Pancreatic cancer affects entosis by regulating the cytoskeleton through mechanical stress

Introduction: Entosis is a type of programed cell death which was firstly defined in 2007. Cell-in-cell (CIC) structures are often formed via entosis and have recently been implicated in the aggressive behavior of pancreatic ductal adenocarcinoma (PDAC). However, how the highly fibrotic and mechanically stiff tumor microenvironment of pancreatic cancer regulates CIC formation remains largely unexplored. The potential mechanotransduction mechanisms underlying this process are still unclear, particularly in clinically relevant models. Rationale: Pancreatic cancer is characterized by pronounced stromal reaction and tissue stiffening. To clarify the relevance of mechanical stress in human disease, we first measured the mechanical properties of surgically resected pancreatic cancer tissues using nanoindentation. Based on these tissue-level findings, we hypothesized that increased tissue stiffness promotes CIC formation via cytoskeleton-associated mechanisms. To test this hypothesis, we integrated mechanical modulation with cellular and organoid models to systematically investigate the relationship between extracellular stiffness and CIC occurrence. Results: Nanoindentation 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. Conclusions: Our study demonstrates that elevated mechanical stiffness in pancreatic cancer tissues is a key driver of CIC formation. The findings suggest that, beyond general cytoskeletal remodeling, microtubule modification may serve as an important mechanistic link connecting mechanical stimuli to CIC formation. This work 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.