Background Emerging data suggested that enzalutamide-treated prostate cancer patients with increased programmed death-ligand 1 (PD-L1) expression may benefit from anti-PD-L1 treatment.1 2 Unfortunately, the Phase III IMbassador250 trial revealed that the combination of atezolizumab (PD-L1 inhibitor) and enzalutamide failed to extend overall survival in patients with castration-resistant prostate cancer (CRPC).3–5 The mechanisms underlying treatment failure remain unknown. In this study, we investigated the regulation of immunosuppressive signaling in enzalutamide resistant prostate cancer and characterized the immune infiltrating cells in murine prostate tumors.
Methods The expression of interferon gamma-related genes was determined using qRT-PCR and/or western blotting. Androgen receptor (AR) and AR-V7 levels were downregulated using specific siRNA. Myc-CaP cells were chronically exposed to increasing concentrations of enzalutamide (5–50 μM) for >12 months, and the cells resistant to enzalutamide were referred to as Myc-CaP MDVR. The gene-regulating mechanisms in drug-resistant prostate cancer cells were determined by RNA sequencing analyses. Myc-CaP and Myc-CaP MDVR xenograft tumors were established in FVB mice, and tumor-infiltrating lymphocytes were isolated using Ficoll. The stained immune cells were determined by flow cytometry, and the data were analyzed using Flowjo.
Results Immune-related signaling pathways (interferon alpha/gamma response, T cell activation, and cell chemotaxis pathways) were suppressed in C4–2B MDVR cells. However, PD-L1 expression was highly upregulated and negatively regulated by AR in C4–2B MDVR cells. Enzalutamide treatment decreased CD8+ T cell but increased monocytic myeloid-derived suppressor cell (M-MDSC) population and PD-L1 expression in murine Myc-CaP tumors. Consistently, chemotaxis and immune response-regulating signaling pathways were suppressed, and PD-L1 expression was increased in enzalutamide-resistant Myc-CaP MDVR cells. Notably, MDSC populations were significantly increased in Myc-CaP MDVR orthotopic tumors compared with those in Myc-CaP parental tumors. Co-culturing bone marrow cells with Myc-CaP MDVR cells significantly promoted MDSC differentiation and polarized macrophages from the M1 to M2 phase.
Conclusions Immunosuppressive signaling is dysregulated in enzalutamide resistant prostate cancer cells. Immunosuppressive alterations in the tumor immune microenvironment can be promoted directly by enzalutamide-resistant CRPC cells, which promote self-immune evasion by inducing immunosuppressive cell infiltration and forming an immunosuppressive tumor microenvironment. The activated immune-related signatures involved in T cells, MDSC, and macrophages may reduce the efficacy of immune checkpoint inhibitors in enzalutamide-resistant prostate cancer.
Acknowledgements This work was supported in part by grants NIH/NCI R37CA249108 (C, Liu), R01CA251253 (C, Liu), and DoD HT94252310144 (C, Liu).
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Ethics Approval All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of UC Davis complied with ethical regulations and humane endpoints.
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