Fig. 6

IGF2BP1 inhibition promote T cell-mediated tumor cell killing and synergizes with PD-1 blockade. a ES-2 cells (low density: 6.500 cells/cm²; or high density: 32.500 cells/cm²) or human PBMCs (32.500 cells/cm²) were treated with indicated concentrations of BT or CUB for 72 h before cell viability was determined by confluence measurements using the Incucyte S3, Cell Titer Glo or propidium iodide staining to determine EC₅₀. Error bars indicate s.d. from N ≥ 3 experiments. b Cocultures of ES-2 cells with HLA-matched PBMCs were cultured for 36 h in the presence of caspase 3/7 green indicating T cell-mediated tumor cell killing. c Cocultures of ES-2 cells and PBMCs treated with BT (10 µM) or CUB (10 nM) for 36 h were analyzed by RNA-seq. Tumor and immune cell content is shown by indicated marker expression as a bubble chart. d GSEA of RNA-seq data from cocultures (c) revealed the enrichment of GOBP gene sets related to T cell activation (cf. Supplementary Table T9). e–g Cocultures of ES-2 cells and PBMCs were treated as in (c). ELISA (e) of cleared supernatants was used to determine IFNγ secretion of T cells. Flow cytometry with indicated antibodies (f) was used to assess alterations in MHC-I and PD-L1 presentation on tumor cells. Intracellular GZMB staining (g) of fixed tumor cells analyzed by flow cytometry are shown as histograms. Numbers indicate MFI (f) or percentage of cells (g). S.d. and statistical significance by unpaired two-tailed t testing was determined for N ≥ 3 experiments. h Indicated T cell activation and exhaustion markers from RNA-seq experiments (c) are shown as a bubble chart. i Cocultures of ES-2 cells with HLA-matched PBMCs were treated with indicated concentrations of Nivolumab in the absence (0.05% DMSO) or presence of BT (5 µM). Relative tumor cell numbers and T cell-mediated tumor cell killing monitored by caspase 3/7 green was used to determine EC₅₀ concentrations. Synergism was evaluated by the combination index (CI)⁴⁵ and the coefficient of drug interaction (CDI).⁴⁶ Synergy is indicated for values <1. j Schematic illustrates treatment groups and regimens of ID8/Trp53-/- cells engrafted in immuno-competent mice (left). Differences in the survival of each treatment group are shown as Kaplan–Meier analyses with HR values < 1 indicating survival benefits (right). Numbers of mice per condition are indicated. Statistical significance was determined by logrank testing. k Ascites fluids from 6 mice per treatment (j) were pooled and analyzed by scRNA-seq. UMAP plot shows identified cell clusters (left). Cluster distribution across treatment conditions (fraction; middle) and proportional cell content (% cells; right) are presented as a bar diagram. l Pearson correlations of IGF2BP1 expression and indicated immune subsets were analyzed in C2 and C5 subtypes of the TCGA-RNAseq cohort (n = 121) and the AOCS dataset (n = 105) using indicated marker genes. Results are presented as a forest plot showing Pearson’s r with 95% confidence intervals; box sizes reflect sample size, and color indicates significance of the correlation (left). Random effect models were applied for each immune subset across datasets to estimate the overall correlation and heterogeneity indicated by I² (right). m Schematic illustrates the potential mechanism, how the IGF2BP1-directed inhibitor BT might synergize with PD-1 blockage to promote T cell-mediated killing of HGSC cells