statistical explanations are not mutually exclusive, and both could be in play.
The method of Yan and Irima would have allowed for the calculation of heritability from sister cell pairs, and the microfluidic techniques described in the Chen papers could have been used in an experiment to calculate heritability in cell populations (by statistical analysis of the phenotypes of a large sample of cell lines each founded from a single progenitor), but this parameter was not estimated by either group, as it was not the focus of the papers.
1.2: The authors claim that "22.7% of cells tracked (2048 individual cells) met the requirements to be used in the analysis." Excluding majority of the data can be biased. Other reviewers raised similar concerns as well. It is recommended that the authors should better explain how they excluded data? For example, the data were excluded for three possible reasons. x% for A reason, y% for B reason, and z% for C reason. Also, if including those excluded data, will it change the conclusion?
We have reworded the MS where appropriate (page 6, lines 104-107) to more clearly explain that our only reason for not including cells in the analysis is that they moved offscreen before sufficient cell generations could be tracked. We therefore analysed all cell families in which it was possible to track at least one pair of "cousin cells", and so we hold no excluded data which could sensibly be re-included in any new analysis that estimated the extent of stably inherited variation in phenotype. We now explain this rationale more clearly and carefully. However, we have still included the 22.7% figure for the sake of transparency.
We have further addressed the underlying issue highlighted by this comment (and also mentioned by reviewer 3; see point 3.1 below) by acknowledging more explicitly (page 7, lines 119-126) that it remains a possibility that faster cells are slightly under-represented in our dataset. We have also included the results of an additional statistical test, comparing the speed of individual cells that divided vs. those that moved offscreen. There was no significant difference, indicating that while we cannot rule out this possibility altogether, its effects will not be large. We have tried to keep the discussion of this point concise, but we hope that we have now explained this limitation sufficiently clearly for a broad readership.
1.3: It is recommended that the authors can discuss the possibility that cellular motility was driven by secreted signals (e.g. secreted proteins/exosome). If secreted signals are the dominant drivers of cell motility. Neighboring cells naturally share similar motility. It may not be caused by heritability as suggested by the authors. Further data analysis maybe necessary to eliminate this concern.