Abstract 1681: Single cell RNA sequencing of primary Ewing sarcoma tumors and identification of circulating tumor cells in patient-matched peripheral blood samples
Authors: Nelson-Taylor Sarah K. , Bodlack Avery , Goodspeed Andrew , Treece Amy , Donaldson Nathan , Cost Carrye , Garrington Tim , Greffe Brian , Luna-Fineman Sandra , Sopfe Jenna , Hayashi Masanori 
Published: June 2022

Abstract
Ewing sarcoma (ES) 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 &gt;70% 5-year survival of those with localized disease. Here, we utilized single-cell RNA sequencing (scRNA-seq) to characterize the transcriptional landscape of primary ES tumors, and to identify circulating tumor cells (CTCs) in peripheral blood at the time of diagnosis in order to further understand ES transcriptional heterogeneity and factors that drive metastasis.
Methods: Viably frozen primary tumor and peripheral blood samples were obtained from 7 ES patients at the time of diagnosis and prior to the initiation of treatment. Tumors were dissociated into a single cell suspension and sorted for viability using FACS (fluorescence activated cell sorting) whereas peripheral blood samples were subjected to a size-based selection with the CellSieve microfiltration system. ScRNA-seq was performed using the 10xChromium platform and count matrices were generated using 10x Genomics Cell Ranger software. Quality control, integration, and cluster analysis was performed with Seurat.
Results: Cluster analysis of integrated, primary tumor samples demonstrated that candidate ES cell clusters express FLI1 and ES marker NKX2-2 and cluster separately from immune cell populations. Additionally, using inferCNV, ES clusters were demonstrated to harbor chromosomal copy number alterations known to be associated with ES and that were previously identified clinically on preliminary pathology reports for each patient; further GSEA analysis showed significant overlap between published ES gene sets and genes upregulated in ES clusters. Further analysis of ES cells demonstrated that cell-cycle phase determined a cluster enriched in pro-proliferation gene signatures, and overall heterogeneity of expression of previously known therapeutic targets. Candidate ES CTCs were identified among the peripheral blood samples among clusters which corresponded with distinct immune cell populations. The candidate ES CTCs expressed NKX2-2 and demonstrated enrichment in oncogenic gene signatures.
Conclusion: ScRNA-seq of primary ES tumors is feasible and demonstrates that ES tumor cells are largely homogeneous in nature; and candidate ES CTCs can be identified in peripheral blood at the time of diagnosis in ES patient and warrant further investigation as to their utility as a biomarker of metastatic disease.
Citation Format: Sarah K. Nelson-Taylor, Avery Bodlack, Andrew Goodspeed, Amy Treece, Nathan Donaldson, Carrye Cost, Tim Garrington, Brian Greffe, Sandra Luna-Fineman, Jenna Sopfe, Masanori Hayashi. Single cell RNA sequencing of primary Ewing sarcoma tumors and identification of circulating tumor cells in patient-matched peripheral blood samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1681.