ECM mimicking tunable 3D hydrogels to model Ewing sarcoma

The extracellular matrix (ECM) is increasingly recognized as a critical regulator of tumor progression and metastasis. Despite its strong influence on tumor fate, the ECM remains poorly characterized within the solid tumor microenvironment. Ewing sarcoma (EwS), an aggressive bone and soft tissue malignancy affecting adolescents and young adults, may be particularly dependent on ECM-mediated cues for progression. Here, we present a synthetic three-dimensional (3D) hydrogel model designed to recapitulate key biochemical features of the EwS tumor microenvironment. Using a polyethylene glycol (PEG) backbone functionalized with ECM-derived integrin-binding peptides, we engineered hydrogels incorporating motifs from collagen I, fibronectin, and the tumor-associated glycoprotein tenascin-C (TNC). Incorporation of TNC-derived peptides, which are heterogeneously distributed in EwS tumors, enabled controlled ECM heterogeneity within the platform. CHLA10 human EwS cells encapsulated in these hydrogels exhibited peptide-dependent proliferation and altered expression of ECM-related and matrix-remodeling genes, highlighting the role of ECM proteins including TNC in influencing cell state. Importantly, the hydrogels were designed with sortase-degradable crosslinkers, enabling gentle and bioorthogonal retrieval of viable cells for transcriptomic and functional analyses-an advance over traditional biomaterial-based systems. This modular 3D system provides a physiologically relevant, animal-free model to study sarcoma-ECM interactions and identify ECM-regulated mechanisms of tumor adaptation and metastasis. Our findings demonstrate that ECM heterogeneity, particularly TNC enrichment, may be a key driver of EwS progression and highlight the potential of biomaterial platforms in uncovering therapeutic targets within the tumor microenvironment.