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  1. Evaluation Summary:

    The study proposes that confined migration renders breast cancer cells resistant to apoptosis via NFkappaB-dependent mechanisms. The technical aspects of the study are impressive and experiments are very well performed and demonstrate the value of mimetic bioengineering approaches, but the postulated central premise would require more rigorous support.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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  2. Reviewer #3 (Public Review):

    Mechanical stress has been emerging as the key factor for activating pro-metastatic features, and authors hypothesize confined migration results in anoikis (death in suspension) and increased invasiveness.

    By clever repurposing of transwell membranes, authors have generated a confined migration assay (CM), in which cells that have crossed the 3 micron-pore membrane are collected, cultured and further analyzed. The intensity of the CM-related response was shown to increase with number of CM rounds, and the response decays and reverts back 5 days after the CM event.

    The resistance to anoikis achieved by CM was not conferred by compressive stress, or migration without constriction through 8-micron pores, and it was further demonstrated to rely on NFkB activation and IAP regulation at post-transcriptional level.
    Using RNASeq, authors next show that while transcription is globally inhibited, resulting in lower nuclear stiffness, components of cell adhesion and regulation of NK-cell cytotoxicity were upregulated. Both of these functions were elegantly and succinctly confirmed by time-lapse measurements of cell velocities and immunofluorescence.

    Finally, in vivo experiments confirmed metastasis was increased in tail-vein injected cells post-CM.

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  3. Reviewer #2 (Public Review):

    The authors present a detailed account of the anti-apoptotic characteristics of breast cancer cells that have undergone confined migration, specifically demonstrating an upregulation of cIAP1, cIAP2 and XIAP that promotes anoikis resistance. These cells were also more migratory and potentially displayed a resistance to immune surveillance.

    On the whole the paper is technically well performed, although the conclusion that confined migration causes these effects needs further work to be validated. A key question is whether confined migration has caused these changes to occur within individual cells, or whether the assay design results in selection of cells with these characteristics.

    For example, it may not be a surprise that selecting cells on the basis of their ability to migrate through a membrane would result in cells with an increased random migratory capacity? Therefore it may also be possible that these innately migratory cells have different expression patterns that include an upregulation of anti-apoptotic proteins? Therefore, there is a need to determine whether these cells were present in the population prior to confined migration, or whether these characteristics were acquired during the process.

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  4. Reviewer #1 (Public Review):

    This manuscript by Fanfone et al., describes the use an in vitro model of confined migration through transwells via a serum gradient, to study how mechanically challenged breast cancer cells acquire IAP-mediated resistance against anoikis. They demonstrate that these mechanically challenged breast cancer cells survive low-attachment culture to form spheroids. Additionally, the authors demonstrate that after confined migration NFkB is activated but potentially dispensable, as when it is blocked, it does not impact anoikis or spheroid formation. Also, mechanically challenged breast cancer cells demonstrate enhanced motility. Finally, the authors also demonstrate in vivo how mechanically challenged breast cancer cells were more metastatic competent to successfully colonize the lungs. This is an impressive manuscript that reveals how developing an in vitro model of confined migration provides much needed insight as to how microenvironmental constriction can in fact aid cancer cells in acquiring a pro-survival and enhanced migratory phenotype to successfully metastasize.

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