Compartmentalized Aldehyde Stress Drives CD8⁺ T Cell Exhaustion and Spatial Exclusion in Breast Cancer

Background Breast cancer has surpassed lung cancer as the most commonly diagnosed malignancy worldwide, yet the mechanistic relationship between cancer stem cell (CSC)-associated aldehyde metabolism and anti-tumor immunity remains poorly defined. ALDH1 enzymes are established CSC markers that catalyze retinaldehyde oxidation to retinoic acid, positioning them at the intersection of stemness maintenance and immune regulation. Methods We integrated six analytical modalities across four independent breast cancer cohorts: bulk transcriptomics (TCGA-BRCA n = 1,081; METABRIC n = 1,979), single-cell RNA-seq (GSE176078, 90,915 cells), spatial transcriptomics (9 Visium specimens), cell-cell communication inference (CellChat), LASSO-Cox prognostic modeling, and pharmacogenomic analysis (GDSC2, n = 805 cell lines). A 70-gene aldehyde metabolism signature was curated from KEGG and MSigDB. A 17-gene Aldehyde-Enriched Signature (AES) was externally validated in three independent cohorts. Immunotherapy response was evaluated in I-SPY2 and IMvigor210. Results ALDE-stress and T cell exhaustion were decoupled at the bulk level (partial rho=-0.001, p = 0.98 after controlling ferroptosis and ROS), confirmed at single-cell resolution by compartment-independent expression of aldehyde enzymes (epithelial ALDE vs CD8 exhaustion: rho=-0.049, p = 0.83). Spatial transcriptomics revealed global tumor-T cell exclusion (Stouffer z=-18.9, p = 1.8e-79), maintained by CSC-derived protein checkpoint signals (MIF-CD74) rather than diffusible aldehyde metabolites. The AES independently predicted overall survival (METABRIC HR = 0.87, 95% CI 0.82–0.92, p = 2.4e-6; nomogram C-index = 0.703 vs PAM50 alone 0.581) but showed no significant immunotherapy response prediction (IMvigor210 AUC = 0.498; I-SPY2 pooled AUC = 0.563), consistent with the compartmental decoupling hypothesis which predicts that a CSC-derived biomarker should not forecast T-cell-mediated immunotherapy outcomes. Conclusions Aldehyde stress and CD8 + T cell exhaustion are compartment-decoupled in breast cancer. CSC niches exclude T cells through spatial segregation and protein checkpoint signaling, and the AES captures CSC-compartment biology rather than T-cell-intrinsic ICI responsiveness. These findings support a dual-compartment therapeutic strategy combining CSC-targeted ALDH inhibition with T-cell-intrinsic metabolic restoration, constituting mechanistically orthogonal and complementary treatment axes.