VEGF induces angiogenesis in a zebrafish embryo glioma model established by transplantation of human glioma cells

  • Authors:
    • Dong Li
    • Xiang-Pen Li
    • Hong-Xuan Wang
    • Qing-Yu Shen
    • Xiang-Ping Li
    • Lu Wen
    • Xiu-Jiao Qin
    • Qiu-Li Jia
    • Hsiang-Fu Kung
    • Ying Peng
  • View Affiliations

  • Published online on: June 12, 2012     https://doi.org/10.3892/or.2012.1861
  • Pages: 937-942
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Zebrafish (Danio rerio) is becoming an increasingly popular vertebrate cancer model. In this study, we established a xenotransplanted zebrafish embryo glioma model to further investigate the molecular mechanisms of tumor angiogenesis. We find that the glioma cell line U87 can survive, proliferate and induce additional SIV branches in zebrafish embryos. In addition, by the means of in situ hybridization and quantitive RT-PCR analyses we find that the transplanted U87 cells can induce the ectopic zebrafish vascular endothelial growth factor A (VEGF A) and its receptor VEGFR2/KDR mRNA expression and increase their expression levels, resulting in additional SIV branches.

Introduction

Glioma is the most common primary brain tumor in adults and the median survival for patients without therapy is up to 3 months (1). Currently the traditional treatment such as resection, chemotherapy and radiotherapy has not greatly improved the median survival for patients. Malignant glioma is notorious for its behavior of rapid proliferation, great invasion and metastasis and it is also among the best vascularized tumors in humans. It is well known that angiogenesis plays a critical role in tumor progression. Therefore new anti-angiogenic treatment strategies are required (26). Revealing of various signaling pathways that lead to activation of angiogenesis will provide molecular insight into developing therapeutic agents to treat glioma. In the past decades, researchers have discovered many molecular mechanisms related to tumor angiogenesis (79).

Traditional rodent cancer models have disadvantages, e.g., the high cost, long developmental phase, and limited availability for high-throughput assays (10), which limited effective ways to find the new molecular mechanisms and monitor the tumor in vivo in real time.

The teleost zebrafish (Danio rerio), a promising alternative vertebrate cancer model has attracted considerable attention in recent years because of compelling advantages of transparency of the embryo, rapid development, fecundity, tractable genetics, great screening efficiency, high levels of physiologic and genetic homology with higher vertebrates (1113). Moreover, the zebrafish embryo within 1 week is immune-free, and showed no immunosuppression to transplanted human glioma cells (14). The transgenic zebrafish strains, e.g., casper transparent mutant line (15), and VEGFR2:G-RCFP line with green fluorescence specifically in blood vessels (16), offered new tools for cancer research.

In this study, we microinjected human U87 glioma cells into zebrafish embryos and developed a xenograft zebrafish glioma model. The U87 cells are first labeled with a red fluorescence protein and then microinjected into perivitelline space of VEGFR2:G-RCFP transgenic zebrafish embryos at 48 h post-fertilization. Then in order to investigate the angiogenesis mechanisms in details, we make use of the staining of endogenous alkaline phosphatase, fluorescent microscopy monitoring, in situ hybridization and quantitive RT-PCR to investigate the change of glioma related molecules in a zebrafish embryo angiogenesis glioma model.

Materials and methods

Glioma cell culture and transfection

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.

Zebrafish incubation

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).

The establishment of zebrafish embryo glioma model

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.

Fluorescent microscopy monitoring

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.

Whole-mount endogenous alkaline phosphatase staining

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.

Whole-mount in situ hybridization

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.

Quantitive RT-PCR analysis

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.

Results

Human glioma cells U87 can survive, proliferate and induce angiogenesis in zebrafish embryos

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).

Human glioma cells U87 can induce the ectopic zebrafish VEGF and VEGFR2 mRNA expression and increase their expression quantity

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).

Discussion

The zebrafish glioma model provides a convenient in vivo system for the study of cancer cell molecular mechanisms. Many other kinds of zebrafish cancer models have already appeared worldwide. These models mainly include three different types. The first type is that Spitsbergen et al(23,24) gained by chemical induction and acquiring several types of cancers in zebrafish through chemical carcinogenesis such as epithelial, mesenchymal, neural neoplasia induced by 7,12-dimethylbenz[a]anthracene and hepatic, mesenchymal neoplasia induced by N-methyl-N′-nitro-N-nitrosoguanidine. The second type of zebrafish cancer model is obtained by genetic manipulation, bmyb mutation causes genome instability and increased cancer susceptibility in zebrafish (25). The third transplanted zebrafish glioma model is that we described herein. Amatruda et al have also reported a study in Cancer Cell(26). A zebrafish cancer model was also presented July 2009 in Spoleto, Italy, recapitulating a number of new zebrafish cancer models (27), indicating that zebrafish model could make a great contribution to cancer research.

Glioma is a notorious malignant cancer in humans and research has been done into its molecular mechanisms in vitro and in vivo for many years. In this study, we found that the U87 human glioma cells can survive, proliferate and induce angiogenesis in a zebrafish embryo. As is known, angiogenesis is a key factor in malignant tumor progression (28) and here these transplanted glioma cells U87 can also induce additional subintestinal vessels and transfer to the distant site through intravasation and extravasation.

Vascular endothelial growth factors (VEGFs) play an important part in angiogenesis (29,30) and currently include VEGF-A,-B,-C,-D,-E and placenta growth factor (PlGF) in its family. They play a crucial role in the process of angiogenesis by binding tyrosine kinase receptors such as VEGF receptor-1,-2, and-3 (31). We carried out an investigation on VEGF A and its receptor VEGFR2/KDR (32,33), we found that the transplanted human U87 glioma cells can not only increase the mRNA expression of zebrafish VEGF A and VEGFR2 but also induce the secretion of VEGF A and VEGFR2 mRNA in different anatomic sites in zebrafish, and these molecules contribute to induction of additional zebrafish SIV branches. This result is consistent with previous published reports. Serbedzija et al have reported that VEGF can induce angiogenesis by the injection of its protein directly into zebrafish embryo (20) and Habeck et al have also shown a very similar result to ours by injecting VEGF plasmid into zebrafish embryo (34). The above evidence proves that VEGF indeed plays an important role in zebrafish embryo angiogenesis. The injected human glioma cells U87 induced zebrafish embryo angiogenesis further confirms that zebrafish angiogenesis-related genes show great conservation with humans, as Liang et al(35) previously showed that zebrafish VEGF’s functional sites.

Acknowledgements

We thank Benping Luo for providing the vector pcDNA3.0-DsRed. This study was supported by the National Natural Science Foundation of China (30973479 to Y. Peng), the National High Technology Research and Development Program of China (863 Program) (2007AA021101 to Y. Peng), and Science and Technology Planning Project of Guangdong Province, China (2009B060700040 and 2011B031800141 to Y. Peng).

Abbreviations:

hpf

hour post-fertilization

dpf

day post-fertilization

hpi

hour post-injection

dpi

day post-injection

DMEM

Dulbecco’s modified Eagle’s medium

SIV

subintestinal vessel

PTU

1-phenyl-2-thiourea

PBS

phosphate buffered saline

PFA

paraformaldehyde

qPCR

quantitative real-time PCR

References

1 

Ng SS, Gao Y, Chau DH, et al: A novel glioblastoma cancer gene therapy using AAV-mediated long-term expression of human TERT C-terminal polypeptide. Cancer Gene Ther. 14:561–572. 2007. View Article : Google Scholar : PubMed/NCBI

2 

Lin ZX, Yang LJ, Huang Q, et al: Inhibition of tumor-induced edema by antisense VEGF is mediated by suppressive vesiculo-vacuolar organelles (VVO) formation. Cancer Sci. 99:2540–2546. 2008. View Article : Google Scholar : PubMed/NCBI

3 

Paez-Ribes M, Allen E, Hudock J, et al: Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell. 15:220–231. 2009. View Article : Google Scholar : PubMed/NCBI

4 

Norden AD, Drappatz J and Wen PY: Novel anti-angiogenic therapies for malignant gliomas. Lancet Neurol. 7:1152–1160. 2008. View Article : Google Scholar : PubMed/NCBI

5 

Vredenburgh JJ, Desjardins A, Herndon JN, et al: Bevacizumab plus irinotecan in recurrent glioblastoma multiforme. J Clin Oncol. 25:4722–4729. 2007. View Article : Google Scholar : PubMed/NCBI

6 

Tuettenberg J, Friedel C and Vajkoczy P: Angiogenesis in malignant glioma- a target for antitumor therapy? Crit Rev Oncol Hematol. 59:181–193. 2006. View Article : Google Scholar : PubMed/NCBI

7 

Gan HK, Lappas M, Cao DX, Cvrljevdic A, Scott AM and Johns TG: Targeting a unique EGFR epitope with monoclonal antibody 806 activates NF-kappaB and initiates tumour vascular normalization. J Cell Mol Med. 13:3993–4001. 2009. View Article : Google Scholar : PubMed/NCBI

8 

Jang FF, Wei W and De WM: Vascular endothelial growth factor and basic fibroblast growth factor expression positively correlates with angiogenesis and peritumoural brain oedema in astrocytoma. J Ayub Med Coll Abbottabad. 20:105–109. 2008.

9 

di Tomaso E, London N, Fuja D, et al: PDGF-C induces maturation of blood vessels in a model of glioblastoma and attenuates the response to anti-VEGF treatment. PLoS One. 4:e51232009.PubMed/NCBI

10 

Huse JT and Holland EC: Genetically engineered mouse models of brain cancer and the promise of preclinical testing. Brain Pathol. 19:132–143. 2009. View Article : Google Scholar : PubMed/NCBI

11 

Lee LM, Seftor EA, Bonde G, Cornell RA and Hendrix MJ: The fate of human malignant melanoma cells transplanted into zebrafish embryos: assessment of migration and cell division in the absence of tumor formation. Dev Dyn. 233:1560–1570. 2005. View Article : Google Scholar

12 

Stoletov K, Montel V, Lester RD, Gonias SL and Klemke R: High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish. Proc Natl Acad Sci USA. 104:17406–17411. 2007. View Article : Google Scholar : PubMed/NCBI

13 

Jesuthasan S: Genetics and development. Zebrafish in the spotlight. Science. 297:1484–1485. 2002. View Article : Google Scholar : PubMed/NCBI

14 

Stoletov K and Klemke R: Catch of the day: zebrafish as a human cancer model. Oncogene. 27:4509–4520. 2008. View Article : Google Scholar : PubMed/NCBI

15 

White RM, Sessa A, Burke C, et al: Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell. 2:183–189. 2008. View Article : Google Scholar : PubMed/NCBI

16 

Cross LM, Cook MA, Lin S, Chen JN and Rubinstein AL: Rapid analysis of angiogenesis drugs in a live fluorescent zebrafish assay. Arterioscler Thromb Vasc Biol. 23:911–912. 2003. View Article : Google Scholar : PubMed/NCBI

17 

Westerfield M: The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio) Eugene, OR: University of Oregon Press; 2007

18 

Nicoli S, Ribatti D, Cotelli F and Presta M: Mammalian tumor xenografts induce neovascularization in zebrafish embryos. Cancer Res. 67:2927–2931. 2007. View Article : Google Scholar : PubMed/NCBI

19 

Nicoli S and Presta M: The zebrafish/tumor xenograft angiogenesis assay. Nat Protoc. 2:2918–2923. 2007. View Article : Google Scholar : PubMed/NCBI

20 

Serbedzija GN, Flynn E and Willett CE: Zebrafish angiogenesis: a new model for drug screening. Angiogenesis. 3:353–359. 1999. View Article : Google Scholar : PubMed/NCBI

21 

Thisse C and Thisse B: High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc. 3:59–69. 2008. View Article : Google Scholar : PubMed/NCBI

22 

Adhikary S and Eilers M: Transcriptional regulation and transformation by Myc proteins. Nat Rev Mol Cell Biol. 6:635–645. 2005. View Article : Google Scholar : PubMed/NCBI

23 

Spitsbergen JM, Tsai HW, Reddy A, et al: Neoplasia in zebrafish (danio rerio) treated with 7,12-dimethylbenz[a]anthracene by two exposure routes at different developmental stages. Toxicol Pathol. 28:705–715. 2000.PubMed/NCBI

24 

Spitsbergen JM, Tsai HW, Reddy A, et al: Neoplasia in zebrafish (Danio rerio) treated with N-methyl-N′-nitro-N-nitrosoguanidine by three exposure routes at different developmental stages. Toxicol Pathol. 28:716–725. 2000.PubMed/NCBI

25 

Shepard JL, Amatruda JF, Stern HM, et al: A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility. Proc Natl Acad Sci USA. 102:13194–13199. 2005.

26 

Amatruda JF, Shepard JL, Stern HM and Zon LI: Zebrafish as a cancer model system. Cancer Cell. 1:229–231. 2002. View Article : Google Scholar : PubMed/NCBI

27 

Mione MC and Trede NS: The zebrafish as a model for cancer. Dis Model Mech. 3:517–523. 2010. View Article : Google Scholar : PubMed/NCBI

28 

Chi A, Norden AD and Wen PY: Inhibition of angiogenesis and invasion in malignant gliomas. Expert Rev Anticancer Ther. 7:1537–1560. 2007. View Article : Google Scholar : PubMed/NCBI

29 

Leung DW, Cachianes G, Kuang WJ, Goeddel DV and Ferrara N: Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 246:1306–1309. 1989. View Article : Google Scholar : PubMed/NCBI

30 

Plate KH, Breier G, Weich HA and Risau W: Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature. 359:845–848. 1992. View Article : Google Scholar : PubMed/NCBI

31 

Roy H, Bhardwaj S and Yla-Herttuala S: Biology of vascular endothelial growth factors. FEBS Lett. 580:2879–2887. 2006. View Article : Google Scholar : PubMed/NCBI

32 

Neufeld G, Tessler S, Gitay-Goren H, Cohen T and Levi BZ: Vascular endothelial growth factor and its receptors. Prog Growth Factor Res. 5:89–97. 1994. View Article : Google Scholar : PubMed/NCBI

33 

Neufeld G, Cohen T, Gengrinovitch S and Poltorak Z: Vascular endothelial growth factor (VEGF) and its receptors. FASEB J. 13:9–22. 1999.PubMed/NCBI

34 

Habeck H, Odenthal J, Walderich B, Maischein H and Schulte-Merker S: Analysis of a zebrafish VEGF receptor mutant reveals specific disruption of angiogenesis. Curr Biol. 12:1405–1412. 2002. View Article : Google Scholar : PubMed/NCBI

35 

Liang D, Xu X, Chin AJ, et al: Cloning and characterization of vascular endothelial growth factor (VEGF) from zebrafish, Danio rerio. Biochim Biophys Acta. 1397:14–20. 1998. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

September 2012
Volume 28 Issue 3

Print ISSN: 1021-335X
Online ISSN:1791-2431

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Li D, Li X, Wang H, Shen Q, Li X, Wen L, Qin X, Jia Q, Kung H, Peng Y, Peng Y, et al: VEGF induces angiogenesis in a zebrafish embryo glioma model established by transplantation of human glioma cells. Oncol Rep 28: 937-942, 2012.
APA
Li, D., Li, X., Wang, H., Shen, Q., Li, X., Wen, L. ... Peng, Y. (2012). VEGF induces angiogenesis in a zebrafish embryo glioma model established by transplantation of human glioma cells. Oncology Reports, 28, 937-942. https://doi.org/10.3892/or.2012.1861
MLA
Li, D., Li, X., Wang, H., Shen, Q., Li, X., Wen, L., Qin, X., Jia, Q., Kung, H., Peng, Y."VEGF induces angiogenesis in a zebrafish embryo glioma model established by transplantation of human glioma cells". Oncology Reports 28.3 (2012): 937-942.
Chicago
Li, D., Li, X., Wang, H., Shen, Q., Li, X., Wen, L., Qin, X., Jia, Q., Kung, H., Peng, Y."VEGF induces angiogenesis in a zebrafish embryo glioma model established by transplantation of human glioma cells". Oncology Reports 28, no. 3 (2012): 937-942. https://doi.org/10.3892/or.2012.1861