Paired Single-Cell Profiling Reveals Glutathione-Dependent Immune Evasion in Breast Cancer Liver Metastasis

Abstract Breast cancer liver metastasis (BCLM) represents a major clinical challenge. Characterizing the differences between primary and metastatic tumors could help identify therapeutic targets for preventing and treating metastasis. Through single-cell transcriptomic sequencing of matched primary tumors and BCLMs from 13 treatment-naïve patients, we uncovered a fundamental paradox in metastatic progression: BCLMs exhibited a highly immune-infiltrated microenvironment characterized by cytotoxic T cell expansion, inflammatory macrophage polarization, and depletion of regulatory T cells, yet they successfully evaded immune-mediated destruction. Metastatic cells retained extensive clonal and genomic diversity from their primary tumors, suggesting a polyclonal metastatic process rather than a stringent metastatic “bottleneck”. Instead, cancer cells featured a metabolic adaptation mechanism in which elevated glutathione (GSH) metabolism in the liver fosters glutamine addiction, creating liver metastasis susceptibility. Integrative regulatory network analyses pinpointed NRF2 as a key regulator associated with GSH metabolism, and functional experiments demonstrated that GSH promoted tumor stemness and attenuated CD8⁺ T cell-mediated antitumor immunity in an NRF2-dependent manner. Inhibition of NRF2 and GSH reduced liver metastasis in vivo and was associated with enhanced antitumor immune responses. Together, this study provides comprehensive single-cell characterization of the immune-metabolic landscape in treatment-naïve BCLM and reveals a potential model in which metastatic success in immune-competent tissues depends on tumor cell-intrinsic metabolic adaptation. These findings provide insights into breast cancer liver metastatic progression and suggest NRF2 and GSH inhibition as a promising therapeutic strategy that could restore immune surveillance in liver metastases.