3D Culture Assays of Murine Mammary Branching Morphogenesis and Epithelial Invasion

Abstract

Epithelia are fundamental tissues that line cavities, glands, and outer body surfaces. We use three-dimensional (3D) embedded culture of primary murine mammary epithelial ducts, called “organoids,” to recapitulate in days in culture epithelial programs that occur over weeks deep within the body. Modulating the composition of the extracellular matrix (ECM) allows us to model cell- and tissue-level behaviors observed in normal development, such as branching morphogenesis, and in cancer, such as invasion and dissemination. Here, we describe a collection of protocols for 3D culture of mammary organoids in different ECMs and for immunofluorescence staining of 3D culture samples and mammary gland tissue sections. We illustrate expected phenotypic outcomes of each assay and provide troubleshooting tips for commonly encountered technical problems.

IRS2 mutations linked to invasion in pleomorphic invasive lobular carcinoma

“…Both of these cell lines are dependent upon IRS2 for invasion and their lack of IRS2 expression permits analysis of the IRS2 mutations without a background of endogenous expression. Cells were embedded within a Matrigel/collagen I gel that mimics the tumor matrix microenvironment (20). Cells with limited invasion capacity or noninvasive cells grow as spherical colonies, and invasive cells form protrusions into the surrounding matrix.…”

Mammary epithelial tubes elongate through MAPK-dependent coordination of cell migration

“…We now seek to understand how these low-polarity cells collectively accomplish ductal elongation. To study RTK-induced branching morphogenesis in real time, we utilize 3D culture (Nguyen-Ngoc et al, 2015) and 3D time-lapse confocal microscopy (Ewald, 2013). Briefly, the mammary gland is surgically removed and pieces of epithelial ducts ('organoids') are isolated through a combination of mechanical disruption, enzymatic digestion and differential centrifugation (Nguyen-Ngoc et al, 2015).…”

“…To study RTK-induced branching morphogenesis in real time, we utilize 3D culture (Nguyen-Ngoc et al, 2015) and 3D time-lapse confocal microscopy (Ewald, 2013). Briefly, the mammary gland is surgically removed and pieces of epithelial ducts ('organoids') are isolated through a combination of mechanical disruption, enzymatic digestion and differential centrifugation (Nguyen-Ngoc et al, 2015). These epithelial organoids are then embedded in ECM proteins characteristic of the basement membrane and the stromal matrix (1:1 Matrigel:collagen I) and induced with FGF2, conditions that support in vivo-like branching morphogenesis (Nguyen-Ngoc and Ewald, 2013).…”

Mammary epithelial tubes elongate through MAPK-dependent coordination of cell migration

“…The organotypic culture methods used here are described in detail by Nguyen-Ngoc et al (2015). In brief, mammary glands were isolated from 8-to 12-week-old female mice and minced with a scalpel.…”

“…Organoids were suspended in a 1:1 mixture of growth factor-reduced Matrigel (BD Biosciences) and rat tail collagen I (Corning) and plated as 400 µl drops containing an average of one organoid/µl. The organoid suspensions were plated on 24-well glassbottom plates (Greiner Bio One) sitting on a 37°C hotplate to promote gel polymerization, as described by Nguyen-Ngoc et al (2015). Plates were incubated for 20 min at 37°C to ensure gelation before addition of culture medium.…”