Fig. 4

Activation of ZEB1 and tumor migration is dependent on 4-1BBL expression and NK cells A uMAP projection of single cell transcriptomics from primary UM tumors (GSE139829, n = 8) showing the expression profile of TNFSF9 and TNFRSF9 in different cell types. B Normalized gene expression of TNFSF9 comparing tumor cells of GEP class 1 over class 2. Mann–Whitney was used to test for significance. C Normalized gene expression of TNFSF9 comparing GEP class 2 tumor cells that are PRAME + over PRAME- cells. Mann–Whitney was used to test for significance. D LEFT: Representative image of realtime NK cell cytotoxicity captured at 48 h. RIGHT: Realtime NK cell cytotoxicity assay over 48 h showing relative increase in green fluorescence due to dye detecting activated caspase-3/7 in tumor cells. Two-way ANOVA was used to test for significance between the two conditions. E LEFT: Representative flow histogram of ZEB1 expression. RIGHT: Mean fluorescence intensity (MFI) of ZEB1 protein expression in parental wild type (WT) and 41BBL-KO OCM-3 cells after 3 days of NK cells co-culture. F LEFT: Representative image of transwell migration showing GFP-OCM3 cells under 5X objective magnification. Scale bar denotes 1 cm. RIGHT: Relative migration of WT and 41BBL-KO OCM-3 tumor cells. Prior to the 48 h transwell migration assay, tumor cells were co-cultured for 3 days with NK cells before harvested for assay. E, F, One-way ANOVA was used to test for significance (n = 4)