The HC11 cell line was kindly provided by Dr. D. Medina (Houston, TX). The authors are grateful to Dr. Andrew Craig of Queen&#39;s University for many reagents and valuable suggestions. EpH4 cells were a gift from Dr. C. Roskelley (UBC, Vancouver). Colleen Schick provided excellent technical assistance for 3D culture studies.
The financial assistance of the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer Foundation (CBCF, Ontario Chapter), the Canadian Breast Cancer Research Alliance, the Ontario Centres of Excellence, the Breast Cancer Action Kingston (BCAK) and the Clare Nelson bequest fund through grants to LR is gratefully acknowledged. BE received grant support from CIHR, CBCF, BCAK and Cancer Research Society Inc. PTG is supported by a Canada Research Chair, Canadian Foundation for Innovation, CIHR, NSERC and Canadian Cancer Society. MN was supported by a studentship from the Terry Fox Training Program in Transdisciplinary Cancer Research from NCIC, a Graduate award (QGA), and a Dean&#39;s Award from Queen&#39;s University. MG was supported by postdoctoral fellowships from the US Army Breast Cancer Program, the Ministry of Research and Innovation of the Province of Ontario and the Advisory Research Committee of Queen&#39;s University. VH was supported by a CBCF doctoral fellowship and a postdoctoral fellowship from the Terry Fox Foundation Training Program in Transdisciplinary Cancer Research in partnership with CIHR. Hanad Adan was the recipient of an NSERC summer studentship. BS was supported by a Queen&#39;s University graduate award.

1. Plating HC11 Cells
<ol>
	<li>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 &#956;g/mL insulin, and 10 ng/mL EGF (see Table of Materials).</li>
	<li>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.
	<ol>
		<li>Aspirate the medium into a flask using a vacuum pump.</li>
		<li>Add 250 &#181;L 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</li>
		<li>Observe under phase contrast microscopy with a 4x objective to make sure the cells have started to detach from the edges before pipetting.</li>
		<li>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.</li>
		<li>Transfer all cells to the bottle with the HC11 medium and swirl.</li>
		<li>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 &#176;C, 5% CO2 incubator.</li>
	</ol>
	</li>
	<li>The next day, or when cells are ~90&#8211;100% confluent, aspirate the medium, and replace it with medium with FBS and insulin but lacking EGF.</li>
</ol>
2. Differentiation Induction, Monitoring, and Quantitation of HC11 Cells
<ol>
	<li>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 &#956;g/mL Hydrocortisone (see Table of Materials), 5 &#956;g/mL Insulin and 5 &#956;g/mL Prolactin (see Table of Materials) for up to 10 days.</li>
	<li>Keep the other ten 3 cm plates as controls: Change to a medium with 10% FBS and 5 &#956;g/mL insulin lacking EGF and HIP.</li>
	<li>Change the medium every 2&#8211;3 days to both HIP-treated cells and controls. Pipette the medium carefully to make sure that cells do not detach.</li>
	<li>To monitor differentiation (i.e., the formation of &#34;domes&#34;), 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).</li>
	<li>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 &#946;-casein (see Table of Materials) (Figure 1B).
	<ol>
		<li>Scrape the cells on ice with 1.5 mL ice-cold phosphate-buffered saline (PBS) into a 1.8 ml plastic centrifuge tube.</li>
		<li>Pellet the cells at 350 x g, 1 min, 4 &#176;C.</li>
		<li>Quickly wash the pellet 2x with ice-cold PBS. Drain any residue.</li>
		<li>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 &#956;g/mL aprotinin, 10 &#956;g/mL leupeptin16. Add 100 &#181;L per 3 cm Petri dish.</li>
		<li>Clarify the lysate by centrifugation at 10,000 x g for 10 min in the cold.</li>
		<li>Determine protein concentration (see Table of Materials) and load 10&#8211;20 &#181;g of protein from each sample onto a 10% polyacrylamide-SDS gel.</li>
		<li>Electrophorese for 15 h at 45 V, or at 100 V for ~2&#8211;2.5 h.</li>
		<li>Transfer onto a nitrocellulose or PVDF membrane using an electroblotting transfer apparatus (see Table of Materials).</li>
		<li>Block with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 (TBST).</li>
		<li>Add the primary antibody, goat anti-&#946;-casein diluted 1:2,000 or mouse anti-&#946; actin diluted 1:1,000 (see Table of Materials).</li>
		<li>Wash 3x with TBST, 5 min each time.</li>
		<li>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.</li>
		<li>Wash 3x with TBST, 5 min each time.</li>
		<li>Add ECL reagents to the membrane according to the manufacturer&#39;s instructions (see Table of Materials).</li>
	</ol>
	</li>
</ol>
3. Plating and 3D Growth of EpH4 Cells in EHS Matrix
NOTE: The matrix is liquid at temperatures &#60;10 &#176;C and solid at temperatures above. Store at -80 &#176;C. Thaw at 4 &#176;C the night before use. Pre-chill the tissue culture plates and pipette tips at -20 &#176;C prior to handling and keep the matrix, plates, and pipette tips on ice to prevent it from solidifying.
<ol>
	<li>Grow EpH4 cells in 2D in RPMI-1640 medium with 10% FBS and 5 &#181;g/mL insulin (EGF is not required).</li>
	<li>Prepare EpH4 growth medium supplemented with 10% (v/v, 3,500 &#181;L) or 20% (v/v 2,000 &#181;L) matrix in two 50 mL conical tubes. Keep on ice until ready to use.</li>
	<li>Coat 10 wells (1 cm2 each) of a 24 well plate with 150 &#956;L 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.</li>
	<li>Incubate the 24 well plate at 37 &#176;C for 1 h to allow the matrix to solidify.</li>
	<li>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.</li>
	<li>Carefully aspirate the medium and place the tube on ice.</li>
	<li>Resuspend cells in 350 &#181;L 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.</li>
	<li>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 &#181;L of cell suspension to each coated well and place in a CO2 incubator at 37 &#176;C for ~1 h to allow the 20% matrix layer to solidify.
	<ol>
		<li>Observe the cells microscopically under phase contrast with a 4x objective. Single cells should be visible.</li>
	</ol>
	</li>
	<li>Add 200 &#181;L of EpH4 medium containing 10% matrix on top of the 350 &#181;L of cells suspended in 20% matrix. Incubate at 37 &#176;C.</li>
</ol>
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.
<ol>
	<li>Begin differentiation induction of EpH4 cells 1 day after plating in the matrix. Carefully remove 150 &#181;L of top medium containing 10% matrix from the wells using a plastic pipette tip. Add 200 &#181;L of EpH4 medium containing HIP and 10% matrix.</li>
	<li>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.</li>
	<li>Monitor mammosphere formation under phase contrast (Figure 3A, lower panel).</li>
</ol>
5. Tubulogenesis Induction of EpH4 Cells Grown in 3D (Figure 3A and 3C)
<ol>
	<li>Prepare 24 well plates and EpH4 cells for growth in matrix as detailed in steps 3.1&#8211;3.9. The day after plating the cells in the matrix, remove 150 &#181;L of EpH4 medium containing 10% matrix from the wells using a plastic pipette tip. Add 200 &#181;L of EpH4 medium containing 10% matrix, HIP, and 20 ng/mL HGF (see Table of Materials).</li>
	<li>Replace the 10% matrix/HIP/HGF medium every 2 days for up to 12&#8211;14 days.</li>
	<li>Monitor tubule formation microscopically using a 20x or 40x objective (Figure 3A, right panel).</li>
</ol>
6. Quantitation of Differentiation: Western Blotting for &#946;-casein
<ol>
	<li>Carefully pipette off the 10% matrix-HIP medium from the wells. Rinse the 20% matrix layer 2x with 350 &#181;L of ice-cold PBS. The spheroids should still be present in the matrix layer.</li>
	<li>Add 700&#8211;1,000 &#181;L 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 &#176;C.</li>
	<li>Carefully transfer the spheroid suspension to a conical tube. Rinse the wells with ~500 &#181;L of PBS-EDTA to recover any remaining spheroids in the wells and add to the conical tube.</li>
	<li>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.</li>
	<li>Centrifuge the solution to pellet the spheroids at 350 x g for 5 min.</li>
	<li>Aspirate the supernatant, lyse the spheroids in ice-cold lysis buffer, and probe for &#946;-casein, cyclin D1, and p120RasGAP by Western blotting as in step 2.5 above16. As primary antibodies, use goat anti-&#946;-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.</li>
</ol>

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The authors have no conflicts to disclose.

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...

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 &#946;-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 differentiation5,6,7. Under these conditions, HC11 cells produce milk proteins such as &#946;-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 &#34;domes&#34; in a stochastic manner. Domes are visible 4&#8211;5 days following induction and gradually increase in size up to day 10, concomitant with an increase in &#946;-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 structures10,11. 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 &#946;-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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30% Acrylamide/0.8% Bis Solution</td>
			<td>Bio Rad</td>
			<td>1610154</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Anti-Cyclin D1 antibody</td>
			<td>rabbit, Santa Cruz</td>
			<td>sc-717</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Anti-p120 antibody</td>
			<td>mouse, Santa Cruz</td>
			<td>sc-373751</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Anti-&#946; actin antibody</td>
			<td>mouse, Cell Signalling technology</td>
			<td>3700</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Anti-&#946; casein antibody</td>
			<td>goat, Santa Cruz Biotechnology</td>
			<td>sc-17971</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Bio Shop</td>
			<td>APR600</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>Bicinchoninic Acid Solution</td>
			<td>Sigma</td>
			<td>B9643-1L-KC</td>
			<td>Protein Determination</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Bio Shop</td>
			<td>ALB007.500</td>
			<td>Protein Determination</td>
		</tr>
		<tr>
			<td>Clarity Western ECL Substrate</td>
			<td>Bio Rad</td>
			<td>170-5061</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Copper(II) sulphate</td>
			<td>Sigma</td>
			<td>C2284-25ML</td>
			<td>Protein Determination</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermofisher Scientific</td>
			<td>D1306</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Calbiochem, Cedarlane Laboratories Ltd</td>
			<td>14952-500</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Bio Shop</td>
			<td>EDT001.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>Sigma</td>
			<td>E9644</td>
			<td>Medium for HC11 Cells</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>PAA</td>
			<td>A15-751</td>
			<td>Cell Culture Medium</td>
		</tr>
		<tr>
			<td>Goat- Anti Rabbit-HRP</td>
			<td>Santa Cruz</td>
			<td>SC-2004</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Hepatocyte Growth Factor recombinant</td>
			<td>Gibco</td>
			<td>PHG0321</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma</td>
			<td>7365-45-9</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Horse Anti-Mouse HRP</td>
			<td>Cell Signalling Technology</td>
			<td>7076</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H0888</td>
			<td>HIP Medium</td>
		</tr>
		<tr>
			<td>insulin</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td>HIP Medium</td>
		</tr>
		<tr>
			<td>Laminar-flow hood</td>
			<td>BioGard Hood</td>
			<td></td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Bio Shop</td>
			<td>LEU001.10</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>Matrix (Engelbreth-Holm-Swarm matrix, Matrigel)</td>
			<td>Corning</td>
			<td>CACB 354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Goat HRP</td>
			<td>Santa Cruz</td>
			<td>sc-8360</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Mowiol 4-88 Reagent</td>
			<td>Calbiochem, Cedarlane Laboratories Ltd</td>
			<td>475904-100GM</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Multi-photon confocal microscope</td>
			<td>Leica TCS SP2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Bio Shop</td>
			<td>SOV850</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>Na4P207</td>
			<td>Sigma</td>
			<td>125F-0262</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bio Shop</td>
			<td>SOD001.1</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>NaF</td>
			<td>Fisher Scientific</td>
			<td>7681-49-4</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>Nikon digital Camera</td>
			<td>Coolpix 995</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Bio Rad</td>
			<td>1620112</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma</td>
			<td>9016-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>30525-89-4</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Phase Contrast Microscope</td>
			<td>Olympus IX70</td>
			<td></td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Phenylmethylsuphonyl fluoride</td>
			<td>Sigma</td>
			<td>329-98-6</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>pMX GFP Rac G12V</td>
			<td>Addgene</td>
			<td>14567</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolactin</td>
			<td>Sigma</td>
			<td>L6520</td>
			<td>HIP Medium</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td>Cell Culture Medium</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish 35</td>
			<td>Sarstedt</td>
			<td>83.3900.</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Tissue Culture Plate-24 well</td>
			<td>Sarstedt</td>
			<td>83.1836.300</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Transfer Apparatus</td>
			<td>CBS Scientific Co</td>
			<td>EBU-302</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Tris Acetate</td>
			<td>Bio Shop</td>
			<td>TRA222.500</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>9002-07-7.</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Bio Shop</td>
			<td>TWN510.500</td>
			<td>Western Blotting</td>
		</tr>
		<tr>
			<td>Veritical Gel Electrophoresis System</td>
			<td>CBS Scientific Co</td>
			<td>MGV-202-33</td>
			<td>Western Blotting</td>
		</tr>
	</tbody>
</table>

differentiation of mouse breast epithelial hc11 and eph4 cells

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 &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
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 &#946;-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 RacV12 increase differentiation, high RacV12 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 RacV12.

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&#8211;6 (IL6) family cytokines and Stat3 (sign...

Watch this Scientific Journal Video about Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells at JoVE.com

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

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.

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 ...

differentiation-of-mouse-breast-epithelial-hc11-and-eph4-cells

Research

JoVE Journal

Cancer Research

8.6K Views.  Queen's University, Kingston. 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.

Video: Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an anti-inflammatory drug to standard cancer regiments may improve patient outcome. One such drug, aspirin (acetylsalicylic acid, ASA), has been explored for cancer chemoprevention and anti-tumor activity. Besides inhibiting the cyclooxygenase 2-prostaglandin axis, ASA&#39;s anti-cancer activities have also been attributed to nuclear factor &#312;B (NF&#312;B) inhibition. Because prolonged ASA use may cause gastrointestinal toxicity, a prodrug strategy has been implemented successfully. In this prodrug design the carboxylic acid of ASA is masked and additional pharmacophores are incorporated.
This protocol describes how we synthesized an aspirin-fumarate prodrug, GTCpFE, and characterized its inhibition of the NF&#312;B pathway in breast cancer cells and attenuation of the cancer stem-like properties, an important NF&#312;B-dependent phenotype. GTCpFE effectively inhibits the NF&#312;B pathway in breast cancer cell lines whereas ASA lacks any inhibitory activity, indicating that adding fumarate to ASA structure significantly contributes to its activity. In addition, GTCpFE shows significant anti-cancer stem cell activity by blocking mammosphere formation and attenuating the cancer stem cell associated CD44+CD24- immunophenotype. These results establish a viable strategy to develop improved anti-inflammatory drugs for chemoprevention and cancer therapy.

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&amp;#954;B Activity and Breast Cancer Stem Cells

This procedure will demonstrate how we synthesized and characterized the anti-NF&#954;B and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...