Fig 2: Justification of the Prognostic Value of the RM-RM in Two BLCA Databases and Real-World Study. A Kaplan–Meier analysis for overall survival (OS) curves of patients in low or high drug resistance and metabolism-related risk score (RM-RS) subgroups from two independent validation cohorts (GSE69795, GSE31684). B The receiver operating characteristic (ROC) curves of 1-, 3-, and 5 year OS of patients in GSE69795 and of 3-, 5-, and 10 year OS of patients in GSE31684 were predicted based on RM-RS. (C) The ROC curve of RM-RS was compared with that of only other clinical features in GSE69795 and GSE31684. D Univariate and multivariate Cox regression analyses of RM-RS and only other clinical features in GSE69795 and GSE31684. E, F Immunohistochemical (IHC) staining was used to detect the protein expression of metabolism-related differentially expressed genes (RM-DEGs) (FASN, MAP2, BMP6, GPC2, CNOT6L, GALNT12 and CARD10) in 60 normal tissues and 170 tumor tissues. The immunohistochemical staining immune response score (IRS) score was statistically analyzed and the violin diagram shows a representative image. G, H pRM-RS was obtained by IRS and RM-RM. The median pRM-RS was divided into a high-risk group and a low-risk group, and KM analysis and difference analysis of other clinical features between the two subgroups were performed. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig 3: Upregulation of FASN promotes drug resistance and poor prognosis in BLCA. A The lipid content of bladder cancer cells and gemcitabine-resistant cells was quantified by BODIPY staining corrected total cell fluorescence (CTCF). B The contents of free fatty acids (FFAs), triglycerides (TGs) and total cholesterol (T-CHO) were used as intracellular lipid indexes. C The expression of FASN in bladder cancer cells resistant to different concentrations of gemcitabine was detected by Western blotting (WB). Density measurement and statistical analysis. Representative images are shown. D The expression of FASN in gemcitabine-resistant bladder cancer cells after FASN knockdown was detected by WB. E, F Cell viability and sensitivity to gemcitabine under all conditions were determined by CCK-8 assay. G The tumorigenic ability of single cells under all conditions was determined by colony formation assay. H, I The lipid content of gemcitabine-resistant cells after FASN knockdown was quantitatively detected by BODIPY staining corrected total cell fluorescence (CTCF) and the contents of free fatty acids (FFAs), triglycerides (TGs) and total bilirubin (T-CHO). J, K, and L Mice with stable knockdown expression of T24-R xenografts were treated with vector control or gemcitabine (50 mg/kg. IP. QOD) for approximately 5 weeks. Tumor volumes were measured every 5 days (n = 5 per group). Tumors were weighed after resection. The graphs show the means ± SEMs. One-way ANOVA followed by Tukey’s multiple comparison test. α = 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****p < 0.0001; ns, no significance

Fig 4: | TVB-3166 inhibited BLCA progression and reversed gemcitabine resistance. A, B Cell viability and sensitivity to gemcitabine under all conditions were determined by CCK-8 assay. C The tumorigenic ability of single cells under all conditions was determined by colony formation assay. D, E The lipid content of gemcitabine-resistant cells after treatment with TVB-3166 was quantitatively detected by BODIPY staining corrected total cell fluorescence (CTCF) and the contents of free fatty acids (FFAs), triglycerides (TGs) and total cholesterol (T-CHO). F, G, and H The mice were divided into 4 groups with 5 mice in each group: Group I (DMSO: DMSO), group II (DMSO: gemcitabine), group III (DMSO: TVB-3166) and group IV (gemcitabine: TVB-3166). Tumor volumes were measured every 5 days (n = 5 per group). Tumors were weighed after resection. The graphs show the means ± SEMs. One-way ANOVA followed by Tukey’s multiple comparison test. α = 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****p < 0.0001; ns, no significance. I Enzyme-linked immunosorbent assay (ELISA) was used to determine the FASN content of xenograft tumors in each group. J Statistical analysis was performed on the rate of KI67- and TUNEL-positive cells in each group of xenograft tumors by immunohistochemical staining (IHC). The corrected total cell fluorescence (CTCF) of BODIPY staining was used to quantitatively detect the lipid content of xenograft tumors in each group. Representative images are shown