促结缔组织增生性小圆细胞肿瘤中上皮样和神经样肿瘤表型及其细胞邻域的空间组织

Desmoplastic small round cell tumor (DSRCT) is a rare and aggressive sarcoma driven by the EWSR1::WT1. Histologically, DSRCT is characterized by distinct spatial heterogeneity of tumor nests surrounded by a desmoplastic stroma. Our previous study showed that DSRCT exhibits heterogeneous expression of androgen receptor (AR)-associated and neuron-specific enolase (NSE)-associated markers, highlighting these as important lineage features of the disease. However, how these AR (Epithelial-like)- and NSE (Neural-like)- associated phenotypes are organized at the single-cell level and contribute to tumor biology remains poorly understood. This study aims to define these phenotypes and delineate their spatial and neighborhood patterns within the DSRCT microenvironment. We used the Lunaphore COMET system to evaluate 12 DSRCT patient specimens, comprising a 20-marker panel on nine slides and a separate 24-marker panel on three slides, which collectively covered epithelial, neural, fibroblast, endothelial, and immune cell types. Using Visiopharm, we applied deep-learning algorithms to identify individual cells and quantify protein expression data. Four custom RNAscope probes targeting EWSR1::WT1-associated neogenes were used to detect tumor cells and identify EWSR1::WT1 activity. Tumor cells were distributed along an AR-NSE expression spectrum, including AR-high, AR-low, double-negative (AR-NSE-), NSE-positive, top 1% NSE-strong, and hybrid phenotype (AR+NSE+). Among AR-associated phenotypes,gradient analysis revealed that AR-high tumor cells were enriched at the tumor nest center and gradually decreased in abundance toward the tumor-stroma interface, where AR-low tumor cells were more prevalent and in closer proximity to fibroblast-rich stromal regions. Among NSE-associated phenotypes, NSE-positive tumor cells were positioned closer to fibroblast-rich stromal regions. In contrast, both the top 1% NSE-strong and AR+NSE+ hybrid phenotypes localized deeper within the tumor region and were farther from fibroblasts. Our work identified novel conserved neighborhoods across samples: tumor-centered, transitional, and fibroblast-enriched neighborhoods. AR-high phenotypes predominantly mapped to tumor-centered neighborhoods, whereas AR-low phenotypes were enriched in transitional and stromal-interacting neighborhoods. These spatial distributions suggest distinct microenvironmental contexts at the center and periphery of the tumor nests, where stromal interactions are more pronounced. Future work will elucidate how stromal cues mediate phenotypic changes in DSRCT.