Experimental strategy
In order to investigate the role of the Notch pathway in the development of the mammary gland, we examined the effect of agonists and antagonists of Notch signaling on cell-fate determination utilizing mammary stem/progenitor cells cultured in suspension, as mammospheres, and differentiated mammary cells, obtained by passaging these cells on a collagen substratum. The experimental strategy is outlined in Fig. 1. The activators and inhibitors of Notch signaling were applied at three different stages of in vitro development, which we have previously characterized [4]. First, the treatment was applied in suspension culture, which is enriched in stem/progenitor cells (self-renewal of stem cells and the proliferation of progenitor cells occur in these conditions) (Fig. 1a). Second, the treatment was applied in cultures on collagen substratum, under conditions that promote differentiation of mammosphere-derived cells (multipotent progenitors commit to a specific lineage) (Fig. 1b). Finally, the treatment was applied in cultures on a collagen substratum, using differentiated cells passaged on a collagen substratum (lineage-restricted progenitors undergo terminal differentiation) (Fig. 1c).
A synthetic peptide derived from the DSL (Delta-Serrate-LAG 2) domain conserved in all Notch ligands [9] and a previously described recombinant Delta 1 ligand fused to the immunoglobulin Fc fragment (used in combination with an anti-human Fc antibody, for ligand clustering) [20] were utilized as agonists of Notch signaling. A Notch4-specific antibody or a GSI that blocks the intramembranous cleavage of Notch, required for signaling [21], was used as the antagonist. The synthetic DSL peptide, the recombinant Delta fused to Fc and GSI are all well characterized with respect to their effect on Notch signaling [9, 20]. We utilized a reporter assay in MCF-7 cells, in which luciferase expression was driven by the Hes-1 promotor, a gene regulated by Notch signaling [22], to confirm the ability of activating or inhibiting ligands to affect Notch signaling.
As shown in Fig. 2a, DSL peptide upregulated Hes-1-driven luciferase expression. These results are consistent with previous characterization of these Notch signaling agonists in reporter assays and in experiments using other cell types [9]. Notch signaling was blocked using a Notch4-specific antibody, or a GSI, which abrogates the intramembranous cleavage of Notch, required for signaling [19, 21]. Both of these inhibitors blocked endogenous Hes-1-driven luciferase expression, as well as the increase in luciferase expression induced by the DSL ligand (Fig. 2a). The specificity of the blocking antibody was confirmed by successful competition with the antigenic peptide used to produce the antibody (data not shown). Furthermore, these effects were dose dependent (Fig. 2b).
Effects of Notch signaling on self-renewal of mammary stem cells
We examined the effects of these agonists and antagonists of Notch signaling on primary, secondary and tertiary mammosphere formation (Fig. 1a). This assay has previously been used for the in vitro study of neural stem cell self-renewal [19] and relies on the observation that neurosphere formation is initiated clonally by neural stem cells [5].
Utilizing retroviral marking studies, we also have demonstrated that mammospheres are clonally derived and do not result from cellular aggregation [4]. Primary, secondary, and tertiary generation mammospheres were formed in the presence or absence of the Notch-activating DSL peptide. Although DSL peptide had only modest effects on primary mammosphere formation, it increased secondary and tertiary mammosphere formation 10-fold, compared with control cultures (Fig. 3a,3b,3d,3e,3g). If this effect was due to stem cell self-renewal, we would predict that cells derived from these spheres would retain their multipotent differentiation ability. We therefore examined the ability of secondary and tertiary mammosphere-derived cells to clonally differentiate along the multiple lineages present in the adult mammary gland.
The adult mammary gland has a lobuloalveolar structure composed of three cell lineages: myoepithelial cells that form the basal layer of ducts and alveoli, ductal epithelial cells that line the lumen of ducts, and alveolar epithelial cells that synthesize milk proteins [23]. Mammospheres grown in the presence or absence of Notch agonists or antagonists were dissociated into single cells and plated at clonogenic densities on collagen substrata in a medium that promotes differentiation [4]. After 7 days of cultivation, the clonally derived colonies were immunostained using lineage-specific markers (epithelial-specific antigen and cytokeratin 18 for ductal epithelial cells, and cytokeratin 14 and smooth muscle actin for myoepithelial cells). The number of multipotent cells after one passage was seven times higher, and that after two passages 100 times higher, in mammospheres cultured in the presence of DSL, compared with mammospheres cultured without added DSL. These calculations are based on the number of spheres (Fig. 3g) and the corresponding percentage of multilineage colonies, in each experiment (Fig. 3h).
The ability of Notch activating ligands to increase mammosphere formation, as well as the production of multilineage progenitors, suggests that Notch activation promotes mammary stem cell self-renewal. Similar effects to the DSL peptide on mammosphere formation were obtained with recombinant Delta Fc fragment (data not shown). In addition to increasing the number of mammosphere initiating cells and bipotent progenitor cells, treatment with Notch agonists increased their proliferation potential, as shown by the increased size of mixed lineage colonies, generated clonally on collagen culture by these cells (Fig. 3i). Treatment with Notch agonists was applied in suspension culture only, and was not applied to sphere-derived cells subsequently cultured on collagen substrata, suggesting that the effect on proliferation is irreversible. This positive effect of Notch activation on proliferation potential of stem/progenitor cells also occurs in the mammospheres, as demonstrated by an increased size of the primary and secondary mammosphere diameter in the DSL-treated suspension cultures (mean ± standard error of the mean, 245 ± 11.5 μM versus 130 ± 3 μM; n = 69 and n = 89). This is due to an increased number of cells per sphere, as demonstrated by cell counts that are approximately double in DSL-treated mammospheres compared with control cultures. The viability of cells within a mammosphere was similar in the presence of and the absence of DSL. These results indicate that the Notch activation increases proliferation of progenitor cells.
In order to further investigate the role of Notch signaling, and in particular Notch4, in mammary stem cell self-renewal, we assayed the effect of Notch inhibitors on mammosphere formation. The addition of a Notch4 blocking antibody to primary cultures had no effect on primary sphere formation, but completely abolished secondary mammosphere formation (Fig. 3a,3c,3d,3f,3h). A similar effect was obtained by blocking Notch activation with a GSI at 1–5 μM, a concentration found to effectively block Notch activation in the luciferase reporter assay (Fig. 2a). In contrast to the effect of adding Notch4 blocking antibody at the time of mammosphere formation, this treatment had no effect when added 24–48 hours after mammosphere formation. This suggests that the effects of Notch activation on mammary stem cell self-renewal may occur during the initial stages of mammosphere formation. The cells responding to modulation of Notch signaling are the sphere initiating cells, capable of survival and proliferation in suspension.
Effect of Notch signaling on lineage specification of mammary progenitor cells
In other developing systems, Notch signaling has been shown to effect lineage commitment as well as stem cell self-renewal [20, 24–26]. The inability of previously described culture systems of mammary cells to maintain progenitor cells in an uncommitted state has limited the in vitro study of lineage commitment. Since we have previously demonstrated that, in addition to mammary stem cells, mammospheres contain mammary progenitors capable of multilineage differentiation, we utilized this system to determine whether Notch signaling could also act on mammary progenitor cells to affect lineage-specific commitment. In order to test this, we added the DSL peptide to mammosphere cultures (Fig. 1a), to cultures of mammosphere-derived cells on collagen substrata in conditions that promote their differentiation (Fig. 1b) or to cultures of differentiated cells (Fig. 1c).
Cells were assayed for lineage-specific commitment, using the clonogenic assay described earlier. As shown in Fig. 3h (DSL1), the percentage of myoepithelial progenitor cells generated by DSL-treated mammospheres was increased approximately 13-fold compared with cells from mammospheres cultured in the absence of DSL ligand. The percentage of myoepithelial progenitor cells generated by mammosphere-derived cells treated with DSL starting from the moment of their plating on collagen culture (DSL2) generated sevenfold more myoepithelial progenitor cells compared with controls. The significant effect of DSL peptide, when added only to suspension-cultured mammospheres, suggests that Notch activation increased the number of myoepithelial progenitors and/or their rate of proliferation in an irreversible manner. The increased size of myoepithelial and bilineage colonies (Fig. 3i) formed in the presence of DSL peptide suggests that Notch stimulation may promote proliferation of myoepithelial and bilineage progenitors.
In order to determine whether the increase in colony size results from an effect of Notch on cell survival, we used propidium iodide staining in the same clonogenic assay. Treatment with DSL peptide did not have a discernable effect on the cell-death rate of either ductal epithelial cells or myoepithelial cells. To assess more accurately the increase in total number of myoepithelial cells upon treatment with DSL in suspension culture, we repeated the same experiment described earlier, utilizing flow cytometry analysis, to quantitate the cell lineage (Fig. 3j). The results showed a 15-fold increase in myoepithelial cells upon addition of DSL peptide to the culture.
Effect of Notch signaling on differentiated mammary epithelial cells
In contrast to the significant effects of Notch signaling on uncommitted mammary cells, modulation of the Notch pathway had no significant effect on differentiated mammary epithelial cells. The addition of DSL peptide to differentiated cells cultured on collagen substrata (Fig. 1c) had no effect on lineage specification, as shown by flow-cytometry analysis using lineage-specific markers (Fig. 3k). Furthermore, the addition of Notch antibody or the GSI at 1–10 μM had no significant effect on cell number or lineage commitment (data not shown). This also demonstrates that Notch activation is not necessary for the survival of fully differentiated mammary cells.
Effect of Notch signaling on branching morphogenesis
It has previously been demonstrated that culture of human or rodent mammary cells in reconstituted basement membrane (Matrigel) promotes morphogenic differentiation [18, 27]. Since mammospheres are composed of undifferentiated mammary cells, we have utilized three-dimensional Matrigel cultures to explore the morphogenic differentiation potential of these cells. Following 3–4 weeks of cultivation in Matrigel, mammospheres develop extensive ductal lobuloalveolar structures similar in morphology to those found in vivo (Fig. 4a,4b). We utilized this system to examine the role of Notch signaling in morphogenesis. Secondary mammospheres were imbedded in Matrigel and treated with DSL peptide, Notch4 antibody or GSI. Treatment with DSL peptide promoted earlier development, as well as increased length and number of branching structures, compared with control cultures (Fig. 4c,4d). In contrast, the Notch4 antibody completely inhibited branching morphogenesis (Fig. 4c,4d). Similar effects were produced by blocking Notch signaling with GSI (data not shown).
Expression of Notch4 in human mammary epithelial cells in vitro
We and other workers have found that expression of Notch receptors in the adult human mammary gland was below the level of detection using immunostaining of tissue sections, but was detectable by RT-PCR [28]. Moreover, expression of Notch4 in dissociated mammary tissue was below the level of detection using flow cytometry. The experiments already described, however, suggested that Notch receptors are present in mammary stem cells and progenitor cells, and are downregulated in differentiated cells. Previous gene expression profile analysis by microarray and real-time RT-PCR showed that mammosphere-derived cells passaged in suspension culture express all Notch receptors at a higher level (twofold to fourfold) than the same cells cultured under conditions that induce differentiation [4].
These results suggest that Notch receptors are expressed at higher levels in more primitive mammary cells, such as those found in the developing mammary gland, compared with the mature gland. To test this hypothesis, we immunostained structures generated by mammospheres in Matrigel culture, at different stages of growth (between 4 days and 2 weeks). We found that cells expressing Notch4 were uniformly distributed in spherical structures at the beginning of cultivation (Fig. 5a,5b). As branching morphogenesis occurred, Notch4 expression became limited to the subterminal end of branches (Fig. 5c,5d). The corresponding areas in vivo have previously been postulated to contain mammary stem cells and progenitor cells [23].