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Fig. 8 VPM1002 induction of bladder cancer cell clearance and apoptosis depends on TNF signaling. (A) Representative confocal images of NMIBC-RT112 control and VPM1002-treated xenografts exposed to either DMSO or pentoxifylline (PTX), in which human cancer cells were labelled with the Deep Red Cell Tracker lipophilic stain (not shown) and for the apoptosis marker activated caspase-3 (green) at 4 dpi. (B) Representative confocal images of infiltrating macrophages (red) in NMIBC-RT112 control and VPM1002+booster-treated xenografts exposed to either DMSO or PTX at 4 dpi. (C) Representative confocal images of Tnfa expression (green) and macrophages (red) in NMIBC-RT112 control and VPM1002+booster-treated xenografts exposed to either DMSO or PTX at 4 dpi. In A-C, 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. (D) Quantification of the percentage of clearance in NMIBC-RT112 control and VPM1002+booster-treated xenografts exposed to either DMSO or PTX at 4 dpi (**P=0.0031; 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. (E) Quantification of the percentage of activated caspase-3-positive (apoptotic) cells in NMIBC-RT112 control and VPM1002-treated xenografts exposed to either DMSO or PTX at 4 dpi (*P=0.0165; ***P=0.0002; ****P<0.0001). Each dot represents one xenograft pooled from three independent experiments. (F) Quantification of absolute numbers of infiltrating macrophages in NMIBC-RT112 control and VPM1002-treated xenografts exposed to either DMSO or PTX at 4 dpi (***P=0.0002; ****P<0.0001). Macrophages were quantified using Tg(mpeg1:mCherry) and Tg(csf1ra:GFP). Each dot represents one xenograft pooled from three independent experiments. (G) Quantification of the percentage of M1-like (Tnfa- or NFκB-positive macrophages, Mpeg1+) and M2-like (Tnfa- or NFκB-negative macrophages, Mpeg1+) in the TME of NMIBC-RT112 control and VPM1002-treated xenografts exposed to either DMSO or PTX at 4 dpi (**P=0.0025; ****P<0.0001). Each dot represents one xenograft pooled from two independent experiments. (H) Representative confocal images of control and zebrafish Tnfa (zTnfa)-treated NMIBC-RT112 cells stained for the actin filament marker phalloidin (green), activated caspase-3 (white) and nuclei (DAPI counterstaining, blue). Scale bars: 50 µm. (I) Relative in vitro gene expression levels of human TNFA, LTA, TNFRSF1A, TNFRSF1B, TNFRSF21 and C1QTNF6 in the NMIBC-RT112 cell line. Bars indicate the fold change of expression relative to that of the housekeeping gene. Each dot represents the average of two or three technical replicates of one independent experiment. (J) Quantification of the percentage of activated caspase-3-positive cells per field in control and zTnfa-treated NMIBC-RT112 cells at 8 and 24 h post treatment. Each dot represents one random field of view (****P<0.0001). (K) Quantification of the mean absolute number of cells per field in control and zTnfa-treated NMIBC-RT112 cells at 8 and 24 h post treatment. Each dot represents one quantified well (**P<0.01). In J,K, data were pooled from two independent experiments. In D-G,I-K, bars indicate the results as mean±s.d. Data sets with a Gaussian distribution (F,G,J,K) 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 (E). Unless stated otherwise, each experimental data set was challenged to the respective control. Additionally, data sets in E-G were analyzed with Welch's one-way ANOVA with Games–Howell post hoc test in which P<0.0001 for the three conditions. ns, not significant, P≥0.05. Note that several transgenic backgrounds were used (see Table S1).