FGFR4 is overexpressed in a highly aggressive GBM subgroup, associated with tumor recurrence. AFGFR4 mRNA levels in non-tumor brain (n = 28) and GBM (n = 219) in the REMBRANDT dataset are shown as violin plots. B Kaplan Meier survival analysis of GBM patients from the REMBRANDT dataset stratified for FGFR4 expression (n = 99.) CFGFR4 mRNA levels in non-malignant brain (yellow, n = 5), low- (blue, n = 147), and high- (red, n = 22) expressing GBM subgroups of the TCGA-GBM RNA sequencing data. DFGFR4 mRNA levels detected by qRT-PCR in GBM (n = 40), glioblastoma stem cells (n = 2, white) models, and non-malignant brain tissue extracts (n = 3, yellow) are shown relatively to Hep3B positive control (2^−ddCT). FGFR4^high and FGFR4^low GBM models selected for further analyses are highlighted, respectively. E Detection of FGFR4 (several bands due to increasing glycosylation) in membrane-enriched fractions from selected GBM cell models and Hep3B by Western blot, with β-actin as loading control. F FGFR4 IHC staining of BTL1376 tumor material is opposed to haematoxilin eosin (HE) stain. Scale bars: 50 µm. GFGFR4 mRNA data (TCGA-GBM-HG-U133A) of primary (n = 497) and recurrent (n = 16) GBM are visualized. HFGFR4 mRNA levels in patient-matched primary GBM (n = 14) and sequential recurrences (rec1, n = 13; rec2, n = 2; rec4, n = 1). Statistical analyses: log-rank test B; Wilcoxon test C; maximization of the t-statistics D; Student’s t-tests A,G,H;. n.s. = not significant,  *p < 0.05, **p < 0.01, ***p < 0.001

FGFR4 overexpression promotes GBM aggressiveness. A Volcano blot showing differentially expressed genes (DEGs) of TCGA-GBM RNA sequencing data in FGFR4^high (red) versus FGFR4^low (blue) GBM. Top 15 genes are annotated and FGF19 is highlighted. Adjusted p-value < 0.05. B Gene sets significantly enriched in the FGFR4^high GBM subgroup are indicated. GSEA of DEGs (in A) were performed. Selected gene ontologies are plotted. C Scheme of the FGFR4-388Gly-GFP protein. (D + E) Clonogenicity D and proliferation capacity E of the FGFR4-388Gly-overexpressing and GFP-transduced, endogenously FGFR4^low GBM models are shown (means ± SEM from three independent experiments). F Filter-migration capacities of FGFR4-388Gly-overexpressing normalized to GFP-transduced endogenously FGFR4^low GBM models are shown (mean ± SEM from three experiments). Representative photographs are shown. G Wound-healing capacity of FGFR4-388Gly-overexpressing and GFP-transduced U251-MG cells in response to FGF19 stimulation (50 ng/ml) is shown at the indicated time points. Results were normalized to the respective FGF19-unstimulated conditions (means ± SD from three experiments). Red asterisks: Significance FGF19-stimulated versus -unstimulated. Statistical analyses: 2-way ANOVA/Bonferroni correction in D,E,G; Student ‘s t-tests in (F). *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant

Inactivation of FGFR4 reduces proliferative capacity and promotes cell death. A Schemes of the kinase domain-truncated, CFP-coupled tFGFR4 (left) and of the point-mutated (mut), kinase-dead FGFR4-KD(K504M) (right), GFP-coupled proteins are shown. (B + F) Clonogenicity upon tFGFR4B, FGFR4-KD(K504M)(F) compared to GFP(B + F, set to 1) transduction was analyzed in endogenously FGFR4^high (BTL1376, BTL1528) or FGFR4^low (U251-MG, BTL1529, BTL53) cell models (mean ± SD from three experiments). C Sphere formation potential of GSC models upon tFGFR4 or GFP transduction (mean ± 25%CI). D Survival/proliferation capacities over time are shown for tFGFR4- and GFP-transduced GBM models. For each model, one representative out of three experiments in duplicates is depicted. E Cell death induction upon tFGFR4 or GFP transduction in GBM models after 3 days. Annexin/PI staining was analyzed by flow cytometry. Significance levels were calculated comparing tFGFR4- to GFP-transduced cells on the respective living or dead fraction. Statistical analyses: 2-way ANOVA/Bonferroni correction (mean ± SD) (B,D,E,F); Student’s t-tests (C). *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant

FGFR4 inactivation attenuates GBM cell migration, endothelial invasion, and adhesion. A Filter-migration of FGFR4-KD(K504M)- and GFP-transduced GBM sublines is shown (mean ± SEM from three experiments). Representative photographs are depicted. B Wound-healing capacities of BTL1528 FGFR4-KD(K504M)- and GFP-transduced cells were evaluated (means ± SEM of one representative experiment in triplicates). C Trans-endothelial invasion capacity of FGFR4-KD(K504M)- and GFP-transduced BTL1376 neurospheres into m-cherry-tagged blood endothelial cells (BEC) was evaluated by live-cell microscopy (left). Generated “wounds” after 6 h co-culture were normalized to the respective sphere sizes (middle). Representative photomicrographs of tumor spheres (GFP), BEC (m-cherry) and invasion wounds (highlighted in turquoise) are depicted (right).DKEGG_FOCAL_ADHESION gene set from GSEA analyses of BTL1528 GFP versus FGFR4-KD(K504M) cells is shown. E FAK expression and phosphorylation and talin expression in the indicated FGFR4-modulated GBM sublines compared to GFP-transduced controls was detected by Western blotting with β-actin as loading control. Ratios were calculated by normalization to β-actin and are shown as fold change to GFP-transduced cells. F Adhesion capacities of BTL1528 FGFR4-KD(K504M) and GFP control cells as percentage of well surfaces covered with cells (means ± SEM of three experiments) (left). Outspread cells (120 min) were counted microscopically (normalized to GFP control cells) (right).G Re-differentiation capacity of the indicated GBM spheroids from FGFR4-KD(K504M) cells normalized to the respective GFP controls are shown (mean ± SEM from three experiments). Statistical analyses: Student’s t-tests (A),(C middle; F right); area under the ROC curve analysis (B); 2-way ANOVA/Bonferroni correction (F left, G). *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant; NES = normalized enrichment score, padj = adjusted p-value

FGFR4 blockade results in loss of GBM cell adhesion and sensitizes towards the RGD-mimetic cilengitide. A-C,EREACTOME gene sets enriched in BTL1528 GFP versus FGFR4-KD(K504M) gene expression data based on GSEA. NES = normalized enrichment score, padj = adjusted p-value. D Adhesion capacities of BTL1528 FGFR4-KD(K504M) and respective GFP cells towards collagen as percentage of cell-coated well surfaces (means ± SEM of three experiments (left). Exemplary photomicrographs are shown. Outspread cells after 60 min were counted microscopically and data normalized to GFP control cells (right).F Integrin-mediated cell adhesion arrays of FGFR4-KD(K504M)- and GFP-transduced BTL1528 subclones are shown (means of two experiments). (G + H) Viability of BTL1528 FGFR4-KD(K504M)- and GFP-expressing GBM cells in response to single-agent cilengitide (G), combined-agent cilengitide + ponatinib (pon.) (H left) and cilengitide + BLU554 (H middle) was evaluated by MTT assays. For each panel, one representative out of three experiments is shown as mean ± SD from triplicates. Combination indices (CI) are given for selected drug concentrations combining cilengitide with ponatinib (blue) or BLU554 (black) (H right). CI values < 0.9 were considered synergistic [22]. Statistical analyses: 2-way ANOVA/Bonferroni correction (D left, F–H), Student’s t-tests (D right). *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant, n.d. = not detected

FGFR4 impacts on tumorigenicity and tumor growth. A-F SCID/CB17 mice were subcutaneously injected with wild-type FGFR4-388Gly-expressing, endogenously FGFR4^low U251-MG cells or GFP-transduced controls (n = 4/group) (A-C) or kinase-dead FGFR4-KD(K504M)-expressing, endogenously FGFR4^high BTL1528 cells or GFP-transduced controls (n = 7/group) D-F. Tumor growth curves (mean tumor volume ± SEM). +  = mouse sacrificed (A/D), Kaplan Meier survival curves (B/E), tumor take (C/F, left), and representative cryo-sections of DAPI stained tumor xenografts (C/F, right) are shown. G Western blot analysis of the indicated proteins in U251-MG (left) or BTL1528 (right) FGFR4-altered xenografts (compare panels A-F). β-actin served as loading control. H Orthotopic tumor formation of BTL1528 GFP and FGFR4-modulated sublines in mice (n = 5/group) is shown. (I) Representative photomicrographs of a BTL1528 GFP orthotopic tumor stained with HE (left panels) or GFP by IHC (right panels). J Primary tumor area over time (left) and extra-primary tumor cell migration in zebrafish larvae (right) of BTL1528 GFP- or FGFR4-KD(K504M)-expressing models one-day post injection (dpi). Data from the independent experiments are shown. Representative photomicrographs of zebrafish larvae are depicted in the lower panels. black arrows: primary tumors, white arrows: extra-primary site tumor cell clusters; 2-way ANOVA (A + D; J left), log-rank (Mantel-Cox) test (B + E), or Student’s t-test (J right). n.d. = no detectable tumors, n.s. = not significant, *p < 0.05, ***p < 0.001. GLY = FGFR4-388Gly, KD = FGFR4-KD(K504M), s.c. = subcutaneous, short/long: exposure times