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

SIMPLE SUMMARY: Retinoblastoma is a rare malignant eye tumor with early childhood occurrence and high invasive potential. Animal models of retinoblastoma have multiple limitations, but recently published zebrafish models are promising. The transparent nature of zebrafish allows visualization of migrating cells in the living young fish under the microscope. Previous zebrafish models have analyzed only side views of the heads whereas the aim of our study is to analyze the timing and the metastatic trajectories of different retinoblastoma cell lines from two angles, the side view and dorsal view. In our zebrafish model, migrating retinoblastoma cells are found along the optic nerve and adjacent regions of the brain or its ventricles. These trajectories of migrating cells resemble the pattern of metastasis in human patients. The zebrafish model may facilitate pharmacological studies for treatment of retinoblastoma in the future. Our results provide new insights into the metastatic behavior of this complex tumor. 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.