Orthotopic transplantation of retinoblastoma cells into vitreous cavity of zebrafish for screening of anticancer drugs

Molecular Cancer, Jul 2013

Background With high throughput screening, novel therapeutic agents can be efficiently identified. Unfortunately, researchers only resort to in vitro cell viability assays for screening of anticancer drugs for retinoblastoma, the most common intraocular cancer in the childhood. Current available animal models of retinoblastoma require more than 2 weeks for tumour formation and the investigation of the efficacy of therapeutic agents. In this study, we established a novel orthotopic transplantation model of retinoblastoma in zebrafish as an in vivo animal model for screening of anticancer drugs. Methods We injected retinoblastoma cells into the vitreous cavity of zebrafish at 48 hours after fertilization. Eyeballs of zebrafish were scanned daily under the confocal laser microscope, and the tumor population was quantitatively analyzed by measuring the mean intensity of green fluorescent protein (GFP). Transplanted retinoblastoma cells were isolated to perform further analyses including Western blotting and reverse transcriptase-polymerase chain reaction to confirm that retinoblastoma cells maintained their characteristics as tumor cells even after transplantation and further isolation. To figure out the potential of this model for screening of anticancer drugs, zebrafish were cultured in Ringer’s solution containing carboplatin and melphalan after the injection of retinoblastoma cells. Results The degree of the tumor population was dependent on the number of retinoblastoma cells injected and maintained stably for at least 4 days. Transplanted retinoblastoma cells maintain their proliferative potential and characteristics as retinoblastoma cells after isolation. Interestingly, systemic application of carboplatin and melphalan demonstrated significant reduction in the tumor population, which could be quantitatively analyzed by the estimation of the mean intensity of GFP. Conclusions This orthotopic retinoblastoma model in zebrafish is expected to be utilized for the screening of anticancer drugs for the treatment of retinoblastoma.

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Orthotopic transplantation of retinoblastoma cells into vitreous cavity of zebrafish for screening of anticancer drugs

Dong Hyun Jo 0 Dain Son 1 Yirang Na 1 Manyoung Jang Jae-Hoon Choi Jin Hyoung Kim 0 Young Suk Yu 0 Seung Hyeok Seok 1 Jeong Hun Kim 0 0 Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital , Seoul , Republic of Korea 1 Department of Microbiology and Immunology and Institute of Endemic Disease, College of Medicine, Seoul National University , Seoul , Republic of Korea Background: With high throughput screening, novel therapeutic agents can be efficiently identified. Unfortunately, researchers only resort to in vitro cell viability assays for screening of anticancer drugs for retinoblastoma, the most common intraocular cancer in the childhood. Current available animal models of retinoblastoma require more than 2 weeks for tumour formation and the investigation of the efficacy of therapeutic agents. In this study, we established a novel orthotopic transplantation model of retinoblastoma in zebrafish as an in vivo animal model for screening of anticancer drugs. Methods: We injected retinoblastoma cells into the vitreous cavity of zebrafish at 48 hours after fertilization. Eyeballs of zebrafish were scanned daily under the confocal laser microscope, and the tumor population was quantitatively analyzed by measuring the mean intensity of green fluorescent protein (GFP). Transplanted retinoblastoma cells were isolated to perform further analyses including Western blotting and reverse transcriptase-polymerase chain reaction to confirm that retinoblastoma cells maintained their characteristics as tumor cells even after transplantation and further isolation. To figure out the potential of this model for screening of anticancer drugs, zebrafish were cultured in Ringer's solution containing carboplatin and melphalan after the injection of retinoblastoma cells. Results: The degree of the tumor population was dependent on the number of retinoblastoma cells injected and maintained stably for at least 4 days. Transplanted retinoblastoma cells maintain their proliferative potential and characteristics as retinoblastoma cells after isolation. Interestingly, systemic application of carboplatin and melphalan demonstrated significant reduction in the tumor population, which could be quantitatively analyzed by the estimation of the mean intensity of GFP. Conclusions: This orthotopic retinoblastoma model in zebrafish is expected to be utilized for the screening of anticancer drugs for the treatment of retinoblastoma. - Background For the effective treatment of cancer, it is crucial to select proper regimens of anticancer drugs, which are based on the robust development of various regimens to improve efficacy and minimize toxicity. Last few decades are blossomed with the introduction of novel therapeutics such as targeted therapy or immunotherapy for cancers [1,2]. In the pediatric cancers, there are also many attempts for the introduction and actual uses of novel therapeutics [3]. Unfortunately, that was not the case in the treatment of retinoblastoma, the most common intraocular malignancy in childhood but an uncommon disease of the incidence of 1/20,000 births worldwide [4,5]. Currently, carboplatin-based regimens are widely utilized in the systemic chemotherapy and melphalan is commonly employed in the intraarterial chemotherapy, which addresses the tumor by the administration of anticancer drugs to ophthalmic artery via catheterization [6,7]. Although these approaches have yielded satisfactory clinical outcomes, there are still patients who are compelled to undergo enucleation, the complete removal of the eyeball, resulting in irreversible vision loss for the lifetime. Novel therapeutic agents can enhance the efficacy of currently utilized administration modalities including intravenous, intraarterial, and intravitreal injection [8]. For the development and screening of novel therapeutic agents, the effective screening tools are desperately required. As for retinoblastoma, a previous attempt on multiple screening of anticancer drugs simply utilized in vitro cell viability assays and measurements of chemosensitivities in the human tumor clonogenic assay using primary retinoblastoma cells and established cell lines [9]. However, there is no effective in vivo animal model for multiple screening of anticancer drugs at one time. Currently available animal models including mice with genetic aberrations and murine orthotopic transplantation models require more than 2 weeks to form tumors; therefore, they are not suitable for rapid and high throughput screening of anticancer drugs [10,11]. In this study, we transplanted retinoblastoma cells into the vitreous cavity of zebrafish to establish a novel orthotopic transplantation model of retinoblastoma in zebrafish that can be utilized for high throughput screening of anticancer drugs. Zebrafish are suitable for extensive testing of multiple drugs because of relatively low maintenance cost, accessibility of in (...truncated)


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Dong Jo, Dain Son, Yirang Na, Manyoung Jang, Jae-Hoon Choi, Jin Kim, Young Yu, Seung Seok, Jeong Kim. Orthotopic transplantation of retinoblastoma cells into vitreous cavity of zebrafish for screening of anticancer drugs, Molecular Cancer, 2013, pp. 71, 12, DOI: 10.1186/1476-4598-12-71