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doi: 10.1242/10.1242/jcs.00164

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Retinoids induce lumen morphogenesis in mammary epithelial cells

Department of Morphology, University of Geneva Medical Center, CH-1211 Geneva 4, Switzerland

View larger version (84K): [in a new window]   Fig. 1. Retinoids substitute for serum in inducing lumen formation by mammary epithelial cells. (A) J3B1A cells suspended in a collagen gel at a concentration of 3x104 cells/ml and grown in serum-free, chemically defined medium for 8 days form small solid colonies devoid of a discernible lumen. (B) J3B1A cells grown for 8 days in defined medium supplemented with 0.1% DCS form cystic structures containing a central cavity. (C) Addition of 1 nM RA to the defined medium mimics the lumen-inducing activity of DCS, resulting in the development of small, irregularly shaped cystic structures. (D) At higher (1 µM) concentration, RA elicits the formation of large spheroidal cysts. (E,F) J3B1A cells were grown for 7 days in defined medium to allow the formation of compact multicellular colonies and subsequently incubated in the presence or absence of 1 µM RA for a further 3 days. Whereas control colonies remain solid (E), RA treatment induces lumen formation (F). (G,H) Effect of RA on preclustered cells. J3B1A cells were grown in suspension on agarose for 3 days to obtain discrete cell aggregates, which were subsequently embedded in collagen gels. After 48 hours of incubation in the absence of RA, the aggregates extend branching cords in the collagen matrix but do not form cystic structures (G). Addition of RA (100 nM) at the time of embedding reduces the extent of branching and induces cavitation of the aggregates (H). Bar, 100 µm.

View larger version (143K): [in a new window]   Fig. 5. When added together with factors that promote branching morphogenesis, RA induces the formation of hollow tubular structures. (A) Serum-free conditioned medium from confluent monolayer cultures of J3B1A cells induces the formation of solid branching cords by J3B1A cells grown in collagen gels (10 day culture). (B) When incubated with conditioned medium for 3 days to allow initial cord formation and subsequently treated with 1 µM RA for a further 7 days, J3B1A cells form branching tubes enclosing a widely patent lumen. (C) Addition of 10 µM LPA stimulates colony branching without inducing lumen formation. (D) Concomitant treatment with 10 µM LPA and 10 nM RA results in the formation of arborized tubular structures that recapitulate the 3D organization of mammary gland ducts. Bar, 100 µM.

View larger version (16K): [in a new window]   Fig. 2. Time course and dose-dependence of retinoic acid effect on lumen formation. J3B1A cells were suspended in collagen gels at 3x104 cells/ml and incubated with either 0.1% DMSO (control) or a wide range of RA concentrations. (A,B) Lumen formation was quantified after 3, 6 and 9 days as described in Materials and Methods. Results are expressed as a mean percentage of cysts (i.e. colonies containing a central cavity) in 25 randomly selected photographic fields (five fields from each of five separate experiments) per experimental condition. In A, values are plotted against time of treatment, whereas in B, the same values are plotted against RA concentration. P=0.01 for values of 100 pM RA at 3 days compared with the control; P<0.025 for values of 100 pM RA at 9 days compared with 3 days; P<0.01 for values of 1 nM and 10 nM RA at 9 days compared with 3 days; P<0.0025 for values of 10 µM RA at 6 days compared with 3 days and at 9 days compared with 6 days. (C) Lumen diameter was quantified on the micrographs taken at day 9 by measuring the minor axis of the central cavity in all cystic colonies. Data are expressed as mean±s.e.m. from five separate experiments. *P<0.01 compared with 100 pM RA; **P<0.0005 compared with 100 nM RA.

View larger version (90K): [in a new window]   Fig. 3. Structural organization of RA-induced cysts. (A) Semi-thin sections of a 12-day-old collagen gel culture incubated with solvent (DMSO) alone show that the colonies of J3B1A cells consist of compact cell aggregates. (B) Semi-thin sections of cultures treated with 10 nM RA show cystic structures enclosing a wide lumen. (C) Thin sections of the wall of a cyst. The cells have a clearly defined polarity, with a microvilli-bearing surface facing the lumen (lu) and a basal surface in contact with the collagen matrix (col). (D) Detail of an apically located junctional complex (arrows). (A,B) Bar, 50 µm. (C) Bar, 2 µm. (D) Bar, 0.5 µm.

View larger version (96K): [in a new window]   Fig. 4. Retinoic-acid-induced lumen morphogenesis in a collagen gel sandwich assay. (A,B) Phase contrast micrographs (asterisks indicate cell-free gel areas). (C,D) Semi-thin (1 µm thick) sections perpendicular to the culture plane. When sandwiched between two collagen layers (see Materials and Methods) for 48 hours, untreated J3B1A cells form flattened cell cords containing only a few small lumina (A,C). By contrast, in the presence of 1 µM RA (B,D), J3B1A cells form large lumina (arrows in B). (A,B) Bar, 200 µm. (C,D) Bar, 100 µm.

View larger version (16K): [in a new window]   Fig. 6. Induction of lumen formation by RAR-pan-reactive and RAR-selective synthetic retinoids. (A) J3B1A cells were suspended in collagen gels and incubated either with 0.1% DMSO (control) or with a wide range of concentrations of the RAR-selective agonist TTNPB. (B) J3B1A cells were suspended in collagen gels and incubated either with 0.1% DMSO (control) or with different concentrations of Am-580, a selective RAR ligand. Lumen formation was quantified after 6 days as described in Materials and Methods. Results are expressed as mean percentage of cysts±s.e.m. in 20 randomly selected photographic fields (five fields from each of four separate experiments) per experimental condition. In A, 1=P<0.05; 2=P<0.025; 3=P<0.0005 (compared with control values); in B, *=P<0.0005 (compared with control values).

View larger version (14K): [in a new window]   Fig. 7. An RAR-selective antagonist inhibits both RA- and DCS-induced lumen formation. (A) J3B1A cells suspended in collagen gels were either treated with RA alone (1 nM) or cotreated with RA and different concentrations of Ro 41-5253, a selective RAR antagonist. (B) J3B1A cells were suspended in collagen gels and incubated either with 0.1% DCS alone or with 0.1% DCS plus different concentrations of Ro 41-5252. Lumen formation was quantified after 6 days. *P<0.025 and **P<0.0005 compared with control values.

View larger version (46K): [in a new window]   Fig. 8. RA induces the production of MMP-9 and uPA. (A) Effect of RA on gelatinase activity. Gelatin zymography of conditioned medium from monolayer cultures of J3B1A cells incubated with 10,100 and 1000 nM RA. The larger band of gelatin lysis corresponds to the reported molecular weight of latent MMP-9, whereas the smaller lower molecular weight band corresponds to the activated MMP-9 species. The zymogram is representative of five distinct experiments. (B) RA induces a dose- and time-dependent increase in uPA activity, as detected by casein zymography of conditioned media. (C,D) RA induces a dose-dependent increase in MMP-9 mRNA but does not modulate MT1-MMP mRNA. Confluent J3B1A cell monolayers were incubated with or without various concentrations of RA for 24 or 48 hours, after which RNA samples (5-10 µg per lane) were prepared and hybridized with either MMP-9 or MT1-MMP cRNA probes, as described in Materials and Methods. A bovine P0 ribosomal phosphoprotein cRNA probe was used to assess uniformity of loading.

View larger version (15K): [in a new window]   Fig. 9. RA-induced lumen formation requires metalloproteinase activity. (A,B) Effect of the synthetic MMP inhibitor BB94 on RA-induced lumen formation. J3B1A cells suspended in collagen gels were treated with RA alone (1 µM), co-treated with RA (1 µM) and BB94 (30 nM to 3 µM) or co-treated with RA (1 µM) and the inactive isomer BB1268 (3 µM). In A, cyst formation was quantified after 6 days as described in Materials and Methods and results are expressed as mean percentage of cysts±s.e.m. in 20 randomly selected photographic fields (five fields from each of four separate experiments) per experimental condition. Cyst formation is inhibited in a dose-dependent manner by BB94 but is not affected by the inactive isomer BB1268 (*P<0.05 and **P<0.01 compared to cultures incubated with RA alone). In B, lumen diameter was quantified after 6 days by measuring the minor axis of the central cavity in all cystic colonies. Lumen diameter is reduced in a dose-dependent manner by BB94, but is not significantly decreased by BB1268 (*P<0.01 compared with cultures incubated with RA alone; **not significantly different from cultures incubated with RA alone).