Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Summary

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Abstract

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.

The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins β-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed "domes".

The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce β-casein and WAP.

Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated Rac^V12 increase differentiation, high Rac^V12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of Rac^V12.

Introduction

In normal tissues or tumors, cells have extensive opportunities for adhesion to their neighbors in a three-dimensional organization, and this is mimicked in culture by high density cell growth. Cell-to-cell adhesion is mediated mainly through cadherin receptors, which define cell and tissue architecture. Interestingly, it was recently demonstrated that cadherins also play a powerful role in signal transduction, especially in survival signaling¹. Paradoxically, some of these cell-to-cell adhesion signals emanating from cadherins were recently found to be shared by both differentiation and neoplasia². Here, we describe methods of induction and assessment of differentiation in two representative types of mouse breast epithelial cell lines, HC11 and EpH4.

The HC11 mouse breast epithelial cell line can provide a useful model for the study of epithelial cell differentiation. HC11 cells are a COMMA-1D-derived cell line, originating from the mammary gland of a mid-pregnant Balb/c mouse³. In contrast to other COMMA-1D derivative clones, the HC11 clone has no requirement for exogenously added extracellular matrix or cocultivation with other cell types for the in vitro induction of the endogenous β-casein gene by lactogenic hormones³. This cell line has been used extensively in differentiation studies because it has retained important characteristics of the normal mammary epithelium: HC11 cells can partially reconstitute the ductal epithelium in a cleared mammary fat pad⁴. Moreover, they can differentiate in a two-dimensional (2D) culture when grown to confluence attached to a plastic Petri dish surface in the presence of a steroid such as Hydrocortisone or Dexamethasone, in addition to Insulin and Prolactin (HIP medium) lacking epidermal growth factor (EGF), an inhibitor of differentiation^5,6,7. Under these conditions, HC11 cells produce milk proteins such as β-casein and WAP, which are detectable by Western blotting within 4 days following induction. At the same time, a portion of HC11 cells forms rudimentary mammary gland-like structures termed "domes" in a stochastic manner. Domes are visible 4–5 days following induction and gradually increase in size up to day 10, concomitant with an increase in β-casein production⁸. Interestingly, HC11 cells possess mutant p53⁹, and therefore represent a preneoplastic state. For this reason, the HC11 model is ideally suited to study signaling networks of differentiation in conjunction with neoplasia in the same cell system.

EpH4 cells, a derivative of IM-2 cells, are a nontumorigenic cell line originally derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a mid-pregnant Balb/c mouse¹⁰. EpH4 cells form continuous epithelial monolayers in 2D culture, but do not differentiate into glandular-like structures^10,11. However, following 3D growth in a material consisting of a mixture of extracellular matrix proteins produced by EHS mouse sarcoma cells¹² (EHS matrix, matrix, or Matrigel, see Table of Materials), in addition to stimulation with HIP, EpH4 cells can recapitulate the initial stages of mammary gland differentiation. Under these conditions, EpH4 cells form spheroids (also called mammospheres) that exhibit apical-basal polarity and a hollow lumen, and are capable of producing the milk proteins β-casein and WAP, similar to lactating mammary epithelial cells. Contrary to HC11 cells, which are undifferentiated, and some express mesenchymal markers¹³, EpH4 cells exhibit a purely luminal morphology¹⁴. EpH4 cells have also been reported to produce milk proteins in 2D culture through stimulation with dexamethasone, insulin, and prolactin¹⁵. However, this approach precludes the study of regulatory effects that mimic the mammary gland microenvironment in 3D culture.

Protocol

1. Plating HC11 Cells

1. In a laminar flow hood using sterile techniques prepare a bottle with 50 mL of HC11 cell medium: RPMI-1640 with 10% fetal bovine serum (FBS), 5 μg/mL insulin, and 10 ng/mL EGF (see Table of Materials).
2. Plate approximately 400,000 cells per 3 cm Petri dish: Pass two 10 cm, 50% confluent Petri dishes into twenty 3 cm dishes in HC11 medium. The cells must be well spread out but drying must be avoided.
   1. Aspirate the medium into a flask using a vacuum pump.
   2. Add 250 µL of trypsin (see Table of Materials) per 10 cm plate. Swirl and hit the plate from the sides while keeping it horizontal to spread the trypsin and to dislodge the attached cells
   3. Observe under phase contrast microscopy with a 4x objective to make sure the cells have started to detach from the edges before pipetting.
   4. Aspirate approximately 1.5 mL of HC11 medium in a sterile, 9 inch Pasteur pipette and squirt it vertically against the cells. Rotate the Petri dish while squirting to dislodge all cells. You must work fast to avoid drying of the cells.
   5. Transfer all cells to the bottle with the HC11 medium and swirl.
   6. Pipette 2 mL of cell suspension in each 3 cm Petri dish. Rock the Petri dishes crosswise to spread evenly. Place the cells in a 37 °C, 5% CO₂ incubator.
3. The next day, or when cells are ~90–100% confluent, aspirate the medium, and replace it with medium with FBS and insulin but lacking EGF.

2. Differentiation Induction, Monitoring, and Quantitation of HC11 Cells

1. After growing the cells in medium lacking EGF for 24 h, add the differentiation medium (HIP medium) to ten 3 cm plates: RPMI-1640 supplemented with 10% FBS, 1 μg/mL Hydrocortisone (see Table of Materials), 5 μg/mL Insulin and 5 μg/mL Prolactin (see Table of Materials) for up to 10 days.
2. Keep the other ten 3 cm plates as controls: Change to a medium with 10% FBS and 5 μg/mL insulin lacking EGF and HIP.
3. Change the medium every 2–3 days to both HIP-treated cells and controls. Pipette the medium carefully to make sure that cells do not detach.
4. To monitor differentiation (i.e., the formation of "domes"), observe the cells under phase contrast microscopy (Figure 1A, left panel). If the cells are expressing green fluorescence protein (GFP), observe under fluorescence microscopy (excitation = 485/20; emission = 530/25; magnification 240x; 20x objective) (Figure 1A, right panel).
5. To quantitate the degree of differentiation, extract proteins from one Petri dish each of HIP-treated cells and controls, once a day for a total of 10 days and probe Western blots for β-casein (see Table of Materials) (Figure 1B).
   1. Scrape the cells on ice with 1.5 mL ice-cold phosphate-buffered saline (PBS) into a 1.8 ml plastic centrifuge tube.
   2. Pellet the cells at 350 x g, 1 min, 4 °C.
   3. Quickly wash the pellet 2x with ice-cold PBS. Drain any residue.
   4. Make a lysis buffer: 50 mM HEPES (pH = 7.4), 150 mM NaCl, 10 mM EDTA, 10 mM Na₄P₂O₇, 1% NP-40, 100 mM NaF, 2 mM Na₃VO₄, 0.5 mM phenylmethylsulphonyl fluoride (PMSF), 10 μg/mL aprotinin, 10 μg/mL leupeptin¹⁶. Add 100 µL per 3 cm Petri dish.
   5. Clarify the lysate by centrifugation at 10,000 x g for 10 min in the cold.
   6. Determine protein concentration (see Table of Materials) and load 10–20 µg of protein from each sample onto a 10% polyacrylamide-SDS gel.
   7. Electrophorese for 15 h at 45 V, or at 100 V for ~2–2.5 h.
   8. Transfer onto a nitrocellulose or PVDF membrane using an electroblotting transfer apparatus (see Table of Materials).
   9. Block with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 (TBST).
   10. Add the primary antibody, goat anti-β-casein diluted 1:2,000 or mouse anti-β actin diluted 1:1,000 (see Table of Materials).
   11. Wash 3x with TBST, 5 min each time.
   12. Add the secondary antibody, horseradish peroxidase (HRP) linked donkey anti-goat diluted 1:2,500, or HRP linked horse anti-mouse antibody diluted 1:10,000.
   13. Wash 3x with TBST, 5 min each time.
   14. Add ECL reagents to the membrane according to the manufacturer's instructions (see Table of Materials).

3. Plating and 3D Growth of EpH4 Cells in EHS Matrix

NOTE: The matrix is liquid at temperatures <10 °C and solid at temperatures above. Store at -80 °C. Thaw at 4 °C the night before use. Pre-chill the tissue culture plates and pipette tips at -20 °C prior to handling and keep the matrix, plates, and pipette tips on ice to prevent it from solidifying.

1. Grow EpH4 cells in 2D in RPMI-1640 medium with 10% FBS and 5 µg/mL insulin (EGF is not required).
2. Prepare EpH4 growth medium supplemented with 10% (v/v, 3,500 µL) or 20% (v/v 2,000 µL) matrix in two 50 mL conical tubes. Keep on ice until ready to use.
3. Coat 10 wells (1 cm² each) of a 24 well plate with 150 μL of undiluted matrix. Spread the matrix using a pipette tip. Avoid creating bubbles. Gently tap the sides of the plate while holding it horizontally to ensure that the matrix is evenly distributed across the bottom of the well.
4. Incubate the 24 well plate at 37 °C for 1 h to allow the matrix to solidify.
5. Trypsinize EpH4 cells as described above for HC11 cells and count them with a hemocytometer. Transfer 5 x 10⁴ cells per well to a 1.5 mL sterile conical centrifuge tube and spin at 250 x g for 5 min.
6. Carefully aspirate the medium and place the tube on ice.
7. Resuspend cells in 350 µL of EpH4 growth medium supplemented with 20% matrix using a 1 mL pipette tip while keeping the conical tube on ice. Avoid bubbles. It is important to ensure a single cell suspension.
8. Once the bottom layer matrix has solidified (the matrix should appear translucent and lighter in color compared to the initial coating of the wells), add the 350 µL of cell suspension to each coated well and place in a CO₂ incubator at 37 °C for ~1 h to allow the 20% matrix layer to solidify.
   1. Observe the cells microscopically under phase contrast with a 4x objective. Single cells should be visible.
9. Add 200 µL of EpH4 medium containing 10% matrix on top of the 350 µL of cells suspended in 20% matrix. Incubate at 37 °C.

4. Differentiation Induction of EpH4 Cells Grown in 3D (Figure 2)

NOTE: Besides differentiation, EpH4 cells can also undergo tubulogenesis when stimulated with HGF (hepatocyte growth factor) in 3D culture. Tubular outgrowths can be seen after 10 days of HIP and HGF stimulation.

1. Begin differentiation induction of EpH4 cells 1 day after plating in the matrix. Carefully remove 150 µL of top medium containing 10% matrix from the wells using a plastic pipette tip. Add 200 µL of EpH4 medium containing HIP and 10% matrix.
2. Replace the 10% matrix-HIP medium every 2 days for up to 10 days. Grow control cells in the same 10% matrix medium without HIP.
3. Monitor mammosphere formation under phase contrast (Figure 3A, lower panel).

5. Tubulogenesis Induction of EpH4 Cells Grown in 3D (Figure 3A and 3C)

1. Prepare 24 well plates and EpH4 cells for growth in matrix as detailed in steps 3.1–3.9. The day after plating the cells in the matrix, remove 150 µL of EpH4 medium containing 10% matrix from the wells using a plastic pipette tip. Add 200 µL of EpH4 medium containing 10% matrix, HIP, and 20 ng/mL HGF (see Table of Materials).
2. Replace the 10% matrix/HIP/HGF medium every 2 days for up to 12–14 days.
3. Monitor tubule formation microscopically using a 20x or 40x objective (Figure 3A, right panel).

6. Quantitation of Differentiation: Western Blotting for β-casein

1. Carefully pipette off the 10% matrix-HIP medium from the wells. Rinse the 20% matrix layer 2x with 350 µL of ice-cold PBS. The spheroids should still be present in the matrix layer.
2. Add 700–1,000 µL of ice-cold PBS with 1 mM ethylenediaminetetraacetic acid (EDTA) directly into the wells. Gently detach the bottom layer of the 100% matrix from the well with a pipette tip to recover spheroids present in that layer. Shake gently for 30 min at 4 °C.
3. Carefully transfer the spheroid suspension to a conical tube. Rinse the wells with ~500 µL of PBS-EDTA to recover any remaining spheroids in the wells and add to the conical tube.
4. Rock on ice for an additional 30 min. Make sure the matrix has fully dissolved. If visible matrix clumps are seen, add more PBS-EDTA or shake longer.
5. Centrifuge the solution to pellet the spheroids at 350 x g for 5 min.
6. Aspirate the supernatant, lyse the spheroids in ice-cold lysis buffer, and probe for β-casein, cyclin D1, and p120RasGAP by Western blotting as in step 2.5 above¹⁶. As primary antibodies, use goat anti-β-casein diluted 1:2,000, rabbit anti-cyclin D1 diluted 1:2,000, and mouse anti-p120 diluted 1:2,000. For secondary antibodies, use HRP linked donkey anti-goat diluted 1:2,500, HRP linked donkey anti-rabbit diluted 1:2,500, and HRP linked horse anti-mouse diluted 1:10,000.

Representative Results

It has long been known that the differentiation of epithelial cells and adipocytes requires confluence and engagement of cadherins². We and others demonstrated that cell-to-cell adhesion and engagement of E- or N-cadherin and cadherin-11, as occurs with the confluence of cultured cells, triggers a dramatic increase in the activity of the small GTPases Rac and cell division control protein 42 (Cdc42), and this process leads to activation of interleukin–6 (IL6) family cytokines and Stat3 (signal transducer and activator of transcription-3^16,17)^1,18,19. Because many components of the cadherin/Rac/IL6/Stat3 pathway may participate in both differentiation and neoplastic transformation, they provide molecular handles for the study of the interrelatedness of these two diametrically opposed processes.

Using the techniques described above, we demonstrated a striking dependence of differentiation upon the strength of the Rac signal in HC11 cells: While endogenous cRac1 is required for differentiation, and at low levels mutationally-activated Rac^V12 causes a distinct increase in differentiation capacity, high Rac^V12 levels trigger a dramatic block of differentiation while inducing neoplasia (Figure 1B). In contrast, even low levels of Rac^V12 expression blocked differentiation in EpH4 cells². Furthermore, even though Rac^V12 activates Stat3 in many cell systems, including HC11¹⁶, mutationally-activated Stat3C blocked differentiation while inducing neoplastic transformation²⁰.

We further explored the differentiation properties of EpH4 cells in a 3D matrix culture. Whereas β-casein production peaked at 8–10 days, cyclin D-1 expression was maximal at 4–6 days after HIP stimulation (Figure 3B). These findings support an inverse relationship between proliferation and differentiation in the EpH4 model. Using DAPI (nuclear) staining and confocal microscopy to determine the positioning of individual epithelial cells, we observed inner cell death and the formation of hollow lumens in mammospheres (Figure 3A,C). We further showed that addition of HGF (20 ng/mL) at 48 h to the HIP medium resulted in the formation of tubular structures (Figure 3A,C), consistent with earlier electron microscopy studies²¹. These approaches using the EpH4 model allow the characterization of specific features of mammary epithelial differentiation and their association with distinct signal transduction pathways.

Figure 1: Assessment and quantitation of HC11 cell differentiation. (A) A dome formed in HC11 cells expressing low levels of GFP-Rac^V12 at 10 days following induction of differentiation. Left: Phase-contrast; Right: Fluorescence of the same field (photos not published before). Scale bar = 100 µm; Magnification = 240x; 20x objective. (B) Effect of Rac^V12 upon HC11 differentiation: Low (lanes 19–24), intermediate (lanes 13–18), or high (lanes 7–12) levels of a Rac^V12-GFP fusion construct were expressed in HC11 cells. Following growth in the absence of EGF for 24 h, cells were induced to differentiate with HIP addition, or not, as indicated. Detergent extracts were prepared at the indicated number of days later and probed for β-casein or β-actin as a loading control. Note the dramatic increase in β-casein in low Rac^V12-GFP expressing cells (lanes 21–24 vs. 3–6), and the dramatic reduction upon expression of high Rac^V12-GFP (lanes 9–12). NE = Control cultures, grown in the absence of EGF, but not induced to differentiate (Niit et al.², reproduced with permission). Please click here to view a larger version of this figure.

Figure 2: Schematic of overlay method for growing EpH4 cells in 3D matrix culture. (A) The wells of a 6 well plate were initially coated with 100% EHS matrix that was allowed to solidify at 37 °C forming a gelled bed of basement membrane measuring approximately 2–5 mm in thickness. EpH4 cells were seeded onto this bed as a single cell suspension in HIP medium with 20% matrix. This was overlayed with 10% matrix in HIP medium. The 10% matrix medium was replaced every other day. Cells proliferated and began to form mammospheres after 4–5 days in culture (see Protocols section). (B) Phase contrast images of EpH4 cells in 2D (monolayer) and 3D (mammosphere) culture were captured using a microscope equipped with a digital camera (300x magnification). Representative images at each stage are shown. Please click here to view a larger version of this figure.

Figure 3: Events in 3D acinar morphogenesis of EpH4 in 3D culture. (A) EpH4 cells were grown in matrix-coated 6 well plates and maintained in complete 3D medium as in Figure 2. During the early stages of morphogenesis, cells proliferated, formed clusters, and organized into two groups: (1) an outer layer of polarized cells and (2) an inner cluster of disorganized cells which underwent cell death, corresponding to apoptosis as shown previously¹². The latter led to the formation of a hollow lumen, characterized by the darkening of the center of the mammospheres as seen by phase contrast microscopy. Addition of HGF (20 ng/mL) to the medium resulted in the formation of tubular structures. More than 60% of the HGF-induced aggregates showed tubulogenesis, compared to untreated control aggregates, which did not. A representative phase contrast photograph of cells at each stage was captured using a microscope equipped with a digital camera (300x magnification; Scale bar = 100 µm). Results are representative of at least three experiments. Schematic was adapted from Starova et al.²². (B) EpH4 cells grown in 3D were harvested on the indicated days. Protein concentrations were normalized and equal protein amounts (20 μg) were subjected to SDS-PAGE, followed by Western blotting and probing with the indicated antibodies. A homogenized mammary gland from a lactating mouse (MGT) was used as an in vivo comparison. p120RasGAP was used as an independent loading control. Cyclin D1 expression increased transiently coinciding with proliferating cells over the first 3–4 days. In contrast, β-casein expression peaked at 8–10 days, corresponding to the formation of mature mammospheres. Results are representative of two experiments. (C) Lumen formation and tubulogenesis of mammospheres was confirmed using DAPI staining of nuclear DNA (fluorescent blue) in aggregates grown on matrix-coated glass coverslips. Cells were fixed in 3% paraformaldehyde, permeabilized with 25 μg/mL digitonin, and nonspecific binding was blocked with 3% BSA. Cells were then stained for nuclear DNA with DAPI (blue). Coverslips were mounted onto glass slides using a mounting medium. Photomicrographs were taken of the central XY-axis plane using a multiphoton confocal microscope (Scale bar = 100µm). Images in Panels B and C are adapted from Starova et al.²². Please click here to view a larger version of this figure.

Discussion

HC11 cells are ideally suited for the study of differentiation in conjunction with neoplastic transformation. An added advantage is that HC11 cells are easily infectable with Mo-MLV-based retroviral vectors to express a variety of genes. In our hands, EpH4 cells were more difficult to infect with the same retroviral vectors than HC11².

Cell-to-cell contact and growth arrest are key prerequisites for HC11 cell differentiation. Thus, to achieve uniform differentiation across the cell layer, it is important to achieve a uniform distribution of cells in the Petri dish at the time of seeding. This is especially important if quantitation of differentiation is desired. Trypsinization must be optimized so that a single cell suspension is achieved and cells do not lose viability because of the manipulations.

Cells being trypsinized must be subconfluent (50–70%), because cells grown to high densities tend to strongly adhere to each other, and separating them is difficult. To avoid drying the cells, aspirate with the Petri dish flat on the floor of the laminar flow hood, then tilt to drain quickly. Cover the Petri dish with the lid. If necessary, you can accelerate the action of the trypsin by placing the Petri dish in a 37 °C incubator.

Because the cells are very confluent during the 10 days following induction, it is important to replace the nutrients that are depleted. When the medium is changed, care should also be taken to avoid detachment of the cells, which may not adhere very well to the plastic due to high cell density. Still, in our experience it is not necessary to use Petri dishes coated with fibronectin, collagen, or CellTak to obtain good differentiation results, unlike differentiating preadipocytes such as 3T3L1²³ or Balb/c3T3 derivatives.

Accurate protein determination for the blotting experiments is critical. Because serum proteins tend to attach to the tissue culture grade plastic, it is important to scrape and wash the cells in a 1.5 mL plastic tube that does not attract proteins and to add the lysis buffer on the cell pellet rather than lysing the cells directly on the Petri dish²⁴.

Regarding protein extraction, it is very important to keep all washing and lysis solutions ice-cold and the Petri dishes or EpH4 mammospheres on ice throughout. This is because in the absence of calcium in the PBS wash the cadherins are not engaged and as a result Stat3-ptyr705 is rapidly dephosphorylated²⁵.

The size of the Petri dishes described for HC11 cells is sufficient for 3–4 Western blots (i.e., the amount of protein recovered per Petri dish is approximately 50 µg per 3 cm dish). We prefer to use 3 cm Petri dishes for differentiation experiments to facilitate protein extraction. If a 6 well tray is used instead, it is difficult to extract proteins from one well in the cold while keeping the rest of the wells sterile. Besides, the CO₂ concentration is important for differentiation and it is difficult to maintain it for the rest of the wells as proteins are being extracted from one well on the bench.

In our experience, unlike the differentiation of preadipocytes²³, several lots of fetal calf serum are able to support the growth as well as the differentiation of HC11 or EpH4 cells. One lot of newborn calf serum tested did not support differentiation, although it could support normal growth: the cells appeared to differentiate at first but lost their ability to differentiate after three passages in newborn calf serum.

In contrast to HC11 cells, differentiation of EpH4 cells is tightly linked to the surrounding 3D matrix architecture. We describe the steps required for EpH4 differentiation in 3D culture modified from previous studies^26,27,28 and subsequent protein extraction for analysis of β-casein production. The EHS matrix is liquid at low temperatures (<10 °C) and solidifies at higher temperatures. It is normally stored at -80 °C, but working aliquots can be stored at -20 °C. Thaw the matrix at 4 °C the night before use and pre-chill tissue culture plates and pipette tips at -20 °C prior to handling. For an accurate assessment of protein concentration it is important to eliminate the matrix completely before extraction with cold PBS washing because the matrix is rich in protein, which may confound the protein determination results.

The EpH4 dependence upon the matrix for differentiation has been demonstrated in multiple experimental models: Reichman et al.¹⁰ showed that EpH4 cells induced by lactogenic hormones produced β-casein within areas of laminin deposition and intensified cytokeratin expression. Somasiri et al.¹¹ demonstrated that PI3-kinase is required for adherens junction-dependent spheroid formation and differentiative milk protein gene expression. Furthermore, Brinkmann et al.²¹ demonstrated that expression of transfected HGF cDNA in EpH4 cells induced branching tubulogenesis of spheroids in a 3D culture model, analogous to the observed HGF-induced tubule formation seen here (Figure 3A,C). These findings illustrate the advantages of the EpH4 cell line model for studying signaling events that regulate mammary gland differentiation.

EpH4 cells can also form mammospheres when plated at concentrations of fetal calf serum lower than the 10% described. Interestingly, they can form mammospheres at lower concentrations, or even in the absence of 20% or 10% matrix, when plated on top of a layer of 100% matrix^22,27. In the absence of matrix in the cell suspension, cooling down the cells is avoided. An added advantage is that all cells are found in a single plane, so they are easier to photograph.

Significance: Cadherin engagement in cultured cells, which approximates the physiological state of a cell in vivo, can activate the Rac/IL6/Stat3 pathway. This may be especially important in epithelial cell differentiation. Using the techniques described, our results exposed the intensity of the signal emanating from this pathway as a central determinant in the balance between cell proliferation vs. differentiation, two fundamentally opposed processes². The in-depth investigation of the E-cadherin/Rac/Stat3 axis may uncover novel amphibolous components of the pathway depending upon the level of expression, that could be exploited as targets for cancer therapy. For such targets, complete inhibition would not be necessary to reverse the neoplastic phenotype. Partial inhibition would even be beneficial, because the residual amounts may actually block transformation and promote differentiation. This may vary with the target as well as the type of tumor in question, as shown from the different behavior of Rac^V12 vs. Stat3C, in HC11 vs. EpH4 cells^2,20.

Future applications: The cadherin/Rac/IL6/Stat3 pathway may play a similar role in the differentiation of other epithelial cells, such as the human breast MCF10A or the canine kidney MDCK²⁹ cell lines, as well as other types of differentiation which depend upon cell confluence, such as of preadipocytes and myoblasts³⁰, which express different types of cadherins. Finally, this technique can be exploited for the examination of the role of Stat5³¹ and other components of differentiative pathways.

Materials

Name	Company	Catalog Number	Comments
30% Acrylamide/0.8% Bis Solution	Bio Rad	1610154	Western Blotting
Anti-Cyclin D1 antibody	rabbit, Santa Cruz	sc-717	Western Blotting
Anti-p120 antibody	mouse, Santa Cruz	sc-373751	Western Blotting
Anti-β actin antibody	mouse, Cell Signalling technology	3700	Western Blotting
Anti-β casein antibody	goat, Santa Cruz Biotechnology	sc-17971	Western Blotting
Aprotinin	Bio Shop	APR600	Lysis Buffer
Bicinchoninic Acid Solution	Sigma	B9643-1L-KC	Protein Determination
Bovine serum albumin	Bio Shop	ALB007.500	Protein Determination
Clarity Western ECL Substrate	Bio Rad	170-5061	Western Blotting
Copper(II) sulphate	Sigma	C2284-25ML	Protein Determination
DAPI	Thermofisher Scientific	D1306	Staining
Digitonin	Calbiochem, Cedarlane Laboratories Ltd	14952-500	Staining
EDTA	Bio Shop	EDT001.500
Epidermal Growth Factor	Sigma	E9644	Medium for HC11 Cells
Fetal calf serum	PAA	A15-751	Cell Culture Medium
Goat- Anti Rabbit-HRP	Santa Cruz	SC-2004	Western Blotting
Hepatocyte Growth Factor recombinant	Gibco	PHG0321
Hepes	Sigma	7365-45-9	Cell Culture
Horse Anti-Mouse HRP	Cell Signalling Technology	7076	Western Blotting
Hydrocortisone	Sigma	H0888	HIP Medium
insulin	Sigma	I6634	HIP Medium
Laminar-flow hood	BioGard Hood		Cell Culture
Leupeptin	Bio Shop	LEU001.10	Lysis Buffer
Matrix (Engelbreth-Holm-Swarm matrix, Matrigel)	Corning	CACB 354230
Mouse Anti-Goat HRP	Santa Cruz	sc-8360	Western Blotting
Mowiol 4-88 Reagent	Calbiochem, Cedarlane Laboratories Ltd	475904-100GM	Staining
Multi-photon confocal microscope	Leica TCS SP2
Na3VO4	Bio Shop	SOV850	Lysis Buffer
Na4P207	Sigma	125F-0262	Lysis Buffer
NaCl	Bio Shop	SOD001.1	Western Blotting
NaF	Fisher Scientific	7681-49-4	Lysis Buffer
Nikon digital Camera	Coolpix 995
Nitrocellulose	Bio Rad	1620112	Western Blotting
NP-40	Sigma	9016-45-9
Paraformaldehyde	Fisher Scientific	30525-89-4	Staining
Phase Contrast Microscope	Olympus IX70		Cell Culture
Phenylmethylsuphonyl fluoride	Sigma	329-98-6	Lysis Buffer
pMX GFP Rac G12V	Addgene	14567
Prolactin	Sigma	L6520	HIP Medium
RPMI-1640	Sigma	R8758	Cell Culture Medium
Tissue Culture Dish 35	Sarstedt	83.3900.	Cell Culture
Tissue Culture Plate-24 well	Sarstedt	83.1836.300	Cell Culture
Transfer Apparatus	CBS Scientific Co	EBU-302	Western Blotting
Tris Acetate	Bio Shop	TRA222.500	Western Blotting
Trypsin	Sigma	9002-07-7.	Cell Culture
Tween-20	Bio Shop	TWN510.500	Western Blotting
Veritical Gel Electrophoresis System	CBS Scientific Co	MGV-202-33	Western Blotting