Cell-in-cell-mediated entosis reveals a progressive mechanism in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with high intratumoral heterogeneity. 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 remains unclear. In this study, we evaluated CICs using immunohistochemistry and immunofluorescence staining. The formation of CICs was induced by suspension culture. Through fluorescence-activated cell sorting and single-cell RNA sequencing (scRNA-seq), we collected entosis-forming cells and analyzed their differential gene expression. Cell functional assays and mouse models were used to investigate malignant phenotypes. Clinical correlations between entosis and PDAC were established by retrospective analysis. We found that entosis was associated with an unfavorable PDAC patient prognosis and was more prevalent in liver metastases than in primary tumors. The scRNA-seq results revealed that several oncogenes were upregulated 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. The formation of CICs was induced by suspension culture. We mixed an equal number of EGFP- and mCherry-stably expressing cancer cells and then induced CIC formation. After CICs induction, we collected the suspended cells and prepared them for FACS. Cells that exhibited both fluorescence signals in the FACS analysis were defined as double-positive (DP; EGFP+/mCherry+ cells), while those exhibiting one fluorescence signal were defined as single-positive (SP; EGFP+/mCherry- or EGFP-/mCherry+ cells). We performed scRNA-seq on DP cells, SP cells, and parental cells (NC).