UNIT 22.3 Preparation of Epithelial and Mesenchymal Stem Cells from Murine Mammary Gland

 

Introduction

The interest in mammary gland epithelial cells (epithelial cells in many cancers) arises from accumulating evidence that it is these cells that play a significant, if not predominant role, in tumor development. One of the current theories on the origin of cancer in both the hematopoietic system and solid tumors implicates tissue-specific stem cells as the tumor initiating cells (Hermann et al., 2010). Breast, prostate, and colon cancers are among those solid cancers in which the epithelial cell fraction has been shown to contain a cancer stem cell (population) that has self-renewing and tumor-initiating potential. This hierarchal model of cancer, as opposed to the stochastic model, presumes that only a small fraction of the tumor cells has the potential for tumor induction (Johnsen et al., 2009). The evidence is now very convincing that most cancers are clonal in origin and although the stem cells from which these clones derive may not be completely characterized, it is widely accepted that stem cells are the culpable cells (Sell, 2010).

The field of mammary gland physiology has been particularly fruitful in stem cell biology due to the historical interest in the remarkable cyclical changes of proliferation, lactation, and involution that occurs in the breast tissue throughout life and pregnancy. Research in the 1950s and 1960s laid the groundwork for the exploration of epithelial stem cell function in the mammary gland (Deome et al., 1959; Daniel et al., 1968). Two seminal papers, indicating the ability of a single cell to regenerate the entire functional mammary gland of a mouse (Shackleton et al., 2006) and the presence of a highly enriched population of cancer-initiating cells in the CD44⁺CD24^– fraction of human breast cancer (Al-Hajj et al., 2003), have stimulated investigations into tissue-specific (cancer) stem cells. For recent reviews on cancer stem cells, see Sell (2010), Shackleton (2010), and Bohl et al. (2011).

NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

 

Basic Protocol: Isolation and Flow Cytometric Sorting of Epithelial and Mesenchymal Stem Cells from Mammary Gland Tumors of PyVT Mice

This descriptive approach to isolate epithelial and mesenchymal stem cells from mammary gland tumors is applicable to several different transgenic models of mammary gland cancer in mice, including the FVB/N-Tg(MMTV-PyVT)634Mul/J (PyVT; Jackson Laboratories, stock no. 002374) and the FVB/N-Tg(MMTVneu)202Mul/J (neu; Jackson Laboratories, stock no. 002376) models, but each model has distinct characteristics. For example, female PyVT mice present mammary gland tumors at 6 to 8 weeks of age, while males are 20 to 24 weeks of age before tumors of adequate size manifest. Thus, in the PyVT mouse, the age of tumors has a high degree of correlation with sex. In the neu mouse, only females get mammary gland tumors and these do not occur until 32 to 36 weeks of age. As tumors increase in size, necrosis and hemolysis will occur within the tumors. IACUC guidelines will dictate how large tumors are allowed to grow (typically 1.5 to 2 cm³), and although necrotic areas do not prevent isolation of usable cells, the younger and typically smaller the tumor, the greater the proportion of the tissue that will be made up of solid tissue and viable cells, and so the better the yield.

Female PyVT mice do not lactate, so male hemizygous PyVT mice are bred with wild-type FVB females to maintain the line, and because of this, the offspring must be genotyped. The primer sequences and the PCR protocol provided by Jackson Laboratories (from where the mice are obtained) work well. FVB mice are also used for clearance of fat pads (see Support Protocol 3) in preparation for transplant of potential mammary gland (tumor) stem cells and must be between 3 and 3.5 weeks of age at the time of surgery. Do not use older mice, as complete removal of all host epithelial cells is unlikely to be achieved. Both male and female neu mice will breed successfully, but breeding should be confined to mice that are 2 to 4 months of age for best results, as both males and females show increasing rates of aggression with aging that will interfere in timely pregnancies.

If age or genealogy is not a critical factor, the grossly dissected glands from two to three animals can be pooled. This may be required since in any one mouse, tumors at each gland can vary enormously in size at any particular time and not all tumors will yield useful tissue. The mouse mammary tumor virus (MMTV) promoter essentially confines tumor expression to the mammary gland, although metastasis to lungs does occur in 80% to 90% of females. Another FVB model, the Wnt1 transgenic mouse (Jackson Laboratories, stock no. 002934), has also been used successfully to derive mammary gland cancer stem cells (Cho et al., 2008). Although other models with different promoters do exist, these are unlikely to yield mammary gland tumors with such high penetrance, and tumors at other sites may dictate sacrifice or death before mammary gland tumors are sufficiently large enough for cell recovery. The use of isolated epithelial and mesenchymal cells can be applied in various fields, including biochemical, developmental, and genetic studies and is not limited to cancer, as the method described below can be used in the isolation of cells from normal mammary glands. This protocol describes the separation of mammary gland epithelial and mesenchymal cells, allowing for assessment of their individual actions and interactions.

 Materials

• Mice: FVB/N-Tg(MMTV-PyVT)634Mul/J (PyVT) (Jackson Laboratories, stock no. 002374)
• CO₂ source
• 70% (v/v) ethanol (see recipe)
• Betadine solution
• Dulbecco's modified Eagle's medium (DMEM; see recipe)
• 0.25% (w/v) trypsin (see recipe), sterile
• Fetal bovine serum (FBS; see recipe)
• Trypan blue solution (see recipe)
• Staining medium (see recipe): Hank's balanced salt solution with and without 2% heat-inactivated FBS, cold
• Antibodies (Biolegend): anti-CD24-fluorescein isothiocyanate (cat. no. 101805), anti-CD44-Pacific Blue (cat. no. 103019), anti-CD49f-AlexaFluor 647 (cat. no. 313609), anti-CD45-phycoerythrin (cat. no. 103105), anti-CD31-phycoerythrin (cat. no. 102407), anti-Ter119-allophycocyanin (cat. no. 116211), anti-Sca1-phycoerythrin (cat. no. 108109)

• Surgical scissors, tweezers, and scalpels
• Disposable 35- and 100-mm petri dishes
• Sterile 1-, 5-, 10-, and 25-ml sterile serological pipets
• 37°C, CO₂-regulated tissue culture incubator
• Sterile 70-µm cell strainers
• 15- and 50-ml screw-capped sterile centrifuge tubes
• Refrigerated centrifuge
• Hemacytometer
• Flow cytometer with sorting capacity (e.g., FACSVantage, Becton Dickinson)

 Harvest tumors

1.	Euthanize mice by CO2 exposure and cervical dislocation. Choose mice with obvious mammary gland tumors that have not reached maximum size permitted by the IACUC, as this will yield the best chance of useful tissue. p type = annotation Mammary gland tumors are heterogeneous and rarely achieve similar size in more than two glands at a time; therefore, pooling several glands is standard practice. p type = annotation All mice should be ordered at 3 weeks of age in the first instance. PyVT mice are the first choice for mammary gland tumors, due to their rapid development. Plan to harvest tumors when female PyVT mice reach 6 weeks of age, although this time frame can vary by 1 week. Only male PyVT mice are used to maintain the line; their breeding performance is most successful between 2 and 5 months of age. The wild-type FVB mouse will be the source of control tissue for pathological studies and FVB females are used in breeding PyVT mice. The two transgenic mice, PyVT and neu, are recommended until one becomes adept at cell recovery, manipulation, and animal husbandry. Experienced investigators may apply these techniques to other cancer or animal models. Investigators should use the same distributor consistently, as colonies founded by different companies may have diverged to the point of histoincompatibility, which would complicate interpretation of transplant experiments.
2.	Lay mouse down in supine position and secure legs to prevent movement.
3.	Swab entire abdomen with 70% ethanol, followed by a betadine solution swab, and then another 70% ethanol wash.
4.	Make an incision from pubis to just under the chin through the skin and fascia but do not penetrate the muscle layer. p type = annotation There are 5 pairs of laterally symmetrical mammary glands in the mouse; thoracic glands (pairs 1 to 3) and inguinal glands (pairs 4 and 5). Pair 1 occurs between the shoulder and the head and pair 2 occurs just distal to the front legs and in a normal mouse these pairs are too small for routine collection of tissue, although tumors readily occur here. Pairs 3 (proximal to axillae) and 4 (just above the hip) are the glands frequently used for normal cell collection. Pair 4 are the glands traditionally removed when one prepares a cleared fat pad, due to their convenient location and size, and therefore they are used most commonly for receipt of injections. Pair 5 glands occur just distally and proximal to pair 4, on either side of the genitalia.
5.	Make four lateral incisions through the skin from sternum right and left to just under axillae and from pubis right and left to just above each hip joint.
6.	Grasp the skin layer with tweezers or forceps and gently but firmly pull each lateral skin flap fully to the right or left. Use the blunt edge of scissors to aid in separating the skin from the muscle.
7.	With blunt dissection, remove tumor from fascia, gently pulling towards edge of opened skin flap while cutting away connective tissue between the tumor and the fascia. p type = annotation Determining which glands will have large enough tumors is unpredictable. Tumors will vary in size and some may contain obvious necrotic or hemolyzed areas, which should be avoided if possible. Blunt rather than fine excision of solid tumor tissue is adequate at this stage.
8.	Immediately place the excised tumors into a 100-mm petri dish kept on a layer of crushed ice and containing 10 ml DMEM. When all tumors have been collected, move the petri dish to a laminar flow hood. p type = annotation Maintaining a sterile field is critical for any long-term culture of recovered cells. Although the mammary gland tumors of these mice contain mixed pathology including areas of sometimes extensive hemolysis, the recovered cells are usually uncontaminated. However, all isolation steps beyond the initial gross dissection of the tumors should be performed in a biological laminar flow cabinet.

 Prepare cell suspension

9.	Mince tissue in the petri dish into 1- to 2-mm sized pieces, using sterile scissors, scalpel, or razor blade.
10.	Carefully remove most of the DMEM with a pipet, leaving the tissue pieces behind.
11.	Add 10 ml sterile 0.25% trypsin, mix, and incubate 30 min at 37°C, with gentle stirring every 10 min.
12.	Inactivate the trypsin by adding an equal volume of DMEM/10% FBS to the petri dish and mix.
13.	Pipet contents of dish through a 70-µm cell strainer into a new 50-ml screw-capped sterile centrifuge tube. p type = annotation All subsequent steps should be with cells on ice and solutions at 4°C.
14.	Centrifuge 5 min at 500 × g, 4°C. Discard supernatant.
15.	Resuspend cells in 10 ml DMEM/10% FBS and place on ice. Remove a small aliquot of cells, stain with trypan blue and count cells on a hemacytometer (expected viability > 95%).

 Label cells with antibodies

16.	For staining, resuspend cells at 1 × 10^6 cells/100 µl in cold HBSS/2% heat-inactivated FBS (staining medium).
17.	Set aside 1 ml of unlabeled cells and keep on ice. Add appropriate concentrations of antibodies per manufacturer's instructions. Incubate 30 min on ice in the dark, gently shaking tubes every 10 min; keep light exposure (especially UV light) to a minimum. p type = annotation Fluorescent labels can be altered to suit investigator preferences or flow cytometer capabilities.
18.	Wash cells by centrifuging two times for 5 min at 500 × g, 4°C, resuspending in staining medium between washes. Resuspend cells in staining medium at 5–10 × 10^6 cells/ml.

 Perform sterile cell sorting

19.

Maintain cells on ice and proceed with flow cytometry and cell sorting. Set initial gates to exclude hematopoietic (CD45^+, Ter119^+) and endothelial cells (CD31^+) and sort based on CD24 expression. Mammary gland epithelial cells are CD24^+CD49f^+CD44^lowCD29^+Sca-1^lowCD45^–CD31^–Ter119^–. Mesenchymal cells are CD24^–CD49f^–CD44^highCD29^+Sca-1^highCD45^–CD31^–Ter119^–. p type = annotation Flow cytometry and cell sorting is typically done by a core facility within an institution, since the equipment and expertise are major investments typically beyond the means of a standard laboratory. The flow cytometry facility will advise on appropriate controls, e.g., aliquots of unlabeled cells will be required to set forward and side-scatter gates and propidium iodide–labeled cells will be needed to exclude dead cells. p type = annotation At this point, cells are ready for culture, transplant, or other manipulations, as the interest of the investigator dictates. For culture, see Support Protocol 1. For transplant studies, see Support Protocols 3 and 4.

 

Alternate Protocol: Alternate Method for Mesenchymal Cell Isolation from Bone Marrow

The mesenchymal cell population is made up of multiple cell types, and their influence on tumor development is now widely recognized and is rapidly becoming a new discipline. Mesenchymal cell phenotypes are tailored to their organ of residence; the best known mesenchymal cell population is that of the bone marrow. Extensive in-depth analysis has explored the potential of these cells to differentiate into multiple cell types, including adipocytes, chondrocytes, and myocytes (Friedenstein et al., 1968; Jiang et al., 2002). Given this potential, it may be of interest to examine if mesenchymal cells from organs other than the breast might interact with or influence mammary gland epithelial cell behavior. Recent reports document bone marrow–derived mesenchymal cells playing a significant role in breast cancer metastasis to bone (Goldstein et al., 2010) and in prostate regrowth after cancer, in which the mesenchymal cells fuse with the prostate epithelial cells (Placencio et al., 2010). This protocol describes an established, flow cytometric-based method to enrich and culture mesenchymal cells from mouse bone marrow. Ex vivo culture is necessary as these cells make up <1 in 10⁶ bone marrow cells (Phinney et al., 1999), so initial yield will be low. These can then be co-injected with epithelial cells in any transplantation study or studied on their own. This description is based on the methods of Bonnet (Anjos-Afonso and Bonnet, 2008).

 Additional Materials (also see Basic Protocol)

• FVB mice (Jackson Laboratories, stock no. 001800)
• Red blood cell lysing buffer (Sigma; store 2 years at room temperature)
• Mesencult complete medium (see recipe)
• Anti-CD11b-FITC (Biolegend, cat. no. 101205)

• Bone scissors
• 3-ml syringes with 25-G needles
• Tissue culture flasks (25-, 75-, and 175-ml filter-top flasks)

 Prepare bone marrow cell suspension

1.	Euthanize FVB mouse as in Basic Protocol, step 1, and prepare for bone marrow extraction by dipping the entire rear legs in a 70% ethanol solution, swab with betadine solution, and then dip legs in 70% ethanol.
2.	Incise skin from ankle to hip, cut through tibia at ankle and femur at hip joints, and remove legs, without skin, from body using bone scissors. Place legs in a 100-mm petri dish containing 5 to 10 ml HBSS on ice. p type = annotation The operation should now be moved to a laminar flow hood.
3.	Remove all tissue from bones and then separate at knee joint.
4.	Fill a 3-ml syringe with HBSS and attach a 25-G needle.
5.	Grasp tibia or femur with tweezers, insert needle into end of bone at knee joint, and push syringe contents gently through bone, collecting extruded marrow/cells into a 50-ml capped centrifuge tube. p type = annotation Removing the patella and gently rotating needle while entering the ends of bone will aid in penetration.
6.	Flush bones several times with more HBSS and pool extractions. Pool the bone marrow from a minimum of five mice for mesenchymal cell culture and purification. p type = annotation Two femurs and two tibias from one mouse will yield on average 30–50 × 10^6 bone marrow cells.
7.	Centrifuge cells 5 min at 500 × g, 4°C.
8.	Discard supernatant, agitate tube to break up pellet of cells, and add 1 ml red blood cell lysing buffer.
9.	Incubate on ice 1 min, add 10 to 20 ml HBSS, and centrifuge as in step 7.
10.	Resuspend cells in HBSS/2% heat-inactivated FBS and count on a hemacytometer.

 Culture cells

11.	Centrifuge cells as in step 7 and resuspend in Mesencult complete medium at 5 × 10^6 cells/ml.
12.	Dispense into 25-ml tissue culture flasks at a density of 1 × 10^6 cells/cm^2.
13.	Discard non-adherent cells on day 3 and replenish medium every 4 to 5 days.
14.	When cells have achieved ~70% confluence, remove medium, rinse cells once with 5 to 10 ml HBSS, and then discard HBSS.

 Passage cells and expand cultures

15.	Passage cells by exposing to 0.25% trypsin for 5 min at 37°C. Warm trypsin solution to 37°C before infusion into flasks and use ~2 ml/25 cm^2.
16.	Remove trypsin solution and detach cells by pipetting flask contents up and down, then decant cells into a 50-ml centrifuge tube containing 1 ml cold FBS (for each 25-cm^2 flask) to inactivate the trypsin.
17.	Rinse flask with ~5 ml Mesencult medium (without supplements) and add to cells in centrifuge tube.
18.	Centrifuge cells 5 min at 500 × g, 4°C.
19.	Resuspend cells in fresh Mesencult complete medium and split 1:3 into new 75-ml flasks.

 Label cells for flow cytometry

20.	At passage 3, trypsinize cells, resuspend in staining medium and label with anti CD11b and anti CD45 antibodies, according to manufacturer's instructions.
21.	Incubate 30 min on ice, with regular agitation, then wash and resuspend cells in staining medium as in Basic Protocol, step 18.
22.	Proceed with cell sorting, collecting double negative CD11b^–CD45^– cells.
23.	Reculture cells in Mesencult complete medium for expansion. Cryopreserve at least two aliquots of cells (see Support Protocol 2). Confirm differentiation potential (e.g., adipocytes, myoblasts) of these mesenchymal cells by exposing them to specific inducing media; see Phinney et al. (1999) for details. Cells are now ready for transplantation or other studies.

 

Support Protocol 1: Culture of Epithelial and Mesenchymal Cells

Expansion of epithelial and mesenchymal cells in culture allows for derivation of sufficient numbers of cells that could otherwise be a limiting factor in initial transplantation studies. Epithelial cells are readily grown in a variety of media and should give no pause to any investigator. Mesenchymal cells are more stringent in their requirements. If one has obtained a pure or highly enriched population of mesenchymal cells by flow cytometry sorting, it is essential to use mesenchymal cell–specific media. This is available from several manufacturers. Cell density plays another key role. If cells show very slow growth using specific media, doubling or tripling cell density at initial seeding should increase chances of obtaining healthy cultures.

 Materials

• Isolated and sorted cells (see Basic Protocol or Alternate Protocol)
• Dulbecco's Modified Eagle's Medium (DMEM; see recipe) or Mesencult complete medium (see recipe)
• 10% FBS (see recipe)
• Epidermal growth factor (see recipe)
• Insulin (see recipe)
• HBSS (see recipe)

• 37°C, 5% CO₂ tissue culture incubator
• Sterile pipets (1-, 5-, 10-, and 25-ml serological pipettes)
• Sterile 0.22-µm membrane filters (Millipore)

1a.	For epithelial cell culture: Infuse single cell suspensions of mammary gland epithelial cells into tissue culture flasks (seed between 5 × 10^4 and 5 × 10^5 cells/cm^2) in DMEM/10% FBS supplemented with epidermal growth factor and insulin. Place in a 37°C, 5% CO2 incubator.
1b.	For mesenchymal cell culture: Infuse single cell suspensions of mesenchymal cell sorted fractions into tissue culture flasks (seed between 5 × 10^4 and 5 × 10^5 cells/cm^2) in Mesencult complete medium and place in a 37°C, 5% CO2 incubator.
2.	Change medium every 2 to 3 days thereafter.
3.	When cells have achieved ~70% confluence, remove medium and rinse cells once with 5 to 10 ml HBSS and discard HBSS.
4.	Passage cells by exposing to 0.25% trypsin, as described in Basic Protocol, steps 11 to 14.
5.	Resuspend cells in fresh culture medium and split 1:3 into new flasks. p type = annotation Frozen stocks of early passage (2 to 5) should be cryopreserved (see Support Protocol 2).

 

Support Protocol 2: Cryopreservation of Cells

When one is successful in obtaining a healthy culture of epithelial or mesenchymal cells, it is prudent to store early passage cells in liquid nitrogen. This is particularly applicable to cell cultures from mammary gland tumors, as it is desirable to preserve any stem cells that might be present with as much differentiation potential as possible. Cryopreservation of cells from the second or third passage enables archiving of unmanipulated cells with optimal potential for growth or differentiation. In vitro culture may induce genetic alterations that can lead to changes from the original phenotype.

 Materials

• Epithelial or mesenchymal cells (70% confluent)
• DMEM medium (see recipe) or Mesencult complete medium (see recipe)
• Cryopreservation medium (see recipe)

• Refrigerated centrifuge
• 1-ml cryotubes
• Freezing container (e.g., Mr Frosty, Nalgene)

1.	Trypsinize cells in flasks when 70% confluent, as described in Basic Protocol, steps 11 to 14.
2.	Resuspend cells in multiples of 5–10 × 10^6(depending upon availability) in culture medium, i.e., DMEM for epithelial cells or Mesencult complete medium for mesenchymal cells.
3.	Centrifuge cells 5 min at 500 × g, 4°C.
4.	Decant culture medium and add 1 ml (dropwise, with constant tube agitation) of cryopreservation medium per 5–10 × 10^6 cells.
5.	Place cells on ice for 30 min.
6.	Dispense 1-ml aliquots into cryotubes.
7.	Place cryotubes in freezing container and place overnight in a –80°C freezer.
8.	Move cryotubes to a liquid nitrogen storage tank within 24 to 48 hr.

 

Support Protocol 3: Clearance of Mammary Gland Fat Pad and Transplantation of Cells

This protocol is based on the pioneering work of Deome, which is still widely used today (Deome et al., 1959). It was originally designed to detect the repopulating ability of epithelial cells but is now most commonly employed to detect cancer stem cells. The mammary gland fat pad (typically number 4) is cleared of host epithelial cells. The fat pad remains as a receptacle for transplanted cells, allowing for demonstration of any mammary gland repopulation potential or interaction with host cells.

 Materials

• FVB mice (must be no older than 3 and 1/2 weeks)
• Ketamine/xylazine mix (see recipe)
• 70% ethanol (see recipe)
• Betadine
• Prepared cells ready for transplant in PBS (see recipe)

• Surgical instruments
• Cauterizer (e.g., Roboz Surgical Instrument, model RS-320)
• 25- to 50-µl microsyringes (Hamilton) and 27-G needles
• Wound closing clips and applicator (Clay Adams Autoclips)

1.	Anesthetize FVB mouse with a 500 µl per 20 g body weight i.p. injection of ketamine (100 mg/kg)/xylazine (10 mg/kg) mixture in PBS.
2.	Lay mouse down in supine position and secure legs.
3.	Swab all of the abdomen with 70% ethanol, followed by a betadine solution swab then another 70% ethanol wash.
4.	Make a vertical incision from mid-abdomen (at a point between the number 4 nipples) to the sternum and then two lateral incisions from the bottom of the mid-abdominal incision, ending between the number 4 and number 5 mammary glands on either side (the resultant incision will have an inverted Y shape).
5.	Retract skin and fascia from the abdominal muscle wall to clear the surgical field. p type = annotation The mammary glands appear as grayish or pinkish bodies with a slightly denser appearance and darker color than the surrounding fat or fascia and will be attached to the underside of the dermis. Careful inspection will distinguish glandular material from the surrounding fat, which can be identified by the presence of shiny, tightly packed spherical lipid globules.
6.	Cauterize the nipple, the blood vessel proximal to the lymph node, and at the point where the number 4 and number 5 fat pads touch. Excise the mammary gland between the cautery points from the nipple to the associated lymph node using fine scissors and precise incisions. p type = annotation The mammary gland anlage at this age should not have grown beyond the lymph node. p type = annotation One can use a single mouse as its own control by removing the number 4 gland from one side only. This depleted side will receive cells prepared for transplant. Alternatively, one can perform complete removal of both glands and use sham-operated mice as controls.
7.	Transplant cells into the fat pad in a volume of 10 to 15 µl of PBS or HBSS using a Hamilton microsyringe and a 27-G needle. p type = annotation Ideally, cells to be transplanted are injected at this time, while the mammary gland fat pad is exposed. This avoids a second operation.
8.	Close incisions with wound clips.
9.	Remove clips 7 days later. p type = annotation If cells were not transplanted at the time of clearance, the above surgical procedure is repeated when cells are ready for transplantation into the residual fat pad.

 

Support Protocol 4: Ectopic Transplantation of Isolated Cells

If the tumorigenicity of cells is to be tested and not epithelial mammary gland function, a second surgery to expose the cleared fat pad can be avoided and cells can be injected subcutaneously in the rear legs of a host. This procedure is best performed with two people to avoid anesthesia.

 Materials

• Mice
• 70% ethanol
• Cells
• 1-ml syringe and 26-G, 5/8-in. needle

1.	Gently restrain mouse and extend rear leg.
2.	Swab injection site with 70% ethanol
3.	Inject cells in a volume of 25 to 50 µl using a 1-ml syringe and 26-G, 5/8-in. needle. p type = annotation Needle should be inserted at a 10° to 20° angle relative to the femur; as cells are injected, a bleb should become visible under the skin. Use the full length of the needle and wait 5 to 10 sec after injection before withdrawal, to avoid leakage of cells.
4.	Monitor mice daily by palpation and note time to first evidence of tumor growth.

sect1 type = reagents

 

Reagents and Solutions

p type = annotation

Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.

 Cryopreservation medium

Prepare a solution of 70 ml heat-inactivated FBS (see recipe), 20 ml DMEM (or Mesencult if mesenchymal cells are being frozen; see recipes), and 5 ml DMSO. Dispense into 10-ml aliquots and store up to 2 years at –20°C.

 Dulbecco's modified Eagle's medium (DMEM)

Prepare in four 500-ml bottles. Leave 1 liter untreated (store up to 12 months at 4°C). Prepare 1 liter for cell culture by adding 10% heat-inactivated fetal bovine serum (see recipe), 50 U/ml penicillin, 50 µg/ml streptomycin (see recipe). Store in dark at 4°C. This volume is a compromise between having a sufficient amount for 1 to 2 weeks of culture maintenance but not so much in one bottle that the loss due to contamination is a serious setback. Ordering DMEM as powder (Gibco BRL) and preparing it in the laboratory is much more economical. Sterile reusable glass bottles, a pH meter, and a vacuum filtration system are required. Ideally, cultures should be maintained without antibiotics, but this requires constant vigilance that comes with experience; therefore, initial cultures can contain penicillin and streptomycin and one can gradually stop supplementing the culture medium with these antibiotics. Store supplemented DMEM up to 6 months at 4°C.

 Epidermal growth factor

Make a stock solution at 10 µg/ml in HBSS by adding 100 µg epidermal growth factor (Sigma-Aldrich) to 10 ml HBSS (see recipe). Filter sterilize and store in 500-µl aliquots in cryovials up to 2 years at –80°C. Add one vial per 500 ml bottle of DMEM.

 Ethanol solution

Make up 70% (v/v) ethanol in purified water. Store 12 months in closed container at room temperature.

 Fetal bovine serum (FBS)

Heat-inactivate fetal bovine serum (FBS; Gibco BRL) by placing sealed bottles in a water bath for 60 min at 56°C. There is no need for further sterilization. Store in 100-ml aliquots up to 2 years at –20°C.

 Hank's balanced salt solution (HBSS)

Prepare in 1-liter bottles from HBSS powder (Gibco BRL), filter sterilize using a 0.22-µm filter, and store up to 12 months at 4°C.

 Insulin

Prepare a stock solution at 5 mg/ml in HBSS (see recipe) by adding 50 mg insulin to 10 ml HBSS. Filter sterilize and store up to 2 years at –80°C in 500-µl aliquots in cryovials. Add one vial per 500-ml bottle of DMEM.

 Ketamine/xylazine

Prepare a working solution by mixing 1 ml of ketamine stock solution (100 mg/ml), 0.5 ml of xylazine stock solution (20 mg/ml), and 8.5 ml of PBS (see recipe). Inject 100 µl per 10 g body weight by i.p. injection using a 1-ml syringe and a 25- or 26-G, 1/2–in. needle. Anesthesia will last ~20 to 30 min and sedation ~60 to 90 min. If more anesthesia is required, use only a ketamine solution (make up as above, omitting xylazine). Store stock solutions up to 18 to 24 months (expiry dated by manufacturer). Discard working solution every month.

 Mesencult complete medium

Mix Mesencult MSC basal medium (Stem Cell Technologies, cat. no. 05501) and Mesenchymal stem cell stimulatory supplements (Stem Cell Technologies, cat. no. 05502) at a ratio of 1 part supplements and 4 parts basal medium. This has a limited shelf-life based on date of receipt; mix only the amount needed for 1 to 2 weeks. Store supplements at –20°C and basal medium at 4°C.

 Penicillin/streptomycin

100× concentrated solutions are widely available and inexpensive. Store up to 2 years at –20°C.

 Phosphate buffered saline (PBS)

• For a 10× solution, prepare the following for 1 liter:
• 80 g NaCl
• 2 g KCl
• 11.5 g Na₂HPO₄.7H₂O
• 2 g KH₂PO₄
• 1 liter H₂O

Adjust pH to 7.4 with 1 N HCl or 1 N NaOH. then autoclave. Dilute 1:10 before use. Store up to 12 months at room temperature in sealed bottles.

 Staining medium

Prepare HBSS (see recipe) with 2% (v/v) heat-inactivated FBS (see recipe). Store up to 6 months at 4°C.

 Trypan blue

Prepare a 0.25% (w/v) Trypan blue solution in PBS (see recipe). This solution does not have to be sterile; store indefinitely at room temperature.

 Trypsin, 0.25%

Prepare a 0.25% (w/v) trypsin solution in sterile PBS (see recipe) or HBSS (see recipe), sterilize using a 0.22-µm syringe filter and store up to 1 year at –20°C in 10- to 25-ml aliquots. Keep on ice when not in use; unused portions can be refrozen multiple times, but will gradually lose activity.

sect1 type = commentary

 

Commentary

 

Background Information

The mammary gland derives from the embryonic ectoderm, from which a small population of cells invades surrounding stroma to form branching ducts that terminate in lobules (Daniel and Silberstein, 1987; Stingl et al., 1998). This terminal duct lobular unit (TDLU) is the basic functional unit of the mammary gland and it is within this structure that the mammary gland stem cells reside (Woodward et al., 2005; Villadsen et al., 2007). The mammary gland has two epithelial layers; an outer myoepithelial/basal layer and a luminal layer lining the ducts. In addition, there are a variety of non-epithelial cells, including mesenchymal cells, endothelial cells, lymphocytes, adipocytes, neurons, and myocytes (Sleeman et al., 2006). In 2006, it was shown that a single cell lacking hematopoietic (CD45 and TER119) and endothelial (CD31) antigens, positive for CD24 and expressing high levels of CD29 (i.e., Lin^-CD24⁺CD29^hi), could generate the entire mammary gland (Shackleton et al., 2006). Mammary gland stem cells have subsequently been shown to play key roles in both regeneration of the mammary gland and in the development of mammary gland tumors.

In the normal human breast, there are three populations within the epithelial progenitors: luminally restricted, basal myoepithelial restricted, and bipotential cells (LaBarge et al., 2007). The luminally restricted cells are sialomucin (MUC)1⁺ and epithelial cell adhesion molecule (EpCAM)⁺ (also known as epithelial specific antigen—ESA); these cells subsequently develop cytokeratin (CK)8, CK18, and CK19 antigens. The myoepithelial cells demonstrate CK5, CK14, and alpha smooth muscle actin (-SMA) antigens; this is the population enriched for stem cells (Stingl et al., 1998; Clarke, 2005). The bipotential progenitors can be found as a core of cells expressing CK19 surrounded by myoepithelial cells expressing CK14 (Stingl et al., 2001). This stem cell–enriched pool of myoepithelial cells correlates highly with cells expressing low levels of CD24; it is these cells that demonstrate robust repopulation of cleared mammary gland fat pads while CD24^high cells show limited repopulation (Sleeman et al., 2006; Fillmore and Kuperwasser, 2008).

The CD44/CD24 antigens are the most commonly cited antigens identifying mammary gland cancer stem cells. CD44 is a transmembrane hyaluronan receptor with a role in cell migration, chemotaxis, and adhesion (Sleeman and Cremers, 2007; Vigetti et al., 2008). CD44 has been used as a marker of cancer-initiating cells in various cancers, including prostate, pancreas, and colon (Collins and Gibson, 1999; Dalerba et al., 2007; Li et al., 2007). CD44⁺ CD24^– ESA⁺ cells have been found to be highly enriched for human breast cancer-initiating ability; this report was one of the first demonstrations of a cancer-initiating stem cell in solid organ tumors (Al-Hajj et al., 2003).

The CD24 antigen, a glycosylphosphatidylinositol of heterogeneous molecular weight, was established as a mammary gland tumor marker in 1999 (Fogel et al., 1999). The expression of CD24 correlates with tumor stage and metastasis (Baumann et al., 2005; Bircan et al., 2006) and it has also been identified as being required for self-renewal regulation in transit-amplifying cells (the stage between stem cells and differentiated cells) (Nieoullon et al., 2007). Recent investigation of human breast cancer cell lines has revealed the riguing finding that invasive CD44⁺CD24^– mesenchymal cells can be derived from a single non-invasive epithelial CD44⁺CD24⁺ cell (Meyer et al., 2009). For a recent review of mammary gland stem cells, see Stingl (2009).

The mouse is an excellent model in which to investigate mammary gland physiology and pharmacology. An extensive body of literature exists on surgical manipulation and transplant of cells to the mammary gland in wild-type and transgenic mice. Investigation of potential mammary gland stem cell function is typically performed by injecting or transplanting cells into a cleared mammary gland fat pad, for which innate epithelial cells from the mammary gland have been surgically removed from the host at 3 weeks of age. There are several transgenic strains, such as the Polyoma Middle T antigen mouse and the Her2/neu mouse, that have been engineered to spontaneously develop mammary gland cancers, which closely reproduces the cellular pathology that is seen in human breast cancer (Cardiff and Wellings, 1999; Lim et al., 2010). These and other mice are available commercially and allow for a relatively simple isolation of (cancer) stem cells as well as non-epithelial cells such as mesenchymal cells. When these donor cells are labeled with antibodies and subjected to cell sorting, the population can be broken down into specific cell types, and when transplanted into a host animal, the influence of the non-epithelial cell population on tumor growth can be studied. Epithelial and mesenchymal cells can be injected together in defined ratios into the cleared mammary gland fat pad or the contribution of the host mesenchymal cells can be studied by transplanting only epithelial donor cells (Guest et al., 2010).

There is a great deal of current interest in the role that mesenchymal and other stromal cells play in cancer maintenance and progression. It has been known for some time that the mammary gland stroma plays an important role in mediating breast tissue response to hormones (Woodward et al., 1998), but more recently the function of stromal/mesenchymal cells in mammary gland tumor development has been recognized. For example, mesenchymal stem cells not only promote migration, invasiveness, and metastasis, but also play roles in tumor hormone independence and regulation by cytokine pathways (Goldstein et al., 2010; Rhodes et al., 2010; Halpern et al., 2011; Liu et al., 2011). In some cases, stromal cells have fused with and transformed mammary gland cancer epithelium (Jacobsen et al., 2006). Stromal fibroblasts have also been found to promote other cancers, including pancreatic cancer (Hwang et al., 2008). For reviews of tumor-associated fibroblasts, see Xouri and Christian (2010) and Franco et al. (2010).

 

Critical Parameters

Transplantation of cells into the cleared mammary gland fat pad is best done immediately after removal of the gland while the mouse remains anesthetized. This avoids a second surgery, which would otherwise be required because precise delivery of cells into the fat pad by injection through the skin is not possible. The age of the recipient dictates the surgery schedule, not the availability of cells, because after 3 and 1/2 weeks to 4 weeks of age, epithelial growth within the mammary gland fat pad is too extensive to permit complete removal. Proliferation of residual host epithelial cells would then complicate any interpretation of transplanted cell growth.

Mesenchymal cells require specific media; do not substitute traditional culture medium (e.g., DMEM), as this will permit growth of other cell types. It must be cautioned that there is as yet no universally accepted distinct antigen profile for mesenchymal cells, so any purification procedures may include cells or populations with other lineage fates. It would benefit the investigator to monitor the literature for reports that may suggest a new and distinct antigen profile for mesenchymal cells in the context of mammary gland biology. Several groups have established procedures to isolate mesenchymal cells from different sources and these populations differ in their antigenic profiles.

 

Troubleshooting

The establishment of cancer stem cell clones (immortal cell lines) from PyVT mammary gland tumors may take multiple attempts, due to the rarity of epithelial stem cells in any given tissue sample. Initial cell fractions from these tumors should be very conservatively sorted, such that there is no reasonable chance of including ungated cells. Sorting with high stringency allows for the greatest confidence in data generated by the cells in later experiments. One must confirm differentiation of mesenchymal stem cells early on in passaging to prevent expansion and investigation of stem cell or other potential of cell cultures that may not be enriched enough.

When collection of bone marrow cells from multiple femurs and tibias is desired, first form holes in all bone ends with one 25-G needle mounted onto a syringe. Penetrating the bone ends with a needle frequently plugs the needle orifice with bone fragments or damages the bevel, which blocks dispensing of syringe contents (but does not prevent one from drilling the holes). This will avoid switching to a new needle with each bone.

 

Anticipated Results

The collection of only one or two mammary gland tumors from a PyVT mouse can be enough to yield a stem cell line. On the other hand, several mice may be sampled with no long-term cultures surviving. In general, sampling of ten mice with tumors should result in the successful establishment of at least one immortal cell line from which epithelial cells can be isolated. Mesenchymal cells from mammary gland typically grow more slowly than epithelial cells, but the chances of establishing a line from, e.g., ten mice, are similar. Transplanted cells in high numbers (e.g., >10⁵) usually yield tumors within 6 to 8 weeks. Lower numbers can take weeks to months, depending upon stem cell activity and dose. Bone marrow–derived mesenchymal cells are typically slow to expand and obtaining numbers sufficient for sorting and purifying (i.e., passage 3) can take 2 to 3 months.

 

Time Considerations

If a colony of PyVT mice is to be established by an investigator, it will take a minimum of 12 to 14 weeks between receipt of mice to the point of obtaining useful mammary gland tumors. Dissection of mammary gland tumors, dissociation into single cell suspensions, labeling, and flow cytometry/sorting can be achieved in 1 day. If yield of sorted cells is sufficient, cells could be transplanted immediately. However, if large numbers of cells are required, culturing for 4 to 8 weeks may be necessary. This is particularly true for mesenchymal cells, which grow more slowly. The development of transplanted cells into tumors is strictly dependent upon the tumorigenic potential and cell number. Injecting cells in moderate to large numbers (on the order of 1 × 10⁴ to 1 × 10⁶) can yield tumors in a matter of weeks, while injecting single cells could conceivably take months to develop into tumors. The surgical preparation of cleared mammary gland fat pads in 10 to 20 mice and the transplant of cells into the cleared pad can be accomplished in 1 day.

 

Acknowledgments

The authors are very grateful for support provided by NIH grants 1RO1AGO23510-01A1 and 1RO1CA112481-01A1.