Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

Summary

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

Abstract

Despite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a "side population" (SP). As the SP cells represent a low percentage (<5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44^high) and the over-expression of aldehyde dehydrogenase (ALDH^high). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of β-catenin.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy worldwide and despite progress in current treatments, patients with advanced disease have a poor prognosis. The overall 5 year survival rate of the patient is around 30% despite the combination of therapeutic approaches including surgery, chemo-radiotherapy and targeted-therapies. Recent studies attribute local recurrence and distant metastasis to the survival of cancer stem-like cells (CSCs) following anticancer therapies¹. There is accumulating evidence supporting the existence of cells presenting stem cells properties (undifferentiated status, self-renewal and differentiation capacities, and telomerase activity) in various solid tumors including breast, brain, prostate, lung, colon, pancreas, liver and skin^2-10. However, the origin of CSCs remains unclear^11,12. They may result from the malignant transformation of normal stem cells^3,13 or dedifferentiation of tumor cells that acquire CSCs-like features^14,15. Therefore, understanding distinctive pathways relating to CSCs will provide insight into early diagnosis and treatment of resistant HNSCC.

It has been proposed that CSCs also possess resistant phenotypes that evade standard chemotherapy and radiotherapy, resulting in tumor relapse compared to the bulk of tumor cells^16-19 and are localized into hypoxic niches²⁰. Numerous factors have been proposed to explain these resistances of CSCs, such as propensity to quiescence, enhanced DNA repair, up-regulated cell cycle control mechanisms, and free-radical scavenging²¹. Moreover, several oncogenic molecular pathways may be specifically activated in CSCs¹⁷. In order to improve knowledge of CSCs for further targeted-therapies, we need reliable methods for the identification and isolation of CSCs, owing to the heterogeneity of stem cell-related markers in various types of cancers²².

In HNSCC, stem-like tumor-initiating cells have been isolated from primary patient tumors by sorting cells expressing different CSC biomarkers (such as drug efflux transporters expression²³, CD44^high, CD24^low CD133^high, c-Met⁺ phenotype^10,24,25, or ALDH^high activity²⁶) or cultivating primary patient tumor to form squamospheres that have CSC properties. Nevertheless, the number of squamospheres decreases dramatically after two passages, thus giving a small sample size for further characterization studies²⁷. Therefore, in vitro assays starting from well-established cell lines is an easier solution to design experiments in order to improve knowledge of CSCs.

The aim of this article is to propose a method to isolate CSCs from HNSCC cell lines using multiparametric flow cytometric analysis and cell sorting. The expression of CD44 in correlation with several CSCs properties including ALDH activity, Side Population (SP) phenotype, spheroid formation ability and tumorigenicity are used to isolate and characterize this sub-population of CSCs. CD44, a cell-surface glycoprotein, is involved in cell adhesion and migration. CD44 is highly expressed in many solid tumors CSCs²⁸, including in head and neck cancer models^29-31. Moreover, CD44^high cells can generate in vivo a heterogeneous tumor whereas CD44^low cells cannot¹⁰. The SP assay is based on the differential potential of cells to efflux the Hoechst dye²² via the ATP-binding cassette (ABC) family of transporter proteins overexpressed within the CSC membrane. This assay includes the use of ABC transporter inhibitors such as verapamil in control samples. Aldehyde dehydrogenase (ALDH) is an intracellular enzyme that is involved in converting retinol to retinoic acid during early stem cell differentiation^25,26. Cells that exhibit high ALDH activity show stem-like cell behavior in HNSCC²⁶ and a very few number of ALDH^high cells are able to generate tumors in vivo^26,32.

The combination of these markers and properties was successfully used by Bertrand et al. to study the resistance in vitro and in vivo of these CSCs to photon and carbon ion radiation¹⁹. Their results clearly showed that the combination of various cell markers and properties are more selective for useful studies on HNSCC CSCs populations than single-marker approaches.

Protocol

All animal procedures were performed according to local guidelines on animal care. All the details of this study were approved by the CECCAPP, a French ethics committee.

1. Selection of a Side Population (SP) by the Hoechst Dye Efflux Assay

1. Staining 50 million cells with Hoechst 33342 dye.
   1. Prepare two 15 ml sterile tubes with a conical bottom: one tube labeled "Hoechst" and one labeled "Hoechst and Verapamil". Prepare 10 ml of 5 mM Verapamil hydrochloride solution in sterile water. Prepare the culture medium (CM) for stem cells.
      1. To prepare CM for CSC (CM-CSC), combine the following: Dulbecco's modified Eagle medium (DMEM):F12 (1:3, v:v), 5% of fetal calf serum (FCS), 0.04 mg/L of hydrocortisone, 100 U/ml of penicillin, 0.1 g/L of streptomycin, and 20 µg/L of Epidermal Growth Factor (EGFR).
   2. Under a laminar flow hood, trypsinize cells. Remove media, wash with sterile phosphate buffered saline (PBS) and add 1 ml of trypsin-EDTA (0.5 g/L) for a 75 cm² flask. Incubate 3 min at 37 °C. Stop the reaction by adding culture medium used for the parental cell line. Count the number of cells using a cell counter.
      1. To prepare CM for parental cell line (CM-P), supplement DMEM with 10% FCS, 0.4 mg/L of hydrocortisone, 100 U/ml of penicillin and 0.1 g/L of streptomycin).
   3. Dilute the cell suspension obtained in the step 1.1.2 to obtain 10⁷ cells/ml in CM-P. Split the cell suspension in the 2 tubes prepared: put 100 µl (10⁶ cells) in the tube "Hoechst and Verapamil" and 4 ml (4 x 10⁷ cells) in the tube "Hoechst".
   4. In the sample "Hoechst and Verapamil", add 10 µl of 5 mM Verapamil hydrochloride solution (final concentration: 0.5 mM) and mix gently. Add 5 µl of 1 g/L Hoechst solution (final concentration: 0.1 g/L). In the sample "Hoechst", add 5 µl of 1 g/L Hoechst solution per 10⁶ cells (200 µl total). Henceforth, keep samples protected from direct light exposure using aluminum foil.
   5. Incubate all tubes in a water bath at 37 °C for 1 hr 30 min. Mix gently every 15 min to prevent cells from settling down.
   6. Centrifuge tubes at 250 x g for 5 min at 4 °C. Remove supernatant and resuspend each pellet with 2 mL of 1x PBS.
   7. Centrifuge tubes at 250 x g for 5 min at 4 °C. Remove supernatant. Resuspend "Hoechst and Verapamil" pellet in 500 µl of 5 mg/L propidium iodide (PI) diluted in PBS buffer and "Hoechst" pellet in 4 ml of 5 mg/L PI diluted in PBS buffer.
   8. Transfer sample solutions through a 70 µm cell strainer to remove aggregates and collect single cells in tubes for sample uptake on the flow cytometer. Keep samples on ice and protect from direct light exposure using aluminum foil.
2. Isolation of the Side Population Excluding Hoechst dye by Cell Sorting.
   1. Perform Hoechst negative cell separation on a flow cytometry sorter with the following parameters: UV laser (355 nm), 2 detectors on the UV laser path with blue Hoechst (450/50 BP) and red Hoechst (610/20 BP) filters, and 2 collectors.
      1. First, analyze sample "Hoechst" that serves as a positive control for staining and flow cytometer set-up.
      2. Using the cytometer software, on the "Global Worksheet" window, click on "Dot Plot" (fifth top right picture) and create a chart on the global worksheet. On ordinate, with a right click, select FSC-A (forward scatter) and on abscissa, SSC-A (side scatter) (Figure 1A). In the same way, create a SSC-W versus SCC-H dot plot. On this second dot plot, to create a P1 region, click "Polygon gate" (fourteenth top right picture) (Figure 1B)³³.
         Note: The P1 region will encompass single cells and discriminate doublets.
      3. Optional: Exclude PI positive cells by creating a FSC-A versus PI gated on P1. On this dot plot, select the PI negative population (P2) to exclude PI positive dead cells.
      4. Using the cytometer software, on the "Global Worksheet" window, click on "Dot Plot" and create a chart on the global worksheet. On ordinate, with a right click, select blue Hoechst-A and on abscissa, red Hoechst-A. With a right click on the population, select "Show population" and P1. On this dot plot, create a region (P2) to select the negative Hoechst dye side population (SP) cells that appears as a side arm on the left of the main population of cells (Figure 1C).
   2. Analyze the sample "Hoechst" and collect 10,000 events. To ensure that the gate P2, which represents the SP population, is well positioned, analyze the sample "Hoechst and Verapamil" (10,000 events) to observe the disappearance of SP population (Figure 1D).
   3. Collect the SP Hoechst dye negative cells in a 15 ml tube containing 1 ml of CM-CSC prepared in 1.1.1.1.
   4. At the end of the cell sorting, centrifuge the cell suspension at 250 x g for 5 min, remove the supernatant and resuspend the pellet with 1 ml of CM-CSC. Count the number of sorted cells using a cell counter and transfer appropriate number of cells to a culture flask (see Table 1). Add CM-CSC and incubate at 37 °C and 5% CO₂ atmosphere.
   5. Maintain cells in culture under the same conditions for a maximum of 2 passages until there is a minimum of 5 x 10⁷ cells (see step 3, "Cell culture method").

2. Selection of the CD44^high/ALDH^high Sub-population in the Side Population Sorted

1. Staining 50 million of SP Cells with the ALDH Detection Kit and CD44 Antibody.
   1. Prepare seven 15 ml sterile tubes labeled as follows: "Unstained" (Tube a), "CD44-APC" (Tube b), "IgG1-APC" (Tube c), "ALDH" (Tube d), "ALDH and DEAB" (Tube e), "ALDH and CD44-APC" (Tube f), "ALDH, DEAB and CD44-APC" (Tube g). Keep all tubes and reagents at 4 °C during the staining.
   2. Prepare buffer A with 4.5 ml of the buffer 1 (contained in the kit) and 45 µl of CD44-APC (Allophycocyanin) antibody (dilution 1:100). Prepare buffer B with 100 µl of buffer 1 and 1 µl of IgG1-APC antibody (dilution 1:100). Prepare buffer C with 4 ml of buffer 1 and 20 µl of the reagent of the kit.
   3. Prepare a cell suspension of the previously sorted cells (step 1.2.5) at 10⁷ cells /ml. Add 100 µL (10⁶ cells) of the cell suspension to tubes a, b and c, and 4 ml (4 x 10⁷ cells) to tube f. For the moment do not put cells in tubes d, e and g.
   4. Centrifuge the tubes containing the cells at 250 x g for 5 min. Remove the supernatant and resuspend the pellets in tubes a, b and c in 100 µl of buffer 1. Add 5 µl of diethylaminobenzaldehyde (DEAB), an ALDH inhibitor to tubes e and g.
      1. Resuspend cells in tube f with 4 ml reagent C and immediately transfer 100 µl into tubes d, e and g. Incubate all tubes in a water bath at 37 °C for 30 min and protect from direct light exposure using aluminum foil. Mix the cell suspension gently by vortexing after 15 min to prevent cells from settling.
   5. From this moment, keep the tubes on ice and protected from direct light exposure using aluminum foil. Centrifuge all tubes at 250 x g for 5 min at 4 °C and remove the supernatant.
   6. Resuspend tube f in 4 ml and tubes b and g with 100 µl of buffer A. Resuspend tube c with 100 µl of buffer B. To the other tubes, add 100 µl of buffer 1. Incubate 10 min at 4 °C.
   7. Centrifuge all tubes at 250 x g for 5 min at 4 °C and remove the supernatant. Rinse once with 1 ml of buffer 1 (4 ml for tube f) and centrifuge again at 250 x g for 5 min at 4 °C. Remove the supernatant. Re-suspend tube f pellet in 4 ml and all other tubes in 1 ml of buffer 1.
   8. Transfer the sample solutions through 70 µm cell strainers to remove aggregates and collect single cells in appropriate tubes for sample uptake on flow cytometer.
2. Isolation of the CD44^high/ALDH ^high Cell Population by Fluorescence Activated Cell Sorting.
   1. Perform CD44^high/ALDH ^high cell sorting on a flow cytometry sorter with the following parameters: Blue laser (488 nm) and red laser (633 nm), 1 detector on the blue laser path with a FITC filter (530/30), 1 detector on the red laser path with an APC filter (660/20) and 2 collectors.
   2. Using the cytometer software, click on "Dot Plot" (the fifth top right picture) to create a FSC-A versus SSC-A dot plot as described in section 1.2.1.2, to check cell morphology and select a population composed of single cells with a SSC-W versus SSC-H dot plot.
   3. Using the cytometer software, on the "Global Worksheet" window, click on "Dot Plot" and create a chart on the global worksheet. On ordinate, with a right click, select APC-A and on abscissa, FITC-A in order to select the double stained population. Create a gate using tubes d and e to select ALDH^high cells (Figure 2A). Note: Positive cells disappear in tube e treated with DEAB (Figure 2B).
      1. On the same chart, create a second gate using tubes b and c in order to select CD44^high cells (Figure 2C and 2D). Positive cells disappear in the tube containing IgG1-APC. Analyze tubes f and g and create a third gate that includes CD44^high/ALDH^high cells (Figure 2E and 2F).
         Note: If positive cells are present in the tube containing IgG1-APC (Figure 2D), the interaction between cells and the APC-stained antibody is not specific.
   4. Collect CD44^high/ALDH^high cells into a 15 ml tube containing 1 ml of CM-CSC. Collect also CD44^low/ALDH^low into a 15 ml tube containing 1 ml of CM¹⁹. Note: Prepare CM-CSC as described in step 1.1.1.1.
   5. Centrifuge the cell suspension at 250 x g for 5 min, remove the supernatant and resuspend the pellet with 1 ml of CM-CSC. Count the number of sorted cells.

3. Cell Culture Method

1. After the double-sorting of the cells as described above, plate the sorted cells into an appropriate culture flask (Table 1) with CM-CSC at 37 °C with 5 % CO₂. After 18-24 hr, check if the cells have adhered and change the culture medium. Change the culture medium every 3 days until the expanding colonies are greater than 50% confluent.
2. Use the appropriate volume of trypsin 0.5 g/L - EDTA (Table 1) to trypsinize cells from the culture flask. Incubate cells 3 to 5 min at 37 °C. Add the appropriate volume of CM-CSC (Table 1) to stop the action of trypsin.
3. Count the number of cells obtained and plate 4 x 10⁵ cells in a 175 cm² culture flask with CM-CSC. Incubate at 37 °C and 5 % CO₂. At this concentration, cells with a doubling time of 24 hr will be 70% confluent in 7 days.
4. Use CSCs for in vitro or in vivo experiments before they have undergone 3 passages.

4. Confirmation of Tumor Potential and CSC Characteristics

1. Tumor Sphere Formation to Confirm the tumor Potential of the CD44^high/ALDH^high Cells.
   1. Trypsinize cells as described in 3.2. Add 1 x 10⁶ cells to a 15 ml tube and centrifuge at 250 x g for 5 min. Remove the supernatant and re-suspend the pellet in a DMEM:F12 (3:1) medium FCS free, 20 ng/ml of rhEGF, 4 mg/L of heparin and 1x B27. Incubate cells in a 6 well low anchorage plates culture flask at 37 °C and 5% CO₂.
      Note: Use DMEM:F12 (3:1) medium containing 5% of FCS, 20 ng/ml of rhEGF, 4 mg/L of heparin and 1x B27 if cell do not grow without FCS.
   2. Observe the tumor sphere formation with an optical microscope from 4 to 10 days after seeding (Figure 3A).
      Note: Tumor sphere diameter should be more than 35 µm. CD44^high/ALDH^high cells will show a faster and more enhanced tumor sphere formation in terms of number and size compared to CD44^low/ALDH^low.
2. Evaluation of In Vivo Tumorigenicity After Subcutaneous Injection of CD44^high/ALDH^high Cells in NOD-SCID Mice.
   1. After sorting, re-suspend cells in PBS at three different concentrations (10⁴ cells/ml, 10⁵ cells/ml, and 10⁶ cells/ml). Inject subcutaneously 100 µl of 10⁴ cells/ml (10³ cells) in the right flank region of 6 mice. Do the same for 10⁵ cells/ml (10⁴ cells), and 10⁶ cells/ml (10⁵ cells).
      Note: Lower dilution than 10³ cells can be tested.
   2. Inject the same concentration of CD44^low/ALDH^low cells in the left flank region. Monitor injected mice for up to 10 weeks to see tumor progression¹⁹.

Results

The isolation of CSCs from HNSCC cell lines required two successive sorting because of the very low percentage of CSCs in the parental cell line. The first sorting was based on the ability of CSCs to exclude the Hoechst dye due to drug efflux transporters. This resulted in acquisition of 1-5% of the total cell population sorted (Figure 1). During the Hoechst dye negative cell sorting, check the size and granulation of sorted cells by looking at the FSC-A versus SSC-A dot ...

Discussion

This protocol describes a reliable method for the successful isolation of CSCs from a specific cell line that is applicable to other HNSCC cell lines. Isolated head and neck CSCs are then suitable for further molecular characterization in vitro and functional validation by transplantation in immunodeficient mice¹⁹. However, some modifications can be tested depending on the side population or the CD44^high/ALDH^high percentages present in the parental cell line. For example, if the ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal Calf Serum Gold	GE Healthcare	A15-151
Hydrocortisone water soluble	Sigma-Aldrich	H0396-100MG
Penicillin/Streptomycin 100x	Dominique Dutscher	L0022-100
DMEM	Gibco	61965-026
F12 Nut Mix (1x) + GlutaMAX-I	Gibco	31765-027
EGF	Promega	G5021	The solution must be prepared just before use because it is very unstable
Heparin	StemcellTM Technologies	7980
B-27 Supplement (50x), minus vitamin A	Gibco	12587-010
Hoechst 33342	Sigma-Aldrich	14533	Corrosive, acute toxicity (oral, dermal, inhalation) category 4
Verapamil hydrochloride	Sigma-Aldrich	V-4629	Acute toxicity (oral, dermal, inhalation) category 3
Propidium Iodide	Sigma-Aldrich	P4170	Acute toxicity (oral, dermal, inhalation) category 4
ALDEFLUOR Kit	Stem Cell	1700
CD44-APC, human antibody	Miltenyi Biotech	130-095-177
IgG1-APC, human antibody	Miltenyi Biotech	130-093-189
Z1 coulter particle	Beckman Coulter	6605698
Optical microscope	Olympus	CKX31
SQ20B cell line	Gift from the John Little’s Laboratory	-
FaDu cell line	ATCC	HTB-43
Low anchorage plates	Thermo Fischer Scientific	145383
BD FACSDiva software v8.0.1	BD Biosciences	-