LAYN Serves as a Prognostic Biomarker and Downregulates Tumor-Infiltrating CD8⁺ T Cell Function in Hepatocellular Carcinoma

Abstract

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.

Graphical Abstract

Keywords:

• hepatocellular carcinoma
• layilin
• CD8⁺ T cell exhaustion
• prognosis

Abbreviations

HCC, Hepatocellular carcinoma; HBV, Hepatitis B virus; HCV, Hepatitis C virus; LAYN, Layilin; IL, Interleukin; IFN, Interferon; TNF, Tumor necrosis factor; GZMB, GranzymeB; PRF-1, Perforin; PD1, Programmed cell death 1; PD-L1, Programmed cell death-ligand 1; CTLA4, Cytotoxic T-lymphocyte antigen; TIM3, T cell immunoglobulin domain and mucin domain-3; LAG3, Lymphocyte activation gene 3; TIGIT, T cell immunoreceptor with Ig and ITIM domains; TME, Tumor microenvironment; MDSCs, Myeloid-derived suppressor cells; CTL, Cytotoxic T cell; T_reg, Regulatory T-cells; T_DE, Terminally differentiated T cells; DCs, Dendritic cells; PBMC, Peripheral blood mononuclear cell; PB, Peripheral blood; TILs, Tumor-infiltrating lymphocytes; OS, Overall survival; PFS, Progression-free survival; DFS, Disease-free survival; DSS, Disease-specific survival; ORR, Objective response rate; ICIs, Immune checkpoint inhibitors; IRs, Inhibitory receptors; E/T, Effector-to-target; CRC, Colorectal cancer; NSCLC, Non-small cell Lung carcinoma; COAD, Colon adenocarcinoma; BRCA, Breast invasive carcinoma; CHOL, Cholangiocarcinoma; LUAD, Lung adenocarcinoma; GO, Gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BP, Biological process; CC, Cell component; MF, Molecular function.

Data Sharing Statement

Data are available upon reasonable request. All data generated that are relevant to the results presented in this article are included in this article or Supplementary Materials.

Ethics Approval and Consent to Participate

The study was approved by the Clinical Research Ethics Committee of Zhongshan Hospital of Fudan University (B2020-262). Written informed consent was obtained from each patient included and this study was performed in accordance with the Declaration of Helsinki.

Consent for Publish

All authors agree to publish this manuscript.

Disclosure

All authors declared no competing interests in this work.

Additional information

Funding

This project was supported by grants from Shanghai Shenkang Hospital Development Center, Special Disease Queue Database Construction Project (SHDC2020CR6006), Zhongshan Hospital Youth Development Fund (2021ZSQN28) and the Sailing Project of “Scientific and Technological Innovation Action Plan” of Shanghai Science and Technology Commission (20YF1406100).