Figure 4

Exosomes derived from EGCG-treated tumor cells suppresses NF-κB pathway and inhibits M2 polarization of tumor-associated macrophages, which is dependent on up-regulated miR-16 in EGCG-treated tumor exosomes. (A) Tumor-associated macrophages were isolated from established murine breast cancer 4T1 cell lines, and treated with exosomes extracted from EGCG (100 μM)-treated and untreated 4T1 cells for 24 hours. Whole cell lysates were prepared and submitted to Western blot using anti-IKKα, anti-I-κB or anti-actin antibodies. (B) 4T1 cells were incubated with EGCG (100 μM) and simultaneously (24 hours) transfected with scramble or miR-16 inhibitor for 24 hours. Exosomes were then extracted from each tumor cells. Tumor-associated macrophages isolated from established murine breast cancer were treated with each exosome as indicated at the concentration of 50 μg/ml for 24 hours, and whole cell lysates were prepared and submitted to Western blot using anti-IKKα or anti-actin antibodies. The numbers in Figure 4A and 4B report the quantification of bands by imaging analysis with KODAK Molecular Imaging Software (MI). (C) 4T1 cells were incubated with EGCG (100 μM) and were simultaneously (24 hours) transfected with scramble or miR-16 inhibitor for 24 hours. Exosomes were then extracted from each tumor cells. Tumor-associated macrophages isolated from established murine breast cancer were treated with each exosome at the concentration 50 mg/ml for 24 hours, and total cellular RNA was extracted and submitted to RT-qPCR analysis for M2^high cytokines (IL-6 and TGF-β) and M2^low cytokine (TNF-α). Histogram shows the relative expression of molecules compared to GAPDH as internal control. (D) Tumor cells were isolated and total RNA from cells were extracted and subjected to RT-qPCR to measure the miR-16 level. Histogram shows the relative expression of miR-16 compared to U6 as an internal control.