Construction of TLS-related gene signature for predicting prognosis and immunotherapy response in hepatocellular carcinoma

BACKGROUND: Tertiary lymphoid structures (TLSs) are critical components of the tumor microenvironment in HCC. However, the role of TLS-related molecules in predicting HCC outcomes and guiding immunotherapy remains unexplored. This study aimed to develop TLS-related gene signature (TLSRS) to predict prognosis and immunotherapy response in patients with HCC​​​​​​. METHODS: TLS-related genes (TLSRGs) were identified through differential gene expression analysis combined with weighted correlation network analysis. TLSRGs were selected to take the intersection and LASSO regression to construct a TLSRS. Following validation of the associations between clinical prognosis and TLS-related gene features, analyses were further expanded to GSEA, HCC molecular classes, tumor microenvironment characteristics, immune-related molecular features and immunotherapy responsiveness. TLSRS was further explored using single-cell, spatial transcriptomics, immunohistochemistry, and multiplex immunofluorescence to determine their relationship with TLS. RESULTS: TLSs are identified as a favorable prognostic factor in HCC. Three TLSRGs (ACKR1, CCR7, and IL7R), which were associated with both TLS presence and prognosis, were constructed with TLSRS. The TLSRS-high group, which was associated with inflammation-related HCC molecular subtypes, exhibited significantly improved clinical outcomes, along with enhanced immune cell infiltration, enriched immune response pathways, and a higher probability of response to immunotherapy. Results from single-cell, spatial transcriptomics, immunohistochemistry, and multiplex immunofluorescence revealed that ACKR1, CCR7, and IL7R shared similar expression patterns and spatial localization with TLS. CONCLUSIONS: The TLSRS shows a correlation with both prognosis and immunotherapy response in patients with HCC, suggesting its potential value in advancing precision medicine for HCC.