Abstract
Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM) hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.
Highlights
The mammary gland is a useful model system for studying developmental processes such as branching morphogenesis and lineage commitment due to extensive post-natal development during puberty and successive cycles of remodeling during pregnancy, lactation and post-lactational regression [1]
In 2D co-cultures of 3T3-L1 and KIM-2, de novo synthesis of basement membrane proteins is regulated by adipogenic differentiation and the localization of cell type (Fig. S1A-F). 3T3-L1 and KIM-2 mammary epithelial cells organized into monotypic islands (Fig. S1A) that further supported the differentiation of KIM-2 cells with lactogenic hormones (Fig. S1B)
Laminin protein levels were raised under adipogenic conditions when epithelial and stromal cell lines were overlaid for 7 days, demonstrating that direct contact between the epithelial and stromal cells is optimal
Summary
The mammary gland is a useful model system for studying developmental processes such as branching morphogenesis and lineage commitment due to extensive post-natal development during puberty and successive cycles of remodeling during pregnancy, lactation and post-lactational regression (involution) [1]. Tertiary branching and formation of lobuolaveolar milk producing structures (acini) takes place in response to estrogen, progesterone and prolactin (Prl). Such processes are largely dependent on the concerted movement of cells [2], often in response to reciprocal signaling between the epithelium and underlying mesenchyme. Such features should be recapitulated in the development of enhanced in vitro 3D models that support ductal-alveolar morphogenesis. Model systems utilizing rBM could be considered more pertinent to end-stage developmental analyses highlighted by the tendency for mammary organoids to form cyst-like structures which fail to differentiate between ductal and alveolar mammary epithelium. ln this context it is noteworthy that most breast cancers arise in ducts [4]
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