3D culture and analysis of the expression of cancer stem cell markers from gastric cancer cell line mkn45

Nội dung text: 3D culture and analysis of the expression of cancer stem cell markers from gastric cancer cell line mkn45

1. JOURNAL OF MEDICAL RESEARCH 3D CULTURE AND ANALYSIS OF THE EXPRESSION OF CANCER STEM CELL MARKERS FROM GASTRIC CANCER CELL LINE MKN45 Ngo Thu Ha¹, Le Thi Thanh Huong¹, La Thi Huyen², Nguyen Phu Hung¹,³ ¹Thai Nguyen University of Sciences ²Institute of Biotechnology - Vietnam Academy of Science and Technology ³French National Institute of Health and Medical Research U1235, Nantes, France Gastric cancer is the fourth leading cause of worldwide cancer mortality, Vietnam belongs to the specified high-risk group of developing gastric cancer. Gastric cancer stem cells (CSCs) are considered to be the origin of gastric adenocarcinoma. 3D cell culture is an important step of identification of stem cell as well as CSC. In this study, a non-adherent cell culture model (3D) was developed with a culture medium that had growth factors selective for the development of gastric CSCs from gastric cancer cell line MKN45. The result showed that only 6% single cancer cells developed into tumorspheres during culture process. Then, flow cytometry and immunofluorescence analysis was carried out to evaluate the expression of CSC markers CD44 and ALDH in samples taken from tumorspheres which arose from gastric CSCs. The results showed that gastric CSCs formed tumorspheres in 3D culture condition with a high expression of 92% and 40,1% for markers CD44 and ALDH, respectively. Keywords: 3D culture, gastric cancer stem cell, CD44, ALDH, MKN45 I. INTRODUCTION Cancer is the leading cause of death family history, chronic gastritis, smoking and globally. Gastric cancer has the fourth high- Helicobacter pylori (H. pylori) infection [2]. est mortality, accounting for 723,000 deaths Epidemiological studies show the close re- in 2012 according to World Health Organi- lationship between chronic atrophic gastritis zation [1]. Vietnamese are at high risk of and H. pylori infection [3]. In atrophic gastri- developing gastric cancer. Important risk tis, the reduction of parietal cells and chief factors for development of gastric cancer in- cells lead to decrease HCl secretion which clude smoked foods, salted fish and meat, is a favorable environment for H. pylori de- velopment. H. pylori overgrowth increases Corresponding author: Nguyen Phu Hung, genetic mutation in epithelial cells of stom- Thai Nguyen University of Sciences ach which can cause gastric cancer [4]. Email: hungnguyenphu@gmail.com Lauren's gastric cancer classification Received: 10 July 2017 system subdivided gastric adenocarcinoma Accepted: 09 Octorber 2017 into two main types: intestinal type accounts JMR 111 E2 (2) - 2018 53
2. JOURNAL OF MEDICAL RESEARCH for 54% and diffuse type is about 32%. The methods in CSC studies [13]. remaining 14% belongs to indeterminate CD44 (Cluster of differentiation 44), a type (mixture of two main types above) [5; receptor of hyaluronic acid, was used to 6]. While intestinal type tumors are typically identify gastric CSCs for the first time by found in the gastric antrum of older individu- Takaishi et al. in 2009 [14]. ALDH (aldehyde als, diffuse type tumors are more common dehydrogenase) is an important marker of in the gastric corpus of younger individuals CSCs in solid tumors and gastric carcinoma [7; 8]. in particular. It is involved in cell detoxifica- Recent research indicated that although tion, regulation of cellular proliferation, and cancer stem cells (CSCs) frequency in tu- relates to drug tolerance in cancer [15]. mors is low, CSCs are responsible for pro- Studies on stem cells and CSCs have liferation and differentiation of tumor cells. not been done previously in Vietnam, and Gastric stem cells which are located in the 3D cell culture is an emerging technique in isthmus or progenitor zone of gastric gland cancer research in general and gastric can- and differentiate into mucous, parietal, chief cer in particular. In this study, MKN45 gas- and endocrine cells can be considered the tric cancer cells were seeded in 3D culture origin of gastric CSCs [9]. In in-vitro exper- condition. The existence of gastric CSCs iments, if one cell can form tumorsphere in was evaluated by measuring tumorsphere non-adherent culture condition (3D) with formation and the expression of CD44 and medium containing growth factors, it can ALDH gastric cancer stem cell markers. qualify as a CSCs. CD44 and ALDH expression was measured Cancer cells grown in 3D cell culture using flow cytometry and immunofluores- condition were more similar to cells in tu- cence analysis. mors when compared with cells that were II. MATERIALS AND METHODS cultured in adherent (2D) condition [10]. CSC identification and culture are important 1. Materials: Gastric cancer cell line parts of cancer research; single cell culture MKN45 to form tumorspheres (3D culture) is a sig- Gastric cancer cell line MKN45 was a nificant tool in CSCs culture. generous gift from Inserm U1053 Laborato- CD44 and ALDH are two popular mark- ry – French National Institute of Health and ers used for evaluation and isolation of Medical Research in Bordeaux. Cells were CSCs in many different types of cancers maintained in RPMI media supplemented [11]. In gastric adenocarcinoma, these with 10% Fetal Bovine Serum (FBS), and markers expression identifies gastric can- 1% ampicillin/streptomycin (P/S). The seed- cer stem cell markers and is related to drug ing density was 1 x 10⁵ cells/well in 3.8 cm² tolerance of tumor [12]. There are many wells using adherent cell culture condition different methods to identify expression of (2D). Cells were incubated at 37oC in the CD44 and ALDH markers, but Flow cytom- presence of 5% CO2. After 2 days, culture etry and immunofluorescence are common 54 JMR 111 E2 (2) - 2018
3. JOURNAL OF MEDICAL RESEARCH medium was replaced. After 4 days, cells monoclonal antibody 1:50 (from BD Bio- were passaged into a new culture well. sciences) and with ALDH substrate 1:100 2. Methods in 500 µl ALDEFLUOR buffer (ALDEFLU- OR kit from Stem cell Technology) at 37oC 3D cell culture method for 20 min. Tumorspheres were washed 2 To create non-adherent culture plates, times with ALDEFLUOR buffer by centrifu- 12 well culture plate (area of 3,8 cm² for gation. The second wash used ALDEFLU- each well) from BD Bioscience were sup- OR buffer contains 10 µg/ml Hoechst 33342 plemented with 500 µM Poly-HEMA solution for nuclei staining (from Sigma). (Poly(2-hydroxy-ethyl methacrylate from After tumorspheres were incubated with Sigma) and 10 mg/ml of concentration. Cul- CD44 monoclonal antibody or stained with ture plates were then dried surface in clean ALDH substrate in ALDEFLUOR kit, images benches overnight. Plate were washed were taken using NIKON fluorescence mi- 3 times using PBS 1X buffer before com- croscope at magnification of 200 and 400 mencing cell culture. times with specialized software. 3D culture: Trypsinized cells were col- Flow cytometry analysis lected from 2D cell culture plate and di- Tumorspheres collected on the 10th day luted in PBS 1X buffer until reaching the of culture were trypsinized and separated concentration 1000 cell/µl. A 2 µl solution into single cells. Single cells were incubat- containing 2000 cells was resuspended in ed with CD44-APC specific antibody (from 2 ml of specific stem cell culture medium in BD Biosciences) and specific ALDH sub- a non-adherent plate (treated by Poly-HE- strate in ALDEFLUOR kit (from Stem cell MA). This medium contains basic medium technology, Canada) according to manufac- DMEMF12/Glutamax (from Invitrogen) sup- tory instruction for 20 min at 37oC. Samples plemented with 1% P/S, Epithelial Growth were washed 2 times by ALDEFLUOR buf- Factor (EGF) 20 ng/ml, Fibroblast Growth fer (500 µl for each) before being placed in Factor (FGF) 20 ng/ml, glucose 0.3%, and specialized 4 ml glass tube (from BD Biosci- insulin 5 µg/ml (all components from Sig- ence) and analysed by FACS Canto II flow ma). 1 ml of medium was replaced with new cytometry system (BD - Biosciences) at the medium each 2 days. Cells were incubated compatible wavelength of APC (Allophyco- at 37oC, 5% CO2. The formation of tumor- cyanin) and FITC (Fluorescein isothiocy- spheres was evaluated for from the 5th day anate) for CD44 and ALDH, respectively. to 14th day of the culture process. Tumor- 20,000 events recorded in flow cytometry spheres were collected for cell analysis. were analyszed using DIVA 6.1 software. Immunofluorescence Collected tumorspheres were centri- 3. Ethics fuged at 1300 rpm for 3 min and washed All MKN45 cells were provided by In- 2 times with PBS 1X buffer. Tumorsphere serm U1053 Laboratory – French National were stained with anti-human CD44-PE Institute of Health and Medical Research in JMR 111 E2 (2) - 2018 55
4. JOURNAL OF MEDICAL RESEARCH Bordeaux. There is no intervention to pa- and percentage of MKN45 single cell that tients and animals. formed tumorspheres is presented in Figure 1. The result showed that only 6% ± 1,2% III. RESULTS single cancer cells developed into tumor- 1. Formation of tumorsphere from spheres by the 5th days of culture process. MKN45 single cell in 3D cell culture This means that for every 100 single cul- condition tured cells, only 6 of them have ability to Images of the tumorsphere formation form tumorsphere. Figure 1. Tumorsphere formation from a single cell of gastric cancer cell line MKN45 A) Tumorsphere formation at the 1st, 5th and 12th day of observation B) Tumorsphere forming percentage (estimated in the 5th day of culture process) Scale bar (to estimate tumorsphere size): 50µm. 2. The expression of CD44 and ALDH using immunofluorescence method As shown in Figure 2, the proportion of CD44 marker labeled with red fluorochrome was high at 90%. Similarly, the proportion of ALDH maker labeled with green fluorochrome was 50%. The immunofluorescence image indicated that CD44 was expressed in the cell mem- brane, whereas ALDH was located in the cytosol. Figure 2. Tumorsphere immunofluorescence after 7 days of culture process CD44 (red), ALDH (green) and Hoechst (blue). Scale bar: 50µm. 56 JMR 111 E2 (2) - 2018
5. JOURNAL OF MEDICAL RESEARCH 3. The expression of CD44 and ALDH using Flow cytometry analysis To determine correctly the number of cells expressing CD44 or ALDH and to confirm the immunofluorescence result above, we used flow cytometry analysis. The result is shown in Figure 3. The dot blot data in P7 range shows that most MKN45 gastric cancer cell expressed CD44 at a proportion of 92,0% ± 2,7% (Figure 3A(b)). On the other hand, the expression of ALDH was 40,1% ± 2,5%, much lower than CD44 (Figure 3B(b)). Figure 3. Result of Flow cytometry analysis (A) for CD44 and (B) for ALDH marker. (a) Control (cells only) (b) Sample (CD44 antibody or ALDEFLUOR™ reagent added) IV. DISCUSSION In the 2D cell culture, the connection between cells occurs on one side of the cell (on the surface of cultured plate). However, in 3D culture condition, cell attachments occur all around the surface of the cell. This influences the cell proliferation and differentiation [16]. The roles of Epithelial Growth Factor (EGF) and Fibroblast Growth Factor (FGF) in stem cell self-renewal have been demonstrated [17; 18]. Therefore, the single cancer cells which were cultured in medium supplied with the above growth factors should have self-renewal capacity allowing cancer stem cells to form tumorspheres. Our result showed that only 6% ± 1,2% single cancer cells formed tumorspheres, this is compatible with the report of Jianming et al. in 2013 about tumorsphere formation of gastric cancer cell line MKN45 [19]. Tumor is comprised of different cells which present distinct phenotypic and functional profiles. The heterogenous tumorsphere which arose from a single cell in 3D cell culture conditions indicate that different tumor cells can originate from a single unique stem cell. JMR 111 E2 (2) - 2018 57
6. JOURNAL OF MEDICAL RESEARCH The result of immunofluorescence analysis we demonstrated that gastric cancer cell showed 20% of the colored cells displayed populations include a small rate of gas- the nucleus dyed Hoechst (blue). Hoechst, tric CSCs. These gastric CSC had the ca- also called bis-Benzimide, is a cytotoxic or- pacity of tumorsphere formation. Besides, ganic compound and tends to bind to dou- immunofluorescence and flow cytometric ble-strained AT-rich regions of DNA [20]. results confirmed tumorspheres contain a Hoechst has been used to identify side pop- large amount of gastric CSC, as measured ulations - which comprise stem cell-like cells by gastric CSC markers CD44 and ALDH. exhibiting specific markers - in research of These results show 3D cell culture is a good stem cell in multiple mammalian species model for gastric CSC assays. The expres- and many different tumor types [21; 22]. sion level of CD44 and ALDH marker was Cells have the ability to be unstained by this confirmed by using both immunofluores- dye because they are able to actively pump cence and flow cytometry analysis simul- Hoechst out of the cell. Besides, ALDH is taneously. The results from the 2 methods a significant enzyme in cell detoxification indicated consistent expression proportions through oxidation of aldehydes to carboxylic for each marker. In Vietnam, 3D cell cul- acids, ALDH participates in ABC (ATP bind- ture has not been widely used in research, ing cassette) transport system. In addition, therefore deploying this method will contrib- side population occupies only a small pro- ute to later studies assessing the effects of portion of tumor cells, but it contains a large drugs on tumor cells. amount of CSC [23], and ALDH+ cells did Acknowledgements not incorporate the Hoechst dye, whereas ALDH is a selective gastric cancer stem cell This work was supported by Research marker. It claims that tumorsphere compris- Grant 55/B2017-TNA-55 from the Ministry es a large proportion of gastric cancer stem of Science and Technology in Vietnam. cell and these can exclude chemicals out REFERENCES of the cell through ABC membrane trans- 1. Ferlay J, Soerjomataram I, Ervik M, port proteins. The lower expression level of et al. (2013). GLOBOCAN 2012 cancer in- ALDH in comparison with CD44 shown in cidence and mortality worldwide: IARC can- Flow cytometry analysis result indicate that cerbase No. 11. Lyon, France. International ALDH is more selective than CD44 (40,1% Agency for Research on Cancer. ± 2,5% in comparison with 92,0% ± 2,7%), 2. El-Omar E.M., Carrington M., Chow this also is compatible with previous re- W.H., et al (2000). Interleukin-1 polymor- search [14; 15]. phisms associated with increased risk of V. CONCLUSION gastric cancer. Nature, 404, 398 - 402. By culturing MKN45 gastric cancer 3. Weck M.N., Gao L., Brenner H cells in 3D condition to form tumorspheres (2009). Helicobacter pylori infection and that have in-vivo tumor characteristics, chronic atrophic gastritis: associations ac- 58 JMR 111 E2 (2) - 2018
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