Exosome derived from epigallocatechin gallate treated breast cancer cells suppresses tumor growth by inhibiting tumor-associated macrophage infiltration and M2 polarization

Background: Tumor-associated macrophages (TAM) play an important role in tumor microenvironment. Particularly, M2 macrophages contribute to tumor progression, depending on the expression of NF-kappa B. Tumor-derived exosomes can modulate tumor microenvironment by transferring miRNAs to immune cells. Epigallocatechin gallate (EGCG) has well known anti-tumor effects; however, no data are available on the influence of EGCG on communication with cancer cells and TAM. Methods: Murine breast cancer cell lines, 4T1, was used for in vivo and ex vivo studies. Exosome was extracted from EGCG-treated 4T1 cells, and the change of miRNAs was screened using microarray. Tumor cells or TAM isolated from murine tumor graft were incubated with exosomes derived from EGCG-treated and/or miR-16 inhibitor-transfected 4T1 cells. Chemokines for monocytes (CSF-1 and CCL-2), cytokines both with high (IL-6 and TGF-beta) and low (TNF-alpha) expression in M2 macrophages, and molecules in NF-kappa B pathway (IKK alpha and I kappa-B) were evaluated by RT-qPCR or western blot. Results: EGCG suppressed tumor growth in murine breast cancer model, which was associated with decreased TAM and M2 macrophage infiltration. Expression of chemokine for monocytes (CSF-1 and CCL-2) were low in tumor cells from EGCG-treated mice, and cytokines of TAM was skewed from M2-into M1-like phenotype by EGCG as evidenced by decreased IL-6 and TGF-beta and increased TNF-alpha. Ex vivo incubation of isolated tumor cells with EGCG inhibited the CSF-1 and CCL-2 expression. Ex vivo incubation of TAM with exosomes from EGCG-treated 4T1 cells led to IKK alpha suppression and concomitant I-kappa B accumulation; increase of IL-6 and TGF-beta; and, decrease of TNF-alpha. EGCG up-regulated miR-16 in 4T1 cells and in the exosomes. Treatment of tumor cells or TAM with exosomes derived from EGCG-treated and miR-16-knock-downed 4T1 cells restored the above effects on chemokines, cytokines, and NF-kappa B pathway elicited by EGCG-treated exosomes. Conclusions: Our data demonstrate that EGCG up-regulates miR-16 in tumor cells, which can be transferred to TAM via exosomes and inhibits TAM infiltration and M2 polarization. We suggest a novel mechanism by which EGCG exerts anti-tumor activity via regulation of TAM in tumor microenvironment.