Abstract 6440: Spatial transcriptomics of Ewing sarcoma: evolution of tumor associated extracellular matrix from localized to metastasis-associated primary Ewing sarcomas

Abstract Objective: To date, the response rate of immunotherapy in Ewing sarcoma (EwS) has been poor. The tumor immune microenvironment (TME) has a vital role in cancer survival and progression with implications in drug resistance and immune escape. However, there is a lack of understanding of the TME of EwS. Although recent single-cell RNA sequencing of EwS and immune cells have revealed a heterogenous transcriptional landscape with distinct subsets of immune cell populations in EwS, little is known about the spatial organization and spatial microenvironmental niches of the TME of EwS. Methods: We performed Spatial Transcriptomics (ST) on 16 primary pre-treatment biopsied EwS tumors (10 with localized EwS (L-EwS); 6 with metastatic EwS (M-EwS)). Results: Integrated analysis revealed inter- and intra-tumoral heterogeneity. We inferred the stromal regions of tumors transcriptionally and noted that tumors from L-EwS had significantly more stromal signature than M-EwS (p-value <2.2e-16). Gene set enrichment analysis using Hallmark gene sets revealed that L-EwS tumors were enriched in epithelial mesenchymal transition (EMT) and inflammatory signatures (adjusted p-value <0.05) and M-EwS tumors were enriched in proliferative signatures (adjusted p-value <0.05). We identified a set of 46 unique genes enriched in L-EwS that overlapped with genes in EMT hallmark pathways and were predominantly extracellular matrix related (ECM-r) genes such as COL3A1, ITGB1, MMP2, ACTA2, TNC, TGFB1. These ECM-r genes spatially overlapped with the stromal regions of individual tumors. Furthermore, gene composite analysis demonstrated spatial colocalization of macrophages and cancer-associated fibroblasts (CAFs) within the ECM-r regions of the tumors. Spatially informed cell type deconvolution revealed EwS tumor regions that correlated well with histology. Moreover, majority of macrophages/monocytes in L-EwS tumors colocalized with CAFs. Deconvolution analysis also uncovered previously unreported B cell infiltrates and CD8 T cell infiltrative tumors. Spatial cellular microenvironment analysis revealed immune microenvironmental signaling throughout all tumors and unique ECM microenvironmental signaling predominantly in L-EwS compared to M-EwS (p-value <0.05) (TGF-B, cell adhesion and ECM remodeling signals). Conclusion: L-EwS tumors are enriched in ECM and ECM-related microenvironmental signals compared to M-EwS. Spatial cellular microenvironmental signals reveal that components of the ECM may serve as anchors for tumors to eventually metastasize, nutrients for EwS to proliferate, and most importantly as a communication network with host immune cells. Further studies will be performed on a novel immunocompetent zebrafish genetic EwS model to identify conserved mechanisms of host-tumor interactions and further dissect the role of ECM in EwS. Citation Format: Christopher Kuo, Krinio Giannikou, Nuoya Wang, Mikako Warren, Andrew Goodspeed, Masanori Hayashi, Micha S. Raredon, James F. Amatruda. Spatial transcriptomics of Ewing sarcoma: evolution of tumor associated extracellular matrix from localized to metastasis-associated primary Ewing sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6440.