Cancer Biology Research Test-Bed Unit 1: Effects of cell-intrinsic and cell-extrinsic signaling and mechanics on metastasis patterns of pediatric sarcomas

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Ewing sarcoma, a malignant tumor of bone and soft tissue affecting children, adolescents, and young adults. For the one-third of Ewing sarcoma patients who develop metastasis, the long-term survival rate remains less than 30%. Decades of clinical trials with ever-increasing intensity of chemotherapy have increased the toxicity of treatment but have not affected the poor outcome of metastatic disease. This failure to adequately treat metastases indicates that new approaches are needed to better understand the genesis of metastatic cells from the primary tumor and behavior of these cells in the in vivo microenvironment. Though there has been substantial progress in genomic profiling of tumors, these assays are unlikely to identify major determinants of metastatic behavior. This is because i) Ewing sarcomas typically have “quiet” genomes with few identifiable driver mutations; and ii) adverse outcomes may arise due to the functional adaptations of a small population of cells to the tumor microenvironment, driven by epigenetic, metabolic, morphologic or non-cell autonomous mechanisms. The broad goal of this project is thus to determine how extrinsic and intrinsic factors influence Ewing sarcoma cell fates at the metastatic site. A significant barrier to better understanding has been the lack of experimental systems that can probe heterogeneity of cell functional states, at whole-organism, single-cell and subcellular levels. Recent findings suggest that modulation of cell-mechanical features via the caveolin-1 and WNT signaling pathways may contribute to Ewing sarcoma metastasis, however the mechanisms of this adaptation are not known. As a system to visualize the heterogeneous functional properties of the metastatic cell population shed from a primary tumor, we leverage the zebrafish embryo as a host organism for human tumor xenografts. Ewing sarcoma cells readily engraft into zebrafish embryos and directly interact with the microenvironment of fully functional organs. The optical clarity of the fish allows us to perform multi-modal imaging to 1) identify host tissues associated with recurrent metastatic events; 2) define morphologic changes in cells undergoing metastatic adaptation in vivo; and 3) probe the activity of cancer cell signaling pathways in metastatic cells at subcellular resolution. Using powerful genetic tools and specific biosensors, we will exploit the quantitative imaging technology developed by TDU-1 to probe the role of caveolin-1 and WNT-dependent signaling in Ewing sarcoma metastasis. These studies will be complemented by parallel assays in mouse models and human Ewing sarcoma tumors, enabled by collaboration with TDU-2. Ultimately, these findings will inform strategies aimed at preventing or eliminating metastasis via targeting signaling mechanisms. We will determine effects of microenvironmental interactions on morphology and signaling of metastatic tumor cells; test the contribution of Caveolin-1 to metastatic cell adaptation to host environments; and probe the role of WNT signaling in Ewing sarcoma metastasis in genetic models. Ultimately these results will inform novel strategies to prevent or ameliorate metastasis in patients with Ewing sarcoma.