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.
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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)