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Fig. 4 Zebrafish bladder cancer xenografts are susceptible to immunotherapy with the conventional and genetically modified BCG strains. (A) Representative confocal images of NMIBC-RT112 control and BCG+booster-treated or VPM1002+booster-treated xenografts. Human cancer cells were labelled with the Deep Red Cell Tracker lipophilic stain (not shown) and were stained for the apoptosis marker activated caspase-3 (green) at 4 dpi. (B) Quantification of the percentage of clearance in NMIBC-RT112 control and treated xenografts at 4 dpi (****P<0.0001; Fisher's exact test). Each dot represents a full round of injections in which N represents the number of xenografts without tumors at 4 dpi relative to the total number of xenografts at 4 dpi. (C) Quantification of the percentage of activated caspase-3-positive (apoptotic) cells in NMIBC-RT112 control and treated xenografts at 4 dpi (****P<0.0001). (D) Representative confocal images of infiltrating macrophages (red) in NMIBC-RT112 control and treated xenografts at 4 dpi. (E) Quantification of absolute numbers of infiltrating macrophages in NMIBC-RT112 control and treated xenografts at 4 dpi (**P=0.0032; ****P<0.0001). (F) Representative confocal images of Tnfa expression (green) and macrophages (red) in NMIBC-RT112 control and treated xenografts at 4 dpi. Human cancer cells were labelled with the Deep Red Cell Tracker lipophilic stain (not shown). In A,D,F, white dashed regions outline the tumor. BCG+booster-treated and VPM1002+booster-treated xenografts were labelled with either the Deep Red Cell Tracker or the Vybrant CM-DiI lipophilic stain (not shown). In all images, anterior is to the left, posterior to the right, dorsal up and ventral down. Scale bars: 50 µm. (G) Quantification of the percentage of Tnfa-positive and Tnfa-negative macrophages in the tumor microenvironment (TME) of NMIBC-RT112 control and BCG+booster-treated or VPM1002+booster-treated xenografts at 4 dpi (****P<0.0001). Each dot represents one xenograft pooled from two independent experiments. (H) Representative confocal image of macrophages (red) and NMIBC-RT112 cells labelled with the Deep Red Cell Tracker lipophilic stain. (I) Quantification of the number of phagocytic macrophages in NMIBC-RT112 control and treated xenografts at 4 dpi (****P<0.0001). Each dot represents one xenograft pooled from two independent experiments. (J) Relative gene expression levels of zebrafish tnfa, il1b, il6, il10, ifng1 and tgfb1b at 4 dpi in the TME of NMIBC-RT112 control and VPM1002+booster-treated xenografts. Bars indicate the fold change of expression to that in the control relative to expression of the housekeeping gene. Each dot represents the average of two or three technical replicates of one independent experiment. In B,C,E,G,I,J, bars indicate the results as mean±s.d. For C,E,G, the numbers of analyzed xenografts are indicated in A,D,F. Data sets with a Gaussian distribution (G) were analyzed by parametric unpaired two-tailed t-test, and data sets that did not pass the D'Agostino–Pearson omnibus and Shapiro–Wilk normality tests were analyzed by nonparametric unpaired Mann–Whitney test (C,E). Unless stated otherwise, each experimental data set was challenged to the respective control. Additionally, the data sets in C,E,I were analyzed with Welch's one-way ANOVA with Games–Howell post hoc test in which P<0.0001, P=0.0005 and P<0.0001, respectively. ns, not significant, P≥0.05. Note that the quantification presented in E is also shown in Fig. 7F, as these data concern the same sets of experiments and xenografts. Data from J are used for the mRNA expression comparison shown in Fig. 7H. Note that several transgenic backgrounds were used (see Table S1).