3D printing-biomimetic local stiff niche enhances glycolysis to boost PDAC cell stem-like phenotype via N6-methyladenosine-suppressed YAP1 mRNA decay.

Cancer stem cells (CSCs), the primary source of therapy resistance in pancreatic ductal adenocarcinoma (PDAC), exist in a dynamic equilibrium through tumor microenvironment (TME)-driven plasticity. However, the stiffness heterogeneity of TME within PDAC functions on tumor cell stem-like phenotypes remains unclear. Bioinformatics, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, of CSCs identified from spatial transcriptomic and single-cell datasets of PDAC lesions exhibited activated mechanical and glycolytic pathways. Detected by the nano-indenter, PDAC tissue exhibited significant stiffness heterogeneity (200-6000 Pa). Further, biomimetic local stiffness niches were engineered using the digital light-processing (DLP) 3D-printing technology and the desmoplastic bioink (gelatin methacrylate & hyaluronic acid methacrylate, GelMA&HAMA) encapsulating PDAC cells, which permits modulation of mechanical properties without altering the biochemical ligand density. Stemness markers (NANOG, OCT4), glycolysis genes (HK2, LDHA), YAP1, and N6-methyladenosine (m6A) regulators (METTL14, IGF2BP3) were evaluated via qRT-PCR and immunofluorescence. Functional assays of glycolysis and stem-like phenotype were also conducted. Dot blot, RNA stability assay, western blot, and RIP assay were exploited to clarify the level and the function of m6A modification. The local stiff niche enhanced the stem-like phenotype of PDAC cells via YAP1-boosted glycolysis. Mechanistically, local stiff niche elevated the YAP1 level via m6A (METTL14/IGF2BP3)-stabilized YAP1 mRNA, linking mechanical inputs to glycolytic-stem-like phenotype adaptations. Collectively, the local stiff niches may drive the emergence of CSCs through epigenetic and metabolic reprogramming in PDAC mechanobiology. This provides new insights for developing more precise therapeutic strategies targeting PDAC mechanical heterogeneity.