Figure 3
Breast cancer-derived soluble factors increase HA synthesis of mesenchymal stem cells and impair adipogenic differentiation. BmMSCs were incubated with supernatant derived from the cell lines MDA-MB-231 and U87-MG over a period of 72 h. Untreated culture medium was used as control. (a) Representative microscope pictures of affinity cytochemical stainings of bmMSCs; HA (green) and Hoechst 33,342 (blue). Scale bar = 100 µm. (b) Microscopic pictures were analysed for HA content, measured as integrated density normalised to the number of nuclei. (c) The amount of secreted HA was measured with an ELISA-like HABP-binding assay, and the results were normalised to the protein content of the cell lysates. (d) BmMSCs were treated with MDA-MB-231 cell-derived supernatant and incubated over a period of 48 h. The expression of HA-system related genes was analysed via qRT-PCR. n = 3. Mean ± SEM. *p < 0.05 compared to control treated bmMSCs. (e) and (f) BmMSCs were differentiated into adipocytes with or without the addition of 4-MU (100 µM) over a period of 28 days in the presence of cancer cell line-derived supernatant or untreated medium as control. (e) Lipid vesicles of adipogenic differentiated bmMSCs were stained with Oil Red O. Scale bar = 200 µm. (f) Adipogenic differentiation was quantified by determining the area fraction of Oil Red O-stained lipid vesicles and normalised to the number of nuclei. *p < 0.05 compared to control/adipoDiff; #p < 0.05 compared to MDA-MB-231 SN/adipoDiff. n = 4. Mean ± SEM. (g) PCA-analysis of supernatants derived by MDA-MB-231 and U87-MG cells analysed by LC–MS, n = 5. (h) Venn diagram of secreted and soluble factors in the supernatants of MDA-MB-231 and U87-MG cells investigated via LC–MS. This figure was prepared with GraphPad Prism (v9.2.0.332, www.graphpad.com).
