Fig. 10

Impact of CAFs on the levels of selected proteins in breast cancer cells. Breast cancer cells: A MCF-7 and B MDA-MB-231 cells were incubated with conditioned media (CM) from calcitriol-treated CAFs derived from tumors of patients with different clinical characteristics. Levels of the following proteins were assessed: A1, B1 E-cadherin levels, A2, B2 OPN (osteopontin) levels, A3, B3 ZEB1 (zinc finger E-box binding homeobox 1) levels, and A4, B4 representative cropped blots. Molecular weight of analyzed proteins: E-cadherin—120 kDa (MCF-7) or 40 kDa (intracellular domain, MDA-MB-231), OPN—40 kDa, ZEB1—130 kDa. Full-length blots are presented in Figures S12 and S13 in the Supplementary Materials. CAFs were stimulated with calcitriol (cal) for 72 h prior to CM generation for 24 h. CM was applied to cancer cells for 72 h. Patients were classified into groups according to plasma 25(OH)D₃ levels (VD3, < 30 ng/mL—deficient (n = 10), > 30 ng/mL—normal (n = 6)), plasma FSH levels (< 25.8 mIU/mL—premenopausal (n = 7), > 25.8 mIU/mL—postmenopausal (n = 9)) and regional or distant metastasis presence (if any—metastatic (n = 7), otherwise nonmetastatic (h 9)). Densitometric analysis was performed using ImageJ software. The results were normalized to β-actin levels and untreated cancer cells. Data are presented as the mean ± SEM. Statistical analysis was carried out using Student’s t-test or the Mann‒Whitney U test for single comparisons and one-way ANOVA or the Kruskal‒Wallis test for multiple comparisons. *p ≤ 0.05 compared to untreated cancer cells