Shuxiu Xiao,1,* Lili Lu,1,* Zhiyuan Lin,2,* Xinming Ye,3 Sheng Su,3 Chenlu Zhang,4 Yang You,4 Wei Li,4 Xiaowu Huang,3 Weizhong Wu,5 Yuhong Zhou1,4 

1Clinical Center for Biotherapy, Zhongshan Hospital, Fudan University, Shanghai, 200032, People’s Republic of China; 2Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, 200032, People’s Republic of China; 3Department of Liver Surgery and Transplantation, and Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, 200032, People’s Republic of China; 4Department of Medical Oncology, Zhongshan Hospital, Fudan University, Shanghai, 200032, People’s Republic of China; 5Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, 200032, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Yuhong Zhou; Weizhong Wu, Email zhou.yuhong@zs-hospital.sh.cn; wu.weizhong@zs-hospital.sh.cn

Background: Layilin (LAYN) represents a valuable prognostic biomarker across various tumor types, while also serving as an innovative indicator of dysfunctional or exhausted CD8+ T cells and exhibiting correlation with immune context. However, the immune function and prognostic significance of LAYN in hepatocellular carcinoma (HCC) remain unexplored. Therefore, our objective is to investigate the role of LAYN in CD8+ T cell exhaustion, clinical prognosis, and the tumor microenvironment within HCC.
Methods: TIMER or GEPIA databases were used to analyze LAYN expression level and its correlation with immune infiltration in HCC. Bioinformatics analysis was conducted on TCGA and scRNA-seq cohorts. The evaluation of LAYN expression level in fresh specimens was performed through IF, IHC, and ELISA assays. Flow cytometry and mRNA-seq were employed to investigate co-expressed genes of LAYN, the LAYN+CD8+ T cell exhaustion signature and immune function. Cell proliferation ability and killing activity were assessed using CCK8 and CFSE/PI.
Results: The expression level of LAYN in HCC tumors was significantly higher compared to peri-tumors. Patients with high levels of LAYN exhibited poorer OS. GO or KEGG analysis confirmed that LAYN was involved in immune response and was positively associated with CD8+ T cell immune infiltration levels. Furthermore, LAYN negatively regulated the immune function of CD8+ T cells, leading to dysfunctional phenotypes characterized by elevated levels of CD39, TIM3 and reduced levels of perforin, TNF-α, Ki-67. CFSE/PI assays demonstrated that LAYN+CD8+ T cells displayed decreased cytotoxic activity. Additionally, there was a positive correlation between LAYN and CD146 levels, which are involved in adhesion and localization processes of CD8+ T cells. Interestingly, blocking LAYN partially restored the exhaustion properties of CD8+ T cells.
Conclusion: LAYN exhibits a strong correlation with immune infiltration in the TME and represents a novel biomarker for predicting clinical prognosis in HCC. Moreover, targeting LAYN may hold promise as an effective strategy for HCC immunotherapy.

Keywords: hepatocellular carcinoma, layilin, CD8+ T cell exhaustion, prognosis