BBN Bladder Cancer Mouse Model.

Bladder cancer was induced in 30 seven-week-old C57BL/6J male mice by administering 0.1% BBN in drinking water for 6 wk. The mice were then randomized into DMSO (n = 15) or lometrexol treatment (n = 15, 15 mg/kg) groups and were given intraperitoneal injections of the respective treatments twice a week for 12 wk. At the end of the treatment period, the bladder tissues of the mice were harvested for hematoxylin and eosin stain (H&E) staining and RNA isolation. Each mouse's tumor grade was assessed as we have previously described (41), in alignment with the American Joint Committee on Cancer (AJCC) pathologic staging guidelines for bladder cancer, and mRNA samples were analyzed for mutations in the Kmt2d/Mll4 gene through RNA-seq.