Integrative Single-Cell and Bulk Transcriptomic Analyses with Spatial Validation Identify a Residual Fatty Acid-EMT Subset Driving Chemotherapy Resistance in Triple-Negative Breast Cancer via MIF- and MK-Mediated Ligand-Receptor Signaling.

Chemotherapy resistance in triple-negative breast cancer (TNBC) remains a critical clinical challenge, with a substantial proportion of patients failing to achieve pathological complete response following neoadjuvant chemotherapy (NAC). Using an integrative single-cell RNA sequencing (scRNA-seq), bulk transcriptomic, and spatial proteomic framework, we aimed to identify the malignant epithelial subset driving this resistance and the intercellular signaling axes through which it reprograms the tumor microenvironment (TME). scRNA-seq analysis of NAC-treated breast tumors revealed a Fatty Acid-EMT co-expressing epithelial subset (FA-EMT) that is selectively enriched in the chemotherapy-resistant residuum. Critically, FA-EMT co-expression-rather than either program individually-most powerfully predicted chemotherapy resistance and reduced overall survival across two independent bulk transcriptomic cohorts comprising 277 TNBC patients (p < 0.001). CellChat ligand-receptor analysis established FA-EMT cells as the dominant TME signaling hub, deploying MDK-NCL and MIF-CD74-CXCR4 axes to simultaneously suppress adaptive and innate anti-tumor immunity via T-cell exhaustion, Treg activation, and the expansion of myeloid-derived suppressor cells. Spatial CyCIF validation in a published paclitaxel-resistant TNBC mouse model (n = 69 cores) confirmed significant Metabolic-EMT enrichment in resistant tumor cores (p = 0.0085) with physical co-localization with immunosuppressive MDSC and Treg populations. These findings establish the FA-EMT subset as a key cellular driver of treatment failure in TNBC and nominate MDK-NCL and MIF-CD74-CXCR4 as mechanistically grounded therapeutic targets with the potential to dismantle the FA-EMT-driven immunosuppressive niche and sensitize chemotherapy-resistant TNBC to cytotoxic treatment.