Multi-Omics Integrated Analysis of the Protective Effect of Tertiary Lymphoid Structures and Associated Key Regulatory Genes in Human Gallbladder Cancer

Tertiary lymphoid structures (TLS) are lymphoid structures found in non-lymphoid tissues, which are associated with immune cell activation and have been involved in pathogenesis of multiple chronic diseases including tumors. Growing evidence has revealed that TLS are associated with better prognosis and response to immunotherapy in most types of tumors. However, what are their associations with the prognosis and the exact roles of TLS-related genes in gallbladder cancer are still unclear. Here, we investigated the associations of TLS with the prognosis in 85 gallbladder cancer patients. Our results showed that the presence of intra-tumoral TLS was negatively associated with T stage ( P  = 0.035) and vascular invasion ( P  = 0.048) and predicted a higher rate of overall survival ( P  = 0.004) and a decreased risk of early recurrence ( P  = 0.002). Moreover, using integrative analysis of single cell RNA sequencing, bulk RNA sequencing, and machine learning, we screened 6 TLS-related genes that potentially involved in the progression of gallbladder cancer, including CTSG, FLNC, CCNB1, HSPB8, NR4A1 and MYLK. Futhermore, through evaluation of biofunctions and clinical significance, we found that CTSG, FLNC, CCNB1 and HSPB8 played an important role in immune infiltration, diagonosis and prognosis of gallbladder cancer. In conclusion, we demonstrated that TLS had a protective effect on human gallbladder cancer, and its related genes, CTSG, FLNC, CCNB1 and HSPB8, played an important role in diagonosis, prognosis, immune infiltration and metastasis of gallbladder cancer. Our findings provided a new insight for the research on clinical biomarkers of gallbladder cancer and development of its therapeutic targets.