ww.nature.com/scientificreports/ ting to"Bcta-Gluc ances Sum of relative abundance k0195) NAF from HC(n=6) NAF from BC(n=6) 0 27 ia:o RB41: 231 teroidia;o bacteroidales: 3 unclassified: unclassified 22 P_Actinobacteria; c_Thermoleophi termless un 13 0 _Bacteroidaceae: g Bacteroides;scaccae Table 1. OTUs contributing to the KEGG ortholog Beta-Glucuronidase(K01195) List of the eight OTUs(classified to Greengenes v13_8)whose PICRUSt prediction corresponds to the Kegg ortholog Beta-Glucuronidase(k01195) The"sum of relative abundances "indicates how much of the"Total Beta Glucuronidase" is predicted by that OTU Method Study population. All experiments involving the use of human tissue samples were performed in accord ance with the Common Rule(45 CFR 46), ICH E6 GCP guidance as well as the Western IRBs requirements for consenting subjects. Written informed consent was obtained from all human subjects. All experimental prot were approved by the Western Institutional Review Board (protocol number 20111656)and specimens rec in the Lee laboratory from Dr Susan Love Research Foundation were de-identified and accepted under xemption approved by John Wayne Cancer Institute Regulatory affair 48 women, 23 healthy control women(HC)and 25 with a history of breast cancer(BC), 18 years or older with at least one intact nipple, provided informed consent and were recruited in the Love Army of Women under a protocol approved by the Western Institutional Review Board. All of the breast cancers were ductal carcinomas Subjects were excluded if they had been diagnosed with metastatic breast cancer; taken antibiotic therapy les than six months from the date of consent; taken oral contraceptives, hormone replacement therapy, any form of estrogen, any selective estrogen receptor modulators, or any aromatase inhibitors within 12 months from the date of consent; were currently lactating or had lactated within 12 months from the date of consent; had any known abnormal levels of sex hormones or prolactin; had received chemotherapy or radiation less than 12 months from the date of consent; had any subareolar or other surgery(papilloma resections, biopsies, or fine needle aspira tions)within two centimeters of the nipple; had any active infections or inflammation in the breast; or were Sample collection. Prior to NAF collection, subjects warmed their breasts with a heating pad, placed outside the hospital gown, for approximately 20 minutes and then massaged their breasts for approximately five minutes The pad does not come into contact with the subjects skin. Skin sampling was performed according to the meth ods of Grice et al., as described in their paper and after communication with the authors. First, we collected a swab of the nipple skin by rubbing a sterile cotton swab(Thermo Fisher Scientific, Lexena, KS)over the surface of the nipple and then the areola once in an expanding circular motion. Next, the nipple was de-keratinized using a mild abrasive gel(Nuprep, D.O. Weaver Co, Aurora, CO)followed by the application of Betadine solution(Purdue Products, Wilson, NC)to sterilize the skin surface. After the preparation with Betadine, a sterile cotton swab was used to collect a post-Betadine nipple skin sample. The entire procedure was performed with open surgery level sterility with the surgeon wearing a scrub suit with cap, gown, shoe covers, mask, and gloves. To elicit NAF, a suc- tion cup fitted with a 20 ml syringe was used to create negative pressure. NAF was collected with a sterile cotton swab. For HC, collection of NAF was attempted on both breasts, but for BC, only the contralateral breast was sam- pled since the ductal system for the breast that had previously been treated for breast cancer would be interrupted. Sampling was performed by the same clinical research team at one location and all the samples in the analysis were collected by one physician. The protocol for NAF collection has been included in Supplemental Method. Preparation of samples for 16s rRNA sequencing. Cotton swab samples of the nipple skin and NAF were immediately placed in sterile RNase/DNase free Eppendorf tubes and kept at-80C until genomic DNA(gDNA)extraction gDNA extraction was performed with a QIAamp DNA Mini Kit(Qiagen)accord ing to the manufacturer's instruction Samples were extracted in batches, with random selection to avoid any batch effects. We used empty Eppendorf tube controls running along all preparations to evaluate poten- tial contamination. Isolated gDNA was submitted to Second Genome Inc for 16S-V4 rRNA gene ampli con sequencing DNA from each sample was amplified using Caporaso primers tailed with sequences to SCIENTIFIC REPORTS 6: 28061 DO1: 10.1038/srep28061www.nature.com/scientificreports/ Scientific Reports | 6:28061 | DOI: 10.1038/srep28061 7 Methods Study population. All experiments involving the use of human tissue samples were performed in accord￾ance with the Common Rule (45 CFR 46), ICH E6 GCP guidance as well as the Western IRB’s requirements for consenting subjects. Written informed consent was obtained from all human subjects. All experimental protocols were approved by the Western Institutional Review Board (protocol number 20111656) and specimens received in the Lee laboratory from Dr. Susan Love Research Foundation were de-identified and accepted under an IRB exemption approved by John Wayne Cancer Institute Regulatory affairs. 48 women, 23 healthy control women (HC) and 25 with a history of breast cancer (BC), 18 years or older with at least one intact nipple, provided informed consent and were recruited in the Love Army of Women under a protocol approved by the Western Institutional Review Board. All of the breast cancers were ductal carcinomas. Subjects were excluded if they had been diagnosed with metastatic breast cancer; taken antibiotic therapy less than six months from the date of consent; taken oral contraceptives, hormone replacement therapy, any form of estrogen, any selective estrogen receptor modulators, or any aromatase inhibitors within 12 months from the date of consent; were currently lactating or had lactated within 12 months from the date of consent; had any known abnormal levels of sex hormones or prolactin; had received chemotherapy or radiation less than 12 months from the date of consent; had any subareolar or other surgery (papilloma resections, biopsies, or fine needle aspira￾tions) within two centimeters of the nipple; had any active infections or inflammation in the breast; or were unwilling to sign an informed consent. Sample collection. Prior to NAF collection, subjects warmed their breasts with a heating pad, placed outside the hospital gown, for approximately 20 minutes and then massaged their breasts for approximately five minutes. The pad does not come into contact with the subject’s skin. Skin sampling was performed according to the meth￾ods of Grice et al., as described in their paper and after communication with the authors. First, we collected a swab of the nipple skin by rubbing a sterile cotton swab (Thermo Fisher Scientific, Lexena, KS) over the surface of the nipple and then the areola once in an expanding circular motion. Next, the nipple was de-keratinized using a mild abrasive gel (Nuprep, D.O. Weaver & Co., Aurora, CO) followed by the application of Betadine® solution (Purdue Products, Wilson, NC) to sterilize the skin surface. After the preparation with Betadine, a sterile cotton swab was used to collect a post-Betadine nipple skin sample. The entire procedure was performed with open surgery level sterility with the surgeon wearing a scrub suit with cap, gown, shoe covers, mask, and gloves. To elicit NAF, a suc￾tion cup fitted with a 20 ml syringe was used to create negative pressure. NAF was collected with a sterile cotton swab. For HC, collection of NAF was attempted on both breasts, but for BC, only the contralateral breast was sam￾pled since the ductal system for the breast that had previously been treated for breast cancer would be interrupted. Sampling was performed by the same clinical research team at one location and all the samples in the analysis were collected by one physician. The protocol for NAF collection has been included in Supplemental Method. Preparation of samples for 16S rRNA sequencing. Cotton swab samples of the nipple skin and NAF were immediately placed in sterile RNase/DNase free Eppendorf tubes and kept at −80 °C until genomic DNA (gDNA) extraction. gDNA extraction was performed with a QIAamp DNA Mini Kit (Qiagen) accord￾ing to the manufacturer’s instruction. Samples were extracted in batches, with random selection to avoid any batch effects. We used empty Eppendorf tube controls running along all preparations to evaluate poten￾tial contamination. Isolated gDNA was submitted to Second Genome Inc. for 16S-V4 rRNA gene ampli￾con sequencing. DNA from each sample was amplified using Caporaso primers tailed with sequences to OTUs contributing to “Beta-Glucuronidase” (k01195) Sum of relative abundances NAF from HC (n=6) Sum of relative abundances NAF from BC (n=6) p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Roseburia; unclassified 0 27 p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Rikenellaceae; unclassified; unclassified 0 265 p_Acidobacteria; c_Chloracidobacteria; o_RB41; f_Ellin6075; unclassified; unclassified 74 231 p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g_Bacteroides; s_uniformis 0 1 p_Firmicutes; c_Bacilli; o_Bacillales; f_ Paenibacillaceae; g_Paenibacillus; s_amylolyticus 0 3 p_FBP; unclassified; unclassified; unclassified; unclassified; unclassified 0 226 p_Actinobacteria; c_Thermoleophilia; o_ Solirubrobacterales; unclassified; unclassified; unclassified 13 0 p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g_Bacteroides; s_caccae 13 0 Total Predicted Beta-Glucuronidase 99 753 Table 1. OTUs contributing to the KEGG ortholog Beta-Glucuronidase (K01195). List of the eight OTUs (classified to Greengenes v13_8) whose PICRUSt prediction corresponds to the KEGG ortholog Beta-Glucuronidase (k01195). The “sum of relative abundances” indicates how much of the “Total Beta￾Glucuronidase” is predicted by that OTU