Abstract
The involvement of necroptosis in the immunosuppressive tumor microenvironment has been established and has been shown to contribute to the growth of pancreatic ductal adenocarcinoma, indicating its role in promoting tumor development. However, the relationship between necroptosis and bladder urothelial carcinoma (BUC) has yet to be fully understood. To shed light on this issue, our study aimed to uncover the impact of necroptosis on immune cell infiltration and immunotherapy response in BUC patients. We conducted an analysis of 67 necroptosis genes to assess their expression and genomic changes across pan-cancer and identified 12 necroptosis genes that are prognostically relevant and associated with immune subtypes and tumor stemness in BUC. Using a public database of 1841 BUC samples, we then performed Unsupervised Cluster Analysis and discovered two distinct necroptotic phenotypes in BUC. These phenotypes showed significant differences in molecular subtypes, immune infiltration patterns, and gene mutation profiles. We confirmed this discovery in BUC through qPCR and WB experiments. To evaluate the impact of necroptosis on prognosis, chemotherapy sensitivity, and immunotherapy response (such as anti-PD-L1), we developed a principal component analysis model called NecroScore. Finally, we validated the effects of RIPK3 and MLKL through a nude mouse transplantation model for BUC. Our study has uncovered that necroptosis plays a role in shaping the tumor immune microenvironment in BUC. The high necroptosis phenotype (Cluster B) was characterized by a higher abundance of tumor immunosuppressive cells and more key biological processes driving tumor progression, while the low necroptosis group (Cluster A) had higher FGFR3 mutations. We found that the infiltration levels of immune cells, including CD8+ T cells, were significantly different between FGFR3 mutated and wild-type (WT) samples. Our results confirmed the reliability of NecroScore as a comprehensive assessment tool for evaluating the immunotherapeutic effect and prognosis of BUC patients, with high NecroScore values favoring basal-like differentiation and lower FGFR3 alterations. We also observed that high expression of MLKL had a significant inhibitory effect on tumor growth and increased neutrophil infiltration in vivo. In our study, we uncovered the regulation pattern of necroptosis in the tumor immune microenvironment of BUC. Additionally, we developed a scoring tool called NecroScore that can be utilized to predict the most suitable chemotherapy and immunotherapy strategy for bladder urothelial carcinoma patients. This tool can effectively guide the chemotherapy and immunotherapy regimens for patients with advanced BUC.