Breast Tissue Sampling Protocol for Microbiome Analysis

Biopsies were consistently obtained from each breast for every study patient. During Surgery #1, and prior to the conclusion of mastectomy, ≥3 sq cm biopsies of the skin and underlying breast parenchyma were obtained (Fig. 2A) at least 3 cm away from a known tumor margin to avoid compromising pathologic assessment. After Surgery #1, one suction drain was aseptically collected in the plastic surgery outpatient clinic at the time of removal and only the portion within the breast tissue was retained for future analyses (Fig. 2B). All patients matriculated, uneventfully, to Surgery #2 where biopsies from the skin (≥3 sq cm), ADM (≥3 sq cm), TE (≥30 sq cm), capsule (≥3 sq cm), and subcutaneous tissues (≥ 3 sq cm) were obtained (Fig. 2C). Immediately upon removal, specimens intended for DNA extraction and 16S rRNA sequencing were stored in 200 proof ethanol (Fisher Scientific; BP2818500), while remaining samples were immediately placed in 4% phosphate buffered solution (PBS) for bacterial culturing and immunofluorescence staining as previously described (25 (link), 93 (link)) (also see Bacterial Culture, Bacterial Identification, Immunofluorescence Sample Processing, and Microbiome Sample Processing sections below).