FIGURE 2.

Effect of TIMP‐1 and MMPs on ECM expression in tumor spheroids. (a) Collagen type‐1 (green) and fibronectin (red) staining of tumor spheroids A:M, B:M, and F:M. Scale bars, 100 μm. (b) Quantifications of fluorescence intensity for Collagen type‐1 and fibronectin in the tumor spheroids. Values were normalized to the intensity of DAPI. *p < 0.05, **p < 0.01 (one‐way ANOVA), n = 3 per group. (c) Protein expression of collagen type‐1 and fibronectin in the tumor spheroids. mRNA expression of (d) Collagen type‐1 alpha 1, fibronectin, and (e) MMPs in the tumor spheroids (one‐way ANOVA), n = 3 per group. (f) mRNA expression and protein secretion of TIMP‐1 in the tumor spheroids. **p < 0.01, ***p < 0.001 (one‐way ANOVA), n = 3 per group. (g) Enzymatic activity of MMP‐1 and MMP‐9 secreted from the tumor spheroids. *p < 0.05, ***p < 0.001 (one‐way ANOVA), n = 3 per group. (h) TIMP‐1 protein secreted from 2D‐ and 3D‐cultured stromal cells and breast cancer cells. **p < 0.01, ****p < 0.0001 (one‐way ANOVA), n = 3 per group. (i) TIMP‐1 protein secreted from 2D‐co‐cultured stromal cells and breast cancer cells. **p < 0.01 (one‐way ANOVA), n = 3 per group. For RT‐qPCR analysis, values were normalized to GAPDH. All data are presented as mean ± SEM. Col I, Col1a1, 3D—a, b, f, or m; monocellular spheroid of hASCs, hBMSCs, hDFs, or MDA‐MB‐231, respectively. 2D—a, b, f, or m; monolayer culture of hASCs, hBMSCs, hDFs, or MDA‐MB‐231, respectively. 2D, two dimension; 3D, three dimension; DAPI, 4′,6‐diamidino‐2‐phenylindole; Col I, Collagen type‐1; Col1a1, Collagen type‐1 alpha 1; ECM, extracellular matrix; hASCs, human adipose‐derived stromal cells; hBMSCs, human bone marrow stromal cells; hDFs, human dermal fibroblasts; SEM, standard error of the mean; MMPs, metalloproteinases; NS, not significant; TIMP‐1, tissue inhibitor of metalloproteinases‐1