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

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.

Grant Number: 5U54CA268072-05
NIH Institute/Center: NIH
Principal Investigator: JAMES AMATRUDA