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Fig. 3 Macrophages are essential for susceptibility of zebrafish bladder cancer xenografts to BCG immunotherapy. (A) Representative confocal images of infiltrating macrophages (red) in BCG/L-clodronate experiments. (B) Representative confocal images of NMIBC-RT112 xenografts stained for the apoptosis marker activated caspase-3 (green) in BCG/L-clodronate experiments. In A,B, human cancer cells were labelled with the Deep Red Cell Tracker lipophilic stain (not shown), and BCG were labelled with either the Deep Red Cell Tracker or the Vybrant CM-DiI lipophilic stain (not shown). White dashed regions outline the tumor. In all images, anterior is to the left, posterior to the right, dorsal up and ventral down. Scale bars: 50 µm. (C) Quantification of the absolute numbers of infiltrating macrophages in BCG/L-clodronate experiments (***P=0.0001). Each dot represents one xenograft pooled from two independent experiments. (D) Quantification of the percentage of clearance in BCG/L-clodronate experiments at 4 dpi (**P<0.01; ****P<0.0001; Fisher's exact test). Each dot represents a full round of injections and N represents the number of xenografts without tumors at 4 dpi relative to the total number of xenografts at 4 dpi. (E) Quantification of the percentage of activated caspase-3-positive (apoptotic) cells in BCG/L-clodronate experiments at 4 dpi (*P=0.0102; ****P<0.0001). Each dot represents one xenograft pooled from three independent experiments. Bars indicate the results as mean±s.d. and the numbers of analyzed xenografts are indicated in A,B. 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 were analyzed with Welch's one-way ANOVA with Games–Howell post hoc test in which P<0.0001. Xenografts represented in A,B correspond to the same sets of experiments and genetic background in which transgenic larvae were also labeled for activated caspase-3. Quantitative data shown in C are also shown in Fig. 2B as these data concern the same sets of experiments and xenografts. The experiments in this figure were performed in parallel with those in Fig. S5; thus, they share the same controls. Note that several transgenic backgrounds were used (see Table S1).