3D culture and analysis of the expression of cancer stem cell markers 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.

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 globally Gastric cancer has the fourth highest mortality, accounting for 723,000 deaths in 2012 according to World Health Organization [1] Vietnamese are at high risk of developing gastric cancer Important risk factors for development of gastric cancer include smoked foods, salted fish and meat, Corresponding author: Nguyen Phu Hung, Thai Nguyen University of Sciences Email: hungnguyenphu@gmail.com Received: 10 July 2017 Accepted: 09 Octorber 2017 JMR 111 E2 (2) - 2018 family history, chronic gastritis, smoking and Helicobacter pylori (H pylori) infection [2] Epidemiological studies show the close relationship between chronic atrophic gastritis and H pylori infection [3] In atrophic gastritis, the reduction of parietal cells and chief cells lead to decrease HCl secretion which is a favorable environment for H pylori development H pylori overgrowth increases genetic mutation in epithelial cells of stomach which can cause gastric cancer [4] Lauren's gastric cancer classification system subdivided gastric adenocarcinoma into two main types: intestinal type accounts 53 JOURNAL OF MEDICAL RESEARCH for 54% and diffuse type is about 32% The remaining 14% belongs to indeterminate type (mixture of two main types above) [5; 6] While intestinal type tumors are typically found in the gastric antrum of older individuals, diffuse type tumors are more common in the gastric corpus of younger individuals [7; 8] Recent research indicated that although cancer stem cells (CSCs) frequency in tumors is low, CSCs are responsible for proliferation and differentiation of tumor cells Gastric stem cells which are located in the isthmus or progenitor zone of gastric gland and differentiate into mucous, parietal, chief and endocrine cells can be considered the origin of gastric CSCs [9] In in-vitro experiments, if one cell can form tumorsphere in non-adherent culture condition (3D) with medium containing growth factors, it can qualify as a CSCs Cancer cells grown in 3D cell culture condition were more similar to cells in tumors when compared with cells that were cultured in adherent (2D) condition [10] CSC identification and culture are important parts of cancer research; single cell culture to form tumorspheres (3D culture) is a significant tool in CSCs culture CD44 and ALDH are two popular markers used for evaluation and isolation of CSCs in many different types of cancers [11] In gastric adenocarcinoma, these markers expression identifies gastric cancer stem cell markers and is related to drug tolerance of tumor [12] There are many different methods to identify expression of CD44 and ALDH markers, but Flow cytometry and immunofluorescence are common 54 methods in CSC studies [13] CD44 (Cluster of differentiation 44), a receptor of hyaluronic acid, was used to identify gastric CSCs for the first time by Takaishi et al in 2009 [14] ALDH (aldehyde dehydrogenase) is an important marker of CSCs in solid tumors and gastric carcinoma in particular It is involved in cell detoxification, regulation of cellular proliferation, and relates to drug tolerance in cancer [15] Studies on stem cells and CSCs have not been done previously in Vietnam, and 3D cell culture is an emerging technique in cancer research in general and gastric cancer in particular In this study, MKN45 gastric cancer cells were seeded in 3D culture condition The existence of gastric CSCs was evaluated by measuring tumorsphere formation and the expression of CD44 and ALDH gastric cancer stem cell markers CD44 and ALDH expression was measured using flow cytometry and immunofluorescence analysis II MATERIALS AND METHODS Materials: Gastric cancer cell line MKN45 Gastric cancer cell line MKN45 was a generous gift from Inserm U1053 Laboratory – French National Institute of Health and Medical Research in Bordeaux Cells were maintained in RPMI media supplemented with 10% Fetal Bovine Serum (FBS), and 1% ampicillin/streptomycin (P/S) The seeding density was x 10⁵ cells/well in 3.8 cm² wells using adherent cell culture condition (2D) Cells were incubated at 37oC in the presence of 5% CO2 After days, culture JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH medium was replaced After days, cells were passaged into a new culture well Methods 3D cell culture method To create non-adherent culture plates, 12 well culture plate (area of 3,8 cm² for each well) from BD Bioscience were supplemented with 500 µM Poly-HEMA solution (Poly(2-hydroxy-ethyl methacrylate from Sigma) and 10 mg/ml of concentration Culture plates were then dried surface in clean benches overnight Plate were washed times using PBS 1X buffer before commencing cell culture 3D culture: Trypsinized cells were collected from 2D cell culture plate and diluted in PBS 1X buffer until reaching the concentration 1000 cell/µl A µl solution containing 2000 cells was resuspended in ml of specific stem cell culture medium in a non-adherent plate (treated by Poly-HEMA) This medium contains basic medium DMEMF12/Glutamax (from Invitrogen) supplemented with 1% P/S, Epithelial Growth Factor (EGF) 20 ng/ml, Fibroblast Growth Factor (FGF) 20 ng/ml, glucose 0.3%, and insulin µg/ml (all components from Sigma) ml of medium was replaced with new medium each days Cells were incubated at 37oC, 5% CO2 The formation of tumorspheres was evaluated for from the 5th day to 14th day of the culture process Tumorspheres were collected for cell analysis Immunofluorescence Collected tumorspheres were centrifuged at 1300 rpm for and washed times with PBS 1X buffer Tumorsphere were stained with anti-human CD44-PE JMR 111 E2 (2) - 2018 monoclonal antibody 1:50 (from BD Biosciences) and with ALDH substrate 1:100 in 500 µl ALDEFLUOR buffer (ALDEFLUOR kit from Stem cell Technology) at 37oC for 20 Tumorspheres were washed times with ALDEFLUOR buffer by centrifugation The second wash used ALDEFLUOR buffer contains 10 µg/ml Hoechst 33342 for nuclei staining (from Sigma) After tumorspheres were incubated with CD44 monoclonal antibody or stained with ALDH substrate in ALDEFLUOR kit, images were taken using NIKON fluorescence microscope at magnification of 200 and 400 times with specialized software Flow cytometry analysis Tumorspheres collected on the 10th day of culture were trypsinized and separated into single cells Single cells were incubated with CD44-APC specific antibody (from BD Biosciences) and specific ALDH substrate in ALDEFLUOR kit (from Stem cell technology, Canada) according to manufactory instruction for 20 at 37oC Samples were washed times by ALDEFLUOR buffer (500 µl for each) before being placed in specialized ml glass tube (from BD Bioscience) and analysed by FACS Canto II flow cytometry system (BD - Biosciences) at the compatible wavelength of APC (Allophycocyanin) and FITC (Fluorescein isothiocyanate) for CD44 and ALDH, respectively 20,000 events recorded in flow cytometry were analyszed using DIVA 6.1 software Ethics All MKN45 cells were provided by Inserm U1053 Laboratory – French National Institute of Health and Medical Research in 55 JOURNAL OF MEDICAL RESEARCH Bordeaux There is no intervention to patients and animals III RESULTS Formation of tumorsphere from MKN45 single cell in 3D cell culture condition Images of the tumorsphere formation and percentage of MKN45 single cell that formed tumorspheres is presented in Figure The result showed that only 6% ± 1,2% single cancer cells developed into tumorspheres by the 5th days of culture process This means that for every 100 single cultured cells, only of them have ability to form tumorsphere Figure 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 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 membrane, whereas ALDH was located in the cytosol Figure Tumorsphere immunofluorescence after days of culture process CD44 (red), ALDH (green) and Hoechst (blue) Scale bar: 50µm 56 JMR 111 E2 (2) - 2018 JOURNAL OF MEDICAL RESEARCH 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 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 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 JOURNAL OF MEDICAL RESEARCH The result of immunofluorescence analysis showed 20% of the colored cells displayed the nucleus dyed Hoechst (blue) Hoechst, also called bis-Benzimide, is a cytotoxic organic compound and tends to bind to double-strained AT-rich regions of DNA [20] Hoechst has been used to identify side populations - which comprise stem cell-like cells exhibiting specific markers - in research of stem cell in multiple mammalian species and many different tumor types [21; 22] Cells have the ability to be unstained by this dye because they are able to actively pump Hoechst out of the cell Besides, ALDH is a significant enzyme in cell detoxification through oxidation of aldehydes to carboxylic acids, ALDH participates in ABC (ATP binding cassette) transport system In addition, side population occupies only a small proportion of tumor cells, but it contains a large amount of CSC [23], and ALDH+ cells did not incorporate the Hoechst dye, whereas ALDH is a selective gastric cancer stem cell marker It claims that tumorsphere comprises a large proportion of gastric cancer stem cell and these can exclude chemicals out of the cell through ABC membrane transport proteins The lower expression level of ALDH in comparison with CD44 shown in Flow cytometry analysis result indicate that ALDH is more selective than CD44 (40,1% ± 2,5% in comparison with 92,0% ± 2,7%), this also is compatible with previous research [14; 15] V CONCLUSION By culturing MKN45 gastric cancer cells in 3D condition to form tumorspheres that have in-vivo tumor characteristics, 58 we demonstrated that gastric cancer cell populations include a small rate of gastric CSCs These gastric CSC had the capacity of tumorsphere formation Besides, immunofluorescence and flow cytometric results confirmed tumorspheres contain a large amount of gastric CSC, as measured by gastric CSC markers CD44 and ALDH These results show 3D cell culture is a good model for gastric CSC assays The expression level of CD44 and ALDH marker was confirmed by using both immunofluorescence and flow cytometry analysis simultaneously The results from the methods indicated consistent expression proportions for each marker In Vietnam, 3D cell culture has not been widely used in research, therefore deploying this method will contribute to later studies assessing the effects of drugs on tumor cells Acknowledgements This work was supported by Research Grant 55/B2017-TNA-55 from the Ministry of Science and Technology in Vietnam REFERENCES Ferlay J, Soerjomataram I, Ervik M, et al (2013) GLOBOCAN 2012 cancer incidence and mortality worldwide: IARC cancerbase No 11 Lyon, France International Agency for Research on Cancer El-Omar E.M., Carrington M., Chow W.H., et al (2000) Interleukin-1 polymorphisms associated with increased risk of gastric cancer Nature, 404, 398 - 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Materials: Gastric cancer cell line MKN45 Gastric cancer cell line MKN45 was a generous gift from Inserm U1053 Laboratory – French National Institute of Health and Medical Research in Bordeaux Cells