A Robust Mammary Organoid System to Model Lactation and Involution-like Processes

Abstract

The mammary gland is a highly dynamic tissue that changes throughout reproductive life, including growth during puberty and repetitive cycles of pregnancy and involution. Mammary gland tumors represent the most common cancer diagnosed in women worldwide. Studying the regulatory mechanisms of mammary gland development is essential for understanding how dysregulation can lead to breast cancer initiation and progression. Three-dimensional (3D) mammary organoids offer many exciting possibilities for the study of tissue development and breast cancer. In the present protocol derived from Sumbal et al., we describe a straightforward 3D organoid system for the study of lactation and involution ex vivo. We use primary and passaged mouse mammary organoids stimulated with fibroblast growth factor 2 (FGF2) and prolactin to model the three cycles of mouse mammary gland lactation and involution processes. This 3D organoid model represents a valuable tool to study late postnatal mammary gland development and breast cancer, in particular postpartum-associated breast cancer. Graphic abstract: Mammary gland organoid isolation and culture procedures.

Keywords: 3D organoid; Ex vivo; Involution; Lactation; Mammary gland; Mouse.

Video 1.. Mammary gland harvesting.

This video was made at Pasteur Institute. according to guidelines from the regulations of Institut Pasteur Animal Care Committees (CETEA). on Animal Care and approved by the French legislation in compliance with European Communities Council Directives (A 75-15-01-3).

Figure 1.. Key steps of mammary gland collection for organoid isolation and 3D culture.

A,B. Images of mouse dissection to access the mammary gland. A. Needles 1–4 represent the points at which to pin the mouse. Needles 5–7 and 8–10 represent the points at which to pin the skin of the mouse. Letters A–F with the blue dotted lines indicate the cuts. B. Green dotted lines denote the mammary gland. The lymph node is denoted in red and must be removed. C. Mammary gland before (left panel) and after (middle panel) mincing with a scalpel. Mammary organoids after transfer to dissociation medium (right panel). D. Example of mammary organoid counting. Left panel: organoids are surrounded by dotted lines. Star represents nerves. Right panel: organoids after embedding in Matrigel^®. Arrow represents the edge of the Matrigel^® dome. Scale bar = 500 μm. E. Freshly isolated primary organoid. Left panel: image of a mammary organoid on day 1 post-isolation. Middle panel: Hematoxylin & eosin staining of an organoid on day 1 post-isolation. Right panel: immunofluorescence staining showing the distribution of myoepithelial (keratin 5+, green) and luminal cells (keratin 8+, red) in organoids on day 1 post-isolation. Hoechst, blue (nuclei). Scale bar = 100 μm.

Figure 2.. Modeling lactation and involution-like processes in primary mammary organoids.

A. Scheme depicting the experimental design. B. Morphology of primary mammary organoids during lactation and involution-like processes. Bright-field images of organoid morphology following morphogenic and lactogenic stimulation and on days 4 or 8 after hormonal withdrawal. Scale bar = 100 μm.

Figure 3.. Passage of involution-like organoids.

A. Scheme depicting the experimental design. PRL: Prolactin; BOM: basal organoid medium. B. Morphology of passaged organoids during the lactation and involution-like processes. Brightfield images of passage 1 (upper panel) and passage 2 (lower panel) organoids following morphogenic and lactogenic stimulation and on days 4 or 8 after hormonal withdrawal. Scale bar = 100 μm.