Integrated single-cell and spatial transcriptomic analysis characterizes a TNBC-enriched EP5-associated malignant epithelial program and prioritizes REEP4 in breast cancer

Triple-negative breast cancer (TNBC) exhibits pronounced epithelial-state plasticity, aggressive clinical behavior, and heterogeneous responses to immune checkpoint blockade (ICB)-based neoadjuvant therapy. Bulk transcriptomic analyses cannot resolve epithelial-state heterogeneity or spatial organization, limiting the interpretation of subtype-associated malignant programs. Integrating single-cell RNA sequencing (scRNA-seq) with spatial transcriptomics (ST) provides an opportunity to characterize TNBC-enriched epithelial programs within their tissue context. We integrated scRNA-seq profiles from 96 breast specimens across multiple breast cancer subtypes with ST datasets to characterize epithelial heterogeneity, pathway activity, transcriptional programs, and spatial organization. EP5-associated signatures were evaluated in independent bulk transcriptomic cohorts for clinicopathological, prognostic, and immunological associations. Non-negative matrix factorization (NMF), survival analyses, and in vitro siRNA-mediated knockdown assays in TNBC cell lines were used to prioritize candidate mediators. EP5 was characterized as a TNBC-enriched epithelial program associated with cell-cycle activation, malignant-like CNV features, spatially pathway-active regions, adverse clinicopathological characteristics, and unfavorable survival-related patterns in retrospective cohorts. Spatial transcriptomic analysis suggested that EP5-high regions were associated with proliferative and pathway-active tissue niches, including regions with higher MKI67 expression. EP5-high tumors also showed increased immune checkpoint expression, higher tertiary lymphoid structure scores, and an observational association with pathological complete response after ICB plus neoadjuvant chemotherapy. Within EP5, NMF identified MP1 as an adverse-prognosis transcriptional program, and REEP4 was prioritized as a candidate functional mediator. siRNA-mediated REEP4 knockdown reduced TNBC cell proliferation and migration in vitro. This study refines prior single-cell and spatial transcriptomic observations by characterizing EP5 as a spatially heterogeneous, TNBC-enriched epithelial program associated with adverse clinicopathological, prognostic, and immune-related features. The MP1/ REEP4 axis may represent a candidate vulnerability within this program. Further subtype-adjusted, patient-derived, prospective, and mechanistic validation is required before EP5- or REEP4 -related strategies can inform TNBC risk assessment or therapeutic guidance.