Unveiling the role of regulatory T cells in the tumor microenvironment of pancreatic cancer through single-cell transcriptomics and in vitro experiments

BACKGROUND: In order to investigate the impact of Treg cell infiltration on the immune response against pancreatic cancer within the tumor microenvironment (TME), and identify crucial mRNA markers associated with Treg cells in pancreatic cancer, our study aims to delve into the role of Treg cells in the anti-tumor immune response of pancreatic cancer. METHODS: The ordinary transcriptome data for this study was sourced from the GEO and TCGA databases. It was analyzed using single-cell sequencing analysis and machine learning. To assess the infiltration level of Treg cells in pancreatic cancer tissues, we employed the CIBERSORT method. The identification of genes most closely associated with Treg cells was accomplished through the implementation of weighted gene co-expression network analysis (WGCNA). Our analysis of single-cell sequencing data involved various quality control methods, followed by annotation and advanced analyses such as cell trajectory analysis and cell communication analysis to elucidate the role of Treg cells within the pancreatic cancer microenvironment. Additionally, we categorized the Treg cells into two subsets: Treg1 associated with favorable prognosis, and Treg2 associated with poor prognosis, based on the enrichment scores of the key genes. Employing the hdWGCNA method, we analyzed these two subsets to identify the critical signaling pathways governing their mutual transformation. Finally, we conducted PCR and immunofluorescence staining in vitro to validate the identified key genes. RESULTS: Based on the results of immune infiltration analysis, we observed significant infiltration of Treg cells in the pancreatic cancer microenvironment. Subsequently, utilizing the WGCNA and machine learning algorithms, we ultimately identified four Treg cell-related genes (TRGs), among which four genes exhibited significant correlations with the occurrence and progression of pancreatic cancer. Among them, CASP4, TOB1, and CLEC2B were associated with poorer prognosis in pancreatic cancer patients, while FYN showed a correlation with better prognosis. Notably, significant differences were found in the HIF-1 signaling pathway between Treg1 and Treg2 cells identified by the four genes. These conclusions were further validated through in vitro experiments. CONCLUSION: Treg cells played a crucial role in the pancreatic cancer microenvironment, and their presence held a dual significance. Recognizing this characteristic was vital for understanding the limitations of Treg cell-targeted therapies. CASP4, FYN, TOB1, and CLEC2B exhibited close associations with infiltrating Treg cells in pancreatic cancer, suggesting their involvement in Treg cell functions. Further investigation was warranted to uncover the mechanisms underlying these associations. Notably, the HIF-1 signaling pathway emerged as a significant pathway contributing to the duality of Treg cells. Targeting this pathway could potentially revolutionize the existing treatment approaches for pancreatic cancer.