A live single-cell state reporter assay links intra-tumor heterogeneity to metastatic proclivity in Ewing sarcoma

Targeting of the most aggressive tumor cell subpopulations is key for effective management of most solid malignancies. However, the metastable nature of tumor heterogeneity, which allows cells to transition between strong and weak tumorigenic phenotypes and the lack of reliable markers of tumor promoting properties hamper identification the most relevant cells. To overcome these obstacles, we designed a functional, cell state-dependent microRNA (miR)-based live cell reporter assay to identify highly tumorigenic cells in xenotransplants of primary Ewing sarcoma (EwS) organoids. Leveraging the inverse relationship between cell pluripotency and miR-145 expression, we successfully separated highly tumorigenic, metastasis-prone (miR-145low) cells from poorly tumorigenic, non-metastatic (miR-145high) counterparts. Gene expression and functional studies of the two cell populations identified EPHB2 receptor as a prognostic biomarker in EwS patients and a major promoter of metastasis. Our study provides a simple and powerful means to identify and isolate tumor cells in states associated with aggressive behavior. Overall design: Cells from two Ewing sarcoma organoids were sorted into GFP+ and GFP- fractions, corresponding to low- and high-miR-145 expression, respectively. Genes differentially expressed between the two fractions were identified.