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Fig. 4 Culture medium composition has a significant effect on tumor cell localization in transplanted zebrafish embryos. (A) Representative images of fli1:EGFP zebrafish embryos 48 hpf transplanted with tumor cell lines cultured in complete neural stem cell medium (UW228-3+, D425 Med+, and MDA-MB-231+). Medium for UW228-3+ was supplemented with 1% FBS for maintenance and removed before transplantation. (B) Brain/body area distribution of tumor cell lines cultured in complete neural stem cell medium (+) compared to normal culture medium in 48 h old embryos. Mean ± SEM, ****P < .0001, Student t-test. (C) Schematic heatmaps of the tumor cell location of tumor cell lines cultured in complete neural stem cell medium (+) compared to normal culture medium in 48 h old embryos. 1 = hindbrain area; 2 = mid/forebrain area; 3 = pericardium area; 4 = yolk sac area; 5 = body and tail area. *P < .05, **P < .01, ****P < .0001, 2-way ANOVA with Šídák post-hoc test. (D) Proliferation rate of UW228-3, D425 Med, and MDA-MB-231 cells cultured in medium with FBS or in neural stem cell medium (+) evaluated by cell counting. Mean ± SD, n = 3 independent experiments. (E) Overall survival analysis of zebrafish transplanted with UW228-3, D425 Med, and MDA-MB-231 cells cultured in medium with FBS or in neural stem cell medium (+). Kaplan–Meier curves depict differences in survival and statistical differences were determined using the Log-rank Mantel-Cox test.