Cell isolation and culture
Mouse ovarian surface epithelial (MOSE) cells were isolated as described by Roby and Paul et al [1-3], and modified by us. Briefly, ovaries from female C57BL/6 mice (Experimental Animal Center for Soochow University, ) were resected and, were incubated for 20 min in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12, Invitrogen, Carlsbad, CA) supplemented with 0.25% trypsin (Invitrogen, Carlsbad, CA). Single cells and clumps of MOSE were collected, resuspended in MOSE cell growth medium, and seeded onto 35 mm dishes in a humidified atmosphere of 5% CO2 at 37 ℃. The MOSE cell growth medium consisted of DMEM/F12 medium supplemented with 5% fetal bovine serum (FBS), 10 ng/ml mouse epidermal growth factor (mEGF), 100 U/mL penicillin and 100 µg/mL streptomycin (Beyotime Biotechnology, shanghai, China), 1% insulin-transferrin-selenium (Invitrogen, Carlsbad, CA). During early passages of cells (pass 1 through 20), medium was changed with the mixed medium containing a 1:1 of the fresh growth medium and culture supernatant. Intermediated (pass 21-90) and late passage (great than 90) cells were routinely passaged. This animal experiment was performed according to the protocols of the Institutional Animal Care and Use Committee of the Soochow University (IACUCSU).
Cell proliferation assay
Cell proliferation was detected by Cell Count Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). In brief, cells were plated in 96-well plates at a density of 1000 cells per well and cultured in growth medium. At specific points in time after plating (0, 7, 24, 48, 72, 96, and 120h), the cell counts were determined according to the manufacturer’s instructions.
For clonogenic survival assay, five hundreds cells were seeded into dishes 35 mm in diameter. After two weeks, the colonies formed were fixed with formaldehyde, stained with Giemsa, and counted using a colony counter soft at GBOX XR-5 (Gene Company Limited, HongKong, China). The plating efficiencies (PE) were determined using the following formula: PE (%) = number of colonies formed / 500 plated cells ×100%. All data points in figures represent three independent experiments.
Soft agar colony formation assay
Two thousand cells were suspended in 1 mL of 0.35% agarose in the growth medium, plated into 35 mm dishes with a bottom layer of 0.7% agarose. Cells were fed every 3 days with 1 ml growth medium. Colonies (>15mm) were counted two or three weeks after seeding. Data are presented as CFU (percent colony forming units in soft agar assay) = mean of the number of colonies per dish/number of cells seeded per dish ´ 100%. Data points in figures represent three independent experiments.
In vivo tumorigenicity assays
Ten female BALB/c nude mice (SPF, 4-6weeks, 18-20g) were bought from Experimental Animal Center for Soochow University, and they were housed under temperature-controlled conditions, underwent a reverse dark-light cycle, and provided with standard mouse pellets and tap-water ad libitum. The mice were randomly divided into three groups (n=3) including M-E group, M-I group and M-L group for injection MOSE cells. After animals were lightly anesthetized with isoflurane, 5×106 of cells were injected subcutaneously into the left and right flanks of each animal. Animals were palpated weekly for tumor formation. Tumor size was measured once per week and tumor volume was calculated using the formula, tumor volume = 0.5 × length × width2. Animals were killed as soon as tumor nodules reached a size of 1.2–1.5 cm. The euthanasia/sacrifice of the mice was used by excessive 3% pentobarbital sodium (0.1 ml/10 g), following cervical dislocation to ensure the death of mice. This animal experiment was performed according to the protocols of the IACUCSU.
The total RNA was isolated from 106 cells using TRIZOL (Life Technologies). cDNA was synthesized from 500 ng of the total RNA using the RT2 first strand fit (SABiosciences, Frederick, MD). After all the control tests, the samples were analyzed using the mouse EMT RT2 profiler PCR array (PAMM-090Z, SABiosciences, Frederick, MD) that profiles the expression of 84 key genes. Altogether 84 different genes were simultaneously amplified in each sample. Five house-keeping genes (B2M, HPRT1, RPL13A, GAPDH, and ACTB), genomic DNA contamination control, reverse transcription control and positive PCR control were included in each PCR array. Briefly, the reaction mix was prepared from 2× SABiosciences RT2 qPCR master mix and 102 ml of sample cDNA. Ten ml of this mixture was added into each well of the PCR array. PCR arrays were performed in 384-well plates on a 7500 real time PCR system (Applied Biosystem, Foster City, CA).
Sphere formation assay was performed using serum-free DMEM/F12 Medium, including 20 ng/mL mEGF, 20 ng/mL basic fibroblast growth factor, 100 U/mL penicillin and 100 µg/mL streptomycin, 2 mg/ml insulin, 4 mg/mL heparin sodium, and 6 mg/mL glucose. Late MOSE cells were plated at a density of 1000 cells in 35 mm dish and cultured for 10 days. Round cell clusters larger than 100 mm were judged as spheres. Data was presented as sphere formation efficiency (%) = (number of spherical formation/plated cells × 100%). Data points in figures represent three independent experiments.
Limiting dilution assay
Limiting dilution assay was performed to measure the number of cells required to generate at least 1 tumor sphere/well as previously described . Briefly, serial two fold dilutions of M-I (from 0 to 5000 cells) and M-L cells (from 0 to 500 cells) were sorted into ultra-low 96-well plates with 6 wells per dilution, respectively. Cultures were fed 50 ml of CSCs medium every 2 days until day 10. Fraction of wells without spheres (y-axis) was plotted against the number of cells plated per well (x-axis). Regression lines were plotted and x-intercept values calculated, which represent the number of cells required to form at least 1 tumor sphere in every well.
Proteins were extracted with lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration of lysate was quantified using a bicinchonininc acid protein assay kit (Beyotime Biotechnology). Equal amounts of total proteins (30 µg) were electrophoretically transferred onto a polyvinylidene fluoride membrane (Millipore, Boston, MA). After being blocked with 5% skimmed milk, the membranes were incubated with primary antibodies of pan-keratin, vimentin, E-cadherin, b-catenin, Slug, Snail, Nanog, and β-actin (Cell Signaling Technology, Danvers, MA) at a 1:1000 dilution. Secondary antibodies were purchased from Amersham Biosciences (Piscataway, NJ) and the immune complexes were detected using an enhanced chemiluminescence (Millipore, Boston, MA) method according to the manufacturer’s instructions.
Cells were plated into micro-slides, and then fixed in 4% paraformaldehyde. Cells were permeabilized with 0.5% Triton followed by blocking with 5% BSA for at least 30 minutes at room temperature (RT). Incubations with primary antibodies were carried out for overnight at 4℃. Appropriate secondary antibodies conjugated to either AlexaFluor 488, AlexaFluor 543, or AlexaFluor 635 were carried out for 30 min at RT. Nuclear was counterstained with 4',6-diamidino-2-phenylindole (DAPI). Image was observed using SP5 confocal laser scan microscope (Leica, Wetxlar, Germany).
Flow cytometry assay
For stem cell marker analysis, 106 cells were resuspended in PBS, incubated with fluorescence-conjugated monoclonal antibodies against CD44-FITC and CD117-PE (BD Pharmingen) at 4℃ for 10 min, and washed by PBS for 3 times. The samples were analyzed by FC500 flow cytometer (Beckman Coulter, Fullerton, CA).
Histology and immunohistochemistry
Resected ovaries and tumors were collected from mice, fixed in 4% paraformaldehyde. Routine paraffin embedding of tissues and hematoxylin and eosin staining of tissue sections were performed by histopathological laboratories of Soochow University (Suzhou, China). For immunohistochemistry, tissues were cut into 5 µm sections. Slides were deparaffinized and rehydrated in an ethanol series. The sections were treated with 3% H2O2/MET for 20 min and boiled with citrate buffer (0.01 M, pH 6.00), then incubated overnight with indicated primary antibodies at 4℃. After that, followed with PBS washed for 3 times, the slides were then incubated with secondary antibodies with HRP. The slides were incubated with DAB solution, and then counterstained with hematoxylin according to standard protocols.
Data were presented as mean ± standard deviation (SD), and were analyzed with one way analysis of variance (ANOVA) and Student’s t-test. P <0.05 were considered statistically significant.