Primary mammary organoid model of lactation and involution

Abstract

Mammary gland development occurs mainly after birth and is composed of three successive stages: puberty, pregnancy and lactation, and involution. These developmental stages are associated with major tissue remodeling, including extensive changes in mammary epithelium as well as surrounding stroma. Three-dimensional (3D) mammary organoid culture has become an important tool in mammary gland biology and enabled invaluable discoveries on pubertal mammary branching morphogenesis and breast cancer. However, a suitable 3D organoid model recapitulating key aspects of lactation and involution has been missing. Here, we describe a robust and straightforward mouse mammary organoid system modeling lactation and involution-like process, which can be applied to study mechanisms of physiological mammary gland lactation and involution as well as pregnancy-associated breast cancer.

Establishment and characterization of mammary organoids from non-traditional model organisms

“…Lastly, we stimulated 3-day old EqMaOs, cultured in regular organoid media supplemented with 5 nM of EGF, as described above, by replacing the regular organoid media with media supplemented with prolactin and devoid of EGF, and culturing for another 3 days. This approach is based on a previously established protocol (Sumbal et al 2020), and we then stained these prolactin-stimulated, 6-day old EqMaOs with a marker for β-lactoglobulin, which is an abundant whey protein in equine milk (Sharifi-Rad et al 2013, Wodas et al 2020). We observed clear β-lactoglobulin-positive signal in EqMaOs irrespective of culture condition or culture period, but an additional positive stain was seen in the lumens of EqMaOs grown in the prolactin media, suggestive of milk secretion (Fig.…”

“…In contrast, we observed many protrusions and disseminating cells on our MaOs from non-traditional model organisms grown in Matrigel. Compared to Matrigel, collagen is known to induce "invasive" phenotypes in MaOs such as cell dissemination and protrusions (Ngyuen-Ngoc et al 2012, Sumbal et al 2020.…”

“…In contrast, we observed many protrusions and disseminating cells on our MaOs from non-traditional model organisms grown in Matrigel. Compared to Matrigel, collagen is known to induce “invasive” phenotypes in MaOs such as cell dissemination and protrusions (Ngyuen-Ngoc et al 2012, Sumbal et al 2020). Interestingly, when we tried to grow mouse MaOs using our protocol involving Matrigel, we did observe some protrusions, albeit not nearly as much as on the MaOs we established from the non-traditional model organisms (data not shown).…”

“…However, cell and tissue culture models from additional non-traditional species, both domesticated and wild, are lacking. Currently available models to study non-traditional model organisms are 3D suspension cultures called mammospheres (Rauner et al 2018, Sumbal et al 2020) and 2D adherent cultures established from these mammospheres, called mammosphere-derived epithelial cells (MDECs) (Spaas et al 2012, Bussche et al 2016, Ledet et al 2018, Ledet et al 2018, Ledet et al 2020). Mammospheres are a culture of free-floating heterogenous mammary epithelial cell colonies enriched for stem/progenitor cells, which are generated using ultra-low attachment plates (Dontu et al 2003).…”

“…While these mammospheres have cellular heterogeneity and may be easily genetically manipulated, they lack in vivo -like mammary gland tissue structures and do not recapitulate cell-to-cell contact and signaling accurately (Srivastava et al 2020). However, all these traits can be recapitulated in mammary organoids (MaOs), which are self-organizing tissues grown in a 3D matrix (Fatehullah et al 2018, Sumbal et al 2020, Srivastava et al 2020). These MaOs can be established from several sources including primary explants, such as single cells or tissue fragments, reconstituted aggregates, suspension cultures, cell lines such as normal mammary epithelium or mammary cancer cell lines, mammary stem cells, and lastly, embryonic stem cells or induced-pluripotent stem cells (Sumbal et al 2020, Fatehullah et al 2018, Srivastava et al 2020).…”