Figure 6.

Education of osteoprogenitors via RM1-BM-EVs promotes tumor cell metabolic activity and migration in vitro. (a) Schematic representation of the vicious cycle between osteoprogenitors and PCa cells mediated by EVs (image was created with BioRender.com). (b) RM1-BM metabolic activity was evaluated based on CellTiter Blue assay comparing untreated tumor cells (CO), cells treated with supernatant from unconditioned osteoprogenitors (OB), or cells treated with supernatant from EVs-conditioned osteoprogenitors (OB + RM1-BM EVs) (n = 3–12). Circles: control, squares: osteoblast supernatant, triangle: osteoblast supernatant with RM1-BM EVs. (c) Representative bright-field images (20×) of the scratch assay of RM1-BM cells treated with supernatant from unconditioned osteoprogenitors or EVs-conditioned osteoprogenitors after 0, 4, and 8 h. (d) Quantification of migration of RM1-BM cells indicated as percentages of scratch closure and calculated as follows: % of scratch closure = x−y/x, where (x) is a distance between edges of the wound, and (y) is the distance which remained cell-free during cell migration. Results are representative of more than three independently executed experiments. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test (b) or unpaired Student’s t-test (d). Data are presented as mean ± SD. Statistical significance is denoted: * p < 0.05; *** p < 0.001vs RM1-BM cells treated with EVs-free osteoblast supernatant.