Conditioned medium from asbestos-exposed fibroblasts affects proliferation and invasion of lung cancer cell lines

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 didn't show as similar effect as asbestos exposure. An enzyme-linked immunosorbent assay (ELISA)-based cytokine array identified interleukin (IL)-6 and IL-8, 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).

Fig 1. RTCA traces showing the effect of three types of asbestos-related media on NCI-H358, Calu-3, A549 lung cancer cells.

Following 24 h of seeding, NCI-H358, Calu-3 cells, A549 cells were cultured with derived media from (A) IMRM, IMRM-C, IMRM-A, and IMRM-D, (B) NCIM, NCIM-C, NCIM-A, NCIM-D, CALM, CALM-C, CALM-A, CALM-D, A549M, A549M-C, A549M-A, and A549M-D (C) direct exposure to 50 mg/L of three types of asbestos (chrysotile, amosite, and crocidolite). Cells were also grown in EMEM normal medium. (D) Comparison of the percentage difference in the mean NCI compared to control (EMEM) at 96 h. Error bars represent SD (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared to EMEM, and #P < 0.05, ##P < 0.01, ###P < 0.001 for comparisons between two groups.

Fig 2. Monitoring the migration of A549 cells.

Migration of A549 cells in various media were monitored for 24 h using the RTCA system. Representative graph of migration of A549 cells towards (A) IMRM, IMRM-C, IMRM-A, IMRM-D, and EMEM, (B) A549M, A549M-C, A549M-A, A549M-D, and EMEM, (C) chrysotile, amosite, crocidolite, and EMEM. (D) Comparison of calculated NCI values of migration in all media at 24 h. Experiments were performed in quadruplet and presented as mean ± SD. Quantification of observed migration revealed significance., **P < 0.01 compared to EMEM and #P < 0.05, ##P < 0.01 compared to IMRM.

Fig 3. Effects of media derived from H₂O₂ and UV-stressed lung fibroblasts on lung cancer cells.

(A) RTCA sensing profile of CI values over time for (A) NCI-H358, (B) Calu-3, and (C) A549 cells exposed to H₂O₂ or UV-treated IMR-90 cell-derived media. The experiment was also conducted in EMEM and IMRM as controls. Data show mean ± SD (n = 3).

Fig 4. Asbestos-exposed IMR-90 cells show increased cytokine secretion.

Cytokines in the supernatants of IMR-90 cells exposed or unexposed to asbestos for 24 h were measured using ELISA. The results are expressed as mean ± SD after subtracting negative value from each value. *P < 0.05, vs. IMRM group, n = 4.