The Application Of Single-cell Sequencing Technologies In The Investigations Of Pancreatic Tumors

Background:Pancreatic cancer is known as the "king of cancers".Early diagnosis of pancreatic cacner is difficult and there is a lack of effective drugs.Although traditional genomic,transcriptomic,and epigenetic studies have identified key mutated genes and aberrant signaling pathways in pancreatic cancer,one of the reasons why these findings have not been successfully translated into clinical applications is the high heterogeneity of pancreatic cancer.The traditional batch sequencing technology can only measure the mixed information of tissue cells and cannot further explore the characteristics of pancreatic cancer tumor cells,and it is difficult to clarify the diagnosis and treatment targets.Single-cell sequencing technology can probe tumor cells at the single-cell level.Existing studies have used single-cell sequencing technology to analyze the tumor microenvironment and stromal cell function in head and neck cancer,liver cancer and other tumors.Because pancreatic tissue is rich in digestive enzymes,single-cell isolation of pancreatic tissue remains the rate-limiting step for conducting single-cell studies of pancreatic cancer.Objective:To explore the construction of single-cell isolation technology suitable for a variety of pancreatic tumor tissues and normal pancreatic tissue and combine single-cell transcriptome sequencing technology to construct a complete pancreatic tumor single-cell research method.And this method was applied to pancreatic cancer heterogeneity analysis and therapeutic target mining.Methods:This study first optimized the steps of pancreatic tissue collection,preservation,enzymatic digestion,and single-cell purification,and explored whether this method could improve the yield of pancreatic cancer single-cell samples and be applied to other types of pancreatic tumors.Then,we used this method to perform single-cell transcriptome sequencing of pancreatic cancer tissue to identify cell groups in pancreatic cancer tissue.Malignant cells were identified by chromosomal copy number variation analysis and differentially expressed gene pathway enrichment analysis.Further subpopulation analysis of tumor malignant cells identifies malignant cell subsets and functions.Finally,based on pancreatic cancer single-cell transcriptome data,we explored the activation state of IL-17 pathway and NOTCH pathway in malignant cells of pancreatic cancer tissue,and verified the effect of IL-17 pathway and NOTCH pathway on pancreatic cancer in vitro cell lines and in vivo mouse models.Cancer cell proliferation,migration and other functional effects.And further use STING network analysis and small molecule inhibitors to explore the regulatory relationship between IL-17 pathway and NOTCH pathway.Finally,the effect of co-inhibiting the IL-17 pathway and NOTCH pathway on tumor growth was explored in a subcutaneous tumorigenic mouse model.Results:We constructed a set of single-cell isolation methods suitable for a variety of pancreatic tissues and normal pancreatic tissues,which improved the single-cell yield of pancreatic cancer tissues and preserved the viability of single-cell samples of normal pancreatic tissues.After the single-cell samples obtained by this method are sequenced by the singlecell transcriptome library,the obtained data of each sample are comparable in quality.Based on this,we mapped the single-cell transcriptome of pancreatic cancer and found that pancreatic cancer tissue contains 10 types of cell groups,including type I ductal cells,type II ductal cells,and acinar cells.Type Ⅱ ductal cells have high levels of chromatin variation and high expression of genes related to tumor characteristics and are identified as malignant cells.We further divided type Ⅱ ductal cells into 7 cell subgroups,and each cell subgroup specifically expressed migration,proliferation,and other function-related genes.The proportion of type Ⅱ ductal cell subsets with high expression of proliferation-related genes varies in pancreatic cancer patients.Pancreatic cancer patients can be divided into three groups according to the expression levels of genes related to proliferation subgroups.Survival analysis shows that pancreatic cancer patients with high expression of genes related to proliferation subgroups have poor prognosis.Gene interaction network analysis identified CDK1,PLK1 and AURKA genes that are centrally regulated in proliferating cell subsets,and inhibition of these genes inhibited pancreatic cancer cell proliferation in vitro.Based on the analysis of pancreatic cancer single-cell transcriptome,it was found that some malignant cells of pancreatic cancer tissue up-regulated IL-17-related signaling pathway and NOTCH signaling pathway.In vitro and in vivo experiments confirmed that IL-17 and NOTCH can promote pancreatic cancer tumor cell function,occurrence and development,respectively.Mechanistic analysis verified that IL-17 activates NOTCH pathway through NFκB pathway.In a subcutaneous tumorigenic mouse model,the growth rate of tumors in the co-inhibition of IL-17 pathway and Notch signaling pathway was significantly slower than that in the inhibition of IL-17 pathway or Notch signaling pathway alone.Conclusions:1.The single-cell isolation method developed in this study can obtain high-quality pancreatic tissue single-cell samples,which can be used for single-cell transcriptome sequencing to obtain comparable quality data for pancreatic tumor research.2.There are 10 types of cells in pancreatic cancer tissue,among which type Ⅱ ductal cells are malignant cells,which are composed of multiple cell subsets with different functions and have strong heterogeneity.3.Pancreatic cancer malignant cell species contain proliferation-related cell subsets.It is found that pancreatic cancer patients with high expression of proliferation subset genes have poor prognosis.CDK1,PLK1 and AURKA,which regulate proliferation characteristics,are potential therapeutic targets for pancreatic cancer.4.The IL-17 pathway and NOTCH signaling pathway can synergistically promote the occurrence and development of pancreatic cancer,and the combined inhibition of the two pathways can limit tumor growth and enhance the effect of drug therapy on pancreatic cancer.