A cancer stem cell-directed nano-chemotherapy enhances anti-PD-L1 immunotherapy in breast cancer by inducing immunogenic cell death.

Immune checkpoint blockade, exemplified by anti-PD-L1 antibody (αPD-L1), has revolutionized cancer immunotherapy. Nevertheless, its therapeutic potential is constrained by the inherently low tumor immunogenicity and the persistence of cancer stem cells (CSCs), which drive immune escape and tumor recurrence. Here, a CSC-targeted, pH-sensitive liposomal nano-cocktail of doxorubicin and bufalin (PSLB/D), was demonstrated to elicit robust immunogenic cell death (ICD), thereby potentiating the efficacy of αPD-L1 in immunologically 'cold' triple-negative breast cancer (TNBC) models. The PSLB/D itself demonstrated potent synergistic tumor-inhibitory effects in both cell lines and 3-D tumor spheroids, and effectively eliminated CSCs, with 85-90% reductions in both primary and secondary sphere formation. Deep penetration and robust ICD-mediated immune response were observed in patient-derived organoids, highlighting its translational potential. The therapeutic efficacy was validated in immunocompromised MDA-MB-231 and immunocompetent 4T1 orthotopic mouse models. Notably, in immunocompetent 4T1 models, PSLB/D induced strong expression of calreticulin, leading to the recruitment of dendritic cells and cytotoxic CD8⁺ T cells. Activation of these immune cells converted the immunosuppressive 'cold' tumors into immunogenic 'hot' ones, substantially amplifying the effectiveness of αPD-L1. This chemo-immunotherapy also generated memory T cells, suggesting the potential for longer antitumor immune responses. STATEMENT OF SIGNIFICANCE: Cancer recurrence and immune evasion in triple-negative breast cancer (TNBC) are largely driven by resilient cancer stem cells (CSCs). This study introduces a pH-sensitive liposomal nano-cocktail composed of doxorubicin and a CSC inhibitor, specifically engineered to overcome the immunosuppressive tumor microenvironment. We demonstrate that this nanococktail triggers potent immunogenic cell death (ICD) in patient-derived organoids and orthotopic models, exceeding the efficacy of conventional formulations or monotherapy. By successfully converting "cold" TNBC tumors into immunologically "hot", the nano-cocktail significantly amplifies anti-PD-L1 immunotherapy outcomes. These findings offer a compelling evidence-based framework for integrating targeted nanomedicine with clinical chemo-immunotherapy protocols to prevent recurrence.