Temozolomide-Mediated Radiosensitization of Human Glioma Cells in a Zebrafish Embryonic System

Abstract

The zebrafish (Danio rerio) is a popular vertebrate model for biomedical research. The rapid development, transparency, and experimental accessibility of the embryo offer opportunities for assessing the developmental effects of anticancer treatment strategies. We therefore systematically investigated parameters for growing U251 human glioma cells expressing red fluorescent protein (U251-RFP) in zebrafish embryos. Factors optimized include injection volume, number of cells injected, anatomic site of injection, age of the embryo at the time of injection, and postinjection incubation temperature. After injection into the embryos, the U251-RFP cells proliferated and the resultant tumors, and even individual cells, could be visualized in real-time via fluorescence microscopy without the need for sacrifice. These tumors recruited host zebrafish vasculature, suggesting cancer cell-host tissue interactions. Having optimized parameters for introducing and growing these human cells in the zebrafish embryos, we exposed both embryos and transplanted cancer cells to ionizing radiation and temozolomide, either alone or in combination. The human tumors in each embryo were substantially diminished following exposure to ionizing radiation and the decrease was further enhanced by pretreatment with temozolomide. In contrast, temozolomide had no discernible effects on embryonic development. These results together support the relative safety of temozolomide during embryonic development, as well as its anticancer efficacy when combined with radiation. These results suggest the value of the zebrafish model for in vivo testing of the efficacy and safety of anticancer strategies, especially on the very young. [Cancer Res 2008;68(9):3396-404]

Developing a Novel Embryo–Larval Zebrafish Xenograft Assay to Prioritize Human Glioblastoma Therapeutics

“…The opacity of the mammalian head makes directly observing how potential therapeutics influence individual invading glioblastoma cells problematic. [4][5][6][7][8][9] Yet, research suggests that migrating cells will drastically alter morphology and mechanisms of movement in three-dimensional versus two-dimensional environments and the migrating cells receive signals from the microenvironment that dictate direction making studies on migration and invasion in an intact brain desirable. 10 While investigating therapeutic efficacy in mammalian models is an important step in the drug development pipeline, testing during the early stages of discovery can be challenging.…”

“…11 Zebrafish xenograft models hold potential for studying human cancer progression, migration, invasion, metastases, and microenvironment interactions to aid in identifying new treatments. 4,6,8,9,12 The embryo-larval zebrafish model has proved useful in quickly identifying and prioritizing the screening of promising new pharmaceuticals. 13 The small size, high fecundity, and transparency of the embryo-larval zebrafish makes it amenable to conducting exposures in multiwell plates and noninvasively studying exposure-dependent effects on cancer progression using microscopy.…”

“…13 The small size, high fecundity, and transparency of the embryo-larval zebrafish makes it amenable to conducting exposures in multiwell plates and noninvasively studying exposure-dependent effects on cancer progression using microscopy. 4,6,8,9,12 Furthermore, embryo-larval zebrafish lack a fully functional adaptive immune system until *28 days making it possible to implant human cells without rejection. 14 Existing embryo-larval zebrafish xenograft models have already demonstrated that human cancer cells, including glioma, can grow, divide, metastasize, and induce angiogenesis in zebrafish similarly to rodent xenograft models.…”

“…14 Existing embryo-larval zebrafish xenograft models have already demonstrated that human cancer cells, including glioma, can grow, divide, metastasize, and induce angiogenesis in zebrafish similarly to rodent xenograft models. 5,6,8,9,[15][16][17] A particular advantage of the zebrafish xenograft model is that only a small number of cancer cells are required for xenotransplantation, which better simulates earlier stages of cancer progression. 8,9 Many embryo-larval zebrafish xenograft glioma models involve transplanting the cells into the yolk sac or perivitelline space.…”

“…8,9 Many embryo-larval zebrafish xenograft glioma models involve transplanting the cells into the yolk sac or perivitelline space. 5,6,9,15 These transplant locations lack a complex brain microenvironment, which is filled with glial cells, neuronal processes, extracellular matrix, and a functional blood-brain barrier important for studying brain cancer. 4 By transplanting glioblastoma cells into the 48-72 h postfertilization zebrafish brain, which possess a functioning blood-brain barrier similar to mammalian models, our laboratory has developed an efficient and relevant assay using high-content imaging to study glioblastoma progression and prioritize the development of new glioblastoma therapies.…”