Abstract
The tumor necrosis factor (TNF) ligand superfamily plays a critical role in immune regulation and has emerged as a promising target in cancer immunotherapy. By binding to its receptor OX40 (CD134), TNFSF4 promotes the proliferation and survival of T cells and plays an important role in the tumor immune microenvironment, but its role in pancreatic cancer is unclear. This study aimed to investigate the expression patterns and prognostic significance of TNF ligand family members in pancreatic cancer (PC), with a specific focus on TNFSF4. We analyzed single-cell RNA sequencing data from the GSE212966 dataset to assess the expression of TNFSF ligands across immune cell types. TCGA-PAAD bulk RNA-seq data were used for non-negative matrix factorization (NMF) clustering to identify molecular subtypes based on TNFSF ligand expression profiles. Immune infiltration was quantified using single-sample gene set enrichment analysis (ssGSEA), and Kaplan-Meier survival curves were used to compare overall survival (OS) and progression-free survival (PFS) between subtypes. Immunotherapy response prediction was evaluated using tumor mutational burden (TMB), immunophenoscore (IPS), and tumor immune dysfunction and exclusion (TIDE) scores. Gene expression validation was performed using qRT-PCR. TNFSF ligands were predominantly expressed in antigen-presenting cells, particularly B cells and macrophages. NMF clustering identified two molecular subtypes of PC, with cluster 2 associated with significantly better OS and PFS (p < 0.05). TNFSF4, highly enriched in B cells, was found to regulate immune-related pathways such as B cell receptor signaling and cytokine-cytokine receptor interaction, as revealed by KEGG pathway analysis. TNFSF4 expression also correlated with favorable immunotherapy markers, suggesting its potential role as a predictive biomarker. These findings were supported by qRT-PCR validation. This study provides a TNFSF ligand-based molecular classification of pancreatic cancer and highlights the immunoregulatory role of TNFSF4. Its association with patient prognosis and immunotherapy responsiveness suggests potential clinical utility in guiding treatment strategies.