Progestin-regulated luminal cell and myoepithelial cell-specific responses in mammary organoid culture

Abstract

Normal mammary gland development requires the coordinated proliferation and morphogenesis of both mammary luminal epithelial cells (LECs) and myoepithelial cells (MECs). Cell proliferation in cultured mammary organoids containing both LECs and MECs is not increased by progestin (R5020) or 17beta-estradiol (E2) alone or R5020+E2 but is increased by E2-regulated, mammary stroma-derived Hepatocyte growth factor (HGF) and further increased by HGF+R5020. We investigated the effects of HGF and/or R5020 on morphology and LEC- and MEC-specific in vitro proliferation in organoids. HGF-induced tubulogenesis was initiated and carried out by LECs starting with cellular extensions, followed by the formation of chains and cords, and culminating in tubule formation. MECs did not appear to have an active role in this process. Whereas HGF by itself caused maximal proliferation of LECs, HGF+R5020 produced a synergistic and specific increase in MEC proliferation. Because only LECs expressed progesterone receptors (PRs), we investigated the role of receptor activator of nuclear factor-kappaB ligand (RANKL), a progestin-induced paracrine factor, in mediating increased MEC proliferation. Quantitative RT-PCR showed that RANKL mRNA was induced by R5020 or HGF+R5020 and RANKL protein colocalized with PRs in LECs. The increased proliferation of MECs in response to HGF+R5020 could be blocked by neutralizing antibody to RANKL and reproduced by treatment with HGF plus exogenous RANKL in place of R5020. Neither R5020, nor exogenously administered RANKL increased proliferation of LECs. These results led us to conclude that RANKL, induced by progestin in PR-positive cells, is secreted and interacts with HGF to specifically increase proliferation of PR-negative MECs.

Figure 1

Organoid, LEC, and MEC morphology after treatment with BM, R5020, HGF, or HGF+R5020. Organoids were treated with BM control or R5020 (20 nm) (A), HGF (50 ng/ml) (B and C), or HGF+R5020 (50 ng/ml + 20 nm) (C). After 24–72 h, organoids were subjected to in situ double-immunofluorescence antibody labeling with anti-SMA (red) and anti-K18 antibodies (green), and images were captured by laser-scanning confocal microscopy as described in Materials and Methods. Green arrows, LECs, red arrows, MECs; dotted circles, lumens. A, Comparison of morphologies after BM or R5020 treatment. B, Sequential events during HGF-induced tubule formation. The formation of LEC cytoplasmic extensions (green arrow) after 24 h was followed by chains of LECs after 48 h (48 h chain). Some LECs have progressed to form a bilayered cord of cells by 48 h (48 h cord). By 72 h tubule formation by LECs nears completion with the development of a lumen (72 h tubule). C, Comparison of organoid morphology after 72 h treatment with HGF or HGF+R5020. Note longer tubules in HGF-treated organoids and elongated appearance of MECs (red). In HGF+R5020-treated organoids, tubules are shorter and MECs (red) are rounded and concentrated at the organoid body. Scale bar, 25 μm.

Figure 2

Regulation of proliferation of LECs and MECs. Organoids were treated for 72 h with control BM, HGF (50 ng/ml), R5020 (20 nm), or HGF+R5020 (50 ng/ml + 20 nm), treated with BrdU (10 nm), fixed, and processed for immunohistochemistry (A) and quantitated for percentage of total LECs and total MECs proliferating by fluorescence (B) microscopy as described in Materials and Methods. A, Proliferating LECs are indicated by red staining nuclei (yellow arrows), and proliferating MECs are indicated by the presence of SMA (green cytoplasm) and red nuclei (white arrows); nonproliferating LECs and MECs are indicated by blue nuclei. B, The values represent the mean ± sem from five separate experiments. *, P < 0.05; the percent of BrdU-positive LECs and MECs in HGF-treated organoids was greater than LECs and MECS of BM-treated organoids; **, P < 0.05; the percent of BrdU-positive MECs in HGF+R5020-treated organoids was greater than MECs in HGF-treated organoids.

Figure 3

c-Met expression in organoids or intact mammary glands. Organoids were treated with control BM, HGF (50 ng/ml), R5020 (20 nm), or HGF+R5020 (50 ng/ml + 20 nm). Sections prepared from cultured organoids (A and B) or intact mammary glands (C) from 5- or 10-wk-old virgin, pregnant, or lactating mice were analyzed for c-Met immunofluorescence staining intensity as described in Materials and Methods. A, c-Met immunofluorescence staining is greater in MECs that express SMA (white arrowheads, red staining cytoplasm) than in LECs (no SMA). B, c-Met expression levels in organoids at 72 h after various treatments. C, c-Met levels in tissue sections from intact mammary glands at various stages of development. The values represent the mean ± sem from five separate culture experiments or from three animals per developmental stage; a minimum of 1000 LECs and 500 MECs per treatment or animal were analyzed. *, P < 0.05; staining intensity of c-Met MECs was significantly lower in ducts during pregnancy and lactation.

Figure 4

PRA expression in organoids. Organoids were treated for 24, 48, or 72 h with control BM, HGF (50 ng/ml), R5020 (20 nm), HGF+R5020 (50 ng/ml + 20 nm), or E (10 nm). Organoid sections were double labeled with anti-PRA (green arrow) and anti-SMA (red arrow) antibodies (A) or anti-PRA (green arrow) and anti-ERα antibodies (red arrow) (B) and analyzed for immunofluorescence staining as described in Materials and Methods. Yellow arrow, PRA+ERα coexpression in the same cells; nuclei were stained with 4′,6′-diamino-2-phenylindole (blue). Scale bar, 25 μm. C and D, The values represent the mean ± sem from five separate experiments; a minimum of 1000 LECs and 500 MECs per treatment or animal were analyzed. *, P < 0.05; staining intensity of PRA was significantly lower in R5020- and HGF+ R5020-treated organoids.

Figure 5

RANKL and RANK expression and the effect of RANKL inhibition on MEC proliferation. Organoids were treated for 24 or 72 h with control BM, HGF (50 ng/ml), R5020 (20 nm), HGF+R5020 (50 ng/ml + 20 nm). A, Real-time RT-PCR analysis of RANKL mRNA expression in mammary organoids after 24 h. B, After 72 h of culture, organoid sections were double labeled with anti-PRA (red arrow) and anti-RANKL (green arrow) antibodies or anti-RANK antibody (C) and analyzed for immunofluorescence staining as described in Materials and Methods. White arrows show punctuate RANK antibody staining consistent with the pattern of RANK membrane expression. Mammary stromal cells in tissue section from intact mammary gland serve as a negative control for RANK (C, inset). Scale bar, 25 μm. D, Organoids were treated with control BM, HGF (50 ng/ml), R5020 (20 nm), HGF+R5020 (50 ng/ml + 20 nm) with (red text) or without RANKL neutralizing antibody (10 μg/ml medium), with RANKL (200 ng/ml), or with HGF + RANKL (50 ng/ml + 200 ng/ml) (green text). After 72 h organoid sections were quantitated for percentage of total LECs and total MECs proliferating by fluorescence microscopy as described in Materials and Methods. *, P < 0.05; the percentage of MEC proliferation in HGF+R5020+antibody (Ab)-treated organoids was significantly lower than the HGF+R5020-treated organoids.