Fig. 6

The effect of the phosphatidyl‐3‐kinase (PI3K) inhibitor everolimus in zebrafish embryos. The effect of the mammalian target of rapamycin (mTOR) inhibitor everolimus on xenografted DsRed‐labelled canine mammary tumor cell line (CMT)‐U27 cells in zebrafish embryos. Three days post‐injection (3 dpi) of untreated cells in the yolk sac (A) cell proliferation in the yolk sac and cells in the tail veins were observed, whereas after pre‐treatment with 10 μM everolimus (B), even more CMT RED cells were observed, indicating that the cells survived, proliferated and intravasated (arrows). Additionally, extravasation was more pronounced when CMT RED cells were directly injected into the circulation through the duct of Cuvier (C), showing tumour cells that formed a clump halfway through the tail, and the cells with everolimus resulted in tail vein tumours and extravasation (D). Statistical analysis was performed using (E, F) Fisher's exact test. *P < 0.05 was considered significant