Invasiveness and metastasis of retinoblastoma in an orthotopic zebrafish tumor model.

Xiaoyun Chen,Ziquan Cao,Lasse Jensen,Kayoko Hosaka,Yuping Sun,Yihai Cao,Huasheng Yang,Jian Wang,Yizhi Liu,Rujie Zhuang

doi:10.1038/srep10351

Abstract

Retinoblastoma is a highly invasive malignant tumor that often invades the brain and metastasizes to distal organs through the blood stream. Invasiveness and metastasis of retinoblastoma can occur at the early stage of tumor development. However, an optimal preclinical model to study retinoblastoma invasiveness and metastasis in relation to drug treatment has not been developed. Here, we developed an orthotopic zebrafish model in which retinoblastoma invasion and metastasis can be monitored at a single cell level. We took the advantages of immune privilege and transparent nature of developing zebrafish embryos. Intravitreal implantation of color-coded retinoblastoma cells allowed us to kinetically monitor tumor cell invasion and metastasis. Further, interactions between retinoblastoma cells and surrounding microvasculatures were studied using a transgenic zebrafish that exhibited green fluorescent signals in blood vessels. We discovered that tumor cells invaded neighboring tissues and blood stream when primary tumors were at the microscopic sizes. These findings demonstrate that retinoblastoma metastasis occurs at the early stage and antiangiogenic drugs such as Vegf morpholino and sunitinib could potentially interfere with tumor invasiveness and metastasis. Thus, this orthotopic retinoblastoma model offers a new and unique opportunity to study the early events of tumor invasion, metastasis and drug responses.

Highlights

Retinoblastoma often exhibits an invasive and metastatic phenotype at the early stage of tumor development[1]
To recapitulate the clinical situation of retinoblastoma development, we developed an embryonic zebrafish model that would fulfill the following criteria: 1) Developing zebrafish to resemble the pediatric situation in human patients; 2) Immune privilege to allow implantation of human and mouse retinoblastoma tumors; 3) Orthotopic implantation to recapitulate the clinical origin of retinoblastoma; 4) Transparent visualization of implanted primary and metastatic tumors at the single cell level; 5) Quantitatively monitoring and assessing tumor cell behavior in the living body of zebrafish; 6) Interaction between retinoblastoma cells and host structural and cellular components in a non-invasive manner; and 7) Assessment of therapeutic effects of drugs that interfere with tumor invasion and metastasis
As vasculatures plays a crucial role in tumor invasion and metastasis and VEGF is an essential mediator for tumor angiogenesis and vascular remodeling, we studied the function of VEGF using a specific morpholino targeting zebrafish Vegf-aa mRNA as previously described[14]

Summary

Introduction

Retinoblastoma often exhibits an invasive and metastatic phenotype at the early stage of tumor development[1]. Invasion of the optic nerve and subsequent spreading to the circulating subarachnoid fluid that further carry tumor cells to the spinal cord is an alternative pathway of metastasis. Several lines of transgenic mouse models are available in the scientific community These genetically manipulated mouse retinoblastomas often carry overexpression of a particular oncogene such as SV40-T antigen or loss of a tumor suppressor gene such as p535. These oncogene-driven models are far from clinical relevance as activation of oncogenes and inactivation of tumor suppressor genes may not exist in human retinoblastomas. Our zebrafish retinoblastoma model offers a unique opportunity to study the mechanisms underpinning metastasis and to assess therapeutic efficacies of drugs that block retinoblastoma invasion

Methods

Results

Conclusion