Stiffness Measurements in Human Lung Slices Can Evaluate the Therapeutic and Preventive Potentials of Anti-fibrotic Drugs

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Abstract RATIONALE: A significant focus of Idiopathic Pulmonary Fibrosis (IPF) drug discovery is to reduce or pre-emptively slow progressive lung stiffening. However, lung stiffness is not readily accessible in conventional IPF drug discovery assays. METHODS: We focused on fibrotic and fibrosis-induced precision cut lung slices (PCLS) from human lungs and treated the slices with approved anti-fibrotic therapeutics, Nintedanib or Pirfenidone. We utilized our patented (US-20230408376-A1) method to measure the macroscale stiffness value per PCLS (Kim et al Sci. Adv. 2023). To elaborate heterogeneity of lung tissue, we developed a method to map local microscale stiffness of PCLS at a spatial resolution of 15um. RESULTS: (1) Compared to the healthy PCLS, the IPF PCLS was ∼3 fold stiffer with a ∼30-fold greater local microscale stiffness variance, suggesting a massively increased spatial heterogeneity in IPF (n=7-9 PCLS per donor per group). (2) Nintedanib (0.3µM, 96 hours) softened the IPF PCLS by 39% (Fig. A and B), reduced width of microscale stiffness distribution by 64% (Fig F), and reduced stiffness variance by 84% (Fig. G). (3) Pirfenidone (100µM, 96 hours) softened the IPF PCLS by 34% (Fig. A and B), reduced width of microscale stiffness distribution by 58% (Fig F), and reduced microscale stiffness variance by 86% (Fig. G). (4) Compared to the healthy PCLS treated with a control cocktail, the healthy PCLS treated with a fibrotic cocktail (TGF-β (5 ng/mL), PDGF-AB (10 ng/mL), TNF-α (10 ng/mL), LPA (5 µM), 96 hours) was 50% stiffer with ∼2-fold wider distribution and ∼6-fold greater microscale stiffness variance. (5) Nintedanib co-treatment (1µM, 96 hours) inhibited fibrotic cocktail induced PCLS stiffening and elevated variance. CONCLUSIONS: We demonstrate that macro- and microscale PCLS stiffness measurements can evaluate the therapeutic and preventive potentials of anti-fibrotic drugs.In comparison to traditional collagen assays and/or measurements of ECM gene expression changes, the key advantage is that PCLS stiffness measurements are non-invasive, longitudinal, suitable for multiplexing with biological changes, and importantly, directly correlates with lung function, and hence, patient comfort, quality of life and, ultimately, survival.

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