DCMiC: a double-cylinder micro-chamber platform for high-throughput drug screening and modeling of microenvironmental resistance in Ewing sarcoma.

We fabricated a double-cylinder micro-chamber (DCMiC) platform using stereolithography-printed master molds, followed by PDMS replica molding and integration into a 96-well plate format for scalable and reproducible generation of Ewing sarcoma spheroids. The simple yet novel DCMiC design stabilizes spheroids during media exchange, enabling reliable long-term culture and high-throughput drug screening. Using this platform, we screened 11 small-molecule compounds previously shown to target vulnerabilities relevant to Ewing sarcoma, including epigenetic regulators, DNA damage response, growth signaling and metabolic pathways. As a result, we identified Torin 2, talazoparib, and trabectedin as top 3 candidates with potent anti-Ewing sarcoma activity. To more accurately model the metastatic tumor microenvironment, we incorporated human lung fibroblasts to generate heterotypic spheroids, which consistently conferred resistance to all 3 compounds. Transcriptomic profiling revealed that fibroblasts reprogram Ewing sarcoma cells by activating pro-survival NFκB and TGF-β1/SMAD signaling while repressing tumor-suppressive programs, highlighting how stromal cues promote therapy resistance. Mechanistically, exogenous TGF-β1 was sufficient to induce resistance in tumor-only spheroids, whereas pharmacological inhibition of TGF-β1 signaling restored drug sensitivity in heterotypic spheroids. These findings establish the DCMiC platform as a low-cost, physiologically relevant system for modeling tumor-stroma interactions and enabling scalable drug discovery in clinically relevant contexts for Ewing sarcoma and other solid tumors.