Original Article 323
Authors
Y. Wang, G.-H. Zhang, S.-L. Li
Affiliation
Department of Radiation Medicine, Chongqing Medical University, Chongqing, China
Key words ▶ chemotherapy ● ▶ cancer stem cells ● ▶ tumor recurrence ● ▶ side population cells ● ▶ ABC transporters ●
Abstract
received 29.05.2014 accepted 04.06.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382077 Published online: September 10, 2014 Drug Res 2015; 65: 323–326 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S.-L. Li Department of Radiation Medicine Chongqing Medical University No. 1 Yixueyuan Road Yuzhong District Chongqing 400016 China Tel.: + 86/23/68485 060 Fax: + 86/23/68485 060 [email protected]
▼
Introduction: Recent studies reported that cancer stem cells (CSCs) are responsible for treatment failure and tumor recurrence. CSCs have been isolated and characterized in several solid tumors. However, the information regarding the CSCs from nasopharyngeal carcinoma (NPC) is very less. Therefore, in the current study we aimed to isolate and characterize the cancer stem like SP cells from NPC. Methods: We have used FACS based Hoechst 33342 dye exclusion technique to identify the cancer stem like SP cells from NPC. The FACS sorted SP cells are further subjected to drug resistance assay, clonogenic formation assay and
Introduction
▼
Nasopharyngeal carcinoma (NPC) is one of the most common cancers originate from the upper region of pharynx, occurs in both men and women among worldwide. Although recent advances in treatment strategy, still treatment failure and tumor recurrence occurs in individuals after chemo-and radiotherapy. According to CSCs theory, CSCs are the small sub-population of cells within the tumor responsible for therapeutic resistance and tumor relapse. Moreover, the current conventional treatment strategy are killing most of the neoplastic cells within the tumor by leaving the cancer stem cells unaffected and thus results in minimal residual disease (MDR). Therefore it is necessary to target and kill the CSCs to eradicate the complete refractory of the tumor and for long term disease free survival. There are 2 different methods to isolate the cancer stem cells based on i) expression of stem cells surface markers such as CD133 and CD44 [1] and ii) Hoechst 33342 dye exclusion technique [2]. In the later method, the cancer cells which exclude this
immunocytochemistry analysis for the expression of CD44 and ABCG2. Results: We have identified 3.9 % of cancer stem like SP cells from NPC. Upon treatment with verapamil, the percentage of SP cells was reduced to 0.9 %. The FACS sorted SP cells showed enhanced expression of CD44 and ABCG2. Further, these SP cells are highly resistant to multi-drugs and high potent of sphere formation when compared to the non-SP cells. Conclusions: Our data clearly suggest that presence of cancer stem like SP cells from nasopharyngeal carcinoma might responsible for chemotherapeutic drug resistance, tumor recurrence and invasion.
dye, falls on the side of the dot plot analysis of FACS and hence they are named as “side population (SP) cells” which shares the remarkable features of CSCs such as chemotherapy and apoptosis resistance, over expression of ABC transporter proteins, self-renewal, expression of stem cell surface markers, highly tumorigenic and differentiation potential [3–5]. Therefore, SP cells are considered as enriched cancer stem cells. Hence, isolation and characterization of SP cells helps to target the CSCs effectively. Recently, cancer stem like SP cells has been well characterized in several tumors such as small cell lung cancer, glioma, prostate cancer, leukemia, neuroblastoma, hepatoma, nasopharyngeal carcinoma, colorectal cancer, thyroid cancer and lung cancer [2, 6–13]. However, there are few or insufficient information about the CSCs of nasopharyngeal carcinoma. Consequently, in the current study, we aimed to isolate and characterize the CSCs like SP cells from NPC based on Hoechst 33342 dye exclusion technique. The results of this paper are described in detail as follows.
Wang Y et al. Nasopharyngeal Cancer Stem Cells … Drug Res 2015; 65: 323–326
Downloaded by: Rutgers University. Copyrighted material.
Isolation and Phenotypic Characterization of Cancer Stem Like Cells from Nasopharyngeal Carcinoma
324 Original Article
▼
Sample collection and cell culture Nasopharyngeal carcinoma samples were collected at the time of surgery according to the ethical approval. The details of NPC samples: number of patients (n = 9); stage – IV; region – infratemporal. The cancer tissues were washed extensively in PBS solution containing antibiotics and incubated overnight in DMEM/ F12 (GIBCO) containing penicillin (500 U/ml), streptomycin (500 μg/ml), and amphotericin B (1.25 μg/ml) (GIBCO). Enzymatic digestion was performed using collagenase (1.5 mg/ml) (GIBCO) and hyaluronidase (20 μg/ml) in PBS for 1 h. Cells were cultured in DMEM with 10 % FBS, supplemented with antibiotics and maintain in T-75 flasks at 37 °C in a humidified 5 % CO2 & 95 % air atmosphere. Cells on becoming 90 % confluent are removed from culture flask using Trypsin-EDTA (0.25 % – 53 mM EDTA) washed, cells suspended in 10 % DMEM. Cell count was taken using hemocytometer.
Labeling of Hoechst 33342 dye Study groups a. Control – cells labeled with Hoechst 33342 dye alone (n = 5) b. Drug treated – cells treated with Verapamil drug and Hoechst 33342 dye (n = 5). Cells were counted by hemocytometer ad approximately 106 cells/ml of 10 % DMEM were labeled with Hoechst 33342 stock (sigma)-bis-benzimide (5 μl/ml) either with dye alone or in combination with drug (verapamil – 0.8 μl/ml). Further, cells were counter stained with PI (propidium iodide) 2 μg/ml. The cells were sorted using a flow cytometer and the sorted cells were cultured and maintained in DMEM/F-12 supplemented with 10 % FBS. The Hoechst 33342 dye was excited at 355 nm and its dual-wavelength fluorescence was analyzed (blue, 450 nm; red, 675 nm).
for 30 min at RT. Cells counterstained with hematoxylin and mounted with Glycerol vinyl alcohol aqueous mounting solution [14]. Under optical microscope, the red colour ABCG2 + cells were observed. All images were processed with adobe photoshop CS4.
Cell resistance assay Approximately 1 × 103 cells/plate were cultured in 96-well plates and treated with the drugs such as 5-fluorouracil (5-FU) with a concentration of 10 μg/ml, cisplatin (20 μmol/L), paclitaxel (2 μmol/L) and docetaxel (2 μg/ml 100). The mean value of OD450 obtained was represented as a graph. Cell resistance in both groups was calculated using the following formula: Cell resistance rate ( %) = (experimental group OD450 value/control group OD450 value) × 100, as described previously [15].
Sphere formation assay Sphere formation assay was performed exactly as described previously [16]. The sorted SP cells and non-SP cells were placed at a density of 1 000 cells/ml resuspended in tumor sphere medium consisting of serum-free 1:1 mixture of Ham’s F-12/DMEM, N2 supplement, 10 ng/ml human recombinant bFGF, and 10 ng/ml EGF and subsequently cultured in ultra-low attachment plates for about 2 weeks. SP and non-SP cells sorted were seeded at a low density of 20 cells/L and the number of generated spheres ( > 100 lm) was counted after 7 days of culture.
Statistical analysis One-way analysis of variance (ANOVA) and student T-test was performed to determine the significant difference between the treatment and control groups. A probability level of p < 0.01 was considered as statistically significance.
Results Immunofluorescent staining
▼
The FACS sorted SP cells were fixed onto glass slides in ice-cold 4 % paraformaldehyde (4 °C, 10 min), and blocked with 1 % bovine serum albumin for 30 min at RT to block nonspecific binding of IgG. After PBS wash, cells were incubated with FITC anti-human CD44 at 4 °C for 30 min in the dark. After extensive PBS wash, cell nuclei were counterstained with Hoechst 33342 and viewed under confocal microscope. For ABCG2 expression, cells were incubated with mouse antihuman ABCG2 antibody at 4 °C for overnight. After PBS wash, cells were added with goat anti-mouse IgG-HRP and incubated
During FACS analysis, live cells were selected against the propid▶ Fig. 1). We have identiium iodide staining (P1 gated region of ● fied about 3.9 % of cancer stem like SP cells from NPC (P2 gated ▶ Fig. 1a), which excludes Hoechst 33342 dye out of region of ● the cells. Upon treatment with verapamil, a MDR1 transporter inhibitor the presence of SP cells was reduced to 0.9 % (P2 gated ▶ Fig. 1b), which confirms the dye exclusion process region of ● actively, involves MDR1 transporter proteins. The sorted SP cells showed enhanced expression of ABC transporter protein ABCG2 (belongs to MDR1 transporter family) and stem cell surface proFig. 1 A representative image for dot plot analysis of FACS. Live cells were selected in P1 region by propidium iodide staining. a Graph showing 3.9 % of SP cells from nasopharygeal carcinoma (NPC) in the P2 gated region which excludes Hoechst 33342 dye. b NPC cells after treatment with verapamil shows reduced percentage of SP cells (0.9 %) in the P2 gated region.
Wang Y et al. Nasopharyngeal Cancer Stem Cells … Drug Res 2015; 65: 323–326
Downloaded by: Rutgers University. Copyrighted material.
Materials and Methods
Original Article 325
tein CD44 which might be responsible for chemotherapeutic ▶ Fig. 2a, drug resistance and tumor metastasis, respectively (● b). The drug resistance assay and clonogenic formation assay revealed that NPC SP cells are highly resistance to multi-drugs, ▶ Fig. 3) and they form tumor have increased cell survival rate (● ▶ Fig. 4). Hence, spheres significantly higher than non-SP cells (● our data clearly suggest that presence of CD44 and ABCG2 positive SP cells might be the major cause for drug resistance and tumor relapse in nasopharyngeal carcinoma.
Discussion
▼ Fig. 3 The FACS sorted NPC SP cells showed significantly increased cell survival rate than non-SP cells after treatment with drugs such as 5-fluorouracil (5-FU), cisplatin, docetaxel and paclitaxel. The bar represents the standard deviation (**, p < 0.01).
Fig. 4 Clone formation efficiency of CD44 positive SP cells. The total number tumor spheres generated by NPC SP cells are significantly higher than non-SP cells. The bar represents the standard deviation (**, p < 0.01).
Cancer stem cells (CSCs) are proposed as a major factor responsible for multi-drug resistance owing to the over expression of efflux pumps and tumor recurrence. The traditional treatment strategies failed to kill the CSCs which remains dormant and cause for minimal residual disease (MDR) after treatment. Therefore elimination of CSCs is an essential goal to eradicate the tumor refractory and prevent tumor relapse. One of the remarkable methods to identify the CSCs is Hoechst 33342-dye exclusion technique and the cells that expel this dye are referred to as side population (SP) cells [10] that possess the essential features of CSCs. In the present work, by using Hoechst 33342 dye exclusion method we have identified 3.9 % SP cells from NPC whose presence is reduced to 0.9 % after treatment with verapamil. Further, immunocytochemistry data revealed that sorted SP cells have increased expression of ABC transporter protein ABCG2. This confirms the dye exclusion process of SP cells from NPC actively involves ABCG2 over expression. CD44 belongs to the family of cell surface proteoglycans and glycoproteins involved in tumor invasion and metastasis in different tumors and therefore CD44 positive expression can be used as a valuable marker for isolation of CSCs. Also, CD44 positive cells involved in chemoand radiotherapy resistance [17–21]. Studies in NPC also showed that increased expression of CD44 positive cells that might be responsible for high tumorigenesis and metastasis [1]. In line Wang Y et al. Nasopharyngeal Cancer Stem Cells … Drug Res 2015; 65: 323–326
Downloaded by: Rutgers University. Copyrighted material.
Fig. 2 Immunocytochemistry analysis of the sorted NPC SP cells. a SP cells showing enhanced expression of ABCG2 (red colour) when compared to non-SP cells. b Increased experssion of CD44 (green) was observed in SP cells than non-SP cells. Cell nuclei were counterstained with Hoechst 33342 (blue colour).
with these findings, we also showed that sorted cancer stem like SP cells are highly positive to CD44 when compared to non-SP cells. Therefore, together with ABCG2, CD44 also contribute to chemotherapy resistance and tumorigenesis and invasion of NPC cells. We also demonstrated that sorted SP cells are highly resistance to the chemotherapeutic agents such as 5-FU, cisplatin, docetaxel and paclitaxel and the survival rate of SP cells after treatment with these drugs are significantly higher compared to non-SP cells. This could be due to over expression efflux pumps (ABCG2) and might also have increased expression of anti-apoptotic factor Bcl-2 and thus results in reduced apoptosis. Also the sorted SP cells are high potent to generate tumor spheres than non-SP cells. Most likely this is due to the increased expression of stem cell surface protein CD44 and this enables the sorted SP cells are self-renewal. We also hypothesize that the sorted NPC SP cells might have an increased expression of other stem cell surface genes and anti-apoptotic factor and bcl-2 which might also confers the properties of metastasis/self renewal and apoptosis-resistance, respectively. However, these speculations should be studied in detail. Therefore, isolation and characterization of side population might provide valuable information for improving treatment strategy and to design a novel therapeutic agent that effectively targets cancer stem cells and prevent minimal residual disease in NPC.
Acknowledgements
▼
This project work is carried out with the National Science Foundation (NO.81171365). We thank National Science Foundation for funding. We also thank Dr. Wanshan Li, Department of Oral and Maxillofacial Surgery, Chongqing Medical University for sharing the FACS protocol by personal communication.
Conflict of Interest
▼
The authors declared no conflict of interests.
References 1 Su J, Xu XH, Huang Q et al. Identification of Cancer Stem-like CD44þ Cells in Human Nasopharyngeal Carcinoma Cell Line. Archives of Medical Research 2011; 42: 15e21 2 Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996; 183: 1797–1806
Wang Y et al. Nasopharyngeal Cancer Stem Cells … Drug Res 2015; 65: 323–326
3 Bourguignon LY, Peyrollier K, Xia W et al. Hyaluronan-CD44 interaction activates stem cell marker Nanog, Stat-3-mediated MDR1 gene expression, and ankyrin-regulated multidrug efflux in breast and ovarian tumor cells. J Biol Chem 2008; 283: 17635–17651 4 Gao AC, Lou W, Sleeman JP et al. Metastasis suppression by the standard CD44 isoform does not require the binding of prostate cancer cells to hyaluronate. Cancer Res 1998; 58: 2350–2352 5 Wang J, Guo LP, Chen LZ et al. Identification of cancer stem cell-like side population cells in human nasopharyngeal carcinoma cell line. Cancer Res 2007; 67: 3716–3724 6 Salcido CD, Larochelle A, Taylor BJ et al. Molecular characterisation of side population cells with cancer stem cell-like characteristics in small-cell lung cancer. Br J Cancer 2010; 102: 1636–1644 7 Yin B, Yang Y, Zhao Z et al. Arachidonate 12-lipoxygenase may serve as a potential marker and therapeutic target for prostate cancer stem cells. Int J Oncol 2011; 38: 1041–1046 8 Tabuse M, Ohta S, Ohashi Y et al. Functional analysis of HOXD9 in human gliomas and glioma cancer stem cells. Mol Cancer 2011; 10: 60 9 Feuring-Buske M, Hogge DE. Hoechst 33342 efflux identifies a subpopulation of cytogenetically normal CD34( + )CD38(−) progenitor cells from patients with acute myeloid leukemia. Blood 2001; 97: 3882–3889 10 Hirschmann-Jax C, Foster AE, Wulf GG et al. A distinct ‘side population’ of cells with high drug efflux capacity in human tumor cells. Proc Natl Acad Sci USA 2004; 101: 14228–14233 11 Mitsutake N, Iwao A, Nagai K et al. Characterization of side population in thyroid cancer cell lines: cancer stem-like cells are enriched partly but not exclusively. Endocrinology 2007; 148: 1797–1803 12 Haraguchi N, Utsunomiya T, Inoue H et al. Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 2006; 24: 506–513 13 Ho MM, Ng AV, Lam S et al. Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 2007; 67: 4827–4833 14 Zhang H, Liu W, Feng X et al. Identification of ABCG2 + cells in nasopharyngeal carcinoma cells. Oncology Reports 2012; 27: 1177–1187 15 He QZ, Luo XZ, Wang K et al. Isolation and characterization of cancer stem cells from high-grade serous ovarian carcinomas. Cell Physiol Biochem 2014; 33: 173–184 16 Yanamoto S, Kawasaki G, Yamada S et al. Isolation and characterization of cancer stem-like side population cells in human oral cancer cells. Oral Oncol 2011; 47: 855–860 17 Honeth G, Bendahl PO, Ringnér M et al. The CD44þ/CD24" phenotype is enriched in basal-like breast tumors. Breast Cancer Res 2008; 10: 53 18 Richardson GD, Robson CN, Lang SH. CD133, a novel marker for human prostatic epithelial stem cells. J Cell Sci 2004; 117: 3539e3545 19 Li C, Lee CJ, Simeone DM. Identification of pancreatic cancer stem cells. Methods Mol Biol 2009; 568: 161–173 20 Zhang S, Balch C, Chan MW et al. Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res 2008; 68: 4311e4320 21 Okamoto A, Chikamatsu K, Sakakura K et al. Expansion and characterization of cancer stem-like cells in squamous cell carcinoma of the head and neck. Oral Oncol 2009; 45: 633e639
Downloaded by: Rutgers University. Copyrighted material.
326 Original Article
Authors
Y. Wang, G.-H. Zhang, S.-L. Li
Affiliation
Department of Radiation Medicine, Chongqing Medical University, Chongqing, China
Key words ▶ chemotherapy ● ▶ cancer stem cells ● ▶ tumor recurrence ● ▶ side population cells ● ▶ ABC transporters ●
Abstract
received 29.05.2014 accepted 04.06.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382077 Published online: September 10, 2014 Drug Res 2015; 65: 323–326 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S.-L. Li Department of Radiation Medicine Chongqing Medical University No. 1 Yixueyuan Road Yuzhong District Chongqing 400016 China Tel.: + 86/23/68485 060 Fax: + 86/23/68485 060 [email protected]
▼
Introduction: Recent studies reported that cancer stem cells (CSCs) are responsible for treatment failure and tumor recurrence. CSCs have been isolated and characterized in several solid tumors. However, the information regarding the CSCs from nasopharyngeal carcinoma (NPC) is very less. Therefore, in the current study we aimed to isolate and characterize the cancer stem like SP cells from NPC. Methods: We have used FACS based Hoechst 33342 dye exclusion technique to identify the cancer stem like SP cells from NPC. The FACS sorted SP cells are further subjected to drug resistance assay, clonogenic formation assay and
Introduction
▼
Nasopharyngeal carcinoma (NPC) is one of the most common cancers originate from the upper region of pharynx, occurs in both men and women among worldwide. Although recent advances in treatment strategy, still treatment failure and tumor recurrence occurs in individuals after chemo-and radiotherapy. According to CSCs theory, CSCs are the small sub-population of cells within the tumor responsible for therapeutic resistance and tumor relapse. Moreover, the current conventional treatment strategy are killing most of the neoplastic cells within the tumor by leaving the cancer stem cells unaffected and thus results in minimal residual disease (MDR). Therefore it is necessary to target and kill the CSCs to eradicate the complete refractory of the tumor and for long term disease free survival. There are 2 different methods to isolate the cancer stem cells based on i) expression of stem cells surface markers such as CD133 and CD44 [1] and ii) Hoechst 33342 dye exclusion technique [2]. In the later method, the cancer cells which exclude this
immunocytochemistry analysis for the expression of CD44 and ABCG2. Results: We have identified 3.9 % of cancer stem like SP cells from NPC. Upon treatment with verapamil, the percentage of SP cells was reduced to 0.9 %. The FACS sorted SP cells showed enhanced expression of CD44 and ABCG2. Further, these SP cells are highly resistant to multi-drugs and high potent of sphere formation when compared to the non-SP cells. Conclusions: Our data clearly suggest that presence of cancer stem like SP cells from nasopharyngeal carcinoma might responsible for chemotherapeutic drug resistance, tumor recurrence and invasion.
dye, falls on the side of the dot plot analysis of FACS and hence they are named as “side population (SP) cells” which shares the remarkable features of CSCs such as chemotherapy and apoptosis resistance, over expression of ABC transporter proteins, self-renewal, expression of stem cell surface markers, highly tumorigenic and differentiation potential [3–5]. Therefore, SP cells are considered as enriched cancer stem cells. Hence, isolation and characterization of SP cells helps to target the CSCs effectively. Recently, cancer stem like SP cells has been well characterized in several tumors such as small cell lung cancer, glioma, prostate cancer, leukemia, neuroblastoma, hepatoma, nasopharyngeal carcinoma, colorectal cancer, thyroid cancer and lung cancer [2, 6–13]. However, there are few or insufficient information about the CSCs of nasopharyngeal carcinoma. Consequently, in the current study, we aimed to isolate and characterize the CSCs like SP cells from NPC based on Hoechst 33342 dye exclusion technique. The results of this paper are described in detail as follows.
Wang Y et al. Nasopharyngeal Cancer Stem Cells … Drug Res 2015; 65: 323–326
Downloaded by: Rutgers University. Copyrighted material.
Isolation and Phenotypic Characterization of Cancer Stem Like Cells from Nasopharyngeal Carcinoma
324 Original Article
▼
Sample collection and cell culture Nasopharyngeal carcinoma samples were collected at the time of surgery according to the ethical approval. The details of NPC samples: number of patients (n = 9); stage – IV; region – infratemporal. The cancer tissues were washed extensively in PBS solution containing antibiotics and incubated overnight in DMEM/ F12 (GIBCO) containing penicillin (500 U/ml), streptomycin (500 μg/ml), and amphotericin B (1.25 μg/ml) (GIBCO). Enzymatic digestion was performed using collagenase (1.5 mg/ml) (GIBCO) and hyaluronidase (20 μg/ml) in PBS for 1 h. Cells were cultured in DMEM with 10 % FBS, supplemented with antibiotics and maintain in T-75 flasks at 37 °C in a humidified 5 % CO2 & 95 % air atmosphere. Cells on becoming 90 % confluent are removed from culture flask using Trypsin-EDTA (0.25 % – 53 mM EDTA) washed, cells suspended in 10 % DMEM. Cell count was taken using hemocytometer.
Labeling of Hoechst 33342 dye Study groups a. Control – cells labeled with Hoechst 33342 dye alone (n = 5) b. Drug treated – cells treated with Verapamil drug and Hoechst 33342 dye (n = 5). Cells were counted by hemocytometer ad approximately 106 cells/ml of 10 % DMEM were labeled with Hoechst 33342 stock (sigma)-bis-benzimide (5 μl/ml) either with dye alone or in combination with drug (verapamil – 0.8 μl/ml). Further, cells were counter stained with PI (propidium iodide) 2 μg/ml. The cells were sorted using a flow cytometer and the sorted cells were cultured and maintained in DMEM/F-12 supplemented with 10 % FBS. The Hoechst 33342 dye was excited at 355 nm and its dual-wavelength fluorescence was analyzed (blue, 450 nm; red, 675 nm).
for 30 min at RT. Cells counterstained with hematoxylin and mounted with Glycerol vinyl alcohol aqueous mounting solution [14]. Under optical microscope, the red colour ABCG2 + cells were observed. All images were processed with adobe photoshop CS4.
Cell resistance assay Approximately 1 × 103 cells/plate were cultured in 96-well plates and treated with the drugs such as 5-fluorouracil (5-FU) with a concentration of 10 μg/ml, cisplatin (20 μmol/L), paclitaxel (2 μmol/L) and docetaxel (2 μg/ml 100). The mean value of OD450 obtained was represented as a graph. Cell resistance in both groups was calculated using the following formula: Cell resistance rate ( %) = (experimental group OD450 value/control group OD450 value) × 100, as described previously [15].
Sphere formation assay Sphere formation assay was performed exactly as described previously [16]. The sorted SP cells and non-SP cells were placed at a density of 1 000 cells/ml resuspended in tumor sphere medium consisting of serum-free 1:1 mixture of Ham’s F-12/DMEM, N2 supplement, 10 ng/ml human recombinant bFGF, and 10 ng/ml EGF and subsequently cultured in ultra-low attachment plates for about 2 weeks. SP and non-SP cells sorted were seeded at a low density of 20 cells/L and the number of generated spheres ( > 100 lm) was counted after 7 days of culture.
Statistical analysis One-way analysis of variance (ANOVA) and student T-test was performed to determine the significant difference between the treatment and control groups. A probability level of p < 0.01 was considered as statistically significance.
Results Immunofluorescent staining
▼
The FACS sorted SP cells were fixed onto glass slides in ice-cold 4 % paraformaldehyde (4 °C, 10 min), and blocked with 1 % bovine serum albumin for 30 min at RT to block nonspecific binding of IgG. After PBS wash, cells were incubated with FITC anti-human CD44 at 4 °C for 30 min in the dark. After extensive PBS wash, cell nuclei were counterstained with Hoechst 33342 and viewed under confocal microscope. For ABCG2 expression, cells were incubated with mouse antihuman ABCG2 antibody at 4 °C for overnight. After PBS wash, cells were added with goat anti-mouse IgG-HRP and incubated
During FACS analysis, live cells were selected against the propid▶ Fig. 1). We have identiium iodide staining (P1 gated region of ● fied about 3.9 % of cancer stem like SP cells from NPC (P2 gated ▶ Fig. 1a), which excludes Hoechst 33342 dye out of region of ● the cells. Upon treatment with verapamil, a MDR1 transporter inhibitor the presence of SP cells was reduced to 0.9 % (P2 gated ▶ Fig. 1b), which confirms the dye exclusion process region of ● actively, involves MDR1 transporter proteins. The sorted SP cells showed enhanced expression of ABC transporter protein ABCG2 (belongs to MDR1 transporter family) and stem cell surface proFig. 1 A representative image for dot plot analysis of FACS. Live cells were selected in P1 region by propidium iodide staining. a Graph showing 3.9 % of SP cells from nasopharygeal carcinoma (NPC) in the P2 gated region which excludes Hoechst 33342 dye. b NPC cells after treatment with verapamil shows reduced percentage of SP cells (0.9 %) in the P2 gated region.
Wang Y et al. Nasopharyngeal Cancer Stem Cells … Drug Res 2015; 65: 323–326
Downloaded by: Rutgers University. Copyrighted material.
Materials and Methods
Original Article 325
tein CD44 which might be responsible for chemotherapeutic ▶ Fig. 2a, drug resistance and tumor metastasis, respectively (● b). The drug resistance assay and clonogenic formation assay revealed that NPC SP cells are highly resistance to multi-drugs, ▶ Fig. 3) and they form tumor have increased cell survival rate (● ▶ Fig. 4). Hence, spheres significantly higher than non-SP cells (● our data clearly suggest that presence of CD44 and ABCG2 positive SP cells might be the major cause for drug resistance and tumor relapse in nasopharyngeal carcinoma.
Discussion
▼ Fig. 3 The FACS sorted NPC SP cells showed significantly increased cell survival rate than non-SP cells after treatment with drugs such as 5-fluorouracil (5-FU), cisplatin, docetaxel and paclitaxel. The bar represents the standard deviation (**, p < 0.01).
Fig. 4 Clone formation efficiency of CD44 positive SP cells. The total number tumor spheres generated by NPC SP cells are significantly higher than non-SP cells. The bar represents the standard deviation (**, p < 0.01).
Cancer stem cells (CSCs) are proposed as a major factor responsible for multi-drug resistance owing to the over expression of efflux pumps and tumor recurrence. The traditional treatment strategies failed to kill the CSCs which remains dormant and cause for minimal residual disease (MDR) after treatment. Therefore elimination of CSCs is an essential goal to eradicate the tumor refractory and prevent tumor relapse. One of the remarkable methods to identify the CSCs is Hoechst 33342-dye exclusion technique and the cells that expel this dye are referred to as side population (SP) cells [10] that possess the essential features of CSCs. In the present work, by using Hoechst 33342 dye exclusion method we have identified 3.9 % SP cells from NPC whose presence is reduced to 0.9 % after treatment with verapamil. Further, immunocytochemistry data revealed that sorted SP cells have increased expression of ABC transporter protein ABCG2. This confirms the dye exclusion process of SP cells from NPC actively involves ABCG2 over expression. CD44 belongs to the family of cell surface proteoglycans and glycoproteins involved in tumor invasion and metastasis in different tumors and therefore CD44 positive expression can be used as a valuable marker for isolation of CSCs. Also, CD44 positive cells involved in chemoand radiotherapy resistance [17–21]. Studies in NPC also showed that increased expression of CD44 positive cells that might be responsible for high tumorigenesis and metastasis [1]. In line Wang Y et al. Nasopharyngeal Cancer Stem Cells … Drug Res 2015; 65: 323–326
Downloaded by: Rutgers University. Copyrighted material.
Fig. 2 Immunocytochemistry analysis of the sorted NPC SP cells. a SP cells showing enhanced expression of ABCG2 (red colour) when compared to non-SP cells. b Increased experssion of CD44 (green) was observed in SP cells than non-SP cells. Cell nuclei were counterstained with Hoechst 33342 (blue colour).
with these findings, we also showed that sorted cancer stem like SP cells are highly positive to CD44 when compared to non-SP cells. Therefore, together with ABCG2, CD44 also contribute to chemotherapy resistance and tumorigenesis and invasion of NPC cells. We also demonstrated that sorted SP cells are highly resistance to the chemotherapeutic agents such as 5-FU, cisplatin, docetaxel and paclitaxel and the survival rate of SP cells after treatment with these drugs are significantly higher compared to non-SP cells. This could be due to over expression efflux pumps (ABCG2) and might also have increased expression of anti-apoptotic factor Bcl-2 and thus results in reduced apoptosis. Also the sorted SP cells are high potent to generate tumor spheres than non-SP cells. Most likely this is due to the increased expression of stem cell surface protein CD44 and this enables the sorted SP cells are self-renewal. We also hypothesize that the sorted NPC SP cells might have an increased expression of other stem cell surface genes and anti-apoptotic factor and bcl-2 which might also confers the properties of metastasis/self renewal and apoptosis-resistance, respectively. However, these speculations should be studied in detail. Therefore, isolation and characterization of side population might provide valuable information for improving treatment strategy and to design a novel therapeutic agent that effectively targets cancer stem cells and prevent minimal residual disease in NPC.
Acknowledgements
▼
This project work is carried out with the National Science Foundation (NO.81171365). We thank National Science Foundation for funding. We also thank Dr. Wanshan Li, Department of Oral and Maxillofacial Surgery, Chongqing Medical University for sharing the FACS protocol by personal communication.
Conflict of Interest
▼
The authors declared no conflict of interests.
References 1 Su J, Xu XH, Huang Q et al. Identification of Cancer Stem-like CD44þ Cells in Human Nasopharyngeal Carcinoma Cell Line. Archives of Medical Research 2011; 42: 15e21 2 Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996; 183: 1797–1806
Wang Y et al. Nasopharyngeal Cancer Stem Cells … Drug Res 2015; 65: 323–326
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