Holm Nielsen Signe, Willumsen Nicholas, Leeming Diana Julie, Daniels Samuel Joseph, Brix Susanne, Karsdal Morten Asser, Genovese Federica, Nielsen Mette Juul
Nordic Bioscience A/S, Herlev, Denmark; Disease Systems Immunology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs. Lyngby, Denmark.
Nordic Bioscience A/S, Herlev, Denmark.
Transl Oncol. 2019 Feb;12(2):368-374. doi: 10.1016/j.tranon.2018.11.004. Epub 2018 Nov 30.
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.
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.
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.
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.
细胞外基质（ECM）重塑是不同肺部疾病（如纤维化和癌症）中的关键事件。结缔组织中最常见的细胞类型是成纤维细胞，激活后会转分化为肌成纤维细胞。所有肌成纤维细胞均表达α - 平滑肌肌动蛋白（α - SMA），已发现其在肺纤维化和癌症中上调。我们评估了α - SMA作为肺纤维化和癌症中活化成纤维细胞的非侵入性生物标志物的潜力。
制备了针对α - SMA N端的单克隆抗体，并开发并对一种测量α - SMA的新型竞争性酶联免疫吸附测定（ELISA）进行了技术表征。在成纤维细胞模型“瓶中瘢痕”以及来自两个不同队列的特发性肺纤维化（IPF）、慢性阻塞性肺疾病（COPD）和非小细胞肺癌（NSCLC）患者的血清中测量α - SMA水平。
开发并验证了新型α - SMA测定法，其技术性能稳健。基于“瓶中瘢痕”结果，α - SMA仅存在于经转化生长因子 - β（TGF - β）激活的成纤维细胞中。在队列1中，IPF、COPD和NSCLC患者的α - SMA水平显著高于健康对照（分别为P = 0.04、P = 0.001和P <0.0001）。用于区分健康对照与IPF患者的受试者工作特征曲线下面积（AUROC）为0.865，区分健康对照与COPD患者的为0.892，区分健康对照与NSCLC患者的为0.983。在队列2中，NSCLC患者的α - SMA水平也显著高于健康对照（P = 0），区分NSCLC与健康对照的AUROC为0.715。
在本研究中，我们开发并验证了一种针对α - SMA N端的稳健竞争性ELISA测定法。添加TGF - β时α - SMA水平上调，表明活化成纤维细胞中α - SMA增加。与健康对照相比，IPF、COPD和NSCLC患者循环中的α - SMA水平显著更高
