Fig. 5.

CAF-activated, polarized monocytes lose their tumoricidal properties and their derived exosomes enhance the proliferation and migration of BC cells and up-regulate the expression of EMT markers. A The cytotoxic activity of monocytes alone or incubated with either CAF- or NF-CM was evaluated by measuring the luciferase activity of MDA-MB-231 BC cells expressing luciferase (MDA-MB-231-luc) in a co-culture condition. Bar graphs represent the relative luciferase activity, indicative of BC viability. The reduced luciferase activity of BC cells co-cultured with control monocytes pointed to the effective anti-tumoral functions of monocytes. In contrast, when monocytes were treated with CAF-CM, these cells lost their tumoricidal properties and instead promoted BC proliferation. Columns, mean of three different experiments; bars, SD. **P-value < 0.01. B MDA-MB-231 BC cells were incubated with exosomes derived from all differentiated monocytes. Results showed that different concentrations of TEMo-Exo (25, 50, and 100 μg/mL) have promoting effects on the proliferation rate of MDA-MB-231 BC cells in a dose- and time-dependent manner. In contrast, BC cell proliferation was not substantially affected by incubation with 100 µg/mL NEMo-Exo as compared to PBS-treated BC cells. As expected, BC cells displayed the lowest rate of proliferation when incubated with 100 µg/mL control monocyte exosomes at the indicated time points. Points, mean of three different experiments, bars, SD. ***P-value < 0.001. C Representative photomicrographs of MDA-MB-231 BC cells cultured with 100 μg/mL exosomes derived from TEMo, NEMo, or control monocytes as well as in standard medium containing PBS at 24 h after scratch wounding. BC cells incubated with 100 μg/mL TEMo-Exo exhibited a considerably higher migration potential compared with those incubated with exosomes derived from control monocytes or NEMo. D, E Enhanced migration of MDA-MB-231 BC cells incubated with 100 μg/mL of TEMo-Exo, compared with the corresponding control cells. D Representative photomicrographs of the migration potential of MDA-MB-231 BC cells in different conditions after 24 h of incubation, assessed using the transwell assay. E Quantitative assessment of migrated cells showed that MDA-MB-231 BC cells treated with 100 μg/mL TEMo-Exo exhibited a significantly higher migration potential compared with cells incubated with exosomes derived from control monocytes or NEMo as well as cells cultured in standard medium containing PBS. Columns, mean of three different experiments; bars, SD. ***P-value < 0.001. F Western blot analysis showed up-regulation of EMT protein markers (N-cadherin, Vimentin, and Snail) and down-regulation of epithelial marker E-cadherin in MDA-MB-231 BC cells, 48 h after treatment with 100 μg/mL TEMo-Exo compared with the corresponding control cells. Actin was used as an endogenous loading control. Western blot images are representative of at least three independent experiments