Differential Response of Non-cancerous and Malignant Breast Cancer Cells to Conditioned Medium of Adipose tissue-derived Stromal Cells (ASCs)

Abstract

Background: The application of adipose tissue-derived stromal cells (ASCs) in regenerative medicine has become a growing trend due to its abundance and differentiation potentials. However, several breast cancer studies indicated that ASCs promote tumor progression, therefore, the use of ASCs for reconstruction after oncological surgery poses potential risks. In this study, we aimed to examine whether cancerous or non-cancerous breast cells will exhibit different responses to ASC-derived CM. Methods: ASCs were isolated from residuals of subcutaneous adipose tissue obtained from patients undergoing surgery. Cancerous MCF-7, MDA-MB231, and MDA-MB468 cell lines and one non-cancerous M10/H184B5F5 cell line were cultured with variant concentrations of ASC-derived conditioned medium (CM) for analysis. Results: ASC-derived CM significantly reduced cell viability by triggering apoptosis in MCF-7, MDA-MB231, and MDA-MB468 cell lines. ATM-Chk2-dependent DNA damage response was activated early in cancer cells when exposed to ASC-derived CM. By contrast, prompted cell proliferation instead of cell death was detected in M10/H184B5F5 cells under the treatment of lower CM concentration. Even when exposed to the highest concentration of CM, only cell cycle arrest accompanied by a weak DNA damage response were detected in M10/H184B5F5 cells, no cell deaths were observed. Conclusions: Overall, this study demonstrated that cancerous and non-cancerous breast cells respond differently to ASC-derived CM. ASC-derived CM triggered significant cell death in breast cancer cell lines, however non-cancerous breast cells exhibited dissimilar response to ASC-derived CM.

Keywords: ASCs; adipose tissue-derived stromal cell; apoptosis; breast cancer cell line.

Figure 1

ASC-derived conditioned medium (CM) suppressed cell proliferation and viability in MCF-7, MDA-MB231 and MDA-MB468 breast cancer cells. MCF7, MDA-MB231 and MDA-MB468 cells were cultured with control culture medium or conditioned K-medium (CM) for 72 hours. A) Cell morphology was observed and recorded through microscopy analysis. Magnification: MCF-7 and MDA-MB231: ×200; MDA-MB468: ×400. B) Cell proliferation and viability were examined using the MTT assay. 1×10⁵ cells were cultured with control or ASC-derived CM as indicated concentrations for 72 hours. Presented values of bar graphs represent the mean of three independent experiments ± s.d. *p < 0.05 compared with control cells.

Figure 2

ASC-derived conditioned medium (CM) enhanced apoptosis in MCF-7 and MDA-MB231 breast cancer cells. A) MCF7 and MDA-MB231 cells were cultured with control culture medium or 100% conditioned K-medium (CM) for indicated time points before harvested, and the cell cycle distribution was analyzed using flow cytometry. In each panel, cell cycle profiles are presented together with bar graphs, indicating the cell distribution in each phase of the cell cycle. Presented values represent the mean of three independent experiments ± s.d. *p < 0.05 compared with control. B) Western blot analysis of activated-caspase-3 in MCF-7 or MDA-MB231 cells cultured with 100% ASC-derived CM for 72 hours. C) Western blot analysis of activated-caspase-3 in MCF-7 or MDA-MB231 cells cultured with 50% ASC-derived CM as indicated time points. GAPDH levels are presented as loading controls.

Figure 3

ASC-derived conditioned medium (CM) induced DNA damage in MCF-7 and MDA-MB231 breast cancer cells. A) MCF-7 and MDA-MB231 cells were cultured with 100% ASC-derived CM for 72 hours before being fixed and stained for DNA content and phosphorylated pSer139 H2AX (γ-H2AX). Images from the immunofluorescence analysis of γ-H2AX positive cells in MCF-7 and MDA-MB231 are presented together with bar graphs, indicating the percentage of the mean of three independent experiments ± s.d. *p < 0.05 compared with control. B) Western blot analysis of γ-H2AX in extracts prepared from MCF-7 and MDA-MB231 cells cultured with 100% ASC-derived CM for 72 h. C) Western blot analysis of γ-H2AX in extracts prepared from MCF-7 and MDA-MB231 cells cultured with 50% ASC-derived CM for indicated time points. GAPDH levels are presented as loading controls.

Figure 4

ASC-derived conditioned medium (CM) failed to induce apoptosis in M10/H184B5F5 cells. A) M10/H184B5F5 cells were cultured with control culture medium or conditioned K-medium (CM) as a dose-dependent manner for 72 hours before MTT analysis. Presented values represent the mean of three independent experiments ± s.d. *p < 0.05 compared with control. B) M10/H184B5F5 cells were cultured with control culture medium or 100% conditioned K-medium (CM) for indicated time points before harvest, and the cell cycle distribution was analyzed using flow cytometry. In each panel, cell cycle profiles are presented together with bar graphs, indicating the cell distribution in each phase of the cell cycle. Presented values represent the mean of three independent experiments ± s.d. C) Western blot analysis of γ-H2AX and activated- caspase-3 in M10/H184B5F5 cells cultured with ASC-derived CM for indicated time points. GAPDH levels are presented as loading controls.

Figure 5

ASC-derived conditioned medium (CM) induced fully activated ATM-Chk2 cascades in MCF-7 and MDA-MB231, but not in M10/H184B5F5 cells. MCF-7, MDA-MB231, and M10/H184B5F5 cells were cultured with ASC-derived CM for up to 72 hours before the harvest and preparation of cell-free extracts. Phosphorylated pSer1981 ATM and pThr68 Chk2 and the total protein level of Chk2 were analyzed using Western blot. GAPDH levels are presented as loading controls.