Engineering tunable synthetic fibrotic niches to model acinar-to-ductal metaplasia in 2D pancreatic culture systems

Abstract

Acinar-to-ductal metaplasia (ADM) is an early pancreatic cell-state transition associated with injury, inflammation, and progression toward pancreatic intraepithelial neoplasia (PanIN). Although genetic and inflammatory drivers of ADM are well established, the contribution of extracellular-matrix mechanics is difficult to isolate because tissue stiffening is accompanied by changes in matrix composition and soluble signaling. Here, we describe a two-dimensional polyacrylamide (PAA) hydrogel platform that independently varies substrate stiffness and adhesive ligand presentation for murine pancreatic acinar 266-6 cells. Hydrogels approximate soft pancreatic tissue (0.50 kPa), a transitional mechanical condition (4.48 kPa), and fibrotic tissue (18.29 kPa). RGD provides a conventional adhesive cue relevant across healthy-to-fibrotic extracellular-matrix states, whereas incorporation of laminin-derived isoleucine-lysine-valine-alanine-valine (IKVAV) creates a more basement-membrane-like environment consistent with preservation of acinar identity. The platform was also adapted to include gelatin, a collagen-derived protein used here to model a more collagen-rich fibrotic matrix context. Peptide coupling is calibrated using fluorescently labeled RGD, and responses after 3 days are assessed by morphology, alpha-amylase 2 (AMY2), Yes-associated protein (YAP) nuclear-to-cytoplasmic localization, and sex-determining region Y-box transcription factor 9 (SOX9). Increasing stiffness increased nuclear YAP localization and reduced AMY2 expression. IKVAV-containing conditions supported greater attachment on softer matrices and partially preserved acinar-like phenotype on stiff gels despite elevated nuclear YAP. Across all ligand conditions, increasing stiffness reduced roundness and geometric regularity/solidity and increased aspect ratio; on gelatin-modified hydrogels, these phenotypic changes corresponded to increased cell-area-normalized SOX9 fluorescence. Together, these readouts identify a stiffness-dependent shift from an acinar-associated state toward an ADM-associated ductal phenotype, while ligand context modulates how this mechanical response is expressed. This protocol provides a controllable, adaptable system for separating mechanical and biochemical extracellular-matrix inputs during pancreatic phenotypic plasticity.