Cell-in-Cell-Mediated Entosis Reveals a Progressive Mechanism in Pancreatic Cancer

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with high intratumoral hetero-geneity. There is a lack of effective therapeutics for PDAC. Entosis, a form of nonapoptotic regulated cell death mediated by cell-in-cell structures (CICs), has been reported in multiple cancers. However, the role of entosis in PDAC progression re-mains unclear. METHODS: CICs were evaluated using immu-nohistochemistry and immunofluorescence staining. The formation of CICs was induced by suspension culture. Through fluorescence-activated cell sorting and single-cell RNA sequencing, entosis-forming cells were collected and their dif-ferential gene expression was analyzed. Cell functional assays and mouse models were used to investigate malignant pheno-types. Clinical correlations between entosis and PDAC were established by retrospective analysis. RESULTS: Entosis was associated with an unfavorable prognosis for patients with PDAC and was more prevalent in liver metastases than in pri-mary tumors. The single-cell RNA sequencing results revealed that several oncogenes were up-regulated in entosis-forming cells compared with parental cells. These highly entotic cells demonstrated higher oncogenic characteristics in vitro and in vivo. NET1, neuroepithelial cell transforming gene 1, is an entosis-related gene that plays a pivotal role in PDAC progression and is correlated with poor outcomes. CONCLUSIONS: Entosis is correlated with PDAC progression, especially in liver metastasis. NET1 is a newly validated entosis-related gene and a molecular marker of poor outcomes. PDAC cells generate a highly aggressive subpopulation marked by up-regulated NET1 via entosis, which may drive PDAC progression.