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.

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 (sign...

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 acr...

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