Abstract 5462: Single-cell spatial proteomic analysis to explore the spatial heterogeneity of desmoplastic small round cell tumors
Authors: Truong Danh , Krishnan Sandhya , Agyemang Clement , Ingram Davis , Lascano Rossana , Basi Akshay , Gomez Javier , Burks Jared , Lazar Alexander , Ludwig Joseph 
Published: March 2024

Abstract
Desmoplastic small round cell tumor (DSRCT) is a rare and usually incurable aggressive sarcoma subtype. All malignant cells harbor a pathognomonic EWSR1::WT1 fusion protein (FP). However, FP-targeted agents are nonexistent; less than 20% of patients survive beyond five years. The spatial organization of DSRCT, consisting of tumor nests surrounded by desmoplastic stroma, is a hallmark of this disease. Given this context, we seek to understand the spatial heterogeneity within DSRCT and the tumor-stroma interactions to reveal potential therapeutic vulnerabilities. We generated a 20-panel marker developed to interrogate the cellular constituents of the tumor microenvironment (TME) and to characterize the multilineage expression DSRCT, including androgen receptor (AR) and neuroendocrine signatures (NE). The following markers were used: 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), AR/NE markers (AR, neural-specific enolase, synaptophysin). We explored the expression of these markers in a nine-patient cohort from 9 x 9 mm2 tissue sections imaged by the Lunaphore COMET. We used a deep learning model in Visiopharm to extract high-quality areas and segment cells based on their nucleus. The spatial image data was converted into matrix data, which could be further analyzed in R. We confirmed that the tumor nests are surrounded by dense desmoplasia by collagen expression. We identified tumor cells, fibroblasts, immune cells, and endothelial cells by thresholding for classical cell markers. ST6GALNAC, a sialyltransferase that was found to be highly expressed in DSRCT based on RNA-seq data, marked the tumor nests. Previous data from single-nucleus RNA-sequencing (snRNA-seq) showed that DSRCT exhibited three possible subtypes: AR+/NE-, AR-/NE+, and Hybrid AR+/NE+. We found concordance between the prior transcriptomic signatures and the proteomic markers. In tumor nests that were positive for AR, we found AR localized to the nucleus, suggesting downstream activation of this pathway. High AR expression was also correlated with positive expression of pan-cytokeratin in the tumor nests. Future work will focus on quantifying spatial relationships and patterns. This includes characterizing the spatial distribution of all cell types found in DSRCT, including fibroblasts, endothelial cells, and immune cells. We will also address whether tumor nest characteristics are associated with cell phenotype or DSRCT subtype.
Citation Format: Danh Truong, Sandhya Krishnan, Clement Agyemang, Davis Ingram, Rossana Lascano, Akshay Basi, Javier Gomez, Jared Burks, Alexander Lazar, Joseph Ludwig. Single-cell spatial proteomic analysis to explore the spatial heterogeneity of desmoplastic small round cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5462.