Abstract 2074: Single-cell RNA-seq reveals heterogeneity for stem cell markers, LRG5 and CD271 to predict a subpopulation in Ewing sarcoma cells with lower levels of BRAF

Abstract

Abstract Introduction: Ewing Sarcoma (ES) is an aggressive bone and soft tissue tumor, which has a high rate of recurrence among all childhood cancers. This suggests the need for a better understanding of heterogeneous cancer populations. In many cancers, recurrence and therapy resistance has been suggested to be due to the relative state of quiescent cells. Our previous work has shown that low EWSR1-FLI1 expressing cells have a significantly lower proliferative activity, which is similar to a quiescent state. Therefore, we used cells with low EWSR1-FLI1 expression to model and predict cells with a quiescent state.Methods: Using siEWSR1 to suppress EWSR1-FLI1, we studied proliferative quiescence. The Fluidigm C1 system was used to perform SMART-Seq whole transcriptome amplification using EW8 cells. We sequenced single- cell RNA-Seq libraries prepared from 700 ES cells. We developed a computational analysis to profile single cells. Then, we used gene expression signatures derived from the quiescent population to classify parental cells with ensemble classifiers. Results: Results revealed that EW8 cells show considerable heterogeneity at the transcriptome level in control (scrambled RNAi) as well as in the quiescent populations. We identified a subpopulation of EW8 cells with a negative correlation between the expression of LGR5 and Nerve Growth Factor Receptor (NGFR/CD271). Our preliminary data from 255 single cells indicate that LGR5 (0-43.57 TPM, median 0) and CD271 (0-80.05 unique transcripts, median 6.33) were correlated (r= -0.12, P<0.0001) in EW8 cells. CD271 is a surface membrane protein, and a mesenchymal stem cell marker, that regulates stemness, and heterogeneity. A subpopulation of cells with low CD271 was distinguished and studied for its co-expression pattern with LRG5 and BRAF. Conclusions: Our results suggest that there are multiple subpopulations within EW8 parental (Control) cells. We can characterize them with proliferation, stemness, motility and dormancy-associated markers. Ongoing studies are aimed to determine, if EW8 cells having elevated LGR5 exhibit different sensitivity to cytotoxic agents. Data derived from this work are expected to provide insights into the extent to which CD271 contributes to dormancy, and ES heterogeneity. Whether CD271 regulates proliferation to promote a dormant state in this subpopulation is being studied. Citation Format: Roxane Khoogar, Peter Houghton. Single-cell RNA-seq reveals heterogeneity for stem cell markers, LRG5 and CD271 to predict a subpopulation in Ewing sarcoma cells with lower levels of BRAF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2074.