Salmon-aligned RNA-seq of DFMO-treated and untreated Ewing Sarcoma PDXs meta_tab.csv: Metadata mapping sample IDs to experimental conditions. salmon_quant.zip: Salmon-quantified expression profiles of DFMO-treated and untreated Ewing Sarcoma PDXs, aligned to a combined human-mouse reference transcriptome. The main analysis retains only reads aligned to human genes. Abstract Polyamine metabolism and signaling play important roles in multiple cancers but have not previously been studied in Ewing sarcoma. Here, we show that blocking polyamine synthesis with D, L-alpha-difluoromethylornithine (DFMO) causes a G1 cell cycle arrest, dose-dependent decreases in sarcosphere formation from Ewing sarcoma cell lines growing in non-adherent conditions and a decrease in clonogenic growth in soft agar. Further, we utilized our orthotopic implantation/amputation model of Ewing sarcoma metastasis to demonstrate that DFMO slowed primary tumor growth in addition to limiting metastasis. RNA sequencing demonstrated gene expression patterns consistent with induction of ferroptosis caused by polyamine depletion. Induction of ferroptosis was validated in vitro by demonstrating that ferrostatin-1, an inhibitor of ferroptosis, allows sphere formation even in the presence of DFMO. Collectively, these results reveal a novel mechanism by which DFMO prevents metastasis – induction of ferroptosis due to polyamine depletion. Our results provide preclinical justification to test the ability of DFMO to prevent metastatic recurrence in Ewing sarcoma patients at high risk for relapse.