Cell lines and culture
BeWo cells were obtained from the JCRB (Japanese Collection of Research Bioresources) Cell Bank (Tokyo, Japan). JAR and JEG-3 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HTR-8/SVneo cells, which are human first-trimester trophoblasts immortalized with the Simian virus 40 large T antigen, were kindly provided by Dr. Charles Graham (Queen’s University, Kingston, Canada). Choriocarcinoma cells and HEK293 cells were maintained in Ham’s F-12 medium (Nacalai Tesque, Inc., Kyoto, Japan) or Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Inc.), respectively, supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. HTR-8/SVneo cells were cultured in a 1:1 mixture of DMEM/Ham’s F-12 containing 10% FBS and antibiotics. All cells were cultured at 37˚C in a humidified atmosphere with 5% CO2.
To induce DNA demethylation, choriocarcinoma cells were treated with either 5-aza-2’-deoxycytidine (5azadC; Sigma-Aldrich, St. Louis, MO, USA) at different doses (from 1 to 50 µM) for 3 days or with 10 µM 5azadC for 5 days. Dimethyl sulfoxide (DMSO; Sigma-Aldrich) was used as a control vehicle. Culture media were changed daily to maintain the stability of 5azadC during treatment.
For the hypoxia experiments, choriocarcinoma cells were subjected to 5azadC treatment for 5 days. After changing to fresh medium, the cells were exposed to 2% O2, 5% CO2, and 93% N2 for 24 h in a MultiGas incubator (WAKEN 9000EX; WAKEN B TECH Co., Ltd, Kyoto, Japan).
Isolation and culture of human villous cytotrophoblasts
After obtaining informed consent, normal term placentas were collected from healthy pregnant women at 37 to 40 weeks of gestation at cesarean delivery due to breech presentation and previous cesarean section. Human villous cytotrophoblasts were isolated from the villous tissues of the placentas as described previously . Purified cytotrophoblasts were cryopreserved at −80 ˚C in a Cellbanker (ZENOAQ, Fukushina, Japan) until further use.
Thawed cytotrophoblasts were suspended in a 1:1 mixture of DMEM/Ham’s F-12 containing 10% FBS and antibiotics. The cells were seeded into type I collagen-coated culture plates or dishes (Sumilon Celltight; Sumitomo Bakelite Co., Ltd., Tokyo, Japan) at a density of approximately 2.3 × 105 cells/cm2 and then incubated for 24 h (cytotrophoblast preparation). After replacement of the culture media, the cytotrophoblasts were incubated for an additional 48 h for syncytium formation (syncytiotrophoblast preparation), and conditioned media were collected for Western blotting.
After obtaining informed consent, a choriocarcinoma tissue was obtained from a cancer patient at the University of Tokyo Hospital. The collected tissue was stored at −80 °C for qRT-PCR and Bisulfite sequencing.
Quantitative real-time polymerase chain reaction (qRT-PCR)
RNA was extracted and mRNA expression was assessed using qRT-PCR as described previously . All data were normalized to β-actin expression or GAPDH expression. Each value was obtained from the mean of three independent experiments. The oligonucleotide primer sequences are listed in Additional file 1: Table S1.
Cell lysates were prepared with lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1% Triton X-100) supplemented with a protease inhibitor cocktail (Nacalai Tesque, Inc.). Protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). Proteins were separated and transferred to polyvinylidene fluoride membranes by gel electrophoresis and electroblotting, respectively. The following primary antibodies were used: anti-human FLT1 N-terminal region (1:1000)  and anti-β-actin (1:500; Cell Signaling Technology, Beverly, MA, USA). Bands were visualized using an ECL Western Blotting Detection System (GE Healthcare, Uppsala, Sweden) on a chemiluminescence imaging system (KETA C Plus; Wealtec Corp., Sparks, NV, USA).
The cell lysates were subjected to immunoprecipitation using Dynabeads Protein G (Invitrogen) bound to anti-human FLT1 monoclonal antibody reacted with the first Ig-like domain of FLT1 (KM1730) as described previously . The precipitated proteins were subjected to Western blotting as described above.
Heparin-affinity pull-down for concentrating sFLT1 proteins
Secreted sFLT1 isoforms within the conditioned medium were concentrated using a previously described pull-down method with Heparin-Sepharose beads .
Genomic DNA was extracted from uncultured cytotrophoblasts, cultured cell lines and a tumor specimen using a NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany). The bisulfite modification procedure was carried out using a MethylEasy Xceed Rapid DNA Bisulphite Modification Kit (Human Genetic Signatures Pty, Randwick, Australia) according to the manufacturer’s instructions. Amplification was performed using EpiTaq HS (Takara Bio) and PCR primers for the detection of 5’ region in FLT1 gene (forward 5’-GTAGGAGGAGGGGTAAGGGTAA-3’ and reverse 5’-ACTCCAACCAAAAAACAACCA-3’). These promers were designed using Methyl Primer Express Software v1.0 (Applied Biosystems Inc. Foster City, CA, USA). The thermal cycling conditions consisted of an initial activation cycle (98°C for 20 s), followed by 40 cycles of denaturation (98°C for 10 s), annealing (55°C for 30 s), and amplification (72°C for 30 s). The PCR products were then cloned into pGEM-T easy vectors (Promega), and DNA sequencing was performed on 20 clones from each sample.
Enzyme-linked immunosorbent assay (ELISA)
The concentration of sFLT1 and VEGF-A in the conditioned media was quantitatively measured by ELISA kits which are commercially available (R&D Systems Inc., Minneapolis, MN, USA). To normalize the sFLT1 and VEGF-A secretion volumes according to cell number, the number of cells was counted using a hemocytometer or estimated from the measured cell lysate protein content.
Establishment of sFLT1-i13- or GFP-expressing JEG-3 cells
To construct expression vectors for sFLT1-i13 or green fluorescent protein (GFP), DNA fragments encoding these molecules were digested using pVL-6N-Flt  and pEGFP-N1 (Clontech, Mountain View, CA, USA), respectively, then cloned into a bovine papilloma virus-based plasmid vector pBCMGSneo . To establish stable sFLT1-i13- or GFP-expressing JEG-3 cells, each vector was transfected into JEG-3 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were selected in the presence of 600 µg/mL G418 (Nacalai Tesque, Inc.) and then maintained in a growth medium containing 200 µg/mL G418. These cells were designated as JEG3-i13 and JEG3-GFP, respectively.
In vivo tumorigenicity assay
All animals were bred at the Bioresource Center of Gunma University. JEG-3 cells expressing sFLT1-i13 or GFP (6 x 106 cells) were resuspended in 200 μL of Ham’s F-12 medium without FBS and antibiotics, then injected into the subcutaneous space of 6-week-old BALB/c-nu/nu female nude mice (Charles River Laboratories Japan, Inc., Kanagawa, Japan). Tumor size was measured every third day using a digital vernier caliper and their volume was calculated according to the following formula: (length × width2) × 0.5.
Quantitative analysis of microvessel density
Tumor tissues were fixed with 4% paraformaldehyde solution (Muto Pure Chemicals, Tokyo, Japan). The specimens were embedded in paraffin and sectioned. For immunostaining of the host microvessels, specimens were treated with a 1:250 dilution of rat anti-mouse CD31 monoclonal antibody (clone SZ31) (Dianova GmbH, Hamburg, Germany), and visualized using a Histofine Simple Stain Mouse MAX PO detection kit (Nichirei Bioscience, Tokyo, Japan). The nuclei were counterstained with hematoxylin. Images of the tumor sections were obtained using a microscope (IX70; Olympus, Tokyo, Japan) equipped with a C5810 color chilled 3CCD camera system (Hamamatsu Photonics, Shizuoka, Japan). Microvessels were defined as any CD31 positive endothelial cell or endothelial cell cluster with or without a definable lumen. The immunostained tumor sections were prepared from each implanted mouse at five sites and the number of microvessels was counted in independently three times per tumor section in a blinded fashion using a 20 × objective lens. The results were expressed as the average number of microvessels per mm2 area.
Data are expressed as the mean ± standard deviation (SD) and parametric data were analyzed using an unpaired t-test. Statistical analyses were performed using Excel 2011 (Microsoft, Seattle, WA, USA) with the Statcel4 (OMS, Tokyo, Japan) “add-in” software. A P value < 0.05 was considered statistically significant.