Entosis Facilitates Pancreatic Cancer Progression By Generating Pro-metastatic Subclones

Background&ObjectivePancreatic cancer is a deadly malignancy of the digestive system,with atypical early clinical symptoms and metastatic potentials,as well as drug resistance to chemotherapy and low response rate to targeted therapy and immunotherapy,resulting in the worse prognosis and low five-year overall survival rate which is only 12%.Entosis,a nonapoptotic programmed cell death mediated by cell-in-cell(CIC)structures,has been shown to be associated with the progression of multiple malignancies,including pancreatic cancer.However,the function and mechanism of Entosis in the progression of pancreatic cancer is currently unknown.The purposes of this study were:(1)to investigate whether Entosis exists in pancreatic cancer tissues and cell lines,and the correlation between Entosis and prognosis;(2)to screen key regulators of Entosis in pancreatic cancer progression,as well as the effect on phenotype after cells experienced Entosis;and(3)to validate whether Entosis and its key regulators are risk factors of promoting pancreatic cancer progression.MethodsWe investigated the expression levels of key regulators of programmed cell death in pancreatic cancer samples and the correlation with patient’s prognosis by analyzing RNAsequencing data and clinicopathological features of those samples from public datasets.We induced the formation of CIC structures among pancreatic cancer cells via suspended cell culture,and observed the fate outcome of internalized cells in CIC structures through time-lapse microscopy.We used lentiviral vectors to construct stable knockdown or overexpression cells.Fluorescence-activated cell sorting was used to sort cancer cells undergoing entotic CIC.Single-cell RNA sequencing analysis was used to delineate gene expression changes of CIC-forming cancer cells.Next,we examined the ability of cell proliferation,migration,invasion,anoikis-resistance,and direct competition among cancer cells by using CCK-8 assay,Transwell assay,anoikis assay and cell competition assay.Real-time fluorescence quantitative PCR and Western blot assays were used to validate mRNA and protein levels of targeted genes.Immunohistochemical staining and overall survival analysis were used to assess the correlation between the expression levels of target genes and the prognosis of pancreatic cancer patients.We further performed subcutaneous xenograft and orthotopic xenograft to assess the tumorigenesis and metastatic potential of cancer cells in vivo respectively.ResultsIn this study,we found that Entosis-related regulators were commonly overexpressed in pancreatic cancer tissues compared with other cancers and associated with worse prognosis of pancreatic cancer patients.CIC structures detected in pancreatic cancer specimens have a significant correlation with liver metastases,suggesting that CIC phenomenon in pancreatic cancer may represent an aggressive pathological feature.The results of our single-cell transcriptome analysis revealed that several oncogenes,such as NET1,ALDH3A1,PLAT,GALNT5,and FAM3C,were upregulated in CIC-forming cancer cells compared with non-CIC-forming cancer cells.Moreover,the results of functional assays demonstrated that these CIC-forming cancer cells had enhanced oncogenic characteristics,for instance,cell proliferation,migration,invasion and anoikis resistance in vitro.The results of nude mice xenograft also showed that these cells had enhanced tumorigenic and metastatic abilities in vivo.Furthermore,NET1(neuroepithelial cell transforming gene 1)overexpression promoted the formation of CIC structures among pancreatic cancer cells,and cancer cells with higher NET1 expression levels exhibited enhanced malignant behaviors.Finally,we found that NET1 protein level was associated with unfavorable prognosis for patients,which may play a pro-tumorigenic role in pancreatic cancer progression.ConclusionThese findings support the hypothesis that entosis may play a vital oncogenic role in pancreatic cancer progression.Pancreatic cancer cells generate a highly "progressive"subpopulation marked by increased expression of NET1 via entotic CIC.NET1 is a newly validated gene involved in entosis.Targeting entotic processes may be a potential therapeutic option for pancreatic cancer.