Abstract 6615: Uncovering the landscape of cellular heterogeneity in low- and high-grade endometrial cancer using single cell profiling

Abstract Introduction: Endometrial cancer (EC) is the most common gynecologic malignancy in women, with rising incidence and mortality. EC is classified into low-grade (I/II), less aggressive, and high-grade (III), associated with poor prognosis and aggressive behavior. Single-cell RNA sequencing (scRNA-seq) offers insights into the interplay between tumor cells and the tumor microenvironment (TME), improving diagnosis and treatment strategies. This study aims to identify unique cell subtypes and factors distinguishing low-grade from high-grade EC, uncovering therapeutic targets to address tumor progression. Methods: Tumors from ten treatment-naïve EC patients (four low-grade endometrioid, six high-grade (three endometrioid, three serous), and two age-matched non-malignant endometrial samples were collected at surgery and frozen. Nuclei were isolated from the frozen samples and single-cell libraries prepared using the Chromium Next GEM Single Cell Kit v3.1 according to the manufacturer’s recommendations. Data were analyzed using Seurat v5 in R v4.3.3. Results: Multiple phenotypic subsets of EC cells, T cells, fibroblasts, macrophages, endothelial, and epithelial cells were identified. Six distinct EC cell subsets were present across tumors, showing significant intratumoral heterogeneity. The MKI67+ EC subcluster predominated in high-grade endometrioid EC, while CHRM2/3+ EC subclusters were exclusive to high-grade serous EC. Both low- and high-grade endometrioid EC exhibited a high proportion of PROM1+ (CD133) cancer stem cells. Epithelial-to-mesenchymal transition (EMT) genes (COL1A2, BNC2, HNF1A-AS1, ITMC2, ZNF492) were upregulated in high-grade EC compared to low-grade, correlating with aggressive phenotypes and poorer prognoses. High-grade EC showed increased activated T cells (PTPN14+), suggesting immune evasion, interestingly, low-grade EC had the highest proportion of exhausted T cells (TIM3+, PD1+) typically associated with aggressive tumors. Consistent macrophage subtypes were observed across EC cells, indicating T-cell-mediated alterations in the TME between EC grades. Conclusion: This study provides the first comprehensive comparison of tumor cell diversity and TME components in low- and high-grade EC (endometrioid and serous). These findings, alongside ongoing in vitro studies, aim to uncover viable, subtype-specific therapeutic targets. *Both authors contributed equally Citation Format: Sabina I. Swierczek, Elizabeth A. Garfinkle, Sophia A. Paxton, Katherine E. Miller, David Doo, Leslie Andriani, Shatovisha Dey, Steven Sieber, Alpana Dholakia, Syeda Rashid, Linus Chuang. Uncovering the landscape of cellular heterogeneity in low- and high-grade endometrial cancer using single cell profiling [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 6615.