Influence of microenvironment on mammary epithelial cell survival in primary culture

Mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix for m multicellular structures termed mammospheres, in which cells and matrix b ecome arranged around a central luminal space. In the presence of lactogeni c hormones, cells within mammospheres become polarized, form tight intercel lular junctions, and secrete milk proteins vectorially into the luminal spa ce. This study examined the mechanism of lumen formation. Histological exam ination of developing mammospheres showed that cavitation was associated sp atially and temporally with the appearance of fragmented nuclear material i n apoptotic bodies, and with the presence of cells positively labeled by te rminal deoxynucleotide transferase-mediated deoxyuridine nick end-labeling (TUNEL). Analysis of [P-32]-deoxynucleotide end-labeled genomic DNA by elec trophoresis and autoradiography showed DNA laddering indicative of apoptosi s. A transient increase in laddering coincided with both lumen formation an d the presence of TUNEL-positive cells. Lumen formation, DNA laddering, and detection of TUNEL-positive cells were all accelerated when matrix composi tion was altered. They were also impaired coordinately when caspase inhibit or was present during the first two days of culture. Therefore, lumen forma tion in mammosphere cultures is due to selective apoptosis of centrally loc ated cells. Mammosphere cavitation was accompanied by redistribution of mat rix constituents to the mammosphere periphery. Western blotting and Western ligand blotting of culture medium showed that lumen formation was also ass ociated with a transient increase in insulin-like growth factor binding pro tein-5 (IGFBP5), a factor implicated in mammary apoptosis in vivo. We propo se that epithelial cell survival during mammosphere development is induced selectively through stabilization by basement membrane constituents, which may act directly on the epithelial cell or confer protection against autocr ine apoptotic factors. (C) 1999 Wiley-Liss, Inc.