Abstract 3531: Discrete extracellular matrix-secreting tumor cell subpopulations remodel the Ewing sarcoma tumor microenvironment to promote invasion
Authors: Wrenn Emma D. , Apfelbaum April A. , Ganapathi Shireen , Rudzinski Erin R. , Deng Xuemei , Garcia Nicolas , Braun Katherine , Lipson Trisha , Bhise Shruti , Kanaan Sami B. , Waltner Olivia , Newman Erika , Furlan Scott , Lawlor Elizabeth R. 
Published: April 2023

Abstract
Ewing sarcoma (EwS) cells exist along a neuro-mesenchymal transcriptional continuum that is largely determined by transcriptional activity of the EWS::FLI1 fusion oncoprotein. Although EWS::FLI1-low state cells are more metastatic in experimental models, the role of these cells in established EwS tumor ecosystems is unknown. Here we have leveraged multimodal single-cell sequencing, spatial transcriptomics, and immunohistochemical profiling to characterize EwS cell subpopulations in cell lines, PDX models, and patient tumors. We identify CD73 as a marker of mesenchymal-high state EwS cells and show that CD73+ tumor cells share properties of experimentally-induced EWS::FLI1-low cells including altered cytoskeletons, enhanced migration and invasion, and increased expression of EWS::FLI1-repressed genes. However, CD73+ cells retain proliferative capacity and do not down-regulate the fusion or the EWS::FLI1-activated signature. Instead, CD73+ EwS cells selectively express a gene signature that is enriched in extracellular matrix (ECM) genes. Examination of xenograft and EwS patient biopsies confirms the existence of subpopulations of spatially and transcriptionally distinct ECM-producing tumor cells and shows them to be more prevalent in infiltrating regions. Importantly, the local tumor microenvironment (TME) surrounding these cells is rich in tumor-derived TNC, SPARC, and biglycan, ECM proteins that have been widely implicated in cancer metastatic progression. Significantly, CD73+ EwS cells also upregulate expression of ECM-sensing genes, including ITGA5 and ITGB1, suggesting potential to activate autocrine feedback loops. In support of this, interrupting outside-in ECM:integrin signaling cascades, through pharmacologic inhibition of FAK or SRC pathways, strongly reduces the invasive properties of CD73+ tumor spheroids in 3D collagen. Together these studies confirm the presence and functional importance of transcriptionally distinct tumor cell subpopulations in established EwS tumor ecosystems. In particular, our data reveal that highly mesenchymal tumor cells both generate and respond to pro-metastatic ECM proteins to support invasion. Studies are ongoing to determine the role of tumor cell-derived ECM on EwS progression and treatment resistance.
Citation Format: Emma D. Wrenn, April A. Apfelbaum, Shireen Ganapathi, Erin R. Rudzinski, Xuemei Deng, Nicolas Garcia, Katherine Braun, Trisha Lipson, Shruti Bhise, Sami B. Kanaan, Olivia Waltner, Erika Newman, Scott Furlan, Elizabeth R. Lawlor. Discrete extracellular matrix-secreting tumor cell subpopulations remodel the Ewing sarcoma tumor microenvironment to promote invasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3531.