Results
Optimization of environmental temperatures. In laboratory environments, zebrafish embryos commonly are raised at 28.5°C , whereas human cells are maintained at 37°C . To enable engraftment of RMS xenotransplants in zebrafish embryos, it was necessary to establish a temperature level that allows for normal development of both zebrafish embryos and human tumor cells. To determine optimal environmental conditions, zebrafish embryos were exposed to different temperatures. The percentage of surviving embryos at 120 hpf was significantly lower at 35°C compared to the control group maintained at 28.5°C (Fig.1A,p =0.0016). This is consistent with previously published observations . Considering the higher mortality rate at 35°C, we tested a gentler two-step approach using temperatures of 33°C from 3 hpf to 48 hpf, followed by a temperature rise to 35°C until 120 hpf, which did not have a negative impact on the survival rates of the zebrafish embryos compared to those in the control group (Fig.1A, p =0.6244). Next, these temperature settings were applied to RMS cells in culture, to examine possible differences in cell proliferation at 1, 48, 96 and 120 hours after plating. Lower temperatures did not affect RD cell growth, and the number of RD cells kept at 37°C did not differ significantly 120 hours after plating (Fig.1B, 4750 cells/well at 37°C vs. 5000 cells/well at 33/35°C, p =0.6753). In contrast, proliferation of Rh30 cells slowed down significantly at lower temperatures compared to the control cells maintained at 37°C (Fig.1C, 8750 cells/well at 37°C vs. 3438 cells/well at 33/35°C,p <0.0001).
Induction of RMS xenografts in zebrafish embryos. To analyze the development of Rh30 and RD cells in vivo , DiI-labeled RMS cells were transplanted into zebrafish embryos at the 1000-cell stadium (Fig.2). Images of the transplants were taken, and cross-sectional areas were measured at 24, 72 and 120 hpf (Fig.3A-C). RD xenografts showed stable growth from 24 hpf until 120 hpf (Fig. 3D), whereas the cross-sectional areas of Rh30 xenografts decreased during the observation period (Fig. 3D). Histology sections from 72-120 hours-old, successfully transplanted embryos were prepared and stained with hematoxylin and eosin. The presence of tumor cell nests confirmed successful engraftment of xenotransplants (Fig. 3E).
Toxicity screening. Possible toxic effects of the candidate anti-RMS drugs on embryonal and larval development were explored until 120 hpf. Vincristine significantly reduced the proportion of surviving embryos starting at drug concentrations higher than 20 µM (Fig. 4A, 94% surviving embryos in E3 vs. 69% surviving embryos in E3 medium with 25µM vincristine, p <0.0001). Dactinomycin treatment also led to significantly lower survival rates starting at concentrations exceeding 1 µM (Fig. 4B, 88% surviving embryos in E3 vs. 73% surviving embryos in E3 medium with 2µM dactinomycin,p =0.0398). The mitogen-activated protein kinase kinase (MEK) inhibitor trametinib negatively affected the embryos starting at drug-concentrations higher than 50 nM (Fig. 4C, 88% surviving embryos in E3 vs. 67% surviving embryos in E3 medium with 100nM trametinib,p =0.0007).
Testing the effects of candidate drugs on RMS xenografts in zebrafish embryos. Successfully transplanted embryos were exposed to selected drugs to assess their effect on RD-xenotransplants in vivo . Vincristine concentrations of 2 and 20 µM reduced the standardized cross-sectional area (SCSA) to 39% (Fig 5A;p <0.0001) and 15% (Fig. 5A;p <0.0001), respectively, compared to vehicle-treated RMS xenotransplants. A statistically significant, dose-dependent effect of vincristine at 2 and 20 µM could also be observed (Fig. 5A;p <0.0001), further supporting the observation that vincristine has antitumoral effects in zebrafish RMS-xenotransplants.
Dactinomycin showed an analogous inhibitory effect on RMS xenografts in zebrafish with a reduction to 52% of the SCSA of non-treated xenotransplants (Fig. 5B; p <0.0001) at a concentration of 0.1 µM, and to 24% of the SCSA of non-treated xenotransplants (Fig. 5B; p <0.0001) at concentrations of 1µM dactinomycin.
2µM vincristine and 0.1µM dactinomycin in combination showed stronger inhibitory effects compared to 0.1µM dactinomycin monotherapy therapy (Fig. 5C; reduction to 28% of the SCSA of untreated xenografts by 2µM vincristine and 0.1µM dactinomycin in combination vs. 52% by 0.1µM dactinomycin alone; p =0.0055). There were no significant differences in the efficacy of the combination treatment compared to exposure to 2µM vincristine alone (Fig. 5C; p=0.4265). Given that RD cells carry an NRAS proto-oncogene, GTPase (NRAS)Q61H- variant resulting in activation of mitogen-activated protein kinase (MAPK) signaling, the MEK-inhibitor trametinib was also tested for inhibitory effects on RMS xenotransplants in zebrafish embryos. At trametinib concentrations of 5 and 50 nM, we observed a reduction of the SCSA to 57% (p <0.0001) and 21% (p <0.0001) of the SCSA of non-treated RMS-xenotransplants, respectively (Fig. 5D).