Modeling Tenascin-C-rich metastatic tumor niches in engineered hydrogel biomaterials.

The extracellular matrix (ECM) plays a pivotal role in shaping tumor behavior by providing biochemical and biophysical cues to cancer cells. Traditional 2D culture systems fail to recapitulate this complexity, and in vivo systems do not readily allow for interrogation of how individual ECM components influence tumor cell behavior. Here, we introduce a fully synthetic, tunable three-dimensional (3D) hydrogel that mimics a soft tissue tumor microenvironment (TME) to enable mechanistic studies of ECM-tumor interactions. The hydrogel features a proteolytically degradable poly(ethylene glycol) base network functionalized with integrin-binding peptides derived from ECM components collagen I and fibronectin. To model metastatic ECM and further tune the hydrogel, we incorporated a tenascin-C (TNC)-derived peptide. To study the impact of these tunable ECM parameters on cancer cell behavior, we encapsulated Ewing sarcoma (EwS) cells within the hydrogels. EwS is an aggressive bone and soft tissue tumor that commonly metastasizes to lung. Our studies demonstrated matrix-dependent growth and phenotypic variation of EwS cells. Specifically, the TNC peptide drove divergent tumor behaviors and induced cell state changes consistent with alterations induced by the native TNC protein. To facilitate downstream functional assays, the hydrogel incorporates sortase-degradable crosslinkers that enable non-perturbative recovery of encapsulated cells. This platform provides a reductionist and reproducible model for studying the ECM's role in cancer cell biology while addressing long-standing challenges in polymeric hydrogel degradation and cell retrieval. Collectively, this work establishes a biomaterials-based framework to dissect EwS tumor-ECM interactions in a controllable 3D microenvironment. STATEMENT OF SIGNIFICANCE: Engineered biomaterials to model Ewing sarcoma (EwS) have been previously employed to study metastasis to the bone. While important efforts, platforms to study EwS metastasis in lung - the most common site of metastasis - have not been developed. Here, we introduce a user-programmable biomaterial designed to mimic the environment of soft tissue metastases. The platform features several attributes: 1) it is mechanically matched to lung tissue; 2) it is readily functionalized with extracellular matrix protein-derived peptides (e.g., Tenascin-C); 3) encapsulated cells can be retrieved in a "biologically invisible" manner via sortase-mediated gel degradation, enabling expanded downstream analysis. This platform provides a powerful, reproducible tool to dissect tumor behavior and identify new targets for cancer therapy.