www.nature.com/scientificreports SC丨ENTF| C REPORTS OPEN Characterization of the microbiome of nipple aspirate fluid of breast cancer survivors Received: 23 March 2016: Alfred A Chan'', Mina Bashir2, 3,. Magali N Rivas,', Karen Duvall+. Peter A Sieling, Accepted: 31 May 2016: Thomas R. Pieber, Parag A Vaishampayan, Susan M. Love&DelphineJ Lee Published: 21 June 2016 The microbiome impacts human health and disease Until recently, human breast tissue and milk were presumed to be sterile. Here, we investigated the presence of microbes in the nipple aspirate fluid(NAF) with a history of breast cancer(BC) and healthy control women(HO) using 165 rRNA gene amplicon nd their potential association with breast cancer. We compared the NAF microbiome between wom sequencing. The NAF microbiome from BC and HC showed significant differences in community composition. Two Operational Taxonomic Units(OTUs)showed differences in relative abundances between NAF collected from BC and HC. In NAF collected from BC, there was relatively higher incidence of the genus Alistipes. By contrast, an unclassified genus from the Sphingomonadaceae family was relatively more abundant in NAF from HC. These findings reflect the ductal source DNa since there were no differences between areolar skin samples collected from BC and HC. Furthermore the microbes associated with BC share an enzymatic activity, Beta-Glucuronidase, which may promote breast cancer This is the first report of bacterial DNA in human breast ductal fluid and the differences between NAF from HC and BC. Further investigation of the ductal microbiome and its potential role in breast cancer The human microbiome is the term applied to the universe of microbes that inhabit our skin and mucosal surfaces. Epidemiologic studies suggest that the human microflora contributes to 16% or more of worldwide malignancies-3. Increased cancer risk is associated with the presence of chronic, persistent, and dysregulated inflammation.. Many of the studies on microbes in relation to cancer have focused on the gut microflora. For instance, infection with Helicobacter pylori, a gram-negative bacterium that selectively colonizes the gastric pithelium and induces gastric inflammation, is correlated with a higher incidence of gastric cancer. Further, merging evidence suggests that infection with Fusobacterium nucleatum, a common member of the oropharyn geal flora and a pathogenic agent involved in gingival and periodontal disease, is associated with the development of human colorectal cancer.. Bacteria present near the tumor site are part of the tumor microenvironment On one hand, the microbiome might promote malignancy by inducing chronic inflammation, by altering the balance of host cell proliferation and death, and by triggering uncontrolled innate and adaptive immune responses. On the other hand, certain microbes might play a preventative role in breast carcinogenesis by affecting levels of strogen or by promoting antitumor immunity and immune surveillance The National Institutes of Health(NIH) Human Microbiome Project(HMP)has a reference collection of bac- terial genomes associated with multiple body sites from healthy human adults. Although an important number of anatomical sites were sampled for their microbiome, the human breast was omitted, presumably because most would claim that, at steady state, the breast tissue is sterile and devoid of any bacterial presence. However, the presence of six to eight ductal openings at the surface of the human nipple allows microbes from the environment, kin, and mouth( through sexual activity and breast feeding) to access the breast ductal system. In fact, microbes Dirks/ Dougherty Laboratory for Cancer Research, Department of Translational Immunology, John Wayne Cancer Institute at Providence Saint Johns Health Center, Santa Monica, CA, USA. Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, USA. Division of Endocrinology and Metabolism, Medical University of Graz, Graz, Austria. "Breast Center at the University of California Los Angeles (UCLA), Westwood, Los Angeles, CA, USA. Dr Susan Love Research Foundation, Encino, CA, USA. These authors contributed equally to this work. Correspondence and requests for materials should be addressed to D.J. L(email g) SCIENTIFIC REPORTS 6: 28061 DO1: 10.1038/srep28061Scientific Reports | 6:28061 | DOI: 10.1038/srep28061 1 www.nature.com/scientificreports Characterization of the microbiome of nipple aspirate fluid of breast cancer survivors AlfredA. Chan1,* , Mina Bashir2,3,* , Magali N. Rivas1,* , Karen Duvall4,5, PeterA. Sieling1, Thomas R. Pieber3, ParagA.Vaishampayan2, Susan M. Love5 & Delphine J. Lee1 The microbiome impacts human health and disease. Until recently, human breast tissue and milk were presumed to be sterile. Here, we investigated the presence of microbes in the nipple aspirate fluid (NAF) and their potential association with breast cancer. We compared the NAF microbiome between women with a history of breast cancer (BC) and healthy control women (HC) using 16S rRNA gene amplicon sequencing. The NAF microbiome from BC and HC showed significant differences in community composition. Two Operational Taxonomic Units (OTUs) showed differences in relative abundances between NAF collected from BC and HC. In NAF collected from BC, there was relatively higher incidence of the genus Alistipes. By contrast, an unclassified genus from the Sphingomonadaceae family was relatively more abundant in NAF from HC. These findings reflect the ductal source DNA since there were no differences between areolar skin samples collected from BC and HC. Furthermore, the microbes associated with BC share an enzymatic activity, Beta-Glucuronidase, which may promote breast cancer. This is the first report of bacterial DNA in human breast ductal fluid and the differences between NAF from HC and BC. Further investigation of the ductal microbiome and its potential role in breast cancer are warranted. The human microbiome is the term applied to the universe of microbes that inhabit our skin and mucosal surfaces. Epidemiologic studies suggest that the human microflora contributes to 16% or more of worldwide malignancies1–3. Increased cancer risk is associated with the presence of chronic, persistent, and dysregulated inflammation1,3,4. Many of the studies on microbes in relation to cancer have focused on the gut microflora. For instance, infection with Helicobacter pylori, a gram-negative bacterium that selectively colonizes the gastric epithelium and induces gastric inflammation, is correlated with a higher incidence of gastric cancer5 . Further, emerging evidence suggests that infection with Fusobacterium nucleatum, a common member of the oropharyn￾geal flora and a pathogenic agent involved in gingival and periodontal disease, is associated with the development of human colorectal cancer6,7. Bacteria present near the tumor site are part of the tumor microenvironment. On one hand, the microbiome might promote malignancy by inducing chronic inflammation, by altering the balance of host cell proliferation and death, and by triggering uncontrolled innate and adaptive immune responses8 . On the other hand, certain microbes might play a preventative role in breast carcinogenesis by affecting levels of estrogen or by promoting antitumor immunity and immune surveillance9 . The National Institutes of Health (NIH) Human Microbiome Project (HMP) has a reference collection of bac￾terial genomes associated with multiple body sites from healthy human adults. Although an important number of anatomical sites were sampled for their microbiome, the human breast was omitted10, presumably because most would claim that, at steady state, the breast tissue is sterile and devoid of any bacterial presence. However, the presence of six to eight ductal openings at the surface of the human nipple allows microbes from the environment, skin, and mouth (through sexual activity and breast feeding) to access the breast ductal system11. In fact, microbes 1 Dirks/Dougherty Laboratory for Cancer Research, Department of Translational Immunology, John Wayne Cancer Institute at Providence Saint John’s Health Center, Santa Monica, CA, USA. 2 Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, USA. 3 Division of Endocrinology and Metabolism, Medical University of Graz, Graz, Austria. 4 Breast Center at the University of California Los Angeles (UCLA), Westwood, Los Angeles, CA, USA. 5 Dr. Susan Love Research Foundation, Encino, CA, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to D.J.L. (email: LeeDJ@JWCI.org) received: 23 March 2016 accepted: 31 May 2016 Published: 21 June 2016 OPEN