TNF-α induces potent elevation in inflammatory traits in MSCs and Tumor-CM-generated CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or from MCF-7 cells (B) over a prolonged period of time (~30 days). TNF-α (50 ng/ml) or its vehicle (in control cells) was added for the last 24 hours. Expression of CCL2, CXCL8 and CCL5 was then determined in supernatants of the cells. (A) Expression of the chemokines in the four experimental groups included in the study: MSCs grown in culture for ~30 days without any additional stimulus (MSCs); MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells (MSCs + MDA CM); MSCs grown in culture for ~30 days and stimulated by TNF-α at the last 24 hours of culture (MSCs + TNF-α); and MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells and stimulated by TNF-α at the last 24 hours of culture (MSCs + MDA CM + TNF-α). Expression of CCL2 (A1), CXCL8 (A2) and CCL5 (A3) was determined by ELISA, in the linear range of absorbance. (B) Experimental design as in (A), but with MCF-7-derived CM. In each panel, the findings are representatives of at least n = 3 experiments that have shown similar results. In comparisons between MSCs and all other groups: *P <0.05, **P ≤0.01, ***P ≤0.001. NS, not significant. #Differences between the two indicated groups have shown variability in n ≥ 3 independent experiments and overall were not significant.