Serological Assessment of Activated Fibroblasts by alpha-Smooth Muscle Actin (α-SMA): A Noninvasive Biomarker of Activated Fibroblasts in Lung Disorders

Abstract

OBJECTIVES: Remodeling of the extracellular matrix (ECM) is a key event in different lung disorders, such as fibrosis and cancer. The most common cell type in the connective tissue is fibroblasts, which transdifferentiate into myofibroblasts upon activation. All myofibroblasts express α-SMA, which has been found to be upregulated in lung fibrosis and cancer. We evaluated the potential of α-SMA as a noninvasive biomarker of activated fibroblasts in lung fibrosis and cancer. METHODS: A monoclonal antibody was raised against the N-terminal of α-SMA, and a novel competitive enzyme-linked immunosorbent assay (ELISA) measuring α-SMA was developed and technically characterized. Levels of α-SMA were measured in the fibroblast model, “scar-in-a-jar”, and in serum from patients with idiopathic pulmonary fibrosis (IPF), chronic obstructive lung disorder (COPD) and non–small cell lung cancer (NSCLC) belonging to two different cohorts. RESULTS: The novel α-SMA assay was developed and validated as technically robust. Based on the scar-in-a-jar results, α-SMA was only present in the fibroblasts activated by TGF-β. In cohort 1, levels of α-SMA were significantly higher in IPF, COPD and NSCLC patients compared to healthy controls (P = 0.04, P = 0.001 and P <0.0001, respectively). The area under the receiver operating characteristics (AUROC) for separation of healthy controls from IPF patients was 0.865, healthy controls from COPD patients was 0.892 and healthy controls from NSCLC patients was 0.983. In cohort 2, levels of α-SMA were also significantly higher in NSCLC patients compared to healthy controls (P = 0) and the AUROC for separating NSCLC and healthy controls was 0.715. CONCLUSIONS: In this study we developed and validated a robust competitive ELISA assay targeting the N-terminal of α-SMA. The level of α-SMA was upregulated when adding TGF-β, indicating that α-SMA is increased in activated fibroblasts. The level of α-SMA in circulation was significantly higher in patients with IPF, COPD and NSCLC compared to healthy controls. This assay could potentially be used as a novel noninvasive serological biomarker for lung disorders by providing a surrogate measure of activated fibroblasts.