Tumor Extracellular Vesicles Regulate Macrophage-Driven Metastasis through CCL5

SIMPLE SUMMARY: About 10–20 percent of patients with breast cancer are diagnosed with triple-negative breast cancer (TNBC). These tumors are named for their lack of expression of estrogen receptor (ER), progesterone receptor (PR), and amplification of human epidermal growth factor receptor 2 (HER2). These genes are targeted by therapies in other breast cancer patients. However, most TNBC patients recur within 5 years. Understanding how and why these tumors metastasize will help clinicians better treat these underserved cancer patients. TNBC tumors are highly infiltrated by tumor-associated macrophages (TAMs) that promote tumorigenesis and metastasis. Our study elucidates how the tumor co-opts macrophages recruited to the tumor through extracellular vesicles (EVs), further increasing tumor metastasis. Expression of tumor CCL5 regulates EV secretion and cargo that further alters macrophage phenotype to drive tumor metastasis. Together, our data suggest a more extensive role of EVs in the biology of tumor metastasis as well as their potential use as biomarkers. ABSTRACT: Purpose: To understand how tumor cells alter macrophage biology once they are recruited to triple-negative breast cancer (TNBC) tumors by CCL5. Method: Mouse bone marrow derived macrophage (BMDMs) were isolated and treated with recombinant CCL5 protein alone, with tumor cell conditioned media, or with tumor extracellular vesicles (EVs). Media from these tumor EV-educated macrophages (TEMs) was then used to determine how these macrophages affect TNBC invasion. To understand the mechanism, we assayed the cytokine secretion from these macrophages to determine how they impact tumor cell invasion. Tumor CCL5 expression was varied in tumors to determine its role in regulating macrophage biology through EVs. Results: Tumor EVs are a necessary component for programming naïve macrophages toward a pro-metastatic phenotype. CCL5 expression in the tumor cells regulates both EV biogenesis/secretion/cargo and macrophage EV-education toward a pro-metastatic phenotype. Analysis of the tumor EV-educated macrophages (TEMs) showed secretion of a variety of factors including CXCL1, CTLA-4, IFNG, OPN, HGF, TGFB, and CCL19 capable of remodeling the surrounding tumor stroma and immune infiltrate. Injection of tumor cells with macrophages educated by metastatic tumor cell EVs into mice increased tumor metastasis to the lung. Conclusion: These results demonstrate that tumor-derived EVs are key mediators of macrophage education and likely play a more complex role in modulating tumor therapeutic response by regulating the tumor immune infiltrate.