Abstract 3974: Flexible and comprehensive phenotyping of tumor and peripheral blood mononuclear cells in endometrial carcinoma

Abstract Endometrial carcinoma (EC) is the most common form of uterine cancer, with increasing prevalence in developed countries due to population aging and rising obesity rates. Though prognosis is relatively good with an 80% survival at five years after diagnosis, the leading treatment is total removal of the uterus, ovaries and fallopian tubes. Thus, identifying biomarkers that may enable more personalized medicine or immunotherapy discoveries is important, both to create more treatment options as well as to tackle aggressive EC subtypes. To that end, we developed a 58-parameter mass cytometry (CyTOF™) panel that enables deep phenotyping of both immune and non-immune cells in tumors as well as in PBMC. In total, 48 markers were measured for each cell, allowing for deep profiling of cell identity, cytokine production, cytotoxicity, exhaustion and activation. This was achieved by combining pre-optimized panels for pan-immune cell phenotyping, T cell profiling and cytokine and cytotoxic mediator detection with nine drop-in antibodies selected for their relevance in EC as biomarkers or potential immunotherapy and drug targets. Cell proliferation was assessed using IdU incorporation and Ki-67 staining, while tumor cell and tumor-infiltrating leukocytes (TILs) were further defined with EpCAM, Her2, β-catenin, CD69, CD103, PD-L1 and FoxP3. Live-cell universal barcoding with a tellurium-based 7-choose-3 strategy allowed multiplexing of samples during cell staining and acquisition. Matched PBMC and dissociated tumor cells (DTCs) from EC patients and healthy donor PBMC (n=5 each) were stimulated with PMA and ionomycin, then barcoded and pooled. Cells were then stained for surface, cytoplasmic and nuclear targets before acquisition on a CyTOF XT mass cytometer. Data was normalized and debarcoded before analysis to yield sample-specific cell frequencies and marker expression. The tumor-derived T cells from EC patients exhibited a tissue residency phenotype (CD69+CD103+/-), robust staining of immune checkpoint markers TIGIT, LAG-3, TIM-3 and OX-40 and increased staining of CD95/Fas, PD-1 and 4-1BB compared with peripheral blood T cells. Intracellular cytokines were detectable on tumor-derived immune cells despite being rare, with some subsets producing IFNγ, TNFα, IL-2 and IL-17A in response to stimulation. This high-parameter panel allows for deep phenotypic and functional profiling of circulating immune cells, tissue and tumor cells and TILs. In addition to dissociated cells from tumor resections and biopsies, this panel could be used with liquid or fine needle aspirate biopsies from tumor samples when combined with live-cell universal barcoding to multiplex low-input samples. By utilizing a flexible and modular assay designed to characterize both immune and non-immune cells, this CyTOF panel can facilitate biomarker discovery and accelerate drug development in cancer therapeutics. Citation Format: Deeqa Mahamed, Geneve Awong, Gary Impey. Flexible and comprehensive phenotyping of tumor and peripheral blood mononuclear cells in endometrial carcinoma [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 3974.