Factors necessary to produce basoapical polarity in human glandular epithelium formed in conventional and high-throughput three-dimensional culture: example of the breast epithelium

Abstract

BackgroundBasoapical polarity in epithelia is critical for proper tissue function, and control of proliferation and survival. Cell culture models that recapitulate epithelial tissue architecture are invaluable to unravel developmental and disease mechanisms. Although factors important for the establishment of basal polarity have been identified, requirements for the formation of apical polarity in three-dimensional tissue structures have not been thoroughly investigated.ResultsWe demonstrate that the human mammary epithelial cell line-3522 S1, provides a resilient model for studying the formation of basoapical polarity in glandular structures. Testing three-dimensional culture systems that differ in composition and origin of substrata reveals that apical polarity is more sensitive to culture conditions than basal polarity. Using a new high-throughput culture method that produces basoapical polarity in glandular structures without a gel coat, we show that basal polarity-mediated signaling and collagen IV are both necessary for the development of apical polarity.ConclusionThese results provide new insights into the role of the basement membrane, and especially collagen IV, in the development of the apical pole, a critical element of the architecture of glandular epithelia. Also, the high-throughput culture method developed in this study should open new avenues for high-content screening of agents that act on mammary tissue homeostasis and thus, on architectural changes involved in cancer development.

ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity

“…Although our data indicate that integrin 1 is required for this basement membrane-mediated effect, other basement membrane receptors might also be involved. We also cannot exclude a role for the apical PAR complex, but our results are consistent with studies indicating that basolateral polarity is a prerequisite for apical polarity, and that basement membrane-mediated signals are required for the maturation of cell-cell adhesions (Li et al, 2003;Miner and Yurchenco, 2004;Plachot et al, 2009;Weir et al, 2006). Furthermore, our data help to explain why certain cell junction…”

“…A prominent role for cell-cell junctions in the establishment of apicobasal polarity has been identified (Vega-Salas et al, 1987a;Vega-Salas et al, 1987b;Wheelock and Johnson, 2003a;Wheelock and Johnson, 2003b), and basement membrane-mediated signaling is a prerequisite for apical domain establishment and cell-cell junction formation in many developmental and 3D cell-based model systems (Li et al, 2003;Miner and Yurchenco, 2004;Plachot et al, 2009;Weir et al, 2006). The basement membrane underlies epithelial cells at their basal surface; it provides structural support, forms a scaffold for cell adhesion (Sequeira et al, 2010;Yurchenco and Wadsworth, 2004), separates epithelial cells from the surrounding stroma and regulates cell behavior (Kalluri, 2003;.…”

Interconnected contribution of tissue morphogenesis and the nuclear protein NuMA to the DNA damage response

“…To induce the formation of growth-arrested S1 and RT4-2 multicellular spheroids lacking basoapical polarity, cells were seeded in type-I collagen (Cellagen Solution AC-5, ICN Biomedicals) (Chandramouly et al, 2007). For experiments comparing EHS with collagen-I conditions in 3D culture, cells were also embedded in EHS gel, as described previously (Plachot et al, 2009), instead of using the drip method. For flat monolayer (2D) cultures, cells were seeded on glass coverslips in 12-well plates, and incubated with H14 medium for 10 days.…”

“…S1 acinar differentiation and T4-2 tumor nodule formation were achieved after 10 days in 3D culture. Briefly, cells were seeded on a thin layer of EHS-derived hydrogel (Matrigel, BD Biosciences) in H14 that was supplemented with 5% Matrigel drip, as described previously (Plachot et al, 2009). Phenotypic reversion of T4-2 cells in 3D culture was induced by treatment with 100 nM EGFR inhibitor Tyrphostin AG1478 (Calbiochem) (Wang et al, 1998).…”

“…When non-neoplastic HMT-3522 S1 cells are cultured in 3D in the presence of EngelbrethHolm-Swarm (EHS)-derived ECM components, such as Matrigel (used in this study), they differentiate into polarized multicellular structures that are both morphologically and functionally similar to mammary acini. The basoapical polarity axis that is characteristic of normal epithelia is evidenced in S1 acini by the basal expression of a6-and b4-integrins and the formation of apical tight junctions (Petersen et al, 1992;Plachot et al, 2009) (supplementary material Fig. S1A).…”