Abstract 6165: Matrix density shapes early invasion and EWSR1::FLI1 protein dynamics in Ewing sarcoma

Abstract Ewing sarcoma (EwS) is a pediatric bone and soft tissue cancer driven by chromosomal translocations generating fusion oncogenes, primarily EWSR1::FLI1. Metastasis is the most adverse prognostic factor, associated with persistently low survival rates. The low mutational burden in EwS suggests that tumor progression relies more on how cells adapt to their environment than on genetic selection. Invasion represents the first transition where cells leave the primary tumor and face new environmental constraints that can shape their metastatic potential. This study aims to establish a model that captures early EwS adaptation during invasion and to determine how extracellular matrix (ECM) parameters influence this process. To study EwS adaptation, we use cell lines derived from primary tumors or metastases to generate 3D spheroids embedded in collagen I matrices of defined densities, allowing us to monitor invasion and its associated morphological and molecular changes. We notably focus on EWSR1::FLI1 dynamics and its regulatory mechanisms during invasion onset, as this transcription factor is essential for EwS transformation and proliferation, yet reduced activity has been suggested in metastatic contexts. The ECM-embedded spheroid model shows that active invasion is restricted to metastasis-derived cells. Spheroids from primary tumors expand or sense the matrix but display rare cell detachment, whereas metastasis-derived spheroids show robust invasion with elongated, amoeboid, and collective behaviors captured by high-resolution microscopy. Increasing collagen density gradually reduces invasion, highlighting the strong influence of matrix properties. This prompted us to examine EWSR1::FLI1 dynamics, with immunohistochemistry revealing a spatial decrease at spheroid edges and within invading cells. Low heterogeneity is detectable before spheroid embedding, but the extent of EWSR1::FLI1 downregulation markedly expands during invasion. Time-course analyses show that this downregulation precedes active detachment. Moreover, fewer EWSR1::FLI1-low cells are observed at the edge of spheroids embedded in invasion-restrictive high-density matrices, further linking matrix density with invasive behavior and EWSR1::FLI1 dynamics. RNAscope shows that spheroid-edge and invading cells retain high EWSR1::FLI1 transcript levels, suggesting that the regulation occurs at the protein level. In line with this, proteasome inhibition with MG132 prevents invasion onset without inducing major cell death, supporting proteasome-dependent regulation of early EwS invasion. Taken together, these findings identify an ECM-associated EWSR1::FLI1-low state as an early adaptive feature during invasion onset. Our model enables dissection of this regulation -from matrix sensing to active detachment- and suggests a role for proteasome-mediated mechanisms in shaping early metastatic traits in Ewing sarcoma. Citation Format: Manon Watzky, James Amatruda, . Matrix density shapes early invasion and EWSR1::FLI1 protein dynamics in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6165.