Extended Data Fig. 9: Antitumor effects of the Gremlin1 antibody are acted through the FGFR1 inhibition.
(a) Binding specificity of the anti-human Gremlin1 to Gremlin1 is validated by the enzyme-linked immunosorbent assay. Ab is anti-human Gremlin1 in this figure. (b) The antibody against Gremlin1 (100 ng/ml) facilitates the inhibition of in vitro cell proliferation by enzalutamide (10 μg/ml) (n = 3 independently treated cell cultures). (c) Anti-Gremlin1 treatment suppresses the sphere formation ability of LNCaP-R cells (n = 3 biological replicates). (d) Annexin-V/DAPI staining demonstrates that anti-Gremlin1 antibody displays a synergistic effect with enzalutamide in inducing cell death (n = 2 independently treated cell cultures, experiments have been repeated at least 3 times). (e) The activation of FGFR1/MEK/ERK signaling pathway is suppressed by the Gremlin1 antibody in LNCaP-R cells. The experiment was repeated at least 3 times with similar results. (f) Immunoblotting confirms the efficiency of BMPR2 knockout in LNCaP-R cells. The experiment was repeated at least 3 times with similar results. (g) BMPR2 knockout shows no significant influence to the inhibitory effect of Gremlin1 antibody (10 μg/ml) on LNCaP-R cell proliferation (n = 3 independently treated cell cultures) and sphere formation (n = 3 biological replicates). Ab: anti-Gremlin1. ADT: treated with enzalutamide at 10 μg/ml. (h) Anti-Gremlin1 antibody (10 μg/ml) suppresses PCa PDO formation in a serial organoid forming assay (n = 3 biological replicates). (i) Expression levels of FGFR1 in four patient-derived xenograft lines are assessed by immunoblotting. The experiment was repeated at least 3 times with similar results. (j) FGFR inhibitor BGJ398 decreases organoid forming capacity of BM1 and BM61 PDX lines. No additive effect is detected between BGJ398 (1 μM) and Gremlin1 antibody (10 μg/ml) treatment (n = 3 biological replicates). Scale bars= 50 μm. FGFRi, FGFR1 inhibitor BGJ398. (Two-tailed Student’ s t test was used for the statistical analysis and the tumor size at the end timepoint was analyzed. Data are presented as means ± s.e.m.).
