Ferroptosis-induced remodeling of glycosylation the immune microenvironment and improves survival in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is still an aggressive and rapidly progressive malignant tumor of the digestive system. Emerging treatment strategies have yielded limited benefits, primarily due to the high heterogeneity of tumors and the presence of various mechanisms of drug resistance. Ferroptosis is a newly discovered type of cell death that has emerged as a promising anti-tumor strategy, while our understanding of the interaction between ferroptosis and tumor glycosylation and immune remodeling remains limited. METHODS: A ferroptosis model was established in PDAC cells via RSL3 treatment. Using mass spectrometry-based glycoproteomics, transcriptomic analysis, and single-cell RNA sequencing (scRNA-seq), we comprehensively analyzed alterations in protein glycosylation, gene expression, and immune microenvironment composition. The identification of functional pathways was performed through GO/KEGG enrichment analysis, and a clinical relevance assessment was performed through TCGA datasets. Furthermore, the key gene expression changes at both the mRNA and protein levels was validated using qPCR and Western blot. RESULTS: Ferroptosis induction has been shown to significantly reshape O and N-glycosylation patterns, affecting proteins related to membrane integrity, immune regulation, and metabolic processes. We identified nine glycosylation-related genes whose expression correlates with ferroptosis sensitivity, including upregulation of ST3GAL1 and downregulation of ST8SIA1, MGAT4A, and EXTL1. Transcriptomic analysis revealed that ferroptosis activates immune-related pathways, including IL-17, TNF, and chemokine signaling. Single-cell analysis indicated that increased ferroptosis activity was associated with enhanced infiltration of proinflammatory phagocytic tumor-associated macrophages exhibiting M1-like characteristics. Notably, qPCR and WB validation confirmed that ferroptosis suppressed the expression of key ferroptosis-related genes (FADS2, SNCA, AQP3) as well as the glycosylation gene GYG2. These genes have been identified as prognostic markers associated with poor clinical outcomes in PDAC patients. CONCLUSIONS: This study demonstrates that ferroptosis remodels the glycosylation pattern and modulates the immune microenvironment in PDAC, enhancing antitumor immunity through TAM activation while affecting cell survival. The findings offer novel insights into the intricate relationship between ferroptosis, glycosylation, and tumor immunity, providing potential therapeutic approaches by integrating ferroptosis inducers with glycosylation targeting or immunomodulatory strategies in PDAC treatment. STRENGTHS AND LIMITATIONS OF THIS STUDY: Firstly, the dual pathway specific enrichment strategy of O-GlcNAc modified peptides and N-glycosylated peptides was applied to ferroptosis study for the first time, which realized a systematic analysis of glycosylation patterns in the process of cell death. Secondly, high-resolution mass spectrometry combined with multi-platform data processing (MaxQuant/PEAKS) was used to deeply integrate transcriptomes and single-cell transcriptomes to construct a panoramic analysis framework with multi-omics mutual evidence. Thirdly, Scissor method was introduced to map TCGA ferroptosis pathway activity to single-cell data to achieve cross-scale analysis from population level to cell subsets. Fourth, combined with multi-dimensional bioinformatics tools, the characteristics of modification sites, subcellular localization, protein interaction network and functional pathway were annotated. Fifth, on the basis of multi-omics results, double-layer validation by qPCR and Western Blot at the transcriptional and protein levels significantly improved the credibility of the research conclusions. Its limitations are that the research mainly relies on high-throughput omics and computational analysis, and lacks systematic in vitro and in vivo functional verification and combination drug sensitivity experiments, as well as the support of real-world clinical cohorts.