Background: Tertiary lymphoid structures (TLSs) are formed by the aggregation of tumour-infiltrating lymphocytes (TILs), which is driven by chemokines or cytokines in the tumour microenvironment. Studies have shown that TLSs are associated with good prognosis in patients with various solid tumours and can improve patient responses to immunotherapy. However, the role of TLSs in hepatocellular carcinoma (HCC) remains controversial, and the underlying molecular mechanism is unclear. Methods: According to haematoxylin-eosin (HE) staining results, HCC patients in Xijing Hospital data and TCGA data were divided into TLS+ and TLS- groups, and Kaplan–Meier (KM) analysis was performed to assess overall survival (OS) and recurrence-free survival (RFS). Immunofluorescence (IF) and immunohistochemistry (IHC) were used to identify TILs in the TLS+ group. Lymphocyte-specific protein tyrosine kinase (LCK), a molecule involved in TLS formation, was explored in LinkedOmics. TILs were divided into two groups by drawing receiver operating characteristic (ROC) curves to calculate cut-off values. Spearman correlation analysis was used to calculate the correlation between LCK and TILs, and the molecular pathways by which LCK regulates immunotherapy were clarified through enrichment analysis. The half-maximal inhibitory concentration (IC50) distribution of sorafenib was observed in groups that varied in LCK expression. Results: According to the HE results, 61 cases in the Xijing Hospital cohort and 195 cases in the TCGA cohort had TLSs, while 89 cases and 136 cases did not. The KM results showed that TLSs had no effect on the OS of HCC patients but significantly affected RFS. The IF/IHC results showed that higher TIL numbers in TLSs were correlated with better prognosis in HCC patients. Spearman correlation analysis showed that LCK expression was positively correlated with TIL numbers. Enrichment analysis showed that upregulation of LCK expression mainly regulated the cytokine signalling pathway, the chemokine signalling pathway and T-cell activation. The IC50 scores of sorafenib in HCC patients with high LCK expression were lower, and the sensitivity was higher. Conclusion: TLSs mainly affected the early RFS of HCC patients but had no effect on OS. The high expression of the TLS formation-related gene LCK can increase the sensitivity of HCC patients to ICIs.

The prognostic value of immune cells in tertiary lymphoid structures (TLSs) remains unclear in hepatocellular carcinoma (HCC). Here, 59 of 145 patients had TLSs in training set, 48 of 120 patients had TLSs in testing set. Immunohistochemistry (IHC) were used to label CD3+ T cells, CD20+ B cells, CD8+ T cells, CD208+ dendritic cells, and CD21+ follicular dendritic cells in TLSs. High CD20+, CD208+, and CD8+ cell densities were favorable prognostic factors for overall survival (OS). High CD3+, CD20+, CD208+, and CD8+ cell densities were significantly associated with reduced early recurrence. TLSs were divided into three grades (A, B, and C) based on immune cell density. Patients with grade C or B had significantly improved OS. Patients with grade C had the lowest recurrence rate, followed by those with grade B, while patients with grade A had the highest recurrence rate. The stromal, immune, and ESTIMATE scores derived from the ESTIMATE package were significantly higher and tumor purity was significantly lower in patients with TLSs. Patients with TLSs had significantly higher relative numbers of memory B cells, plasma cells, CD8+ T cells, NK cells, and dendritic cells and lower relative numbers of Treg cells, macrophages, and M2 macrophages according to the CIBERSORT assessment. Bioinformatics analysis and experiments confirmed that KLRK1 and GZMA expression are associated TLSs formation and can predict TLSs existence. Grade B and grade C were favorable prognostic factors for OS and recurrence and could represent immuneactive tumors.

Background ARPC3 is associated with poor prognosis in patients with various cancers. However, the mechanisms by which it affects immunotherapy and prognosis in patients with hepatocellular carcinoma (HCC) remain unclear. Method The expression difference in ARPC3 between normal and HCC tissues and the effect of ARPC3 on prognosis were evaluated by using multiple databases. GSEA was used to predict the pathway by which ARPC3 affects HCC progression. Using TCGA database, the First Affiliated Hospital of Anhui Medical University (AHMU) database and ICGC database, the correlation between ARPC3, tumor infiltrating lymphocytes (TILs) and immune checkpoints was studied. To explore the effect of ARPC3 on immune checkpoint inhibitors (ICIs), We investigated the association of ARPC3 with immunotherapy-associated ferroptosis genes. Results The expression of ARPC3 in normal tissues was lower than that in tumor tissues, and as an independent prognostic risk factor for HCC, patients with HCC whose ARPC3 expression was high had a worse prognosis. GSEA suggested that the upregulation of ARPC3 mainly affected immune-related pathways. Three databases showed that ARPC3 expression levels affected the infiltration levels of B cells, T cells, macrophages, neutrophils, and NK cells in tumors. In addition, we confirmed that ARPC3 may influence the efficacy of ICI therapy by influencing the expression of immune checkpoints and ferroptosis-related genes in HCC. Conclusions ARPC3 is an independent prognostic risk factor for HCC patients and may influence the immunotherapy of HCC by influencing the expression of immune checkpoints and ferroptosis-related genes.