Entosis, first identified as a non-apoptotic cell death program, in which Cell-in-cell (CIC) structures are often formed, involves one cell engulfing another, causing the death of the internalized cell. However, the exact molecular mechanisms and factors controlling entosis are unclear. Entosis have recently been implicated in the aggressive behavior of pancreatic ductal adenocarcinoma (PDAC). PDAC is a highly lethal disease and characterized by pronounced stromal reaction and tissue stiffening. However, if the highly fibrotic and mechanically stiff tumor microenvironment of pancreatic cancer regulates entosis process remains largely unexplored.

To clarify the relevance of mechanical stress in human disease, we measured the mechanical properties of surgically resected pancreatic cancer tissues using nanoindentation. By using live cell immunofluorescence staining and continuous imaging, we observed the real time dynamic morphological changes of inner cell and outer cell during entosis process. We integrated mechanical modulation with cellular and organoid models to systematically investigate the relationship between extracellular stiffness and CIC occurrence.

Nanoindentation analysis revealed significantly elevated tissue stiffness in pancreatic cancer tissues compared with non-tumor pancreatic tissues. In vitro, by constructing a matrigel concentration gradient to simulate different mechanical environments, we found that CIC formation in pancreatic cancer cells and patient-derived pancreatic cancer organoids increased in a stress-dependent manner, whereas CIC structures were rarely observed in normal pancreatic organoids. Dual-fluorescence competition assays further showed that cells pre-exposed to higher mechanical stress were more prone to undergo entosis and become the inner cell of CIC structures. Pharmacological intervention indicated that microtubule-related processes contribute to CIC regulation. Immunofluorescence analysis revealed differential microtubule modification patterns between inner and outer cells within CIC structures. This study provides a novel mechanistic framework for understanding the interplay between tissue mechanics, cytoskeletal regulation, and entosis in pancreatic cancer and offers potential avenues for targeting mechanically driven tumor adaptation.

This study demonstrates that elevated mechanical stiffness in pancreatic cancer tissues is a key driver of entosis. Beyond general cytoskeletal remodeling, microtubule modification may serve as an important mechanistic link connecting mechanical stimuli to CIC formation.

National High Level Hospital Clinical Research Funding(2025-PUMCH-C-042), CAMS Innovation Fund for Medical Sciences(CIFMS, 2025-I2M-XHXX-012).