Spatial and Single-Cell Dissection of Fibroblast Subpopulation Reprogramming Driving Stromal Collapse in Breast Cancer Lymph Node Metastasis

Objective: This study aimed to define conserved molecular drivers of breast cancer lymph node metastasis (LNM) through an unbiased, multi-omics approach, resolving spatial and cellular mechanisms of stromal reprogramming. Methods: We integrated three bulk RNA-seq datasets (GSE110590, GSE193103, GSE209998) to identify differentially expressed genes (DEGs) in LNMs versus primary tumors. Tissue-specific genes were excluded where possible. Single-cell (scRNA-seq; GSE225600) and spatial transcriptomics (Visium) from primary/metastatic tissues were analyzed using Seurat and spacexr. Fibroblast subclusters were isolated for functional enrichment. Clinical validation included genetic alteration profiling (cBioPortal), survival analysis (KM Plotter), and stage-specific expression dynamics (TNMplot). Therapeutic candidates were screened via CTD. Findings: Bulk RNA-seq identified three genes consistently downregulated in lymph node metastases compared to primary tumors: MAB21L1, F2RL2, and COL6A6 (log₂FC ≤ -2.62; adj. p < 0.05). Single-cell RNA-seq analysis revealed that all three DEGs converged exclusively within specific fibroblast subpopulations in primary tumors; these DEGs were not enriched in other cell types. DEG-expressing fibroblasts orchestrated complementary protective barriers: F2RL2⁺ fibroblasts mediated immune crosstalk (via CXCL14, C1S) and TGF-β balance; COL6A6⁺ fibroblasts reinforced ECM structure (COL6A6/COL6A3) and inhibited Wnt signaling (SFRP2); MAB21L1⁺ fibroblasts suppressed SMAD signaling (through NR2F1 upregulation) and blocked cell migration. Metastatic niches exhibited significant depletion of protective fibroblast subpopulations (F2RL2⁺: reduced from 1.9% to 0.1%; COL6A6⁺: reduced from 3.1% to 1.3%); this depletion coincided with ECM dissolution and disrupted spatial coordination of immune markers (proximity-dependent HLA-DRA/CXCL9 expression). High expression of these DEGs correlated with improved recurrence-free survival (e.g., COL6A6: HR = 0.19, p = 0.002); genomic deletions of the DEGs were significantly more frequent in metastases versus primary tumors (e.g., MAB21L1 deep deletion: 9.13% vs. 0.57%). Conclusion: An unbiased transcriptomic approach revealed specialized fibroblast subpopulations as central gatekeepers against LNM via ECM, immune, and signaling barriers. Their erosion drives stromal collapse in metastasis. Epigenetic modulators (Valproic Acid, Vorinostat) were prioritized for therapeutic restoration of suppressive stroma.