A Microcavity-Based Sarcoma Spheroid Model for Drug Efficacy and Mechanism Analysis Using Cost-effective 3D Printing Technology

Ewing sarcoma is a pediatric bone cancer with poor survival in relapsed cases, highlighting the need for better preclinical models. Here, we developed a scalable 3D spheroid platform using SLA-printed microcavity arrays to generate uniform Ewing sarcoma spheroids. High-throughput screening of 11 compounds identified Torin 2, Talazoparib, and Trabectedin as potent inhibitors. Incorporating lung fibroblasts into hybrid spheroids revealed fibroblast-induced drug resistance, mimicking the metastatic tumor microenvironment. Transcriptomic analysis identified activation of NF-κB and TGF-β signaling pathways as key mediators of this resistance. This platform offers a cost-effective, biologically relevant system for drug testing and mechanistic studies in Ewing sarcoma and other stromal-influenced cancers. Hybrid or monoculture spheroids were digested into single cells using trypsin. GFP positive Ewing sarcoma cells were sorted using BD Symphony S6 (CWRU Cytometry & Imaging Microscopy Core), and subject to total RNA extraction using TRIzol according to the manufacturer’s instructions. 250ng of total RNA was used to construct RNA-seq library using the NEBNext UltraExpress RNA Library Prep Kit following manufacturer’s instructions. Briefly, RNA was polyA enriched using NEBNext Poly(A) mRNA Magnetic Isolation Module and enzymatically fragmented. cDNA was synthesized followed by end repair and adapter ligation, and PCR enrichment with indexed primers. Library was pair end sequenced for 76bp using Illumina NovaSeq platform. Sequencing reads were mapped to reference genome hg38 using RNA STAR with default parameters. Read count tables were created using FeatureCounts with a custom GTF file containing protein coding genes only. Differentially expressed genes were analyzed using DESeq2 with two independent replicates using default parameters. TPM values was calculated using Kallisto.