ABSTRACT: Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric soft tissue sarcoma with poor relapse-free survival, especially in metastatic cases. Tumor-initiating cells (TICs) are implicated in recurrence and therapy resistance in ARMS, but their metabolic adaptations remain poorly understood. Emerging evidence suggests that lipid metabolism supports stem-like properties in cancer, yet the role of lipid remodeling enzymes in TIC function has not been defined. Here, we investigate the transcriptomic and metabolic profiles of ARMS TICs using tumorsphere-derived and adherent cells from cell line and patient-derived xenograft models. Gene expression and lipid species differences were analyzed using RNA sequencing, gene set enrichment analysis, qPCR, and mass spectrometry. Lipid droplet accumulation was assessed using Oil Red O and BODIPY staining, and functional dependence on PLA2 activity was evaluated using Darapladib. Seahorse assays were used to examine glycolytic and mitochondrial flux, and in vitro and in vivo assays assessed tumorsphere formation, migration, and tumorigenicity. We found that tumorspheres exhibited upregulation of lipid metabolism, especially multiple PLA2 isoforms, and downregulation of glycolytic gene expression. Lipidomic profiling revealed enrichment of linoleic acid-derived triglycerides and lysophospholipids, alongside increased PLA2 activity. Tumorspheres accumulated lipid droplets and upregulated lipid storage regulators PPARG and CD36. Inhibition of PLA2 with Darapladib impaired TIC self-renewal, migration, and in vivo tumor growth, without affecting mouse body weight. These effects were rescued by exogenous linoleic acid supplementation, implicating PLA2-mediated lipid signaling in TIC maintenance. Despite suppressed glycolysis and partial sensitivity to fatty acid oxidation blockade, TICs remained functionally intact under metabolic stress, demonstrating high metabolic flexibility. Collectively, these findings identify PLA2-driven lipid remodeling as a key metabolic adaptation in ARMS TICs that supports stem-like function independently of canonical energy pathways, highlighting PLA2 as a promising therapeutic target in pediatric sarcomas.
