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Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996
Biochemical and Biophysical Research Communications 419 (2012) 728–734
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996 Xin-xing Li a, Jian Wang a,⇑, Hao-lu Wang a, Wei Wang a, Xiao-bin Yin a, Qi-wei Li a, Yu-ying Chen b, Jing Yi b a b
Division of General Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China Department of Biochemistry and Molecular Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
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Article history: Received 14 February 2012 Available online 24 February 2012 Keywords: Gallbladder carcinoma Cancer stem cell Side population cell
a b s t r a c t The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substrate Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Gallbladder carcinoma (GBC) is the fifth most common malignancy of the gastrointestinal tract [1]. The prognosis of GBC cancer is extremely poor due to the presence of unresectable or metastatic disease in the majority of patients, with an overall 5-year survival rate of less than 10% [1,2]. Chemotherapy is the most commonly available treatment for this malignancy [3]. However, GBC is known to be non-sensitive to routine chemotherapy, such as 5-Fu, cisplatin (CDDP) and gemcitabine. Therefore, it is crucial to discover new strategies which could enhance the chemotherapeutic efficacy of GBC cells. It is generally believed that conventional chemotherapy does not kill all tumor cells, and a small subpopulation of cells, which are resistant to the chemotherapy and have the capability of initiating tumors, might be the source of recurrence. These cells are referred to as cancer stem cells (CSCs) [4], which have been found in many types of solid tumors including breast [5], brain [6], pancreatic [7], gastric cancers [8] and so on. CSCs might share
⇑ Corresponding author. Fax: +86 21 58393018. E-mail address: [email protected] (J. Wang). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.090
many properties of normal stem cells, such as self-renewal, unlimited proliferative potential and so on. Moreover the tumorinitiating and chemotherapy-resistant capability of CSCs may be attributed to the expressions of several ATP-binding cassette (ABC) transporters [9–11]. High levels of ATP-binding cassette transporters (ABC-T) family members in CSCs, such as ABCG2 and MDR1, are responsible for the extrusion of both Hoechst 33342 and many other drugs. In fact, the ABC-T has been used as important markers for isolation and analysis of stem cells [12]. On the basis of the ability to efflux the fluorescent DNA-binding dye Hoechst 33342, Goodell [13] first identified SP cells from mouse bone marrow as a small cell population that was highly enriched for hematopoietic stem cells and endowed with long-term repopulating capacity. Ever since their discovery, SP cells have been detected in many tumor tissues, such as esophageal carcinoma [14], glioblastoma [15]. The existence of SP cells has been proven to play an important role in tumor growth and relapse in many solid tumors [9,16]. Identification of cancer stem-like cells will greatly facilitate the investigation of this critical population of cells. In this study, we sorted SP cells from a human gallbladder carcinoma cell line, SGC-996 and aimed to preliminarily identify the biological characteristics of SP cells from the cell line.
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2. Materials and methods
2.5. Cell migration and invasion assays
2.1. Cell culture
Cell migration assay was performed using the Boyden chamber containing an 8 lm pore size membrane (BD Bioscience). In cell invasion studies the membranes were pre-coated with Matrigel (1 mg/ml; BD Bioscience). In both cases, cells were seeded in wells at a density of 0.2 105 cells/100 lL in RPMI-1640 medium containing 0.2% FBS in the upper chamber. In the lower chamber, 500 lL of RPMI-1640 media containing 0.2% FBS were added. After 24 h, the chamber was removed, fixed, and stained with trypan blue. Cells in the upper chamber were removed using the alcohol. Cells migrating through the membrane and cells invading the matrix were photographed in three randomly-selected fields and counted in average.
The human gallbladder cancer cell line SGC-996 and GBC-SD was provided by Academy of Life Sciences, Tongji University (Shanghai, China). SGC-996 cells were maintained in RPMI-1640 medium (GibcoBRL, Gaitherburg, MD, USA). GBC-SD cells were maintained in DMEM medium (GibcoBRL, Gaitherburg, MD, USA). The media were supplemented with antibiotics and 10% newborn calf serum. Cells were cultured in a humidified atmosphere with 5% CO2 at 37 °C.
2.2. Side population analysis using flow cytometry (FCM) The protocol was based on Goodell et al. [13]. Briefly, when cells were in a logarithmic growth phase, they were detached from the culture flask, washed, centrifuged and suspended in culture media supplemented 2% newborn calf serum at a concentration of 1 106 cells/ml at 37 °C. Hoechst 33342 (Sigma, USA) was added at a final concentration of 5 lg/ml in the presence or absence of 50 lg/ml verapamil (Sigma, USA), and the samples were incubated at 37 °C for 90 min. During the incubation time, cells were gently tapped every 15 min. Afterwards, the cells were washed and re-suspended in Hank’s Balanced Salt Solution (HBSS) (Invitrogen, USA) with 2% newborn calf serum at 1 106 cells/ml and kept at 4 °C. Propridium iodide (Sigma, USA) was added at a concentration of 1 lg/mL to exclude dead cells before flow cytometry (Becton Dickson, San Diego, CA). Hoechst 33342 was excited with an ultraviolet laser at 350 nm, and fluorescence emission was measured with DF424/44 (Hoechst blue) and DF630/22 (Hoechst red) optical filters.
2.3. Repopulation assay An entire SGC-996 cell population was stained with Hoechst 33342 to determine whether SP cells have the ability to repopulate in vitro. SP and non-SP cells separated by FACS were cultured for 7 days in RPMI 1640 with 10% newborn calf serum. After 7 days, SP cells and non-SP cells were restained with Hoechst 33342 and analyzed using FACS.
2.4. Cell proliferation and clone formation assays For cell proliferation study, growth curves of SP and non-SP cells were determined by the MTT assay (sigma), as described by Huang [17]. Approximately 2.0 103 cells per well were seeded in 96well plates. After incubation, 10 lL MTT (5 mg/ml) was added to each well and incubated at 37 °C for 4 h. Afterwards, the culture medium was removed and 100 lL DMSO (dimethyl sulphoxide) (Invitrogen, USA) was added to each well. After shaking thoroughly for 10 min, the absorbance of each well was determined with a spectrophotometer at a wavelength of 570 nm. Triplicate wells were used for each group. To examine clonogenic ability, SP and non-SP cells were plated at 300 cells per well in six-well culture dishes in RPMI 1640 with 10% newborn calf serum. Triplicate wells were performed for each group. After 2 weeks, cells were washed with phosphate buffered saline, fixed in methanol for 15 min and then stained with crystal violet (Sigma, USA) for 30 min. Clones with P50 cells were scored, and the clone formation efficiency (CFE) was calculated according to the formula: (the clone number/the plated cell number) 100%.
2.6. Chemotherapeutic drug sensitivity assays 8.0 103 cells per well were seeded in 96-well plates. Cells were incubated with CDDP and doxorubicin at increasing concentrations for 24 h. The absorbance of each well was determined with a spectrophotometer at a wavelength of 570 nm using MTT assay. The viability rate was calculated according to the formula: absorbance of experimental well/absorbance of control well 100%. 2.7. Analysis of the expression of MDR1, MRP1, MRP2, and ABCG2 Expressions of MDR1, MRP1, MRP2 and ABCG2 genes were monitored by Real-time PCR. Total mRNA was extracted separately from SP and non-SP cells using Trizol reagent (Invitrogen Life Technologies). The cDNA for each sample was synthesized using 1 lg of total RNA and SuperScript reverse transcriptase according to the manufacturer’s protocol. Detection of PCR products was performed on a LightCycler system (Roche Applied Science) using the SYBR Green I kit (TaKaRa Biotechnology, Dalian, China), according to the manufacturer’s instructions. The primers for MDR1 were 50 -GAGGAAGACATGACCAGGTA-30 and 50 -CTGTCGCATTATAGCATGAA-30 . The primers for MRP1 were 50 -GAGGAAGGGAGTTCAGTCTT-30 and 50 -ACAAGACGAGCTGAATGAGT-30 . The primers for MRP2 were 50 -AAATATTTTGCCTGGGAACC-30 and 50 -TGTGACCACAGATACCAGGA-30 . The primers for ABCG2 were 50 -ACCTGAAGGCATTTACTGAA -30 and 50 -TCTTTCCTTGCAGCTAAGAC-30 . The primers for GAPDH (internal control) were 50 -ATCCCTCCAAAATCAAGTGG -30 and 50 -GTTGTCATGGATGACCTTGG-30 . To determine whether ABCG2 expression was a marker for the SP phenotype, immunocytochemistry (ICC) was performed. Freshly sorted cells were cytospun onto positively charged slides. Rat antihuman polyclonal ABCG2 antibody (IgG2a; 1:40; Abcam) was used to detect the expression of ABCG2. Sections were finally counterstained with hematoxylin and then examined under an Axioplan 2 fluorescent microscope. The ratio of the positive area for ABCG2 was quantified by Zeiss KS400 software. 2.8. Tumorigenicity assay in vivo Approval for the animal experiments was obtained from Shanghai Experimental Animal Center. To explore the tumorigenic capacity, SP and non-SP cells sorted from human gallbladder carcinoma SGC-996 cells, ranging from 500 to 5.0 106, suspended in 100 lL RPMI 1640, were injected subcutaneously into 6-week old BALB/c-nu/nu female mice. The mice were monitored every week for palpable tumor formation and were sacrificed at 4–12 weeks after transplantation for detecting the tumor formation. Tumors were measured using vernier caliper and then photographed. Tumor cells were also histologically examined by hematoxylineosin staining.
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2.9. Statistical analysis Data were shown as mean values ± S.D. SPSS17.0 software was used for statistical analysis. ANOVA (analysis of variance) was applied for comparison of the means of two or multiple groups. A value of p < 0.05 was considered significant.
3. Results
3.3. Cell proliferation and clone formation of SP and non-SP cells from SGC-996 MTT assay was used to evaluate the proliferation abilities of SP and non-SP cells. The cells were cultured for 7 days and the cell viabilities were tested by MTT assay on each day. After 4 days, proliferation of SP cells was much higher than that of non-SP cells (Fig. 2A). Clone formation assays of SP and non-SP cells were examined. After having cultured for about 2 weeks, most clones reached a size
3.1. SP analysis in human gallbladder carcinoma cell lines The proportion of SP cells was examined by staining cells with Hoechst 33342 dye to generate a Hoechst Blue–Red profile. As a control, the ABC transporter inhibitor verapamil was added to inhibit the efflux of Hoechst 33342. The proportion of SP cells from the commonly studied human gallbladder carcinoma cell lines SGC-996 and GBC-SD were 1.0% and 0.8%, respectively (Fig. 1A–a, A-c), which decreased significantly in the presence of verapamil (Fig. 1A-b, A–d).
3.2. Self-renewal activity of SP cells from SGC-996 To compare the repopulating abilities of SP and non-SP cells from SGC-996, SP cells were sorted, collected, and cultured in RPMI 1640 with 10% newborn calf serum. After 7 days of culturing, analysis by FCM revealed the renewal capability of SP cells. The cultured SP cells contained 1.9% of SP cells (Fig. 1B-c). Conversely, the cultured non-SP cells yielded very few SP cells (Fig. 1B-b). In addition, SP cells generated both SP and non-SP cells showed a fraction size comparable with the original population (Fig. 1B-a).
Fig. 1. The proportions of SP cells from SGC-996 and GBC-SD were 1.0% (A-a) and 0.8% (A-c) respectively, which decreased significantly in the presence of verapamil (A-b, A-d). After 7 days of culturing, the cultured SP cells contained 1.9% of SP cells (B-c) comparable with the original population (B-a). Conversely, the cultured nonSP cells yielded very few SP cells (B-b).
Fig. 2. Cell proliferation (A), clone formation (B, C), migration assay (D, E), invasion assay (F, G) and chemotherapeutic drug sensitivity assay (H) of SP and non-SP cells. Lines, columns; mean of three experiments; bars, S.D. ⁄p < 0.05.
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of more than 50 cells. We counted the clone number and found that the number of colony formation in SP cells from SGC-996 was 55.8%, which was significantly higher than that of corresponding non-SP cells, 19.6% (Fig. 2B and C).
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invasive capacity than non-SP cells (Fig. 2D–G). Hence, the stronger ability of metastasis in gallbladder carcinoma might largely be attributed to the existence of SP cells. 3.5. Chemotherapeutic drug sensitivity assays
3.4. Cell migration and invasion The metastatic behavior is an important feature of gallbladder carcinoma, so it is definitely worthwhile examining if SP cells are more migratory and invasive than non-SP cells. In this study although SP and non-SP cells were both migratory on uncoated and coated surfaces, SP cells exhibited stronger migratory and
To investigate possible differences in drug resistance between SP and non-SP cells, we chose two commonly used anti-tumor drugs, such as CDDP and doxorubicin. After 24 h treatment, the sensitivities to chemotherapeutic drugs were assessed with the MTT assay. This experiment revealed that non-SP cells were more sensitive to CDDP and doxorubicin than SP cells (Fig. 2H).
Fig. 3. Relative mRNA expressions of ABC transporters genes in SP and non-SP cells were monitored by Real-time PCR (A). ICC was performed to detect the expression of ABCG2 (B). The ratio of the positive area for ABCG2 was quantified by Zeiss KS400 software (C). Columns; mean of three experiments; bars, S.D. ⁄p < 0.05.
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3.6. SP cells show higher expression levels of the ABCG2 molecule ABC transporters have the capacity to export many cytotoxic drugs and are up-regulated in SP cells derived from other tumor cell lines [17]. In particular, ABCG2 has been implicated in high Hoechst 33342 dye efflux capacity that marks the SP phenotype. Therefore, Real-time PCR assay was used to measure the mRNA expression of ABC transporters MDR1, MRP1, MRP2, and ABCG2 in SP and non-SP cells. In addition, ICC assay was used to determine the protein expression and location of ABCG2 in this study. We found that expression of ABCG2 was higher in SP than non-SP cells (Fig. 3), meanwhile there were no differences of MDR1, MRP1 and MRP2 between SP and non-SP cells. 3.7. SP cells display higher tumorigenicity in vivo As shown in Table 1, SP cells from SGC-996 were injected into BALB/c-nu/nu female mice subcutaneously. One out of five mice formed tumors when inoculated with 5.0 103 SP cells; all five mice formed tumors when 5.0 105 SP cells were inoculated. However, only one out of five mice formed tumors when inocu-
Table 1 SP Cells and Non-SP Cells Inoculated into Nude Mice. Injected cell number
SP cells Non-SP cells
5 102
5 103
5 104
5 105
5 106
0/5 –
1/5 –
4/5 0/5
5/5 2/5
– 5/5
To explore the tumorigenic capacity, SP and non-SP cells sorted from human gallbladder carcinoma SGC-996 cells, ranging from 500 to 5.0 106, were injected subcutaneously into 6-week old BALB/c-nu/nu female mice.
lated with 5.0 105 non-SP cells, but when inoculated with 5.0 103 non-SP cells tumor formation was not observed in mice. Therefore, it was apparent that the tumor formation ability of SP cell was much higher than that of non-SP cells. By performing hematoxylin-eosin histological staining, we observed apparent pleomorphism of nuclei in the tumor tissue derived from SP and non-SP cells displaying typical cancer cell morphology. However, as compare with the tumors from non-SP cells, the tumors from SP cells showed central necrosis (Fig. 4).
4. Discussion The CSC hypothesis proposes that CSCs which are rich in primitive and undifferentiated cells can renew themselves, proliferate infinitely, and escape chemotherapy [4,18,19]. This small population of cells becomes the root of recurrence and metastasis [20]. Therefore it is desired to isolate and to characterize CSCs for effective therapeutic strategies of targeting these cells. As to the methods of detecting CSCs, identification of specific markers for CSCs is the preferred one [21,22]. But in most malignant tumors, investigators still have not identified specific CSC markers. Toward this direction, sorting SP cells using FCM by a dye Hoechst 33342 exclusion method has recently become a new means of CSC research. In this present study, we have isolated a small population of stemlike cells SP cells from the cell line, SGC-996, which is a malignant cell line from a female patient who suffered from human gallbladder papillary adenocarcinoma, and was established by Medical College of Tongji University of China. We have previously reported that together with platinum therapeutics, some adjucts can demonstrate enhanced anti-cancer effects in the SGC-996 cells [23]. Therefore, we have further explored SP cells from the SGC-996 cells, with the purpose of identification of new strategies of a more effective therapy for gall bladder cancer.
Fig. 4. The tumor formation ability of SP cell was much higher than that of non-SP cells. After 5.0 104 SP and Non-SP cells injected into BALB/c-nu/nu female mice subcutaneously, four-fifth mice formed tumors derived from SP cells (A, B), however non-SP cells tumor formation was not observed in mice. One out of five mice formed tumor when inoculated with 5.0 103 SP cells (B). Tumors cells were stained by hematoxylin-eosin (C).
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We detected SP cells from a human gallbladder carcinoma cell line, SGC-996, and found that the percentage is approximately 1.0%. In addition we detected similar percentage of SP cells (0.8%) from another internationally commonly studied gallbladder cancer cell line GBC-SD. Consistent with observations from others [14,15], the proportions of SP cells were rare and decreased in the presence of verapamil, which can effectively block ABCG2 from pumping out the Hoechst dye. After 2 weeks culture, SP cells of SGC-996 were able to reproduce the entire cancer cell line phenotype that exhibited a stem-like characteristic, and the SP fraction was a higher population than that from the original cell line SGC-996. In sharp contrast, non-SP cells scarcely had the capacity to produce SP cells. These findings imply that SP cells follow the so-called hierarchical CSC model (ref) and possibly have the characteristics of asymmetrical cell division. Our findings support the note that SP cells are likely responsible for tumor metastasis and recurrence because they are very powerful in proliferation, clonogenicity, tumorigenicity and invasion [9,16,24]. In this study, we found that SP cells demonstrate a higher proliferation rate, a higher clonal-generating capability, more migratory and invasive abilities than non-SP cells. Furthermore, our experiments revealed that SP cells were more tumorigenic than non-SP counterparts when injected into immunodeficient mice. As few as 5.0 103 SP cells could form tumor while only 5 105 non-SP cells produced the same phenomenon, which is in agreement with what reported in hepatocellular carcinoma [25], laryngeal cancer [26], esophageal carcinoma [14] and head and neck cancer [24] where SP cells also show a higher capacity to initiate tumor formation. Furthermore, compare with the tumor from non-SP cells, the tumor from SP cells displayed apparent central necrosis. Taken together, SP cells appear to be able to sustain their tumor initiation and to have a strong tumorigenicity. This may be a result of a population of amplifying cells that have tumor initial ability by their renewal, rapid proliferation, high clonogenicity and strong metastasis capabilities. Experimental studies suggest that although cytotoxic reagents may seem to be effective to the bulk of cancerous cells, SP cells appear to be more resistant to chemotherapy. Hirshmann-Jax and colleagues first demonstrated that SP cells from neuroblastoma were less sensitive to mitoxantrone [27]. Similar results were obtained in ovarian cancer [28] and Ewing’s sarcoma [29]. Consistently, we found in the present study that SP cells from the cell line SGC-996 were more resistant to two frequently-used chemotherapeutic agents, CDDP and doxorubicin. ABCG2 is a member of the ABC transporters, which can pump a wide variety of metabolites and drugs out of cells [30]. Widely expressed in CSCs, ABCG2 is also found to confer the SP phenotype [11]. Recent studies have shown that ABCG2 is a molecular determinant of the SP phenotype in a wide range of solid tumors [14,15,24,26,29]. Along this line, we have discovered in the present study that the mRNA and protein expression levels of ABCG2 were higher in SP cells from SGC-996 cells. In addition, in sharp contrast to ABCG2 which could pump out drugs and some materials from the cytoplasm of the cells, no difference was found between SP and non-SP cells in terms of mRNA expression of the other ABC transporters, such as MDR1, MRP1 and MRP2. Therefore, similar to the studies on other tumors [11], ABCG2 may be marker for CSCs and an attractive candidate therapeutic target for gallbladder carcinomas. In summary, SP cells from a human gallbladder carcinoma cell line, SGC-996, can be effectively sorted by using FACS with a dye Hoechst 33342 exclusion. SP cells possess the characteristics of CSC, such as asymmetrical cell division, rapider proliferation, higher clonogenicity, stronger tumorigenicity, more migratory and invasive abilities. Also SP cells are more resistant to chemotherapy
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and express high levels of ABCG2 in vivo. Hence, these results collectively suggest that SP cells are progenitor/stem cells and ABCG2 may be a candidate marker for SP cells and an attractive therapeutic target for human gallbladder cancer. Acknowledgments We thank Professor Wei-Qiang Gao for his very helpful editing this manuscript. This work was supported by grants from Shanghai science and technology commission, China (09411960800, J. Wang). References [1] S. Gourgiotis, H.M. Kocher, L. Solaini, A. Yarollahi, E. Tsiambas, N.S. Salemis, Gallbladder cancer, Am. J. Surg. 196 (2008) 252–264. [2] S. Misra, A. Chaturvedi, N.C. Misra, I.D. Sharma, Carcinoma of the gallbladder, Lancet Oncol. 4 (2003) 167–176. [3] J. Valle, H. Wasan, D.H. Palmer, D. Cunningham, A. Anthoney, A. Maraveyas, S. Madhusudan, T. Iveson, S. Hughes, S.P. Pereira, M. Roughton, J. Bridgewater, Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer, N. Engl. J. Med. 362 (2010) 1273–1281. [4] T. Reya, S.J. Morrison, M.F. Clarke, I.L. Weissman, Stem cells, cancer, and cancer stem cells, Nature 414 (2001) 105–111. [5] M. Al-Hajj, M.S. Wicha, A. Benito-Hernandez, S.J. Morrison, M.F. Clarke, Prospective identification of tumorigenic breast cancer cells, Proc. Natl. Acad. Sci. USA 100 (2003) 3983–3988. [6] H.D. Hemmati, I. Nakano, J.A. Lazareff, M. Masterman-Smith, D.H. Geschwind, M. Bronner-Fraser, H.I. Kornblum, Cancerous stem cells can arise from pediatric brain tumors, Proc. Nat. Acad. Sci. USA 100 (2003) 15178–15183. [7] P.C. Hermann, S.L. Huber, T. Herrler, A. Aicher, J.W. Ellwart, M. Guba, C.J. Bruns, C. Heeschen, Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer, Cell Stem Cell 1 (2007) 313–323. [8] S. Takaishi, T. Okumura, S.P. Tu, S.S.W. Wang, W. Shibata, R. Vigneshwaran, S.A.K. Gordon, Y. Shimada, T.C. Wang, Identification of Gastric Cancer Stem Cells Using the Cell Surface Marker CD44, Stem Cells 27 (2009) 1006–1020. [9] L. Moserle, M. Ghisi, A. Amadori, S. Indraccolo, Side population and cancer stem cells: therapeutic implications, Cancer Lett. 288 (2010) 1–9. [10] H. Ishii, M. Iwatsuki, K. Ieta, D. Ohta, N. Haraguchi, K. Mimori, M. Mori, Cancer stem cells and chemoradiation resistance, Cancer Sci. 99 (2008) 1871–1877. [11] X.W. Ding, J.H. Wu, C.P. Jiang, ABCG2: a potential marker of stem cells and novel target in stem cell and cancer therapy, Life Sci. 86 (2010) 631–637. [12] H.E. Schwabedissen, H.K. Kroemer, In vitro and in vivo evidence for the importance of breast cancer resistance protein transporters BCRP/MXR/ABCP/ ABCG2, Handb. Exp. Pharmacol. 201 (2011) 325–371. [13] M.A. Goodell, K. Brose, G. Paradis, A.S. Conner, R.C. Mulligan, Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo, J. Exp. Med. 183 (1996) 1797–1806. [14] D. Huang, Q. Gao, L. Guo, C. Zhang, W. Jiang, H. Li, J. Wang, X. Han, Y. Shi, S.H. Lu, Isolation and identification of cancer stem-like cells in esophageal carcinoma cell lines, Stem Cells Dev. 18 (2009) 465–473. [15] R. Fukaya, S. Ohta, M. Yamaguchi, H. Fujii, Y. Kawakami, T. Kawase, M. Toda, Isolation of cancer stem-like cells from a side population of a human glioblastoma cell line, SK-MG-1, Cancer Lett. 291 (2010) 150–157. [16] C. Wu, B.A. Alman, Side population cells in human cancers, Cancer Lett. 268 (2008) 1–9. [17] X.Z. Huang, J. Wang, C. Huang, Y.Y. Chen, G.Y. Shi, Q.S. Hu, J. Yi, Emodin enhances cytotoxicity of chemotherapeutic drugs in prostate cancer cells: the mechanisms involve ROS-mediated suppression of multidrug resistance and hypoxia inducible factor-1, Cancer Biol. Ther. 7 (2008) 468–475. [18] L.E. Ailles, I.L. Weissman, Cancer stem cells in solid tumors, Curr. Opin. Biotechnol. 18 (2007) 460–466. [19] J.B. Spillane, M.A. Henderson, Cancer stem cells: a review, ANZ. J. Surg. 77 (2007) 464–468. [20] C.E. Eyler, J.N. Rich, Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis, J. Clin. Oncol. 26 (2008) 2839–2845. [21] S. Bomken, K. Fiser, O. Heidenreich, J. Vormoor, Understanding the cancer stem cell, Br. J. Cancer 103 (2010) 439–445. [22] A. Sengupta, J.A. Cancelas, Cancer stem cells: a stride towards cancer cure?, J Cell Physiol. 225 (2010) 7–14. [23] W. Wang, Y.P. Sun, X.Z. Huang, M. He, Y.Y. Chen, G.Y. Shi, H. Li, J. Yi, J. Wang, Emodin enhances sensitivity of gallbladder cancer cells to platinum drugs via glutathion depletion and MRP1 downregulation, Biochem. Pharmacol. 79 (2010) 1134–1140. [24] M.H. Tabor, M.R. Clay, J.H. Owen, C.R. Bradford, T.E. Carey, G.T. Wolf, M.E. Prince, Head and neck cancer stem cells: the side population, Laryngoscope 121 (2011) 527–533. [25] G.M. Shi, Y. Xu, J. Fan, J. Zhou, X.R. Yang, S.J. Qiu, Y. Liao, W.Z. Wu, Y. Ji, A.W. Ke, Z.B. Ding, Y.Z. He, B. Wu, G.H. Yang, W.Z. Qin, W. Zhang, J. Zhu, Z.H. Min, Z.Q. Wu, Identification of side population cells in human hepatocellular carcinoma
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Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996 Xin-xing Li a, Jian Wang a,⇑, Hao-lu Wang a, Wei Wang a, Xiao-bin Yin a, Qi-wei Li a, Yu-ying Chen b, Jing Yi b a b
Division of General Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China Department of Biochemistry and Molecular Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
a r t i c l e
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Article history: Received 14 February 2012 Available online 24 February 2012 Keywords: Gallbladder carcinoma Cancer stem cell Side population cell
a b s t r a c t The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substrate Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Gallbladder carcinoma (GBC) is the fifth most common malignancy of the gastrointestinal tract [1]. The prognosis of GBC cancer is extremely poor due to the presence of unresectable or metastatic disease in the majority of patients, with an overall 5-year survival rate of less than 10% [1,2]. Chemotherapy is the most commonly available treatment for this malignancy [3]. However, GBC is known to be non-sensitive to routine chemotherapy, such as 5-Fu, cisplatin (CDDP) and gemcitabine. Therefore, it is crucial to discover new strategies which could enhance the chemotherapeutic efficacy of GBC cells. It is generally believed that conventional chemotherapy does not kill all tumor cells, and a small subpopulation of cells, which are resistant to the chemotherapy and have the capability of initiating tumors, might be the source of recurrence. These cells are referred to as cancer stem cells (CSCs) [4], which have been found in many types of solid tumors including breast [5], brain [6], pancreatic [7], gastric cancers [8] and so on. CSCs might share
⇑ Corresponding author. Fax: +86 21 58393018. E-mail address: [email protected] (J. Wang). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.090
many properties of normal stem cells, such as self-renewal, unlimited proliferative potential and so on. Moreover the tumorinitiating and chemotherapy-resistant capability of CSCs may be attributed to the expressions of several ATP-binding cassette (ABC) transporters [9–11]. High levels of ATP-binding cassette transporters (ABC-T) family members in CSCs, such as ABCG2 and MDR1, are responsible for the extrusion of both Hoechst 33342 and many other drugs. In fact, the ABC-T has been used as important markers for isolation and analysis of stem cells [12]. On the basis of the ability to efflux the fluorescent DNA-binding dye Hoechst 33342, Goodell [13] first identified SP cells from mouse bone marrow as a small cell population that was highly enriched for hematopoietic stem cells and endowed with long-term repopulating capacity. Ever since their discovery, SP cells have been detected in many tumor tissues, such as esophageal carcinoma [14], glioblastoma [15]. The existence of SP cells has been proven to play an important role in tumor growth and relapse in many solid tumors [9,16]. Identification of cancer stem-like cells will greatly facilitate the investigation of this critical population of cells. In this study, we sorted SP cells from a human gallbladder carcinoma cell line, SGC-996 and aimed to preliminarily identify the biological characteristics of SP cells from the cell line.
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2. Materials and methods
2.5. Cell migration and invasion assays
2.1. Cell culture
Cell migration assay was performed using the Boyden chamber containing an 8 lm pore size membrane (BD Bioscience). In cell invasion studies the membranes were pre-coated with Matrigel (1 mg/ml; BD Bioscience). In both cases, cells were seeded in wells at a density of 0.2 105 cells/100 lL in RPMI-1640 medium containing 0.2% FBS in the upper chamber. In the lower chamber, 500 lL of RPMI-1640 media containing 0.2% FBS were added. After 24 h, the chamber was removed, fixed, and stained with trypan blue. Cells in the upper chamber were removed using the alcohol. Cells migrating through the membrane and cells invading the matrix were photographed in three randomly-selected fields and counted in average.
The human gallbladder cancer cell line SGC-996 and GBC-SD was provided by Academy of Life Sciences, Tongji University (Shanghai, China). SGC-996 cells were maintained in RPMI-1640 medium (GibcoBRL, Gaitherburg, MD, USA). GBC-SD cells were maintained in DMEM medium (GibcoBRL, Gaitherburg, MD, USA). The media were supplemented with antibiotics and 10% newborn calf serum. Cells were cultured in a humidified atmosphere with 5% CO2 at 37 °C.
2.2. Side population analysis using flow cytometry (FCM) The protocol was based on Goodell et al. [13]. Briefly, when cells were in a logarithmic growth phase, they were detached from the culture flask, washed, centrifuged and suspended in culture media supplemented 2% newborn calf serum at a concentration of 1 106 cells/ml at 37 °C. Hoechst 33342 (Sigma, USA) was added at a final concentration of 5 lg/ml in the presence or absence of 50 lg/ml verapamil (Sigma, USA), and the samples were incubated at 37 °C for 90 min. During the incubation time, cells were gently tapped every 15 min. Afterwards, the cells were washed and re-suspended in Hank’s Balanced Salt Solution (HBSS) (Invitrogen, USA) with 2% newborn calf serum at 1 106 cells/ml and kept at 4 °C. Propridium iodide (Sigma, USA) was added at a concentration of 1 lg/mL to exclude dead cells before flow cytometry (Becton Dickson, San Diego, CA). Hoechst 33342 was excited with an ultraviolet laser at 350 nm, and fluorescence emission was measured with DF424/44 (Hoechst blue) and DF630/22 (Hoechst red) optical filters.
2.3. Repopulation assay An entire SGC-996 cell population was stained with Hoechst 33342 to determine whether SP cells have the ability to repopulate in vitro. SP and non-SP cells separated by FACS were cultured for 7 days in RPMI 1640 with 10% newborn calf serum. After 7 days, SP cells and non-SP cells were restained with Hoechst 33342 and analyzed using FACS.
2.4. Cell proliferation and clone formation assays For cell proliferation study, growth curves of SP and non-SP cells were determined by the MTT assay (sigma), as described by Huang [17]. Approximately 2.0 103 cells per well were seeded in 96well plates. After incubation, 10 lL MTT (5 mg/ml) was added to each well and incubated at 37 °C for 4 h. Afterwards, the culture medium was removed and 100 lL DMSO (dimethyl sulphoxide) (Invitrogen, USA) was added to each well. After shaking thoroughly for 10 min, the absorbance of each well was determined with a spectrophotometer at a wavelength of 570 nm. Triplicate wells were used for each group. To examine clonogenic ability, SP and non-SP cells were plated at 300 cells per well in six-well culture dishes in RPMI 1640 with 10% newborn calf serum. Triplicate wells were performed for each group. After 2 weeks, cells were washed with phosphate buffered saline, fixed in methanol for 15 min and then stained with crystal violet (Sigma, USA) for 30 min. Clones with P50 cells were scored, and the clone formation efficiency (CFE) was calculated according to the formula: (the clone number/the plated cell number) 100%.
2.6. Chemotherapeutic drug sensitivity assays 8.0 103 cells per well were seeded in 96-well plates. Cells were incubated with CDDP and doxorubicin at increasing concentrations for 24 h. The absorbance of each well was determined with a spectrophotometer at a wavelength of 570 nm using MTT assay. The viability rate was calculated according to the formula: absorbance of experimental well/absorbance of control well 100%. 2.7. Analysis of the expression of MDR1, MRP1, MRP2, and ABCG2 Expressions of MDR1, MRP1, MRP2 and ABCG2 genes were monitored by Real-time PCR. Total mRNA was extracted separately from SP and non-SP cells using Trizol reagent (Invitrogen Life Technologies). The cDNA for each sample was synthesized using 1 lg of total RNA and SuperScript reverse transcriptase according to the manufacturer’s protocol. Detection of PCR products was performed on a LightCycler system (Roche Applied Science) using the SYBR Green I kit (TaKaRa Biotechnology, Dalian, China), according to the manufacturer’s instructions. The primers for MDR1 were 50 -GAGGAAGACATGACCAGGTA-30 and 50 -CTGTCGCATTATAGCATGAA-30 . The primers for MRP1 were 50 -GAGGAAGGGAGTTCAGTCTT-30 and 50 -ACAAGACGAGCTGAATGAGT-30 . The primers for MRP2 were 50 -AAATATTTTGCCTGGGAACC-30 and 50 -TGTGACCACAGATACCAGGA-30 . The primers for ABCG2 were 50 -ACCTGAAGGCATTTACTGAA -30 and 50 -TCTTTCCTTGCAGCTAAGAC-30 . The primers for GAPDH (internal control) were 50 -ATCCCTCCAAAATCAAGTGG -30 and 50 -GTTGTCATGGATGACCTTGG-30 . To determine whether ABCG2 expression was a marker for the SP phenotype, immunocytochemistry (ICC) was performed. Freshly sorted cells were cytospun onto positively charged slides. Rat antihuman polyclonal ABCG2 antibody (IgG2a; 1:40; Abcam) was used to detect the expression of ABCG2. Sections were finally counterstained with hematoxylin and then examined under an Axioplan 2 fluorescent microscope. The ratio of the positive area for ABCG2 was quantified by Zeiss KS400 software. 2.8. Tumorigenicity assay in vivo Approval for the animal experiments was obtained from Shanghai Experimental Animal Center. To explore the tumorigenic capacity, SP and non-SP cells sorted from human gallbladder carcinoma SGC-996 cells, ranging from 500 to 5.0 106, suspended in 100 lL RPMI 1640, were injected subcutaneously into 6-week old BALB/c-nu/nu female mice. The mice were monitored every week for palpable tumor formation and were sacrificed at 4–12 weeks after transplantation for detecting the tumor formation. Tumors were measured using vernier caliper and then photographed. Tumor cells were also histologically examined by hematoxylineosin staining.
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2.9. Statistical analysis Data were shown as mean values ± S.D. SPSS17.0 software was used for statistical analysis. ANOVA (analysis of variance) was applied for comparison of the means of two or multiple groups. A value of p < 0.05 was considered significant.
3. Results
3.3. Cell proliferation and clone formation of SP and non-SP cells from SGC-996 MTT assay was used to evaluate the proliferation abilities of SP and non-SP cells. The cells were cultured for 7 days and the cell viabilities were tested by MTT assay on each day. After 4 days, proliferation of SP cells was much higher than that of non-SP cells (Fig. 2A). Clone formation assays of SP and non-SP cells were examined. After having cultured for about 2 weeks, most clones reached a size
3.1. SP analysis in human gallbladder carcinoma cell lines The proportion of SP cells was examined by staining cells with Hoechst 33342 dye to generate a Hoechst Blue–Red profile. As a control, the ABC transporter inhibitor verapamil was added to inhibit the efflux of Hoechst 33342. The proportion of SP cells from the commonly studied human gallbladder carcinoma cell lines SGC-996 and GBC-SD were 1.0% and 0.8%, respectively (Fig. 1A–a, A-c), which decreased significantly in the presence of verapamil (Fig. 1A-b, A–d).
3.2. Self-renewal activity of SP cells from SGC-996 To compare the repopulating abilities of SP and non-SP cells from SGC-996, SP cells were sorted, collected, and cultured in RPMI 1640 with 10% newborn calf serum. After 7 days of culturing, analysis by FCM revealed the renewal capability of SP cells. The cultured SP cells contained 1.9% of SP cells (Fig. 1B-c). Conversely, the cultured non-SP cells yielded very few SP cells (Fig. 1B-b). In addition, SP cells generated both SP and non-SP cells showed a fraction size comparable with the original population (Fig. 1B-a).
Fig. 1. The proportions of SP cells from SGC-996 and GBC-SD were 1.0% (A-a) and 0.8% (A-c) respectively, which decreased significantly in the presence of verapamil (A-b, A-d). After 7 days of culturing, the cultured SP cells contained 1.9% of SP cells (B-c) comparable with the original population (B-a). Conversely, the cultured nonSP cells yielded very few SP cells (B-b).
Fig. 2. Cell proliferation (A), clone formation (B, C), migration assay (D, E), invasion assay (F, G) and chemotherapeutic drug sensitivity assay (H) of SP and non-SP cells. Lines, columns; mean of three experiments; bars, S.D. ⁄p < 0.05.
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of more than 50 cells. We counted the clone number and found that the number of colony formation in SP cells from SGC-996 was 55.8%, which was significantly higher than that of corresponding non-SP cells, 19.6% (Fig. 2B and C).
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invasive capacity than non-SP cells (Fig. 2D–G). Hence, the stronger ability of metastasis in gallbladder carcinoma might largely be attributed to the existence of SP cells. 3.5. Chemotherapeutic drug sensitivity assays
3.4. Cell migration and invasion The metastatic behavior is an important feature of gallbladder carcinoma, so it is definitely worthwhile examining if SP cells are more migratory and invasive than non-SP cells. In this study although SP and non-SP cells were both migratory on uncoated and coated surfaces, SP cells exhibited stronger migratory and
To investigate possible differences in drug resistance between SP and non-SP cells, we chose two commonly used anti-tumor drugs, such as CDDP and doxorubicin. After 24 h treatment, the sensitivities to chemotherapeutic drugs were assessed with the MTT assay. This experiment revealed that non-SP cells were more sensitive to CDDP and doxorubicin than SP cells (Fig. 2H).
Fig. 3. Relative mRNA expressions of ABC transporters genes in SP and non-SP cells were monitored by Real-time PCR (A). ICC was performed to detect the expression of ABCG2 (B). The ratio of the positive area for ABCG2 was quantified by Zeiss KS400 software (C). Columns; mean of three experiments; bars, S.D. ⁄p < 0.05.
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3.6. SP cells show higher expression levels of the ABCG2 molecule ABC transporters have the capacity to export many cytotoxic drugs and are up-regulated in SP cells derived from other tumor cell lines [17]. In particular, ABCG2 has been implicated in high Hoechst 33342 dye efflux capacity that marks the SP phenotype. Therefore, Real-time PCR assay was used to measure the mRNA expression of ABC transporters MDR1, MRP1, MRP2, and ABCG2 in SP and non-SP cells. In addition, ICC assay was used to determine the protein expression and location of ABCG2 in this study. We found that expression of ABCG2 was higher in SP than non-SP cells (Fig. 3), meanwhile there were no differences of MDR1, MRP1 and MRP2 between SP and non-SP cells. 3.7. SP cells display higher tumorigenicity in vivo As shown in Table 1, SP cells from SGC-996 were injected into BALB/c-nu/nu female mice subcutaneously. One out of five mice formed tumors when inoculated with 5.0 103 SP cells; all five mice formed tumors when 5.0 105 SP cells were inoculated. However, only one out of five mice formed tumors when inocu-
Table 1 SP Cells and Non-SP Cells Inoculated into Nude Mice. Injected cell number
SP cells Non-SP cells
5 102
5 103
5 104
5 105
5 106
0/5 –
1/5 –
4/5 0/5
5/5 2/5
– 5/5
To explore the tumorigenic capacity, SP and non-SP cells sorted from human gallbladder carcinoma SGC-996 cells, ranging from 500 to 5.0 106, were injected subcutaneously into 6-week old BALB/c-nu/nu female mice.
lated with 5.0 105 non-SP cells, but when inoculated with 5.0 103 non-SP cells tumor formation was not observed in mice. Therefore, it was apparent that the tumor formation ability of SP cell was much higher than that of non-SP cells. By performing hematoxylin-eosin histological staining, we observed apparent pleomorphism of nuclei in the tumor tissue derived from SP and non-SP cells displaying typical cancer cell morphology. However, as compare with the tumors from non-SP cells, the tumors from SP cells showed central necrosis (Fig. 4).
4. Discussion The CSC hypothesis proposes that CSCs which are rich in primitive and undifferentiated cells can renew themselves, proliferate infinitely, and escape chemotherapy [4,18,19]. This small population of cells becomes the root of recurrence and metastasis [20]. Therefore it is desired to isolate and to characterize CSCs for effective therapeutic strategies of targeting these cells. As to the methods of detecting CSCs, identification of specific markers for CSCs is the preferred one [21,22]. But in most malignant tumors, investigators still have not identified specific CSC markers. Toward this direction, sorting SP cells using FCM by a dye Hoechst 33342 exclusion method has recently become a new means of CSC research. In this present study, we have isolated a small population of stemlike cells SP cells from the cell line, SGC-996, which is a malignant cell line from a female patient who suffered from human gallbladder papillary adenocarcinoma, and was established by Medical College of Tongji University of China. We have previously reported that together with platinum therapeutics, some adjucts can demonstrate enhanced anti-cancer effects in the SGC-996 cells [23]. Therefore, we have further explored SP cells from the SGC-996 cells, with the purpose of identification of new strategies of a more effective therapy for gall bladder cancer.
Fig. 4. The tumor formation ability of SP cell was much higher than that of non-SP cells. After 5.0 104 SP and Non-SP cells injected into BALB/c-nu/nu female mice subcutaneously, four-fifth mice formed tumors derived from SP cells (A, B), however non-SP cells tumor formation was not observed in mice. One out of five mice formed tumor when inoculated with 5.0 103 SP cells (B). Tumors cells were stained by hematoxylin-eosin (C).
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We detected SP cells from a human gallbladder carcinoma cell line, SGC-996, and found that the percentage is approximately 1.0%. In addition we detected similar percentage of SP cells (0.8%) from another internationally commonly studied gallbladder cancer cell line GBC-SD. Consistent with observations from others [14,15], the proportions of SP cells were rare and decreased in the presence of verapamil, which can effectively block ABCG2 from pumping out the Hoechst dye. After 2 weeks culture, SP cells of SGC-996 were able to reproduce the entire cancer cell line phenotype that exhibited a stem-like characteristic, and the SP fraction was a higher population than that from the original cell line SGC-996. In sharp contrast, non-SP cells scarcely had the capacity to produce SP cells. These findings imply that SP cells follow the so-called hierarchical CSC model (ref) and possibly have the characteristics of asymmetrical cell division. Our findings support the note that SP cells are likely responsible for tumor metastasis and recurrence because they are very powerful in proliferation, clonogenicity, tumorigenicity and invasion [9,16,24]. In this study, we found that SP cells demonstrate a higher proliferation rate, a higher clonal-generating capability, more migratory and invasive abilities than non-SP cells. Furthermore, our experiments revealed that SP cells were more tumorigenic than non-SP counterparts when injected into immunodeficient mice. As few as 5.0 103 SP cells could form tumor while only 5 105 non-SP cells produced the same phenomenon, which is in agreement with what reported in hepatocellular carcinoma [25], laryngeal cancer [26], esophageal carcinoma [14] and head and neck cancer [24] where SP cells also show a higher capacity to initiate tumor formation. Furthermore, compare with the tumor from non-SP cells, the tumor from SP cells displayed apparent central necrosis. Taken together, SP cells appear to be able to sustain their tumor initiation and to have a strong tumorigenicity. This may be a result of a population of amplifying cells that have tumor initial ability by their renewal, rapid proliferation, high clonogenicity and strong metastasis capabilities. Experimental studies suggest that although cytotoxic reagents may seem to be effective to the bulk of cancerous cells, SP cells appear to be more resistant to chemotherapy. Hirshmann-Jax and colleagues first demonstrated that SP cells from neuroblastoma were less sensitive to mitoxantrone [27]. Similar results were obtained in ovarian cancer [28] and Ewing’s sarcoma [29]. Consistently, we found in the present study that SP cells from the cell line SGC-996 were more resistant to two frequently-used chemotherapeutic agents, CDDP and doxorubicin. ABCG2 is a member of the ABC transporters, which can pump a wide variety of metabolites and drugs out of cells [30]. Widely expressed in CSCs, ABCG2 is also found to confer the SP phenotype [11]. Recent studies have shown that ABCG2 is a molecular determinant of the SP phenotype in a wide range of solid tumors [14,15,24,26,29]. Along this line, we have discovered in the present study that the mRNA and protein expression levels of ABCG2 were higher in SP cells from SGC-996 cells. In addition, in sharp contrast to ABCG2 which could pump out drugs and some materials from the cytoplasm of the cells, no difference was found between SP and non-SP cells in terms of mRNA expression of the other ABC transporters, such as MDR1, MRP1 and MRP2. Therefore, similar to the studies on other tumors [11], ABCG2 may be marker for CSCs and an attractive candidate therapeutic target for gallbladder carcinomas. In summary, SP cells from a human gallbladder carcinoma cell line, SGC-996, can be effectively sorted by using FACS with a dye Hoechst 33342 exclusion. SP cells possess the characteristics of CSC, such as asymmetrical cell division, rapider proliferation, higher clonogenicity, stronger tumorigenicity, more migratory and invasive abilities. Also SP cells are more resistant to chemotherapy
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and express high levels of ABCG2 in vivo. Hence, these results collectively suggest that SP cells are progenitor/stem cells and ABCG2 may be a candidate marker for SP cells and an attractive therapeutic target for human gallbladder cancer. Acknowledgments We thank Professor Wei-Qiang Gao for his very helpful editing this manuscript. This work was supported by grants from Shanghai science and technology commission, China (09411960800, J. Wang). References [1] S. Gourgiotis, H.M. Kocher, L. Solaini, A. Yarollahi, E. Tsiambas, N.S. Salemis, Gallbladder cancer, Am. J. Surg. 196 (2008) 252–264. [2] S. Misra, A. Chaturvedi, N.C. Misra, I.D. Sharma, Carcinoma of the gallbladder, Lancet Oncol. 4 (2003) 167–176. [3] J. Valle, H. Wasan, D.H. Palmer, D. Cunningham, A. Anthoney, A. Maraveyas, S. Madhusudan, T. Iveson, S. Hughes, S.P. Pereira, M. Roughton, J. Bridgewater, Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer, N. Engl. J. Med. 362 (2010) 1273–1281. [4] T. Reya, S.J. Morrison, M.F. Clarke, I.L. Weissman, Stem cells, cancer, and cancer stem cells, Nature 414 (2001) 105–111. [5] M. Al-Hajj, M.S. Wicha, A. Benito-Hernandez, S.J. Morrison, M.F. Clarke, Prospective identification of tumorigenic breast cancer cells, Proc. Natl. Acad. Sci. USA 100 (2003) 3983–3988. [6] H.D. Hemmati, I. Nakano, J.A. Lazareff, M. Masterman-Smith, D.H. Geschwind, M. Bronner-Fraser, H.I. Kornblum, Cancerous stem cells can arise from pediatric brain tumors, Proc. Nat. Acad. Sci. USA 100 (2003) 15178–15183. [7] P.C. Hermann, S.L. Huber, T. Herrler, A. Aicher, J.W. Ellwart, M. Guba, C.J. Bruns, C. Heeschen, Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer, Cell Stem Cell 1 (2007) 313–323. [8] S. Takaishi, T. Okumura, S.P. Tu, S.S.W. Wang, W. Shibata, R. Vigneshwaran, S.A.K. Gordon, Y. Shimada, T.C. Wang, Identification of Gastric Cancer Stem Cells Using the Cell Surface Marker CD44, Stem Cells 27 (2009) 1006–1020. [9] L. Moserle, M. Ghisi, A. Amadori, S. Indraccolo, Side population and cancer stem cells: therapeutic implications, Cancer Lett. 288 (2010) 1–9. [10] H. Ishii, M. Iwatsuki, K. Ieta, D. Ohta, N. Haraguchi, K. Mimori, M. Mori, Cancer stem cells and chemoradiation resistance, Cancer Sci. 99 (2008) 1871–1877. [11] X.W. Ding, J.H. Wu, C.P. Jiang, ABCG2: a potential marker of stem cells and novel target in stem cell and cancer therapy, Life Sci. 86 (2010) 631–637. [12] H.E. Schwabedissen, H.K. Kroemer, In vitro and in vivo evidence for the importance of breast cancer resistance protein transporters BCRP/MXR/ABCP/ ABCG2, Handb. Exp. Pharmacol. 201 (2011) 325–371. [13] M.A. Goodell, K. Brose, G. Paradis, A.S. Conner, R.C. Mulligan, Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo, J. Exp. Med. 183 (1996) 1797–1806. [14] D. Huang, Q. Gao, L. Guo, C. Zhang, W. Jiang, H. Li, J. Wang, X. Han, Y. Shi, S.H. Lu, Isolation and identification of cancer stem-like cells in esophageal carcinoma cell lines, Stem Cells Dev. 18 (2009) 465–473. [15] R. Fukaya, S. Ohta, M. Yamaguchi, H. Fujii, Y. Kawakami, T. Kawase, M. Toda, Isolation of cancer stem-like cells from a side population of a human glioblastoma cell line, SK-MG-1, Cancer Lett. 291 (2010) 150–157. [16] C. Wu, B.A. Alman, Side population cells in human cancers, Cancer Lett. 268 (2008) 1–9. [17] X.Z. Huang, J. Wang, C. Huang, Y.Y. Chen, G.Y. Shi, Q.S. Hu, J. Yi, Emodin enhances cytotoxicity of chemotherapeutic drugs in prostate cancer cells: the mechanisms involve ROS-mediated suppression of multidrug resistance and hypoxia inducible factor-1, Cancer Biol. Ther. 7 (2008) 468–475. [18] L.E. Ailles, I.L. Weissman, Cancer stem cells in solid tumors, Curr. Opin. Biotechnol. 18 (2007) 460–466. [19] J.B. Spillane, M.A. Henderson, Cancer stem cells: a review, ANZ. J. Surg. 77 (2007) 464–468. [20] C.E. Eyler, J.N. Rich, Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis, J. Clin. Oncol. 26 (2008) 2839–2845. [21] S. Bomken, K. Fiser, O. Heidenreich, J. Vormoor, Understanding the cancer stem cell, Br. J. Cancer 103 (2010) 439–445. [22] A. Sengupta, J.A. Cancelas, Cancer stem cells: a stride towards cancer cure?, J Cell Physiol. 225 (2010) 7–14. [23] W. Wang, Y.P. Sun, X.Z. Huang, M. He, Y.Y. Chen, G.Y. Shi, H. Li, J. Yi, J. Wang, Emodin enhances sensitivity of gallbladder cancer cells to platinum drugs via glutathion depletion and MRP1 downregulation, Biochem. Pharmacol. 79 (2010) 1134–1140. [24] M.H. Tabor, M.R. Clay, J.H. Owen, C.R. Bradford, T.E. Carey, G.T. Wolf, M.E. Prince, Head and neck cancer stem cells: the side population, Laryngoscope 121 (2011) 527–533. [25] G.M. Shi, Y. Xu, J. Fan, J. Zhou, X.R. Yang, S.J. Qiu, Y. Liao, W.Z. Wu, Y. Ji, A.W. Ke, Z.B. Ding, Y.Z. He, B. Wu, G.H. Yang, W.Z. Qin, W. Zhang, J. Zhu, Z.H. Min, Z.Q. Wu, Identification of side population cells in human hepatocellular carcinoma
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