Abstract
Rationale: Hormonal therapy is fundamental to prostate cancer (PCa) management; however, its long-term efficacy is compromised by enzalutamide resistance (ENZR), which is fuelled by prostate cancer stem-like cells (PCaSCs) and an immunosuppressive microenvironment. Methods: A CD44-targeted nanoactivator (EC@HNA) was engineered to co-deliver ENZ and siCAMK1D. Its physicochemical properties, cellular uptake and gene-silencing efficiency were characterized in vitro. Functional and mechanistic assays were used to assess PCaSCs expansion, cytokine modulation, immune cell dynamics, and CREB-dependent regulation of stemness genes. Therapeutic efficacy and safety were validated in ENZR cell cultures, murine tumor models, and patient-derived organoids. Results: EC@HNA efficiently delivered siCAMK1D and achieved potent CAMK1D silencing, thereby significantly suppressing the expansion and self-renewal of PCaSCs. This treatment downregulated the immunosuppressive cytokines IL-10 and TGF-β, decreased regulatory T cell (Treg) infiltration, promoted M1-like polarization of tumor-associated macrophages, and enhanced CD8⁺ T cell infiltration and cytotoxicity in ENZR prostate tumors, thereby reprogramming the tumor immune microenvironment. Mechanistically, EC@HNA suppressed CREB phosphorylation at Ser133, which transcriptionally repressed key stemness regulators, including CD44, CD133, and NR4A1, thereby attenuating tumor stemness and immune evasion. These effects have been validated using in vitro cell models, ENZR xenografts, and patient-derived organoids. Collectively, EC@HNA dismantled the stemness-immunity axis sustaining ENZR and restored robust anti-tumor immunity with minimal systemic toxicity. Conclusions: Overall, the CD44-targeted EC@HNA nanoplatform disrupted stemness programs and restored tumor-immune surveillance, representing a promising strategy to reverse ENZR and potentiate immunotherapy in clinical ENZR PCa patients.