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Xiaoyun Chen, Wei Xiao, Yizhi Liu; Invasiveness and metastasis of retinoblastoma in an orthotopic zebrafish tumor model. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):73.
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© ARVO (1962-2015); The Authors (2016-present)
Retinoblastoma is a highly invasive malignant tumor that often invades the brain and metastasizes to distal organs through the blood stream. Invasiveness and metastasis of retinoblastoma can occur at the early stage of tumor development. However, an optimal preclinical model to study retinoblastoma invasiveness and metastasis in relation to drug treatment has not been developed. In this study, we developed an orthotopic zebrafish model in which retinoblastoma invasion and metastasis can be monitored at a single cell level.
Human and mouse retinoblastoma cell lines were labeled with fluorescent DiI dye before injection. At 48 hours after fertilization, wild type and fli1: EGFP-transgenic zebrafish embryos which exhibit green fluorescent signals in blood vessels were anesthetized and injected about 100-200 retinoblastoma cells into the vitreous cavities using the Pneumatic PicoPump under the stereomicroscope. Zebrafish embryos were examined every 2 days under a fluorescent microscope to monitor tumor cell growth, invasion, metastasis, and the interactions between retinoblastoma cells and surrounding microvasculatures. Further, to prove this method can be used to evaluate the efficacy of new therapies, sunitinib was added directly to the aquaria water after tumor cells implantation to attain a final concentration of 1 μM, and zebrafish embryos were examined with a fluorescent microscopy after 4 days.
After 2 days implantation, dissemination of tumor cells from the primary sites could be detected using fluorescent microscopy. Total numbers of metastatic foci was progressively increased and reached the maximal level at day 6 after tumor implantation. Tumor cells could disseminate to the heads, the lateral healthy eyes and the tails of zebrafish. Additionally, the primary tumor masses could stably maintain in the vitreous cavity of zebrafish for 2 days after implantation, but progressively decreased after 4 days. We also found that most tumor cells formed clusters around the hyaloid vessels attached to lens at day 2 after tumor cell implantation. Finally, treatment with retinoblastoma-bearing zebrafish embryos with 1 μM of sunitinib could significantly attenuate retinoblastoma invasion and metastasis.
Thus, this orthotopic retinoblastoma model in zebrafish offers a new and unique opportunity to study the early events of tumor invasion, metastasis and drug responses.
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