Isolation of head and neck squamous carcinoma cancer stem-like cells in a syngeneic mouse model and analysis of hypoxia effect

  • Authors:
    • Sónia Duarte
    • Agnès Loubat
    • David Momier
    • Majlinda Topi
    • Henrique Faneca
    • Maria C. Pedroso de Lima
    • Georges F. Carle
    • Valérie Pierrefite-Carle
  • View Affiliations

  • Published online on: July 6, 2012     https://doi.org/10.3892/or.2012.1904
  • Pages: 1057-1062
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Abstract

The incidence of oral tumors is increasing around the world and despite recent advances in early detection and diagnosis, current treatments are still unsatisfactory. Recent data suggest that tumor persistence and recurrence could be due to the presence of a rare cell population called cancer stem cells (CSCs), which are generally spared by traditional treatments. Therefore, identification and characterization of CSCs are extremely important to develop novel and effective treatment strategies for cancer. The aim of this study was to identify and isolate CSCs in an established murine head and neck squamous cell carcinoma (HNSCC) cell line and to investigate the influence of hypoxic conditions on the isolated cell popul­ation. Using the expression of the aldehyde dehydrogenase 1 (ALDH1) enzymatic activity, which is now recognized as a CSC marker in various tumors, we isolated a cell population expressing high levels of ALDH1 (ALDH1high) representing 1±0.6% in the murine SCC-VII cell line. These cells were injected subcutaneously in syngeneic animals to evaluate their tumorigenic properties. For the lowest injected cell dose (250 injected cells), tumor occurrence and median tumor size were higher in ALDH1high injected mice than in ALDH1low injected mice. Following an in vivo passage and culture in serum-free medium, the percentage of ALDH1high cells increased by 3‑fold in SCC-VII CSCs (oral spheres) compared to the SCC-VII cell line. This percentage was further increased when oral spheres were cultured under hypoxic conditions. In conclusion, this study reports for the first time the isolation of HNSCC CSCs in a syngeneic mouse model and the use of hypoxia as a method to further enrich the ALDH1high cell population.

Introduction

Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common cancer in developed countries and is responsible for approximately 350,000 cancer deaths per year (1). In addition, the incidence of oral tumors is increasing around the world (2). Despite recent advances in early detection and diagnosis, current treatments of oral cancer are unsatisfactory mainly due to the development of distant metastasis and the emergence of treatment-resistant recurrences, resulting in an unchanged 5-year survival rate over the last two decades (3,4). It is now established that tumor persistence and recurrence could be due to the presence of cancer stem cells (CSCs) in some cancers. The cancer stem cell theory of carcinogenesis postulates that normal stem or progenitor cells can eventually become malignant because of the occurrence of genetic and epigenetic alterations (5). Based on their similarity to normal stem cells, CSCs exhibit extensive capability of self-renewal and are likely to be more resistant to therapy (5). Moreover, they have the ability to generate a tumor after injection of a very small number of cells and to reconstitute the cellular heterogeneity typical of the original tumor (6,7). The presence of CSCs has now been evidenced in solid tumors from various origins including head and neck (5,8,9). This rare HNSCC CSC population can be isolated by using cell surface antigens such as CD44 (8) or CD133 (10). The propensity of CSCs to efflux the vital DNA binding Hoechst 33342 dye, due to an increased expression of multiple drug resistance transporter proteins, has also been used as a means to isolate them as a side population (SP) (6). Alternatively, HNSCC CSCs can be isolated on the basis of the expression of the aldehyde dehydrogenase 1 (ALDH1) enzymatic activity (11,12), which is now recognized as a CSC marker in tumors from various origins (13). This last method was described as more specific as the isolated cells appear to be more tumorigenic than the CD44+ CSCs (12).

Although primary cancer cells obtained from patients represent the most physiologically relevant material to isolate CSCs, this source presents two main disadvantages. First, it constitutes a very limited resource due to the relatively small size of head and neck tumors and the small percentage of CSCs within the tumor. Second, the human cells need to be administered in immunocompromised animals and, depending on the degree of host immunocompetence, these xenotransplantation assays have been shown to underestimate or overestimate the frequency of cells with tumorigenic potential (14).

In the present study, the objective was to isolate ALDH1high cells in a murine established HNSCC cell line and to determine if these cells would exhibit cancer stem cell-like properties. In addition, we examined the effect of a hypoxic environment on the CSC population in vitro.

Materials and methods

Cell culture

The SCC-VII cell line is derived from murine oral squamous cell carcinoma and is able to induce progressive tumors in syngeneic hosts. SCC-VII cells were maintained at 37°C under 5% CO2, in Dulbecco’s modified Eagle’s medium (Invitrogen, Cergy Pontoise, France) supplemented with 10% fetal calf serum (FCS) (Dutscher, Brumath, France), penicillin (100 U/ml) and streptomycin (100 μg/ml).

The oralspheres (SCC-VII CSCs) were cultured on ultra-low attachment flasks (Corning, Sigma-Aldrich) in serum-free DMEM/F12 medium (Invitrogen) with penicillin (100 U/ml) and streptomycin (100 μg/ml) supplemented with 10 ng/ml bFGF (Peprotech), 20 ng/ml EGF (Peprotech), 4 μg/ml, heparin (Sigma-Aldrich), 4 mg/ml bovine serum albumin (Sigma-Aldrich), 20 μg/ml insulin (Sigma-Aldrich) and N2 supplement (Invitrogen). Oralspsheres were dissociated mechanically and enzymatically, using Accumax reagent (Sigma-Aldrich). This reagent that combines protease, collagenolytic and DNase activities was used to generate single cell suspension for ALDH1 labeling and flow cytometry analysis.

Hypoxia/normoxia

In normoxia, the oralspheres were grown in a humidified 21% O2 and 5% CO2 environment. For hypoxia experiments, the oralspheres were grown in a humidified 1% O2 and 5% CO2 environment using a MiniGalaxy A incubator (RS Biotech, Ltd). The medium was replaced twice per week after being prewarmed and equilibrated for 4 h in 1% O2.

ALDH1high cell selection

The ALDEFLUOR kit (StemCell Technologies SARL, Grenoble, France) was used to isolate the cell population with a high aldehyde dehydrogenase 1 (ALDH1) enzymatic activity. Single cell suspensions obtained from freshly trypsinized SCC-VII cells or dissociated oralspheres were suspended in ALDEFLUOR assay buffer. Then, 5 μl of ALDH substrate (BAAA) were added to the cell suspension and the samples were incubated during 40 min at 37°C. As a negative control for each sample of cells, an aliquot was treated with 5 μl of diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor. Fluorescence-activated cell sorting (FACS) was used to analyze the ALDH1high cell population. The sorting gates were established using the ALDEFLUOR-stained cells treated with DEAB.

Animals and tumor model

Six- to eight-week-old female C3H/HeOuJ mice (Charles River, L’arbresle, France) were used to assess the in vivo stem cell properties of the ALDH1high population, compared to the ALDH1low population. All the surgical procedures and the care given to the animals were performed in accordance with institutional guidelines. After anesthetization of the mice, 1,000 or 250 ALDH1high or ALDH1low SCC-VII cells (50 μl) mixed with 50 μl of Matrigel (BD Biosciences, NJ, USA) were injected subcutaneously using a 25-gauge needle. Tumor growth was monitored and after visual detection, the mice were sacrificed and tumor formation was assessed. Tumor volumes were estimated using the formula: length × width2 × π/6 (15).

Tumor dissociation

After excision, the tumors were washed in PBS and DMEM-F12 containing the penicillin/streptomycin (500 U/ml) and amphotericine B (1.25 μg/ml). Then, each tumor was cut into small fragments using sterile scissors and incubated at 37°C with DMEM F-12 containing 1.5 mg/ml collagenase I, 20 μg/ml hyaluronidase and 0.006% of DNase I. After 1 h of digestion, cells were filtered first through a 100-μm nylon sieve, followed by centrifugation at 1,000 rpm for 5 min and then filtered through a 40-μm nylon sieve and centrifuged again. The pellet was suspended in supplemented serum-free DMEM/F12 medium, as described above for culture of oralspheres.

Statistical analysis

The results are expressed either as median [95% confidence intervals (CI)] or as mean ± standard error. Comparison of tumor volumes between different mice groups was performed using the Mann-Whitney test, which is a non-parametric, two-tailed probability test. P-values were considered to be statistically significant when <0.05.

Results

Isolation of ALDH1high cells from the murine SCC-VII squamous cell carcinoma cell line

Using the ALDEFLUOR assay and FACS analysis, ALDH1high and ALDH1low cells were isolated from the SCC-VII cell line. Untreated cells exhibited some autofluorescence and the entry of the fluorescent ALDEFLUOR substrate within the cells shifted them to the P1 gate (Fig. 1A). In the presence of the ALDH1 enzyme, substrate cleavage increased its fluorescence, shifting the cells to the P5 gate, which was not observed in the presence of DEAB, the ALDH1 inhibitor. As expected, the majority of SCC-VII cells exhibited low ALDH1 activity, with the percentage of ALDH1high cells being 1±0.6%. Following sorting, ALDH1high cells were cultured in suspension in serum-free medium with bFGF and EGF. As shown in Fig. 1B, the ALDH1high cells were able to grow as spheres in this medium.

Tumorigenicity of ALDH1high and ALDH1low cells in syngeneic animals

To evaluate the tumorigenicity of ALDH1high and ALDH1low cells, the two cell populations were injected subcutaneously in two different doses (1,000 or 250 cells) in syngeneic C3H mice immediately after sorting. Fig. 2A shows tumor occurrence according to ALDH1 expression and number of injected cells. At the highest cell number, both ALDH1high and ALDH1low cells were able to induce tumors in 100% of cases. The median tumor volumes were not statistically different with 226.1 and 267.9 mm3 when generated by ALDH1low and ALDH1high cells, respectively (Fig. 2B). At the lowest number of injected cells, tumors were developed in 3 out of 5 mice after administration of ALDH1low cells and in 6 out of 7 mice in the case of ALDH1high cells. Although the differences in tumor size were not statistically significant, a 5-fold increase in the median volume of tumors generated by ALDH1high cells (368.4 mm3; 95% CI, 118–784) compared to the ones generated by ALDH1low cells (74.2 mm3; 95% CI, 0–333.4) was observed (Fig. 2C and D). Taken together, these results suggest that ALDH1high cell population is enriched in CSCs.

In vitro selection of oralspheres from ALDH1high derived-tumors

Following tumor dissociation, the cell suspension was cultured in serum-free medium with bFGF and EGF. Spheres, which we called oralspheres, formed by cells aggregates were able to grow exponentially in these conditions (Fig. 3A). To determine the differentiation potential of these cells, oralspheres were then cultured in standard medium in the presence of 10% serum. After 24 h of culture, undifferentiated cells in suspension attached to the plastic, gradually migrating from oralspheres and differentiating into large and adherent cells (Fig. 3B).

The percentage of ALDH1high cells was determined in oralspheres, following an in vivo passage, after 3 months of culture in serum-free medium. As shown in Fig. 3C, a slight increase in this percentage was observed with averages of 1% for the SCC-VII cell line and 3% for oralspheres.

Effect of a hypoxic environment on oralspheres in vitro

As CSCs are able to grow under extreme conditions, we then analyzed the effect of hypoxia on the growth of this population. To this aim, oralspheres were cultured under either normoxia (21% O2) or hypoxia (1% O2) for 8 weeks. As observed in Fig. 4A, after 15 days oralspheres did not exhibit large spheres under hypoxia as compared to those in normoxia. In addition, under hypoxic conditions, the number of cells decreased and some of them appeared to degenerate and die. The cells finally recovered after 4 weeks under hypoxia, and some spheres could be observed (Fig. 4B). As shown in Fig. 4C and D, comparison of the percentage of ALDH1high cells in oralspheres maintained in normoxia and hypoxia revealed a significant increase of the percentage of ALDH1high cells under the latter conditions with a mean of 6.7% (±1.3) vs. 2.6% (±1.9), under the former conditions (P<0.05).

Discussion

It has been suggested that CSCs could be one of the key determinants of treatment failure in HNSCC like in other cancer types (16,17). So far, most studies regarding HNSCC CSCs have been carried out in vitro with only a few studies performed in an in vivo environment. Developing in vivo models is needed to obtain a comprehensive understanding of the biology of these cells (18). In vivo experiments have generally been performed in human cancer cells and immunocompromised animals, and these xenotransplantation assays have been described to result in a great variability in the frequency of cells with tumorigenic potential, depending on the degree of host immunocompetence (14). For these reasons, we decided to isolate CSCs in a syngeneic HNSCC murine model. Although different markers are available for CSC isolation, their detection within the total tumor bulk remains a challenge (18). ALDH1 was shown to select HNSCC cells exhibiting high self-renewal capacity, chemo- and radioresistance (16), and ALDH1high cells appear to be more tumorigenic than the CD44+ CSCs (12). Using this approach in the SCC-VII cell line, we showed that ALDH1high cells represent approximately 1±0.6% of the population, which is in accordance with previously published results (12), while the majority of the cell population presented a low ALDH1 activity. Growth in serum-free conditions has been demonstrated as a suitable method by which HNSCC CSCs can be efficiently cultured in vitro in an undifferentiated state (6). This was indeed the case for the SCC-VII ALDH1high cells, in agreement with previously reported results (7,19). Following selection of ALDH1high and ALDH1low cells, the tumorigenic potential of both populations was evaluated in a syngeneic C3H immunocompetent mouse model for oral cancer. For the injection of 1,000 cells, tumor occurrences and volumes were not significantly different between ALDH1high and ALDH1low cells, suggesting that the highest tested cell dose did not discriminate the tumorigenic potential of the two populations. This observation also indicates that cells with a high tumorigenic potential were present in the ALDH1low cell population, strengthening the need for the identification of additional markers of the CSCs. The 250 cell transplantation assay allowed the discrimination between ALDH1high and ALDH1low cells for both tumor volumes and occurrences, suggesting that ALDH1high cells were enriched in CSCs. As expected, these cells were able to grow as spheres in serum-free medium and serum supplementation led to sphere adhesion and differentiation (20).

Stem cell niches are often located in anatomical regions characterized by hypoxic conditions, which are crucial for the maintenance of an undifferentiated state for stem cells from various origins (21). Das et al have shown that CSCs localize in hypoxic areas of solid tumors in vivo and that they migrate to areas of hypoxia in nude mice (22). These data reinforce the hypothesis that a CSC niche is characterized by a hypoxic environment. According to these findings, we analyzed the effect of hypoxia on the growth of the CSC population. Although SCC-VII CSCs lost their capacity to grow as spheres and some of them even degenerated during the first 15 days, they were then able to recover after an adaptation period. Finally, we compared the ALDH1high cell percentage after an 8-week culture in normoxia or hypoxia. The percentage of ALDH1high cells was significantly higher when the oralspheres were maintained in a hypoxic instead of a normoxic environment, which demonstrated that culturing these cells in hypoxia favoured the enrichment in ALDH1high cells. These results suggest that, as for colorectal cell line-derived CSCs (23), hypoxia is involved in the maintenance of the stem-like phenotype.

Overall, this study reports for the first time the isolation of HNSCC CSCs in a syngeneic mouse model and the use of hypoxia as a method to further enrich the ALDH1high cell population. These cells appear to be a suitable model to develop new therapeutic strategies aiming at eradicating relapses in HNSCC.

Acknowledgements

We would like to thank Dr Nathalie Mazure and Dr Jacques Pouyssegur (CNRS UMR 6543, Nice) for the opportunity to use the MiniGalaxy A incubator. We also thank Dr Pascal Staccini (Département Informatique Médicale, Faculté de Médecine, Nice) for statistical analysis of the results. This study was supported by the CNRS and the PESSOA-EGIDE Program (2010–2011), the Portuguese Foundation for Science and Technology and France Cancer. S.D. is recipient of a fellowship from the Portuguese Foundation for Science and Technology (SFRH/BD/39727/2007).

Abbreviations:

ALDH

aldehyde dehydrogenase

CSCs

cancer stem cells

DEAB

diethylaminobenzaldehyde

DMEM

Dulbecco’s modified Eagle’s medium

FACS

fluorescence-activated cell sorting

FCS

fetal calf serum

HNSCC

head and neck squamous cell carcinoma

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September 2012
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Spandidos Publications style
Duarte S, Loubat A, Momier D, Topi M, Faneca H, Pedroso de Lima MC, Carle GF and Pierrefite-Carle V: Isolation of head and neck squamous carcinoma cancer stem-like cells in a syngeneic mouse model and analysis of hypoxia effect. Oncol Rep 28: 1057-1062, 2012
APA
Duarte, S., Loubat, A., Momier, D., Topi, M., Faneca, H., Pedroso de Lima, M.C. ... Pierrefite-Carle, V. (2012). Isolation of head and neck squamous carcinoma cancer stem-like cells in a syngeneic mouse model and analysis of hypoxia effect. Oncology Reports, 28, 1057-1062. https://doi.org/10.3892/or.2012.1904
MLA
Duarte, S., Loubat, A., Momier, D., Topi, M., Faneca, H., Pedroso de Lima, M. C., Carle, G. F., Pierrefite-Carle, V."Isolation of head and neck squamous carcinoma cancer stem-like cells in a syngeneic mouse model and analysis of hypoxia effect". Oncology Reports 28.3 (2012): 1057-1062.
Chicago
Duarte, S., Loubat, A., Momier, D., Topi, M., Faneca, H., Pedroso de Lima, M. C., Carle, G. F., Pierrefite-Carle, V."Isolation of head and neck squamous carcinoma cancer stem-like cells in a syngeneic mouse model and analysis of hypoxia effect". Oncology Reports 28, no. 3 (2012): 1057-1062. https://doi.org/10.3892/or.2012.1904