The orthotopic MB49 model

The tumorigenic cell line MB49 (Mouse Bladder-49) was kindly provided by Dr K. Esuvaranathan, National University of Singapore, Singapore. The original stock vial was expanded in culture to a low passage clone and cryopreserved in liquid nitrogen as a primary cell line stock for rederivation purposes. Fig 2B, 2C and 2E shows the expression pattern of known cell adherence, urothelial and immune associated markers on the MB49 cell line. Cells used in the experiments were thawed and cultured for not more than 7 passages. The cell culture and the orthotopic MB49 model instillation were performed as previously described [54], the animals were sacrificed and tumors were harvested after 5 days. The anesthesia used was 75 mg/kg ketamine and 1 mg/kg medetomidine administered intraperitoneally and reversal was induced by 1 mg/kg atipamezole administered subcutaneously, both at 0.1 ml per 10 g of body weight. During the duration of the experiment the animals were weighed and examined twice weekly. Urine and serum were collected 1 day prior to and 5 days post intravesical instillation of MB49 cells. The humane endpoint for the orthotopic MB49 model was the same as for carcinogen induced bladder cancer: 10 days of visible low hematuria or 5 days of complete hematuria or 15% body weight loss. In the experiments performed no MB49 tumor bearing animals reached the humane endpoint.

(A) For cell line generation, female Hgf-Cdk4^R24C mice were exposed for 10–11 weeks to OH-BBN in drinking water. After OH-BBN discontinuation, mice were monitored for 1, 5 or 10 weeks and sacrificed. Six single cell colonies were randomly selected and characterized by (B) expression of surface markers by flow cytometry or (C) qRT-PCR to determine the cell origin and molecular profile of the cells in comparison to established cell lines or healthy bladder urothelium. (D) Only the 74/7 cell line formed tumors after subcutaneous inoculation to C57BL/6 or Hgf-Cdk4^R24C mice in doses of 5 or 2x10⁶ cells, respectively. The new cell lines and MB49 cells were cultured with or without IFNγ (10 ng/ml) for 24h and (E) the expression of cell surface immune recognition markers was analyzed by flow cytometry (normalized to unstained control cells).