Abstract PHB01: The zebrafish as a mechanical filter: Using zebrafish xenografts to model Ewing sarcoma metastasis in vivo

Ewing Sarcoma (ES), a pediatric cancer driven by the oncogenic fusion protein EWS-FLI1, recurs or metastasizes in 1 in 3 patients with no clear correlation to accumulation of secondary mutations, raising the possibility that environmental cues may drive metastasis. Previous work in the field has observed distinct cellular morphologies of ES cells in cell culture as a function of EWS-FLI1 level and posited the idea that this may correlate with distinct functional cellular states. In this work, we use zebrafish xenografts to investigate the effect of variable microenvironments on cellular morphologies of ES cells. We use high resolution light-sheet microscopy to focus on specific recurring seeding sites within the 3-day old zebrafish larvae. We then implement a unique cell-shape classification method to distinguish discrete 3D cellular morphologies in these datasets. Using this workflow, we observe a variety of cellular morphologies within the fish, including elongated cellular protrusions, blebbing, and rounded cells. We demonstrate that ES cells take on distinct morphological states as a function of the region in which they reside. We find cell proliferation, but not cell death, vary greatly in the different regions, suggesting differences in the functional state of the ES cells. Interestingly, knockdown of EWS-FLI1 in xenografted cells leads to discrete switches between cellular morphologies, but only as a function of seeding site within the fish, suggesting a previously undescribed environmental cue that modulates cellular effects of EWS-FLI1. Overall, the xenograft model for ES in zebrafish embryos can be viewed as a “mechanical filter”, which provides unique, complex 3-dimensional environments to study cell behavior within an organism, while leveraging the advantages of the zebrafish system, namely higher throughput and high resolution in vivo imaging capabilities. This work establishes a pipeline for classification and distinction of morphological states within a living organism, and, more specifically, helps our understanding of cellular plasticity of ES cells in response to microenvironment. Citation Format: Dagan Segal, Bo-Jui Chang, Hanieh Mazloom-Farsibaf, Reto Fiolka, James Amatruda, Gaudenz Danuser. The zebrafish as a mechanical filter: Using zebrafish xenografts to model Ewing sarcoma metastasis in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PHB01.