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.

Figure 1

Persistence of human malignant glioma cells within, and lack of developmental effects on, the developing zebrafish embryo. U251-RFP cells growing within the developing zebrafish embryo for at least 7 d at 30°C do not perturb development. Lateral views of a representative zebrafish embryo after transplantation of U251-RFP human glioma cells, imaged under fluorescence alone (right column images) or merged bright field and fluorescent images (left column images). Leftmost column of text, embryonic age at time of imaging. 12 hpf, embryo imaged at 2 h after transplantation. Bar, 300 μm. The remainder of embryos were imaged at 1, 2, 3, 5, or 7 dpf. Bar, 200 μm.

Figure 2

Visualization of individual human glioma cells proliferating within a live zebrafish embryo. Lateral views of a single, live zebrafish embryo over a 9-d period following transplantation of individual U251-RFP cells. At baseline (2 dpf), two individual U251-RFP cells are observed. At 3 dpf, three individual U251-RFP cells observed. At 4 dpf, five individual U251-RFP cells are noted. At 6 dpf, at least seven individual U251-RFP cells are notable. At 9 dpf, there is a further increase in the number of U251-RFP cells. Scale bar, 200 μm.

Figure 3

Visualization of angiogenesis within zebrafish embryos following transplantation of human glioma cells. A to C, lateral views of live zebrafish embryos after transplantation of U251-RFP cells in fli1:EGFP zebrafish. A, representative zebrafish at 2 dpf showing transplanted human glioma cells (RFP) located separately from the zebrafish endothelium (GFP). B, a representative zebrafish at 5 dpf showing the developing zebrafish endothelium, with portions of the vasculature approaching the U251-RFP tumor cell masses (arrowheads). Scale bar for A and B, 50 μm. C, an image at higher magnification showing a single distinct GFP-expressing zebrafish blood vessel growing into a U251-RFP tumor cell mass. Scale bar, 10 μm in C.

Figure 4

Exposure to IR reduces the size and fluorescence emitted by human glioma cells growing in zebrafish embryos. Embryos containing U251:RFP tumors were exposed to DMSO (as in Fig. 5) or irradiated with 10 Gy and imaged on sequential days. A to C, images taken under fluorescence of representative zebrafish embryos after transplantation of U251:RFP cells, and imaged immediately before exposure to DMSO (left column) or 10 Gy irradiation (right column) at 1 dpf (A), 3 dpf (B), or 5 dpf (C). The tumor masses in the embryos treated with DMSO have visibly grown in size at both 3 and 5 dpf, while the tumor masses in the irradiated embryo show regression in size at both time points. D, the fluorescence emitted by the tumor masses of either treatment group was measured at all three time points, and the mean levels of emitted fluorescence are plotted in the histograms in arbitrary fluorescence units (bars, SE). These data were derived from three replicate experiments encompassing the following numbers of embryos: n = 22 for the “DMSO” group and n = 18 for the “10 Gy” group.

Figure 5

Combined temozolomide and IR results in the greatest inhibition of tumor growth and fluorescence emitted from human malignant glioma cells grown in zebrafish embryos. Embryos containing U251:RFP tumors were exposed to temozolomide alone (TMZ Only), irradiated with 10 Gy, or temozolomide followed by 10 Gy IR (10 Gy and 10 Gy + TMZ, respectively) and imaged on sequential days. A to C, images taken under fluorescence of representative zebrafish embryos at 1 dpf (A), 3 dpf (B), or 5 dpf (C). D, the fluorescence emitted by the tumor masses of each treatment group was measured at all three time points. Columns, mean levels of emitted fluorescence in arbitrary fluorescence units; bars, SE. These data were derived from three replicate experiments encompassing the following numbers of embryos: n = 16 for the “TMZ only” group, n = 18 for the “10 Gy” group, and n = 19 for the “10 Gy + TMZ” group.

Figure 6

Comparison of the effects of treatments on the cumulative changes in fluorescence emitted by human malignant glioma cells in zebrafish embryos. The cumulative changes in the mean fluorescence emitted by U251-RFP tumor masses in each treatment group between the beginning of the experiment at 24 hpf and the conclusion at 120 hpf. The treatment groups included embryos treated with DMSO or temozolomide alone (TMZ Only), 5 Gy and 10 Gy IR, and temozolomide combined with 10 Gy IR (10 Gy + TMZ). Columns, mean percentage changes in each treatment group; bars, SE. The values were determined from three replicate experiments involving a total of 93 embryos. *, P < 0.001 compared with embryos treated with DMSO only. ‡, P < 0.05 compared with embryos treated with 10 Gy.