Tumor Biology and Immunology Transfer of miRNA in Macrophage-Derived Exosomes Induces Drug Resistance in Pancreatic Adenocarcinoma Yoav Binenbaum 1 , Eran Fridman 1 , Zvi Yaari 2 , Neta Milman 1 , Avi Schroeder 2 , Gil Ben David 1 , Tomer Shlomi 3 , and Ziv Gil 1,4 Abstract Pancreatic ductal adenocarcinoma (PDAC) is known for its resistance to gemcitabine, which acts to inhibit cell growth by termination of DNA replication. Tumor-associated macro- phages (TAM) were recently shown to contribute to gemcita- bine resistance; however, the exact mechanism of this process is still unclear. Using a genetic mouse model of PDAC and electron microscopy analysis, we show that TAM communi- cate with the tumor microenvironment via secretion of approximately 90 nm vesicles, which are selectively internal- ized by cancer cells. Transfection of artificial dsDNA (barcode fragment) to murine peritoneal macrophages and injection to mice bearing PDAC tumors revealed a 4-log higher concen- tration of the barcode fragment in primary tumors and in liver metastasis than in normal tissue. These macrophage-derived exosomes (MDE) significantly decreased the sensitivity of PDAC cells to gemcitabine, in vitro and in vivo. This effect was mediated by the transfer of miR-365 in MDE. miR-365 impaired activation of gemcitabine by upregulation of the triphospho-nucleotide pool in cancer cells and the induction of the enzyme cytidine deaminase; the latter inactivates gemcitabine. Adoptive transfer of miR-365 in TAM induced gemcitabine resistance in PDAC-bearing mice, whereas immune transfer of the miR-365 antagonist recovered the sensitivity to gemcitabine. Mice deficient of Rab27 a/b genes, which lack exosomal secretion, responded significantly better to gemcitabine than did wildtype. These results identify MDE as key regulators of gemcitabine resistance in PDAC and demonstrate that blocking miR-365 can potentiate gemcita- bine response. Significance: Harnessing macrophage-derived exosomes as conveyers of antagomiRs augments the effect of chemo- therapy against cancer, opening new therapeutic options against malignancies where resistance to nucleotide analogs remains an obstacle to overcome. Cancer Res; 78(18); 5287–99. Ó2018 AACR. Introduction Pancreatic ductal adenocarcinoma (PDAC) ranks fourth among cancer-related deaths. Despite decades of research, the cure rate of the disease remains disappointingly low (<5%; ref. 1). This dismal prognosis is due to late detection and to resistance of tumors to all known systemic therapies. Gemcitabine, the first-line drug for the treatment of PDAC, is a cytidine analog that acts to inhibit cell growth by arrest of DNA replication. Resistance to gemcitabine develops within weeks of initiation of therapy, as a result of intrinsic resistance and envi- ronmental factors (2). Gemcitabine is metabolized intracellularly by deoxycytidine kinase (dCK), to active phospho-nucleosides; the incorporation of these nucleosides into DNA and RNA leads to replication arrest. Among the mechanisms known to cause gem- citabine resistance are loss of membranal transporters, deficiency of dCK, competition with de novo CTP, and upregulation of cytidine deaminase (CDA), the enzyme that metabolizes gemci- tabine to its inactive form. Treatment with nab-paclitaxel was shown to reduce CDA expression and potentiate gemcitabine efficacy; this highlights the importance of CDA in mediating drug resistance (3). Macrophages are associated with poor prognosis in PDAC (4) and were shown to secrete soluble factors that induce gemci- tabine resistance of PDAC cells (5). We hypothesized that tumor- associated macrophages (TAM) secrete vesicles that transfer molecular signals to cancer cells, thus inducing drug resistance. Here, we demonstrate a mechanism by which resistance to chemotherapy is mediated through shuttling of miRNAs between TAM and cancer cells, via exosomes. Materials and Methods Animals All animal experiments were approved by The Institutional Animal Care and Use Committee at the Technion, approval# IL-086-07-2013 and IL-124-12-2012. Wild-type (WT) C57/bl 1 The Laboratory for Applied Cancer Research, Department of Otolaryngology Head and Neck Surgery, Clinical Research Institute at Rambam Healthcare Campus, Haifa, Israel. 2 Laboratory for Targeted Drug Delivery and Personalized Medicine Technologies, Technion, Israel Institute of Technology, Haifa, Israel. 3 Departments of Computer Science and Biology, Technion, Israel Institute of Technology, Haifa, Israel. 4 Technion Integrated Cancer Center, Rappaport Institute of Medicine and Research, Technion, Israel Institute of Technology, Haifa, Israel. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Ziv Gil, Rambam Medical Center, Haifa 3525408, Israel. Phone: 972-4-7772480; E-mail: G_Ziv@rambam.health.gov.il doi: 10.1158/0008-5472.CAN-18-0124 Ó2018 American Association for Cancer Research. 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