CD4~+T Cell Confers Chemoresistance Of Bladder Cancer Through Hypoxia Mediated TNF Signaling

Bladder cancer is the most commonly diagnosed cancer in male urinary system tumor with the characteristics of easy recurrence and poor prognosis.Since cisplatin was approved for clinical use in 1978,platinum drugs have been widely used as one of the first-line clinical treatment drugs.Treatment with cisplatin often causes cells to become resistant,which becomes one of the most important factors in the failure of chemotherapy.The role of tumor microenvironment(TME)in chemotherapy response is complex,which has dual effects of chemoprotection and chemosensitization.Although there is compelling evidence that activating immune cells is a promising therapy,the exact role of TME including immune cells in chemoprotective effects is unknown.Hypoxia is a common feature of solid tumor microenvironment and an important regulator of the microenvironment.Therefore,the study of tumor microenvironment hypoxia and chemoresistance mechanism is very important for clinical treatment.To further analyze the heterogeneity of chemotherapy resistance in bladder cancer at the single-cell level,we used N-butyl-N-(4-hydroxybutyl)-nitrosamine(BBN)to induce orthotopic bladder cancer in male mice.Single-cell sequencing analysis of tumor tissue samples obtained from cisplatin-treated and control mice revealed that: The proportion of bladder cancer cells increased after treatment,which proved the resistance to cisplatin treatment.The distribution of bladder cancer cell types also changed significantly before and after treatment.The proportion of KRT14+ cells in the cisplatin group increased,which was defined as the cisplatin resistant subpopulation.By comparing this cluster with the treatment-sensitive cluster,it was found that the chemotherapy-resistant cluster(KRT14+)enriched TNF signaling and HYPOXIA signaling pathways.We further performed hypoxia scoring in the microenvironment,and found that hypoxia signals were highly enriched in CD4+T cells,and the expression level of hypoxia-inducible factor HIF1α was the highest in CD4+T cells.Cell communication analysis revealed enhanced interaction between CD4+T cells and resistant cluster via TNF axis after cisplatin treatment.The combination of HIF1α inhibitor and cisplatin significantly increased the level of apoptosis of tumor cells co-cultured with CD4+T,indicating that HIF1α inhibitor could inhibit the promotion of drug resistance of tumor cells by CD4+T.We demonstrated that hypoxia in the tumor microenvironment led to chemoprotection of CD4+T cells,which mediated TNFα-NF-κB-signaling in tumor cells through the TNF axis to promote tumor cell survival and lead to cisplatin resistance.The elucidation of this resistance mechanism provides a new idea for the clinical treatment of bladder cancer.