| Objective:Bladder urothelial carcinoma is one of the most common cancers,ranking in the top ten for both morbidity and mortality in men worldwide.The purpose of this study is to explore the feasibility and effectiveness of CD39 as a new therapeutic target for bladder urothelial carcinoma by clarifying the expression and distribution of CD39 in bladder urothelial carcinoma,and lay a foundation for the clinical application of CD39 targeting.Methods:Single-cell transcriptomic sequencing analysis,tissue microarray staining and reanalysis of public data sets were performed to determine the expression and distribution of CD39 in bladder urothelial carcinoma and its relationship with the efficacy of targeted PD-L1 therapy.Then,the therapeutic effect of sodium polyoxotungstate（POM-1,a CD39inhibitor/CD39i）on bladder urothelial carcinoma was investigated in animal experiments.Then,the infiltrated immune cells in tumor tissues of mice were isolated by CD45immunomagnetic sphere for single-cell transcriptome sequencing,and the panoramic map of the changes of CD39i mediated immune cell subsets in tumor infiltration was constructed.The mechanism of CD39i inhibiting the progression of bladder urothelial carcinoma was elucidated and validated by Batf3-/-mouse model（classical dendritic cell type 1（c DC1）deletion model）and NK cell depletion model.Finally,a series of animal experiments were conducted to explore the efficacy of the combination of CD39i and current commonly used drugs for bladder urothelial carcinoma treatment.Results:Single-cell transcriptomic sequencing,tissue microarray staining in bladder urothelial carcinoma and paracancer tissues,and reanalysis of the public dataset Imvigor210RNA-seq showed that CD39 expression was elevated in bladder urothelial carcinoma and was associated with depletion of tumor-infiltrating CD8+T cells.Animal experiments showed that CD39i could significantly inhibit the progression of bladder urothelial carcinoma.Flow cytometry and single-cell transcriptome sequencing revealed that CD39i increased the proportion of proliferative CD8+T cells,NK cells,and c DC1 in intra tumor infiltration in mice.Cell communication network analysis suggested that Xcl1 secreted by NK cells could bind to Xcr1 on the surface of c DC1 and proliferating c DC1,while Il15 and Il12b secreted by c DC1 and mature c DC1（m DC1）could bind to Il15ra and Il12rb1/2 on the surface of proliferating CD8+T cells.Batf3-/--c DC1 depletion model and NK cell depletion model validation experiments showed that the anti-tumor effect of CD39i was significantly inhibited under both c DC1 depletion and NK cell depletion conditions.An exploration of combination therapy based on CD39i showed that CD39i had a significant synergistic effect with cisplatin,but not with PD1 or PD-L1 targeting inhibitors,in the treatment of bladder urothelial carcinoma,and the combination of CD39i and cisplatin significantly increased the proportion of intratumoral c DC1 invasion.Conclusions:CD39 is overexpressed in bladder urothelial carcinoma and predicts depletion of CD8+T cell function.CD39 is a potential therapeutic target for bladder urothelial carcinoma.Blocking CD39 by CD39i can significantly inhibit the progression of bladder urothelial carcinoma in mice.CD39i promotes the proliferation and activation of NK cells,and then recruits c DC1/proliferative c DC1 into the tumor,and activates the anti-tumor activity of CD8+T cells in the proliferating stage,thus achieving the anti-tumor effect.NK cells and c DC1 are the key to CD39i mediated anti-tumor effect.In addition,CD39i had a significant synergistic effect with cisplatin,but not with PD1 or PD-L1 targeting inhibitors,in the treatment of bladder urothelial carcinoma,and this synergistic effect may be related to the cytotoxicity of cisplatin inducing tumor cell death followed by the release of large amounts of ATP,and the combination of the two can significantly increase the proportion of intratumoral invasion of c DC1. |
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