Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease in which tissue stiffens because of excessive extracellular matrix (ECM). This mechanical stiffening reinforces profibrotic behavior and accelerates matrix remodeling. ECM-based hydrogels retain native biochemical cues but are difficult to tune mechanically to replicate healthy and fibrotic lung stiffness. Donor-to-donor heterogeneity further complicates preclinical evaluation. A mechanically tunable, physiologically accurate ECM platform is therefore needed to study stiffness-dependent fibroblast activation and variable drug responses. Methods Decellularized pigs lung ECM (dECM) was processed into pre-gel solutions and reinforced with functionalized nanoparticles to control stiffness. Mechanical testing showed that soft hydrogels (≈1.6 ± 0.4 kPa) matched healthy lungs (1-3 kPa), while stiff hydrogels (≈14.8 ± 2.6 kPa) reflected fibrotic lungs (8-20 kPa) (Fig. A-B). Primary human fibroblasts from were cultured within the hydrogels. Preventative and therapeutic regimens of pirfenidone, the αv-integrin inhibitor CWHM-12, and the ROCK inhibitor Y-27632 were applied. α-Smooth muscle actin (α-SMA), Yes-associated protein (YAP) localization, and single-cell α-SMA intensity were analyzed (Fig. C-I). Results Stiff dECM hydrogels significantly increased α-SMA intensity by 2.8- to 4.1-fold relative to soft gels (Fig. C) and promoted nuclear localization of YAP in all donors. Drug treatment reduced α-SMA expression in a donor-dependent manner (Fig. D-G). Pirfenidone achieved the strongest suppression, lowering α-SMA by 60-70 % across donors (p 0.0001), whereas CWHM-12 and Y-27632 reduced α-SMA by 25-45 % (p 0.001). Conditioning fibroblasts within stiff matrices before treatment amplified the stiffness effect but preserved the same drug-response ranking. IPF-derived fibroblasts displayed higher baseline α-SMA yet followed the same pattern of drug sensitivity. Pooled analysis (Fig. H) confirmed that overall α-SMA expression decreased significantly in all treated groups compared with stiff vehicle controls (p 0.0001). Quantification of matrix-associated TGF-β (Fig. I) showed a 3.5 % ± 0.4 increase in TGF-β storage in stiff versus soft hydrogels, while pirfenidone treatment reduced this to ∼1.9 % ± 0.3 above the soft baseline, indicating suppression of stiffness-driven TGF-β accumulation and demonstrating that the hydrogel network functions as a dynamic ECM reservoir. Conclusions Mechanically tunable lung dECM hydrogels reproduce physiological stiffness ranges and capture donor-specific drug responses. Stiffness enhances α-SMA and TGF-β retention, while pirfenidone provides the most consistent suppression across donors. The 3D matrix environment dominates over cell origin, supporting this platform as a tissue-mimetic tool for antifibrotic drug evaluation and integration of patient variability into preclinical testing. This abstract is funded by: CIHR
Dabaghi et al. (Fri,) studied this question.