Zebrafish as an Orthotopic Tumor Model for Retinoblastoma Mimicking Routes of Human Metastasis

Abstract

Background: Retinoblastoma (RB) is the most common eye cancer in children that has a high mortality rate when left untreated. Mouse models for retinoblastoma have been established but are time- and cost-intensive. The aim of this work was to evaluate an orthotopic transplantation model of retinoblastoma in zebrafish that also allows for tracking migratory routes and to explore advantages and disadvantages with respect to drug testing.

Methods: Three fluorescence-labeled retinoblastoma cell lines (RB355, WERI-RB-1, Y79) were injected into the left eye of two-day-old zebrafish, while the un-injected right eye served as control. The migratory trajectories of injected retinoblastoma cells were observed until 8 days post injection (dpi), both in lateral and dorsal view, and measuring fluorescence intensity of injected cells was done for RB355 cells.

Results: Time until the onset of migration and routes for all three retinoblastoma cell lines were comparable and resulted in migration into the brain and ventricles of the forebrain, midbrain and hindbrain. Involvement of the optic nerve was observed in 10% of injections with the RB355 cell line, 15% with Y79 cells and 5% with WERI-RB-1 cells. Fluorescence intensity of injected RB355 cells showed an initial increase until five dpi, but then decreased with high variability until the end of observation.

Conclusion: The zebrafish eye is well suited for the analysis of migratory routes in retinoblastoma and closely mirrors patterns of retinoblastoma metastases in humans.

Keywords: RB355; WERI-RB-1; Y79; childhood eye cancer; malignant retinal tumor; orthotopic transplantation; retinoblastoma; zebrafish.

Figure 1

Migration of WERI-RB-1 cells in the first 5 days after injection of 80–100 suspension cells into the vitreous cavity of the left eye of a 2-day-old zebrafish (A) on day of injection, (B) on day 1, (C) on day 2 and (D) on day 5 post injection in lateral view; (E) on the day of injection with transmitted light, (F) on day 1, (G) on day 2 and (H) on day 5 post injection in dorsal view. Only the dorsal view shows the initiation of migration of WERI-RB-1 cells in the brain already after one day post injection. After 5 days post injection, only a few cells are found in the eye and most have migrated to the brain. On day 8 post injection no tumor cells were visible (not shown).

Figure 2

Mean GFP-fluorescence intensity of 20 zebrafish injected with RB355 cells in comparison of the left side view with the dorsal view. Determination of GFP-intensity was done with microscopic photographs from the left side view (left eye injected) and the dorsal view with the software ImageJ by marking the tumor margins. One zebrafish died at 1 dpi; 19 RB355-injected zebrafish could be observed until day 5 post injection (5 dpi) but the tumor cells could be observed until day 7 only in 10 cases. In cases where no fluorescence could be measured, the quantity was set to 12, which was the quantity of the background. The amount of analyzed zebrafish with measured fluorescence is given for each column in parentheses: 0 dpi (20), but could not be measured for dorsal view, 1 dpi (19), 2 dpi (19), 5 dpi (19), 7 dpi (10) and on 8 dpi (9), only one zebrafish could be measured on 9 dpi (not shown). Bars represent the standard error of measured fluorescence.

Figure 3

Positions of WERI-RB-1 cells in the first 5 days after injection of 80–100 suspension cells into the vitreous cavity of the left eye of a 2-day-old zebrafish (A) on day of injection, (B) on day 1, (C) on day 2 and (D) on day 5 post injection in lateral view; (E) on day 1, (F) on day 2, (G) on day 5 and (H) on day 8 post injection in dorsal view. The time interval includes the complete migration of the WERI-RB-1 cells out of the left eye into the brain.

Figure 4

Retinoblastoma cells migrated towards the contralateral eye but not outside of the central nervous system. (A–E) shows 5 pictures of a z-stack in the dorsal head view taken with the Zeiss LSM 800 confocal microscope one day post injection (1 dpi) of 80–100 Y79 cells in the left eye. (F) shows the maximum intensity projection (MIP) of all five layers. The cells start to migrate from the retina of the left eye postulated along the optic nerve and reached near the right eye.

Figure 5

Retinoblastoma cells migrate into the ventricles at longer observation. Injection of 80–100 RB355 cells in a 2-day-old zebrafish eye. (A) on day of injection in lateral view, (B). on day 5, (C) on day 7 and (D) on day 9 post injection in dorsal view. (E) a second zebrafish on day of injection in lateral view, (F) on day 5, (G) on day 7 and (H) on day 9 post injection in dorsal view. Both injections show a migration of tumor cells into the ventricles after 5 days post injection. From 5 dpi until 9 dpi, the cells marked the ventricles of the forebrain, midbrain and hindbrain.

Figure 6

Demonstration of ventricles through direct cell-injection into the hindbrain ventricle. (A,B) demonstrates the injection of a methylene blue solution directly into the hindbrain ventricle. (A) shows a side view, (B) shows the dorsal view and (C,D) demonstrates the ventricles through direct injection of fluorescence-marked RB355 cells into the hindbrain ventricle of three-day-old zebrafish. The pattern of cell distribution after direct injection into the hindbrain ventricle (C,D) reflects the pattern of cell distribution after injection of the cells in the eye and postulated metastasis into the ventricles of fore-, mid- and hindbrain (as shown in Figure 5B–D,F–H and Figure 7B).

Figure 7

Metastatic spread of retinoblastoma cells into the brain ventricles. Injection of 80–100 Y79 cells into the left eye of a 2-day-old zebrafish. Figures are showing lateral and dorsal views by the confocal microscope Zeiss LSM800. Both views show metastasis of the tumor cells in the ventricle already after 4 days post injection: (A) on day 4 post injection in lateral view; (B) on day 5 post injection in dorsal view. The trapezoid shape of the forebrain ventricle after cell migration into this ventricle is visible (A,B) are from the same embryo). (C) a second embryo 4 days post injection showing one migration event in the anterior and a second one into the posterior region of the head. In both embryos, no cells can be seen in the injected eye after 4 days.

Figure 8

Hematoxylin-Eosin (H.E.) staining confirmed the localization of metastatic retinoblastoma cell line cells. Figure 8 shows frontal sections of different zebrafish heads in the region of the retina (A,B) Without cell injection and (C–F) after RB355 cell injection. (A,C–F) With objective magnification 20× and one eye; (B) in higher magnification (40×). (A) Zebrafish head of a six-day-old zebrafish without cell injection and the following marked structures: 1. lens, 2. pharyngeal cartilage, 3. brain at the forebrain-midbrain margin, 4. retina. (B) Retinal layers of a 6-day-old zebrafish: 1. retinal pigment epithelium (RPE), 2. photoreceptor layer (PRL) and outer nuclear layer (ONL), 3. outer plexiform layer (OPL) 4. inner nuclear layer (INL), 5. inner plexiform layer (IPL), 6. ganglion cell layer (GCL), 7. exit of the optic nerve (ON). (C) Two-day-old zebrafish at the day of RB355 cell injection, RB355 cells are visible only in the vitreous cavity. (D) Four-day-old zebrafish, two days after RB355 cell injection. Tumor cells have left the vitreous cavity and margin of the eye cup, and have migrated towards the brain. (E) Seven-day-old zebrafish, five days after RB355 cell injection. RB355 cells have entered the forebrain ventricle. (F) Nine-day-old zebrafish, seven days after RB355 cell injection. RB355 cells have entered the forebrain ventricle and some are visible near the location of the optic nerve and optic chiasma. (C–F) Injected RB355 cells are marked with a dashed line.

Figure 9

Higher magnifications of Figure 8C–F showing migration of injected RB355 cells in detail. (A) Two-day-old zebrafish at the day of RB355 cell injection. RB355 cells can be identified by the much larger size compared to cells of the retina layers of the host. Most of the cells are found in the vitreous cavity and some have migrated up to the photoreceptor layer. (B) Two days after RB355 cell injection, most of the injected cells have migrated through the retina of the injected eye and are situated at the margin of the epithelium of the eye cup. Some cells have migrated near the contralateral eye, but do not enter the contralateral eye (C) Five days after RB355 cell injection, migrated cells are found in the forebrain ventricle. (D) Seven days after RB355 cell injection. Asterisk marks axons of the optic nerve. RB cells are found immediately near the axons and migrated into the brain near to the contralateral eye, but have not entered the un-injected eye either. A second group of cells is visible in the forebrain ventricle.