Atomic Force Microscopy‐Based Nanomechanical Signatures for Staging Classification and Drug Response in Pulmonary Fibrosis

Abstract Current therapies for pulmonary fibrosis (PF) show high variability in effectiveness, while the lack of specific biomarkers hinder early diagnosis, and treatment monitoring. With few approved drugs and inconsistent treatment responses, developing personalized therapies is essential. Since PF progression involves changes in tissue structure and mechanics, mechanobiology plays a central role. It is hypothesized that Atomic Force Microscopy (AFM) can identify unique nanomechanical fingerprints (NMFs) to characterize fibrosis stages and monitor treatment response. AFM is first used to assess NMFs in human biopsy samples, followed by evaluation in bleomycin‐induced murine PF models. NMFs are also studied in mice treated with pirfenidone, a drug known to reduce collagen I. AFM data are supported by histopathological staining, polarized and second harmonic generation (SHG) microscopy, and real‐time PCR analysis of collagen I expression. AFM measurements detected distinct NMFs in human samples and tracked alterations during PF progression in mice. Changes in NMFs correlated with collagen I content, histology, and SHG microscopy. In silico analysis supported NMFs’ potential as diagnostic biomarkers. Furthermore, AFM‐based NMFs assessed pirfenidone treatment outcomes. These findings provide the first evidence that AFM‐based NMFs can serve as biomarkers for PF staging and treatment monitoring, offering a powerful diagnostic tool to complement standard biopsies.