A Novel Potential Immunotherapy Target In Prostate Cancer Microenvironment:PD-L1 Positive Nerves

Background and Objective:Prostate cancer is the most common malignant disease in men,accounting for 19%of all new tumors in the United States in 2017,rating the third highest mortality.Radical prostatectomy(RP)and endocrine therapy are the mainstream therapies for early localized and advanced metastatic prostate cancer,respectively.These treatments can delay the progression of the disease in early treatment stages,however,most patients will eventually develop into castration resistant prostate cancer(CRPC),leading to a poor prognosis.On the other hand,in 2013,tumor-infiltrating nerve fibers were demonstrated to have a pronounced promotion to both early development and advanced metastasis process of prostate cancer,and were associated with poor clinical outcomes.Unlike with conventional radiotherapy and chemotherapy,which result in a nonspecific eradication of both normal and cancerous cells,tumor immunotherapy can induce specific immune antitumor responses.In recent years,anti-PD-1/PD-L1 pathway tumor immunotherapy developed in clinical trials has shown an unprecedented anti-cancer effect,however,this effect is limited to a small number of patients.And the antineurogenic therapies are only in the exploratory stage.This study aims to explore the roles of PD-1/PD-L1 pathway and nerve in contributing to the formation of an immunosuppressive tumor microenvironment in prostate cancer,with which tumor immunotherapy and antineurogenic therapies are creatively combined.Materials:1.Human prostate tissues:Consecutive slides of 74 cases of human primary prostate cancer tissues and 7 cases of castration resistant prostate cancer tissues,which obtained from patients treated with radical prostatectomy and transurethral resection of prostate from the tissue bank at MGH from 2001 to 2004.Human placenta and tonsil as positive controls.2.Animals:Two male and two female 3-week old Pten lox/lox mice were purchased from Jackson Laboratories;One 9-week old male PB-Cre + mice were purchased from the NCI-Frederick Cancer Institute.Methods:1.Antibody specificity was validated using two PD-L1 clones targeting the N-terminal and C-terminal antigenic determinants of PD-L1 molecules.2.The expression of PD-L1 in prostate cancer tissues was detected by immunohistochemistry.PD-L1 coimmunoactivities with PGP9.5,Vimentin,a-SMA and CD34 were determined by immunofluorescence,respectively.3.Analysis of the correlation between PD-L1 positive nerve density and clinicopathological features and prognosis in human with prostate cancer.4.Pten loxp/loxp mice(Strain B6;129S4-Ptentm1 Hwu/J)and PB-Cre4 mice[Strain B6.Cg-Tg(Pbsn-cre)4Prb]were used to construct Pten conditional knockout modles.5.PCR genotyping was applied to obtain homozygous mouse PTEN-/-(PTEN lox/lox;PB-Cre +);HE staining was used to observe the pathological changes of mice prostate.6.Western blot and IHC were utilized to explore the effects of conditional knockout of PTEN on expression of PD-L1 and PGP9.5 in prostate cancer tissues.Statistical analysis:The version 17.0 SPSS for Windows(IBM,USA)was used for statistical analysis.Values are shown as the mean ± standard error.The Kolmogorov-Smirnov test was used to assess whether the data of PD-L1 positive nerve fibers density was normal distribution or not.Statistical analysis of quantitative results of immunohistochemical PD-L1 positive cells was performed using two independent samples of t-test.The comparison of PD-L1 and PGP9.5 in nerve fibers was analysed by Sign test(binomial distribution).Kaplan-Meier method was used for the survival analysis and Cox regression analysis was used for the univariate and multivariate analysis.The associations between the PD-L1 positive fiber density and the clinicopathological features of patients with prostate cancer were analyzed using the Mann-Whitney test(one tail)by GraphPad Prism version 5 for Windows(GraphPad Software,Inc.).Differences were considered statistically significant when the p value was less than 0.05.Results:1.PD-L1 antibody clones E1L3N and E1J2J have the same specific expression in positive control tissues.2.PD-L1 co-stained with pan neuronal marker PGP9.5.3.The positive rate of PD-L1 in primary prostate cancer cells was 1.4%(1/74).In the nerves,the overall positive rates were 93.2%(69/74),6.8%(5/74)were negative,1.4%(1/74)positive in cancerous areas,33.3%(23/69)positive in adjacent benign areas,65.2%(45/69)positive in both cancer and adjacent areas(P<0.0001).PD-L1 was significantly lower in tumor areas than that in benign(P<0.0001).PD-L1 was negative in all of castration-resistant prostate cancers(0/7).4.The associations of PD-L1 positive NFDs with the clinicopathologic characteristics of PCa patients are summarized in Table 3.Higher PD-L1 positive NFDs were significantly associated with the higher Gleason score(P=0.034),positive perineural invasion(P=0.037)and biochemical recurrence(P=0.024).Kaplan-Meier survival analysis of PD-L1 positive NFDs dichotomized by median demonstrated that high PD-L1 positive NFDs was associated with significantly reduced biochemical recurrence-free survival(P=0.016);In subsequent univariate Cox proportional hazards model,PD-L1 positive NFDs was strongly prognostic for biochemical recurrence[P=0.002;OR,1.024;95%confidence interval(CI),1.009-1.039].In multivariate Cox proportional hazards model analysis of all patients,PD-L1 furthermore remained an independent prognostic factor of BCR[P=0.004;OR,1.023;95%confidence interval(CI),1.007-1.040]when analyzed together with Gleason score and pT status.5.The expression of both PD-L1 and PGP9.5 was significantly increased in PTEN knockout mouse prostate when compared to wild type(P<0.05).Conclusions:1.Neuronal PD-L1 may be invoveld in tumor immunosuppressive mechanisms that promote the progression of prostate cancer.2.PD-L1 positive nerve fibers can be used as a predictor of the risk of biochemical recurrence after RP and that of biochemical recurrence-free survival.This may provide a new strategy for prostate tumor immunotherapy.3.The loss function of PTEN may contribute to an immunosuppressive microenvironment by simultaneously increasing the expression of PD-L1 and the density of tumor-infiltrating nerves in prostate cancer.