Abstract B041: Bone microenvironment cues shift Ewing sarcoma cells towards pro-metastatic, ECM-secreting cell states
Authors: Wrenn Emma , Apfelbaum April , Lipson Trisha , Rudzinski Erin , Deng Xuemei , Braun Katherine , Garcia Nicolas , Bhise Shruti , Waltner Olivia , Furlan Scott , Lawlor Beth 
Published: January 2023

Solid tumors are complex ecosystems comprised of subpopulations of heterogeneous cancer cells that are altered by interactions with diverse cellular and acellular factors present in tumor microenvironments (TMEs). Accruing evidence suggests that heterogeneity of tumor cells and the TME, and bidirectional crosstalk between these spatially and temporally dynamic components, contributes to the generation of metastatic and therapy-resistant cell subpopulations. Ewing sarcomas are highly metastatic bone and soft tissue tumors driven by pathognomonic FET:ETS transcription factor fusions, predominantly EWS::FLI1. Despite sharing this single oncogenic driver, we and others have shown that Ewing sarcoma tumor cells are highly plastic cells that exist in transcriptionally and phenotypically distinct states. Notably, the extent of EWS::FLI1 transcriptional activity, ranging from low to high, has been shown to in part define the Ewing sarcoma cell state. Cells with high levels of fusion protein activity are generally proliferative and non-motile, while cells with lower levels of EWS::FLI1 expression or activity display enhanced metastatic potential. In this study we have leveraged single cell mRNA-seq and CITE-seq of Ewing sarcoma cell lines, gene expression and immunohistochemical profiling of primary tumor samples, and 3D culture invasion assays to define the complex Ewing sarcoma ecosystem and to investigate the role of tumor cell subpopulations and tumor: TME cross talk on tumor biology. Our results show cells with low levels of EWS::FLI1 activity activate an epithelial-mesenchymal transition (EMT)-like shift in gene expression. This activation of a partial EMT program is notable for an increase in expression and secretion of extracellular matrix (ECM) proteins by subpopulations of tumor cells and by increased matrix invasion. These ECM-related genes serve as transcriptional markers of the EMT-like cell state in both Ewing sarcoma models and in primary patient tumors. Moreover, the local TME surrounding these subpopulations is rich in tumor cell-derived ECM proteins including Tenascin-C, biglycan, and SPARC, proteins implicated in promotion of metastasis. We propose that subpopulations of ECM-secreting Ewing sarcoma tumor cells remodel the TME in a manner reminiscent of cancer associated fibroblasts (CAFs) in carcinomas. Further, we propose that this CAF-like matrix deposition contributes to local tumor progression and metastatic dissemination. Significantly, this ECM-secreting tumor cell state can be induced by canonical Wnt and TGF-beta ligands, suggesting that factors present in the bone TME can serve as inducers of a metastatic cell state. Together these findings demonstrate that Ewing sarcoma metastasis may be promoted by specific cell subpopulations which respond to and remodel the bone tumor microenvironment.

Citation Format: Emma Wrenn, April Apfelbaum, Trisha Lipson, Erin Rudzinski, Xuemei Deng, Katherine Braun, Nicolas Garcia, Shruti Bhise, Olivia Waltner, Scott Furlan, Beth Lawlor. Bone microenvironment cues shift Ewing sarcoma cells towards pro-metastatic, ECM-secreting cell states [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B041.