Lymph Node Metastasis-Associated Spatiotemporal Mapping of the TFF3-Linked Niche in Breast Cancer: Integrating Radiogenomic Signatures with Immune-Ecosystem Remodeling

Primary breast cancer (PBC) with axillary lymph node metastasis (ALNM+) is associated with distinct clinical outcomes, including reduced survival (The Cancer Genome Atlas/Foshan cohorts, P < 0.05) and an attenuated response to anti-programmed cell death protein 1 antibody/anti-programmed death-ligand 1 antibody (anti-PD-1/anti-PD-L1) therapy. Through ALNM-stratified single-cell RNA sequencing profiling, we identified 3 hallmark immune subsets in ALNM+ PBC: (a) proliferative MKI67 + T cells, (b) exhausted GZMA + CD8 + T cells, and (c) CCL13 / CXCL10 / TOP2A + macrophages. Cross-modal integration of metastasis–epithelial–mesenchymal transition (EMT) signatures with Mendelian colocalization analysis prioritized TFF3 as a central mechanistic regulator. We validated malignant-cell-specific TFF3 expression across pan-cancer single-cell profiles and in PBC lineages. Integration of Mendelian colocalization signatures with pan-cancer spatial atlases established the TFF3 oncogene as a regulator of spatial EMT programs. Radiogenomic modeling that incorporated machine-learning-derived computed tomography features identified a TFF3 -based radiomics risk score. Spatial multi-omics analyses—including bulk RNA sequencing, proteomics, and spatial transcriptomics—established a correlation between TFF3 expression and both MAPK signaling activation and EMT markers. Functional validation demonstrated that TFF3 plays a dual role as an amplifier of the MAPK–EMT axis and a modulator of immune checkpoints. Critically, the prometastatic phenotype driven by TFF3 was rescued upon pharmacological inhibition of MAPK signaling, providing direct evidence of this mechanistic link. In vivo xenograft models confirmed that TFF3 knockdown suppressed metastasis. Pharmacogenomic screening identified 6-mercaptopurine as a novel TFF3 antagonist, which exhibited dose-dependent inhibition of the MAPK–EMT axis. Furthermore, the antimigratory effect of 6-mercaptopurine was reversed by TFF3 overexpression, confirming the functional specificity of this drug–target interaction. Notably, tumors with high TFF3 expression ( TFF3 hi) exhibited elevated resistance to PD-1 inhibitors but heightened sensitivity to MAPK inhibitors, suggesting a potential theranostic framework for ALNM stratification.