System for study of diverse cellular composition in alveolar rhabdomyosarcoma

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric soft tissue sarcoma often driven by PAX3::FOXO1 or PAX7::FOXO1 fusions, for which therapy can be limited if metastatic. ARMS pathology shows pronounced cellular heterogeneity, including malignant subpopulations at distinct myogenic differentiation stages. Detailed subpopulation transcriptional programs and microenvironmental influences are of ongoing interest biologically and therapeutically. Using a genetically engineered mouse model and single-cell RNA sequencing (scRNA-seq), we characterized tumor composition and cellular states. Histology and immunohistochemistry confirmed diverse morphologies and differential expression of myogenic markers. scRNA-seq identified 17 clusters, including 11 malignant, immune and stromal subpopulations. Transcriptomic analysis revealed precursor-like cells expressed epithelial/EMT-like programs, while rhabdomyoblast-like cells upregulated extracellular matrix and mesenchymal regulators. Allografts in immunodeficient mice showed that immune context shapes cellular composition. Lineage-specific reporters and Symphony mapping validated spatial segregation of precursor and rhabdomyoblast-like cells. These findings demonstrate transcriptionally distinct malignant subpopulations within an immune-influenced microenvironment. These ARMS genetically engineered mouse model and allograft systems provides a platform to study tumor heterogeneity and drug responses.