Xiao Shuxiu, Lu Lili, Lin Zhiyuan, Ye Xinming, Su Sheng, Zhang Chenlu, You Yang, Li Wei, Huang Xiaowu, Wu Weizhong, Zhou Yuhong
Clinical Center for Biotherapy, Zhongshan Hospital, Fudan University, Shanghai, 200032, People's Republic of China.
Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, 200032, People's Republic of China.
J Hepatocell Carcinoma. 2024 Jun 6;11:1031-1048. doi: 10.2147/JHC.S464806. eCollection 2024.
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.
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 LAYNCD8 T cell exhaustion signature and immune function. Cell proliferation ability and killing activity were assessed using CCK8 and CFSE/PI.
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 LAYNCD8 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.
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.
层黏蛋白（LAYN）是多种肿瘤类型中一种有价值的预后生物标志物，同时也是功能失调或耗竭的CD8 T细胞的创新指标，并与免疫环境相关。然而，LAYN在肝细胞癌（HCC）中的免疫功能和预后意义仍未被探索。因此，我们的目的是研究LAYN在HCC中CD8 T细胞耗竭、临床预后和肿瘤微环境中的作用。
使用TIMER或GEPIA数据库分析LAYN表达水平及其与HCC中免疫浸润的相关性。对TCGA和scRNA-seq队列进行生物信息学分析。通过免疫荧光（IF）、免疫组织化学（IHC）和酶联免疫吸附测定（ELISA）检测新鲜标本中LAYN表达水平。采用流式细胞术和mRNA测序研究LAYN的共表达基因、LAYN⁺CD8 T细胞耗竭特征和免疫功能。使用CCK8和CFSE/PI评估细胞增殖能力和杀伤活性。
与癌旁组织相比，HCC肿瘤中LAYN的表达水平显著更高。LAYN水平高的患者总生存期较差。基因本体（GO）或京都基因与基因组百科全书（KEGG）分析证实LAYN参与免疫反应，并与CD8 T细胞免疫浸润水平呈正相关。此外，LAYN负向调节CD8 T细胞的免疫功能，导致功能失调的表型，其特征为CD39、T细胞免疫球蛋白和粘蛋白结构域3（TIM3）水平升高，穿孔素、肿瘤坏死因子-α（TNF-α）、Ki-67水平降低。CFSE/PI检测表明LAYN⁺CD8 T细胞的细胞毒性活性降低。此外，LAYN与CD146水平呈正相关，CD146参与CD8 T细胞的黏附和定位过程。有趣的是，阻断LAYN可部分恢复CD8 T细胞的耗竭特性。
LAYN与肿瘤微环境（TME）中的免疫浸润密切相关，是预测HCC临床预后的新型生物标志物。此外，靶向LAYN可能是一种有前景的HCC免疫治疗有效策略。
