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