3D culture and analysis of the expression of cancer stem cell markers from gastric cancer cell line MKN45

JOURNAL OF MEDICAL RESEARCH<br /> <br /> 3D CULTURE AND ANALYSIS OF THE EXPRESSION<br /> OF CANCER STEM CELL MARKERS FROM GASTRIC<br /> CANCER CELL LINE MKN45<br /> Ngo Thu Ha¹, Le Thi Thanh Huong¹, La Thi Huyen², Nguyen Phu Hung¹,³<br /> ¹Thai Nguyen University of Sciences<br /> ²Institute of Biotechnology - Vietnam Academy of Science and Technology<br /> ³French National Institute of Health and Medical Research U1235, Nantes, France<br /> Gastric cancer is the fourth leading cause of worldwide cancer mortality, Vietnam belongs to the<br /> specified high-risk group of developing gastric cancer. Gastric cancer stem cells (CSCs) are considered<br /> to be the origin of gastric adenocarcinoma. 3D cell culture is an important step of identification of stem<br /> cell as well as CSC. In this study, a non-adherent cell culture model (3D) was developed with a culture<br /> medium that had growth factors selective for the development of gastric CSCs from gastric cancer<br /> cell line MKN45. The result showed that only 6% single cancer cells developed into tumorspheres<br /> during culture process. Then, flow cytometry and immunofluorescence analysis was carried out<br /> to evaluate the expression of CSC markers CD44 and ALDH in samples taken from tumorspheres<br /> which arose from gastric CSCs. The results showed that gastric CSCs formed tumorspheres in 3D<br /> culture condition with a high expression of 92% and 40,1% for markers CD44 and ALDH, respectively.<br /> <br /> Keywords: 3D culture, gastric cancer stem cell, CD44, ALDH, MKN45<br /> <br /> I. INTRODUCTION<br /> Cancer is the leading cause of death<br /> globally. Gastric cancer has the fourth highest mortality, accounting for 723,000 deaths<br /> in 2012 according to World Health Organization [1]. Vietnamese are at high risk of<br /> developing gastric cancer. Important risk<br /> factors for development of gastric cancer include smoked foods, salted fish and meat,<br /> Corresponding author: Nguyen Phu Hung,<br /> Thai Nguyen University of Sciences<br /> Email: hungnguyenphu@gmail.com<br /> Received: 10 July 2017<br /> Accepted: 09 Octorber 2017<br /> <br /> JMR 111 E2 (2) - 2018<br /> <br /> family history, chronic gastritis, smoking and<br /> Helicobacter pylori (H. pylori) infection [2].<br /> Epidemiological studies show the close relationship between chronic atrophic gastritis<br /> and H. pylori infection [3]. In atrophic gastritis, the reduction of parietal cells and chief<br /> cells lead to decrease HCl secretion which<br /> is a favorable environment for H. pylori development. H. pylori overgrowth increases<br /> genetic mutation in epithelial cells of stomach which can cause gastric cancer [4].<br /> Lauren's gastric cancer classification<br /> system subdivided gastric adenocarcinoma<br /> into two main types: intestinal type accounts<br /> 53<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> for 54% and diffuse type is about 32%. The<br /> remaining 14% belongs to indeterminate<br /> type (mixture of two main types above) [5;<br /> 6]. While intestinal type tumors are typically<br /> found in the gastric antrum of older individuals, diffuse type tumors are more common<br /> in the gastric corpus of younger individuals<br /> [7; 8].<br /> Recent research indicated that although<br /> cancer stem cells (CSCs) frequency in tumors is low, CSCs are responsible for proliferation and differentiation of tumor cells.<br /> Gastric stem cells which are located in the<br /> isthmus or progenitor zone of gastric gland<br /> and differentiate into mucous, parietal, chief<br /> and endocrine cells can be considered the<br /> origin of gastric CSCs [9]. In in-vitro experiments, if one cell can form tumorsphere in<br /> non-adherent culture condition (3D) with<br /> medium containing growth factors, it can<br /> qualify as a CSCs.<br /> Cancer cells grown in 3D cell culture<br /> condition were more similar to cells in tumors when compared with cells that were<br /> cultured in adherent (2D) condition [10].<br /> CSC identification and culture are important<br /> parts of cancer research; single cell culture<br /> to form tumorspheres (3D culture) is a significant tool in CSCs culture.<br /> CD44 and ALDH are two popular markers used for evaluation and isolation of<br /> CSCs in many different types of cancers<br /> [11]. In gastric adenocarcinoma, these<br /> markers expression identifies gastric cancer stem cell markers and is related to drug<br /> tolerance of tumor [12]. There are many<br /> different methods to identify expression of<br /> CD44 and ALDH markers, but Flow cytometry and immunofluorescence are common<br /> 54<br /> <br /> methods in CSC studies [13].<br /> CD44 (Cluster of differentiation 44), a<br /> receptor of hyaluronic acid, was used to<br /> identify gastric CSCs for the first time by<br /> Takaishi et al. in 2009 [14]. ALDH (aldehyde<br /> dehydrogenase) is an important marker of<br /> CSCs in solid tumors and gastric carcinoma<br /> in particular. It is involved in cell detoxification, regulation of cellular proliferation, and<br /> relates to drug tolerance in cancer [15].<br /> Studies on stem cells and CSCs have<br /> not been done previously in Vietnam, and<br /> 3D cell culture is an emerging technique in<br /> cancer research in general and gastric cancer in particular. In this study, MKN45 gastric cancer cells were seeded in 3D culture<br /> condition. The existence of gastric CSCs<br /> was evaluated by measuring tumorsphere<br /> formation and the expression of CD44 and<br /> ALDH gastric cancer stem cell markers.<br /> CD44 and ALDH expression was measured<br /> using flow cytometry and immunofluorescence analysis.<br /> <br /> II. MATERIALS AND METHODS<br /> 1. Materials: Gastric cancer cell line<br /> MKN45<br /> Gastric cancer cell line MKN45 was a<br /> generous gift from Inserm U1053 Laboratory – French National Institute of Health and<br /> Medical Research in Bordeaux. Cells were<br /> maintained in RPMI media supplemented<br /> with 10% Fetal Bovine Serum (FBS), and<br /> 1% ampicillin/streptomycin (P/S). The seeding density was 1 x 10⁵ cells/well in 3.8 cm²<br /> wells using adherent cell culture condition<br /> (2D). Cells were incubated at 37oC in the<br /> presence of 5% CO2. After 2 days, culture<br /> <br /> JMR 111 E2 (2) - 2018<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> medium was replaced. After 4 days, cells<br /> were passaged into a new culture well.<br /> 2. Methods<br /> 3D cell culture method<br /> To create non-adherent culture plates,<br /> 12 well culture plate (area of 3,8 cm² for<br /> each well) from BD Bioscience were supplemented with 500 µM Poly-HEMA solution<br /> (Poly(2-hydroxy-ethyl methacrylate from<br /> Sigma) and 10 mg/ml of concentration. Culture plates were then dried surface in clean<br /> benches overnight. Plate were washed<br /> 3 times using PBS 1X buffer before commencing cell culture.<br /> 3D culture: Trypsinized cells were collected from 2D cell culture plate and diluted in PBS 1X buffer until reaching the<br /> concentration 1000 cell/µl. A 2 µl solution<br /> containing 2000 cells was resuspended in<br /> 2 ml of specific stem cell culture medium in<br /> a non-adherent plate (treated by Poly-HEMA). This medium contains basic medium<br /> DMEMF12/Glutamax (from Invitrogen) supplemented with 1% P/S, Epithelial Growth<br /> Factor (EGF) 20 ng/ml, Fibroblast Growth<br /> Factor (FGF) 20 ng/ml, glucose 0.3%, and<br /> insulin 5 µg/ml (all components from Sigma). 1 ml of medium was replaced with new<br /> medium each 2 days. Cells were incubated<br /> at 37oC, 5% CO2. The formation of tumorspheres was evaluated for from the 5th day<br /> to 14th day of the culture process. Tumorspheres were collected for cell analysis.<br /> Immunofluorescence<br /> Collected tumorspheres were centrifuged at 1300 rpm for 3 min and washed<br /> 2 times with PBS 1X buffer. Tumorsphere<br /> were stained with anti-human CD44-PE<br /> JMR 111 E2 (2) - 2018<br /> <br /> monoclonal antibody 1:50 (from BD Biosciences) and with ALDH substrate 1:100<br /> in 500 µl ALDEFLUOR buffer (ALDEFLUOR kit from Stem cell Technology) at 37oC<br /> for 20 min. Tumorspheres were washed 2<br /> times with ALDEFLUOR buffer by centrifugation. The second wash used ALDEFLUOR buffer contains 10 µg/ml Hoechst 33342<br /> for nuclei staining (from Sigma).<br /> After tumorspheres were incubated with<br /> CD44 monoclonal antibody or stained with<br /> ALDH substrate in ALDEFLUOR kit, images<br /> were taken using NIKON fluorescence microscope at magnification of 200 and 400<br /> times with specialized software.<br /> Flow cytometry analysis<br /> Tumorspheres collected on the 10th day<br /> of culture were trypsinized and separated<br /> into single cells. Single cells were incubated with CD44-APC specific antibody (from<br /> BD Biosciences) and specific ALDH substrate in ALDEFLUOR kit (from Stem cell<br /> technology, Canada) according to manufactory instruction for 20 min at 37oC. Samples<br /> were washed 2 times by ALDEFLUOR buffer (500 µl for each) before being placed in<br /> specialized 4 ml glass tube (from BD Bioscience) and analysed by FACS Canto II flow<br /> cytometry system (BD - Biosciences) at the<br /> compatible wavelength of APC (Allophycocyanin) and FITC (Fluorescein isothiocyanate) for CD44 and ALDH, respectively.<br /> 20,000 events recorded in flow cytometry<br /> were analyszed using DIVA 6.1 software.<br /> 3. Ethics<br /> All MKN45 cells were provided by Inserm U1053 Laboratory – French National<br /> Institute of Health and Medical Research in<br /> 55<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> Bordeaux. There is no intervention to patients and animals.<br /> <br /> III. RESULTS<br /> 1. Formation of tumorsphere from<br /> MKN45 single cell in 3D cell culture<br /> condition<br /> Images of the tumorsphere formation<br /> <br /> and percentage of MKN45 single cell that<br /> formed tumorspheres is presented in Figure<br /> 1. The result showed that only 6% ± 1,2%<br /> single cancer cells developed into tumorspheres by the 5th days of culture process.<br /> This means that for every 100 single cultured cells, only 6 of them have ability to<br /> form tumorsphere.<br /> <br /> Figure 1. Tumorsphere formation from a single cell of gastric cancer cell line MKN45<br /> A) Tumorsphere formation at the 1st, 5th and 12th day of observation<br /> B) Tumorsphere forming percentage (estimated in the 5th day of culture process)<br /> Scale bar (to estimate tumorsphere size): 50µm.<br /> 2. The expression of CD44 and ALDH using immunofluorescence method<br /> As shown in Figure 2, the proportion of CD44 marker labeled with red fluorochrome was<br /> high at 90%. Similarly, the proportion of ALDH maker labeled with green fluorochrome was<br /> 50%. The immunofluorescence image indicated that CD44 was expressed in the cell membrane, whereas ALDH was located in the cytosol.<br /> <br /> Figure 2. Tumorsphere immunofluorescence after 7 days of culture process<br /> CD44 (red), ALDH (green) and Hoechst (blue). Scale bar: 50µm.<br /> 56<br /> <br /> JMR 111 E2 (2) - 2018<br /> <br /> JOURNAL OF MEDICAL RESEARCH<br /> 3. The expression of CD44 and ALDH using Flow cytometry analysis<br /> To determine correctly the number of cells expressing CD44 or ALDH and to confirm the<br /> immunofluorescence result above, we used flow cytometry analysis. The result is shown in<br /> Figure 3. The dot blot data in P7 range shows that most MKN45 gastric cancer cell expressed<br /> CD44 at a proportion of 92,0% ± 2,7% (Figure 3A(b)). On the other hand, the expression of<br /> ALDH was 40,1% ± 2,5%, much lower than CD44 (Figure 3B(b)).<br /> <br /> Figure 3. Result of Flow cytometry analysis<br /> (A) for CD44 and (B) for ALDH marker. (a) Control (cells only)<br /> (b) Sample (CD44 antibody or ALDEFLUOR™ reagent added)<br /> <br /> IV. DISCUSSION<br /> In the 2D cell culture, the connection between cells occurs on one side of the cell (on<br /> the surface of cultured plate). However, in 3D culture condition, cell attachments occur all<br /> around the surface of the cell. This influences the cell proliferation and differentiation [16].<br /> The roles of Epithelial Growth Factor (EGF) and Fibroblast Growth Factor (FGF) in stem cell<br /> self-renewal have been demonstrated [17; 18]. Therefore, the single cancer cells which were<br /> cultured in medium supplied with the above growth factors should have self-renewal capacity<br /> allowing cancer stem cells to form tumorspheres. Our result showed that only 6% ± 1,2%<br /> single cancer cells formed tumorspheres, this is compatible with the report of Jianming et al.<br /> in 2013 about tumorsphere formation of gastric cancer cell line MKN45 [19].<br /> Tumor is comprised of different cells which present distinct phenotypic and functional<br /> profiles. The heterogenous tumorsphere which arose from a single cell in 3D cell culture<br /> conditions indicate that different tumor cells can originate from a single unique stem cell.<br /> JMR 111 E2 (2) - 2018<br /> <br /> 57<br /> <br />