(D) Comparison of calculated NCI values of migration in all media at 24 h. cells/well. Media derived from lung fibroblasts were added. The optimum number of (A) NCI-H358 (B) Calu-3, and (C) A549 cells were 10,000 cells/well, 40,000 cells/well, and 4,000 cells/well, respectively.(TIF) pone.0222160.s004.tif (173K) GUID:?F6772751-9214-4598-9C7D-D8E2E807F36B S5 Fig: Viability of IMR-90 cells treated with asbestos, H2O2, and UV. ATP production of viable cells was determined using the CellTiter-Glo luminescence assay HIV-1 inhibitor-3 (Promega, Southampton, UK). (A) Viability of IMR-90 cells exposed to 50 mg/L asbestos (chrysotile, amosite, and crocidolite) for 24 h. (B) Viability of 24 h-cultured IMR-90 cells after exposure to 0.01, 0.1, 1, and 10 mM H2O2 for 3 h. (C) Viability of 24 h-cultured IMR-90 cells after UV irradiation (10, 25, 50, and 100 J/m2).(TIF) pone.0222160.s005.tif (191K) GUID:?4842B7C3-2751-49E5-8C59-81B03DA679CE S6 Fig: Titration of lung cancer cells for migration in RTCA. (A) NCI-H358 and (B) HIV-1 inhibitor-3 Calu-3 cells could not migrate toward CIM-plate 16. (C) A549 cells showed different rates of migration according to the cell seeding numbers.(TIF) pone.0222160.s006.tif (107K) GUID:?96ACE331-8DD7-4030-98DD-18EA39E00345 Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract The importance of the role of fibroblasts in cancer microenvironment is well-recognized. However, the relationship between fibroblasts and asbestos-induced lung cancer remains underexplored. To investigate the effect of the asbestos-related microenvironment on lung cancer progression, lung cancer cells (NCI-H358, Calu-3, and A549) were cultured in media derived from IMR-90 lung fibroblasts exposed to 50 mg/L asbestos (chrysotile, amosite, and crocidolite) for 24 h. The kinetics and migration of lung cancer cells in the presence of asbestos-exposed lung fibroblast media were monitored using a real-time cell analysis system. Proliferation and migration of A549 cells increased in the presence of media derived from asbestos-exposed lung fibroblasts than in the presence of media derived from normal lung fibroblasts. We observed no increase in proliferation and migration in lung cancer cells cultured in asbestos-exposed lung cancer cell medium. In contrast, increased proliferation and migration in lung cancer cells exposed to media from asbestos-exposed lung fibroblasts was observed for all types of asbestos. Media derived from lung fibroblasts exposed to other stressors, such as hydrogen peroxide and UV radiation didnt show as similar effect as asbestos exposure. An enzyme-linked immunosorbent assay (ELISA)-based cytokine array identified interleukin (IL)-6 and IL-8, TNF which show pleiotropic regulatory effects on lung cancer cells, to be specifically produced in higher amounts by the three types of asbestos-exposed lung fibroblasts than normal lung fibroblasts. Thus, the present study demonstrated that interaction of lung fibroblasts with asbestos may support the growth and metastasis of lung cancer cells and that chrysotile exposure can lead to lung cancer similar to that caused by amphibole asbestos (amosite and crocidolite). Introduction Lung cancer, one of the respiratory diseases caused by asbestos inhalation, is estimated to cause higher annual deaths than other asbestos-related diseases. Asbestos-induced lung cancer is further aggravated by pulmonary fibrosis, which provides a favorable environment for lung cancer development [1]. Indeed, radiographic and HIV-1 inhibitor-3 histological evidence shows that most patients with lung cancer employed in the asbestos cement and asbestos insulation industries were affected by pulmonary fibrosis. These reports demonstrated that excessive asbestos can act as a lung carcinogen because of its fibrogenicity [2]. Fibroblasts, the end effector cells of fibrosis in fibrotic lungs, are embedded within the interstitium of all epithelial tissues and play important roles in organogenesis, wound healing, inflammation, and fibrosis [3]. In particular, fibroblasts that have acquired an activated phenotype (activated fibroblasts and cancer-associated fibroblasts), characterized by the expression of -smooth-muscle actin (-SMA) and secretion of increased amounts of extracellular matrix (ECM) components and growth factors such as transforming growth factors- (TGF-), promote tumor growth and progression. These types of fibroblasts are often referred to as myofibroblasts because of the expression of -SMA, a myofibroblast marker [4]. Asbestos fibers deposited in interstitial spaces are phagocytosed by macrophages and epithelial cells [5, 6], which subsequently alter the morphology and biochemistry of fibroblasts during fibrogenesis [7]. As myofibroblasts are the predominant sources of collagen and fibrogenic cytokines in fibrotic lesions, previous studies showing that direct exposure of lung fibroblasts to asbestos increases deposition of collagen or ECM constituents, including hydroxyproline [8], have postulated that asbestos-treated lung fibroblasts have the potential to activate or differentiate into myofibroblasts and consequently cause fibrosis [9]. These fibroblasts also continually modify their interactions with the lung microenvironment and are capable of supporting the dynamic complexity of tumor microenvironment [10]. For example,.