Abstract 5323: Spatial heterogeneity of tumor and stromal cells in desmoplastic small round cell tumors

Abstract Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive sarcoma subtype that harbors a pathognomonic EWSR1::WT1 fusion protein (FP). DSRCT is usually incurable and FP-targeted agents are nonexistent. The five-year survival rate is below 20%. Tumor nests are classically surrounded by desmoplastic stroma in DSRCT, yet the interactions between the tumor cells and the stromal cells are poorly understood. Given this context, our study aims to understand the spatial heterogeneity within DSRCT and the tumor-stroma interactions. We utilized a 20-panel marker to study the cellular constituents of the tumor microenvironment (TME) in DSRCT and to characterize its multilineage expression, including epithelial and neural (NE) signatures. The markers included: DSRCT (ST6GALNAC, pan-Cytokeratin), Fibroblasts (Collagen, α-smooth muscle actin), Endothelial Cells (CD31, α-smooth muscle actin), T-cells (CD45, CD4, CD8), Macrophages (CD45, CD68, CD163, CD11c), and AR/NE markers (androgen receptor, neural-specific enolase, synaptophysin). Marker expression was explored in a nine-patient cohort using 9 x 9 mm2 tissue sections imaged by the Lunaphore COMET system. A deep learning model, Visiopharm, extracted high-quality regions and segmented cells based on nuclear staining. Spatial data were transformed into matrix format for analysis in Python, where UMAP and Leiden clustering identified distinct clusters consisting of tumoral cells, fibroblasts, and endothelial cells. Our analysis revealed two distinct fibroblast clusters—one with high α-SMA expression and the other with high collagen expression. These two clusters exhibited distinct spatial distributions and proportions across patient samples, indicating heterogeneity in stromal composition within the DSRCT microenvironment. Our previous study identified three DSRCT subtypes: AR+/NE-, AR-/NE+, and Hybrid AR+/NE+. The current spatial analysis suggests that AR+/NE- tumor nests are consistently surrounded by α-SMA-high fibroblasts, hinting at subtype-specific stromal interactions. Preliminary findings from co-culture experiments of DSRCT cells with human mesenteric fibroblasts suggest that fibroblasts enhance the proliferative capacity of DSRCT cells. These findings provide novel insights into the stromal heterogeneity of the DSRCT microenvironment and its association with tumor subtypes. Our future work will focus on quantifying spatial relationships and patterns, which includes how tumor nest characteristics are associated with fibroblast cell type or DSRCT subtype. Proximity and neighborhood analysis in python and Visiopharm analysis will be applied. Ultimately, our results might reveal novel drug targets that stop oncogenic tumor-stroma signaling. Citation Format: Jiaqian Fan, Adewale A. Adebayo, Diana Shamsutdinova, Davis Ingram, Clement Agyemang, Roberto Cardenas-Zuniga, Alexander Lazar, Danh Truong, Joseph Ludwig. Spatial heterogeneity of tumor and stromal cells in desmoplastic small round cell tumors [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 5323.