Targeting CAMK1D-engineered nanoactivator suppresses cancer stem cell maintenance and immune evasion in enzalutamide-resistant prostate cancer

<b>Rationale:</b> 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. <b>Methods:</b> 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 <i>in vitro</i>. 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. <b>Results:</b> 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 <i>in vitro</i> 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. <b>Conclusions:</b> 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.