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

ABSTRACT Tertiary lymphoid structures (TLSs) are lymphoid structures found in non‐lymphoid tissues, which have been involved in the pathogenesis of multiple chronic diseases including tumors. TLSs are associated with better prognosis and response to immunotherapy in most types of tumors. However, what their associations with prognosis and the exact roles of TLS‐related genes in gallbladder cancer (GBC) are still unclear. Here, we investigated the associations of TLS with the prognosis in 85 patients with GBC. 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 deep machine learning, we screened six TLS‐related candidate genes in GBC, including CTSG, FLNC, CCNB1, HSPB8, NR4A1, and MYLK. Furthermore, through the evaluation of biofunctions and clinical significance, we found that CTSG, FLNC, CCNB1, and HSPB8 played an important role in immune infiltration, diagnosis, and prognosis of GBC. Finally, cell‐based experimental validation indicated that either knockdown of CTSG or overexpression of HSPB8 promoted CD8+ T cell apoptosis and exhaustion. In conclusion, TLS had a protective effect on human GBC, and its related genes, played a potential role in diagnosis, prognosis, immune infiltration, and metastasis of GBC. Our findings provided a new insight for the research on clinical biomarkers of GBC and development of its therapeutic targets.