Unraveling the microenvironmental influences on the normal mammary gland and breast cancer

Abstract

The normal mammary gland and invasive breast tumors are both complex 'organs' composed of multiple cell types as well as extracellular matrix in three-dimensional (3D) space. Conventionally, both normal and malignant breast cells are studied in vitro as two-dimensional monolayers of epithelial cells, which results in the loss of structure and tissue function. Many laboratories are now investigating regulation of signaling function in the normal mammary gland using 3D cultures. However, it is also important to assay malignant breast cells ex vivo in a physiologically relevant environment to more closely mimic tumor architecture, signal transduction regulation and tumor behavior in vivo. Here we present the potential of these 3D models for drug testing, target validation and guidance of patient selection for clinical trials. We also argue that in order to get full insight into the biology of the normal and malignant breast, and to create in vivo-like models for therapeutic approaches in humans, we need to continue to create more complex heterotypic models to approach the full context the cells encounter in the human body.

Fig. 1

The normal and malignant breast. (A) Hematoxylin and eosin staining of tissue sections from a normal adult breast and an invasive ductal carcinoma. The normal mammary gland is a highly organized structure. The acini have a central lumen and are lined by an inner layer of luminal epithelial and an outer layer of myoepithelial cells. The bi-layer of epithelial cells is separated by a BM from the surrounding stroma, which is comprised of stromal ECM and stromal cells such as fibroblasts and adipocytes. Breast carcinomas have lost organized tissue architecture. Cancer-associated fibroblasts are the major cell type of the tumor stroma. (B) Cells from the normal and malignant mammary gland form indistinctive monolayers when plated on plastic substratum. (left) In 3D lrECM, normal mammary epithelial cells form spherical structures with a hollow lumen. (right) Breast cancer cells form disorganized tumor-like structures in 3D lrECM, which can be reverted to near-normal morphology by normalizing aberrant signaling pathways.

Fig. 2

Morphologies of breast cancer cell lines cultured in 2D and 3D. (top) Images of 4 representative breast cancer cell lines cultured as 2D monolayer, (middle) and in 3D lrECM grouped by 3D morphological classification: Round, Mass, Grape-like and Stellate. (bottom) 3D cultures were stained for F-actin and nuclei were counterstained with DAPI. Scale bars: top panel, 100 μm; middle panel, 50 μm; bottom panel, 20 μm. Adapted from [77].

Fig. 3

Myoepithelial cells confer correct polarity to luminal epithelial cells. (a, b, c) In lrECM, luminal cells form acini with correct polarity, (a′, b′, c′) whereas in collagen I (coll I), they form inside-out acini with reversed polarity. (a″, b″, c″) By addition of human myoepithelial cells (MEP) the reversed polarity of luminal cells in collagen I is corrected. (a, a′, a″) Apical marker sialomucin (red) and basal marker epithelial specific antigen (green). (b, b′, b″) Sialomucin (red) and occludin (green), which is expressed at apical cell-cell contacts. (c, c′, c″) nuclear stain (red) and basal marker β4-integrin (green). Adapted from [109] with permission of the Company of Biologists.