Cancer-associated fibroblasts (CAFs) are the major components of the tumor microenvironment. cells. Thus, we provided evidence for the first time of the role of CAF exosomes and their miRs in the induction of the stemness and EMT phenotype in different breast cancer cell lines. Indeed, CAFs strongly promote the development of an aggressive breast cancer cell phenotype. Keywords: exosomes, breast cancer, microenvironment, cancer-associated fibroblasts, microRNAs INTRODUCTION Breast cancer is the most common cancer in women, and is only second to lung cancer for cancer-related mortality [1]. Tumor epithelial cells coexist in carcinomas with different stromal cell types that together create the microenvironment of cancer cells. Cancer-associated fibroblasts (CAFs), the major components of tumor stroma, are active fibroblasts that, similarly to myofibroblasts, are highly heterogeneous, acquire contractile features, and express -smooth-muscle MLL3 actin (-SMA) [2]. Active fibroblasts play similar roles in wound healing and in cancer, which may be considered as a wound that Phenytoin (Lepitoin) does not heal [3]. CAFs represent 80% of the resident fibroblasts in breast tumors. CAFs release high levels of growth factors, cytokines, chemokines, and metalloproteases that may affect either other stroma cells or cancer cells. Accumulated evidence indicates that they play an important role in cancer initiation, angiogenesis, invasion, and metastasis of breast cancer [4C6]. Thus, CAFs represent an attractive target for cancer therapy. Exosomes are small (40C100 Phenytoin (Lepitoin) nm) vesicles that have emerged as important mediators of intercellular communication in Phenytoin (Lepitoin) cancer. They have been identified in most body fluids, including urine, amniotic fluid, serum, saliva, breast milk, cerebrospinal fluid, and nasal secretions [7]. Exosomes mediate local and systemic cell communication through the horizontal transfer of information, such as microRNAs, mRNAs, and proteins. Over the last decade, a number of studies has revealed that exosomes influence major tumor-related pathways, such as invasion, migration, epithelial-to- mesenchymal transition (EMT), metastasis, and therapy resistance [8C12]. MicroRNAs (miRs) are a class of non-coding 17C24 nucleotide-long RNAs that mediate post-transcriptional gene silencing. miRs are involved in many biological activities such as cell proliferation, cell differentiation, cell migration, disease initiation, and progression. Their deregulation plays an essential role in the development and progression of cancer: miRs are up- or down-regulated in malignant tissues compared to the normal counterpart, and so can be either oncogenes or tumor suppressors. Recently, microRNAs have been identified in exosomes, which can be taken up Phenytoin (Lepitoin) by neighboring or distant cells and subsequently promote oncogenic signaling in recipient cells upon delivery of the cargo [13C17]. Here, we analyze whether the release of CAF exosomes and their specific miR cargo could dictate an aggressive phenotype in breast cancer. Our results demonstrate that three miRs (miRs -21, -143, and -378e) are released from CAF exosomes. When loaded into breast cancer cells, they promote important tumorigenic features: stemness, EMT, and anchorage-independent cell growth. Thus, the release of CAF exosomes may be responsible for the delivery of miRs that promote oncogenic signaling in breast cancer cells. RESULTS Identification of oncogenic miRs in CAF exosomes Breast fibroblasts were isolated from human breast biopsies for primary culture. The isolated cultures Phenytoin (Lepitoin) were characterized by immunocytochemistry for CK22 (pan-keratin) and Western blot analysis for e-cadherin and -SMA (Supplementary Figure 1a, b). Exosomes were isolated from breast fibroblast-conditioned media with ExoQuick-TC and characterized by Western blot analysis for the exosomal markers CD63, CD81, Hsp70, and Alix (Supplementary Figure 1c). To identify oncogenic miRs in CAF exosomes, we conducted genome-wide expression profiling of miRs (nCounter miRNA assay, nanoString Technologies, OSU), comparing exosomal miRs derived from two breast CAF cultures (patients #3 and #4) and two normal fibroblast (NF) cultures (patients #1 and #2). We found that three miRs were significantly up-regulated in CAF exosomes respect to NF exosomes: miR-21-5p, miR-378e, and miR-143-3p (Table ?(Table1).1). RT-PCR was conducted to confirm the array data. Interestingly, we found that miR-143-3p was up-regulated in CAF cells as compared to NFs, but we did not observe the same for miR-21-5p or miR-378e (Supplementary Figure 2a, b, c). Furthermore, we analyzed expression levels of miRs -21, -143 and -378e in CAFs from.