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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.

SummaryTriple-negative breast cancers (TNBC) are highly infiltrated by tumor-associated macrophages (TAMs) that promote tumor growth, survival, metastasis and therapeutic resistance. Although cytokines such as CCL5 have been implicated in TAM recruitment to TNBC tumors, the mechanism by which tumor cells educate TAMs is not understood. Here we show that tumor EVs are both necessary and sufficient for programming TAMs toward a pro-metastatic phenotype. The mechanism involves CCL5 regulation of tumor extracellular vesicles (EVs), which activate TLR2 and TLR3, leading to secretion of a common set of cytokines that further stimulate tumor cell invasion and metastasis as well as alter the tumor microenvironment. Cytokine expression is significantly correlated to CCL5 expression and up-regulated in TNBC patient tumors. These results demonstrate for the first time that tumor EVs are key mediators of TAM education, phenocopy the pro-metastatic and drug resistant state of the tumors to TAMs, and illustrate the potential clinical relevance of these findings to TNBC patients.HighlightsTumor extracellular vesicles (EVs) are required for pro-metastatic programming of tumor-associated macrophages (TAMs)Tumor CCL5 and macrophage TLR signaling mediate tumor EV programming of TAMs in TNBCsTumor EVs mediate drug resistance in TAMs and alter recruitment of regulatory T-cells.Cytokines expressed by EV-educated TAMs are enriched and correlate with CCL5 in human TNBC patients.eTOCChemokines such as CCL5 recruit tumor-associated macrophages (TAMs) that are required for metastasis, but TAM programming is not understood. Rabe et al. show that tumor extracellular vesicles (EVs) are required for programming TAMs via Toll-like Receptors (TLRs) to phenocopy the tumor, rewire the microenvironment, drive metastasis and promote immune cell evasion.

Oxidative phosphorylation is an attractive target for cancer therapy. Reprogramming metabolic pathways by promoting oxidative phosphorylation could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options like triple negative breast cancer (TNBC). Here we show that BACH1, a heme-binding transcription factor whose expression is enriched in patients with TNBC, inhibits oxidative phosphorylation through direct transcriptional regulation of electron transport chain (ETC) gene expression. Treatment of cells with hemin, which induces BACH1 degradation, mimics BACH1 depletion with shRNA. Pretreatment of TNBC tumors with BACH1 shRNA or hemin overcame resistance to metformin, an anti-diabetic drug, and abolished the growth of both cell line and patient-derived tumor xenografts. BACH1 gene expression inversely correlated with ETC gene expression in breast cancer patients as well as other tumor types, highlighting the clinical relevance. This study demonstrates that oxidative phosphorylation represents an Achilles heel that can be exploited through targeting BACH1 to sensitize breast cancer and potentially other tumor tissues to mitochondrial inhibitors. Citation Format: Jiyoung Lee, Ali Yesilkanal, Casey Frankenberger, Mohamad Elbaz, Daniel Rabe, Jielin Yan, Felicia Rustandy, Peter Hart, Christie Kang, Elizabeth Grossman, Jason Locasale, Daniel Nomura, Marcelo Bonini, Marsha Rosner. Effective combination therapy for breast cancer targeting BACH1 and mitochondrial metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5497.

Triple-negative breast cancers (TNBC) are highly infiltrated by tumor-associated macrophages (TAMs) that promote tumor growth, survival, metastasis and therapeutic resistance. Although cytokines such as CCL5 have been implicated in TAM recruitment to TNBC tumors, the mechanism by which tumor cells educate TAMs is not understood. Here we show that tumor extracellular vesicles (EVs) are both necessary and sufficient for programming TAMs toward a pro-metastatic phenotype. The mechanism involves CCL5 regulation of tumor EVs, which activate TLR2 and TLR3, leading to secretion of a common set of cytokines that further stimulate tumor cell invasion and metastasis as well as alter the tumor microenvironment. Cytokine expression is significantly correlated to CCL5 expression and up-regulated in TNBC patient tumors. These results demonstrate for the first time that tumor EVs are key mediators of TAM education, phenocopy the pro-metastatic and drug resistant state of the tumors to TAMs, and illustrate the potential clinical relevance of these findings to TNBC patients. Citation Format: Daniel C. Rabe, Jiyoung Lee, Felicia Rustandy, Marsha R. Rosner. Tumor extracellular vesicles are required for tumor-associated macrophage programming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1739.