Abstract: Objective To identify the composition of the tumor microenvironment （TME） for the efficacy of bladder cancer immunotherapy in public databases and to evaluate their expression and distribution in human bladder cancers and N-butyl-N-（4-hydroxybutyl）-nitrosamine （BBN）-driven spontaneous mouse bladder tumors. Methods IMvigor210 database is a clinical trial for PD-L1 blockade treatment in advanced bladder cancer. We used DESeq2 to screen out differentially expressing genes （DEGs） that are associated with immunotherapy efficacy in IMvigor210 database. The differentially enriched pathways were further identified by GO and KEGG pathway enrichment analysis. IOBR R package was utilized to identify the cell composition in the TME. The clinical significance of differential cells gene signatures or associated gene expression were evaluated in TCGA bladder cancer database. By using 32 bladder cancer specimens from Sun Yat-sen Memorial Hospital, these cell infiltrations were confirmed by immunohistochemistry and associated with tumor stage. Furthermore, we established a BBN-driven spontaneous mouse bladder tumor model. H&E staining and immunohistochemistry were applied to compare the similarity of tumor progression and major composition of TME between mouse bladder tumor and human bladder cancer. Results In the IMvigor210 database, 521 DEGs were identified to be associated with efficacy of immunotherapy. Forty-four DEGs of them were enriched in collagen-containing extracellular matrix, suggesting that fibroblasts were engaged in the efficacy of immunotherapy. TME analysis showed that gene sets of histones or cell cycle, NK cells and CD8⁺ T cells gene signatures were enriched in the effective group, while gene signatures of inflammatory cancer-associated fibroblasts were enriched in the ineffective group. In the TCGA bladder cancer database, NK cells and CD8⁺ T cells gene signatures were positively associated with good prognosis of patients, while CD56 expression was positively associated with tumor stage. By contrast, PDGFRB expression was positively associated with poor prognosis and tumor stage. Moreover, PDGFRB⁺ cancer-associated fibroblasts were heavily infiltrated in high-stage bladder cancer tissues. In the BBN mouse model, increasing proportion of mice developed bladder tumors and ultimately into muscle-invasive bladder cancer （MIBC） in a time-dependent manner. BBN-driven mouse tumor recapitulated the histology and manifested similar disease progression to human bladder cancer. There were similar infiltration patterns of NK cells, CD8⁺ T cells and cancer-associated fibroblasts in mouse tumor and human bladder cancer. Conclusion Bioinformatics analysis showed that NK cells, CD8⁺ T cells and caner-associated fibroblasts are associated with the efficacy of immunotherapy and have clinical significance in human bladder cancer. The infiltration patterns of these cells were similar in human cancer and a spontaneous mouse tumor model. These results provide potential targets and a rational mouse model for further research on immunotherapy of bladder cancer.

Key words: bladder cancer, bioinformatics analysis, tumor microenvironment, cancer-associated fibroblast, BBN-driven spontaneous mouse bladder tumor

摘要： 目的 应用生物信息学分析鉴定膀胱癌免疫治疗中起关键作用的肿瘤微环境（TME）组成特征,初步分析这些组分在人膀胱癌组织和BBN（N-butyl-N-（4-hydroxybutyl）-nitrosamine）诱导的自发小鼠膀胱肿瘤中表型的相似性。方法 分析晚期膀胱癌PD-L1阻断治疗IMvigor210数据集,利用DESeq2筛选与疗效相关的差异基因,通过GO和KEGG通路富集分析找出差异富集的通路;采用IOBR分析TME中的细胞浸润特征。通过TCGA数据库分析差异浸润细胞基因集与膀胱癌预后和分期的关系,在我院32例膀胱癌组织标本中进行免疫组化验证。建立BBN诱导的自发小鼠膀胱肿瘤模型,通过H&E染色和免疫组化对BBN诱导的自发小鼠膀胱肿瘤和人膀胱癌进展相似性及其TME主要组分进行比较。结果 在IMvigor210数据集中存在521个与免疫治疗疗效相关的差异基因,其中44个差异基因富集在含胶原细胞外基质上,提示成纤维细胞与免疫治疗疗效相关。TME分析发现细胞周期、组蛋白等肿瘤细胞基因集与NK细胞、CD8⁺ T细胞基因集在膀胱癌免疫治疗有效组中富集,而炎症性肿瘤相关成纤维细胞基因集在免疫治疗无效组中富集。TCGA分析显示：NK细胞和CD8⁺ T细胞基因集与膀胱癌患者总体生存时间呈正相关,CD56表达与膀胱癌病理分期呈正相关;PDGFRB表达与膀胱癌病理分期呈正相关。相比于低分期膀胱癌,PDGFRB⁺成纤维细胞在高分期膀胱癌中数量更多。在BBN小鼠模型中,BBN诱导的自发小鼠膀胱肿瘤发生率随着诱导时间逐渐升高,最终发展为肌层浸润性膀胱癌。免疫组化染色显示：小鼠和人膀胱肿瘤的TME中NK细胞和CD8⁺ T细胞以及成纤维细胞的浸润特征相似。结论 本研究结合生信分析和免疫组化评分发现：膀胱癌中NK细胞、CD8⁺ T细胞和成纤维细胞与免疫治疗疗效和预后具有相关性;这些细胞在BBN诱导的自发小鼠膀胱肿瘤与人膀胱癌组织中的浸润表型相似。这些结果可为研究膀胱癌免疫治疗的机制提供理论依据和动物模型。

关键词: 膀胱癌, 生物信息学, 肿瘤微环境, 肿瘤相关成纤维细胞, BBN诱导的自发小鼠膀胱肿瘤模型