Abstract
Simple SummaryAbout 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.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.
Highlights
Of the women in the United States that develop breast cancer, 15–20% will have basallike or triple-negative breast cancer (TNBC) [1]
To determine whether tumor extracellular vesicles (EVs) secretion plays an active role in macrophage-induced tumor cell invasion, we knocked down Rab27a, a known regulator of the EV secretion machinery (Figure S3A) [22,26]
We observed significantly reduced tumor cell invasion when tumor EVeducated macrophages (TEMs) were educated using EVs from either shRab27a BM1 and shRab27a LMB cells compared to controls (Figure 1D)
Summary
Of the women in the United States that develop breast cancer, 15–20% will have basallike or triple-negative breast cancer (TNBC) [1]. Stromal CCL5 and tumor CCR3 (but not CCR1 or CCR5) expression together are associated with poor outcome in TNBC patients [16] Factors such as CCL5 that enable recruitment of TAMs to mammary tumors have been identified, the process by which recruited macrophages are educated or polarized by tumor cells is not well-understood. CCL5 expression within the tumor increases the secretion of tumor EVs and alters the ability of EVs to educate macrophages This results in TAMs that reflect the metastatic state of the tumor cells. The genes we identified in this study are highly correlated to CCL5, and cluster in a subset of TNBC patient tumors within The Cancer Genome Atlas (TCGA) cohort Taken together, these results indicate that tumor EVs play an integral role in education of non-resident macrophages to a pro-metastatic TAM phenotype in TNBCs, driving tumor metastasis
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