Matrix stiffness remodels the immunosuppressive tumor microenvironment via the PIEZO1-DCLK1-STAT5B pathway

Background Pancreatic ductal adenocarcinoma (PDAC) has an abysmal 5-year survival rate (8%), largely attributed to its dense fibrotic stroma that forms a high-stiffness microenvironment. This biomechanical feature exacerbates immunosuppression and promotes immune escape, but the molecular mechanism by which matrix stiffness-derived mechanical signals convert into immunosuppressive cues remains undefined. This study aims to uncover the key mechanotransduction-immunity crosstalk pathway in PDAC and provide novel therapeutic targets. Methods We utilized our established adjustable extracellular matrix (ECM) stiffness models (in vitro 3D culture and in vivo orthotopic transplantation) as core research platforms. Integrated approaches including CRISPR-Cas9 gene editing, multi-omics (protein modification omics, CUT&Tag-seq), single-cell RNA sequencing, and spectral flow cytometry were employed to dissect the mechanotransduction pathway and its regulatory effect on the tumor microenvironment. Results We identified a novel PIEZO1-Ca²⁺-DCLK1-STAT5B axis mediating stiffness-induced immunosuppression. High matrix stiffness specifically activated the mechanosensitive ion channel PIEZO1, triggering Ca²⁺ influx. Intracellular Ca²⁺ upregulated and activated DCLK1 via dual mechanisms: inhibiting ANAPC5/PSMA7-mediated ubiquitination and promoting HPCAL1-dependent serine phosphorylation. Activated DCLK1 bound to STAT5B through its serine/proline-rich linker domain (DCLK1) and SH2 domain (STAT5B), inducing STAT5B phosphorylation and nuclear translocation. Nuclear STAT5B, under the regulation of super-enhancers (marked by H3K27ac/H3K4me1), transcriptionally activated immunosuppressive genes (TGF-beta, PD-L1, KRAS), ultimately shaping an immunosuppressive microenvironment characterized by increased infiltration of Tregs, MDSCs, and M2 macrophages, and impaired effector T cell function. Preliminary data confirmed that high stiffness upregulated all components of this axis, and STAT5B inhibition significantly reduced TGF-beta/PD-L1/KRAS expression and immunosuppressive cell infiltration. Conclusion This study is the first to uncover a biomechanics-driven immunosuppression pathway in PDAC, establishing a direct link between matrix stiffness and immune escape. The PIEZO1-Ca²⁺-DCLK1-STAT5B axis provides a novel therapeutic target for reversing immunosuppression and improving PDAC therapeutic efficacy.