Epithelial-Mesenchymal Transition is Associated with Altered Immune Composition and Cytotoxic Function in Triple-Negative Breast Cancer

Abstract Epithelial-mesenchymal transition (EMT) is a dynamic process that contributes to breast cancer progression, metastasis, and therapy resistance. EMT also influences the tumor immune microenvironment, shaping immune cell infiltration and function. Although the immunological features of fully epithelial or fully mesenchymal tumor states have been characterized, the immune landscape associated with intermediate or partial EMT states remains poorly understood. To address this gap, we established five single-cell–derived clonal populations from the triple-negative 4T1 mouse mammary tumor cell line, representing a spectrum of EMT phenotypes, and analyzed tumors derived from these clones using single-cell RNA sequencing. We show that tumors derived from these clones retained their relative EMT states in vivo, and that EMT progression is associated with a graded reduction in tumor immunogenicity, accompanied by alterations in immune cell composition and function. Along the EMT spectrum, tumor cells exhibited progressive downregulation of major histocompatibility complex (MHC) class I and II gene expression, along with decreased infiltration of cytotoxic CD8+ T cells, and reduced expression of key effector and trafficking genes (Gzmb, Ccr5, Cxcr6). B cell composition also shifted, with decreased frequencies of IgG1-producing plasma cells and an enrichment of regulatory-like B cells. Natural killer (NK) cells similarly demonstrated progressive functional suppression, marked by reduced expression of cytotoxic molecules and the downregulation of key effector pathways. These findings reveal that EMT is associated with immune cell recruitment and function in a graded manner, providing a rationale for integrating EMT phenotyping into therapeutic strategies to overcome immune resistance in breast cancer. Competing Interest Statement Brock Christensen is a co-founder of Cellintec LLC but declares no non-financial competing interests. Lucas Salas is a co-founder of Cellintec LLC but declares no non-financial competing interests.