ISG15-driven immune modulation and tumor progression in breast cancer metastasis: insights from single-cell and...

Cancer stem cells (CSCs) play a crucial role in breast cancer (BRCA) progression and lymph node metastasis. This study aimed to elucidate how CSCs reshape the immune microenvironment during metastatic dissemination, with a particular focus on macrophage and T-cell regulation.

A mouse orthotopic BRCA model was established to obtain primary tumor (BRCA_PT) and lymph node metastatic (BRCA_LNMT) tissues. Single-cell RNA sequencing and spatial transcriptomics were used to characterize cellular heterogeneity, marker genes, and intercellular communication. TCGA-BRCA data were analyzed for differential expression, functional enrichment, and immune cell infiltration. In vitro, 4T1-S CSCs were used to assess self-renewal, migration/invasion, ISG15-mediated signaling, and interactions with macrophages and T cells. ELISA, western blotting, sphere formation, colony formation, CCK-8, Transwell, luciferase reporter assays, and ChIP were performed. In vivo, subcutaneous and orthotopic mouse models were used to evaluate the effect of ISG15 on tumor growth and lymph node metastasis.

Bioinformatic analyses revealed an elevated proportion of CSCs in BRCA_LNMT, where CSCs likely induced M2 macrophage polarization through TAM-mediated communication. ISG15 was highly expressed in metastatic tumors and associated with M2 polarization and reduced T-cell activation. In vitro, ISG15 enhanced CSC self-renewal and invasiveness, promoted IL-10-mediated M2 polarization, and upregulated PD-L1 via JAK-STAT signaling to suppress T-cell activity. In vivo, ISG15 silencing significantly inhibited tumor growth and lymph node metastasis.

ISG15 in BRCA CSCs promotes lymph node metastasis by driving M2 macrophage polarization and suppressing T-cell activation, highlighting a critical role for ISG15-mediated immunomodulation and a potential therapeutic target.