Abstract A040: Microphysiological models uncover invasive and proteomic shifts in Ewing sarcoma cell subpopulations

Abstract

Epithelial-to-mesenchymal transition (EMT) drives morphological and molecular changes that promote cancer progression. Ewing sarcoma cells, driven by fluctuations in the master oncogene EWS FLI1, can shift along the EMT spectrum. Yet the proteomic changes underlying EMT, including alterations in cell–cell and cell–matrix interactions, remain poorly understood. We use microphysiological systems to identify metastatic drivers and examine how 3D tumor organization and external EMT induction (via Wnt-5a and TGF-β1) reshape cell subpopulation protein expression and invasiveness. TC71 GFP cells were treated with StemXVivo EMT Inducing Supplement for 5 days and assessed by wound healing, viability assays, western blot, and single-cell proteomics. We developed a 3000-cell tumor spheroid micromodel (500–1000 µm diameter) and used it to evaluate invasion and chemoresistance with greater physiological relevance. EMT induction was chemoprotective in 2D monolayers (Doxorubicin IC50: control 8.88±1.57nM (±95%Confidence Interval) vs EMT 12.6±1.67nM) but not in spheroids (control 114.2±28.16nM vs EMT 72.73±16.88nM). To assess invasive potential, spheroids were embedded in Cultrex basement membrane matrix with 100 ng/ml IGF-1—an ignition signal for bone sarcomas that mimics adolescent hormonal conditions. IGF-1 significantly increased the furthest invasive distance from the spheroid centroid (n=15/group, control ± IGF-1 p=0.02; EMT ± IGF-1 p=0.0123). While EMT enhanced wound-healing and migration in 2D (p<0.001), control spheroids showed greater invasion (p=0.0063), indicating that increased motility does not necessarily translate into greater invasiveness. To investigate the structural changes underlying these behaviors, we examined F-actin—a cytoskeletal regulator —and ezrin—a membrane–cytoskeleton crosslinker and EMT regulator. We found a statistically significant increase in bulk ezrin expression by western blot, both with the addition of IGF-1 from control and after EMT induction (n=3 replicates, p=0.015 and p=0.0086, respectively). To investigate the heterogeneity of actin expression and shifts induced by EMT stimulation, we used Single Cell Protein Interaction Fractionation Through Electrophoresis and Immunoassay Readout (SIFTER), a single-cell proteomics tool for quantifying multimeric protein complexes. EMT stimulation significantly increased median F actin levels in monolayers (control median 6.76E4 Arbitrary Fluorescence Units (AFU) (n=760 cells, coefficient of quartile variation CQV=0.74) vs EMT median 8.41E4 AFU (n=324, CQV=1.06) p<0.0001), with a further rise in spheroids (control spheroid median 10.55E4 AFU (n=214, CQV=0.80) vs spheroid EMT median 22.12E3 AFU (n=244, CQV=0.74) p<0.0001). This work represents the first use of a spheroid model for single-cell proteomic analysis in Ewing sarcoma and reveals that EMT stimulation increased invasion, elevated F-actin levels, and broadened single-cell heterogeneity, with stronger shifts in 3D spheroids. Neica Ivens. Joseph, Julea Vlassakis, PhD. Microphysiological models uncover invasive and proteomic shifts in Ewing sarcoma cell subpopulations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Breaking Barriers in the Fight against Rare Cancers; 2026 Jul 18-20; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(14_Suppl):Abstract nr A040.