Integration of Single-cell RNA and Bulk RNA sequencing revealed malignant ductal cell heterogeneity and prognosis signatures in pancreatic cancer

Haiyang Du¹Gao Si²Jiqing Si³Xuejie Song^4*Fuchun Si^1*

• ¹Henan University of Chinese Medicine, Zhengzhou, China
• ²Department of Ophthalmology, Peking University Third Hospital, Beijing, China
• ³Henan Hospital of TCM, The Second Affiliated Hospital of Henan University of traditional Chinese Medicine, Zhengzhou, Henan Province, China
• ⁴Traditional Chinese Medicine (ZhongJing) school, Henan University of Chinese Medicine, Zhengzhou, Henan Province, China

Pancreatic cancer is a highly malignant digestive system tumor with a poor prognosis. Despite recent advances in diagnosis and treatment, the overall survival rate remains extremely low. Understanding the early diagnostic markers and mechanisms of pancreatic cancer is crucial for developing effective treatment strategies. We collected 74 single-cell RNA sequencing (scRNA-seq) samples and identified and characterized the main cell types and their distribution characteristics through unsupervised clustering and gene expression analysis. Large-scale chromosomal copy number variation analysis was used to distinguish malignant from non-malignant ductal cells. Using non-negative matrix factorization analysis, we identified a gene set significantly associated with staging, and combined with TCGA data and machine learning techniques, identified three prognosis-related genes (ANLN, NT5E, and CTSV), which were validated through two independent datasets. High expression of these genes was associated with poorer prognosis and significantly elevated levels of M0 macrophages. Pyscenic and CellPhoneDB analysis revealed that high-expression malignant ductal cells interact with M0 macrophages through CXCL14-CXCR4 and IL1RAP-PTPRF, with SPI1 being an upstream transcription factor of IL1RAP. ANLN, NT5E, and CTSV play crucial roles as prognostic genes in PAC and may provide new targets for treatment strategies. The interaction between CXCL14-CXCR4 in high-expression malignant ductal cells and M0 macrophages emphasizes its potential impact on the tumor microenvironment. In vitro experiments showed that CTSV knockdown could significantly inhibit the proliferation and migration of PAC cells. This finding provides important clues for further research into the pathogenesis of PAC and the development of new treatment strategies.