| Summary: | Emerging evidence suggests that cancer stem-like (CS-like) cells are responsible for cancer progression and relapse. The identification and characterisation of CS-like cells is therefore important to reveal potential targets that could be used to design more effective personalised treatment to improve outcomes. The cell surface marker prominin-1 (CD133) has been used to identify Ewing sarcoma (ES) CS-like cells (ES-CS-like), however some primary ES cells are devoid of CD133 and may be down-regulated by the microenvironment in cell culture. Therefore, alternative approaches are required to identify ES-CS-like cells. Two approaches were compared to enrich for putative ES-CSCs from ES cell lines; isolation of ES-CS-like cells by CD133 expression and using a functional single cell self-renewal assay. The second approach was more reliable for studying the miRNA and mRNA expression profiles in ES-CS-like spheroids compared to ES cells grown as monolayers. In ES-CS-like spheroids, the expression of the stem cell marker EBAF (q < 0.03), miR-210-3p (q < 0.12) and the ABC transporter protein ABCG1 (q < 0.14) were all significantly increased. The phenotypic significance of ABCG1 was investigated using knock-in and knock-out experiments. Overexpressing ABCG1 protein using a lentiviral vector was unachievable as a result of the increased expression of the E3 ligase; NEDD4-1. This ligase prevented the post translational overexpression of ABCG1 mRNA. Interestingly, knock-down of ABCG1 mRNA appeared to stabilise ABCG1 protein and decrease viable number of ES cell line (SK-N-MC), suggesting ABCG1 may have a cell cycle or survival function. In conclusion, growth of ES spheroids from single cells enriches for cells which express stem cell markers, suggesting that the single cell self-renewal assay can be used to enrich for ES-CS-like cells. Furthermore, ABCG1 and miR-210-3p may be drivers of the ES-CS-like phenotype and might be used to select patients at greatest risk and inform design of targeted treatments. These observations require validation using functional assays and confirmation in patient derived cells and tumours. Whether CDKN1A-interacting zinc finger protein 1 (CIZ1) plays a role in the ES-CS-like phenotype is yet to be investigated.
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