Single Nuclear RNA Sequencing Highlights Intra-Tumoral Heterogeneity and Tumor Microenvironment Complexity in Testicular Embryonic Rhabdomyosarcoma

Background: Testicular embryonic rhabdomyosarcoma (ERMS) is a rare soft tissue tumor in children featured with high intra-tumoral heterogeneity. In this study, we aimed to comprehensively delineate the testicular ERMS intra-tumoral heterogeneity and tumor microenvironment. Methods: Cell types and the corresponding marker genes were identified by single-nuclear RNA sequencing (snRNA-seq). Functional states of different clusters were evaluated by uniform manifold approximation and projection and differentially expressed genes. Kaplan- Meier curve analysis was constructed according to the gene expression profile to determine the correlation between candidate marker genes and the overall survival and disease-free survival of patients with osteosarcoma from TCGA. Results: A total of 8868 tumor cells and 10,147 normal cells were obtained from testicular ERMS tissues. The heterogeneous malignant subtype was composed of six subgroups (C1-C6) with differential proliferative and migratory potentials. Cell trajectory analysis revealed the C1 subgroup might be the starting cells of the tumor and transform into two different types of malignant cells, C2 and C5/6, during the development of RMS. The differentially expressed genes were closely related to cell adhesion and extracellular matrix signaling pathways. Furthermore, the interaction analysis between cell subgroups (macrophages and tumor cells, endothelial cells and tumor cells) demon-strated that collagen-related gene COL6A1 plays a key role from the initiation of ERMS to the entire process of malignant transformation. Conclusion: Our findings provide a new insight in the understanding of the initiation and progression of testicular ERMS and have potential value in the development of markers for the diagnosis and stratification of testicular ERMS.