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Cell Biology International (2011) 35, 227–234 (Printed in Great Britain)
Using ABCG2-molecule-expressing side population cells to identify cancer stem-like cells in a human ovarian cell line
Jun Dou*22, Cuilian Jiang†1, Jing Wang‡1, Xian Zhang‡, Fengshu Zhao*, Weihua Hu*, Xiangfeng He*, Xiaoli Li*, Dandan Zou* and Ning Gu§
*Department of Pathogenic Biology and Immunology, Southeast University Medical School, Nanjing 210009, Peoples Republic of China, †Maternal and Child Health, Nanjing 210008, Peoples Republic of China, ‡Zhongda Hospital, Medical School, Southeast University, Nanjing 210009, Peoples Republic of China, and §School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, Peoples Republic of China


CSCs (cancer stem cells) are a small subset of cells within a tumour that possesses the characteristics of stem cells and are considered to be responsible for resistance to chemoradiation. Identification of CSCs through stem cell characteristics might have relevant clinical implications. In this study, SP (side population ) cells were sorted from a human ovarian cancer cell line by FACS to determine whether cancer stem cell-like SP cells were present. A very small fraction of SP cells (2.6%) was detected in A2780 cells. SP cells possessed the following characteristics: highly proliferative activity, marked ability for self-renewal in soft agar and culture medium, high expression of ABCG2, drug resistance to vinblastine in vitro, and strong tumourigenic potential in Balb/c nude mice. It is concluded that there exists in the A2780 cell line a small number of SP cells with high expression of ABCG2. The cells have the characteristics of cancer stem-like cells, and identification and cloning of such human SP cells can help in improving therapeutic approaches to ovarian cancer in patients.


Key words: ABCG2 molecule, cancer stem cell, cancer stem-like cell, ovarian cancer, side population cell

Abbreviations: A2780 cells, human ovarian cell line, ABC, ATP-binding cassette; CM, complete medium, CSCs, cancer stem cells, qRT-PCR, quantitative real-time PCR, R1, region 1, R2, region 2, SP cells, side population cells

1These authors contributed equally to this work.

2To whom correspondence should be addressed (email njdoujun@yahoo.com.cn).


1. Introduction

Epithelial ovarian carcinoma has the third highest incidence rate in women; its mortality, however, is the highest among gynaecological malignancies in the People's Republic of China because early detection remains difficult. There are many therapeutic regimens for ovarian carcinoma, such as operation, chemoradiation, immunotherapy and physiotherapy. These original approaches take ovarian carcinomas as homogeneous masses of cells, and therapeutic goals are set at reducing the quantity of cancer cells and eradicate as much of the main focus of the tumour as possible. Unfortunately, cancer recidivation tends to occur due to the development of resistance of some cells to chemoradiation. Surgery, radiotherapy and chemotherapy can scarcely improve long-term survival rate in patients with ovarian cancer. New therapies are urgently needed to increase recovery rate (Hakkarainen et al., 2003; Pan et al., 2008; Ferrandina et al., 2009). Much attention has been recently focused on the role of CSCs (cancer stem cells) in the initiation and progression of solid malignancies. Since CSCs can proliferate and self-renew extensively, sustaining tumour growth, the identification of CSCs might have relevant clinical implications (Perillo et al., 2004; Neethan et al., 2007; Santin et al., 2009).

At present, it is difficult to differentiate CSCs morphologically from the stem cells or tumour cells, and differentiation can only be based on the cell surface-specific markers (Zhang et al., 2008; Baba et al., 2009; Kusumbe et al., 2009) or biological function for isolating and identifying CSCs (Galli et al., 2004; Wang et al., 2007; Jun et al., 2009a; Hu et al., 2010). Therefore, much attention has been given to laboratory techniques that are used to isolate CSCs from clinic specimens and cultured cancer cell lines (Kim et al., 2005; Lubna et al., 2005; Jun et al., 2007). SP (side population) cells may comprise cells endowed with stem cell features such as higher proliferative rate, much lower apoptosis rate and higher tumourigenesis than non-SP cells. Therefore, SP cell analysis may be used for identification of CSC population (Ouyang et al., 2006; Moserle et al., 2008). Clone-forming assays in soft agar media and serum-free culture media have been used for measuring the proliferative activity of tumour cells and clone-forming capability (Al-Hajj et al., 2004; Henriksen et al., 2005; Wang et al., 2007).

Therapeutics-resistant tumour cells often overexpress one of several ABC (ATP-binding cassette) transporters that can mediate the efflux of several classes of anticancer drugs, including multidrug resistance (MDR1 /ABCB1), the multidrug resistance protein 1(ABCC1) and ABCG2/ BCRP1 (breast cancer resistance protein 1), etc. Also, since the SP phenotype has often been correlated with expression of ABCG2, this may be one of CSCs markers that has been analysed in different cancer stem-like cells (Matsui et al., 2004; Jun et al., 2009b). Such techniques are adopted as methods for the identification of cancer stem-like cells that are very similar to CSCs (Al-Hajj et al., 2004; Matsui et al., 2004).

In the present study, we have focused on the hypothesis that malignant growth arises from a rare population of CSCs within a tumour that provide it with unlimited regenerative capacity (Baba et al., 2009). We wanted to know if there were cancer stem-like cells in the cultured A2780 tumour cell lines and understand the biological features of this cancer stem cell-like subpopulation, since this might lead to more effective ovarian cancer therapeutic strategies involving the selective targeting and elimination of ovarian CSCs (Pan et al., 2008; Baba et al., 2009).

2. Materials and methods

2.1. Cell lines and animals

A2780 cell line is from ovarian cancer patient of origin, a well-established ovarian cancer model system, purchased from the Cellular Institute. The cells are cultured at 37°C in 5% CO2 in air in CM (complete medium) consisting of RPMI 1640, 2 mM l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 10% FBS (fetal bovine serum). Balb/c nude mice of 5–6 weeks of age were acquired from the Animal Center of Shanghai. Mice were raised in the animal facilities of the Experimental Animal Center, Medical School, Southeast University, under sterile conditions in air-filtered containers. All the experiments were performed in compliance with the guidelines of the Animal Research Ethics Board of Southeast University, People's Republic of China.

2.2. Sorting SP cells

The protocol of sorting SP cells was based on the published reports (Lubna et al., 2005; Wang et al., 2007). Analysis and cell sorting were performed on a FACS. SP cells and non-SP cells were sorted simultaneously (Lubna et al., 2005).

2.3. Isolation of ABCG2 molecule phenotype cells

ABCG2 molecule phenotype cells were isolated from 1×107 A2780 cells, following the protocol previously described (Matsui et al., 2004; Jun et al., 2009b). ABCG2+ cells was the designation given to A2780 cells expressing the ABCG2 molecule and ABCG2 cells for A2780 cells not expressing the ABCG2 molecule.

2.4. Proliferative assay of SP and non-SP cells

SP cell or non-SP cell suspensions, 2×103, seeded into 96-well plates, were assayed for proliferative activity in triplicate wells. To assay cell viability, the SP cell or non-SP cell suspensions were mixed with 0.4% Trypan Blue (Sigma) at 24, 48, 72, 96 ,120 and 144 h after incubation, and the mean values of the viable count were counted using a Neubauer haemocytometer chamber (Maranon et al., 2000).

2.5. Colony formation assay

For colony formation, the assay protocol followed was that previously described by Müller-Tidow et al. (2001) and Ignatova et al. (2002).

2.6. In vivo tumour study

For the tumourigenic experiment, nude mice were injected subcutaneously. in the flank with 5×104 SP cells or 5×104 non-SP cells. For the experiment with ABCG2+ cells, mice were injected s.c in the flank of mice with 5×105 ABCG2+ cells or 5×105 ABCG2 cells, with six mice/group (Jun et al., 2009b).

2.7. RNA isolation and quantitative RT-PCR

The PCR sense primer sequence for ABCG2 gene was 5′-GGCTTATACGGCCAGTTCCA-3′, and the anti-sense was 5′-GTCCGTTACATTGAATCCTGGAC-3′. PCR sense primer sequence for the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene was 5′-CATCCTGCACCACCAACTGCTT-3′, andthe anti-sense was 5′-ACAGCCTTGGCAGCACCAGT-3′. Total cellular RNA was extracted from 1×106 SP cells or non-SP cells by using Rneasy Mini Kit (Qiagen), according to the manufacturer's instructions. qRT-PCR (quantitative real-time PCR) was performed using the Light Cycler RNA Master SYBR Green kit and the Light Cycler 480 (Roche Biochemicals) as described in the published papers (Ouyang et al., 2006).

2.8. Western blotting

Cells, 1×106, were collected and lysed in protein extraction buffer (Novagen). Mouse anti-human Ab (antibody) was added to the membrane for 1 h at room temperature, and the membrane was rinsed for 5 min with Ab wash solution three times before adding goat anti-mouse secondary Ab, which was incubated for a further 1 h, and subsequent steps were performed according to Jun et al. (2006).

2.9. Resistance to chemotherapeutic agents in SP cells and ABCG2+ cells

SP cells or non-SP cells suspensions, 3×104, or 3×104 ABCG2+ cells or ABCG2 cells suspensions were seeded into a 96-well plate with 0.5 unit μl/ml vincristine in each well (Baiyunshang Company). Cellular resistance to chemotherapeutic agents was calculated according to the method of Mosmann et al. (1983).

2.10. Detection of SP cells in ABCG2+ cells

ABCG2+ or ABCG2 cells, 1 × 104, were plated on 24-well plates for 24 h; the supernatant was discarded, and 1 ml CM was added to the plates. The cell suspension was stained with Hoechst 33342 dye at 5 μg/ml at 37°C for 90 min and washed with PBS twice. To each well, 1 ml of CM was added again and then visualized by light microscope and immunofluorescence microscope. SP cells with ABCG2+ cells percentage (%): [the number of SP cells in the mean of six visual fields visualized by light microscopy−the number of SP cells in the mean of six visual fields visualized by immunofluorescence microscopy]/the number of SP cells in the mean of six visual fields visualized by light microscopy (Cuilian et al., 2009).

2.11. Statistical analysis

Statistical comparisons were performed using the Student's t test method, and P<0.05 was considered significant statistically.

3. Results

3.1. Isolation of SP cells and ABCG2+ cells in A2780 cell line

We first isolated SP cells from A2780 cells by FACS, which revealed verapamil-sensitive SP cells accounted for 0.1±0.01% in R1 (region 1). SP cells nearly disappeared in the R1 of Figure 1(A), due to the presence of verapamil. Since verapamil blocked the fluorescent dye Hoechst 33342 from being pumped out of SP cells, the SP cells were stained with the dye. As a result, the SP cells were invisible to the FACS in R1 of Figure 1(A). The SP cells were seen in the R2 (region 2) (2.7±0.19%) of Figure 1(B). Thus, true SP cells were 2.6±0.18% of the total A2780 population, and the number of 2.6±0.18% equals 2.7%±0.19% in R2 minus 0.1%±0.01% in R1 (Figure 1).

ABCG2+ cells were next isolated from A2780 cells by FACS, was accounting for ∼2.0%. The sorted SP cells or non-SP cells, or the isolated ABCG2+ cells and ABCG2 cells, were, respectively, cultured in CM for 4 h for the subsequent experiments.

3.2. Evaluation of the capabilities of cellular proliferation and clone formation in the SP cells

Clone formation in soft agar was used to assay the ability of cells to cross tissue barriers and cell invasion, and this ability was also used to measure the cellular proliferative activity.

The cloning efficiency correlated positively with the disease stage of the tumour (Matsui et al., 2004). Therefore, we designed experiments to test whether there were differences in proliferative activity and clone formation capability between the SP and the non-SP cells in vitro. The proliferative activities dynamically every 24 h after 2000 SP and 2000 non-SP cells had been seeded into a 96-well plate in CM (Figure 2A). After 1 week of incubation, the numbers of SP cells reached 15518 cells, whereas non-SP cells were only 10756, a difference that was statistically significant (P<0.05). Since one of the features of stem cells is their ability to form colonies in the soft agar media, colony formation was around 16% for the SP cells and 7% for the non-SP cells when measured at 11 days after incubation in the soft agar media (P<0.05). Morphologically, SP cells appeared to be closely connected clones in the soft agar media, whereas the non-SP cells were looser and more dispersed. The cell counts for non-SP was less than that of SP cells in each clone (Figures 2B and 2C). Eight days after incubation in the CM, the SP cells and non-SP cells had different colony-forming rates (21% in the SP cells compared with 9% in the non-SP cells; see Figures 2D and 2E). The difference (Figure 2 F) was statistically significant (P<0.01). From these results, we concluded that the SP cells had a greater self-renewal capacity in vitro than non-SP cells.

3.3. Evaluation of tumourigenesis of the SP cells and ABCG2+ cells in Balb/c nude mice

In the tumourigenic experiment, 5×104 SP cells and 5×104 non-SP cells, 5×104 or 5×105 ABCG2+ cells and 5×105 ABCG2 cells were injected s.c into the flank of nude mice. Four of the six mice injected with the SP cells in 23 days and two of the six mice injected with the ABCG2+ cells or one of the six mice injected with the ABCG2 cells in 36 days generated tumours, respectively, whereas only one of the six mice injected with 5×104 non-SP cells grew tumours by 60 days of observation. In addition, there was no detected tumour in the rest of the mice injected with the ABCG2+ or ABCG2 cells or 5×104 bulk cell population by 60 days. These results suggested that the SP cells in A2780 cell line had a greater tumourigenic capability than that of the non-SP cells (Figure 3). However, no obvious differences in the tumourigenesis between the ABCG2+ cells and ABCG2 cells were found (data not shown).

3.4. Detecting the ABCG2 expression in the SP cells

Stem cells have many features that distinguish them from mature, differentiated cells, of which one particularly intriguing feature is that stem cells express high levels of specific ABC drug transporters, including ABCB1, ABCG2, etc. (Kim et al., 2002). In order to test ABCG2 gene expression in the SP cells or non-SP cells, qRT-PCR and Western blotting were used. The results showed that the expression of ABCG2 gene was 5.81 times higher in the SP cells than in the non-SP cells (Figure 4), which was statistically significant (P<0.01). Since the drug-transporting property of stem cells conferred by ABC transporters is the basis for the SP cells (Michael et al., 2005), our current results suggested that SP cells with an up-regulated ABCG2 expression represent stem cell-like cancer cells.

3.5. Analysis of resistance to chemotherapeutic drug in SP cells and ABCG2+ cells

According to the CSC hypothesis, CSCs are naturally resistant to chemotherapy through their quiescence, their capacity for DNA repair and ABC transporter expression (Michael et al., 2005). Based on these properties, we selected vincristine as a conventional chemotherapeutic agent principally used for the treatment of solid tumours to test whether SP cells were resistant to vincristine. The resistance of SP cells to vincristine was statistically significant compared with non-SP cells (81% compared with 37%, P<0.01; Figure 5A). Subsequently, we tested the resistance to vincristine of ABCG2+ cells and ABCG2 cells. As predicted, the resistance of ABCG2+ cells was higher than that of ABCG2 cells (79% compared with 35%, P<0.01). Further, ABCG2+ cells were incubated with anti-ABCG2 Ab, and resistance to vincristine was again analysed in the same way. Treated ABCG2+ cells showed a marked decrease in resistance by 38% (Figure 5C), which was statistically significant compared with the ABCG2+ cells not incubated with the Ab (P<0.01). The findings implied that the resistance of ABCG2+ cells to vincristine mainly depended on the ABCG2 molecular transporter.

3.6. Testing SP cells in ABCG2+ cells

After finding that the SP cells expressed higher levels of the ABCG2 molecule, the relationship was analysed between the SP cell phenotype and expression of ABCG2. Light and fluorescence microscopy was used to detect the numbers of SP cells in ABCG2+ cells. The data indicated that the SP cell percentage in ABCG2+ cells was markedly higher than that of ABCG2 cells after these cells had been stained with Hoechst 33342 (Figure 6). The result strongly suggested that the ABCG2 molecule is associated with maintaining the SP phenotype, and particularly with resistance to vincristine.

4. Discussion

It is known that CSCs residing in epithelial ovarian cancers possess CD133+ (Kusumbe et al., 2009) or CD44+CD117+ subpopulation cells (Zhang et al., 2008) and that these specific markers may be potential therapeutic targets in this devastating disease. Our previous study indicated the CD44+CD133+ CD24+ B16F10 cells were stem-like cells of melanoma (Jun et al., 2009a). Therefore, we hypothesized that CD133+, CD44+, CD117+ may be markers of CSC. Whether these molecules are CSC-specific markers or not, however, was unclear. This is because CSCs arising from normal stem cells may share many characteristics (Neethan et al., 2007).

In the present study, we first sorted the SP cells from the A2780 cell line based on their ability to extrude some fluorescent dyes, Hoechst 33342, by FACS. These SP cells were enriched in potential tumourigenic cells, such as stem-like cancer cells (Burkert et al., 2008). To understand the biological characteristics of the SP cells, we explored the proliferative activity and the clone formative capability of the SP with non-SP cells in vitro. The SP cells gradually increased proliferative activity in contrast to the non-SP cells, 3 days after culture (Figure 2A). Theoretically, CSCs have a low rate of division, and proliferation in their niche helps them avoid chemoradiation (Burkert et al., 2006; Zou et al., 2008); thus, CSCs are relatively quiescent proliferative in restrictive niche environments. However, the SP cells were sorted from other tumour cells, and they may become separate from their niche environments and then gain the ability to proliferate faster, in contrast with the non-SP cells (Burkert et al., 2006). Thus, SP cells were typically stem cells capable of self-renewing, including having a highly proliferative capacity and a clone-forming potential greater than non-SP cells.

To evaluate self-renewal characteristics of SP cells in vivo, the tumourigenic experiments undertaken demonstrated that the SP cells had more effective tumourigenic capability than non-SP cells in Balb/c nude mice, which may be good evidence that the SP cells are the cancer stem-like cells in the A2780 cell line (Figure 3). Nevertheless, the ABCG2+ cells did not have obvious tumourigenic capability compared with the ABCG2 cells in vivo (data not shown). These results imply that the ABCG2 molecule may not be associated with self-renewal characteristic of the SP cells.

According to the CSC hypothesis, chemotherapy kills most cells in a tumour; however, it is believed to leave CSCs behind, which might be an important mechanism in the development of resistance to chemotherapy (Zou et al., 2008). If the SP cells had CSC characteristic, it should characteristically be resistant to chemotherapy. Our data showed that the SP cells have a stronger resistance against vincristine than non-SP cells (Figure 5A). As past investigations have shown that the drug-transporting property of stem cells is conferred by these ABC transporters (Lubna et al., 2005), we detected specific ABCG2 drug transporters in the SP cells. The results of RT-PCR, qRT-PCR and Western blotting analyses, a higher level of ABCG2 were particularly expressed by SP cells (Figure 4). The findings suggest that the up-regulation of ABCG2 is closely associated with resistance of SP cells to vincristine, and this was markedly decreased when the ABCG2+ cells were incubated with anti-ABCG2 Ab (Figure 5C).

In summary, our findings show definite evidence of the existence of SP cells with high ABCG2 molecule expression; these cells have the characteristics of cancer stem-like cells in this human ovarian A2780 cell line. The findings suggest an important method for identifying cancer stem-like cells in human ovarian and other cancer cell lines. The findings also suggest that the ABCG2 molecule may be one of the specific markers of cancer stem-like cells and may be a potential target molecule for therapy.

Author contribution

Jun Dou was responsible in designing the study and writing the manuscript. Ning Gu designed the study with Jun Dou and analysed the results of the experiments. Chuilian Jiang and Jing Wang participated in the major experiments, such as detection of SP cells in ABCG2+ cells, cell culture, resistance to chemotherapeutic agents in SP cells, ABCG2+ cells and statistical analysis. Xian Zhang and Fengshu Zhao were responsible for RNA isolation and quantitative RT-PCR, Western blotting. Weihua Hu and Xiangfeng He were responsible for the proliferative assay of SP and non-SP cells, colony formation assay. Xiaoli Li and Dandan Zhou were responsible for the sorting of SP cells, in vivo tumour study and breeding animals.

Funding

This work was supported in part by the National Natural Science Foundation of China (no. 81071769), the Science Foundation of Southeast University (no. 9223001446), the Groundbreaking experimental design of undergraduate student in China (no. G2007059) and in part by the 973 Program of China (no. 2011CB933500).

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Received 2 June 2010/4 October 2010; accepted 26 November 2010

Published as Cell Biology International Immediate Publication 26 November 2010, doi:10.1042/CBI20100347


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ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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