Single cell RNA-sequencing of Ewing sarcoma tumors demonstrates transcriptional heterogeneity and clonal evolution

Abstract

Purpose: Ewing sarcoma (EwS) is the second most common bone cancer in children, accounting for 2% of pediatric cancer diagnoses. Patients who present with metastatic disease at the time of diagnosis have a dismal prognosis, compared to the >70% 5-year survival of those with localized disease. Novel therapeutic approaches that can impact metastatic disease are desperately needed, as well as a deeper understanding of the heterogeneity of EwS tumors. Experimental Design: Here, we utilized single cell RNA-sequencing to characterize the transcriptional landscape of primary EwS tumors and surrounding tumor microenvironment (TME) in a cohort of seven untreated EwS patients, as well as in circulating tumor cells (CTCs). A potential CTC therapeutic target was evaluated through immunofluorescence of fixed CTCs from a separate cohort. Results: Primary tumor samples demonstrate a heterogenous transcriptional landscape with several conserved gene expression programs, including those composed of genes related to proliferation and EWS gene targets, which were found to correlate with overall survival. Copy-number analysis identified subclonal evolution within patients prior to treatment. Analyses of the immune microenvironment reveal an immunosuppressive microenvironment with complex intercellular communication within the tumor and immune cells. Single cell RNA-sequencing and immunofluorescence of CTCs at the time of diagnosis identified TSPAN8 as a potential therapeutic target. Conclusions: EwS tumors demonstrate significant transcriptional heterogeneity as well as a complex immunosuppressive microenvironment. This work evaluates several proposed targets which would warrant further exploration as novel therapeutic strategies.