The human malignant glioma cell line U87 (American Type Culture Collection) was maintained in 90% DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco, NY, USA) and 10% fetal bovine serum (Invitrogen, CA, USA) with 5% CO₂ at 37°C. Plasmid DNA labeled with red fluorescent protein DNA fragment (pcDNA3.0-DsRed) was provided by Dr Ben-Ping Luo and is amplified using Endo-Free Plasmid Mini kit I (Omega, USA). One day before transfection, we plated 0.5–2×10⁵ U87 cells in 500 μl of growth medium without antibiotics in each well of a 24-well plate so that cells are 90–95% confluent at the time of transfection. Then these cells were transfected with pcDNA3.0-DsRed vector using Lipofectamine 2000 (Invitrogen) as per the instructions with slight modification. The U87 glioma cells expressing red fluorescent protein (U87-DsRed glioma cells) were selected using 300 μg/ml G418 and cultured for future use.

The adult AB zebrafish and VEGFR2:G-RCFP (provided by integrated laboratory of Sun Yat-Sen University) transgenic zebrafish were maintained in zebrafish breeding system as previously described (17). The zebrafish embryos were incubated in petri dishes with E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄) at 28°C until they were ready for use. If the zebrafish embryos are used after 48 hpf, it should be placed in E3 buffer with 1-phenyl-2-thiourea (PTU) (Sigma-Alrich, USA) at 24 hpf and the buffer should be changed every day in order to eliminate melanin. The embryos containing chorion should be dechorioned with 1 mg/ml pronase (Sigma-Alrich) and then anesthetized with 0.04 mg/ml tricaine (Sigma-Alrich) before they are microinjected with cells( 18).

Firstly, the prepared U87-DsRed glioma cells were trypsinized and suspended in Matrigel (R&D, USA) at a density of ~10⁸ cells/ml. Then Narishige microinjector was used to inject 10–30 nl suspended U87-DsRed cells into the perivitelline space near SIV of the VEGFR2:G-RCFP transgenic zebrafish embryos at 48 hpf (18,19) to establish this zebrafish embryo glioma model. The blank matrigel injected zebrafish embryos were used as a negative control.

When the zebrafish embryo glioma model was set up a Nikon epifluorescent microscope was used to monitor the progression of glioma cells and the subintestinal vessel (SIV) changes of transgenic VEGFR2-RCFP zebrafish in real time continually for 2 days.

In order to further investigate the angiogenesis ability of glioma cells, we fixed the U87 injected zebrafish embryos at 1 day post-injection (dpi) and 2 dpi in phosphate buffered saline (PBS)-4% paraformaldehyde (PFA) for 2 h at room temperature and then stained for endogenous alkaline phosphatase activity following the protocol of Serbedzija et al(20). Then embryos were observed and photographed under a Nikon stereomicroscope.

We further investigated the molecular mechanisms of glioma angiogenesis by in situ hybridization test of zebrafish VEGF A and VEGFR2/KDR mRNA expression in zebrafish embryo glioma model using VEGF A and KDR digoxigenin-labeled antisense RNA probes. At first, we cloned part of the zebrafish VEGF and KDR sequence which were ~1 kb into the pcDNA3.0 plasmid. The primers were as follows: VEGF (forward: 5′-ttggaattcagcgactcaccgcaacactc-3′, reverse: 5′-ataaagcttcattcgttgttccgctcctg-3′); KDR (forward: 5′-gcagaattcattcccatgccgaacattac-3′, reverse: 5′-gttaagcttagtctg aggcgatcttgagg-3′). Then by using the DIG System nucleic acid labeling kit (Roche, USA), we prepared these two probes with T7 promoter in vitro and then observed the signals by using an NBT/BCIP staining solution (Roche). The details of the high-resolution in situ hybridization protocol have been reported (21). We used a blank Matrigel injected zebrafish as a negative control for zebrafish angiogenesis glioma model in whole-mount in situ hybridization. The experiments were repeated 3 times.

To determine the exact change of zebrafish VEGF A and VEGFR2 mRNA expression in zebrafish embryo glioma model we also carried out quantitive RT-PCR tests of zebrafish VEGF and VEGFR2 mRNA expression. The qRT-PCR primers were as follows: VEGF (forward: 5′-tgctcctgcaaattcacacaa-3′, reverse: 5′-atcttggcttttcacatctgcaa-3′); KDR (forward: 5′-tggagttccagcacccttta-3′, reverse: 5′-cgtccttcttcaccctttca-3′); β-actin (forward: 5′-cgtgacatcaa ggagaagct-3′, reverse: 5′-tcgtggataccgcaagattc-3′). Total RNA was extracted from 40 glioma angiogenesis zebrafish embryos and then treated with DNase I. Promega reverse transcription kit was used to get the zebrafish single-stranded cDNA. Then we used the SYBR GreenER™ qPCR SuperMix Universal kits (Applied Biosystems Inc.) to carry out quantitive PCR in Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems Inc.) according to the manufacturer’s instructions.

Data were normalized with zebrafish β-actin and repeated 3 times with similar results. Ct-value for each sample was calculated with the ^ΔΔCt-method (22) and results were expressed as 2^−ΔΔCT. A blank Matrigel injected zebrafish was used as a negative control.

We established the zebrafish embryo glioma model and we monitored the activity of U87 cells and changes of SIV in zebrafish embryo glioma model by the use of epifluorescent microscopy. The human U87 glioma cells can survive, proliferate and induce angiogenesis in zebrafish embryos from 24 to 48 hpi (Fig. 1). The angiogenesis ability increased from 1 to 2 dpi (Fig. 1D and F). We calculated the percentage of additional SIV branches in the zebrafish embryo and also found that the percentage of positive SIV phenotype increased from 1 to 2 dpi (Fig. 2).

Endogenous alkaline phosphatase staining for zebrafish embryo glioma model showed similar results on additional SIV branches to that observed by the epifluorescent microscope (Fig. 3B and D).

By in situ hybridization and quantitive RT-PCR test of zebrafish VEGF and VEGFR2 mRNA expression in zebrafish embryo glioma model from 1 to 2 dpi, we found that the expression area of VEGF and VEGFR2 mRNA around SIV had enlarged (Fig. 4) and their expression was also increased (Fig. 5).