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 vivo imaging, and tiny size [
12,
13]. Especially, we investigated the potential of orthotopic transplantation of retinoblastoma to mimic the tumor microenvironment as much as possible. In addition, we can quantitatively analyze the degree of the tumor population in this model with a public image processing program. Furthermore, there is no change in the characteristics of tumor cells between before and after the injection, demonstrating the stability of transplanted cells as tumor cells during the study. Also, we identified the possibility of this model as a screening tool for various anticancer drugs with 2 widely utilized anticancer drugs for retinoblastoma, carboplatin and melphalan.
Discussion
The development of novel effective regimens is one of the best parts in the treatment of cancer. In addition, the investigation into proper administration options is required in localized tumors such as retinoblastoma, which originates from the inner layers of the retina and extends into the adjacent structures such as vitreous cavity, optic nerve, and choroid [
18,
19]. In this regard, researchers on the treatment of retinoblastoma have struggled to find novel therapeutic agents and modalities to deliver anticancer drugs to tumors. Particularly, current studies are mainly focused on the development and validation of novel therapeutic modalities such as intraarterial and intravitreal chemotherapy plus conventional intravenous chemotherapy [
8]. With these efforts, tumors of group A to C according to the International Classification of Retinoblastoma (ICR) demonstrate the treatment success rate of over 90% by intravenous chemotherapy with carboplatin, vincristine, and etoposide [
6]; intraarterial chemotherapy with melphalan can be utilized as salvage treatment after chemoreduction failure or primary treatment for advanced stages, group D or E according to the ICR [
8]. However, studies on the novel therapeutic agents for retinoblastoma slightly lag behind compared to rapid increase in the development and introduction of targeted therapy or immunotherapy in other pediatric cancers [
3]. Still, enucleation is the last choice for treatment failure with chemotherapy of various administration modalities; therefore, we speculated that the introduction of novel anticancer drugs might improve the clinical outcomes of current available modalities, minimizing the risk of irreversible vision and eyeball loss.
For the development of novel therapeutic agents, multiple screening of various candidate drugs is required. Once several candidate drugs are selected from in vitro experiments, adequate in vivo studies should be performed for identification of better drugs in animal models. However, current animal models of retinoblastoma are not suitable for high throughput screening of drugs in that tumor is formed after more than 2 weeks and we cannot perform experiments on many mice at one time. For example, the orthotopic transplantation model in mice requires 4 to 8 weeks to demonstrate the efficacy of anticancer drugs, although the tumor formation is evident in all the pups [
10]. Similarly, researchers wait 2 weeks after the birth to form tumors in a genetic mouse model of retinoblastoma (Chx10-Cre;Rb(lox/lox);p107(−/−);p53(lox/lox)) [
11].
In this study, we completed 1 round of experiments within 1 week after the fertilization and performed injection of retinoblastoma cells into the vitreous cavity on more than 200 zebrafish at a sitting. Transplantation of tumor cells to zebrafish has been utilized for investigation of tumor biology [
20,
21]. In addition, a few attempts explored the possibility of zebrafish models as tools for screening of anticancer drugs [
13,
22,
23]. In these models, tumor cells from oral squamous cell carcinoma and leukemia cell lines are injected through yolk sac. We attempted the different approach from these studies in that we injected tumor cells into the vitreous cavity, with which retinoblastoma interacts in real patients. As shown in Figure
1, we successfully injected tumor cells into the vitreous cavity of zebrafish embryos at 48 hpf.
Interestingly, the highest increase in the GFP expression occurred between 0 dpi and 1 dpi and the level of GFP expression maintained stably throughout the study period, when we quantitatively analyzed the data. We speculated the presumptive reasons of this phenomenon as follows: 1) right after the injection, tumor cells might be inevitably packed. The emission of fluorophores might be interfered by other obscuring tumor cells or intraocular structures such as the lens in front of tumor cells when we observed the cells under the confocal laser microscope. 2) In this regard, after the redistribution of tumor cells from 0 dpi to 1 dpi, the expression of GFP was increased. The more important part with the alterations of GFP expression is that there was stable expression of GFP between 1 dpi and 4 dpi. In the normal culture condition, SNUOT-Rb1 cells double their numbers every 24 hours [
14]. However, in the vitreous cavity of zebrafish and previous murine models of mice, doubling of tumor cells is not that fast [
10]. The interaction between the tumor and the vitreous might be different from that between the tumor and the culture media.
The distribution patterns as well as the mean intensity of GFP expression were different according to the number of injected cells. We can observe dispersed cells when we injected only 20 cells (Figure
2A). In contrast, the injection of 100 retinoblastoma cells induced more ordered distribution of tumor cells as shown in Figures
2B and
3E. So we decided to inject 100 cells for further experiments on investigating into the potential of this model for screening of anticancer drugs.
Interestingly, we found the potential of this model as a tool for multiple screening of anticancer drugs with carboplatin and melphalan. We necessarily selected these 2 currently utilized drugs because they are widely utilized in real clinical settings for retinoblastoma patients. Interestingly, both drugs demonstrated effective anticancer activity on this model. These results might provide the possibility of this model as a screening tool for the evaluation of anticancer drugs which are in the development process. Furthermore, as previously mentioned, we can inject more than 200 zebrafish embryos at a sitting; therefore, more than 20 different candidate drugs can be screened with 1 session of experiments. Quantitative analysis of the tumor population yielded stable and reproducible results, demonstrating the usefulness of this model. After the image was captured with the confocal laser microscope, the quantitative data can be easily obtained with the public image processing program, ImageJ, by a few clicks.
Methods
Zebrafish
Adult wild-type zebrafish, purchased from a local aquarium farm and maintained in the laboratory facility, were utilized for producing embryos by breeding. Zebrafish were raised at 28.5°C in alternate dark–light cycles of 13 and 11 hours, respectively. Twenty-four hours after fertilization, zebrafish embryos were placed in fresh Ringer’s solution with 0.2 mM PTU (Sigma-Aldrich, St. Louis, MO) to inhibit pigmentation of eyeballs. Care, use, and treatment of animals were done in agreement with the Association for Research in Vision and Ophthalmology for the use of animals in ophthalmic and vision research and the guidelines established by the Seoul National University Institutional Animal Care and Use Committee.
Cell culture
SNUOT-Rb1 cells, from the previously established retinoblastoma cell line by our group [
15], were maintained in RPMI 1640 medium (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Rockville, MD) and 1% penicillin-streptomycin solution (Invitrogen, Carlsbad, CA) at 37°C in the humidified atmosphere of 95% air and 5% CO
2. The cell line underwent the authentication by DNA fingerprinting analysis with short tandem repeat markers by Korean Cell Line Bank on November 22, 2012. For the visualization of cells with GFP, SNUOT-Rb1 cells were transfected with cop GFP Control Lentiviral Particles (sc-108084; Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer’s instructions. To select stable clones expressing GFP, cells were maintained in the culture media containing 4 μg/ml puromycindihydrochloride (sc-108071; Santa Cruz Biotechnology) for 2 weeks.
Intravitreal injection of retinoblastoma cells
At 48 hpf, zebrafish embryos were anesthetized with 0.042 mg/ml Tricaine (ethyl 3-aminobenzoate methanesulfonate; Sigma-Aldrich). Eight to 10 hours before the injection, zebrafish embryos were dechorionated if necessary. For the stable injection, zebrafish were placed on the 1.7% agarose gel containing 1 ppm methylene blue. Then, using the Pneumatic PicoPump (PV820; World Precision Instruments, Sarasota, FL), cells of the indicated number were injected into the vitreous cavity of zebrafish embryos via glass capillaries attached to the Hamilton syringe (Hamilton Company, Reno, NV) under the stereomicroscope (Leica S6 E; Leica Microsystems, Wetzlar, Germany). Eyes of zebrafish were scanned daily on the Coverglass-Bottom dish (SPL Life Sciences, Pocheon, Republic of Korea) by the confocal laser microscope (Fluoview FV1000; Olympus, Tokyo, Japan) to record the alterations in the tumor population.
Quantification of tumor population
We obtained the images of the eyeballs of zebrafish 1 hour, 24, 48, 72, 96 hours after the injection of retinoblastoma cells with the confocal laser microscope (Fluoview FV1000, Olympus). The mean intensity of GFP expression was measured using the ImageJ program (1.46r; National Institutes of Health, Bethesda, MD) [
24]. After opening the image file captured with the confocal laser microscopy in the program, the color threshold was set using the menu Image>Adjust>Color Threshold. Then, the area over the designated threshold (brightness 48) was selected automatically with the yellow demarcation line or by putting the ‘select’ button on the pop-up. The mean intensity of GFP expression was calculated by the menu Analyze>Tools>Color Histogram or putting the ‘RGB’ button twice on the pop-up in the menu Analyze>Histogram to demonstrate green histogram of the image (Different approaches were required to get the values from version to version). The values were demonstrated as ‘Mean’ or ‘gMean’ on the pop-up. Eight zebrafish were included per group for experiments.
Isolation of retinoblastoma cells
Four days after the injection of retinoblastoma cells, about 150 zebrafish were collected in 4°C phosphate-buffered saline supplemented with 1% penicillin-streptomycin solution (Invitrogen). Then, the zebrafish were digested with 1.5 mg/ml collagenase from Clostridium histolyticum (Sigma-Aldrich) in Hank’s balanced salt solution supplemented with 5% FBS (Gibco BRL) and 1% penicillin-streptomycin solution (Invitrogen) at 37°C for 30 minutes. The isolated cells were suspended and incubated in RPMI 1640 (WelGENE) supplemented with 10% FBS (Gibco BRL), 1% penicillin-streptomycin solution (Invitrogen), and 4 μg/ml puromycindihydrochloride (Santa Cruz Biotechnology). After the sufficient colonies were formed, we proceeded on further analyses.
Immunoblot
The cells were lysed with RIPA buffer containing a protease inhibitor (Roche, Penzberg, Germany). The lysates were centrifuged at 13,000 rpm at 4°C for 20 minutes. Then, the supernatants were delivered to new micro test tubes for further processes. Equal amounts of extracted proteins from the cells were separated by electrophoresis on 7.5% SDS-PAGE and transferred to nitrocellulose membranes (AmershamHybond ECL, GE Healthcare Bio-Sciences, Piscataway, NJ). The membranes were incubated with anti-GFAP antibody (1:1,000, ab53554, Abcam, Cambridge, United Kingdom), anti-NSE antibody (1:1,000, #9536, Cell Signaling Technology, Beverly, MA), and anti-β-actin antibody (1:3,000, A2066, Sigma-Aldrich) at 4°C overnight. Then, the membranes were incubated with species-specific horseradish peroxidase-conjugated secondary antibodies (Pierce, Thermo Scientific, Waltham, MA). After the treatment of membranes with Amersham ECL™ western blotting detection reagent (GE Healthcare Bio-Sciences), the membranes were exposed to the film (AmershamHyperfilm ECL, GE Healthcare Bio-Sciences).
Reverse transcriptase-polymerase chain reaction (PCR)
Total RNA was extracted from the cells using TRIzol (Invitrogen) according to the manufacturer’s instructions. For the synthesis of cDNA, 1 μg of total RNA was mixed and reverse transcribed with oligo(dT)
15 primer, Superscript II reverse transcriptase (Invitrogen Corp.) and dNTPs. Polymerase chain reaction (PCR) was performed with the resultant cDNA, 10X PCR buffer, 2.5 mMdNTPs, 10 mM forward and reverse primers, DNA polymerase (Corebiosystem, Seoul, Republic of Korea), and RNAse-free water. The primers for cellular retinaldehyde-binding protein (CRALBP) were 5′-TGGCAAAGTCAAGAAATCACC-3′ (forward) and 5′-CGTGGACAAAGACCCTCTCA-3′ (reverse) [
25], and the resultant product was 313 bp. PCR was performed with denaturation in 5 minutes at 94°C, followed by 35 cycles of 30 seconds of denaturation at 94°C, 30 seconds of annealing at 60°C, and 30 seconds of elongation at 72°C. The primers for GAPDH were 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse), and the resultant product was 500 bp. PCR was performed with denaturation in 5 minutes at 94°C, followed by 30 cycles of 30 seconds of denaturation at 94°C, 30 seconds of annealing at 65°C, and 30 seconds of elongation at 72°C. The PCR products were electrophoresed on 1% agarose gels containing ethidium bromide in a constant 100 V field.
Preparation and treatment of anticancer drugs
Carboplatin (C2538) and melphalan (M2011) were purchased from Sigma-Aldrich. Zebrafish were cultured in fresh Ringer’s solution containing 200 μM anticancer drugs after the intravitreal injection of retinoblastoma cells. The solutions were changed every 24 hours. Eyes of zebrafishembyos were scanned daily on the Coverglass-Bottom dish (SPL Life Sciences) by the confocal laser microscope (Fluoview FV1000, Olympus).
Cell viability assay
Cell viability was evaluated with 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (water soluble tetrazolium salt, WST-1) assay using EZ-Cytox Cell Viability Assay kit (Itsbio, Seoul, Republic of Korea) according to the manufacturer’s instruction. Briefly, SNUOT-Rb1 cells were plated in 96 well plates and cultured overnight (1 × 104 cells per well). The cells were treated with anticancer drugs of different concentrations (25, 50, 100, 200, 400 μM) for 48 hours. Then, the reagent from EZ-Cytox Cell Viability Assay kit was applied to each well. After 2 hours of additional incubation, 96 well plates were shaken thoroughly on the shaker for 1 minute. Absorbance was measured at 450 nm using the microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA). To confirm the results of WST-1 assay, direct estimation of viable cells using Trypan Blue Stain (Life Technologies, Carsbad, CA) was performed after the treatment with anticancer drugs of different concentrations for 48 hours.
Statistical analysis
Differences of the values between experiments were assessed with the Student’s t-test. All statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA). The mean value and the standard error of the mean were shown in figures. P-values less than 0.05 were considered as statistically significant, and *, **, *** were designated as P < 0.05, P < 0.001, P < 0.0001, respectively, in figures.
Acknowledgements
We thank to Dr. Hyoung Oh Jun for technical help on experiments. This study was supported by the Bio-Signal Analysis Technology Innovation Program (2013–036042), the Pioneer Research Program of MEST/NRF (2013-005321), the Global Core Research Center (GCRC) grant from NRF/MEST, Republic of Korea (2012–0001187), the Ministry of Education, Science and Technology, (2012–0004090) and Seoul National University College of Medicine Research Grant, Republic of Korea (800–20110049).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JHK and SHS designed the research; DHJ and DS performed the research; YN, MJ, JC, JHK and YSY contributed materials and established methods; DHJ and DS collected data; GHJ, DS, JHK and SHS analyzed data; and DHJ, DS, JHK and SHS wrote the paper. All authors read and approved the final manuscript.