Chapter 21 - Springer Link

Chapter 21 In Vitro Assays of Breast Cancer Stem Cells Debangshu Samanta and Gregg L. Semenza Abstract Aldehyde dehydrogenase and mammosphere assays enable the cost-effective quantification and characterization of cancer stem cells (CSCs) from cancer cell lines as well as cancer tissue. Here we describe the quantification of CSCs in breast cancer cell lines using aldehyde dehydrogenase and mammosphere assays under hypoxic (1% O2) and non-hypoxic (20% O2) culture conditions. Using this method, a significant enrichment of CSCs compared to bulk populations is observed when breast cancer cells are exposed to 1% O2 for 72 h. Key words Cancer stem cells (CSC), Tumor-initiating cells, Breast cancer, Hypoxia, Self-renewal, FACS, Hypoxia-inducible factors (HIFs)

1 Introduction Breast cancer mortality usually occurs when cancer cells metastasize and become resistant to chemotherapy. Breast cancer stem cells (BCSCs), which are also called tumor-initiating cells, represent a subpopulation that is central to both of these processes. Although many breast cancer cells enter the circulation, only BCSCs are capable of forming a secondary tumor [1, 2]. Intratumoral hypoxia is common in most solid tumors, including breast, cervical, and head/neck cancers [3]. Hypoxia induces metastatic properties and the BCSC phenotypes through transcriptional activation of target genes by hypoxia-inducible factor 1 (HIF-1) and HIF-2 [4]. The gold standard assay of CSC self-renewal is the demonstration that in vivo transplantation with limiting cell numbers results in tumor formation in recipient mice, an assay that is time-consuming and expensive. Reliable in vitro assays are necessary to efficiently and cost-effectively quantify stem cells. In this chapter, we describe two methods that are used routinely to quantify BCSCs, namely, the aldehyde dehydrogenase [5] and mammosphere [6] assays. These assays identify populations of cells that are enriched by several orders of magnitude for tumor-initiating cells as compared to the bulk cancer cell population.

L. Eric Huang (ed.), Hypoxia: Methods and Protocols, Methods in Molecular Biology, vol. 1742, https://doi.org/10.1007/978-1-4939-7665-2_21, © Springer Science+Business Media, LLC 2018

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One widely accepted method for identifying cancer stem cells in vitro is based on the enzymatic activity of aldehyde dehydrogenase 1 (ALDH1) [7], which was first used to identify hematopoietic stem cells and which may regulate stem cell differentiation through the metabolism of retinol to retinoic acid [8, 9]. The commercially available ALDEFLUOR™ assay employs BODIPY-aminoacetaldehyde (BAAA), which freely diffuses into intact and viable cells and, in the presence of ALDH1, is converted to the fluorescent product BODIPYaminoacetate, which is retained inside the cells. The amount of fluorescent reaction product is proportional to the ALDH1 activity in the cells and is measured using a flow cytometer. The assay can be used to isolate a subpopulation of cells that display stem cell properties from normal human breast tissue or breast carcinomas [5]. The mammosphere assay is a modification of the neurosphere assay, which was developed to quantify neural stem cells [10]. Dontu and colleagues developed an in vitro culture system that allowed for propagation of human mammary epithelial cells (HMECs) under non-adherent culture conditions [6, 11]. Cells capable of surviving and proliferating in such conditions formed discrete clusters of cells termed “mammospheres.” Such spheroids were enriched in progenitor cells capable of differentiating along multiple lineages, including luminal, myoepithelial, and alveolar cells. Secondary mammospheres generated from harvesting, digesting primary mammospheres into single cells, and replating can be used to demonstrate self-renewal capacity [12]. Several other assays have been used to identify or isolate cells with CSC properties, such as side population cells, which are characterized by the exclusion of Hoechst dye, and CD44+CD24− cells, which mark BCSCs in luminal-type breast cancers [13–15]. However, in basal-type cancers and cell lines, the majority of cells are CD44+CD24−, such that this assay is not a reliable marker for BCSCs. Hypoxia has been shown to increase the percentage of BCSCs, both in vitro and in vivo through the activity of HIF-1, which activates the transcription of multiple genes that promote the BCSC phenotype [16–22]. Hypoxia may also induce the BCSC phenotype via HIF-1-independent mechanisms [23, 24].

2 Materials Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18 MΩ cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials. We do not add sodium azide to reagents.

In Vitro Assays of Breast Cancer Stem Cells

2.1 Media

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All breast cancer cells are cultured in their recommended media as published in [15]. 1. DMEM (1×). 2. RPMI-1640 (1×). 3. DMEM/F12 (50:50). 4. Penicillin/Streptomycin. 5. Heat-inactivated BenchMark fetal bovine serum (FBS). 6. Hank’s balanced salt solution (HBSS) containing 2% FBS. 7. 0.05% Trypsin-EDTA (1×). 8. Prepare 1 L of 1× PBS as follows: Start with 800 mL of distilled water and then add the following: 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4. Adjust the pH to 7.4 with HCl. Finally, add distilled water to a total volume of 1 L. Autoclave and cool to room temperature before use. 9. Add appropriate amount of FBS (5–10% v/v, depending on the cell line) and penicillin/streptomycin (1% v/v) to the medium and store at 4 °C. 10. Prior to use, warm the medium and trypsin in a water bath maintained at 37 °C for 10 min.

2.2 ALDH Assay

1. ALDEFLUOR Kit (StemCell Technologies). 2. 5-mL polystyrene round-bottomed tube with cell strainer (35 μm) cap (Corning).

2.3 Mammosphere Assay

1. MammoCult™ Medium (StemCell Technologies). 2. Ultralow adherence dishes (6-well plates). 3. Heparin. 4. Hydrocortisone.

2.4 Hypoxia

1. Modular Incubator Chamber. 2. Gas cylinders containing mixture of 5% CO2, 1% O2, and balance N2.

3 Methods Carry out all procedures at room temperature unless otherwise specified.

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3.1 ALDEFLUOR Kit Preparation

1. Allow kit reagents to come to room temperature (15–25 °C) before use for the first time. 2. Add 25 μL of DMSO to the ALDEFLUOR reagent bottle, pipette up and down, and let it stand for 1 min at room temperature (see Note 1). 3. Add 25 μL of 2 N HCl (supplied) to the ALDEFLUOR reagent vial and pipette up and down several times (see Note 2). 4. Incubate this mixture at room temperature for 15 min. 5. Add 360 μL of ALDEFLUOR Assay Buffer (supplied) to the vial and mix well making the total volume of ALDEFLUOR reagent 410 μL. 6. Aliquot the ALDEFLUOR reagent into separate microcentrifuge tubes (depending on your experimental plans) and store at −20 °C. Avoid repeated freezing and thawing of aliquots (see Note 3).

3.2 Preparation of Complete Mammosphere Medium

3.3 Exposure of Cells to Hypoxia

1. Prepare complete MammoCult Medium (Human) by adding 50 mL of thawed MammoCult Proliferation Supplements to 450 mL of MammoCult Basal Medium. 2. Add the following to obtain complete medium:

(a) Heparin to 4 μg/mL (0.0004%).

(b) Hydrocortisone to 0.48 μg/mL (add 100–500 mL of medium).

1. Plate appropriate number of cells so that they are 85–90% confluent at time of harvesting. Determine the number of cells before so that after induction of hypoxia for 72 h, the cells are not overconfluent (see Note 4). 2. The next day aspirate culture medium from the dish and replenish with fresh medium appropriate to each cell line. 3. Induce hypoxia in the cells by placing them in the modular incubator. All components of the modular incubator are nontoxic and can be alcohol-sterilized. 4. In one of the two outlets of the modular incubator, connect with the nozzle from cylinder with air containing 5% CO2, 1% O2, and balance N2. Keep the other outlet free so that the air goes out from the other nozzle. The air pressure in the cylinder should not exceed 2 p.s.i. Continue flushing the air in the modular incubator containing 20% O2 for 3 min. 5. First close the outlet of the modular chamber which was letting the air out. Only after you have secured this outlet, close the inlet quickly (see Note 5).


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6. Seal the Chamber and place at 37 °C in your laboratory incubator or warm room. 7. For checking the BCSC phenotype, incubate the cells under hypoxia for 72 h. Afterward, take out the modular incubator. First release either one of the two valves. When the pressure is released, release the stainless steel ring clamp and open the incubator (see Note 6). 3.4 Cell Sample Preparation

1. Aspirate culture medium from the dish and wash the cells with 1× PBS at room temperature (see Note 7). 2. Add 1× 0.05% trypsin to cover the entire flask. Gently rock the flask to get complete coverage of the cell layer (see Note 8). 3. Place the flask in the tissue culture incubator for 2 min. 4. Observe the cells under the microscope for detachment. If cells are less than 90% detached, increase the incubation time a few more minutes, checking for dissociation every 30 s. You may also tap the vessel to expedite cell detachment. 5. Add a volume of medium containing FBS that is at least 5 times the volume of trypsin to stop the trypsin from acting. 6. Homogenize the mixture by pipetting up and down several times. 7. Determine the total number of cells and percent viability using a hemocytometer, cell counter and trypan blue exclusion, or the Countess automated cell counter (Invitrogen).

3.5 ALDEFLUOR Assay

1. Aliquot from 2.5 × 105 to 1 × 106 cells into pre-labeled microcentrifuge tubes. (see Notes 9 and 10). 2. Label one “test” and one “control” tube for each sample to be tested. 3. Label an additional tube as the unstained as negative control. 4. Centrifuge the cells 250 × g at room temperature for 5 min. 5. Aspirate the supernatant and wash the cells with 1 mL of PBS. 6. Centrifuge the cells 300 × g at room temperature for 5 min and aspirate the supernatant, ensuring that the cell pellet is not disturbed. Carefully aspirate and discard the supernatant with a pipette. 7. Dissolve the pellet in 500 μL of ALDEFLUOR Assay Buffer. 8. For 2.5 × 105 cells, add 1 μL of the activated ALDEFLUOR reagent to each tube (see Note 11). 9. For the control tubes, add 2 μL of ALDEFLUOR DEAB reagent (see Note 12). 10. Place the cells in a tissue culture incubator at 37 °C for 45 min (see Note 13).

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11. Following incubation, centrifuge all tubes for 5 min at 250 × g and remove the supernatant. Resuspend the cell pellets in 0.5 mL of ALDEFLUOR™ Assay Buffer. 12. Pass the cells through the cell strainer into the 5-mL polystyrene round-bottomed tube (see Note 13). 13. Analyze the cells using a flow cytometer. 3.6 Flow Cytometer Setup

1. Create a forward scatter (FSC) vs. side scatter (SSC) dot plot. 2. In setup mode, place your unstained tube on the cytometer. 3. Adjust the FSC and SSC voltage, such that the majority of the nucleated cell population is at the center of the FSC vs. SSC plot. 4. Gate on all nucleated cells, excluding debris (R1). 5. Create a FITC vs. SSC dot plot, gated on R1. 6. Adjust the FITC photomultiplier tube voltage such that there is significant shift along the FITC axis between the unstained and DEAB stained sample. 7. Align the rightmost edge of the stained DEAB control population with the second log decade on the FITC axis. 8. Keep the FSC, SSC, and FITC voltages constant for the remaining of the experiment. 9. Change the cytometer from setup to active mode, and collect at least 50,000 events in R1 for each DEAB and sample tube, without changing the instrument settings (Fig. 1).

3.7 Primary Mammosphere Assay

1. Resuspend a predetermined number of cells in 2 mL of complete MammoCult Medium (in triplicate) in each well of a 6-well ultralow adherent plate. The seeding density for cell lines is typically in the range of 5000–20,000 cells per well. 2. Incubate cultures in a 5% CO2, humidified incubator at 37 °C for 7 days without replenishing the medium (see Note 14). 3. Photograph the mammospheres under an Olympus TH4-100 microscope with 4× apochromat objective lens (Fig. 2). 4. Determine the mammosphere number and volume using ImageJ software. 5. Count the mammospheres with area > 500 pixels in images of three fields per well in triplicate wells and determine the mean number of mammospheres per field (see Note 15).

3.8 Secondary Mammosphere Assay

1. Harvest mammospheres after 7 days in culture. Collect the entire culture from three wells into a 15-mL conical tube and centrifuge at 87 × g for 5 min. 2. Aspirate as much supernatant as possible with a pipette without disturbing the pellet.

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Fig. 1 Typical FACS plot of ALDEFLUOR Assay. In the top panel shows the plot of Forward Scatter Cells (FSC) vs. Side Scatter Cells (SSC). Only the single cell population is gated P1 for further analysis. In the bottom panel, the gated cells are plotted as SSC vs. FITC, since excitation wavelength of the activated ALDEFLUOR reagent is 488 nm which corresponds to FITC. Note how in the middle panel (control) the gate P2 is put at the rightmost edge of the population. Any cells having more fluorescence would be scored positive. The ALDEFLUOR positive cells are colored green

Fig. 2 Micrographs demonstrating the characterization of mammospheres derived from breast cancer cell lines MDA-231, SUM-159 and MCF-7, (4×) 7 days after plating. 5000 MDA231; SUM159 and 10000 MCF7 cells were plated. All scale bars are 2 mm

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3. Add 0.5 mL of pre-warmed Trypsin-EDTA for each tube. 4. Break up the mammospheres into individual cells by passage in and out of a 25-gauge syringe three times (see Notes 16 and 17). 5. Add 5 mL of cold HBSS containing 2% FBS and centrifuge the cell suspension at 87 × g for 5 min. 6. Aspirate the supernatant and resuspend the pellet in 0.5 mL of complete MammoCult Medium. 7. Perform the cell count using Trypan Blue. 8. Plate the same number of live cells as was plated to generate the primary mammospheres. 9. Incubate cultures in a 5% CO2, humidified incubator at 37 °C for 7 days without replenishing the medium (see Note 14). 10. Photograph and quantify the number of secondary mammospheres as described for the primary mammospheres.

4 Notes 1. The dry ALDEFLUOR reagent is an orange-red powder that changes to a bright yellow-green color upon addition of DMSO. 2. 2 N HCl must be added after addition of DMSO. 3. When frozen aliquots (−20 °C) of the activated ALDEFLUOR reagent are thawed, a small precipitate may be observed. Before use, mix the thawed reagent to resuspend the precipitate. This precipitate does not affect assay performance. 4. Confluent cultures will have increased cell death. 5. If you are not fast, then the pressure might build up in the chamber which might lower the longevity of the incubator. 6. One indicator for the proper hypoxia induction is that the medium would turn orange which denotes the medium slightly turns acidic. 7. HBSS can also be used for washing the cells. 8. Instead of trypsinization, use a sterile cell scraper to gently scrape cells from the dish. 9. Fresh or previously frozen samples can be analyzed for ALDEFLUOR assay. However, ALDH activity will only be detected in viable cells. The assay can also be performed on blood samples and hematopoietic cells (e.g., peripheral blood, apheresis product, and bone marrow or cord blood). 10. If using blood samples where the red blood cell to leukocyte ratio (RBC/WBC) of the specimen is >2:1, lyse the erythrocytes with ammonium chloride solution (StemCell Technologies).


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11. For human breast cancer cell lines MDA-MB-231, MCF-7, SUM159, and SUM-149, this volume of ALDEFLUOR reagent works well. For other cell lines, the optimal volume of ALDEFLUOR reagent used for the experiment should be determined. 12. DEAB (diethylaminobenzaldehyde) is a specific inhibitor of ALDH. It is used to control for background fluorescence. 13. Optimal incubation times may vary between different cell types and should be determined. Do not exceed an incubation time of 60 min. 14. After dissolving the cells in ALDEFLUOR buffer, take measurement as soon as possible. Keep the cells chilled (2–8 °C or on ice) until measurement to slow down the product efflux. 15. The number of days in culture must be kept to a minimum and optimized for each cell type. Plates must not be moved during the culture period. 16. Primary and secondary mammospheres can also be quantitated using the size of the mammospheres, such as greater than 50-μm diameter. 17. More sensitive cells may simply require gentle pipetting rather than syringe passage to obtain a single cell suspension. Make sure that the cells are not overexposed to trypsin.

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