Mentor Name: James Amatruda

This POST project focuses on Ewing sarcoma (EwS), a malignant cancer of bone and soft tissue that occurs in children, adolescents and young adults. At least 1/3 of patients with EwS will develop metastasis, which carries an extremely poor prognosis. To date, it is not known why tumor cells in metastatic sites cannot be eliminated by chemotherapy. Ewing sarcoma is a genomically “quiet” disease, and it is unlikely that metastatic cells will display penetrant secondary mutations that could explain metastasis and provide targets for better therapies. Instead, we believe that non-genomic adaptation of cancer cells to novel environments, mediated by changes in cell morphology, signaling and survival, underlies metastatic cell behavior. To identify and understand these adaptive changes we require a system that allows dynamic, high-resolution imaging in vivo in a physiologic environment. We have adopted the zebrafish for this approach. Specifically, we have developed procedures to implant fluorescently-labeled human EwS cells into transparent zebrafish larvae. The cells readily engraft and can be serially imaged as they adapt to diverse environments, and traverse different stages of the metastatic cascade (intravasation, transit, extravasation, adaptation and survival). Among the three most important questions we are studying are: 1) to which sites in the embryos do implanted cells spread? 2) Are metastatic cells able to proliferate, and does proliferation vary in different tissue/organ environments? 3) Similarly, are there differences in cell survival in different environments? We will implant EwS cells into two-day-old zebrafish embryos. The cells are fluorescently labeled and the transparent embryos lack an adaptive immune system, allowing the EwS cells to engraft and spread. We will examine the migratory patterns and phenotypes of the EwS cells by studying extravasation and proliferation and apoptosis of the four cell lines using fluorescent microscopy. We will establish an immunostaining protocol to quantify proliferation and apoptosis, two essential hallmarks of metastatic behavior.