An Orthotopic Model of Uveal Melanoma in Zebrafish Embryo: A Novel Platform for Drug Evaluation.

Chiara Tobia,Francesco Semeraro,Daniela Coltrini,Elisa Scalvini,Alessandra Loda,Roberto Ronca,Jessica Guerra,Sara Rezzola

doi:10.3390/biomedicines9121873

Abstract

Uveal melanoma is a highly metastatic tumor, representing the most common primary intraocular malignancy in adults. Tumor cell xenografts in zebrafish embryos may provide the opportunity to study in vivo different aspects of the neoplastic disease and its response to therapy. Here, we established an orthotopic model of uveal melanoma in zebrafish by injecting highly metastatic murine B16-BL6 and B16-LS9 melanoma cells, human A375M melanoma cells, and human 92.1 uveal melanoma cells into the eye of zebrafish embryos in the proximity of the developing choroidal vasculature. Immunohistochemical and immunofluorescence analyses showed that melanoma cells proliferate during the first four days after injection and move towards the eye surface. Moreover, bioluminescence analysis of luciferase-expressing human 92.1 uveal melanoma cells allowed the quantitative assessment of the antitumor activity exerted by the canonical chemotherapeutic drugs paclitaxel, panobinostat, and everolimus after their injection into the grafted eye. Altogether, our data demonstrate that the zebrafish embryo eye is a permissive environment for the growth of invasive cutaneous and uveal melanoma cells. In addition, we have established a new luciferase-based in vivo orthotopic model that allows the quantification of human uveal melanoma cells engrafted in the zebrafish embryo eye, and which may represent a suitable tool for the screening of novel drug candidates for uveal melanoma therapy.

Highlights

The zebrafish embryo has been successfully employed as a platform for modeling human diseases and for large-scale screening of new drugs [1,2,3,4]
To evaluate whether the zebrafish embryo eye represents a microenvironment suitable for the grafting of melanoma cells, we first assessed the behavior of the well-characterized model of invasive murine melanoma represented by B16-BL6-DsRed+ cells [2], which were injected into the eye of zebrafish embryos at 48 hpf
Preliminary observations suggest that the displacement of melanoma cells observed at t4 is in part a consequence of the invasive properties of cancer cells and in part due to the remodeling of the eye that occurs during embryo development, which plays a not negligible role in tumor cell localization within the eye

Summary

Introduction

The zebrafish embryo has been successfully employed as a platform for modeling human diseases and for large-scale screening of new drugs [1,2,3,4]. Relatively low costs of maintenance, and optical transparency, combined with the opportunity to perform high-quality imaging, led to an extensive use of this model in cancer research. In this regard, mammalian tumor cell grafting in zebrafish embryos can be achieved in different anatomical sites, giving opportunity to study various aspects of the disease, such as tumor progression, angiogenesis, cancer cell spreading, and metastasis formation. One of the major drawbacks of the use of the zebrafish embryo as a model in oncology is the quantification of tumor xenograft growth in the different anatomical sites, generally performed by measuring the fluorescence signal generated by engrafted fluorescent tumor cells [10,11].

Methods

Results

Conclusion