Integrative analysis of bulk and single-cell sequencing reveals TNFSF9 as a potential regulator in microsatellite instability stomach adenocarcinoma.

Background: Stomach adenocarcinoma (STAD) with microsatellite instability (MSI) is associated with a better prognosis compared to Non-MSI. This study aims to elucidate the differences in the tumor microenvironment (TME) of MSI and explore its underlying mechanisms in STAD.

Methods: TME differences between MSI and Non-MSI were analyzed using single-cell RNA sequencing (MSI = 7, Non-MSI = 19) and bulk RNA sequencing (MSI = 39, Non-MSI = 198). Differentially expressed genes (DEGs) were used to identify enriched pathways and hub genes. TNFSF9 expression was validated by immunohistochemistry (IHC) on 23 STAD sections (MSI = 13, Non-MSI = 10) and confirmed in tumor epithelial cells using SNU-1 (MSI) and AGS (Non-MSI) cell lines through quantitative polymerase chain reaction (qPCR) and Western blot (WB).

Results: The results showed MSI was significantly associated with a better prognosis (P < 0.05). Within the TME, MSI was associated with a higher abundance of antigen-presenting cells, including M1 macrophages (40.1% vs. 27.9%) and activated dendritic cells (22.1% vs. 10.5%), as well as pro-inflammatory Th1-like CD4⁺ T cells (15% vs. 11%). However, MSI also showed an increase in exhausted T cells, indicating a complex immune landscape. Signaling pathway and cell communication analyses revealed an enrichment of cytokine-related pathways in MSI. Hub gene analysis revealed that TNFSF9 was predominantly expressed in stromal cells and partially in tumor epithelial cells in MSI, with its upregulation further confirmed through IHC, qPCR, and WB. Correlation analysis demonstrated a positive relationship between TNFSF9 expression and the abundance of M1 macrophages.

Conclusions: These findings provide new insights into the TME of MSI in STAD, emphasizing the significant role of TNFSF9 in shaping MSI-specific TME, enhancing immunotherapy efficacy, and improving patient survival.