Abstract A05: Deep immunofluorescence imaging of solvent-cleared mouse mammary glands

Abstract We seek to trace the progression of epithelial lesions originating from Lgr5+ cells in mouse mammary glands using the 3DISCO protocol for solvent-cleared tissue. Lineage tracing is increasingly being used to probe different cell types that exist within the mammary gland. Lgr5 is a stem cell marker and it is expressed in several different tissues including colon, stomach, and breast. It has been reported that Lgr5+ cells are necessary for mammary gland organogenesis. However, the cells that initiate tumor formation have remained intangible. Lgr5+ cells reside within the basal cell population of mammary ducts. Lgr5 is a downstream target of Wnt and therefore Lgr5+ cells may have a role in breast cancer progression. Since heterogeneity of tumors is shaped by several factors, including the cell of origin, specific oncogenic events, and interactions between tumor cells and cells of the microenvironment, it is important to obtain 3-dimensional information of tumor development. We used the 3DISCO protocol for solvent-clearing mammary fat pads for deep immunofluorescence staining of transgenic mice harboring a EGFP reporter for Lgr5+ cells (mouse: Lgr5-EGFP-Ires-CreERT2). We performed deep immunofluorescence staining with DAPI and an antibody against the basal cell marker keratin 5 (K5). Mammary fat pads were successfully solvent-cleared and prepared for deep immunofluorescence staining. Epithelial mammary ducts were easily recognized within the fat tissue using DAPI nuclear staining and an antibody against K5. Three-dimensional confocal microscope imaging protocols for whole mammary glands, including epithelial ducts, were established. We optimized the method using tile scans covering the entire fat pad, and multiple z-stacks of selected areas of Lgr5+ cells expressing EGFP were also detected and colocalized with K5+ cells. The whole-organ tissue clearing, assessed in the mouse mammary gland, is suitable to obtain 3-dimensional information of various cell populations using specific molecular markers. This technique enables high-resolution, 3D imaging and phenotyping of tissue and might significantly enhance our understanding of mammary gland tumorigenesis. We will use the established protocol to localize the appearance of early lesions in mouse mammary glands and determine the role of Lgr5+ cells in mammary tumor development. Citation Format: Anna Polec, Jens Henrik Norum, Andreas Brech, Therese Sorlie. Deep immunofluorescence imaging of solvent-cleared mouse mammary glands [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A05.