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Triple-negative breast cancer (TNBC) patients have the highest risk of recurrence and metastasis, cannot be treated with targeted therapies, and many do not respond to chemotherapy, making them a clinically underserved group. While physiological inhibitors of metastasis (metastasis suppressors) play key roles in regulating tumor growth, invasion and metastasis, their role in regulating the tumor microenvironment and immune system is unknown. ,Using species-specific RNAseq we determined that expression of the metastasis suppressor Raf kinase inhibitor protein (RKIP) in tumors markedly reduces the number and metastatic potential of infiltrating TAMs. While TAMs isolated from TNBC xenografts drive in vitro invasion, RKIP+ derived TAMs did not drive invasion and had decreased secretion of pro-metastatic factors including SLPI, OPN, MMP-12, Galectin-3, VEGF-A, VEGF-D, TNFR2, and PGRN. We determined that RKIP regulates TAM recruitment by blocking HMGA2, which activates CCL5 expression. CCL5 rescued pro-metastatic TAM infiltration as well as tumor intravasation. We additionally showed that factors decreased in RKIP-derived TAMs were restored in CCL5-derived TAMs. ,To determine the mechanism of macrophage programming by TNBC cells, we treated bone marrow derived macrophages (BMDMs) with conditioned media (CM) from TNBC cells, CM depleted of exosomes, or tumor exosomes alone. Only exosomes alone showed increased levels of pro-invasive genes. CCL5 expression in tumor cells increased the pro-invasive phenotype of macrophages via tumor exosomes. Additionally, expression of RKIP blocked pro-invasive programming of macrophages through exosomes. Finally, we found that resistance to the CCL5 inhibitor Maraviroc could be transmitted to macrophages through exosomes.,For in vivo validation, mice were injected with exosome-depleted tumor cells using shRab27a. While shRab27a in tumors did not change TAM number, it decreased the ability of TAMs to drive TNBC invasion and reduces expression of pro-invasive cytokines including CCL3, CCL7, CCL19, CXCL1, CXCL3, Flt-1, OPN, SLPI, TGF- β3, TNFSF14, and Thrombospondin. Co-injection of exosome programmed TEMs, which also express these pro-invasive cytokines, resulted in both an increase in number and size of lung metastases. Taken together, these results indicate that exosomes are essential for the pro-metastatic programming of TAMs within the microenvironment as well as transferring therapeutic resistance.


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