Abstract PD7-03: A living biobank of normal mammary organoids derived from patients at low and increased risk of developing breast cancer
Authors: Rosenbluth Jennifer M , Li Carman Man-Chung , Gray Kenneth G , Gao Walter , Schackmann Ron CJ , Zoeller Jason J , Selfors Laura M , Moore Kaitlin , Dillon Deborah , Garber Judy , Brugge Joan S 
Published: February 2021

Abstract
Background: Mutations in BRCA1 and BRCA2 are associated with a high risk of developing breast cancer, but the earliest molecular changes that lead to the transformation of mammary cells in the setting of BRCA1/2 heterozygosity remain unknown. We previously demonstrated that breast organoid cultures derived from histologically normal tissues can preserve all of the major mammary epithelial lineages for further study in vitro1. In addition, organoid culture medium is fully defined, enabling modulation of the medium to enhance the growth of distinct mammary subpopulations. Here, we undertook the development of a large biobank of breast organoids derived from patients with and without increased breast cancer risk, including patients with inherited mutations in BRCA1 and BRCA2. Methods: Breast samples with normal histology (taken from reduction mammoplasties or prophylactic mastectomies), were digested for ~2 hours using collagenase, embedded in basement membrane extract, and grown in a fully-defined organoid medium1. CyTOF profiling was performed using our previously published mammary-specific heavy metal-tagged antibody panel1. Lentiviral transduction was performed to knock-down BRCA1 and/or overexpress mutant p53. Murine engraftment of normal breast organoids was performed by intra-ductal injection of ~1,000 organoids into NCG mice using a modification of protocols for breast cancer cell lines2, with removal of mammary glands after 3 months for detailed immunohistochemistry analysis. Results: A living biobank of &gt; 100 normal breast organoids derived from patients with and without inherited mutations in breast cancer predisposition genes, including BRCA1 and BRCA2, was established. Cultures were generated with high efficiency (&gt;95%) and could be serially passaged with the longest cultures &gt; 16 months. CyTOF profiling of organoids revealed the maintenance of multiple epithelial cell subtypes, with at least one subtype of luminal cells enriched in tissues and organoids from BRCA1/2 mutation carriers. Protein and RNA expression patterns of breast organoids were found to correlate with patient tissues analyzed using a combination of single-cell analyses (CyTOF, single-cell RNA sequencing, and immunohistochemistry). The impact of factors present in the organoid medium on distinct mammary epithelial cell subtypes was assessed by CyTOF, enabling identification of conditions that promote expansion of cell populations that are enriched in tissues from BRCA1/2 mutation carriers. Furthermore, normal breast cell types could be engrafted into the murine mammary gland, and could be modified by lentiviral transduction for gene transfer, enabling future studies of the tumorigenic potential of distinct normal and premalignant epithelial cell subtypes. Conclusion: We have shown that organoid cultures can be used to propagate normal breast tissues with high efficiency, preserve normal as well as potential premalignant breast epithelial cell types, and model methods to inhibit precancerous cells in vitro. Thus, organoids are a useful complement to murine and other models of breast cancer development, and can ultimately be used to identify potential cancer interception strategies for patients at high risk of developing breast cancer.
References:1.Rosenbluth JM, Schackmann RCJ, Gray GK, et al: Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages. Nat Commun 11:1711, 20202.Zoeller JJ, Bronson RT, Selfors LM, et al: Niche-localized tumor cells are protected from HER2-targeted therapy via upregulation of an anti-apoptotic program in vivo. NPJ Breast Cancer 3:18, 2017
Citation Format: Jennifer M Rosenbluth, Carman Man-Chung Li, Kenneth G Gray, Walter Gao, Ron CJ Schackmann, Jason J Zoeller, Laura M Selfors, Kaitlin Moore, Deborah Dillon, Judy Garber, Joan S Brugge. A living biobank of normal mammary organoids derived from patients at low and increased risk of developing breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-03.