| Purpose: Prostate cancer is the second-ranked malignant tumor responsible for male deaths.Among all tumor-specific deaths,PCa ranks fifth.It causes more than 300,000 deaths per year,accounting for approximately 6.6% of cancer deaths of males.Androgen deprivation therapy(ADT)can be used for advanced patients,while the 5-year overall survival rate is only 25.4 %,which can be attributed to the rapid development of castration-resistant prostate cancer(CRPC).Enzalutamide is a new drug for the treatment of CRPC.It functions by inhibiting the androgen receptor(AR)from entering the nucleus.Gene mutations,AR splicing,and tumor microenvironment(TME)play a key role in the occurrence and development of enzalutamide resistance.Substantial academic efforts have been made to explore TME and immune cells.Therefore,studies on the immunophenotyping of prostate cancer and the crosstalk between immune cells and enzalutamide treatment are of significance,which can guide the clinical treatment.Methods: Herein,the gene expression profile and clinical prognosis information of1557 prostate cancer patients were collected.Among them,the pathological tissue sections of 69 patients were stained with immunohistochemistry to verify relevant results.Moreover,the non-negative matrix factorization algorithm and recent template prediction were used to distinguish immune subtype groups.Then,to characteristic the features of immunophenotypes,published signatures representing the immune cell types or the host anti-tumor immunity were collected.Specifically,THP-1 cells were induced to macrophages by 100ng/ml PMA solution.The conditioned medium was harvested with further co-cultivation with prostate cancer.After the removal of impurities and fragments,exosomes were harvested from the conditioned medium and obtained after70 min of ultracentrifugation at 120,000 rpm.Afterward,the transmission electron microscope was used to observe the morphology of exosomes.MTT and Ed U experiments were carried out to determine the cell proliferation representing the response to enzalutamide.The expressions of ATP1B1 and mi RNA-320 b in prostate cancer cells were altered for the observation of their function.In the next step,Realtime quantitative PCR was used to detect the expression of m RNA and mi RNA,and the Ago2 experiment was performed to verify the interaction between mi RNA and m RNA.Moreover,the transcriptional regulation of AR to ATP1B1 was evaluated through the Ch IP experiment and the dual luciferase report experiment.Results: Approximately 14.9% to 24.3% of patients were identified to be the immune activated subtype,which was associated with a good prognosis for relapse-free survival.Patients with immune activation status benefited more from anti-PD-1/PD-L1 treatment.Among the subtypes and immunosuppressive subtypes,macrophages displayed high relatively high characteristics.In further analysis,a poor prognosis was revealed by the high infiltration of M2 macrophages.The infiltration abundance of M2 macrophages increased after exposure to ARSI,and higher infiltration correlated with the shorter effective time of ARSI treatment.The resistance to enzalutamide treatment was verified in C4-2 and C4-2B cells after they were co-cultured with M2 macrophage.The increase expression of the key downstream gene ATP1B1 was found to be regulated by AR;whereas the downregulation of AR was affected by mi R-320 b from the exosome of the M2 macrophage.As found from further exploration,the expression and activity of the PI3K/AKT pathway increased after the treatment with the conditioned medium of M2 macrophages.This increase was inhibited after the knock-down of ATP1B1 or the introduction of an AKT inhibitor.In in vivo experiments,all groups received enzalutamide treatment,among which the macrophage + prostate cancer group presented the largest tumor size.After the addition of mi R-320 b inhibitor,the treatment of enzalutamide became effective again.Conclusion: Our study identifies a novel immune molecular classifier that is closely related to clinical prognosis and provides new insights into immunotherapeutic strategies that anti-PD-1/PD-L1 therapy is applicable to patients in the immuneactivated subgroup.Additionally,immune infiltration of M2 macrophages is related to enzalutamide resistance of prostate cancer cells.M2 macrophages transport mi R-320 b through exosomes and affect the AR/ATP1B1/PI3K/AKT pathway in prostate cancer cells.Overall,inhibiting mi R-320 b or ATP1B1 is a new approach to the treatment of enzalutamide resistance prostate cancer. |
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