Development Of New Models Of Orthotopic Primary Human Retinoblastoma Xenografts

Abstract

Purpose: : To develop preclinical orthotopic models of primary human retinoblastoma suitable for the pharmacological assessment of intravitreal administration of standard and/or new therapeutic compounds.

Methods: : Orthotopic models of human retinoblastoma have been developed from three subcutaneous xenografted models that have previously been established and characterized in our laboratory, i.e. RB102-FER, RB111-MIL, and RB200-GS that have been maintained in nude mice. Mice bearing xenografts were sacrificed and tumors were dissected to obtain a suspension of fresh tumor cells at a concentration of 8000 cells/µl in DMEM serum-free medium. Under intraperitoneal anesthesia, 2µl of cell suspension was injected into the subretinal space of the right eye for 3 groups of mice using a 32G needle via a Hamilton syringe. Each group was constituted by 2 female SQUID mice and 3 male nude mice. After subretinal injection, ophthalmic examination of the mice was done every 15 days with binocular microscope. When tumor cells invaded vitreal cavity and anterior chamber, the mice were sacrificed for ophthalomological pathological analyses.

Results: : Tumor cells developed in all injected eyes, whatever the mouse categories used, 4 weeks after orthotopic transplantation for RB102-FER and 6 weeks after for RB200-GS. In contrast, no tumor growth was observed in injected eyes of the RB111-MIL model. In the 2 first xenografts, the proliferation started with a white hemorrhagic retinal mass; three weeks later, the vitreous was invaded by tumor cells that extended to the anterior chamber. Pathological examination of the injected eyes confirmed the presence of a massive infiltration of the retina, vitreous and anterior chamber by retinoblastoma cells.

Conclusions: : We have then developed two new models of orthotopic primary human retinoblastoma. Considering that our models well reproduce the patient’s tumors from which they have been obtained, these new orthotopic xenografts therefore offer new tools for relevant preclinical assessment.

Keywords: retinoblastoma • transplantation • tumors