Comprehensive analysis of ammonia-induced cell death and GLS1 in gastric adenocarcinoma: implications for prognosis and therapeutic strategies.

Background: Gastric cancer, primarily manifested as gastric adenocarcinoma (STAD), remains one of the leading causes of cancer-related deaths worldwide. Ammonia-induced cell death (AID), a newly discovered form of cell death, has gained attention due to its unique mechanisms, including lysosomal alkalinization and mitochondrial dysfunction. However, the role of AID in STAD, particularly its impact on immune regulation and patient prognosis, remains unclear.

Methods: This study integrated bulk RNA-seq and single-cell RNA-seq (scRNA-seq) data. The Seurat package was used for single-cell gene expression analysis, clustering different cell types, and AID scoring. CellChat software analyzed the ligand-receptor interactions between immune cell subtypes. Differential expression analysis (DEGs) and GO/KEGG enrichment identified key genes associated with AID. We constructed a prognostic risk model based on these findings. Immune cell infiltration was analyzed using the "GSVA" and "xCell" packages. In vitro experiments were conducted on HGC-27 and MKN45 gastric cancer cell lines, where GLS1 was knocked down using siRNA to assess the effects on cell proliferation, migration, invasion, and lysosomal function. Additionally, drug sensitivity tests were used to evaluate the impact of GLS1 overexpression on resistance to various anticancer drugs.

Results: Single-cell clustering analysis revealed that T cells had significantly higher ammonia-induced cell death (AID) scores, leading to the classification of cells into high-AID and low-AID groups. Gene expression analysis and pathway enrichment showed significant enrichment in lysosomal and mitochondrial pathways, consistent with known AID mechanisms. A prognostic risk model based on five key genes (C1QA, MARCKSL1, GLS1, N4BP2L2, and CD68) effectively classified patients into high-risk and low-risk groups, with the high-risk group showing stronger immune cell infiltration, including CD4 + and CD8 + T cells, dendritic cells, and macrophages. Among the model genes, GLS1 was identified as the most significant prognostic factor, the strongest risk factor, and the most central gene in the interaction network. In vitro experiments showed that GLS1 inhibition led to increased ammonia levels, elevated lysosomal pH, and reduced lysosomal function, thereby enhancing AID expression. Furthermore, GLS1 overexpression significantly promoted cell proliferation, migration, and tumor growth. Finally, gastric cancer cells with GLS1 overexpression exhibited resistance to multiple anticancer drugs, highlighting the potential value of GLS1 as a therapeutic target.

Conclusion: The AID model is a promising biomarker for accurately determining survival and predicting the effectiveness of immunotherapy in STAD patients. GLS1 plays a crucial role in driving tumor proliferation and migration and may act as a potential tumor biomarker of STAD.