CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells

The fourth mammary fat pads were harvested from 20 virgin female 10 to 12 week old FVB mice following removal of the intra-mammary lymph nodes. Fat pads were chopped three times with a McIlwain Tissue Chopper (Mickle Laboratory Engineering Company, Gomshall, Surrey, UK) set to cut at 100 μm intervals and the finely minced tissue was transferred to a digestion mix consisting of serum-free Leibowitz L15 medium (Sigma, Poole, Dorset, UK) containing 3 mg/ml collagenase A (Sigma) and 1.5 mg/ml trypsin (Sigma). This was incubated for 1 h at 37°C in a shaking incubator to liberate a mixture of epithelial tissue fragments ('organoids'), non-epithelial fragments (such as pieces of blood vessel or nerve bundles) and non-epithelial single cells. These were washed by pelleting and resuspension in L15/10% FCS to remove the collagenase mix and fatty waste, and then incubated with red blood cell lysis buffer (Sigma) to remove erythrocytes (2 × 5 minutes). Following a further wash in L15/10% FCS, the sample was 'pre-plated' in Dulbecco's modified Eagle's medium (Invitrogen, Paisley, UK) with 10% FCS (Sigma) for 1 h at 37°C/5% CO₂. The majority of the contaminating fibroblasts, which are single cells, attach to the tissue culture plastic in this time, whereas the epithelial organoids do not. The epithelial and non-epithelial fragments, and contaminating single cells such as lymphocytes and some fibroblasts, can then be poured off, leaving the majority of the fibroblasts behind.

To obtain single mammary epithelial cells, the organoid preparations were washed twice in Ca²⁺/Mg²⁺-free PBS/0.02% w/v EDTA and incubated for 15 minutes in Joklik's Modification of Minimal Essential Medium for Suspension Culture (Sigma), to allow cell-cell contacts to begin to break down. The sample was then pelleted and resuspended in 2 ml 0.25% w/v trypsin/0.2% w/v EDTA in Hank's Balanced Salt Solution (Sigma). This was incubated at 37°C for 2 minutes. Release of DNA from damaged cells at this stage causes clumping, so after the 2 minute incubation a P1000 pipette was used to disaggregate DNA clumps and then 5 ml of serum-free L15 medium containing 1 μg/ml type I DNase (Sigma) was added. The sample was incubated for a further 5 minutes at 37°C and then an equal volume of L15/10% FCS added to stop the trypsinisation. Remaining clumps were removed by filtration through a 40 μm cell strainer and the resultant single cells pelleted, resuspended in fresh L15/10% FCS and counted.

Cells were incubated at 10⁶/ml in L15/10% FCS with anti-CD24-fluorescein isothiocyanate (clone M1/69, BD Biosciences, Oxford, UK, 0.5 μg/ml) and anti-CD45-phycoerythrin-Cy5 (clone 30-F11, BD Biosciences, Oxford, UK, 0.25 μg/ml) for 45 minutes at 4°C. Cells were washed in L15/10% FCS and resuspended in L15/10% FCS/0.01% 4',6-diamidino-2-phenylindole dihydrochloride (DAPI).

Analysis was carried out on a BD FACSVantageSE DiVa (BD Biosciences) equipped with two Coherent 90 C-4 argon ion lasers (Coherent, Santa Clara, CA, USA) set at 488 nm and 333.6 to 333.8 nm. Samples were gated on the basis of forward- and side-scatter. Dead cells (DAPI bright) and leukocytes (CD45⁺) were excluded. Doublets and higher order clumps were excluded using a time-of-flight approach, where forward-scatter-height was plotted against forward-scatter-area. Routine examination of sorted cells revealed >99% single cellularity.

Cells were sorted directly onto poly-L-lysine coated slides, air dried, and stored at -20°C. The cells were fixed in 1:1 methanol acetone at -20°C for 5 minutes and stained with antibodies against cytokeratin (CK)14 (clone LL002, Lab Vision, Suffolk, UK, 2.1 μg/ml) or CK8/18 (clone 5D3, Novocastra, Vision Biosystems, Newcastle-upon-Tyne, UK, 2 μg/ml) in addition to a nuclear stain (DAPI). Secondary antibodies were isotype specific goat anti-mouse antibodies (A21127, A21157, A21126; Invitrogen, Paisley, UK). Lack of non-specific staining by secondary antibodies was confirmed using isotype matched control primary antibodies (clones 15H6 and B10, Southern Biotech, Birmingham, Alabama, USA). Lack of cross reactivity in double staining experiments was confirmed with controls incubated with single primary antibodies and both secondary antibodies.

Quantitative real time reverse transcription PCR (qPCR) reactions were carried out to determine fold changes in expression of a selection of genes with known luminal epithelial (Lactotransferrin (Ltf), Milk Fat Globule-EGF Factor 8 protein (Mfge8), CK18 (Krt1-18)), myoepithelial/basal (CK14 (Krt1-14), Myosin Light Polypeptide 6 (Myl6a)) or non-epithelial (Myl6a, Procollagen 3a1 (Col3a1), CD31) distribution, compared to leucocyte-depleted, bulk mammary cells. Populations were freshly sorted into tubes rinsed with FCS, resuspended in Trizol reagent (Invitrogen, Paisley, UK), and stored at -20°C. RNA was extracted according to the manufacturer's instructions. cDNA synthesis was carried out using Sensiscript RT kit (Qiagen, Crawley, UK). Up to 50 ng of RNA was transcribed into cDNA using an oligo dTⁿ primer (Promega, Southampton, UK) per reaction; 1 μl of cDNA was used per qPCR reaction. Each analysis reaction was performed in triplicate. GAPDH was used as an endogenous control throughout all experimental analyses. Gene expression analysis was performed using TaqMan Gene Expression Assays on an ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, California, USA). Analysis was performed using the Δ-ΔCt method, which determines fold changes in gene expression relative to a comparator sample (mammary epithelial cells that had been depleted for CD45⁺ cells but not separated further). Significant deviation of the mean value of each sample group from a fold difference of 1 (no change compared to the comparator sample) was tested using a t-test on Log₁₀ transformed data. Standard curves were derived from sequence verified cDNA IMAGE clones (MRC Geneservice, Cambridge, UK) for each gene of interest confirming that sample amplification was within the linear range of the assay (data not shown).

Transplantation into cleared fourth mammary fat pads of 21-day old syngeneic female mice was carried out as described [5]. All animal work was approved by Local Ethics Committee and carried out under Home Office approval. Eight weeks after transplantation, fat pads were wholemounted and analysed as described [5]. They were scored as negative for outgrowth if no epithelial structures could be observed. They were scored as 'failed clears' if they contained an epithelial ductal network that could be seen to have grown in from one edge of the fat pad and in which the majority of ductal branching had the same directionality. If outgrowths could be seen to have originated from a central region of the cleared fat pad and the directionality of the ductal branching was different in different parts of the fat pad, they were scored as successful transplants. Successful transplants had a region of epithelial outgrowth dissected out under a binocular microscope for paraffin embedding by routine methods and routine immunocytochemistry to detect α-isoform smooth muscle actin (clone 1A4; Sigma).