Abstract 5130: DNA methylation profiling reveals epigenetic dynamics across epithelial-mesenchymal transition states in the triple-negative breast tumor microenvironment

Abstract Epithelial-mesenchymal transition (EMT) is a dynamic, reversible process that facilitates tumor invasion and metastasis. Tumors often exhibit intermediate EMT states with co-expression of epithelial and mesenchymal markers. DNA methylation can regulate the expression of EMT-related genes, influencing the stability of epithelial and mesenchymal cell states. However, DNA methylation changes during the dynamic process as cells transition through intermediate EMT states are not well understood. This study integrated single-cell RNA sequencing (scRNA-seq) and DNA methylation profiling to investigate dynamic DNA methylation changes across the EMT spectrum. Single-cell-derived clonal populations from the heterogeneous 4T1 murine triple-negative breast cancer cell line were isolated for five states representing the spectrum of EMT: an epithelial-like clone (E), three intermediate clones (EM1, EM2, EM3), and a mesenchymal-like clone (M). These clones were orthotopically transplanted into the mammary fat pads of mice, and tumors were harvested to isolate CD45- and CD45+ cell populations for DNA methylation profiling at over 285, 000 CpG sites throughout the mouse genome using an Illumina mouse methylation array. Previous scRNA-seq revealed significantly higher expression of Epcam in the epithelial-like clone and Gpnmb in the mesenchymal-like clone. Consistent with the changes in gene expression, methylation shifts were observed across EMT states in the Epcam and Gpnmb promoter regions, both in vitro and in vivo. Notably, immune cell proportions estimated using DNA-based deconvolution exhibited trends consistent with scRNA-seq data, highlighting the potential of multi-omics approaches to comprehensively study the EMT process. To further investigate the progressive increase in mesenchymal transition, we employed a linear model using the CD45- fraction. Hypomethylated CpGs in mesenchymal states were enriched in EMT-related transcription factors, such as FOXC1 and HOXA6. Compared with DNA methylation data from the tumor CD45+ cell fraction, principal component analysis (PCA) of CD45- samples showed separation by EMT states, suggesting fewer DNA methylation changes in tumor infiltrating immune cells. This study highlights epigenetic alterations as cells transition from epithelial to mesenchymal states, with methylation patterns aligning closely with gene expression profiles. Furthermore, these findings emphasize the opportunity to use DNA-based cell type deconvolution to accurately assess and investigate immune cell infiltration within the tumor microenvironment in association with cancer cell plasticity and immune response in tumor progression. Citation Format: Hanxu Lu, Fred W. Kolling, Lucas A. Salas, Diwakar R. Pattabiraman, Todd W. Miller, Brock C. Christensen. DNA methylation profiling reveals epigenetic dynamics across epithelial-mesenchymal transition states in the triple-negative breast tumor microenvironment [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 5130.