Gupta_2025_Cell.Commun.Signal_23_404

BACKGROUND: Advancements in chemotherapy have improved outcomes for rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. However, relapse-free survival rates remain below 20% in metastatic alveolar rhabdomyosarcoma (ARMS). Tumor-initiating cells (TICs) drive recurrence. Targeting this pool of cells has been shown to reduce relapse rates and metastasis in various cancers. Emerging evidence implicates lipid metabolism in supporting stem-like properties in cancer. However, TICs remain poorly characterized in ARMS. This study investigates the transcriptomic and metabolic profile of TICs in ARMS. METHODS: Transcriptomic and lipidomic profiling were performed on tumorsphere-derived and adherent cells from ARMS cells. Differential expression of metabolic genes and lipid species was assessed via RNA-Sequencing and mass spectrometry. Functional dependence on Phospholipase A2 (PLA2) signaling was tested on TICs in vitro and in vivo. Seahorse assays were used to measure glycolytic and mitochondrial flux. Statistical significance was determined using Student's t-test or one-way ANOVA. RESULTS: Transcriptomic analysis of ARMS tumorspheres showed upregulation of lipid metabolism especially different PLA2 enzyme subtypes, and suppression of glycolytic gene expression. Lipidomic analyses revealed enrichment of linoleic acid-derived triglycerides and lysophospholipids, paralleled by increased PLA2 activity. Tumorspheres also showed elevated expression of lipid storage regulators PPARG and CD36 and correspondingly, significant lipid droplet accumulation. Inhibition of PLA2 with Darapladib reduced tumorsphere formation and cellular motility in vitro and tumor volume in vivo. These defects were rescued by exogenous linoleic acid (LA) supplementation, implicating PLA2-driven lipid signalling in TIC maintenance. Interestingly, TICs display high metabolic flexibility in energy consumption, as neither glycolysis nor fatty acid oxidation (FAO) inhibition significantly impaired tumorsphere formation. CONCLUSIONS: PLA2-mediated lipid remodelling supports TIC by promoting linoleic acid-linked lipid signalling. PLA2 inhibition impairs self-renewal, motility, and tumorigenic potential, highlighting it as a promising therapeutic target in ARMS. These findings uncover lipid remodelling as a key metabolic adaptation in ARMS TICs, independent of canonical energy pathways, with implications for targeting the stem-like compartment in pediatric sarcomas.